Pscaxcd3, cd19xcd3, c-metxcd3, endosialin xcd3, epcamxcd3, igf-1rxcd3 or fap-alpha xcd3 bispecific single-chain antibody with inter-species specificity

FIELD: medicine.

SUBSTANCE: present invention refers to immunology. Presented is a molecule of bispecific single-chain antibody containing a first binding domain able to bind to epitope of CD3-epsilon-chain of human and Callithrix jacchus (tamarin), Saguinus oedipus (cotton-top tamarin) and Saimiri sciureus (squirrel monkey), and a second binding domain able to bind to an antigen specified in a group consisting of: PSCA, CD19, C-MET, endosialin, EGF-like domain 1 EpCAM coded by exon 2, FAP-alpha or IGF-IR (or IGF-1R) or a human and/or a primate. The epitope CD3e contains an amino acid sequence disclosed in the description. Disclosed are a nucleic acid coding the above molecule of the bispecific single-chain antibody, an expression vector, a host cell and a method for producing the antibody, as well as the antibody produced by the method. Described is a based pharmaceutical composition containing the molecule of the bispecific single-chain antibody and a method for preventing, treating or relieving cancer or an autoimmune antibody. Presented is using the above molecule of the bispecific single-chain antibody for making the pharmaceutical composition for preventing, treating or relieving cancer or the autoimmune disease.

EFFECT: using the invention provides the clinical improvement in relation to T-cell redistribution, reducing it, and the improved safety profile.

23 cl, 74 dwg, 17 tbl, 33 ex

 

The present invention relates to a molecule especifismo single-chain antibodies containing the first binding domain capable of contacting epitope of CD3-Epsilon-chain of human and Primate, non chimpanzees, where the specified epitope is part of an amino acid sequence that is a member of the group consisting of SEQ ID NO: 2, 4, 6 and 8, and a second binding domain capable of contacting the antigen is selected from the group consisting of stem cell antigen of the prostate (PSCA), antigen of b-lymphocytes CD19 (CD19), receptor growth factor hepatocyte (C-MET), indocyanine, EGF (epidermal growth factor)-like domain 1 of the adhesion molecule of epithelial cells (Ersam), encoded by exon 2, protein activation of fibroblasts alpha (FAP-alpha) and receptor insulin-like growth factor I (IGF-IR or IGF-1R). According to the proposed invention is also nucleic acids that encode the specified molecule especifismo single-chain antibodies, and vectors and the host cell and the method of their preparation. The invention also relates to pharmaceutical compositions containing the specified molecule especifismo single-chain antibodies, and to medical applications of the indicated molecules especifismo single-chain antibodies.

T-cell recognition mediated clonotypes distributed and�the-beta and gamma-Delta T-cell receptors (TcR), which interact with peptide-loaded molecules peptide-MHC (major histocompatibility complex) (rmns) (Davis &Bjorkman, Nature 334 (1988), 395-402). Antigen-specific chain TcR do not contain signaling domains, but instead are associated with a conservative multi-subunit machine signal transmission CD3 (Call, Cell 111 (2002), 967-979; Alarcon, Immunol. Rev. 191 (2003), 38-46; Malissen Immunol. Rev. 191 (2003), 7-27). The mechanism by which TcR-ligation is directly connected with the device signal transmission, remains a fundamental issue T-cell biology (Alarcon, supra; Davis, Cell 110 (2002), 285-287). It seems obvious that in sustained T-cell responses involved contact with the receptor, the TcR oligomerization and organization of complexes of TcR-pMHC higher order in the immunological synapse (Davis & van der Merwe, Curr. Biol. 11 (2001), R289-R291; Davis, Nat. Immunol. 4 (2003), 217-224). However, the earliest transmission of a signal through the TcR occurs in the absence of these events and can lead to ligand-induced change in the CD3-Epsilon (Alarcon, supra; Davis (2002), supra; Gil, J. Biol. Chem. 276 (2001), 11174-11179; Gil, Cell 109 (2002), 901-912). Epsilon-, gamma-, Delta - and Zeta-subunit signaling complex associated with each other with the formation of the CD3-Epsilon-gamma heterodimer, CD3-Epsilon-Delta heterodimer and CD3-Zeta Zeta of homodimer (Call, see above). Various studies revealed that CD3 molecules important for nadlezhascheyzaschity on the cell surface alpha-beta TcR and normal development of T cells (Berkhout, J. Biol. Chem. 263 (1988), 8528-8536; Wang, J. Exp.Med. 188 (1998), 1375-1380; Kappes, Curr. Opin. Immunol. 7 (1995), 441-447). The study of the structure of fragments of ectodomains murine CD3-Epsilon-gamma heterodimer showed that the Epsilon-gamma-subunits represent a C2-set lg domains that interact with each other with the formation of unusual transverse (side-to-side) dimeric configuration (Sun, Cell 105 (2001), 913-923). Although rich in cysteine residues of the "stem", apparently, plays an important role in triggering the dimerization CD3 (Su, see above, Borroto, J. Biol. Chem. 273 (1998), 12807-12816), through the interaction of the extracellular domains of the CD3-Epsilon and CD3-gamma is sufficient for complex formation of these proteins with TcR-beta (Manolios, Eur. J. Immunol. 24 (1994), 84 to 92; Manolios & Li, Immunol. Cell Biol. 73 (1995), 532-536). Despite the ongoing discussion on this issue, the predominant TcR stoichiometry is likely to contains one alpha-beta TcR, one CD3-Epsilon-gamma heterodimer, one CD3-Epsilon-Delta heterodimer and one CD3-Zeta-Zeta C) an homodimer (Call, see above). Considering the Central role of the human CD3-Epsilon-gamma heterodimer in the immune response was not so long ago the crystal structure of this complex is associated with therapeutic antibody AKT (Kjer-Nielsen, PNAS 101, (2004), 7675-7680).

Many therapeutic strategies based on modulating T-cell immunity by targeting the transmission signal�and through TcR, in particular monoclonal antibodies (mAb) against human CD3, which are widely used in clinical regimen of administration of immunosuppressive drugs. CD3-specific murine mAb ACT was the first mAb, approved for use in humans (Sgro, Toxicology 105 (1995), 23-29), and it finds wide clinical use as an immunosuppressant in the transplantation (Chatenoud, Clin. Transplant 7 (1993), 422-430; Chatenoud, Nat. Rev. Immunol. 3 (2003), 123-132; Kumar, Transplant. Proc. 30 (1998), 1351-1352), while type 1 diabetes (Chatenoud (2003), supra) and in psoriasis (Utset, J. Rheumatol. 29 (2002), 1907-1913). Moreover, anti-CD3 mAb can induce partial T-cell signal transmission and clonal tolerance (Smith, J. Exp.Med. 185 (1997), 1413-1422). ACT described in the literature as a potent T-cell mitogen (Van Wauve, J. Immunol. 124 (1980), 2708-18) and as a potent T-cell killer (Wong, Transplantation 50 (1990), 683-9). OCT shows these types of activity are time-dependent manner, and after an early activation of T cells, leading to release of cytokines, the subsequent introduction OCT blocks all known T-cell function. This helps later to block T-cell function OCT found such widespread use as an immunosuppressant in the courses of therapy with the aim of reducing or even cancelling the rejection of tissue allograft.

ACT reverses the rejection of tissue allograft probably by blocking the function of all t-cells, which plays a major role in acute rejection. OCT interacts with the CD3 complex and blocks the function of the CD3 complex in the membrane of T-cells, which is associated with the antigen recognition structure of T cells (TCR) and is important for signal transduction. The subject of many studies was the question of what is the subunit of TCR/CD3 associated OCT. Some facts demonstrate specificity OCT to the Epsilon-subunit complex of the TCR/CD3 (Tunnacliffe, Int. Immunol. 1 (1989), 546-50; Kjer-Nielsen, PNAS 101, (2004), 7675-7680). Additional evidence suggests that binding ACT with a complex of TCR/CD3 requires the presence of other subunits of this complex (Salmeron, J. Immunol. 147 (1991), 3047-52).

Other well known antibody, with specificity for the CD3 molecule are listed in Tunnacliffe, Int. Immunol. 1 (1989), 546-50. As indicated above, these CD3-specific antibodies are able to induce different T-cell responses such as the production of lymphokines (Von Wussow, J. Immunol. 127 (1981), 1197; Palacious, J. Immunol. 128 (1982), 337), proliferation (Van Wauve, J. Immunol. 124 (1980), 2708-18) and induction of T-cell suppressor (Kunicka, "Lymphocyte Typing II 1 (1986), 223). That is, depending on the experimental conditions CD3-specific monoclonal antibody can either inhibit or to induce cytotoxicity (Leewenberg, J. Immunol. 134 (1985), 3770; Phillips, J. Immunol. 136 (1986) 1579; Platsoucas, Proc. Natl. Acad. Sci. USA 78 (1981), 4500; Itoh, Cell. Immuol. 108 (1987), 283-96; Mentzer, J. Immunol. 135 (1985), 34; Landegren, J. Exp.Med. 155 (1982), 1579; Choi (2001), Eur. J. Immunol. 31, 94-106; Xu (2000), Cell Immunol. 200, 16-26; Kimball (1995), Transpl. Immunol. 3, 212-221).

Although it is known that many antibodies against CD3, described in the field, recognize the CD3-Epsilon-subunit of the CD3 complex, most of them in fact are associated with the conformational epitopes and, therefore, recognize the CD3-Epsilon only in the native environment of the TCR. Conformational epitopes are characterized by the presence of two or more individual amino acid residues that are separated in primary sequence, but come together on the surface of the molecule when the polypeptide folds into the native protein/antigen (Sela, (1969) Science 166, 1365; Laver, (1990) Cell 61, 553-6). Conformational epitopes bound by antibodies against CD3-Epsilon, described in this area can be divided into two groups. In the main group of these epitopes are epitopes formed by the two subunits of CD3, such as CD3-Epsilon-chain and CD3 gamma or CD3-Delta chain. For example, multiple studies, it was found that the most widely used monoclonal antibodies against CD3-Epsilon, namely OCT, WT31, UCHT1, 7D6 and Leu-4, do not bind to cells singly transfected by CD3-Epsilon chain. However, these antibodies stained cells twice transfetsirovannyh combinations�Oia CD3-Epsilon plus or CD3-gamma, either CD3-Delta (Tunnacliffe, supra; Law, Int. Immunol. 14 (2002), 389-400; Salmeron, J. Immunol. 147 (1991), 3047-52; Coulie, Eur. J. Immunol. 21 (1991), 1703-9). The second, smaller group of the conformational epitope is formed within the CD3-Epsilon subunit. A member of this group is, for example, mAb ARA 1/1 induced against denaturated CD3-Epsilon chain (Risueno, Blood 106 (2005), 601-8). In aggregate, the majority of antibodies against CD3-Epsilon, described in the field, recognize conformational epitopes located on two or more CD3 subunits. Individual amino acid residues that form the three-dimensional structure of these epitopes can be localized either on the CD3-Epsilon subunit, or CD3-Epsilon subunit and other subunits of CD3, such as CD3 gamma or CD3-Delta.

Another problem associated with antibodies against CD3, is that many antibodies against CD3, as it was established, are species-specific. Monoclonal antibodies against CD3 operated by highly specific recognition of its target molecules, which is true, generally, for other monoclonal antibodies. They recognize only a single site, or epitope, on their target molecule CD3. For example, one of the most widely used and best-characterized monoclonal antibodies specific to CD3-complex, is OCT-3. This antibody were�deistvuet with chimpanzee CD3, but not with the homologue CD3 other primates, such as macaques, or with CD3 dogs (Sandusky et al., J. Med. Primatol. 15 (1986), 441-451). Similarly, in WO 2005/118635 or WO 2007/033230 described human monoclonal antibodies against CD3-Epsilon, which interact with CD3-Epsilon person, but not with CD3-Epsilon, mouse, rat, rabbit or Primate, non chimpanzees, such as the rhesus monkey, cynomolgus macaque, or baboon. AHTH-CD3 monoclonal antibody UCHT-1 also interacts with CD3 chimpanzees, but not macaque CD3 (own data). On the other hand, there are also examples of monoclonal antibodies that recognize antigens macaque, but not their human copies. One example of this group is a monoclonal antibody FN-18, directed to macaque CD3 (Uda et al., J. Med. Primatol. 30 (2001), 141-147). Interests is the fact that found that peripheral lymphocytes from approximately 12% Javanese macaques do not interact with a monoclonal antibody against CD3 macaque-rhesus (FN-18) due to polymorphism of the CD3 antigen in macaques. Uda and co-authors described the replacement of two amino acids in the sequence CD3 Javanese macaques, which do not interact with the antibody FN-18, compared with CD3 animals that interact with the antibody FN-18 (Uda et al., J. Med. Primatol. 32 (2003), 105-10; Uda et al., J. Med. Primatol. 33 (2004), 34-7).

This discriminating ability, i.e., species specificity, characteristic not only of monoclonal� antibodies against CD3 (and their fragments), but monoclonal antibodies in General, significantly hampers their development as therapeutic agents for the treatment of human diseases. In order to obtain permission to sell any new drug candidate must undergo rigorous testing. This testing can be divided into preclinical and clinical phase and clinical phase is further divided into well-known clinical phase I, II and III, which are conducted on the patients-the people and pre-clinical phase is conducted on animals. The goal of preclinical testing is to prove that a drug candidate has the desired activity and, very importantly, is safe. Only after establishing the safety and possible efficacy of the product candidate in preclinical animal testing is a drug-candidate will be approved by the appropriate regulatory authority for clinical trials in humans. Drug candidates can be tested in terms of safety for the animals in the following three ways: (1) on the relevant form, i.e. the form in the body which drug candidates can recognize ontologicheskie antigens, (2) transgenic animal containing human antigens, and (3) by using the artificial�RA drug candidate, which can contact ontologicheskie antigens present in the body of the animal. Restrictions on transgenic animals are that this technology is usually limited to rodents. Between rodents and man, there are significant differences in physiology, and the results of safety cannot be easily extrapolated to humans. Restrictions on using the simulator of the drug candidate are different composition of a substance compared to real drug candidate, and often the animals used are rodents with the limitation as discussed above. Therefore, preclinical data obtained in rodents, have limited predictive ability in relation to the drug candidate. The approach to solving the problem of selection of security testing is to use relevant species, preferably lower Primate, currently the restriction of monoclonal antibodies suitable for therapeutic intervention in humans and described in this area, is that relevant species are higher primates, in particular chimpanzees. Chimpanzees are considered endangered species, and their anthropomorphic nature of the use of such animals to test the safety of lekarstvennogo banned in European countries and severely restricted in other countries. CD3 has also been successfully used as targets for bespecifically single-chain antibodies with the purpose to redirect cytotoxic T cells to pathological cells and, as a result, patients withdraw cells from an appropriate organism (WO 99/54440; WO 04/106380). For example, Bargou with co-workers (Science 321 (2008): 974-7) recently reported clinical activity design CD19×CD3 especifismo antibodies, called blinatumomab, which has the potential to have contact with all cytotoxic T cells in patients-people for lysis of cancer cells. Low doses, such as from 0.005 milligrams per square meter per day, in patients with non-Hodgkin lymphoma led to the depletion of target cells in the blood. Partial and complete regression of tumors were first observed at a dose level of 0.015 milligrams, and all seven patients who were treated with doses of 0.06 milligrams, there was tumor regression. Blinatumomab also resulted in the clearance of tumor cells from bone marrow and liver. Although the study has provided clinical proof of concept for therapeutic efficacy of the format especifismo single-chain antibodies in the treatment of cancer of the blood cells, there still exists a need in the successful concepts of therapies for other cancers.

It is estimated that in the United States in 2008�do have 186320 men will be newly diagnosed prostate cancer, and about 28660 men will die from the disease. According to most previous reports on mortality from cancer in 2004, the death rate from prostate cancer among American men was 25 per 100,000. In the late 1980s, widespread use of the test on the specific antigen of the prostate (PSA) has played a major role in improving the treatment of prostate cancer. This test allows you to measure the amount of PSA protein in the blood that is often elevated in patients with prostate cancer. In 1986, the Directorate for control of food and drugs U.S. Food and Drug Administration) approved the use of the PSA test to monitor patients with prostate cancer and in 1994 further endorsed its use as a screening test in respect of this condition. Because of universal testing PSA in the U.S. currently, approximately 90 percent of all cases of prostate cancer diagnosed at an early stage and, therefore, men are living longer after diagnosis. However, to determine whether PSA screening saves lives, will need the results of two ongoing clinical trials, and it is sponsored by the National cancer Institute (NCI) Screening tests for pre�titelnoj cancer, lung, colorectal and ovarian (PLCO) and the European study of screening in relation to prostate cancer (ERSPC). In conducted over the past 25 years, clinical trials investigated the efficacy of natural and synthetic compounds in the prevention of prostate cancer. For example, in Test to prevent prostate cancer (RCRT), which was attended by about 19,000 healthy men, it was discovered that finasteride, a drug approved for the treatment of benign prostatic hyperplasia (BPH), which is a benign enlargement of the prostate gland, reduces the risk of developing prostate cancer by 25 percent. Another test, for the prevention of cancer with the use of selenium and vitamin E (SELECT), which involves more than 35,000 men, is conducted to determine whether daily supplements of selenium and vitamin E reduce the incidence of prostate cancer in healthy men. In other trials on the prevention of prostate cancer that are currently assessed the protective potential of multivitamins, vitamins C and D, soy, green tea and lycopene, a natural compound found in tomatoes. One study was about kotarumalos in 2005 showed that specific genes were merged in 60-80% of the analyzed tumors of the prostate. This study represents the first observation of non-random gene rearrangements in prostate cancer. This genetic variation can be used, ultimately, as a biomarker for diagnosis and possibly for the treatment of this disease. Other studies have shown that genetic variation in a specific region of chromosome 8 can increase the risk of developing prostate cancer in humans. These genetic variations explain approximately 25 percent of cases of prostate cancer that occur in white men. They are the first reliable genetic variants that increase the risk of developing prostate cancer and may help scientists better understand the genetic causes of this disease. Conducted a study that aimed to determine how the proteins circulating in the blood of the patient, can be used to improve diagnosis of prostate cancer and other cancers. In 2005 scientists have identified a group of specific proteins produced by the immune system of the patient in response to prostate tumors. These proteins, like antibodies, capable of detecting the presence of cells �aka prostate cancer in blood samples with an accuracy of over 90 percent. When used in combination with PSA these proteins and other blood proteins can, ultimately, be used to reduce the number of false-positive results, obtained only by testing for PSA, and therefore to reduce the many unnecessary prostate biopsies performed each year due to false-positive PSA test results.

In addition to PSA have been identified, several other markers for prostate cancer, including, for example, testimony transmembrane epithelial antigen of prostate (STEAP) (Hubert et al, PNAS 96 (1999), 14523-14528), specific membrane antigen prostate (PSM/PSMA) (Israeli et al., Cancer Res. 53 (1993), 227-230) and stem cell antigen of the prostate (PSCA) (Reiter et al" Proc. Nat. Acad. Sci. 95: 1735-1740, 1998). The stem cell antigen of the prostate (PSCA) is a protein of 123 amino acids, was first identified by searching for genes whose expression increases with the progression of cancer in the model using xenotransplanted LAPC-4 (line of prostate cancer cells) (Reiter et al., see above). It is a glycoside-phosphatidylinositol-anchored cell surface protein that belongs to the family of surface antigens Thy-1/Ly-6. PSCA has a 30% homology to stem cell antigen type 2. Although the function of PSCA sun� still being debated, the PSCA homologues have different activities and are themselves involved in carcinogenesis. It has been shown that the stem cell antigen type 2 prevents apoptosis in immature thymocytes (Classon and Coverdale, PNAS 91 (1994), 5296-5300). Thy-1 activates T-cells by transmitting signals through src tyrosine kinase (Amoui et al., Eur. J. Immunol. 27 (1997), 1881-86). The genes of the Ly-6 are involved in Apocalypse and cell adhesion (Schrijvers et al., Exp.Cell. Res. 196 (1991), 264-69). Initial studies on the messenger RNA (mRNA) and subsequent staining with monoclonal antibody (mAb) revealed that PSCA is expressed on the cell surface of normal and malignant prostate cells (Reiter et al., see above; Gu et al., Opodepe 19 (2000), 1288-96; Ross et al., Cancer Res. 62 (2002), 2546-53). In the normal prostate gland PSCA mRNA is detected in a subpopulation of basal and secretory cells. In carcinoma of the prostate expression of PSCA mRNA is detected in approximately 50-80% of cases of primary and about 70% of cases of metastatic cancer (Reiter et al., see above). There is information about immunohistochemistry 112 cases of primary prostate cancer and nine cases of cancer of the prostate with metastasis to bone (Gu et al., see above). The expression of PSCA detected in 94% of cases, overexpression detected in 40% of cases of clinically established prostate cancer. High levels of PSCA protein expression detection�s well as in nine cases out of nine investigated bone metastases of prostate cancer. Outside PSCA prostate is detected in the epithelial layer of cells of the transitional epithelium, in some renal collecting ducts, the neuroendocrine cells of the stomach and in the placental trophoblast. Importantly, according to the recent researches PSCA overexpression is observed in most cancers of the pancreas and invasive and non-invasive transition but cell carcinomas (Amara et al., Cancer Res. 61 (2001), 4660-4665; Argani et al., Cancer Res. 61 (2001), 4320-24). Since PSCA is expressed on the cell surface and sverkhekspressiya in large parts of various types of cancer, it is regarded as a target for development of cancer treatment strategies. However, to substantiate this possibility will require additional clinical correlation.

The expression of some CD antigens highly limited linespecific lymphohemopoietic cells, and in the last few years, antibodies against lymphoid-specific antigens, effective either in vitro or in animal models used for developing methods of treatment. In this regard, it was shown that CD19 is a very useful target. CD19 is expressed throughout b-cell line differentiation from Pro-b cells to Mature b cells, it is not "are shed", is uniformly expressed on all lympany cells and the lack�attempt to stem cells (Haagen, Clin Exp Immunol 90 (1992), 368-75, 14; Uckun, Proc. Natl. Acad. Sci. USA 85 (1988), 8603-7). CD19 is involved in the development of some mediated b-cell diseases, such as various forms of non-Hodgkin's lymphoma, mediated by b-cells in autoimmune diseases or the depletion of b-cells.

Another example of a molecule that is involved in the progression and spread of many types of human cancer, is the receptor for the growth factor of hepatocytes MET (C-MET). The MET oncogene, encoding a receptor tyrosine kinase (RTK) growth factor hepatocyte (HGF) and scatter factor (SF), controls genetic programs leading to cell growth, invasion and protection from apoptosis. Uncontrolled activation of MET is critical not only to generate oncogenic properties, but also to achieve the invasive phenotype (Trusolino, L & Comoglio, p. M. (2002) Nat. Rev, Cancer 2, 289-300). The role of MET in human tumors revealed experimentally and clearly proven by the opening of the MET-activating mutations in inherited forms of carcinomas (Schmidt et al., Nat. Genet. 16 (1997), 68-73; Kim et al., J. Med. Genet. 40 (2003), e97). Constitutive activation of MET occurs frequently in sporadic cancers, and several studies have shown that the oncogene MET sverkhekspressiya in specific tumors of histiocytes or is activated by autocrine mechanisms (for a list see the website http://www.vai.org/met/). In addition, gene MET amplif�ciruits in hematopoietic metastatic colorectal carcinomas (Di Renzo et al., Clin. Cancer Res. 1 (1995), 147-154).

It turned out that secreted in the culture of fibroblasts of the scattering factor (SF) that has the ability to cause intracellular dissociation of epithelial cells, and growth factor hepatocyte (HGF), a potent mitogen for hepatocytes in culture from blood platelets or blood of patients with acute liver failure, independently identified as a ligand of Met, are one and the same molecule. Met and SF/HGF largely expressed in various tissues. The expression of Met (the receptor) in the norm is limited in its spread by cells of epithelial origin, and the expression of SF/HGF (ligand) is limited to cells of mesenchymal origin.

Met is a transmembrane protein that is produced in the form of single-stranded precursor. This precursor proteoliticeski cleaved in forinovo the website with the formation of a high degree and fully glycosylated extracellular α-subunit of 50 kDa and a β-subunit of 145 kDa with a large extracellular region (involved in the binding of the ligand), a transmembrane segment and an intracellular region (catalytic activity) (Giordano (1989) 339: 155-156). α - and β-chains linked by disulfide bridges. The extracellular site of Met contains a region of homology with semaphorins (Sema domain, which contains the complete α-chain� and N-terminal part of the β-chain of Met), rich in cysteine residues Met-related sequence (MRS), followed by rich glycyrrhetinate residues (G-P) repeats, and four immunoglobulin-like structures (Birchmeier et al., Nature Rev. 4 (2003), 915-25). The intracellular region of Met contains three areas: (1) juxtamembrane segment, which contains: (a) a serine residue (Ser 985), who, when he fosforilirovanii by protein kinase C or CA2+-calmodulin - dependent kinases, reduces the activity of receptor kinases (Gandino et al., J. Biol. Chem. 269 (1994), 1815-20); and (b) tyrosine (Tyr 1003) that binds the ubiquitin ligase Cbl, which is responsible for polyubiquitination Met, endocytosis and degradation (Peschard et al., Mol. Cell 8 (2001), 995-1004); (2) tyrosine kinase domain, which when activated, the receptor undergoes transphosphorylation on Tyr1234 and Tyr1235; (3) the C-terminal region, which contains two key tyrosine (Tyr-1349 and Tyi-1356), inserted in a degenerate motif, which is multisubstrate docking (docking) site, is able to recruit several downstream adaptors containing the Src homologous-2 (SH2) domains of the Met receptor, as most receptor tyrosine kinases (RTK) use different tyrosines to bind specific signaling molecules. It was demonstrated that two tyrosine docking sites is necessary and sufficient for signal transduction in vitro, the� and in vivo (Maina et al., Cell 87 (1996), 531-542; Ponzetto et al., Cell 77 (1994), 261-71).

Although potent and selective preclinical drug candidates have been developed using C-MET as a tumor target, subsequent clinical trials should reveal whether these medicines are safe and demonstrate therapeutic efficacy in humans. In light of these uncertainties, there is still a need for new therapeutic concepts for cancer.

Cancer ahead of heart disease as the main cause of death of Americans under the age of 85 years in 2005. Currently cancer is the second leading cause of death in Germany after cardiovascular diseases. If significant breakthroughs in the prevention of cancer, comparable to the achievements in the field of cardiovascular disease will not be reached in the next few years, cancer will become the leading cause of death in Germany for 15-20 years. Inhibition of tumor angiogenesis is one of anti-cancer strategies, which aroused great interest among clinicians and cancer researchers in the last few years. During these research efforts have identified several tumor endothelial markers. Endothelial markers of tumor (S), such indocyanine (= TEM or CD248), sverkhekspressiya � the process of tumor angiogenesis (St. Croix et al., Science 289 (2000), 1197-1202).

Although their functions are still not thoroughly understood, in studies of developing embryos and tumors clearly established that they are expressed strictly in vascular endothelial cells (Carson-Walter et al., Cancer Res. 61: 6649-6655, 2001). Accordingly, endothelin, a transmembrane protein type I 165 kDa, is expressed on the cell surface of the endothelium of blood vessels of tumors in a variety of types of human cancer, but not detected in blood vessels or other types of cells in many normal tissues. It is actinopodidae molecule C-type signal leader peptide consisting of 757 amino acids, five globular extracellular domains (including the domain of pectin With-type, and one domain that is similar to the pattern Sushi/ccp/scr, and three EGF repeats), followed by mezinarodni region, a transmembrane segment and a short cytoplasmic tail (Christian et al., J. Biol. Chem. 276: 7408-7414, 2001). The crust protein indocyanine bears largely sililirovanie O-linked oligosaccharides and is sensitive to O-sialoglycoproteins, that can be attributed to the group silmutaneously molecules. N-terminal 360 amino acids indocyanine demonstrate homology with thrombomodulin, a receptor involved in the regulation of blood coagulation, and with complete�tare receptor C1qRp. This structural relationship indicates that endothelin functions as a tumor endothelial receptor. Although mRNA indocyanine widely expressed on endothelial cells in normal human and murine somatic tissues, protein endothelin largely limited to the yellow body and vysokolegirovannye fabrics such as granular tissue healing wounds or tumors (Opavsky et al., J. Biol. Chem. 276 (2001, 38795-38807; Rettig et al., PNAS 89 (1992), 10832-36). The protein expression indocyanine subjected to increasing regulation on tumor endothelial cells carcinomas (breast, kidney, lung, colorectal, colon, pancreas mesothelioma), sarcomas, and neuroectodermal tumors (melanoma, glioma, neuroblastoma) (Rettig et al., see above). In addition, endothelin is expressed at a low level on a subpopulation of tumor stromal fibroblasts (Brady et al., J. Neuropathol. Exp.Neurol. 63 (2004), 1274-83; Opavsky et al., see above). Because of its limited distribution in normal tissues and common expression on tumor endothelial cells of many different types of solid tumors, endothelin is considered as a target for antibody anti-angiogenic strategies for cancer treatment. However, until now there is still no effective therapeutic approaches using endos�Alina as tumor endothelial targets.

A molecule that is very common and largely is expressed on most cells of adenocarcinoma and multiple squamose-cell carcinoma, is Eram (CD326) (Went et al., Br. J. Cancer 94 (20006), 128-135). Recent studies have shown that Arcam is a signaling molecule that can activate the expression of the nuclear proto-oncogene C-ICC and tsiklonov (Munz et al., Oncogene 23 (2004), 5748-58). If sverkhekspressiya in resting T-cells Arcam induces cell proliferation, independence from growth factors and growth of colonies in soft agar, which are signs of oncogenic proteins. When blocking the expression of Arcam small interfering RNA (miRNAs) in breast cancer cells, these cells cease to proliferate, to migrate and to be invasive (Osta et al., Cancer Res. 64 (2004), 5818-24). This oncogenic signaling Arcam may explain why overexpression Arcam correlates with poor overall survival in many malignant tumors, including breast cancer and ovarian cancer (Spizzo et al., Gynecol. Oncol. 103 (2006), 483-8). Arcam already used as an antigen target in several therapeutic approaches using antibodies, including antibody (Obemeder et al., Eur. J. Cancer 42 (2006), 2530-8) and Arcam×CD3 bespecifically single-chain antibody, named MT. Characteristics MT recently been �described for (Brischwein et al., 43 (2006), 1129-43). This antibody has activity against various cell lines of human carcinoma expressing the antigen target, and is currently undergoing testing in phase I trials regarding the safety and initial evidence of effectiveness.

More specifically, cancer is ahead of heart disease as the main causes of death among Americans up to 85 years in 2005. Currently cancer is the second leading cause of death in Germany after cardiovascular diseases. If significant breakthroughs in the prevention of cancer, comparable to the achievements in the field of cardiovascular diseases, will not be reached in the next few years, cancer will become the leading cause of death in Germany for 15-20 years. Among more than 100 different types of cancers epithelial cancer is the leading cause of death from cancer in Germany. When epithelial cancer invasion and metastasis of malignant epithelial cells in normal tissues is accompanied by adaptive changes in supporting mesenchymal stroma of the target organ. Altered gene expression in these untransformed stromal cells was discussed in terms of providing potential targets for therapy. Cell surface proteiny protein activation of fibroblasts alpha (FAP-alpha) is one example of �aka the targets of activated tumor fibroblasts. Protein activation of fibroblasts alpha is an inducible cell surface glycoprotein that was originally identified in cultured fibroblasts using monoclonal antibody F19. Immunohistochemical studies showed that FAP-alpha temporarily expressed in some normal fetal mesenchymal tissues, but adult normal tissue and malignant epithelial, neural and hematopoietic cells are usually FAP-alpha-negative. However, most of the common epithelial cancers contain large amounts of FAP-alpha-reactive stromal fibroblasts. Scanlan and others (Proc. Nat. Acad. Sci. 91: 5657-5661, 1994) cloned cDNA (complementary DNA) FAP-alpha from the cDNA expression library of human fibroblast WI-38 by immunoselection using antibody F19. The predicted protein FAP-alpha people, consisting of 760 amino acids, is an integral membrane protein type II with a large C-terminal extracellular domain that contains 6 potential sites M-glycosylation, 13 cysteine residues and 3 segments that correspond to highly catalytic domains of semiprotect, a hydrophobic transmembrane segment and a short cytoplasmic tail. FAP-alpha demonstrates 48% �aminokislotnoi identity with dipeptidylpeptidase IV (DPP4) and 30% homologous with DPP4-related protein (DPPX). In Northern blot analysis has detected in fibroblasts mRNA FAP-alpha 2,8 T. D. of Sarasa is an integral membrane gelatinase 170 kDa, whose expression correlates with invasionist of melanoma cells and human carcinoma. Goldstein al (Biochim. Biophys. Acta 1361: 11-19, 1997) cloned and characterized the corresponding cDNA of saraz. The authors found that sarasa and FAP-alpha represent the same protein and the products of the same gene. Pineiro-Sanchez and co-authors (J. Biol. Chem. 272: 7595-7601 1997) has identified the protein sarasu/FAP-alpha from the cell membranes and ethnoreligious" shed vesicles of LOX human melanoma cell. Inhibitors semipretioase blocked gelatinase activity seprate/FAP-alpha, confirming that sarasa/FAP-alpha contains a catalytically active serine residue (residues). The authors found that sarasa/FAP-alpha consists of a monomer, N-glycosylated subunits of 97 kDa, which proteoliticeski inactive. They concluded that sarasa/FAP-alpha are similar to DPP4 that their proteolytic activity is dependent on Association of the subunits. Based on its activity to the destruction of gelatin and denatured by heating collagen type I and type IV, an assumption was made about the role of seproz/FAP-alpha in the remodeling of extracellular matrix, tumor growth, and metastasis of cancerous tumors. Moreover, Sep�Aza/FAP-alpha shows a restricted expression pattern in normal tissues and its uniform expression in the surrounding stroma of many malignant tumors. Thus, sarasa/FAP-alpha can be used as a target to explore the concept of targeting the tumor stroma for immunotherapy of epithelial human cancer. However, although there have been several clinical trials to study the role seprate/FAP-alpha as a tumor antigen target, conventional immunotherapeutic approaches or inhibition of the enzymatic activity of seproz/FAP-alpha is still not ensured the achievement of therapeutic efficacy (see, for example, Welt et al., J. Clin. Oncol. 12:1193-203, 1994; Narra et al., Cancer Biol. Ther. 6, 1691-9, 2007; Henry et al., Clinical Cancer Research 13, 1736-1741, 2007).

Receptor insulin-like growth factor I (IGF-IR or IGF-1R) is a receptor with tyrosinekinase activity, having 70% homology with the insulin receptor IR. IGF-1R is a glycoprotein having a molecular weight of approximately 350000. He is heterotetrameric receptor, in which each half-linked by disulfide bridges, consists of an extracellular alpha subunit and a transmembrane beta-subunit. IGF-1R binds IGF 1 and IGF 2 with very high affinity, but also able to communicate with the insulin affinity 100 to 1000 times below. On the contrary, 1R insulin binds with very high affinity, although IGF bind to a receptor of insulin only with affinity 100 R�W below. Tyrosine kinase domain of IGF-1R and 1R has a very high homology to the sequences, although the zone of reduced homology, respectively, are related to rich in cysteine residues of the region situated on the alpha subunit and the C-terminal part of beta-subunit. Differences in the sequences observed in the alpha-subunit are located in the area of the ligand binding and thus determine the relative affinity of IGF-1R and 1R to IGF and insulin, respectively. Differences in the C-terminal part of the beta-subunit lead to divergent signaling pathways of these two receptors: IGF-1R mediates the mitogenic effect of differentiation and antiapoptosis effect, whereas activation of IR fundamentally affect the effects at the level of metabolic pathways (Baserga et al., Biochim. Biophys. Acta, 1332: F105-126, 1997; Baserga R. Exp.Cell. Res., 253:1-6, 1999). Cytoplasmic tyrosinekinase proteins are activated as a result of binding of ligand to the extracellular domain of the receptor. In the activation of kinases, in turn, involved the stimulation of various intracellular substrates, including IRS-1, IRS-2, and She Grb 10 (F. Peruzzi et al., J. Cancer Res. Clin. Oncol., 125:166-173, 1999). The two main substrates of IGF-1R are the IRS and She that mediate, via activation of numerous downstream effectors, most of the effects of growth and differentiation, related to the accession to this IGF receptor. Follow�Adelino, the availability of substrates can dictate the final biological effect associated with the activation of IGF-1R. When prevailing IRS-1, the cells tend to proliferate and transform. When She dominates, the cells tend to differentiate (Valentinis B. et al.; J. Biol. Chem. 274:12423-12430, 1999). It appears that the effects of protection from apoptosis involved the way phosphatidyl-Inositol-3-kinase (PI 3-kinase) (Prisco M. et al., Horm. Metab. Res., 31:80-89, 1999; Peruzzi F. et al., J. Cancer Res. Clin. Oncol., 125:166-173, 1999). The role of the IGF system in carcinogenesis has been the subject of intensive research in the last ten years. The interest in this arose after the discovery that in addition to its mitogenic and antiapoptotic properties of IGF-1R, apparently, required to create and maintain the transformed phenotype. In fact, it was firmly established that overexpression or constitutive activation of IGF-1R leads, in a wide variety of cells, growth of cells, not associated with the substrate, in media without fetal calf serum, and to the formation of tumors in Nude thymus of mice. This in itself is not a unique property, because the various products expressed genes can transform cells, including a significant number of receptors of growth factors. But the decisive discovery which clearly demonstrated the major role of IGF-1R in the transformation, was demo�exposure, R-cells in which the gene encoding IGF-1R, inactivated, completely immune to the transformation of different agents, which usually is able to transform cells, such as the E5 protein of bovine papilloma virus, overexpression of EGFR (the receptor for epidermal growth factor) or PDGFR (platelet receptor growth factor), T-antigen of SV 40, activated ras or the combination of these two last factors (Sell S. et al., Proc. Natl. Acad. Sci., USA, 90: 11217-11221, 1993; Sell, S. et al., Mol. Cell. Biol., 14:3604-3612, 1994; Morrione A. J., Virol., 69:5300-5303, 1995; D. Coppola et al., Mol. Cell. Biol., 14:458a-4595, 1994; DeAngelis Tet al., J. Cell. Physiol., 164:214-221, 1995).

IGF-1R is expressed in various tumors and cell lines, tumors, and IGF amplificateur tumor growth through their accession to IGF-1R. Other arguments in favor of the role of IGF-1R in gave carcinogenesis studies using murine monoclonal antibodies against the receptor or a dominant negative IGF-1R. In fact, murine monoclonal antibodies against IGF-1R inhibit the proliferation of numerous cell lines in culture and tumor cell growth in vivo (Arteaga C. et al., Cancer Res., 49:6237-6241, 1989 Li et al., Biochem. Biophys. Res. Corn., 196:92-98, 1993; Zia F, et al., J. Cell. Biol., 24:269-275, 1996; Scotland! K et al., Cancer Res., 58:4127-4131, 1998). In the works of Jiang and co-authors (Oncogene, 18:6071-6077, 1999) has also been shown that dominant negative IGF-1R is able to inhibit the proliferation of tumor.

�the shepherd, what effort has been made to identify new targets for therapeutic approaches to cancer therapy, cancer is still one of the most frequently diagnosed diseases. In this regard, there is still a need for effective cancer therapies.

According to the present invention proposed molecule especifismo single-chain antibodies containing the first binding domain capable of contacting epitope CD3ε(Epsilon) chain of human rights and the primacy of non-chimpanzee, where the specified epitope is part of an amino acid sequence that is a member of the group consisting of SEQ ID NO: 2, 4, 6 and 8, and a second binding domain capable of contacting the antigen is selected from the group consisting of stem cell antigen of the prostate (PSCA), antigen of b-lymphocytes CD19 (CD19), receptor growth factor hepatocyte (C-MET), indocyanine, EGF-like 1 domain of the adhesion molecule of epithelial cells (Ersam), encoded by exon 2, protein activation of fibroblasts alpha (FAP-alpha) and receptor insulin-like growth factor I (IGF-IR or IGF-1R).

Although contact with T-cells bespecifically single-chain antibodies described in this area, have a great therapeutic potential for the treatment of malignant diseases, most of these bespecifically molecules is limited to �they are species-specific and only recognize antigen and due to genetic similarities, in all likelihood chimpanzees. An advantage of the present invention is to create especifismo single-chain antibody containing the binding domain exhibiting cross-species specificity to CD3-Epsilon-chain of human rights and the primacy of non-chimpanzee.

In the present invention was unexpectedly identified N-terminal containing amino acid residues 1-27 of the polypeptide fragment of the extracellular domain of CD3-Epsilon, which, unlike all other known epitopes of CD3-Epsilon, described in this area, retains its three-dimensional structural integrity when removed from its native environment in the CD3 complex (and possibly fusion with a heterologous amino acid sequence, such as Arcam or Fc-region of immunoglobulin).

Thus, according to the present invention proposed molecule especifismo single-chain antibodies containing the first binding domain capable of contacting epitope N-terminal containing amino acid residues 1-27 of the polypeptide fragment of the extracellular domain of CD3-Epsilon chains (CD3-Epsilon chain, for example, removed from its native environment and/or contains (presented on the surface) in T-cell) of a person and at least one of the primacy of non-chimpanzee, where specified e�iTop is part of the amino acid sequence, a member of the group consisting of SEQ ID NO: 2, 4, 6 and 8; and a second binding domain, is able to communicate with specific membrane antigen prostate (PSMA). Preferred primates, non-chimpanzee referred to in this description elsewhere. At least one (or a sample of or all of) rule(s) selected(e) of Callithrix jacchus (marmoset), Saguinus Oedipus (cotton-top tamarins), Saimiri sciureus (squirrel monkey) and Massa fasciculahs (cynomolgus macaques) (SEQ ID NO: or 2225 or 2226 or both), is particularly preferred. Massa mulatta, also known as the rhesus monkey, also regarded as another preferred primacy. It is therefore envisaged that the antibodies of the invention bind with (able contact) independent from the environment epitope N-terminal containing amino acid residues 1-27 of the polypeptide fragment of the extracellular domain of CD3-Epsilon of human and Callithrix jacchus, Saguinus oedipus, Saimiri sciureus and Masasa fascicularis (SEQ ID NO: or 2225 or 2226 or both), and possibly also Masasa mulatta. Molecule especifismo single-chain antibodies containing the first binding domain, as defined herein, can be obtained (available) or may be manufactured in accordance with the Protocol set forth in the examples below (in particular in Example 2). For this purpose, (a) immunization of mice with the N-terminal containing AMI�kislotniy residues 1-27 polypeptide fragment of the extracellular domain of CD3-Epsilon person and/or Saimiri sciureus; (b) the establishment of the immune scFv library mouse antibodies; (b) identification of CD3-Epsilon-specific binding of fragments by testing the ability to contact at least SEQ ID NO: 2, 4, 6 and 8.

Independence from the environment of the CD3 epitope, proposed in this invention corresponds to the first 27 N-terminal amino acids of CD3-Epsilon, or functional fragments of this stretch of 27 amino acids. The phrase "independent of the environment" used in this description in relation to the CD3 epitope, means that the binding described here linking molecules/antibody molecules of the invention does not change or modification of conformation, sequence or structure surrounding the antigenic determinant or epitope. In contrast, the CD3 epitope recognized by traditional CD3-binding molecule (e.g., molecule, described in WO 99/54440 or WO 04/106380) localized on CD3-Epsilon chains in the C-terminal region to the N-terminal amino acids 1-27 independent from the environment epitope, and he adopts the proper conformation only if it is embedded in the rest of the Epsilon-chain and held in the correct steric situation at the expense of heterodimerization Epsilon-chain or gamma-or Delta-chain of CD3.

AHTH-CD3-binding molecules/domains as part of a molecule especifismo single-chain antibodies, which repre�Alena in this description and created (and directed) against independent from the environment of the CD3 epitope, provide unexpected clinical improvement in relation to the redistribution of T cells and, consequently, a more favorable safety profile. Without any connection with theory, since the CD3 epitope does not depend on the environment, making it an Autonomous self-sufficient subdomain without much impact on the rest of the CD3-complex, CD3-binding molecules/domains presented in this description, induce smaller allosteric changes in the conformation of CD3 than traditional CD3-binding molecules that recognize dependent environment CD3 epitopes (e.g. as described in WO 99/54440 or WO 04/106380).

Independence from the environment of the CD3 epitope that is recognized by the CD3-binding domain especifismo single-chain antibodies according to the invention (PSCA×CD3, CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3), is associated with less or with no redistribution of T cells (redistribution of T-cells is identified with an initial episode of reduction and subsequent restoration of the absolute number of T-cells) during the initial phase of treatment indicated bespecifically single-chain antibodies according to the invention. The result is better safety profile bespecifically single-chain antibodies according to the invention compared with conventional CD3 binding molecules known in this field which recognise dependent environment CD3 epitopes. In particular, since the redistribution of T cells during the initial phase of treatment with CD3 binding molecules is a major risk factor of adverse events such as adverse events of the Central nervous system (Central nervous system), bespecifically single-chain antibodies according to the invention have a significant advantage from the point of view of security compared with CD3 binding molecules known in this field, due to the recognition rather independent from the environment than dependent environment epitope of CD3. Patients with these adverse events of the Central nervous system associated with the redistribution of T cells during the initial phase of treatment traditional CD3-binding molecules, usually suffer from confusion and disorientation and in some cases of urinary incontinence. Confusion is a change in the mental condition in which the patient is not able to think with his or her usual level of clarity. The patient is usually hard to concentrate, and the process of thinking is not only blurred and unclear, but is often significantly delayed. Patients with adverse events of the Central nervous system associated with the redistribution of T cells during the initial phase of treatment traditional CD3-binding molecules, may also suffer sweat�ray memory. Frequently confusion leads to loss of ability to recognize people, places, to determine the time or date. The sense of disorientation is common in confusion, and the ability to make decisions is broken. Adverse events of the Central nervous system associated with the redistribution of T cells during the initial phase of treatment traditional CD3-binding molecules can additionally include unclear speech and/or difficulties with the choice of words. This disorder can worsen as expression, and speech perception, and the ability to read and write. In addition to urinary incontinence in some patients, adverse events of the Central nervous system associated with the redistribution of T cells during the initial phase of treatment traditional CD3-binding molecules can also be accompanied by vertigo and dizziness.

The preservation of three-dimensional structures within the mentioned 27-amino-acid N-terminal polypeptide fragment of CD3-Epsilon can be used to create binding domain, preferably a human, who is able to communicate with the N-terminal polypeptide fragment of CD3-Epsilon in vitro and with native CD3 complex (CD3-Epsilon-subunit of the CD3 complex) on T-cells in vivo with the same binding affinity. These data are strictly indicate that the N-terminal fragment, as he description�n here forms a tertiary conformation, which is similar to its structure, normally existing in vivo. It was a very sensitive test demonstrating the importance of structural integrity of amino acids 1-27 of the N-terminal polypeptide fragment of CD3-Epsilon. Individual amino acids from amino acids 1-27 of the N-terminal polypeptide fragment of CD3-Epsilon were replaced with alanine (alanine scan) to test the sensitivity of amino acids 1-27 of the N-terminal polypeptide fragment of CD3-Epsilon to minor violations. CD3-binding domains as part bespecifically single-chain antibodies according to the invention were used to test for binding to alanine mutants of amino acids 1-27 of the N-terminal polypeptide fragment of CD3-Epsilon (see Example 5). Individual replacement first five amino acid residues at the very end of the N-terminal fragment and two amino acids in positions 23 and 25 amino acids 1-27 of the N-terminal polypeptide fragment of CD3-Epsilon were critical for binding of the antibody molecules. Replacement of amino acid residues in the area of regulations 1 to 5, which contain the residue of Q (glutamine at position 1), D (aspartic acid in position 2), G (glycine at position 3), N (asparagine at position 4) and E (glutamic acid in position 5), to alanine abrogated binding bespecifically odnosima�echnik antibodies according to the invention, preferably human, with the specified fragment. At the same time, at least for some bespecifically single-chain antibodies according to the invention, preferably human, two amino acid residues on the C-end of the mentioned fragment T (threonine at position 23) and 1 (isoleucine at position 25) reduced the binding energy with bespecifically single-chain antibodies according to the invention, preferably a human.

Unexpectedly, it was found that the thus-bespecifically single-chain antibodies according to the invention, preferably a human, not only recognize the N-terminal fragment of CD3-Epsilon person, but also the corresponding homologous fragments of CD3-Epsilon of various primates, including cacahuete monkeys (marmoset, Callithrix jacchus; cotton-top tamarins, Saguinus oedipus; squirrel monkey, Saimiri sciureus) and lower narrow-nosed monkeys (Masasa fascicularis, also known as cynomolgus macaques macaques or a crab-eater; or Massa mulatta, also known as the rhesus monkey). Thus, there were found specific for many primates bespecifically single-chain antibodies according to the invention. The following analysis of the sequences confirmed that the man and primates have a common plot with vysokomaslichnoy sequence at N-end of the extracellular domain of CD3-Epsilon.

Amino acid seq�the sequence of the above-mentioned N-terminal fragments CD3-Epsilon presented in SEQ ID NO: 2 (human), SEQ ID NO: 4 (Callithrix jacchus); SEQ ID NO: 6 (Saguinus oedipus); SEQ ID NO: 8 (Saimiri sciureus); SEQ ID NO: 2225 QDGNEEMGSITQTPYQVSISGTTILTC or SEQ ID NO: 2226 QDGNEEMGSITQTPYQVSISGTTVILT (Masasa fascicularis, also known as cynomolgus macaques) and SEQ ID NO: 2227 QDGNEEMGSITQTPYHVSISGTTVILT (Masasa mulatta, also known as the rhesus monkey).

In one embodiment of this invention the second binding domain PSCA×CD3 especifismo single-chain antibody of the invention binds to the antigen of stem cells of the prostate (PSCA). In alternative embodiments the second binding domain binds to CD19, C-MET, endothelina (CD248), Arcam, FAPα or IGF-1R. As shown in the examples below, the second binding domain EpCAM×CD3 especifismo single-chain antibody of the invention binds to amino acid residues 26-61 EGF-like domain 1 Ersam, which is encoded by exon 2 of the gene Ersam. These amino acid residues 26-61 EGF-like domain 1 Ersam person represented in SEQ ID NO: 571. Thus, Hersam-directed bespecifically single-stranded molecules of the present invention are unique in your own class Ersam-binding molecules, which is clearly different from Arcam-binding molecules on the basis of Hersam-binding fragment HD69 described previously. Specified Arcam-binding fragment HD69 binds to another epitope (i.e., epitope, localized in the EGF-like domain 1 Ersam �of a person).

Preferably, the second binding domain PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) especifismo single-chain antibody binds to PSCA (respectively CD19, C-MET, indocyanines, Arcam, IGF-1R or FAPα) person or PSCA (respectively CD19, C-MET, indocyanines, Arcam, IGF-1R or FAPα) the primacy of non-chimpanzee; more preferably it binds to PSCA (respectively CD19, With METH, indocyanines, Arcam, IGF-1R or FAPα) man and PSCA (respectively CD19, C-MET, indocyanines, Arcam, IGF-1R or FAPα) the primacy of non-chimpanzee and, therefore, exhibits species specificity; even more preferably PSCA (respectively CD19, C-MET, indocyanines, Arcam, IGF-1R or FAPα) man and PSCA (respectively CD19, C-MET, indocyanines, Arcam, IGF-1R or FAPα) macaque (and therefore also exhibits species specificity). Particularly preferably, macaque PSCA (respectively CD19, C-MET, endothelin, Arcam, IGF-1R or FAPα) is a PSCA (respectively CD19, C-MET, endothelin, Arcam, IGF-1R or FAPα) cynomolgus macaque and/or PSCA (respectively CD19, C-MET, endothelin, Arcam, IGF-1R or FAPα) macaque-rhesus. It should be borne in mind that the second binding domain EpCAM×CD3 especifismo single-chain antibodies of the present invention preferably binds to EGF-like domain 1 Ersam Primate, non-PWM�Anse, which is encoded by exon 2 of the gene Ersam the primacy of non-chimpanzee. As indicated above, Ersam the primacy of non-chimpanzee, is a preferably Arcam macaque, more preferably Arcam Javanese macaque and/or Arcam macaque-rhesus.

However, from the scope of the present invention is not excluded that the second binding domain may also contact the homologues of PSCA (respectively CDIQ, C-MET, indocyanine, Arcam, IGF-1R or FAPα) other species, such as PSCA (respectively 0019, C-MET, endothelin, Arcam, IGF-1R or FAPα) chimpanzees or homologue of PSCA (respectively CD19, C-MET, endothelin, Arcam, IGF-1R or FAPα) rodents.

It is clear that in the preferred embodiment of the species specificity of the first and second binding domains of the antibody according to the invention are identical.

Prostate cancer is the second highest number of cancer cases in men. It is estimated that in 2008 in the U.S. 186320 men will be newly diagnosed prostate cancer and approximately 28660 men will die from the disease (see, for example, http://www.cancer.gov/cancertopics/types/prostate). The risk of prostate cancer is strictly linked to age: very few cases are registered in men under 50 years old, and three-quarters of cases occur in men over the age of 65 years. The largest number of cases diagnosed in men aged 70-74 years. Currently lying� the rate of growth of the older population is much higher than the growth rate of the total population. It is projected that by 2025-2030 years, the population over 60 years will grow 3.5 times faster than the total population. According to forecasts, the share of seniors will more than double worldwide over the next half-century period, and this means that we should expect a further increase in cases diagnosed prostate cancer. However, PSCA is a target not only for prostate cancer. More precisely, PSCA overexpression was also detected in bladder cancer (Amara et al., Cancer Res 61 (2001): 4660-4665) and pancreatic cancer (Argani et al., Cancer Res 61 (2001): 4320-4324). In light of the above, PSCAxCD3 bespecifically single-chain antibody according to the invention provides a pre-emptive tool for the destruction of PSCA-expressing cancer cells, including, but not limited to, cells prostate cancer, bladder cancer or pancreatic cancer, in humans. As shown in the following Examples, the cytotoxic activity of PSCA×CD3 especifismo single-chain antibodies according to the invention is higher than the cytotoxic activity of antibodies described in this field.

Preferably, according to the present invention also suggested that PSCA×CD3 bespecifically single-chain antibody containing a second binding domain, which �vyzyvaetsya as with human PSCA, and with the homologue of macaque PSCA, i.e. the homologue of the primacy of non-chimpanzee. In a preferred embodiment bespecifically single-chain antibody contains a second binding domain exhibiting cross-species specificity to the antigen of stem cells of the prostate (PSCA) of the person and the primacy of non-chimpanzee. In this case, identical molecule especifismo single-chain antibodies can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and in the quality of medicines in humans. In other words, the same molecule can be used in preclinical animal studies, and clinical studies in humans. Due to this, are achieved to a high degree comparable results and a much higher predictive ability of animal studies compared with a species-specific molecules simulators. As both the CD3-binding domain, and PSCA-binding domain of the PSCA×CD3 especifismo single-chain antibodies according to the invention exhibit cross-species specificity, i.e. interact with antigens of human and Primate, non chimpanzees, it can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic prof�La these binding domains in primates and, in identical form, as drugs in humans.

CD19 is a cell surface molecule, which Express only b-lymphocytes and follicular dendritic cells of the hematopoietic system. He is the earliest b-cell evolutionary stable antigens that are highly expressed and is present on most pre-b-cells and most cells non-T-cell acute lymphocytic leukemia cells and b-cell chronic lymphocytic leukemia.

In a preferred embodiment bespecifically single-chain antibody according to the invention contains a second binding domain exhibiting cross-species specificity to human CD19 and the primacy of non-chimpanzee. In this case, identical molecule especifismo single-chain antibodies can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and as a drug in humans. Due to this, are achieved to a high degree comparable results and a much higher predictive ability of animal studies compared with a species-specific molecules simulators. Since in this embodiment and CD3-binding domain, and CD19-binding domain of CD19×CD3 especificacao odnosima�echnolo antibodies of the invention exhibit species specificity i.e. interact with antigens of human and Primate, non chimpanzees, it can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and, in identical form, as drugs in humans.

As described here above, the MET oncogene, encoding a receptor tyrosine kinase (RTK) growth factor hepatocyte (HGF) and scatter factor (SF), controls genetic programs leading to cell growth, invasion and protection from apoptosis. As shown in the Examples below, C-MET×CD3 bespecifically single-chain antibody according to the invention provides a pre-emptive tool for the destruction of C-MET-expressing cancer cells, such as C-MET-positive cell line MDA-MB-231 breast cancer person. Cytotoxic activity of C-MET×CD3 especifismo single-chain antibodies according to the invention is higher than the cytotoxic activity of antibodies described in this area. Preferably, and as CD3-binding domain, and C-MET-binding domain of C-MET×CD3 especifismo single-chain antibodies according to the invention exhibit cross-species specificity, i.e. interact with antigens of human and Primate, non-chimpanzee, C-MET×CD3 bespecifically single-chain antibody according to the invention can� to be used for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and, in identical form, as drugs in humans.

Preferably, in yet another alternative embodiment of the present invention proposed C-MET×CD3 bespecifically single-chain antibody containing a second binding domain that binds to both the C-MET person, and a homologue of C-MET macaque, i.e. the homologue of the primacy of non-chimpanzee. In a preferred embodiment bespecifically single-chain antibody contains a second binding domain exhibiting cross-species specificity to C-MET of man and the primacy of non-chimpanzee. In this case, identical molecule especifismo single-chain antibodies can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and in the quality of medicines in humans. In other words, the same molecule can be used in preclinical animal studies, and clinical studies in humans. Due to this, are achieved to a high degree comparable results and a much higher predictive ability of animal studies compared with a species-specific molecules simulators. Since preferably� and CD3-binding domain, and C-MET-binding domain of C-MET×CD3 especifismo single-chain antibodies according to the invention exhibit cross-species specificity, i.e. interact with antigens of human and Primate, non chimpanzees, it can be used for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and, in identical form, as drugs in humans.

Angiogenesis, i.e. the formation of new capillaries, is essential for a number of important physiological events, both normal and pathological. Recently, increased attention was focused on the purification and characterization of inhibitors of this process, as angiogenesis inhibitors have potential therapeutic value in controlling solid tumors. Because of its restricted distribution in normal tissues and excess expression on tumor endothelial cells of many different types of solid tumors endothelin can be used as a target for based on anti-angiogenic strategies for cancer treatment. In particular, directional influence on tumor endothelial cells instead of cancer cells has the advantage that expression of the target on untransformed endothelial cells of blood vessels the tumors� more stable than the expression of the targets genetically unstable cancer cells. Endothelin×CD3 bespecifically single-chain antibody according to the invention provides a primary means for inhibiting the formation of capillaries in solid tumors, which plays a major role in the maintenance of tumor growth. In this new and inventive therapeutic approach pressure is not aimed at the tumor cells and blood vessels of the tumor. Endothelin×CD3 bespecifically single-chain antibody according to the invention is recruiting cytotoxic T-cells to endothelin-positive endothelial cells in tumors, including, but not limited to, carcinomas (breast, kidney, lung, colorectal, colon, pancreas mesothelioma), sarcomas, and neuroectodermal tumors (melanoma, glioma, neuroblastoma), causing the depletion endothelin-expressing endothelial cells in the tumor. This occurs through the inhibition of tumor angiogenesis, leading to regression or even the depletion of the tumor. As shown in the following Examples, the cytotoxic activity endothelin×CD3 especifismo single-chain antibodies according to the invention is higher than the activity of antibodies described in this area. Because the growth of solid tumors requires the recruitment support blood� vessels, therapeutic use endothelin×CD3 especifismo single-chain antibodies according to the invention provides a novel and inventive approach for targeting endothelial cells of tumor and destroy them.

Preferably, according to the present invention also proposed endothelin×CD3 bespecifically single-chain antibody containing a second binding domain that binds to both indocyanine person, and with the homologue indocyanine macaque, i.e. the homologue of the primacy of non-chimpanzee. In a preferred embodiment, bespecifically single-chain antibody contains a second binding domain exhibiting cross-species specificity to indocyanine of man and the primacy of non-chimpanzee. In this case, identical molecule especifismo single-chain antibodies can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and as a drug in humans. In other words, the same molecule can be used in preclinical animal studies, and clinical studies in humans. Due to this, are achieved to a high degree comparable results and a much higher predictive capability studies vividly�tion compared with a species-specific molecules simulators. Preferably, and as CD3-binding domain, and endothelin-binding domain endothelin×CD3 especifismo single-chain antibodies according to the invention exhibit cross-species specificity, i.e. interact with antigens of man and the primacy of non-chimpanzee, it can be used for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and, in identical form, as a drug in humans.

It was previously shown that Arcam is expressed on so-called "cancer stem cells" (Dalerba et al., PNAS 104 (2007), 10158-63; Dalerba et al., Ann. Rev. Med. 58 (2007), 267-84). In this regard, EpCAM×CD3 bespecifically single-chain antibody according to the invention not only provides a preferential tool for the destruction of Hersam-expressing cancer cells in epithelial cancer or a minimal residual cancer, but may be useful for the depletion of the alleged perpetrators responsible for tumor relapse after therapy. In addition, the cytotoxic activity of EpCAM×CD3 especifismo single-chain antibodies according to the invention is higher than the cytotoxic activity of antibodies described in this field.

In a preferred embodiment bespecifically single-chain antibody according to the invention contains a second binding domain, about�plausi interspecies specificity to Ersam of man and APE, non chimpanzees. In this case, identical molecule especifismo single-chain antibodies can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and as a drug in humans. Due to this, are achieved to a high degree comparable results and a much higher predictive ability of animal studies compared with a species-specific molecules simulators. Since in this embodiment and CD3-binding domain, and Hersam-binding domain Arcam×CD3 especifismo single-chain antibodies according to the invention exhibit cross-species specificity, i.e. interact with antigens of man and the primacy of non-chimpanzee, it can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and, in identical form, as a drug in humans.

FAP-alpha×CD3 bespecifically single-chain antibody according to the invention provides a primary means for the destruction of the stroma of solid tumors such as epithelial tumors, which plays a major role in maintaining the growth and neovascularization of tumors. In this new and inventive tera�euticalscom approach pressure is not aimed at the tumor cells, and activated stromal fibroblasts. Previous studies have shown that the most common types of epithelial cancers, including more than 90% of primary carcinomas and malignant breast, lung and colorectal, contain redundant FAP-alpha-interacting stromal fibroblasts (Scanlan et al., PNAS 91 (1994), 5657-5661, and references cited in this document). In contrast, normal tissue and benign and predislocation epithelial pathological changes contain only rare FAP-alpha-positive stromal cells. FAP-alpha×CD3 bespecifically single-chain antibody according to the invention is recruiting cytotoxic T-cells to FAP-alpha-positive activated stromal fibroblasts in primary and malignant epithelial tumors, causing depletion of stromal cells in the tumor. In particular, directional influence on tumor stromal cells instead of cancer cells has the advantage that expression of the target on nontransgenic stromal cells are more stable than the expression of the targets genetically unstable cancer cells. As shown in the following Examples, the cytotoxic activity of FAP-alpha×CD3 especifismo single-chain antibodies according to the invention is higher than the activity of antibodies described in this area.Because the growth of solid tumors requires the recruitment of a supporting stroma, therapeutic applications of FAP-alpha×CD3 especifismo single-chain antibodies according to the invention provides a novel and inventive approach for targeting tumor stromal cells and their destruction.

Preferably, according to the present invention also proposed FAP-alpha×CD3 bespecifically single-chain antibody containing a second binding domain that binds to both the FAP-alpha man and a homologue of FAP-alpha macaque, i.e. the homologue of the primacy of non-chimpanzee. In a preferred embodiment bespecifically single-chain antibody contains a second binding domain exhibiting cross-species specificity to FAP-alpha of human rights and the primacy of non-chimpanzee. In this case, identical molecule especifismo single-chain antibodies can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and as a drug in humans. In other words, the same molecule can be used in preclinical animal studies, and clinical studies in humans. Due to this, are achieved to a high degree comparable results and a much higher predictive ability of animal studies compared with in�despecification molecules simulators. Because as CD3-binding domain and FAP-alpha-binding domain of FAP-alpha×CD3 especifismo single-chain antibodies according to the invention exhibit cross-species specificity, i.e. interact with antigens of man and the primacy of non-chimpanzee, it can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and, in identical form, as drugs in humans.

As described here above, IGF-1R is a receptor (and therefore cell surface antigen) with tyrosinekinase activity, having 70% homology with the insulin receptor R. 1 IGF-1R is expressed in various tumors and tumor cell lines, and IGF amplificateur tumor growth through adherence to IGF-1R.

In a preferred embodiment bespecifically single-chain antibody according to the invention contains a second binding domain exhibiting cross-species specificity to IGF-1R of human rights and the primacy of non-chimpanzee. In this case, identical molecule especifismo single-chain antibodies can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and as a drug in humans. �and this is achieved to a high degree comparable results and a much higher predictive ability of animal studies compared with a species-specific molecules simulators. Since in this embodiment and CD3-binding domain, and IGF-1R-binding domain of IGF-1R×CD3 especifismo single-chain antibodies according to the invention exhibit cross-species specificity, i.e. interact with antigens of man and the primacy of non-chimpanzee, it can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and, in identical form, as drugs in humans.

It was discovered in the present invention that there is the possibility of creating PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) especifismo single-chain antibodies, preferably human, with identical molecule can be used in preclinical testing in animals and in clinical studies and even in the treatment of the person. This is possible thanks to the unexpected identification of PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) especifismo single-chain antibodies, preferably human, which, in addition to binding to CD3-Epsilon and PSCA (respectively CD19, C-MET, endothelin, Arcam, IGF-1R or FAPα), respectively (and likely due to the genetic similarity with a copy of chimpanzees), also associated with the homologues listed�s antigens primates, non chimpanzees, including cacahuete monkeys and lower narrow-nosed monkeys. As shown in the Examples below, the specified PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) bespecifically single-chain antibody according to the invention, preferably human, can be used as a therapeutic agent or drug against different diseases, including, without limitation, cancer.

PSCA×CD3 bespecifically single-chain antibody is particularly preferred for the treatment of prostate cancer, bladder cancer or pancreatic cancer.

Specified CD19×CD3 bespecifically single-chain antibody according to the invention, preferably human, can be used as a therapeutic agent against various diseases, including, without limitation, cancer, preferably a b-cell malignancy such as non-Hodgkin's lymphoma, mediated by b-cells in autoimmune diseases or the depletion of b-cells.

The specified C-MET×CD3 bespecifically single-chain antibody according to the invention, preferably human, can be used as a therapeutic agent against various diseases, including, without limitation, cancer, preferably a carcinoma, sarcoma, glioblastoma/astrocytoma, �alanamu, mesothelioma, Wilms tumor or a hematopoietic malignancy such as leukemia, lymphoma or multiple myeloma.

Specified endothelin×CD3 bespecifically single-chain antibody according to the invention, preferably a human, provides a new and inventive approach for targeting tumor endothelium and its destruction, including (but not limited to) carcinomas (breast, kidney, lung, colorectal, colon, pancreas mesothelioma), sarcomas, and neuroectodermal tumors (melanoma, glioma, neuroblastoma). Endothelin×CD3 bespecifically single-chain antibody according to the invention may deprive solid tumors their supporting blood vessels, thereby inhibiting angiogenesis and thus the growth of these tumors.

Specified EpCAM×CD3 bespecifically single-chain antibody according to the invention, preferably human, can be used as a therapeutic agent against various diseases, including, without limitation, cancer, preferably epithelial cancer.

Specified IGF-1R×CD3 bespecifically single-chain antibody according to the invention, preferably human, can be used as a therapeutic agent against various diseases, including, without limitation, cancer, predpochtitel�but bone cancer or soft tissue (e.g. Ewing's sarcoma), breast cancer, liver cancer, lung cancer, cancer in the head and neck, colorectal cancer, prostate cancer, leiomyosarcoma, cervical and endometrial cancer, ovarian cancer, prostate cancer and pancreatic cancer. IGF-1R×CD3 bespecifically single-chain antibody according to the invention can be used as a therapeutic agent against autoimmune diseases, preferably psoriasis.

From the point of view of the above, there is no need to design simulating the PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, Arcam×CD3, IGF-1R×CD3 or FAPα×CD3) bespecifically single-chain antibody for testing in phylogenetically distant (from people). As a result, identical molecule can be used in preclinical testing in animals and, when it is intended for administration to humans in clinical testing and for subsequent permission to sell and therapeutic use as a drug. The ability to use the same molecule for preclinical animal testing and later for introducing people actually eliminates or at least substantially reduces the risk that data obtained in preclinical testing on animals, will have limited applicability to people Briefly, obtaining preclinical safety data in animals using the same molecules, which in reality will be administered to the people, gives greater assurance of applicability to the scenario pertaining to people. On the contrary, in the traditional approaches with the use of molecules imitators need to adapt molecular these molecules simulators to animal test system used for preclinical safety assessment. So, the molecule is intended for use in therapy, it differs from molecules simulator used in preclinical testing, in sequence and probably structure, pharmacokinetic parameters and/or biological activity, and therefore, data obtained in preclinical testing on animals, have limited applicability to humans/tolerability in humans. The use of molecules imitators requires the design, production, purification and characterization of a fully new design. This leads to additional development costs and the need to spend more time to obtain this molecule. In the end, in addition to developing the actual medicinal product which is intended for use in therapy of human rights, it is necessary to separately develop them�Tatary, we have had to spend two lines of development for the two molecules. Thus, the main advantage of the described here PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) especifismo single-chain antibodies according to the invention, preferably a human, exhibiting cross-species specificity, is that the identical molecule can be used for therapeutic treatment of humans and in preclinical testing on animals.

Preferably, at least one of the specified first or second binding domain especifismo single-chain antibodies according to the invention is a CDR (a site that defines complementarity)-transplantirovannam, humanised or human, as detailed below. Preferably, the first binding domain and a second binding domain especifismo single-chain antibodies according to the invention are CDR-transplanted, humanized or human. For PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) especifismo single-chain antibodies according to the invention, preferably a human, the formation of the immune response against these binding molecules are eliminated to the extent possible with the introduction of the molecules of patients-people.

More about�him the main advantage of the PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) especifismo single-chain antibodies according to the invention, preferably a human, is its suitability for preclinical testing of various primates. The behavior of the drug candidate in animals should ideally correspond to the expected behavior of this drug candidate when administered to humans. Thus, it is necessary that the data obtained as a result of such pre-clinical testing, generally had a high predictive power for the people. However, the tragic outcome of previously conducted Phase I clinical trials using TGN1412 (a monoclonal antibody against CD28), when in preclinical testing indicated antibodies was not observed adverse effects or were observed only limited adverse effects in animal studies conducted in cynomolgus rhesus, and after the introduction of the specified antibodies patients-people in six patients-people have developed multiple organ failure (Lancet 368 (2006), 2206-7), indicates that the drug candidate may act differently in primates and in humans. The results of these dramatic unwanted negative phenomena show that the restriction of preclinical testing, only one species (prima�, non chimpanzees) may be insufficient. The fact that PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) bespecifically single-chain antibody of the invention binds some cacahuete and lower narrow-nosed monkeys, can help to solve problems arising in the case referred to above. Accordingly, according to the present invention proposed means and methods to minimize species differences in the effects when the development and testing of medicines for the treatment of a person.

Having PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) bespecifically single-chain antibody according to the invention, preferably a human, exhibiting cross-species specificity, it is no longer necessary to adapt the test animal to the drug candidate intended for administration to humans, for example to create transgenic animals. PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) bespecifically single-chain antibody according to the invention, preferably a human, exhibiting cross-species specificity, according to the uses and methods according to the invention can be directly used for preclinical testing on primates, which are not chimpanzees, without any genetic treatment �jivotnih. As is well known to specialists in this area, the approaches in which the test animal can adapt to a drug-candidate, there is always the risk that the results obtained in preclinical safety testing will be less representative and predictive for humans due to the modification of the animal. For example, the transgenic animal proteins encoded by the transgenes, often sverkhekspressiya in a high degree. Therefore, data obtained on the biological activity of antibodies against this protein antigen, may have limited predictive value for people who have protein is expressed at much lower, more physiological levels.

An additional advantage of exhibiting cross-species specificity PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) especifismo single-chain antibodies according to the invention, preferably a human, is that chimpanzees as endangered species are not used for animal testing. Chimpanzees are the closest relatives of humans, and earlier on the basis of data on human genome sequencing, they were grouped in the family of hominids (Wildman et al., PNAS 100 (2003), 7181). Therefore, it is usually assumed that data obtained using chimpanzees, in�high degree predictable for people. However, because of their endangered status, the number of chimpanzees, which can be used for medical experiments, is extremely limited. As mentioned above, the upkeep and maintenance of chimpanzees for testing on animals is, consequently, expensive, and ethically problematic. The use of PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) especifismo single-chain antibodies according to the invention, preferably human, and avoids ethical objections, and financial costs during preclinical testing without compromising the quality, i.e. the suitability of the data obtained have been tested on animals. In this regard, the use of PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) especifismo single-chain antibodies according to the invention, preferably human, provide a reasonable alternative to research on chimpanzees.

Another advantage of the PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) especifismo single-chain antibodies according to the invention, preferably a human, is the possibility of extraction of multiple blood samples when using them as part of preclinical testing on animals, for example during pharmacokinetic studies on the stomach�'s. Multiple extraction of blood samples is achieved easier with the use of the primacy of non-chimpanzee than with the use of lower animals such as a mouse. The extraction of multiple blood samples makes it possible to continuously test the blood parameters to determine the biological effects induced PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) bespecifically single-chain antibody according to the invention, preferably human. In addition, the extraction of multiple blood samples enables the researcher to assess the pharmacokinetic profile PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) especifismo single-chain antibodies according to the invention, preferably a human, as defined here. In addition, potential side effects that can be induced by the specified PSCA×CD3 (respectively CD19×CD3, C-MET×CD3 endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) bespecifically single-chain antibody according to the invention, preferably a human, as reflected by the blood can be measured in different blood samples extracted during the course of the introduction of the specified antibodies. This provides the possibility of determining the potential toxicity profile PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, ECAM×CD3, IGF-1R×CD3 or FAPα×CD3) especifismo single-chain antibodies according to the invention, preferably a human, as defined in this specification.

Advantages PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) especifismo single-chain antibodies according to the invention, preferably a human, as defined here, exhibiting cross-species specificity, can be briefly summarized as follows:

First, PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) bespecifically single-chain antibody according to the invention, preferably a human, as defined herein, which is used in preclinical testing, is the same antibody that is used in the treatment of the person. So there is no need to develop two independent molecules, which may differ in their pharmacokinetic properties and biological activity. The big advantage is that, for example, the pharmacokinetic results more directly transferable to and applicable to humans than, for example, in the traditional approaches with the use of simulators.

Secondly, the use of PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) especifismo single-chain antibodies p� to the invention, preferably human, as defined herein, to obtain therapeutic agents for humans is less expensive and time-consuming than the approaches with the use of simulators.

Thirdly, PSCA×CD3 (respectively CD19×CD3, C-MET×CD3, endothelin×CD3, EpCAM×CD3, IGF-1R×CD3 or FAPα×CD3) bespecifically single-chain antibody according to the invention, preferably a human, as defined herein, can be used for preclinical testing on only one species of primates, but also on the number of different species of primates, which reduces the risk of potential species differences between primates and man.

Fourthly, chimpanzees as an endangered species for testing on animals can be excluded if desired.

Fifthly, multiple blood samples can be extracted for a comprehensive pharmacokinetic studies.

Sixthly, due to the human origin, preferably human binding molecules according to a preferred embodiment of the invention, the formation of the immune response against these binding molecules is minimized when administered to patients people. Eliminates the induction of an immune response using antibodies specific to drug-candidate, non-human, such as, for example,mouse, leading to the development of human anti-mouse antibody (HAMA) against therapeutic molecules of mouse origin.

And last but not least:

- Therapeutic applications PSCA×CD3 especifismo single-chain antibodies according to the invention provides a novel and inventive therapeutic approach for cancer treatment, including, but not limited to, prostate cancer, bladder cancer or pancreatic cancer. The examples below clearly demonstrate for each construct the potent recruitment of cytotoxic activity of effector cells of human and macaque against cells that are positive for the PSCA.

Therapeutic application of CD19×CD3 especifismo single-chain antibodies according to the invention provides a novel and inventive therapeutic approach for the treatment of cancer, preferably a b-cell zlokacestvennosti, such as non-Hodgkin's lymphoma, mediated by b-cells in autoimmune diseases or the depletion of b-cells. As shown in the following Examples, the cytotoxic activity of the CD19×CD3 especifismo single-chain antibodies according to the invention is higher than the activity of antibodies described in this field.

- Therapeutic applications of C-MET×CD3 especifismo single-chain antibodies according to the invention provides a new and inventor�cue therapeutic approach for the treatment of cancer, preferably carcinoma, sarcoma, glioblastoma/astrocytoma, melanoma, mesothelioma, Wilms tumor or a hematopoietic malignancy such as leukemia, lymphoma or multiple myeloma. As shown in the following Examples, the cytotoxic activity of C-MET×CD3 especifismo single-chain antibodies according to the invention is higher than the activity of antibodies described in this field.

- Therapeutic use endothelin×CD3 especifismo single-chain antibodies according to the invention provides a novel and inventive approach for targeting tumor endothelial cells and their destruction, including (but not limited to) carcinomas (breast, kidney, lung, colorectal, colon, pancreas mesothelioma), sarcomas, and neuroectodermal tumors (melanoma, glioma, neuroblastoma). Endothelin×CD3 bespecifically single-chain antibody according to the invention may deprive solid tumors their supporting blood vessels, thereby inhibiting angiogenesis and thus the growth of these tumors.

- Therapeutic use Arcam×CD3 especifismo single-chain antibody of the invention provide a new and inventive therapeutic approach for cancer treatment, preferably epithelial cancer and/or minimum R�idealnogo cancer. EpCAM×CD3 bespecifically single-chain antibody according to the invention not only provides a preferential tool for the destruction of Hersam-xpressiya cancer in cancer cells, preferably epithelial cancer or a minimal residual cancer, but it may be useful to eliminate the alleged perpetrators responsible for tumor relapse after therapy. In addition, the cytotoxic activity of EpCAM×CD3 especifismo single-chain antibodies according to the invention is higher than the cytotoxic activity of antibodies described in this field.

Therapeutic application of FAP-alpha×CD3 especifismo single-chain antibodies according to the invention provides a novel and inventive approach for targeting tumor stromal cells and their destruction: FAP-alpha×CD3 bespecifically single-chain antibody according to the invention deprives solid tumors such as epithelial tumors, their supporting stroma, thereby inhibiting the growth and neovascularization of solid tumors.

Therapeutic application of IGF-1R×CD3 especifismo single-chain antibodies according to the invention provides a novel and inventive approach for treating cancer (preferably bone cancer or soft tissue (e.g. Ewing sarcoma), breast cancer, liver cancer, lung cancer, cancer in the head isei, colorectal cancer, prostate cancer, uterine leiomyosarcoma, cervical and endometrial cancer, ovarian cancer, prostate cancer and pancreatic cancer) or autoimmune disease (preferably psoriasis).

As noted in the description above, according to the present invention proposed polypeptides, i.e. bespecifically single-chain antibodies containing the first binding domain capable of contacting epitope CD3ε-chain of human rights and the primacy of non-chimpanzee, and a second binding domain capable of contacting PSCA (respectively CD19, C-MET, endothelin, Arcam, IGF-1R or FAPα), and the second binding domain preferably binds to PSCA (respectively CD19, C-MET, Endothelin, Arcam, IGF-1R or FAPα) human and Primate, non chimpanzees. The advantage of molecules bespecifically single-chain antibodies as drugs candidates that meet the requirements of the preferred especifismo single-chain antibodies according to the invention, consists in the use of these molecules in preclinical testing in animals and in clinical studies and even for the treatment of a person. In a preferred embodiment bespecifically single-chain antibody of the invention exhibiting cross-species specificity, the second binding domain, bind�ISA cell surface antigen, is the human. In bespecifically molecule according to the invention with cross-species specificity, binding domain that binds to an epitope of CD3-Epsilon-chain of human rights and the primacy of non-chimpanzee, is in the order VH-VL or VL-VH to the N-end or C-end bespecifically molecules. Examples bespecifically molecules according to the invention exhibiting cross-species specificity, with different arrangements of VH chain and VL chain in the first and second binding domains are described in the examples below.

Used in this description, the term "bespecifically single-chain antibody" means a single polypeptide chain containing two binding domain. Each binding domain comprises one variable region from a heavy chain antibody ("VH region"), where VH-region of the first binding domain specifically binds to the CD3ε molecule, and the VH-region of the second binding domain specifically binds to PSCA, CD19, C-MET, indocyanines, Arcam, IGF-1R or FAPα. These two binding domain may be linked to each other short polypeptide spacer. Non-limiting examples of polypeptide spacer is Gly-Gly-Gly-Gly-Ser (G-G-G-G-S) and its repetitions. Each binding domain may further comprise a single variable region of light chain antibody ("VL-region"), and the VH-region and VL-region in each of the first and veraswami domains linked via a polypeptide linker, for example the type described and claimed in EP 623679 B1, but in any case having a length sufficient to allow the VH-region and VL-region of the first binding domain and a VH-region and VL-region of the second binding domain to form a couple with each other so that together they were able to specifically contact the respective first and second binding domains.

The term "protein" is well known in this field and describes biological compounds. Proteins contain one or more amino acid chains (polypeptides), in which amino acids are linked together by peptide bonds. The term "polypeptide" as used herein, describes a group of molecules that consist of more than 30 amino acids. In accordance with the invention, this group of polypeptides includes "whites" provided that proteins are composed of a single polypeptide chain. In addition, in accordance with the definition of the term "polypeptide" describes fragments of proteins, provided that these fragments are composed of more than 30 amino acids. Polypeptides also can form multimeric, such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide molecule. Polypeptide molecules that form these dimers, trimers, etc., may be identical or non-identical. Appropriate structures �more of a high order such multimeric called, therefore, Homo - or heterodimers, Homo - or heterotrimer, etc. an Example of heteropolymer is an antibody molecule, which, in its existing in nature, form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms "polypeptide" and "protein" also refer to natural modified polypeptides/proteins, in which the modification is carried out, for example, as a result of posttranslational modifications in the type of glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in this field.

The term "binding domain" characterizes in accordance with the present invention, a domain of the polypeptide, which specifically binds to/interacts with the given(s) target(s) structure/antigen/epitope. Thus, the binding domain is a "site of interaction with the antigen. The term "site of interaction with the antigen determines, in accordance with the present invention, the polypeptide motif, which is able to specifically interact with a specific antigen or group of antigens, for example with the same antigen in different species. It is also clear that the binding/interaction defines "specific recognition". The term "specifically recognizing" means in accordance with the USA�AI with the present invention, the fact that the antibody molecule is able to specifically interact and/or communicate with at least two, preferably at least three, more preferably at least four amino acids of the antigen, such as CD3 antigen of human rights, as defined in this description. Such linking can be illustrated with specificity on the "principle of lock and key". Thus, specific motifs in the amino acid sequence of the binding domain and the antigen bind to each other through their primary, secondary, or tertiary structure, and also due to secondary modifications of this structure. Specific interaction site of interaction of the antigen with its specific antigen can also lead to a simple specified binding site to the antigen. Moreover, the specific interaction site of interaction of the antigen with its specific antigen alternative may lead to the initiation signal, for example by induction of changes in the conformation of the antigen oligomerization of the antigen, etc. Preferred example of a binding domain according to the present invention is an antibody. Binding domain may be a monoclonal or polyclonal antibody or may be derived from monoclonal or polyclonal and�of tetela,

The term "antibody" encompasses derivatives or functional fragments which still retain the binding specificity. Methods of obtaining antibodies is well known in this field and are described, e.g. in Harlow and Lane "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane "Using Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, 1999. The term "antibody" also encompasses immunoglobulins (Ig) of different classes (i.e. IgA, IgG, IgM, IgD and IgE) and subclasses (such as lgG1, lgG2, etc.).

The definition of the term "antibody" also includes such embodiments, chimeric, single chain and humanized antibodies as well as fragments of antibodies, such as, inter alia, Fab fragments. Fragments or derivatives of antibodies include fragments F(ab')2, Fv, scFv, or single-domain antibodies, antibody consisting of a single variable domain or immunoglobulin single variable domain that contains only one variable domain, which can be either the VH or VL, that specifically bind to the antigen or epitope independently of other V regions or domains (see, e.g., Harlow and Lane (1988) and (1999), cited above). Such immunoglobulin single variable domain encompasses not only the polypeptide selected single variable domain of the antibody, but also larger polypeptides that contain one and�or more monomers of the polypeptide sequence of a single variable domain of the antibody.

In this area there are various methods that can be used to obtain such antibodies and/or fragments. So, derivative (antibodies) can be also obtained with the use of peptidomimetics. Further, techniques described for obtaining single-chain antibodies (see, among others, U.S. patent 4946778), can be adapted for obtaining single-chain antibodies specific to a selected(s) polypeptide(s). Furthermore, for expression of humanized antibodies specific to the polypeptides and fusion proteins of the present invention, can be used transgenic animals. To obtain monoclonal antibodies, one can use any technique which provides antibodies produced by cultures of stable cell lines. Examples of such techniques include a hybrid technique (Kohler and Milstein, Nature, 256 (1975), 495-497), creamnow technique, hybrid technique using b-cells (Kozbor, Immunology Today 4 (1983), 72) and EBV-hybrid technique (EBV - Epstein-Barr) for the production of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96). Surface plasma resonance, which is used in the BIAcore system can be used to increase the efficiency of phage antibodies that bind to the epitope of the target polypeptide, such as CD3-Epsilon, PSA, CD19, C-MET, endothelin, Arcam, IGF-1R or FAPα (Schier, Human Antibodies Hybridomas, 7 (1996), 97 To 105; Malmborg, J. Immunol. Methods, 183 (1995), 7-13). In the context of this invention also provides that the term "antibody" encompasses structures of antibodies that can be expressed in the host, as described in the description below, for example the design of antibodies that can be transfected by and/or transpulmonary using, among other things, viruses or plasmid vectors.

The term "specific interaction" as used in accordance with the present invention, means that the binding domain does not possess the ability to cross-communicate, or does not have significant cross ability to interact with polypeptides having a structure similar to the structure of the polypeptide with which binding domain binds, and which could be expressed by the same cells, and the polypeptide of interest. Cross the ability to interact with the panel investigated binding domain can be tested, for example, by assessing binding of the specified panel binding domain in standard conditions (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988 and Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999). Examples of specific interaction of the binding domain with concrete�m include antigen specificity of the ligand to its receptor. This definition, in particular, covers the interaction of ligands that induce a signal after binding to its specific receptor. An example of this interaction, which, in particular, also covered by the specified definition, is the interaction of antigenic determinants (epitope) binding domain (binding site) of an antibody.

As used herein, the term "interspecies specificity", or "cross-species specificity" or means the binding of a binding domain described herein, with the same molecule-target in humans and primates, which are not chimpanzees. Thus, "cross-species specificity" or "cross-species specificity", should be understood as inter-specific ability to interact with the same molecule "X" (i.e. the homologue), expressed in various forms, but not with a molecule other than "X". Cross-species specificity of a monoclonal antibody that recognizes, for example, CD3-Epsilon person, in relation to CD3-Epsilon, the primacy of non-chimpanzee, for example, CD3-Epsilon macaque may be determined, for example, FACS-analysis (analysis using cell sorting device with activation of fluorescence (fluorescence-activated cell sorter)). FACS-analysis carried out by a method wherein the corresponding monoclonal antibody test for the hol�ivanie with human cells and Primate, non chimpanzees, such as macaque cells expressing these antigens CD3-Epsilon person and the primacy of non-chimpanzee, respectively. The corresponding analysis is presented in the examples below. The above is applicable, mutatis mutandis, for the antigen PSCA, CD19, C-MET, endothelin, Arcam, IGF-1R and FAPα: cross-species specificity of a monoclonal antibody that recognizes, for example, PSCA, CD19, C-MET, endothelin, Arcam, IGF-1R or FAPα person, in relation to PSCA, CD19, C-MET, indocyanine, Arcam, IGF-1R or FAPα the primacy of non-chimpanzee, for example, PSCA, CD19, C-MET, indocyanine, Arcam, IGF-1R or FAPα macaque, can be determined, for example, FACS-analysis. FACS-analysis carried out by a method wherein the corresponding monoclonal antibody is tested for binding to cells of man and the primacy of non-chimpanzee, for example cells of macaques expressing these antigens PSCA, CD19, C-MET, endothelin, Arcam, IGF-1R or FAPα of man and the primacy of non-chimpanzee, respectively,

Used in this description, the term "CD3-Epsilon" means a molecule, expressed as part of the T-cell receptor, and has the meaning usually attributed to it in the prior art. As for the person, the term includes an individual independently or combined form of all known�tion of CD3 subunits, for example, CD3-Epsilon, CD3-Delta, CD3-gamma, CD3-Zeta, CD3-alpha and CD3-beta. CD3 antigens primates that are not chimpanzees, which are mentioned in this description are, for example, CD3 of Masasa fascicularis and CD3 of Massa mulatta. As for Masasa fascicularis, the term encompasses the CD3-Epsilon FN-18-negative and CD3-apsion FN-18-positive, CD3-gamma and CD3-Delta. As for Massa mulatta, the term encompasses the CD3-Epsilon, CD3-gamma and CD3-Delta. Preferably, the specified CD3, as used in this description, is the CD3-Epsilon.

CD3-Epsilon person listed in GenBank under the number of access NM_000733 and contains SEQ ID NO: 1. CD3-gamma person listed in GenBank under the number of access NM_000073. CD3-Delta man is listed in GenBank under the number of access NM_000732.

CD3-apsion "FN-18-negative" Masasa fascicularis (i.e., CD3-Epsilon, is not recognized by the monoclonal antibody FN-18 due to polymorphism, as described above) are listed in GenBank under the number of access AV.

CD3-Epsilon "FN-18-positive" Masasa fascicularis (i.e., CD3-Epsilon, recognized by monoclonal antibody FN-18) is listed in GenBank under the number of access AV. CD3-gamma Masasa fascicularis listed in GenBank under the number of access AV. CD3-Delta of Masasa fascicularis listed in GenBank under the number of access AV.

Nucleic acid sequence and amino acid sequence corresponding to CD3-Epsilon, gamma and Delta homologues of Massa mulatta can be identified � allocated using recombinant techniques, described in the art (Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 3rdedition, 2001). Applicable, with the necessary amendments, CD3-Epsilon, gamma and Delta homologues of other primates that are not chimpanzees, are defined in this description. Identification of the amino acid sequence of the marmoset (Callithrix jacchus), squirrel monkeys (Saimiri sciureus) and Oedipus Tamarin (Saguinus oedipus) described in the examples below. Amino acid sequence of the extracellular domain of CD3-Epsilon Callithrix jacchus presented in SEQ ID NO: 3, amino acid sequence of the extracellular domain of CD3-Epsilon Saguinus oedipus is presented in SEQ ID NO: 5, and amino acid sequence of the extracellular domain of CD3-Epsilon Saimiri sciureus presented in SEQ ID NO: 7.

PSCA person is listed in GenBank under the number of access NM_005672. The corresponding cDNA and amino acid sequence presented in SEQ ID NO: 444 and 443 respectively. Cloning of a homologue of macaque PSCA demonstrated in the examples below. The corresponding cDNA and amino acid sequence presented in SEQ ID NO: 446 and 445, respectively.

CD19 person is listed in GenBank under the number of access NM_001770. On the basis of this information about sequences, the skilled in the art without any inventive activity clone (and to Express) molecule CD19 macaque. For example, cDNA C19 person or its fragment, specified GenBank under the number of access NM_001770, can be used as probe hybridization to cDNA library screening macaque (e.g. cDNA library Javanese monkey or macaque-rhesus) in appropriate hybridization conditions. Recombinant technology and screening methods (including hybridization approaches in molecular biology are described, for example, in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 3rdedition 2001.

C-MET person listed in GenBank under the number of access NM_000245. The corresponding cDNA and amino acid sequence presented in SEQ ID NO: 776 and 777, respectively. Cloning of a homologue of C-MET macaque demonstrated in the examples below. The corresponding cDNA and amino acid sequence presented in SEQ ID NO: 788 and 789, respectively.

Endothelin person is listed in GenBank under the number of access NM_020404. The corresponding cDNA and amino acid sequence presented in SEQ ID NO: 913 and 914, respectively. Cloning of a homologue of indocyanine macaque demonstrated in the examples below, the corresponding cDNA and amino acid sequence presented in SEQ ID NO: 915 and 916, respectively.

Arcam person is listed in GenBank under the number of access NM_002354. The corresponding cDNA and amino acid sequence presented in SEQ ID NO: 1029 and 1028, respectively. As demonstrated in the following�tion following examples, the second binding domain EpCAM×CD3 especifismo single-chain antibody of the invention binds to an epitope located within amino acid residues 26-61 EGF-like domain 1 Ersam, which is encoded by exon 2 of the gene Ersam. These amino acid residues 26-61 EGF-like domain 1 Ersam person represented in SEQ ID NO: 1130. On the basis of this information about sequences, the skilled in the art without any inventive activity clone (and to Express) molecule Arcam macaque. For example, cDNA Arcam person or its fragment that is listed in GenBank under the number of access NM_002354, can be used as probe hybridization to cDNA library screening macaque (e.g. cDNA library Javanese monkey or macaque-rhesus) in appropriate hybridization conditions. Recombinant technology and screening methods (including hybridization approaches in molecular biology are described, for example, in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 3rdedition 2001.

FAP-alpha man is listed in GenBank under the number of access NM_004460. The corresponding cDNA and amino acid sequence presented in SEQ ID NO: 1149, and 1150, respectively. Cloning of a homologue of FAP-alpha macaque demonstrated in the examples below, the corresponding cDNA and amino acid sequence presented in SEQ ID NO 1151 and 1152, respectively.

IGF-1R person is listed in GenBank under the number of access NM_000875. The corresponding cDNA and amino acid sequence presented in SEQ ID NO: 1988 and 1989 respectively. Coding sequence of IGF-1R macaque published in GenBank (access number HM). The corresponding cDNA and amino acid sequence presented in SEQ ID NO: 1998 and 1999 respectively.

In accordance with the above, the term "epitope" defines an antigenic determinant that specifically binds to/identified binding domain, as defined above. Binding domain can specifically communicate/interact with conformational or continuous epitopes, which are unique to the structure of the target, for example with CD3-Epsilon-HenbK) of man and the primacy of non-chimpanzee, or PSCA, CD19, C-MET, indocyanines, Arcam, IGF-1R or FAPα of man and the primacy of non-chimpanzee. Conformational or continuous epitope is characterized in relation to polypeptide antigens by the presence of two or more discrete amino acid residues which are separated in primary sequence, but come together on the surface of the molecule when the polypeptide folds into the native protein/antigen (Sela, (1969) Science 166, 1365 and Laver, (1990) Cell 61, 553-6). Two or more individual amino acid residues contributing to the epitope are present separately located on teaching�Tkach one or more than one polypeptide chain. These residues are combined on the surface of the molecule when the polypeptide(s) chain(s) is minimized(for example, in a three-dimensional structure with the formation of the epitope. Conversely, continuous or linear epitope consists of two or more individual amino acid residues that are present in a single linear segment of the polypeptide chain. In the present invention dependent from the environment" the CD3 epitope refers to a specified conformation of the epitope. This is dependent on the environment epitope located on CD3-Epsilon chains, can be folded into the correct conformation, only if it is immersed in the rest of the Epsilon-chain and held in proper position by heterodimerization Epsilon-chain or gamma-chain, or with the Delta-chain of CD3. On the contrary, independent from the environment of the CD3 epitope, which is proposed in this application, refers to N-terminal containing amino acid residues 1-27 of the polypeptide or its functional fragment of the CD3-Epsilon chain. This N-terminal containing amino acid residues 1-27 polypeptide or its functional fragment retains its three-dimensional structural integrity and correct conformation when it is removed from the native environment in the CD3 complex. Independence from the environment N-terminal containing amino acid residues 1-27 of the polypeptide or its functional fragment, which is part of unikl�exact domain of CD3-Epsilon, represents, thus, epitope, which is completely different from the epitopes of CD3-Epsilon, described in WO 2004/106380 in connection with the method of producing the human binding molecules. In this method, used separately expressed recombinant CD3-Epsilon. The conformation of this separately expressed recombinant CD3-Epsilon different from the conformation adapted to its natural form, i.e. the form in which the subunit of CD3-Epsilon TCR/CD3-complex exists as part of a non-covalent complex or subunit of CD3-Delta or subunit of CD3-gamma TCR/CD3-complex. If the antigen for selection of antibodies from libraries of antibodies using separately expressed recombinant protein CD3-Epsilon, from the library identify antibodies specific to this antigen, although this library does not contain antibodies with specificity to self antigens/autoantigens. This is because the separately expressed recombinant protein CD3-apsion does not exist in vivo; it is not autoantigen. Consequently, subpopulations of b cells expressing antibodies specific to this protein were depleted in vivo; a library of antibodies constructed from such cells will contain the genetic material for antibodies specific to that individual�of expressed recombinant protein CD3-Epsilon.

However, because independent from the environment N-terminal containing amino acid residues 1-27 polypeptide or its functional fragment is an epitope, which is folded in its native form, the binding domains of the present invention can be identified by methods based on the approach described in WO 04/106380. Therefore, it was possible to verify by tests that binding molecules, described in WO 04/106380, not able to contact with the N-terminal amino acid residues 1-27 CD3-Epsilon chain. Therefore, traditional anti-CD3-binding molecules or molecules of antibodies against CD3 (e.g., described in WO 99/54440) bind to CD3-Epsilon chain in a position that is closer to the C-Terminus than independent from the environment N-terminal containing amino acid residues 1-27 polypeptide or functional fragment, referred to in this description. Molecules of antibodies ACT and UCHT-1 prior art also have the specificity to the Epsilon-subunit TSC/CD3-complex between amino acid residues 35 and 85, and thus the epitope of these antibodies is also located closer to the C-Terminus. In addition to this, UCHT-1 binds to CD3-Epsilon chain in the region between amino acid residues 43 and 77 (Tunnacliffe, Int. Immunol., 1 (1989), 546-50; Kjer-Nielsen, PNAS, 101 (2004), 7675-7680; Salmeron, J. Immunol., 147 (1991), 3047-52). Therefore, anti-CD3-Molek�crystals of the prior art are not associated with a particular in this description is independent of the environment of the N-terminal, containing amino acid residues 1-27 of the epitope (or functional fragment) and not directed against him. In particular, the state of the prior art does not provide anti-CD3-molecules that bind specifically to independent from the environment of the N-terminal containing amino acid residues 1-27 of the epitope, and which have cross-species specificity, i.e. contact CD3-Epsilon person and the primacy of non-chimpanzee.

To create a binding domain, preferably a human, are contained in the molecule especifismo single-chain antibodies according to the invention, can be used, for example, a monoclonal antibody that binds to CD3-Epsilon both human and Primate, non chimpanzees (for example with CD3-Epsilon macaque), or monoclonal antibody that binds to PSCA, respectively CD19, C-MET, indocyanines, Arcam, IGF-1R or FAPα person and/or the primacy of non-chimpanzee.

Used in this description, the terms "human" and "person" refer to the species Homo sapiens. Since we are talking about the medical applications of the constructions described in this description, patients, people should be treated the same molecule.

Preferably, at least one of the specified first or second binding domain especifismo single-chain antibodies according to the invention is�I CDR-transplantirovannam, humanised or human. Preferably, the first and second binding domains especifismo single-chain antibodies according to the invention are CDR-transplanted, humanized or human.

Used in this description, the term "human" antibody means that bespecifically single-chain antibody as defined herein, contains the amino acid(s) sequence(s) contained party in the repertoire of antibody germline of the person. In order to determine a specified bespecifically single-chain antibody can therefore be considered human if it consists of such(their) amino acid(s) sequence(s), i.e. if the(s) amino acid(s) sequence(s) especifismo single-chain antibodies is(are) identical(bubbled) expressed(bubbled) amino acid(bubbled) sequence(s) in the germline of the person. Bespecifically single-chain antibody as defined herein, can be considered human if it consists of a sequence(s) that(s) rejected(are) from its (their) closest () sequence(s) of the germline of the person not more than would be expected on the imprint of somatic hypermutation. Additionally, antibodies of many mammals, reptiles, birds�them non-people, for example rodents, such as mice and rats, contain the amino acid sequence of a VH CDR3, which, as you might expect, also exist in the expressed repertoire of human antibodies. Any(s) so(s) sequence(s) of human or of human origin, which is(s), as you might expect, exist(s) in the expressed human repertoire, may also be considered "human" for the purposes of the present invention.

Used in this description, the terms "humanized", "humanization", "humanoid" or grammatically related variants are used interchangeably and refer to bespecifically single-chain antibody containing at least one of its binding domain of at least one portion that defines complementarity ("CDR") of antibodies are not human or its fragment. Humanization approaches described for example in WO 91/09968 and US 6407213. As non-limiting examples, the term covers the case when a variable region of at least one binding domain contains a single CDR, for example, the third VH CDR in (CDRH3), from another animal, not a person, for example a rodent and a case where the variable region/region contains(at) respective first, second and third CDRs from the specified MS�now, non-human. In the case when all CDR binding domain especifismo single-chain antibodies are replaced by their corresponding equivalents, e.g., a rodent, usually speak of "CDR-transplantation", and this term should be understood as covered by the term "humanized" or grammatically related variants used in this description. The term "humanized" or grammatically related variants also cover cases where, in addition to the replacement of one or more CDR within VH and/or VL of the first and/or second binding domain, an additional(s) mutation(s) (e.g. replacement) at least one single amino acid residue (residues) in frame sections (FR) between CDR-sections performed(s) in such a way that the amino acid(s) in this/these position(s) complies(ly) with the amino acid(s) in this/these position(s) of the animal from which is used to replace the CDR-plots. As is well known in this field, such individual mutations are often made in wireframe plots after CDR-transplantation to restore the original affinity of binding of the antibodies are not human, used as a CDR in the donor to target molecules. The term "humanized" may also encompass amino acid(s) substitution(s) in the CDR regions of animal�, not human, amino acid(s) of the corresponding CDR of the site of human antibodies, in addition to amino acid substitutions in the frame sections, as described above.

Used in this description, the terms "homologue" or "homology" means the following: the conclusion that there are homologies between proteins and DNA is often done on the basis of sequence similarity, especially in bioinformatics. For example, if two or more genes have highly similar DNA sequences, it is likely they are homologous. But the similarity of sequences may arise from different ancestors: short sequences may be similar by chance, and the sequence may be similar since both selected to bind with a particular protein such as a transcription factor. Such sequences similar but not homologous. Region sequences that are homologous, also called conservative. It should not be confused with conservatism in the amino acid sequences in which amino acid in a specific position replaced, but physico-chemical properties of amino acids remain unchanged. Homologous sequences are of two types: ontologicheskie and paralogies. Homologous sequences are ontologicheskie if they finish�dissolved speciation event: when a species diverges into two separate species divergent copies of a single gene in the resulting form is considered ontologicheskie. Orthologs, or ontologicheskie genes, are genes in different species that are similar to each other because they originate from a common ancestor. The most compelling evidence that two similar gene are ontologicheskie, is the result of phylogenetic analysis of lineage genes. Genes that are found in a single monophyletic taxon, are orthologs, peredauschiesya inherited from a common ancestor. Orthologs often, but not always, have the same function. Ontologicheskie sequences provide useful information in studies of taxonomic classification of organisms. The pattern of genetic divergence can be used to determine the relatedness of organisms. Two organisms that are in very close relationship, obviously, will show very similar DNA sequences between the two orthologs. In contrast, an organism that is evolutionarily removed from another organism, is likely to show more divergence in the sequence of the orthologs. Homologous sequences are patalogichesky if they are separated by a duplication event gene: if a gene in the body are duplicated, occupying two different positions in the same genome, t� two copies are patalogichesky. The set of sequences that are patalogichesky, called paralogue each other. Paralogy usually have the same or similar function, but sometimes not: due to the lack of initial selective pressure on duplicated copy of this gene copy freely mutates and acquires a new function. An example can be found in rodents such as rats and mice. Rodents have a pair of paralogistic genes of insulin, although it is unclear whether there was any deviation in the function. Paralogies genes often belong to one and the same species, but it is not required: for example, the gene for hemoglobin people and myoglobin gene chimpanzees are paralogue. This is a common problem in bioinformatics: when the genome sequences of different species is established and homologous genes are found, an expert is unable to immediately come to the conclusion that these genes have the same or similar function, since they can be paralogue, the function of which varies.

Used in this description, the term "primacy of non-chimpanzee" or "the primacy of non-chimp." or its grammatical variants refers to any animal-Primate (i.e. not a person), other than chimpanzees, i.e., other than an animal belonging to the genus Pan, including Pan paniscus (pygmy chimpanzee, Bonobo) and Pan troglodytes (common chimpanzee), also known as Anthropopithecustroglodytes (common chimpanzee) or Simla satyrus (the orangutan). It should be borne in mind, however, that it is possible that the antibodies according to the invention can also contact using their first and/or second binding domain with the corresponding epitopes/fragments, etc. these chimpanzees. The present invention simply allows you to avoid testing on animals using chimpanzees, if desirable. Therefore, it is also envisaged that in another embodiment the antibodies of the present invention are also associated with the use of their first and/or second binding domain with the corresponding epitopes chimpanzees. The terms "Primate", "species of primates", "primates" or grammatical variants mean the detachment of the placental mammals, divided into two suborders of prosimians and anthropoids and including apes, monkeys and lemurs. Specifically, the term "Primate" as used in this description, includes the suborder Strepsiirhini (Micronesia monkeys) (of paleobotany that are not tarsiers), including infraorder Lemurifonnes (glamourouslesya) (including the superfamily Cheirogaleoidea (dwarf lemurs) and Lemuroidea (Lemuria)), infraorder Chiromyiformes (reconocibles) (including the family Daubentoniidae (roanokeva)) and infraorder Lorisiformes (lorianne) (including family Loiisidae (laravie) and Galagidae (ganagobie)). The term "Primate" as used herein, also includes PEDOT�poison Haplorrhini (sugenoya monkeys), including infraorder Tarsiiformes (Dolgopyatova) (including the family Tarsiidae (Dolgopyatova)), infraorder Simiiformes (obezyanoobrazny) (including Platyrrhini (new world monkeys) or cacahuete monkeys and Catarrhini (old world monkeys), including Cercopithecidea (maryshopanne) or lower narrow-nosed monkeys)).

In the framework of the invention species of primates that are not chimpanzees, can be understood as representing a lemur, tarsier, Gibbon, igranka (owned cacahuetes monkeys of the family Cebidae (Capuchins)) or lower narrow-nosed monkey (belonging to the superfamily Cercopithecoidea (dog-like)).

Used in this description, the term "lower narrow-nosed monkey" includes any monkey of the superfamily Cercopithecoidea, which itself is divided into the following families: Cercopithecinae (martyshkova monkeys), which are found mainly in Africa, but includes the other gender macaques found in Asia and North Africa; and Colobinae (Tonkolili), which includes the most common genera in Asia and also the African representatives calstatela monkeys.

Specifically, within the subfamily Cercopithecinae preferred the primacy of non-chimpanzee can occur from the tribe Martynovich (Cercopithecini), within the genus Allenopithecus (swamp monkey Allen, Allenopithecus nigroviridis); within the genus Miopithecus (dwarf marmoset talapoin, Miopithecu talapoin; the Gabonese talapoin, Miopithecus ogouensis); within the genus Erythrocebus (common marmoset hussars, Erythrocebus patas); within the genus Chlorocebus (green monkey, Chlorocebus sabaceus; green monkey grivet, Chlorocebus aethiops; monkey chornobylska vervet, Chlorocebus djamdjamensis; monkey antalosky, Chlorocebus tantalus; vervada, Chlorocebus pygerythrus; malbrouck, Chlorocebus cynosuros); or within the genus Cercopithecus (Dryas monkey or monkey sologaistoa, Cercopithecus dryas; Diana monkey, Cercopithecus diana; monkey soloveyka, Cercopithecus roloway, large belonoha monkey, Cercopithecus nictitans; blue monkey, Cercopithecus mitis; the silver monkey, Cercopithecus doggetti; Golden monkey, Cercopithecus kandti; monkey six, Cercopithecus albogularis; Mona monkey, Cercopithecus top; monkey Mona Campbell, Cercopithecus campbelli; monkey Mona Lowe, Cercopithecus lower, monkey crested Mona, Cercopithecus pogonias; monkey Mona wolf, Cercopithecus wolfi; Mona monkey dent, Cercopithecus denti; monkey small belonoha, Cercopithecus petaurista; white throat monkey, Cercopithecus erythrogaster, monkey of Sclater, Cercopithecus sclateli; slider monkey, Cercopithecus erythrotis; monkey globaliza [mustache], Cercopithecus cephus; red monkey, Cercopithecus ascanius; monkey L Hoest, Cercopithecus Ihoesti; monkey Preuss, Cercopithecus preussi; monkey solncenosca, Cercopithecus solatus; monkey cofinality, Cercopithecus hamlyni; de monkey Brass, Cercopithecus neglectus).

Alternative, preferred the primacy of non-chimpanzee, also within p�of semeistvo Cercopithecinae, but within the tribe Avianova (Papionini), can occur from the kind of macaques (Masasa) (monkey barbariska, Massa sylvanus; macaques linkworthy, Massa silenus; South swingvote macaques or beruk (Beruk), Masasa nemestrina; macaques North swingvote, Massa leonina; macaques agisci or Bokkoi, Massa pagensis; the macaques Siber, Massa siberu; moor macaques, Massa taiga; booted macaques, Massa ochreata; macaques Tonkin, Massa tonkeana; macaques Huck, Massa hecki; macaques gerontology, Massa nigriscens; macaques celebuski or crested black monkey, Massa nigra; macaques cynomolgus macaques or a crab-eater, or long-tailed macaques or Kera (Kera), Masasa fasciculans; short-tailed macaques or macaque bear, Massa arctoides; rhesus monkeys, Massa mulatta; Macaca Formanski mountain, Massa cyclopis; Japanese macaques, Massa fuscata; macaques Ceylon, Massa sinica; Indian macaques, Massa radiata; tailless macaques, Massa sylvanmus; Assamese macaques, Massa assamensis; Tibetan macaques or macaque Milne-Edwards, Massa thibetana; macaques arunachali or "the monkey from the depths of the forest", Masasa munzala); within the genus Lophocebus (mangabey red-necked, Lophocebus albigena; Lophocebus albigena albigena; Lophocebus albigena osmani; Lophocebus albigena johnstoni; mangoba chernokogyi, Lophocebus aterrimus; mangoba Odenbach, Lophocebus opdenboschi; mangoba highland, Lophocebus kipunjl); within the genus Papio (baboon monkey, Papio hamadryas; Sphinx, or Guinea baboon, Papio papio; the Anubis baboon, Papio anubis; baboon baboon or yellow�th baboon, Papio cynocephalus; chakma, or baboon bear, Papio ursinus); within the genus Theropithecus (gelada, Theropithecus gelada); within the genus Cercocebus (mangoba smoky, Cercocebus atys; Cercocebus atys atys; Cercocebus atys lunulatus; mangoba common, Cercocebus torquatus; mangoba prompt, Cercocebus agilis; mangoba zolotodobychi, Cercocebus chrysogaster, Mangala river (Tang), Cercocebus galeritus; mangoba Sanya, Cercocebus sanjei); or within the genus Mandrillus (mandrill, Mandrillus sphinx; driel, Mandrillus leucophaeus).

Most preferred is Masasa fascicularis (also known as macaques cynomolgus macaques or a crab-eater, and so designated in the Examples as "Javanese") and Massa mulatta (the rhesus monkey, designated as "RH").

Within the subfamily Colobinae preferred the primacy of non-chimpanzee can occur from the African group within the genus Colobus (colobus black Colobus satanas; Angolan colobus, Colobus angolensis', colobus Royal, Colobus polykomos; colobus bear, Colobus vellerosus; black-and-white guereza, Colobus guereza); within the genus Piliocolobus (colobus Western red, Piliocolobus badius; Piliocolobus badius bad/us; Piliocolobus badius temminckii; Piliocolobus badius waldronae; colobus pennant Piliocolobus pennantii; Piliocolobus pennantii pennantii; Piliocolobus pennantii epieni; Piliocolobus pennantii bouvieri; the red colobus Preuss, Piliocolobus preussi; red colobus Folon, Piliocolobus tholloni; red colobus, Central African, Piliocolobus foai; Piliocolobus foai foai; Piliocolobus foai ellioti; Piliocolobus foai oustaleti; Piliocolobus foai semlikiensis; Piliocolobus foai parmentierorum;red colobus Ugandan, Piliocolobus tephrosceles; red colobus songwise, Piliocolobus gordonorum; red colobus Zanzibar, Piliocolobus kirkii; colobus red river (Tang), Piliocolobus rufomitratus}; or within the genus Procolobus (olive colobus, Procolobus verus).

Within the subfamily Colobinae preferred the primacy of non-chimpanzee, an alternative may be one of a group of langurs (tanatarov), within the genus Semnopithecus (grey langur Nepali, Semnopithecus schistaceus; gray langur Kashmir, Semnopithecus ajax; the grey langur taraski, Semnopithecus hector, a langur Severianin grey, Semnopithecus entellus; gray langur ceroscopy, Semnopithecus hypoleucos; langur GENERALNYY grey, Semnopithecus dussumieri; langur grey pubescent, Semnopithecus priam); within the group of T. vetulus of the genus Trachypithecus (langur purpuralis, Trachypithecus vetulus; Nilgiri langur, Trachypithecus johnii); within the group of T. chstatus of the genus Trachypithecus (Javanese langur, Trachypithecus auratus; encotel silver, or the silvery langur, Trachypithecus cristatus; langur Indochinese, Trachypithecus germaini; langur tenasserim, Trachypithecus barbei); within the group of T. obscurus of the genus Trachypithecus (langur smoky, or spectacled, Trachypithecus obscurus; langur Feira, Trachypithecus phayrei); within the group of T. pileatus species Trachypithecus (langur saposhnikovia, Trachypithecus pileatus; langur, Shortridge, Trachypithecus shortridgei; the Golden langur, or tehipite gay, Trachypithecus geei); within the group of T. francoisi of the genus Trachypithecus (Francois langur, Trachypithecus francoisi; langur hatinhensis, Trachypithecus hatinhesis; the white-headed langur, Trachypithecus poliocephalus; langur lauriski, Trachypithecus laotum; a langur of Delacour, Trachypithecus delacouri; black langur Indochinese, Trachypithecus ebenus); or within the genus Presbytis (surely Sumatran, Presbytis melalophos; encotel striped, Presbytis femoralis; encotel serwacski, Presbytis chrysomelas; encotel belovedly, Presbytis siamensis; encotel white-fronted, Presbytis frontata; encotel Javanese, Presbytis comata; encotel Thomas, Presbytis thomasi; encotel of Hawes, Presbytis hosei; encotel chestnut, Presbytis rubicunda; langur metawiki or Joja, Presbytis potenziani; encotel natonski, Presbytis natunae).

Within the subfamily Colobinae preferred the primacy of non-chimpanzee, an alternative may be from the group of the day (odd-nosed) monkeys, within the genus Pygathrix (pygathrix krasnoprudny, Pygathrix nemaeus; pygathrix chernobilsky, Pygathrix nigripes; pygathrix seroiusly, Pygathrix cinerea); within the genus Rhinopithecus (snub-nosed monkey Golden, Rhinopithecus roxellana; snub-nosed monkey black, Rhinopithecus bieti; snub-nosed monkey grey, Rhinopithecus brelichi; langur monkey Tonkin snub, Rhinopithecus avunculus); within the genus Nasalis (nose APE ordinary, Nasalis larvatus); or within the genus Simias (langur swingvote, Simias concolor).

Used in this description, the term "marmoset" means any cacahuete monkeys of the genus Callithrix, relating, for example, to Atlantic igranka of the subgenus Callithrix (so!) (common marmoset, Callithrix (Callithrix) jacchus; marmoset chernuha Callithrix (Callithrix) penicillata; marmoset Wieda, Callithrix (Callithrix) kuhlii; white-headed marmoset, Callithrix (Callithrix) geoffroyi; yellow-headed marmoset, Callithrix (Callithrix) flaviceps; marmoset white ears, Callithrix (Callithrix) aurita); non Amazon igranka of the subgenus Mico (silk marmoset) (marmoset Rio-Akari, Callithrix (Mico) acariensis; Manicore marmoset, Callithrix (Mico) manicorensis; silvery marmoset, Callithrix (Mico) argentata; marmoset white, Callithrix (Mico) leucippe; Emilia's marmoset (Emilia''s marmoset), Callithrix (Mico) emiliae; marmoset chernositona, Callithrix (Mico) nigriceps; marmoset Mark (Marca''s marmoset), Callithrix (Mico) marcai; black-tailed marmoset, Callithrix (Mico) melanura; marmoset Steller's sea, Callithrix (Mico) humeralifera; Maues marmoset, Callithrix (Mico) mauesi; marmoset Golden (gold-and-white marmoset), Callithrix (Mico) chrysoleuca; the marmoset Gershkovich (Hershkovitz''s marmoset), Callithrix (Mico) intermedia; marmoset Satéré, Callithrix (Mico) saterei); igranka Roosmalens' Dwarf, belonging to the subgenus Callibella (Callithrix (Callibella) humilis); or dwarf igranka belonging to the subgenus Cebuella (Callithrix (Cebuella) pygmaea).

Other genera cacahuete monkeys include tamarino of the genus Saguinus (which includes a group of S. oedipus group S. midas group S. nigricollis group S. mystax group S. bicolor and a group of S. inustus) and squirrel monkeys genus Saimiri (e.g. Saimiri sciureus, Saimiri oerstedii, Saimiri ustus, Saimiri boliviensis, Saimiri vanzolini).

In a preferred embodiment of molecules especifismo single-chain antibodies according to the invention, the primacy of non-chimpanzee, is a low narrow-nosed monkey. In a more preferred embodiment �ƈ polypeptide of the lower narrow-nosed monkey is a monkey of the genus macaques of the genus Papio. Most preferably, a monkey of the genus of macaques is Assamese macaques (Masasa assamensis), macaques barbariski (Masasa sylvanus), bonnet macaques (Masasa radiata), booted macaques or celebuski booted (Masasa ochreata), macaques celebuski crested (Masasa nigra), macaques formanski mountain (Masasa cyclopsis), snow monkey or Japanese macaque (Masasa fuscata), cynomolgus macaques, macaques or a crab-eater, or long-tailed macaques (Masasa fascicularis), linkworthy macaques (Masasa silenus), swingvote macaques (Masasa nemestnna), the rhesus monkey (Masasa mulatta), Tibetan macaques (Masasa thibetana), macaques Tonkin (Masasa tonkeana), macaques Ceylon (Masasa sinica), short-tailed macaques, macaques or cracolici, or bear macaques (Masasa arctoides), or moor macaques (Masasa maurus). Most preferably, the monkey kind of baboons is the Hamadryas Baboon, Papio hamadryas; Guinea Baboon, Papio papio; Olive Baboon, Papio anubis; Yellow Baboon, Papio cynocephalus; Chacma Baboon, Papio ursinus.

In an alternative preferred embodiment of the molecules especifismo single-chain antibodies according to the invention, the primacy of non-chimpanzee, is cacahuete monkey. In a more preferred embodiment of this polypeptide cacahuete a monkey is a monkey of the genus Callithrix (tamarins)), of the genus Saguinus (Tamarina) or of the genus Saimiri (squirrel monkeys). Most preferably, a monkey of the genus Callithrix is Callithrix jacchus (common marmoset), monkeys�Oh of the genus Saguinus is Saguinus oedipus (cotton-top tamarins), and monkey of the genus Saimiri is Saimiri sciureus (squirrel monkey).

Used in this description, the term "cell surface antigen" means a molecule that is found on the cell surface. In most cases, this molecule will be located in or on the plasma membrane of cells, so that at least part of this molecule remains available in its tertiary form outside the cell. Non-limiting examples of cell surface molecules, which are localized in the plasma membrane, is a transmembrane protein that contains, in its tertiary conformation, regions of hydrophilicity and hydrophobicity. Wherein at least one hydrophobic region makes possible the introduction or insertion of cell surface molecules in the hydrophobic plasma membrane of the cell, and a hydrophilic region extend on either side of the plasma membrane into the cytoplasm and extracellular space, respectively. Non-limiting examples of cell surface molecules present on the plasma membrane are proteins that are modified at the cysteine residue to carry Palmitoyl group, proteins, modified by C-terminal cysteine residue to carry varnishlog group, or proteins that are modified at the C-Terminus to carry glycoside phosphatidylinositol�flax ("GPI") anchor. These groups allow covalent attachment of proteins to the outer surface of the plasma membrane, where they remain available for recognition of extracellular molecules such as antibodies. Examples of cell surface antigens are CD3-Epsilon and PSCA, respectively CD19, C-MET, endothelin, Arcam, IGF-1R or FAPα.

As explained in the above description, PSCA is a cell surface antigen that is a target for the treatment of various types of cancer, including, but not limited to, prostate cancer, bladder cancer or pancreatic cancer.

As also explained in the above description, CD19 is a cell surface antigen that is a target for the treatment of various types of cancer, including, but not limited to, b-cell malignancy such as non-Hodgkin's lymphoma, mediated by b-cells in autoimmune diseases or the depletion of b-cells.

As also explained in the above description, C-MET is a cell surface antigen that is a target for the treatment of various types of cancer, including, but not limited to, carcinoma, sarcoma, glioblastoma/astrocytoma, melanoma, mesothelioma, Wilms tumor or a hematopoietic malignancy such as leukemia, lymphoma or multiple mie�ohms.

In addition, as explained in the above description, endothelin is a cell surface antigen that is a target for the treatment of various types of cancer, including, but not limited to, carcinomas (breast, kidney, lung, colorectal, colon, pancreas mesothelioma), sarcomas, and neuroectodermal tumors (melanoma, glioma, neuroblastoma).

As also explained in the above description, Arcam is a cell surface antigen that is a target for the treatment of various types of cancer, including, but not limited to, epithelial cancer or a minimal residual cancer.

In addition, as explained in the above description, FAP-alpha is a cell surface antigen that is a target for therapy of various cancers, including, without limitation, epithelial cancer.

In addition, as explained in the description above, IGF-1R) is a cell surface antigen that is a target for the treatment of various types of cancer, including, but not limited to, bone cancer or soft tissue (e.g. Ewing sarcoma), breast cancer, liver cancer, lung cancer, cancer in the head and neck, colorectal cancer, prostate cancer, leiomyosarcoma, cervical and endometrial cancer, ovarian cancer, prostatic cancer�Oh cancer and pancreatic cancer, and autoimmune diseases, including, without limitation, preferably psoriasis.

In light of this, PSCA (respectively CD19, C-MET, endothelin, Arcam, IGF-1R or FAPα) can be also described as a tumor antigen. Used in this description, the term "tumor antigen" may indicate those antigens that are presented on the tumor cells. These antigens can be presented on the cell surface by extracellular phase, which is often combined with the transmembrane and cytoplasmic part of the molecule. These antigens can sometimes be presented only by tumor cells and never the normal cells. Tumor antigens can be expressed exclusively on tumor cells or may represent a tumor-specific mutation in contrast to normal cells. In this case, they are called tumor-specific antigens. More common are antigens that are presented by tumor cells and normal cells, and they are called tumor-associated antigens. These tumor-associated antigens can sverkhekspressiya unlike normal cells, or they are available for binding of antibodies in tumor cells due to the less compact structure of the tumor tissue in contrast to normal tissue. Non-limiting example�mi tumor antigens in accordance with the present invention are PSCA, CD19, C-MET, endothelin, Arcam, IGF-1R and FAPα.

As explained in the above description, the molecule especifismo single-chain antibody of the invention binds the first binding domain with an epitope CD3ε(Epsilon) chain of human rights and the primacy of non-chimpanzee, where the specified epitope is part of an amino acid sequence that is a member of the group consisting of 27 amino acid residues as shown in SEQ ID NO: 2, 4, 6 or 8, or a functional fragment.

In accordance with the present invention for molecules especifismo single-chain antibodies according to the invention it is preferable that the specified epitope is the portion of the amino acid sequence containing 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids.

More preferably, the epitope comprises at least the amino acid sequence Gln-Asp-Gly-Asn-Glu (Q-D-G-N-E).

Within the scope of the present invention, the functional fragment of the N-terminal amino acid residues 1-27 indicates that the specified functional fragment remains independent from the environment epitope maintaining their three-dimensional structural integrity if it is removed from its native environment in the CD3 complex (and merge with heterological amino acid sequence, such as Arcam or Fc-�part b immunoglobulin, for example, as shown in Example 3.1). The preservation of three-dimensional structure within a 27-amino-acid N-terminal polypeptide, or its functional fragment, CD3-Epsilon can be used to create a binding domain that bind to the N-terminal polypeptide fragment of CD3-Epsilon in vitro and with native CD3 complex (CD3-Epsilon-subunit of the CD3 complex) on T-cells in vivo with the same binding affinity. In the present invention, the functional fragment of the N-terminal 1-27 amino acid residues means that the proposed CD3-binding domains can still contact such functional fragments, and it does not depend on the environment. Specialist in the art is aware of methods epitope mapping to determine which amino acid residues of the epitope recognized by these anti-CD3-binding domains (for example, alanine scanning; see the examples below).

In one embodiment of the invention, the molecule especifismo single-chain antibodies according to the invention comprises a first binding domain capable of contacting epitope CD3ε-chain of human rights and the primacy of non-chimpanzee, and a second binding domain capable of contacting the cell surface PSCA antigen. In alternative embodiments of the invention specified second link�s domain is able to bind with cell surface antigen CD19, With METH, indocyanines, Arcam, IGF-1R or FAPα.

In the present invention is also preferred that the second binding domain binds to cell surface antigen PSCA, respectively CD19, C-MET, indocyanines, Arcam, IGF-1R or FAPα person and/or PSCA, respectively CD19, C-MET, indocyanines, Arcam, IGF-1R or FAPαa the primacy of non-chimpanzee. Particularly preferably, the second binding domain binds to cell surface antigen PSCA, respectively CD19, C-MET, indocyanines, Arcam, IGF-1R or FAPα person and PSCA, respectively CD19, C-MET, indocyanines, Arcam, IGF-1R or FAPα the primacy of non-chimpanzee, preferably PSCA, respectively CD19, C-MET, endothelin, Arcam, IGF-1R or FAPα macaque. It should be borne in mind that the second binding domain binds to at least one PSCA, respectively CD19, C-MET, indocyanines, Arcam, IGF-1R or FAPα the primacy of non-chimpanzee, however, he may also be contacted with two, three or more, the PSCA homologues, homologues respectively CD19, homologues of C-MET, homologues indocyanine, homologues Ersam, homologues of IGF-1R or homologues of FAPα of the primacy of non-chimpanzee. For example, the second binding domain can bind to PSCA, respectively CD19, C-MET, indocyanines, Arcam, IGF-1R or FAPα Javanese macaque and PSCA, respectively CD19, C-MET, indocyanines, Arcam, IGF-1R or FAP� macaque-rhesus.

To create a second binding domain of the molecule especifismo single-chain antibodies according to the invention, for example especifismo single-chain antibodies as defined herein, can be used a monoclonal antibody that binds to a cell surface antigen, such as PSCA, respectively CDIQ, C-MET, endothelin, Arcam, IGF-1R or FAPα and humans and/or the primacy of non-chimpanzee. Suitable binding domains for especificacao polypeptide, as defined herein, may be formed, for example, monoclonal antibodies with cross-species specificity of the recombinant methods described in this field. A monoclonal antibody that binds to a cell surface antigen of human rights and the homologue of the indicated cell surface antigen from the primacy of non-chimpanzee, can be tested by FACS analysis, as described above. For specialists in this field it is obvious that antibodies with cross-species specificity can also be created hybrid methods described in the literature (Milstein and Kohler, Nature 256 (1975), 495-7). For example, mice can be subjected to alternative immunization cell surface antigen of human rights and the primacy of non-chimpanzee, such as PSCA, respectively CD19, C-MET, endothelin, Arcam, IGF-1R or FAPα.Of these mice on a hybrid technology hybrid allocate cells producing an antibody with cross-species specificity, and analyzed by FACS analysis as described above. Education and analysis bespecifically polypeptides, such as bespecifically single-chain antibodies exhibiting cross-species specificity, as described here, demonstrated in the examples below. Advantages bespecifically single-chain antibodies exhibiting cross-species specificity, include those benefits that are listed in the description.

Particularly preferred in respect of the molecule especifismo single-chain antibodies according to the invention is that the first binding domain capable of contacting epitope CD3ε(Epsilon) chain of human rights and the primacy of non-chimpanzee contains a VL-region containing CDR-L1, CDR-L2 and CDR-L3 selected from the following:

(a) CDR-L1 as shown in SEQ ID NO: 27, CDR-L2 as shown in SEQ ID NO: 28 and CDR-L3 as shown in SEQ ID NO: 29;

(b) CDR-L1 as shown in SEQ ID NO: 117, CDR-L2 as shown in SEQ ID NO: 118 and CDR-L3 as shown in SEQ ID NO: 119; and

(c) CDR-L1 as shown in SEQ ID NO: 153, CDR-L2 as shown in SEQ ID NO: 154 and CDR-L3 as shown in SEQ ID NO: 155.

Variable regions, i.e., the variable light chain ("L" or "VL") and variable heavy chain ("H" or "VH") is defined in this field as providing binding domain of the antibody. These variabel�s area contains parcels defining complementarity.

The term "portion that defines complementarity" (CDR) as well known in this area to determine the antigenic specificity of the antibody. The term "CDR-L" or "LCDR" refers to the CDRs in VL, while the term "CDR-H" or "HCDR" refers to the CDRs in VH.

In an alternative preferred embodiment of the molecules especifismo single-chain antibodies according to the invention the first binding domain capable of contacting epitope CD3ε(Epsilon) chain of human rights and the primacy of non-chimpanzee contains a VH-region containing CDR-H1, CDR-H2 and CDR-H3 selected from the following:

(a) CDR-H1 as shown in SEQ ID NO: 12, CDR-H2 as shown in SEQ ID NO: 13 and CDR-H3 as shown in SEQ ID NO: 14;

(b) CDR-H1 as shown in SEQ ID NO: 30, CDR-H2 as shown in SEQ ID NO: 31, CDR-H3 as shown in SEQ ID NO: 32;

(c) CDR-H1 as shown in SEQ ID NO: 48, CDR-H2 as shown in SEQ ID NO: 49 and CDR-H3 as shown in SEQ ID NO: 50;

(d) CDR-H1 as shown in SEQ ID NO: 66, CDR-H2 as shown in SEQ ID NO: 67, and CDR-H3 as shown in SEQ ID NO: 68;

(e) CDR-H1 as shown in SEQ ID NO: 84, CDR-H2 as shown in SEQ ID NO: 85, and CDR-H3 as shown in SEQ ID NO: 86;

(f) CDR-H1 as shown in SEQ ID NO: 102, CDR-H2 as shown in SEQ ID NO: 103, and CDR-H3 as shown in SEQ ID NO: 104;

(g) CDR-H1 as shown in SEQ ID NO: 120, CDR-H2 as shown in SEQ ID NO: 121, and CDR-H3 as shown in SEQ ID NO: 122;

(h) CDR-H1, �AK is presented in SEQ ID NO: 138, CDR-H2 as shown in SEQ ID NO: 139, and CDR-H3 as shown in SEQ ID NO: 140;

(i) CDR-H1 as shown in SEQ ID NO: 156, CDR-H2 as shown in SEQ ID NO: 157, and CDR-H3 as shown in SEQ ID NO: 158; and

(j) CDR-H1 as shown in SEQ ID NO: 174, CDR-H2 as shown in SEQ ID NO: 175, and CDR-H3 as shown in SEQ ID NO: 176.

Preferred is also that the binding domain capable of contacting epitope CD3ε-chain of human rights and the primacy of non-chimpanzee contains a VL region selected from the group consisting of a VL-region as shown in SEQ ID NO.: 35, 39, 125, 129, 161 or 165.

Alternatively, preferably, the first binding domain capable of contacting epitope CD3ε(Epsilon) chain of human rights and the primacy of non-chimpanzee contains a VH region selected from the group consisting of a VH-region as shown in SEQ ID NO.: 15, 19, 33, 37, 51, 55, 69, 73, 87, 91, 105, 109, 123, 127, 141, 145, 159, 163, 177 181.

More preferably, the molecule especifismo single-chain antibodies according to the invention is characterized by the first binding domain capable of contacting epitope CD3ε(Epsilon) chain of human rights and the primacy of non-chimpanzee, which contains a VL region and a VH region selected from the group consisting of the following:

(a) VL-region as shown in SEQ ID NO: 17 or 21 and a VH-region as shown in SEQ ID NO: 15 or 19;

(b) VL-region as shown in SEQ ID NO: 35 or 39, and a VH-region�ü, as represented in SEQ ID NO: 33 or 37;

(c) VL-region as shown in SEQ ID NO: 53 or 57, and a VH-region as shown in SEQ ID NO: 51 or 55;

(d) VL-region as shown in SEQ ID NO: 71 or 75, and a VH-region as shown in SEQ ID NO: 69 or 73;

(e) VL-region as shown in SEQ ID NO: 89 or 93, and a VH-region as shown in SEQ ID NO: 87 or 91;

(f) VL-region as shown in SEQ ID NO: 107 or 111, and a VH-region as shown in SEQ ID NO: 105 or 109;

(g) VL-region as shown in SEQ ID NO: 125 or 129, and a VH-region as shown in SEQ ID NO: 123 or 127;

(h) VL-region as shown in SEQ ID NO: 143 or 147, and a VH-region as shown in SEQ ID NO: 141 or 145;

(i) VL-region as shown in SEQ ID NO: 161 or 165, and a VH-region as shown in SEQ ID NO: 159 or 163; and

(j) VL-region as shown in SEQ ID NO: 179 or 183, and a VH-region as shown in SEQ ID NO: 177 or 181.

According to a preferred embodiment of the molecule especifismo single-chain antibodies according to the invention a pair of VH-region and VL-region of the first binding domain that binds to CD3-Epsilon, are in the format of single-chain antibodies (scFv). VH and VL-region are arranged in the order VH-VL or VL-VH. Preferably, the VH-region is located N-terminal to linker sequence. VL-region is located on the C-end of the linker sequence. In other words, the arrangement of domains in a CD3-associated�eating domain of the molecule especifismo single-chain antibodies according to the invention is preferably a VH-VL, and specified CD3-binding domain is located C-terminal to the second (cell surface antigen, such as PSCA, CD19, C-MET, endothelin, Arcam, IGF-1R or FAPα) binding domain. Preferably, the VH-VL contains or represents SEQ ID NO: 185.

The preferred embodiment described above molecules especifismo single-chain antibodies according to the invention is characterized by the first binding domain capable of contacting epitope CD3ε(Epsilon) chain of human rights and the primacy of non-chimpanzee containing amino acid sequence selected from the group consisting of SEQ ID NO: 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185 or 187.

The present invention also relates to the above-described bespecifically single-chain antibody, wherein the second binding domain binds to cell surface antigen PSCA, CD19, C-MET, endothelin, Arcam, IGF-1R or FAPα.

PSCA×CD3

According to a preferred embodiment of the invention described above, the molecule especifismo single-chain antibody contains a second binding domain is a group of the following sequences of CDR H1, CDR H2, CDR H3, CDR L1, CDR L2 and CDR-L3 selected from the group consisting of:

(a) CDR H1-3 of SEQ ID NO: 382-384 and CDR L1-3 of SEQ ID NO: 377-379;

(b) CDR H1-3 of SEQ ID NO: 400-402 and CDR L1-3 of SEQ ID NO: 395-397;

(c) CDR H1-3 of SEQ ID NO: 414-416 and CDR L1-3 of SEQ ID NO: 409-411;

(d) CDR H1-is SEQ ID NO: 432-434 and CDR L1-3 of SEQ ID NO: 427-429;

(e) CDR H1-3 of SEQ ID NO: 1215-1217 and CDR L1-3 of SEQ ID NO: 1220-1222;

(f) CDR H1-3 of SEQ ID NO: 1187-1189 and CDR L1-3 of SEQ ID NO: 1192-1194;

(g) CDR H1-3 of SEQ ID NO: 1173-1175 and CDR L1-3 of SEQ ID NO: 1178-1180;

(h) CDR H1-3 of SEQ ID NO: 1229-1231 and CDR L1-3 of SEQ ID NO: 1234-1236;

(i) CDR H1-3 of SEQ ID NO: 1201-1203 and CDR L1-3 of SEQ ID NO: 1206-1208;

k) CDR H1-3 of SEQ ID NO: 1257-1259 and CDR L1-3 of SEQ ID NO: 1262-1264; and

l) CDR H1-3 of SEQ ID NO: 1243-1245 and CDR L1-3 of SEQ ID NO: 1248-1250.

The sequence of the corresponding VL - and VH-regions of the second binding domain of the molecule especifismo single-chain antibodies according to the invention and corresponding scFv is presented in the sequence listing.

In the molecule especifismo single-chain antibodies according to the invention the binding domains are arranged in the order VL-VH-VH-VL, VL-VH-VL-VH, VH-VL-VH-VL or VH-VL-VL-VH, as illustrated in the examples below. Preferably, the binding domains are arranged in the order VH PSCA-VL PSCA-VH CD3-VL CD3 or VL PSCA-VH PSCA-VH CD3-VL CD3.

A particularly preferred embodiment of the invention relates to a polypeptide described above, where the molecule especifismo single-chain antibody contains a sequence selected from the following:

(a) amino acid sequence as shown in any of SEQ ID NO: 389, 421, 439, 391, 405, 423, 441, 1226, 1198, 1184, 1240, 1212, 1268 or 1254;

(b) amino acid sequence encoded by a nucleic acid sequence as represented bluboy of SEQ ID NO: 390,422, 440, 392, 406, 424, 442, 1227, 1199, 1185, 1241, 1213, 1269 or 1255; and

(c) amino acid sequence at least 90% identical, more preferably at least 95% identical, most preferably at least 96% identical to the amino acid sequence (a) or (b).

This invention relates to a molecule especifismo single-chain antibodies containing the amino acid sequence as shown in any of SEQ ID NO: 389, 421, 439, 391, 405, 423, 441, 1226, 1198, 1184, 1240, 1212, 1268 or 1254, as well as to amino acid sequences at least 85% identical, preferably 90%, more preferably at least 95% identical, most preferably at least 96, 97, 98 or 99% identical to the amino acid sequence SEQ ID NO: 389, 421, 439, 391, 405, 423, 441, 1226, 1198, 1184, 1240, 1212, 1268 or 1254. The invention also relates to the corresponding nucleic acid sequences as represented in any of SEQ ID NO: 390, 422, 440, 392, 406, 424, 442, 1227, 1199, 1185, 1241, 1213, 1269 or 1255, as well as to nucleic acid sequences at least 85% identical, preferably 90%, more preferably at least 95% identical, most preferably at least 96, 97, 98 or 99% identical to the nucleic acid sequences, represented in SEQ ID NO.: 390, 422, 440, 392, 406, 424, 442, 1227, 1199 1185, 1241, 1213, 169 or 1255. It should be borne in mind that the sequence identity is determined by the entire nucleotide or amino acid sequence. For sequence alignment can be used, for example, the programs Gap or BestFit (Needleman and Wunsch J. Mol. Biol. 48 (1970), 443-453; Smith and Waterman, Adv. Appl. Math 2 (1981), 482-489), which are included in the software package GCG (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991). It is routine for specialists in this field method for the determination and identification of nucleotide or amino acid sequence having, for example, 85% (90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the nucleotide or amino acid sequences especifismo single-chain antibodies according to the invention. For example, according to the hypothesis of "swing" - F. Crick 5'-base on antikodona is not spatially limited to the same extent as the other two bases, and therefore can have non-standard base pairing. In other words: the third position in the triplet codon can vary in such a way that two of the triplet, which differ in this third position, can encode the same amino acid residue. This hypothesis is known to the person skilled in the art (see, for example, http://en.wikipedia.org/wiki/Wobble_Hypothesis; Crick, J Mol Biol 19 (1966): 548-55).

Preferred locat�Oia domains in PSCA×CD3 bespecifically single-chain antibody constructs of the invention are demonstrated in the examples below.

In a preferred embodiment of the invention bespecifically single-chain antibodies have cross-species specificity in respect of the CD3-Epsilon and against cell surface antigen PSCA of human rights and the primacy of non-chimpanzee, recognized by their second binding domain.

CD19×CD3

According to a preferred embodiment of the invention described above, the molecule especifismo single-chain antibody contains a second binding domain is a group of the following sequences of CDR H1, CDR H2, CDR H3, CDR L1, CDR L2 and CDR-L3 selected from the group consisting of:

(a) CDR H1-3 of SEQ ID NO: 478-480 and CDR L1-3 of SEQ ID NO: 473-475;

(b) CDR H1-3 of SEQ ID NO: 530-532, and CDR L1-3 of SEQ ID NO: 525-527;

(c) CDR H1-3 of SEQ ID NO: 518-520 and CDR L1-3 of SEQ ID NO: 513-515;

(d) CDR H1-3 of SEQ ID NO: 506-508 and CDR L1-3 of SEQ ID NO: 501-503;

(e) CDR H1-3 of SEQ ID NO: 494-496 and CDR L1-3 of SEQ ID NO: 489-491;

(f) CDR H1-3 of SEQ ID NO: 542-544 and CDR L1-3 of SEQ ID NO: 537-539;

(g) CDR H1-3 of SEQ ID NO: 554-556 and CDR L1-3 of SEQ ID NO: 549-551;

(h) CDR H1-3 of SEQ ID NO: 566-568 and CDR L1-3 of SEQ ID NO: 561-563;

(i) CDR H1-3 of SEQ ID NO: 578-580 and CDR L1-3 of SEQ ID NO: 573-575;

j) CDR H1-3 of SEQ ID NO: 590-592 and CDR L1-3 of SEQ ID NO: 585-587;

k) CDR H1-3 of SEQ ID NO: 602-604 and CDR L1-3 of SEQ ID NO: 597-599;

l) CDR H1-3 of SEQ ID NO: 614-616 and CDR L1-3 of SEQ ID NO: 609-611;

m) CDR H1-3 of SEQ ID NO: 626-628 and CDR L1-3 of SEQ ID NO: 621-623;

n) CDR H1-3 of SEQ ID NO: 638-640 and CDR L1-3 of SEQ ID NO: 633-635;

(o) CDR H1-3 of SEQ ID NO: 650-652 and CDR L1-3 of SEQ ID NO: 645-647;

p) CDR H1-3 of SEQ ID NO: 662-664 and CDR L1-3 of SEQ ID NO: 657-659;

q) CDR H1-3 of SEQ ID NO: 674-I CDR L1-3 of SEQ ID NO: 669-671;

r) CDR H1-3 of SEQ ID NO: 686-688 and CDR L1-3 of SEQ ID NO: 681-683;

s) CDR H1-3 of SEQ ID NO: 698-700 and CDR L1-3 of SEQ ID NO: 693-695;

t) CDR H1-3 of SEQ ID NO: 710-712 and CDR L1-3 of SEQ ID NO: 705-707;

u) CDR H1-3 of SEQ ID NO: 722-724 and CDR L1-3 of SEQ ID NO: 717-719;

(v) CDR H1-3 of SEQ ID NO: 734-736, and CDR L1-3 of SEQ ID NO: 729-731;

w) CDR H1-3 of SEQ ID NO: 746-748 and CDR L1-3 of SEQ ID NO: 741-743;

x) CDR H1-3 of SEQ ID NO: 758-760 and CDR L1-3 of SEQ ID NO: 753-755;

y) CDR H1-3 of SEQ ID NO: 1271-1273 and CDR L1-3 of SEQ ID NO: 1276-1278;

z) CDR H1-3 of SEQ ID NO: 1285-1287 and CDR L1-3 of SEQ ID NO: 1290-1292;

aa) CDR H1-3 of SEQ ID NO: 1299-1301 and CDR L1-3 of SEQ ID NO: 1304-1306;

ab) CDR H1-3 of SEQ ID NO: 1313-1315 and CDR L1-3 of SEQ ID NO: 1318-1320;

ac) CDR H1-3 of SEQ ID NO: 1327-1329 and CDR L1-3 of SEQ ID NO: 1332-1334;

ad) CDR H1-3 of SEQ ID NO: 1341-1343 and CDR L1-3 of SEQ ID NO: 1346-1348;

ae) CDR H1-3 of SEQ ID NO: 1355-1357 and CDR L1-3 of SEQ ID NO: 1360-1362;

af) CDR H1-3 of SEQ ID NO: 1369-1371 and CDR L1-3 of SEQ ID NO: 1374-1376;and

ag) CDR H1-3 of SEQ ID NO: 1383-1385 and CDR L1-3 of SEQ ID NO: 1388-1390.

The sequence of the corresponding VL - and VH-regions of the second binding domain of the molecule especifismo single-chain antibodies according to the invention and corresponding scFv is presented in the sequence listing.

In the molecule especifismo single-chain antibodies according to the invention the binding domains are arranged in the order VL-VH-VH-VL, VL-VH-VL-VH, VH-VL-VH-VL or VH-VL-VL-VH, as illustrated in the examples below. Preferably, the binding domains are arranged in the order VH CD19-CD19 VL-VH CD3-VL CD3 or CD19 VL-VH CD19-VH CD3-VL CD3. More preferably, the binding domains for b�s in order CD19 VL-VH CD19-VH CD3-VL CD3.

A particularly preferred embodiment of the invention relates to a polypeptide described above, where the molecule especifismo single-chain antibody contains a sequence selected from the following:

(a) amino acid sequence as shown in any of SEQ ID NO: 481, 485, 483, 533, 521, 509, 497, 545, 557, 569, 581, 593, 605, 617, 629, 641, 653, 665, 677, 689, 701, 713, 725, 737, 749, 761, 1282, 1296, 1310, 1324, 1338, 1352, 1366, 1380 or 1394;

(b) amino acid sequence encoded by a nucleic acid sequence as represented in any of SEQ ID NO: 482, 486, 484, 534, 522, 510, 498, 546, 558, 570, 582, 594, 606, 618, 630, 642, 654, 666, 678, 690, 702, 714, 726, 738, 750, 762, 1283, 1297, 1311, 1325, 1339, 1353, 1367, 1381 or 1395; and

(c) amino acid sequence at least 90% identical, more preferably at least 95% identical, most preferably at least 96% identical to the amino acid sequence (a) or (b).

This invention relates to a molecule especifismo single-chain antibodies containing the amino acid sequence as shown in any of SEQ ID NO: 481, 485, 483, 533, 521, 509, 497, 545, 557, 569, 581, 593, 605, 617, 629, 641, 653, 665, 677,689, 701, 713, 725, 737, 749, 761, 1282, 1296, 1310, 1324, 1338, 1352, 1366, 1380 or 1394, as well as to amino acid sequences, at least 85% identical, preferably 90%, more preferably at least 95% identical, most�more preferably at least 96, 97, 98 or 99% identical to the amino acid sequence SEQ ID NO: 481, 485, 483, 533, 521, 509, 497, 545, 557, 569, 581, 593, 605, 617, 629, 641, 653, 665, 677, 689,701,713, 725, 737, 749, 761, 1282, 1296, 1310, 1324, 1338, 1352, 1366, 1380 or 1394. The present invention also relates to the corresponding nucleic acid sequences as represented in any of SEQ ID NO: 482, 486, 484, 534, 522, 510, 498, 546, 558, 570, 582, 594, 606, 618, 630, 642, 654, 666, 678, 690, 702, 714, 726, 738, 750, 762, 1283, 1297, 1311, 1325, 1339, 1353, 1367, 1381 or 1395, as well as to sequences of the nucleic acid at least 85% identical, preferably 90%, more preferably at least 95% identical, most preferably at least 96, 97, 98 or 99% identical to the nucleic acid sequences presented in SEQ ID NO: 482, 486, 484, 534, 522, 510, 498, 546, 558, 570, 582, 594, 606, 618, 630, 642, 654, 666, 678, 690, 702, 714, 726, 738, 750, 762, 1283, 1297, 1311, 1325, 1339, 1353, 1367, 1381 or 1395. It should be borne in mind that the sequence identity is determined by the entire nucleotide or amino acid sequence. For sequence alignment can be used, for example, the programs Gap or BestFit (Needleman and Wunsch J. Mol. Biol. 48 (1970), 443-453; Smith and Waterman, Adv. Appl. Math 2 (1981), 482-489), which are included in the software package GCG (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991). It is routine for specialists in this field method for the determination and identification of nucleotide or amino acid sequence having, for example, 85% (90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the nucleotide or amino acid sequences especifismo single-chain antibodies according to the invention. For example, according to the hypothesis of "swing" - F. Crick 5'-base on antikodona is not spatially limited to the same extent as the other two bases, and therefore can have non-standard base pairing. In other words: the third position in the triplet codon can vary in such a way that two of the triplet, which differ in this third position, can encode the same amino acid residue. This hypothesis is known to the person skilled in the art (see, for example, http://en.wikipedia.org/wiki/Wobble_Hypothesis; Crick, J Mol Biol 19 (1966): 548-55).

The preferred arrangement of domains in a CD19×CD3 bespecifically single-chain antibody constructs of the invention are demonstrated in the examples below.

In a preferred embodiment of the invention bespecifically single-chain antibodies have cross-species specificity in respect of the CD3-Epsilon and against cell surface antigen CD19 of human rights and the primacy of non-chimpanzee, recognized by their second binding domain.

c-MET×CD3

According to a preferred embodiment of the invention oharakterizovat�tion above molecule especifismo single-chain antibody contains a second binding domain is a group of the following sequences of CDR H1, CDR H2, CDR H3, CDR L1, CDR L2 and CDR-L3 selected from the group consisting of:

(a) CDR H1-3 of SEQ ID NO: 821-823 and CDR L1-3 of SEQ ID NO: 816-818;

(b) CDR H1-3 of SEQ ID NO: 836-838 and CDR L1-3 of SEQ ID NO: 833-835;

(c) CDR H1-3 of SEQ ID NO: 845-847 and CDR L1-3 of SEQ ID NO: 840-842;

(d) CDR H1-3 of SEQ ID NO: 863-865 and CDR L1-3 of SEQ ID NO: 858-860;

(e) CDR H1-3 of SEQ ID NO: 881-883 and CDR L1-3 of SEQ ID NO: 876-878;

(f) CDR H1-3 of SEQ ID NO: 899-901 and CDR L1-3 of SEQ ID NO: 894-896;

(g) CDR H1-3 of SEQ ID NO: 1401-1403 and CDR L1-3 of SEQ ID NO: 1406-1408;

(h) CDR H1-3 of SEQ ID NO: 1415-1417 and CDR L1-3 of SEQ ID NO: 1420-1422;

(i) CDR H1-3 of SEQ ID NO: 1429-1431 and CDR L1-3 of SEQ ID NO: 1434-1436;

j) CDR H1-3 of SEQ ID NO: 1443-1445 and CDR L1-3 of SEQ ID NO: 1448-1450;

k) CDR H1-3 of SEQ ID NO: 1457-1459 and CDR L1-3 of SEQ ID NO: 1462-1464;

l) CDR H1-3 of SEQ ID NO: 1471-1473 and CDR L1-3 of SEQ ID NO: 1476-1478;

m) CDR H1-3 of SEQ ID NO: 1639-1641 and CDR L1-3 of SEQ ID NO: 1644-1646;

n) CDR H1-3 of SEQ ID NO: 1625-1627 and CDR L1-3 of SEQ ID NO: 1630-1632;

(o) CDR H1-3 of SEQ ID NO: 1611-1613 and CDR L1-3 of SEQ ID NO: 1616-1618;

p) CDR H1-3 of SEQ ID NO: 1597-1599 and CDR L1-3 of SEQ ID NO: 1602-1604;

q) CDR H1-3 of SEQ ID NO: 1569-1571 and CDR L1-3 of SEQ ID NO: 1574-1576;

r) CDR H1-3 of SEQ ID NO: 1555-1557 and CDR L1-3 of SEQ ID NO: 1560-1562;

s) CDR H1-3 of SEQ ID NO: 1583-1585 and CDR L1-3 of SEQ ID NO: 1588-1590;

t) CDR H1-3 of SEQ ID NO: 1541-1543 and CDR L1-3 of SEQ ID NO: 1546-1548;

u) CDR H1-3 of SEQ ID NO: 1513-1515 and CDR L1-3 of SEQ ID NO: 1518-1520;

(v) CDR H1-3 of SEQ ID NO: 1527-1529 and CDR L1-3 of SEQ ID NO: 1532-1534;

w) CDR H1-3 of SEQ ID NO: 1499-1501 and CDR L1-3 of SEQ ID NO: 1504-1506; and

x) CDR H1-3 of SEQ ID NO: 1485-1487 and CDR L1-3 of SEQ ID NO: 1490-1492.

The sequence of the corresponding VL - and VH-regions of the second binding domain of the molecule especifismo single-chain antibodies to invent�NIJ, as well as the corresponding scFv is presented in the sequence listing.

In the molecule especifismo single-chain antibodies according to the invention the binding domains are arranged in the order VL-VH-VH-VL, VL-VH-VL-VH, VH-VL-VH-VL or VH-VL-VL-VH, as illustrated in the examples below. Preferably, the binding domains are arranged in the order VH C-MET-VL C-MET-VH CD3-VL CD3 or VL-METH-VH C-MET-VH CD3-VL CD3.

A particularly preferred embodiment of the invention relates to a polypeptide described above, where the molecule especifismo single-chain antibody contains a sequence selected from the following:

(a) amino acid sequence as shown in any of SEQ ID NO: 829, 853, 871, 889, 831, 855, 873, 891, 905, 1412, 1426, 1440, 1454, 1468 or 1482;

(b) amino acid sequence encoded by a nucleic acid sequence as represented in any of SEQ ID NO: 830, 854, 872, 890, 832, 856, 874, 892, 906, 1413, 1427, 1441, 1455, 1469 or 1483; and

(c) amino acid sequence at least 90% identical, more preferably at least 95% identical, most preferably at least 96% identical to the amino acid sequence (a) or (b).

This invention relates to a molecule especifismo single-chain antibodies containing the amino acid sequence as shown in any of SEQ ID NO: 829,853, 871, 889, 831, 855, 873, 891, 905, 1412, 1426, 1440, 1454, 1468 or 1482, at least 85% identical, preferably 90%, more preferably at least 95% identical, most preferably at least 96, 97, 98 or 99% identical to the amino acid sequence SEQ ID NO: 829, 853, 871, 889, 831, 855, 873, 891, 905, 1412, 1426, 1440, 1454, 1468 or 1482. The present invention also relates to the corresponding nucleic acid sequences as represented in any of SEQ ID NO: 830, 854, 872, 890, 832, 856,874, 892, 906, 1413,1427, 1441, 1455, 1469, or 1483, and to nucleic acid sequences at least 85% identical, preferably 90%, more preferably at least 95% identical, most preferably at least 96, 97, 98 or 99% identical to the nucleic acid sequences, represented in SEQ ID NO.: 830, 854, 872, 890, 832, 856, 874, 892, 906, 1413, 1427, 1441, 1455, 1469 or 1483. It should be borne in mind that the sequence identity is determined by the entire nucleotide or amino acid sequence. For sequence alignment can be used, for example, the programs Gap or BestFit (Needleman and Wunsch J. Mol. Biol. 48 (1970), 443-453; Smith and Waterman, Adv. Appl. Math 2 (1981), 482-489), which are included in the software package GCG (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991). It is routine for specialists in this field method for the determination and identification of the nucleotide or amine�acid sequence, having, for example, 85% (90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the nucleotide or amino acid sequences especifismo single-chain antibodies according to the invention. For example, according to the hypothesis of "swing" - F. Crick 5'-base on antikodona is not spatially limited to the same extent as the other two bases, and therefore can have non-standard base pairing. In other words: the third position in the triplet codon can vary in such a way that two of the triplet, which differ in this third position, can encode the same amino acid residue. This hypothesis is known to the person skilled in the art (see, for example, http://en.wikipedia.org/wiki/Wobble_Hypothesis; Crick, J Mol Biol 19 (1966): 548-55).

The preferred arrangement of domains in C-MET×CD3 bespecifically single-chain antibody constructs of the invention are demonstrated in the examples below.

In a preferred embodiment of the invention bespecifically single-chain antibodies have cross-species specificity in respect of the CD3-Epsilon and against cell surface antigen C-MET human rights and the primacy of non-chimpanzee, recognized by their second binding domain.

Endothelin×CD3

According to a preferred embodiment of the invent�ment described above, the molecule especifismo single-chain antibody contains a second binding domain is a group of the following sequences of CDR H1, CDR H2, CDR H3, CDR L1, CDR L2 and CDR-L3 selected from the group consisting of:

CDR H1-3 of SEQ ID NO: 910-912 and CDR L1-3 of SEQ ID NO: 907-909.

Since these CDR sequences of heavy and light chains of the second binding domain, a specialist in this field can obtain a molecule especifismo single-chain antibodies according to the invention without any further inventive activity. In particular, the CDR sequences can be located in the frame of the VL and VH chains are in the form of scFv.

According to a preferred embodiment of the invention described above, the molecule especifismo single-chain antibody contains a second binding domain is a group of the following sequences of CDR H1, CDR H2, CDR H3, CDR L1, CDR L2 and CDR-L3 selected from the group consisting of:

(a) CDR H1-3 of SEQ ID NO: 1653-1655 and CDR L1-3 of SEQ ID NO: 1658-1660;

(b) CDR H1-3 of SEQ ID NO: 1667-1669 and CDR L1-3 of SEQ ID NO: 1672-1674;

(c) CDR H1-3 of SEQ ID NO: 1681-1683 and CDR L1-3 of SEQ ID NO: 1686-1688; and

(d) CDR H1-3 of SEQ ID NO: 1695-1697 and CDR L1-3 of SEQ ID NO: 1700-1702;

(e) CDR H1-3 of SEQ ID NO: 1709-1711 and CDR L1-3 of SEQ ID NO: 1714-1716; and

(f) CDR H1-3 of SEQ ID NO: 1723-1725 and CDR L1-3 of SEQ ID NO: 1728-1730.

In the molecule especifismo single-chain antibodies according to the invention the binding domains are arranged in the order VL-VH-VH-VL, VL-VH-VL-VH, VH-VL-VH-VL or VH-VL-VL-VH, as illustrated in the examples below. Preferably, the binding domains are arranged in the order VH endothelin-VL e�goselin-VH CD3-VL CD3 or VL endothelin-VH endothelin-VH CD3-VL CD3.

A particularly preferred embodiment of the invention relates to a polypeptide described above, where the molecule especifismo single-chain antibodies described above contains the first binding domain capable of contacting epitope CD3ε of man and the primacy of non-chimpanzee, and a second binding domain capable of contacting endothelina and containing CDR-H1, 2 and 3 of SEQ ID NO: 910-912 and CDR L1, 2 and 3 of SEQ ID NO: 907-909, or the amino acid sequence of a CDR of at least 50%, 60%, 70%, 75%, 80%, 85% or 90% identical, more preferably at least 95% identical, most preferably at least 96%, 97%, 98% or 99% identical to each of the corresponding CDR amino acid sequences as defined above. It should be borne in mind that the sequence identity is determined by the entire amino acid sequence or CDRH cdrl stock.

A particularly preferred embodiment of the invention relates to a polypeptide described above, where the molecule especifismo single-chain antibody contains a sequence selected from the following:

(a) amino acid sequence as shown in any of SEQ ID NO: 1664, 1678, 1692, 1706, 1720, or 1734;

(b) amino acid sequence encoded by a nucleic acid sequence as represented in any of SEQ I NO: 1665, 1679, 1693, 1707, 1721 or 1735; and

(c) amino acid sequence at least 90% identical, more preferably at least 95% identical, most preferably at least 96% identical to the amino acid sequence (a) or (b).

This invention relates to a molecule especifismo single-chain antibodies containing the amino acid sequence as shown in any of SEQ ID NO: 1664, 1678, 1692, 1706, 1720 or 1734, as well as to amino acid sequences at least 85% identical, preferably 90%, more preferably at least 95% identical, most preferably at least 96, 97, 98 or 99% identical to the amino acid sequence SEQ ID NO: 1664, 1678, 1692, 1706, 1720 or 1734. The present invention also relates to the corresponding nucleic acid sequences as represented in any of SEQ ID NO: 1665, 1679, 1693, 1707, 1721 or 1735, as well as to nucleic acid sequences at least 85% identical, preferably 90%, more preferably at least 95% identical, most preferably at least 96, 97, 98 or 99% identical to the nucleic acid sequences presented in SEQ ID NO: 1665, 1679, 1693, 1707, 1721 or 1735.

Unless otherwise indicated, it should be borne in mind that identity to the sequence defined�is throughout nucleotide or amino acid sequence. For sequence alignment can be used, for example, the programs Gap or BestFit (Needleman and Wunsch J. Mol. Biol. 48 (1970), 443-453; Smith and Waterman, Adv. Appl. Math 2 (1981), 482-489), which are included in the software package GCG (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991). It is routine for specialists in this field method for the determination and identification of nucleotide or amino acid sequence having, for example, 85% (90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the nucleotide or amino acid sequences or CDR sequences especifismo single-chain antibodies according to the invention. For example, according to the hypothesis of "swing" - F. Crick 5'-base on antikodona is not spatially limited to the same extent as the other two bases, and therefore can have non-standard base pairing. In other words: the third position in the triplet codon can vary in such a way that two of the triplet, which differ in this third position, can encode the same amino acid residue. This hypothesis is known to the person skilled in the art (see, for example, http://en.wikipedia.org/wiki/Wobble_Hypothesis; Crick, J Mol Biol 19 (1966); 548-55).

The preferred arrangement of the domains in endothelin×CD3 bespecifically single-chain antibody constructs of the invention cont�monsterboy in the examples below.

In a preferred embodiment of the invention bespecifically single-chain antibodies have cross-species specificity in respect of the CD3-Epsilon and against cell surface antigen indocyanine of man and the primacy of non-chimpanzee, recognized by their second binding domain.

Arcam×CD3

According to a preferred embodiment of the invention described above, the molecule especifismo single-chain antibody contains a second binding domain is a group of the following sequences of CDR H1, CDR H2, CDR H3, CDR L1, CDR L2 and CDR-L3 selected from the group consisting of:

(a) CDR H1-3 of SEQ ID NO: 940-942 and CDR L1-3 of SEQ ID NO: 935-937;

(b) CDR H1-3 of SEQ ID NO: 956-958 and CDR L1-3 of SEQ ID NO: 951-953;

(c) CDR H1-3 of SEQ ID NO: 968-970 and CDR L1-3 of SEQ ID NO: 963-965; and

(d) CDR H1-3 of SEQ ID NO: 980-982 and CDR L1-3 of SEQ ID NO: 975-977;

(e) CDR H1-3 of SEQ ID NO: 992-994 and CDR L1-3 of SEQ ID NO: 987-989;

(f) CDR H1-3 of SEQ ID NO: 1004-1006 and CDR L1-3 of SEQ ID NO: 999-1001;

(g) CDR H1-3 of SEQ ID NO: 1028-1030 and CDR L1-3 of SEQ ID NO: 1023-1025;

(h) CDR H1-3 of SEQ ID NO: 1040-1042 and CDR L1-3 of SEQ ID NO: 1035-1037;

(i) CDR H1-3 of SEQ ID NO: 1052-1054 and CDR L1-3 of SEQ ID NO: 1047-1049;

j) CDR H1-3 of SEQ ID NO: 1074-1076 and CDR L1-3 of SEQ ID NO: 1069-1071;

k) CDR H1-3 of SEQ ID NO: 1086-1088 and CDR L1-3 of SEQ ID NO: 1081-1083;

l) CDR H1-3 of SEQ ID NO: 1098-1000 and CDR L1-3 of SEQ ID NO: 1093-1095;

m) CDR H1-3 of SEQ ID NO: 1110-1112 and CDR L1-3 of SEQ ID NO: 1105-1107;

n) CDR H1-3 of SEQ ID NO: 1122-1124 and CDR L1-3 of SEQ ID NO: 1117-1119;

(o) CDR H1-3 of SEQ ID NO: 1016-1018 and CDR L1-3 of SEQ ID NO: 1011-1013; and

p) CDR H1-3 of SEQ ID NO: 1765-1767 � CDR L1-3 of SEQ ID NO: 1770-1772.

The sequence of the corresponding VL - and VH-regions of the second binding domain of the molecule especifismo single-chain antibodies according to the invention and corresponding scFv is presented in the sequence listing.

In the molecule especifismo single-chain antibodies according to the invention the binding domains are arranged in the order VL-VH-VH-VL, VL-VH-VL-VH, VH-VL-VH-VL or VH-VL-VL-VH, as illustrated in the examples below. Preferably, the binding domains are arranged in the order VH EpCAM-VL EpCAM-VH CD3-VL CD3 or VL Arcam-VH EpCAM-VH CD3-VL CD3. More preferably, the binding domains are arranged in the order VL EpCAM-VH EpCAM-VH CD3-VL CD3.

A particularly preferred embodiment of the invention relates to a polypeptide described above, where the molecule especifismo single-chain antibody contains a sequence selected from the following:

(a) amino acid sequence as shown in any of SEQ ID NO: 944, 948, 946, 960, 972, 984, 996, 1008, 1032, 1044, 1056, 1078, 1090, 1102, 1114, 1126, 1020 or 1776;

(b) amino acid sequence encoded by a nucleic acid sequence as represented in any of SEQ ID NO: 945, 949, 947, 961, 973, 985, 979, 1009, 1033, 1045, 1057, 1079, 1091, 1103,1115, 1127, 1021 or 1777; and

(c) amino acid sequence at least 90% identical, more preferably at least 95% identical, most preferred�flax at least 96% identical to the amino acid sequence (a) or (b).

This invention relates to a molecule especifismo single-chain antibodies containing the amino acid sequence as shown in any of SEQ ID NO: 944, 948, 946, 960, 972, 984, 996, 1008, 1032, 1044, 1056, 1078, 1090, 1102, 1114, 1126 1020 or 1776, as well as to amino acid sequences at least 85% identical, preferably 90%, more preferably at least 95% identical, most preferably at least 96, 97, 98 or 99% identical to the amino acid sequence SEQ ID NO: 944, 948, 946,960,972, 984, 996, 1008, 1032, 1044, 1056, 1078, 1090, 1102, 1114, 1126, 1020 or 1776. The present invention also relates to the corresponding nucleic acid sequences as represented in any of SEQ ID NO: 945, 949, 947, 961, 973, 985, 979, 1009, 1033, 1045, 1057, 1079, 1091, 1103, 1115, 1127, 1021 or 1777, as well as to nucleic acid sequences at least 85% identical, preferably 90%, more preferably at least 95% identical, most preferably at least 96, 97, 98 or 99% identical to the nucleic acid sequences presented in SEQ ID NO: 945, 949, 947, 961, 973, 985, 979, 1009, 1033, 1045, 1057, 1079, 1091, 1103, 1115, 1127, 1021 or 1777. It should be borne in mind that the sequence identity is determined by the entire nucleotide or amino acid sequence. For sequence alignment can be used, for example, PR�grams Gap or BestFit (Needleman and Wunsch J. Mol. Biol. 48 (1970), 443-453; Smith and Waterman, Adv. Appl. Math 2 (1981), 482-489), which are included in the software package GCG (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991). It is routine for specialists in this field method for the determination and identification of nucleotide or amino acid sequence having, for example, 85% (90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the nucleotide or amino acid sequences especifismo single-chain antibodies according to the invention. For example, according to the hypothesis of "swing" - F. Crick 5'-base on antikodona is not spatially limited to the same extent as the other two bases, and therefore can have non-standard base pairing. In other words: the third position in the triplet codon can vary in such a way that two of the triplet, which differ in this third position, can encode the same amino acid residue. This hypothesis is known to the person skilled in the art (see, for example, http://en.wikipedia.org/wiki/Wobble_Hypothesis; Crick, J Mol Biol 19 (1966): 548-55).

The preferred arrangement of the domains in EpCAM×CD3 bespecifically single-chain antibody constructs of the invention are demonstrated in the examples below.

In a preferred embodiment of the invention bespecifically single-chain antibodies have IU�species specificity in respect of the CD3-Epsilon and against cell surface antigen Ersam of man and APE, non chimpanzees, recognized by their second binding domain.

FAPα×CD3

According to a preferred embodiment of the invention described above, the molecule especifismo single-chain antibody contains a second binding domain is a group of the following sequences of CDR H1, CDR H2, CDR H3, CDR L1, CDR L2 and CDR-L3 selected from the group consisting of:

CDR H1-3 of SEQ ID NO: 1137-1139 and CDR L1-3 of SEQ ID NO: 1132-1134.

The sequence of the corresponding VL - and VH-regions of the second binding domain of the molecule especifismo single-chain antibodies according to the invention and corresponding scFv is presented in the sequence listing.

According to a preferred embodiment of the invention described above, the molecule especifismo single-chain antibody contains a second binding domain is a group of the following sequences of CDR H1, CDR H2, CDR H3, CDR L1, CDR L2 and CDR-L3 selected from the group consisting of:

(a) CDR H1-3 of SEQ ID NO: 1137-1139 and CDR L1-3 of SEQ ID NO: 1132-1134;

(b) CDR H1-3 of SEQ ID NO: 1849-1851 and CDR L1-3 of SEQ ID NO: 1854-1856;

(c) CDR H1-3 of SEQ ID NO: 1835-1837 and CDR L1-3 of SEQ ID NO: 1840-1842; and

(a) CDR H1-3 of SEQ ID NO: 1779-1781 and CDR L1-3 of SEQ ID NO: 1784-1786;

(e) CDR H1-3 of SEQ ID NO: 1793-1795 and CDR L1-3 of SEQ ID NO: 1798-1800;

(f) CDR H1-3 of SEQ ID NO: 1863-1865 and CDR L1-3 of SEQ ID NO: 1868-1870;

(g) CDR H1-3 of SEQ ID NO: 1807-1809 and CDR L1-3 of SEQ ID NO: 1812-1814;

(h) CDR H1-3 of SEQ ID NO: 1821-1823 and CDR L1-3 of SEQ ID NO: 1826-1828;

(i) CDR H1-3 of SEQ ID NO: 1891-1893 and CDR L1-3 �h SEQ ID NO: 1896-1898;

j) CDR H1-3 of SEQ ID NO: 1877-1879 and CDR L1-3 of SEQ ID NO: 1882-1884;

k) CDR H1-3 of SEQ ID NO: 1961-1963 and CDR L1-3 of SEQ ID NO: 1966-1968;

l) CDR H1-3 of SEQ ID NO: 1947-1949 and CDR L1-3 of SEQ ID NO: 1952-1954;

m) CDR H1-3 of SEQ ID NO: 1975-1977 and CDR L1-3 of SEQ ID NO: 1980-1982;

n) CDR H1-3 of SEQ ID NO: 1933-1935 and CDR L1-3 of SEQ ID NO: 1938-1940;

(o) CDR H1-3 of SEQ ID NO: 1919-1921 and CDR L1-3 of SEQ ID NO: 1924-1926; and

p) CDR H1-3 of SEQ ID NO: 1905-1907 and CDR L1-3 of SEQ ID NO: 1910-1912.

In the molecule especifismo single-chain antibodies according to the invention the binding domains are arranged in the order VL-VH-VH-VL, VL-VH-VL-VH, VH-VL-VH-VL or VH-VL-VL-VH, as illustrated in the examples below. Preferably, the binding domains are arranged in the order VH FAP-alpha-VL FAP-alpha-VH CD3-VL CD3 or VL FAP-alpha-VH FAP-alpha-VH CD3-VL CD3. More preferably, the binding domains are arranged in the order VL FAP-alpha-VH FAP-alpha-VH CD3-VL CD3.

A particularly preferred embodiment of the invention relates to a polypeptide described above, where the molecule especifismo single-chain antibody contains a sequence selected from the following:

(a) amino acid sequence as shown in any of SEQ ID NO: 1143, 1147, 1145, 1860, 1846, 1790, 1804, 1874, 1818 or 1832;

(b) amino acid sequence encoded by a nucleic acid sequence as represented in any of SEQ ID NO: 1144, 1148, 1146, 1861, 1847, 1791, 1805, 1875, 1818 or 1833; and

(c) amino acid sequence at least 90% �dentina, more preferably at least 95% identical, most preferably at least 96% identical to the amino acid sequence (a) or (b).

This invention relates to a molecule especifismo single-chain antibodies containing the amino acid sequence as shown in any of SEQ ID NO: 1143, 1147, 1145, 1860, 1846, 1790, 1804, 1874, 1818 or 1832, as well as to amino acid sequences at least 85% identical, preferably 90%, more preferably at least 95% identical, most preferably at least 96, 97, 98 or 99% identical to the amino acid sequence SEQ ID NO: 1143, 1147, 1145, 1860, 1846, 1790, 1804, 1874, 1818 or 1832. The present invention also relates to the corresponding nucleic acid sequences as represented in any of SEQ ID NO: 1144, 1148, 1146, 1861, 1847, 1791, 1805, 1875, 1818 or 1833, as well as to nucleic acid sequences at least 85% identical, preferably 90%, more preferably at least 95% identical, most preferably at least 96, 97, 98 or 99% identical to the nucleic acid sequences presented in SEQ ID NO: 1144, 1148, 1146, 1861, 1847, 1791, 1805, 1875, 1818 or 1833. It should be borne in mind that the sequence identity is determined by the entire nucleotide or amino acid sequence. Lavirovaniya sequences can be used, for example, the programs Gap or BestFit (Needleman and Wunsch J. Mol. Biol. 48 (1970), 443-453; Smith and Waterman, Adv. Appl. Math 2 (1981), 482-489), which are included in the software package GCG (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991). It is routine for specialists in this field method for the determination and identification of nucleotide or amino acid sequence having, for example, 85% (90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the nucleotide or amino acid sequences especifismo single-chain antibodies according to the invention. For example, according to the hypothesis of "swing" - F. Crick 5'-base on antikodona is not spatially limited to the same extent as the other two bases, and therefore can have non-standard base pairing. In other words:

third position in the triplet codon can vary in such a way that two of the triplet, which differ in this third position, can encode the same amino acid residue. This hypothesis is known to the person skilled in the art (see, for example, http://en.wikipedia.org/wiki/Wobble_Hypothesis; Crick, J Mol Biol 19 (1966): 548-55).

The preferred arrangement of the domains in FAP-alpha×CD3 bespecifically single-chain antibody constructs of the invention are demonstrated in the examples below.

In a preferred embodiment of the invention �specificheskie single-chain antibodies have cross-species specificity in respect of the CD3-Epsilon and against cell surface antigen FAP-alpha man and APE, non chimpanzees, recognized by their second binding domain.

IGF-1R×CD3

According to a preferred embodiment of the invention described above, the molecule especifismo single-chain antibody contains a second binding domain is a group of the following sequences of CDR H1, CDR H2, CDR H3, CDR L1, CDR L2 and CDR-L3 selected from the group consisting of:

(a) CDR H1-3 of SEQ ID NO: 2016-2018 and CDR L1-3 of SEQ ID NO: 2021-2023;

(b) CDR H1-3 of SEQ ID NO: 2030-2032 and CDR L1-3 of SEQ ID NO: 2035-2037;

(c) CDR H1-3 of SEQ ID NO: 2044-2046 and CDR L1-3 of SEQ ID NO: 2049-2051;and

(d) CDR H1-3 of SEQ ID NO: 2058-2060 and CDR L1-3 of SEQ ID NO: 2063-2065;

(e) CDR H1-3 of SEQ ID NO: 2072-2074 and CDR L1-3 of SEQ ID NO: 2077-2079;

(f) CDR H1-3 of SEQ ID NO: 2086-2088 and CDR L1-3 of SEQ ID NO: 2091-2093;

(g) CDR H1-3 of SEQ ID NO: 2100-2102 and CDR L1-3 of SEQ ID NO: 2105-2107;

(h) CDR H1-3 of SEQ ID NO: 2114-2116 and CDR L1-3 of SEQ ID NO: 2119-2121;

(i) CDR H1-3 of SEQ ID NO: 2128-2130 and CDR L1-3 of SEQ ID NO: 2133-2135;

j) CDR H1-3 of SEQ ID NO: 2142-2144 and CDR L1-3 of SEQ ID NO: 2147-2149:

k) CDR H1-3 of SEQ ID NO: 2156-2158 and CDR L1-3 of SEQ ID NO: 2161-2163;

l) CDR H1-3 of SEQ ID NO: 2170-2172 and CDR L1-3 of SEQ ID NO: 2175-2177;

m) CDR H1-3 of SEQ ID NO: 2184-2186 and CDR L1-3 of SEQ ID NO: 2189-2191;

n) CDR H1-3 of SEQ ID NO: 2198-2200 and CDR L1-3 of SEQ ID NO: 2203-2205; and

(o) CDR H1-3 of SEQ ID NO: 2212-2214 and CDR L1-3 of SEQ ID NO: 2217-2219.

The sequence of the corresponding VL - and VH-regions of the second binding domain of the molecule especifismo single-chain antibodies according to the invention and corresponding scFv is presented in the sequence listing.

In the molecule especif�ical single-chain antibodies according to the invention the binding domains are arranged in the order VL-VH-VH-VL, VL-VH-VL-VH, VH-VL-VH-VL or VH-VL-VL-VH, as illustrated in the examples below. Preferably, the binding domains are arranged in the order VH IGF-1R-VL IGF-1R-VH CD3-VL CD3 or VL IGF-1R-VH IGF-1R-VH CD3-VL CD3.

A particularly preferred embodiment of the invention relates to a polypeptide described above, where the molecule especifismo single-chain antibody contains a sequence selected from the following:

(a) amino acid sequence as shown in any of SEQ ID NO: 2027, 2041, 2055, 2069, 2083, 2097, 2111, 2125, 2139, 2153, 2167, 2181, 2195, 2209 or 2223;

(b) amino acid sequence encoded by a nucleic acid sequence as represented in any of SEQ ID NO: 2028, 2042, 2056, 2070, 2084, 2098, 2112, 2126, 2140, 2154, 2168, 2182, 2196, 2210 or 2224; and

(c) amino acid sequence at least 90% identical, more preferably at least 95% identical, most preferably at least 96% identical to the amino acid sequence (a) or (b).

This invention relates to a molecule especifismo single-chain antibodies containing the amino acid sequence as shown in any of SEQ ID NO: 2027, 2041, 2055, 2069, 2083, 2097, 2111, 2125, 2139, 2153, 2167, 2181, 2195, 2209 or 2223, as well as to amino acid sequences at least 85% identical, preferably 90%, more preferably at �'ere 95% identical, most preferably at least 96, 97, 98 or 99% identical to the amino acid sequence SEQ ID NO: 2027, 2041, 2055, 2069, 2083, 2097, 2111, 2125, 2139, 2153, 2167, 2181, 2195, 2209 or 2223. The present invention also relates to the corresponding nucleic acid sequences as represented in any of SEQ ID NO: 2028, 2042, 2056, 2070, 2084, 2098, 2112, 2126, 2140, 2154, 2168, 2182, 2196, 2210 or 2224, as well as to nucleic acid sequences at least 85% identical, preferably 90%, more preferably at least 95% identical, most preferably at least 96, 97, 98 or 99% identical to the nucleic acid sequences, represented in SEQ ID NO.: 2028, 2042, 2056, 2070, 2084, 2098, 2112, 2126, 2140, 2154, 2168, 2182, 2196, 2210 or 2224. It should be borne in mind that the sequence identity is determined by the entire nucleotide or amino acid sequence.

For sequence alignment can be used, for example, the programs Gap or BestFit (Needleman and Wunsch J. Mol. Biol. 48 (1970), 443-453; Smith and Waterman, Adv. Appl. Math 2 (1981), 482-489), which are included in the software package GCG (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991). It is routine for specialists in this field method for the determination and identification of nucleotide or amino acid sequence having, for example, 85% (90%, 95%, 96%, 97%, 98% or 99%) identity posledovatelnosti nucleotide or amino acid sequences especifismo single-chain antibodies according to the invention. For example, according to the hypothesis of "swing" - F. Crick 5'-base on antikodona is not spatially limited to the same extent as the other two bases, and therefore can have non-standard base pairing. In other words: the third position in the triplet codon can vary in such a way that two of the triplet, which differ in this third position, can encode the same amino acid residue. This hypothesis is known to the person skilled in the art (see, for example, http://en.wikipedia.org/wiki/Wobble_Hypothesis; Crick, J Mol Biol 19 (1966): 548-55).

The preferred arrangement of the domains in the IGF-1R×CD3 bespecifically single-chain antibody constructs of the invention are demonstrated in the examples below.

In a preferred embodiment of the invention bespecifically single-chain antibodies have cross-species specificity in respect of the CD3-Epsilon and against cell surface antigen IGF-1R of human rights and the primacy of non-chimpanzee, recognized by their second binding domain.

In an alternative embodiment of the present invention proposed a nucleic acid sequence that encodes the molecule described above especifismo single-chain antibodies according to the invention.

The present invention also relates to a vector containing a molecule of nucleic� acid of the present invention.

Many suitable vectors are known to those skilled in molecular biology, and their choice will depend on the desired function, and they include plasmids, Comedy. viruses, bacteriophages and other vectors conventionally used in genetic engineering. To construct various plasmids and vectors can be used in methods well known to those skilled in the art, e.g., techniques described in Sambrook et al. (see above) and in Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989), (1994). Alternatively, the polynucleotides and vectors of the invention can be incorporated into liposomes for delivery to target cells. As described in more detail below, to highlight individual DNA sequences used cloning vector. If necessary the expression of a specific polypeptide, the relevant sequences can be transferred into expression vectors. Typical cloning vectors include pBluescript SK, pGEM, pUC9, pBR322 and pGBT9. Typical expression vectors include pTRE, pCAL-n-EK, pESP-1, pOP13CAT.

Preferably, the vector contains a nucleic acid sequence that is a regulatory sequence functionally associated with the specified nucleic acid sequence, which is defined in this specification.

The term "regulator�tial sequence" refers to DNA sequences are necessary to carry out the expression of the coding sequences with which they legirovanyh. The nature of such regulatory sequences differs depending upon the host body. In prokaryotes, the regulatory sequences generally include promoter, the binding site of ribosomes and terminators. In eukaryotes regulatory sequences generally include promoters, terminators and, in some cases, enhancers, transactivator or transcription factors. The term "regulatory sequence" includes, as a minimum, all components whose presence is necessary for expression, and may also include additional preferred components.

The term "functionally linked" refers to the adjacent position, where it is described thus components are in such a positional relationship that allows them to function properly. The regulatory sequence functionally linked" to the coding sequence, Legerova in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence. In the case when the regulatory sequence is a promoter, the specialist it is obvious that it is preferable to use double-stranded nucleic acid.

T�Kim, mentioned vector is preferably an expression vector. "Expressing vector" is a construct that can be used to transform a selected host and provides the expression of the coding sequence in the selected host. Expression vectors can be, for example, cloning vectors, binary vectors or integrating vectors. Expression includes transcription of the nucleic acid molecule is preferably in broadcast (region) of mRNA. Regulatory elements ensuring expression in prokaryotes and/or eukaryotic cells, known to those skilled in the art. In the case of eukaryotic cells they contain normal promoters ensuring initiation of transcription, and possible poly-A signals ensuring termination of transcription and stabilization of the transcript. Possible regulatory elements permitting expression in prokaryotic cells-owners, include, for example, the promoter PL, lac, trp or tac in E. coli, and examples for regulatory elements permitting expression in eukaryotic cells-the owners are the promoter AOH or in yeast GAL1 or promoters on the basis of CMV, SV40 (simian virus 40), RSV (the rous sarcoma virus), CMV-enhancer, SV40-enhancer or globinemia introns in the cell�Kah mammals and other animals.

In addition to the elements responsible for the initiation of transcription such regulatory elements may also contain signals termination of transcription, such as the site of the SV40-poly-A or website tk-poly-A, located below the polynucleotide. In addition, depending on the expression system, to the coding sequence described nucleic acid sequences can be added leader sequence capable of directing the polypeptide to a cellular compartment or secreting it into the medium, and they are well known in the art (see also the Examples below). Leader(s) sequence(s) ar(s)is conducted in the appropriate phase with sequences of initiation and translation termination, and preferably, the leader sequence capable of directing secretion of translated protein or area in the periplasmic space or the extracellular environment. Perhaps, the heterologous sequence can encode a protein that includes the N-terminal identifying a peptide that provides the desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product (see above). In this context, in the art known suitable expression vectors, such�to Okayama-Berg cDNA-expressing vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, with pcDNA3 (In-vitrogene), pEF-DHFR, pEF-ADA or pEF-neo (Mack et al. PNAS (1995) 92, 7021-7025, and Raum et al. Cancer Immunol. Immunother. (2001) 50(3), 141-150) or pSPORT1 (GIBCO BRL).

Preferably, expression regulatory sequences will be eukaryotic promoter systems in vectors capable of transforming transfinitely eukaryotic cells-owners, but the regulatory sequences of prokaryotic hosts may also be used. After the vector will be included in the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and, if desirable, can continue to follow the collection and purification of the polypeptide of the invention (see, e.g. the Examples below).

An alternative expression system that can be used for protein expression, active against the cell cycle is the system of insects. In one such system as a vector for expression of foreign genes in cells of Spodoptera frugiperda or Trichoplusia larvae used the nuclear polyhedrosis virus Autographa califomica (AcNPV). Coding sequence of the described nucleic acid molecules can be cloned in a nonessential region of the virus, such as poliakrilovye gene, and placed under the control polietilenovogo about�otara. Successful insertion of the specified coding sequence to inactivate poliakrilovye gene and produce recombinant virus lacking the protein shell. These recombinant viruses are then used to infect cells of S. frugiperda or Trichoplusia larvae in which the protein is expressed according to the invention (Smith, J. Virol. 46 (1983), 584; Engelhard, Proc. Nat. Acad. Sci. USA, 91 (1994), 3224-3227).

Additional regulatory elements may include transcriptional as well as translational enhancers. Preferably, the above-described vectors of the invention contain selected and/or measurable marker.

Selectable marker genes useful for selection of transformed cells and, for example, plant tissue and plants, well known to those skilled in the art and attached, for example, resistance to antimetabolites as the basis of selection for dhfr, which provides resistance to methotrexate (Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994), 143-149); npt, which provides resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J. 2 (1983), 987-995); and hygro, which provides resistance to hygromycin (Marsh, Gene 32 (1984), 481-485). Described additional selectable genes, namely trpB, which allows cells to utilize indole instead of tryptophan; hisD, which allows cells Ooty�'izirovat gastinel instead of histidine (Hartman, Proc. Natl. Acad. Sci. USA, 85 (1988), 8047); mannose-6-fortismere, which allows cells to utilize mannose (WO 94/20627); and ODC (intendencias), which provides resistance to the inhibitor emitintermediate 2-(deformity)-DL-ornithine (DFMO) (McConlogue, 1987, in: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.); or deaminase from Aspergillus terreus, which provides resistance to blasticidin S (Tamura, Biosci. Biotechnol. Biochem., 59 (1995), 2336-2338).

Useful measurable markers are also known to those skilled in the art and are commercially available. Preferably, the specified marker is a gene encoding luciferase (Giacomin, PI. Sci., 116 (1996), 59-72; Scikantha, J. Bad, 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett., 389 (1996), 44-47) or β-glucuronidase (Jefferson, EMBO J. 6 (1987), 3901-3907). This embodiment, in particular, is useful for simple and rapid screening of cells, tissues and organisms containing the vector.

As described above, the nucleic acid molecule can be used by itself or as part of a vector for expression of the polypeptide of the invention in cells, for example, for cleaning and for gene-therapeutical targets. The nucleic acid molecule or vectors containing a sequence(I) DNA encoding(I) any polypeptide described above according to the invention, is introduced into cells that in turn produce INTA�asousi polypeptide. Gene therapy, is based on introducing therapeutic genes into cells using techniques ex vivo or in vivo, represents one of the most important applications of gene transfer. Suitable vectors, methods or gene delivery system for gene therapy in vitro or in vivo are described in the literature and known to those skilled in the art (see, e.g., Giordano, Nature Medicine, 2 (1996), 534-539; Schaper, Circ. Res., 79 (1996), 911-919; Anderson, Science, 256 (1992), 808-813; Verma, Nature, 389 (1994), 239; Isner, Lancet, 348 (1996), 370-374; Muhlhauser, Circ. Res., 77 (1995), 1077-1086; Onodera, Blood, 91 (1998), 30-36; Verma, Gene Ther., 5 (1998), 692-699; Nabel, Ann. N. Y. Acad. Sci., 811 (1997), 289-292; Verzeletti, Hum. Gene Ther., 9 (1998), 2243-51; Wang, Nature Medicine, 2 (1996), 714-716; WO 94/29469; WO 97/00957, US 5580859; US 5589466; or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640). The aforementioned nucleic acid molecules and vectors can be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell. Preferably, said cell is a germ line cell, embryonic cell or an egg or comes from them; most preferably, said cell is a stem cell. An example of an embryonic stem cell can be, among other things, stem cell, which is described in Nagy, Proc. Natl. Acad. Sci. USA, 90 (1993), 8424-8428.

According to the invention also suggested the owner put�th transformation or transfection vector according to the invention. The specified host can be obtained by introducing the vector described above according to the invention described above or nucleic acid molecule of the invention into the host. The presence of at least one vector or at least one nucleic acid molecule in the host may mediate the expression of a gene encoding the above described construction of single-chain antibodies.

The described nucleic acid molecule or vector according to the invention, which is injected into the host, or can be integrated into the host genome, or may be supported extrachromosomal.

The host may be any prokaryotic or eukaryotic cell.

The term "prokaryotes" means all bacteria that can be transformed or transfected by the DNA molecules or RNA for the purpose of expression of the protein according to the invention. Prokaryotic hosts can include gram-negative and gram-positive bacteria, such as, for example, E. coli, S. typhimuiium, Serratia marcescens and Bacillus subtilis. The term "eukaryotic" encompasses cells of yeast, higher plant, insect and preferably mammalian. Depending on the host used in the procedure obtain recombinant protein encoded by the polynucleotide of the present invention may be glycosylated or may be deglycosylated. Especially preferred is a m�is using plasmid or virus, containing(his) encoding the polypeptide sequence of the invention, genetically fused with N-terminal FLAG-tag and/or C-terminal His-tag. Preferably, the length of the indicated FLAG-tagged is about 4 to 8 amino acids, most preferably 8 amino acids. Described above, the polynucleotide can be used for transformation or transfection of a host using any of the techniques generally recognized by experts in the field of technology. In addition, methods of producing the fusion, functionally related genes and their expression, for example in mammalian cells and bacteria, are well known in the art (Sambrook, see above).

Preferably, the specified host is a bacterium or cell of insects, fungi, plants or animals.

In particular, it is envisaged that the specified host can be a mammal cells. In particular, preferably the host cell include cells of SSS (cells Chinese hamster ovary), COS cells (fibroblasts, African green monkey), lines of myeloma cells such as SP2/0 or NS/0. As illustrated in the examples below, as hosts of the particularly preferred cells SNO.

More preferably, the specified host is a human cell or human cell line, such as per.C6(Kroos, Biotechnol. Prog., 2003, 19: 163-168).

In yet another embodiment the present invention relates to a method of producing molecules especifismo single-chain antibodies according to the invention, comprising culturing the host according to the invention in conditions that ensure the expression of this molecule especifismo single-chain antibodies according to the invention, and the allocation of the obtained polypeptide from the culture.

The transformed hosts can be grown in fermenters and cultivated in accordance with methods known in the art to achieve optimal cell growth. Then the molecule especifismo single-chain antibodies according to the invention can be isolated from the growth medium, cellular lysates, or cellular membrane fractions. The isolation and purification, for example, expressed by microbes molecules especifismo single-chain antibodies according to the invention can be carried out by any conventional methods, such as, for example, preparative chromatographic methods of separation and immunological separation methods, for example separation methods using monoclonal or polyclonal antibodies directed, e.g., against the tag molecules especifismo single-chain antibody of the invention or as described in the examples below.

In this area �Ethniki know, the culturing conditions of the host, providing the expression depend on the system of the host and expressing/overexpressing vector used in this method. The parameters to be modified to achieve the conditions that provide for expression of the recombinant polypeptide, are known in the art. Therefore, suitable conditions can be determined by the specialist in the art without further inventive contribution.

Immediately after the expression of the molecule especifismo single-chain antibodies according to the invention can be purified according to standard in the art methods, including precipitation with ammonium sulfate, a method using affinity columns, column chromatography, electrophoresis in the gel, and the like (see Scopes, "Protein Purification", Springer-Verlag, N.Y. (1982)). For pharmaceutical applications preferred essentially pure polypeptides with a homogeneity of at least about 90-95%, and most preferred with homogeneity 98-99% or more. After cleaning, partially or to homogeneity, if desired, the molecule especifismo single-chain antibodies according to the invention can then be used for therapeutic purposes (including in vitro) or for the design and implementation of analysis methods. In addition, in any given�e examples described methods for isolating molecules especifismo single-chain antibodies according to the invention from the culture. The allocation can also be implemented by a method of separating molecules especifismo single-chain antibodies according to the invention, capable of contacting epitope of CD3-Epsilon (CD3ε) of man and the primacy of non-chimpanzee, comprising the following stages:

(a) alignment of polypeptide(s) in contact with the N-terminal fragment of the extracellular domain of CD3ε, consisting of up to 27 amino acids containing the amino acid sequence Gln-Asp-Gly-Asn-Glu-Glu-Met-Gly (SEQ ID NO: 341) or Gln-Asp-Gly-Asn-Glu-Glu-lle-Gly (SEQ ID NO: 342), fixed via its C-end to the solid phase;

(b) elution of the bound(s) polypeptide(s) from the specified section; and

(c) isolation of the polypeptide(s) from the eluate of step (b).

Preferably, the polypeptide(s) selected(s) by the above method according to the invention is(are) a polypeptide(s) of the person.

This method of separating molecules especifismo single-chain antibodies according to the invention is a method of separating one or more different polypeptides with the same specificity for the fragment of the extracellular domain of CD3ε, containing at its N-end amino acid sequence Gln-Asp-Gly-Asn-Glu-Glu-Met-Gly (SEQ ID NO: 341) or Gln-Asp-Gly-Asn-Glu-Glu-lle-Gly (SEQ ID NO: 342), from a variety of polypeptides candidates, as well as the method of purification of the polypeptide from solution. Non-limiting examples of the last spasibochki molecules especifismo single-chain antibodies from a solution is, for example, clearing recombinantdna molecules expressed especifismo single-chain antibodies from culture supernatant or mixture of such a culture.

As mentioned above, the fragment used in this way, represents the N-terminal fragment of the extracellular domain of CD3ε molecule of the primacy. Amino acid sequence of the extracellular domain of CD3ε molecule of different species represented in SEQ ID NO: 1, 3, 5 and 7. Two forms of the N-terminal of octamer presented in SEQ ID NO: 341 and 342. Preferably, the N-end is freely available for binding of the polypeptides to be identified by the method of the invention. The term "freely available" in the context of the invention means not having additional motifs, such as Mis-label. Interference of such a His-tag with a binding molecule identified by the method of the invention described in the following examples 6 and 20.

According to this method, the specified portion is fixed via its C-end to the solid phase. Specialist in the art can easily and without any inventive activity choose a suitable solid phase substrate depending on the embodiment of the method according to the invention. Examples of solid substrates include, but are not limited to, matrix-type granules (for example agarose pellets, sepharose pellets, policy�ironnie pellets, dextran granules), tablets (culture plates or multi-well tablets), as well as chips, such as Biacore®. The choice of means and methods of fixation/immobilization of a fragment specified on a solid substrate depends on the choice of the solid substrate. The commonly used method of fixation/immobilization-linking via N-hydroxysuccinimidyl (NHS) ester. Underlying this binding chemistry, and alternative methods of fixation/immobilization are known to the person skilled in the art, for example from Hermanson "Bioconjugate Techniques", Academic Press, Inc. (1996). For fixation/immobilization on chromatographic substrates typically use the following tools: NHS-activated sepharose (e.g. HiTrap-NHS from GE Life Science-Amersham), CnBr-activated sepharose (for example, GE Life Science-Amersham), NHS-activated dextran pellets (Sigma) or activated polymethacrylate. These reagents can also be used in the periodic approach. In addition, in the periodic approach can be used dextran pellets containing iron oxide (available, e.g., from Miltenyi). These granules can be used in combination with a magnet to separate the granules from the solution. The polypeptides can be immobilized on the Biacore chip (e.g., on the chip CM 5) due to the use of NHS-activated carboxymethylcysteine. Additional examples of origina�Dasha solid substrate are multi-well plates with reactive amino groups (e.g., tablets Nunc Immobilizer™).

According to this method, the specified portion of the extracellular domain of CD3-Epsilon may be associated with a solid substrate directly or through a stretch of amino acids that may represent a linker or other protein/polypeptide group. Alternatively, the extracellular domain of CD3-Epsilon may be linked via one or more than one adaptor molecule.

Means and methods of elution of the peptide or polypeptide bound to the immobilized epitope are well known in this field. The same is true for the allocation identified(s) or polypeptide(s) of the eluate.

A method of separating one or more molecules especifismo single-chain antibody with the same specificity for the fragment of the extracellular domain of CD3ε, containing at its N-end amino acid sequence Gln-Asp-Gly-Asn-Glu-Glu-X-Gly (where X represents Met or Not), from a variety of polypeptides candidates may include one or more stages of the following methods of selection of antigen-specific structures:

CD3ε-specific binding domains can be selected from antibody repertoires. The phage display library can be constructed by standard techniques as described, for example, in "Phage Display: A Laboratory Manual"; Ed. Barbas, Burton, Scott & Silverman; Cold Spring Harbor Laboratory Press, 2001. The format of the fragments of anti�l in the library of antibodies can be scFv, but usually can also be a Fab-fragment, or even a fragment of single-domain antibodies. For the isolation of fragments of antibodies can be used not subjected to any impact of the library fragments of antibodies. For the selection of binding structures with potentially low immunogenicity upon the subsequent therapeutic applications, the preferred choice for direct selection of fragments of human antibodies can be libraries of fragments of human antibodies. In some cases, they can be the basis for synthetic libraries of antibodies (Knappik et al. J. Mol. Biol., 2000, 296: 57 ff). The appropriate format may be Fab, scFv (as described below) or domain antibodies (dAb, the review of which is presented in Holt et al., Trends Biotechnol., 2003, 21:484ff).

It is also known in this field that in many cases a suitable source of human immune antibodies against the antigen target no. Therefore, animals are subjected to immunization with the antigen-targeted, and from animal tissues, such as spleen or RVMS (mononuclear cells peripheral blood), allocate the appropriate library of antibodies. N-terminal fragment can be is biotinylated or covalently linked to the protein type KLH (keyhole lymph snails) or bovine serum albumin (BSA). According to the General approaches used for immunization of rodents. Some of the immune repertoires of antibodies �e of human origin can be particularly useful for other reasons, for instance, due to the presence of single-domain antibodies (VHH) derived from a species of one-humped camel (camelid species (as described in Muyldermans, J. Biotechnol., 74: 277; De Genst et al., Dev. Como. Immunol., 2006, 30: 187 ff). Thus, the appropriate format of the library of antibodies can be Fab, scFv (as described below) or single-domain antibodies (VHH).

In one possible approach 10-week-old mice from F1 crosses of balb/c × C57black can be immunized with whole cells, for example expressing transmembrane Arcam showing on the N-end as a translational fusion of the N-terminal amino acids 1-27 of the Mature CD3ε-chain. Alternatively, mice can be immunized fused protein 1-27 CD3-Epsilon-Fc (corresponding to the approach described in the Example 2 below). After booster(s) of immunization(s) can be taken blood samples, and serum titer of antibodies against CD3-positive T cells can be tested, for example, in FACS-analysis. Usually serum titers significantly higher in vaccinated than in unvaccinated animals.

Immunized animals can be the basis for the creation of immune libraries of antibodies. Examples of such libraries include phage display library. Such libraries usually can be constructed by standard techniques as described, for example, in "Phage Display: A Laboratory Manual"; Ed. Barbas, Burton, Scott & Silverman;Cold Spring Harbor Laboratory Press, 2001.

Antibodies are not the person can also be humanized by phage display with the establishment of libraries more antibody variable, which can later be enriched in relation to the linking of fragments in the process of selection.

In the approach using phage display any of the pools of phages, which exhibits a library of antibodies, forms the basis for the selection of binding structures with the appropriate antigen as target molecules. The Central stage at which secrete antigen-specific, antigen-bound phages, called panningen. Thanks to the exposure of fragments of antibodies on the surface of phages, this General method is called rahovym display. One of the preferred ways of selection is to use small proteins, such as N2 domain of filamentous phage, translational fusion with the N-end of the scFv exhibited by this phage. Another display method known in the art, which can be used to separate connecting structures, is the method of ribosomal display (see review in Groves & Osbourn, Expert. Opin. Biol. Ther., 2005, 5: 125 ff; Lipovsek & Pluckthun, J. Immunol. Methods, 2004, 290: 52 ff). To demonstrate the binding of scFv-containing phage particles with the fused protein 1-27 CD3ε-Fc phage library carrying the cloned scFv-repertoire, can be�ü collected from the respective culture supernatant using PEG (polyethylene glycol). scFv-containing phage particles can be subjected to incubation with immobilized fused protein CD3ε-Fc. Immobilized protein CD3ε-Fc may be deposited on the solid phase. Linking patterns can be-eluted, and the eluate can be used to infect a fresh batch of uninfected bacterial hosts. Bacterial hosts, subjected to successful transduction fahmideh copy, encoding a human scFv-fragment, can be subjected to selection for resistance to carbenicillin and after that may be infected with, for example the helper phage VCMS 13, to start the second round of exposure of antibodies and in vitro selection. Normal conduct in the amount of from 4 to 5 cycles of selection. Linking the selected linking structures can be tested on D3-positive Jurkat cells, cells HPBall, PBMC or transfected eukaryotic cells that carry N-terminal CD3ε-sequence, merged with the exposed surface Ersam, in a flow-cytometric analysis (see below, Example 4).

Preferably, the above method may be a method in which a fragment of the extracellular domain of CD3e consists of one or more fragments of the polypeptide having the amino acid sequence of any one, is presented in SEQ ID NO: 2, 4, 6 or 8. B�more preferably, the specified portion is a 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 amino acid residues in length.

This method of identifying molecules especifismo single-chain antibodies may represent a method of screening a plurality of molecules bespecifically single-chain antibody containing a binding domain that binds to an epitope of human CD3ε and the primacy of non-chimpanzee, with interspecies specificity. Alternatively, the identification method is a method for cleaning/separation of molecules especifismo single-chain antibody containing a binding domain that binds to an epitope of human CD3ε and the primacy of non-chimpanzee, with interspecies specificity.

In addition, according to the present invention proposed a composition comprising a molecule especifismo single-chain antibody of the invention or bespecifically single-chain antibody obtained by the method described above. Preferably, said composition is a pharmaceutical composition.

According to the present invention also suggested that the molecule especifismo single-chain antibodies as defined herein, or obtained by the method as defined herein, where the specified molecule especifismo single-chain antibodies are designed to�is destined for use in the prevention, treatment or reduction of symptoms of cancer or autoimmune diseases.

For molecules PSCA×CD3 especifismo single-chain antibodies specified cancer preferably represents prostate cancer, bladder cancer or pancreatic cancer.

For molecules CD19×CD3 especifismo single-chain antibodies specified cancer preferably is a b-cell malignancy, such as B-NHL (b-cell non-Hodgkin's lymphoma), B-ALL (acute lymphoblastic b-cell leukemia, B-CLL (chronic lymphocytic b-cell leukemia or multiple myeloma (where the molecule especifismo single-chain antibodies according to the invention preferably depletes CD19-positive cancer stem cells/initiating cells, cancer). However, this molecule especifismo single-chain antibodies are also intended for use in the prevention, treatment or reduction of symptoms mediated by b-cells in autoimmune diseases or autoimmune diseases involving pathogenic b-cells, such as rheumatoid arthritis or depletion of b-cells.

For molecule c-MET×CD3 especifismo single-chain antibodies specified cancer preferably is a carcinoma, sarcoma, glioblastoma/astrocytoma, melanoma, mesothelioma, Wilms tumor or a hematopoietic sloc�chastenest, such as leukemia, lymphoma or multiple myeloma. An exhaustive list of different types of cancer that can be treated bespecifically single-chain antibody can be found, for example, on the website http://www.vai.org/met/.

For molecules endothelin×CD3 especifismo single-chain antibodies specified cancer preferably includes, but is not limited to, carcinomas (breast, kidney, lung, colorectal, colon, pancreas mesothelioma), sarcomas, and neuroectodermal tumors (melanoma, glioma, neuroblastoma).

For molecules Arcam×CD3 especifismo single-chain antibodies specified cancer preferably is an epithelial cancer or a minimal residual cancer.

For molecules FAPα×CD3 especifismo single-chain antibodies specified cancer preferably is an epithelial cancer.

For molecule IGF-1R×CD3 especifismo single-chain antibodies specified cancer preferably is a cancer of bone or soft tissue (e.g. Ewing sarcoma), breast cancer, liver cancer, lung cancer, cancer in the head and neck, colorectal cancer, prostate cancer, leiomyosarcoma, cervical and endometrial cancer, ovarian cancer, prostate cancer and pancreatic cancer. Alternatively, the molecule IGF-1R×CD3 especificacao of ADNOC�renal antibodies according to the invention is also used in the prevention, treating or reducing symptoms of autoimmune diseases, preferably psoriasis.

A preferred molecule is especifismo single-chain antibodies, further comprising suitable carriers, stabilizers and/or excipient. In addition, preferred is a specified molecule especifismo single-chain antibodies, suitable for administration in combination with an additional drug. Specified drug can be a non-proteinaceous compound or a proteinaceous compound, and can be administered simultaneously or asynchronously with the molecule especifismo single-chain antibody as defined elsewhere herein.

In accordance with the invention the term "pharmaceutical composition" refers to compositions for administration to a patient, preferably a patient person. Specific preferred pharmaceutical composition of the present invention comprises bespecifically single-chain antibody directed against and generated against independent from the environment of CD3 epitopes. Preferably, the pharmaceutical composition comprises suitable carriers, stabilizers and/or excipient. In a preferred embodiment of the pharmaceutical composition is a composition for parenteral, transdermal, Nutripro�swetnam, intra-arterial, intrathecal, and/or intranasal administration or administration by direct injection into the tissue. In particular, it is envisaged that the specified composition is administered to the patient by infusion or injection. The introduction of the suitable compositions may be effected in various ways, for example intravenous, intraperitoneal, subcutaneous, intramuscular, local or intradermal injection. In particular, according to the present invention provides for the continuous introduction of appropriate composition. As a non-limiting example, a continuous, i.e., continuous infusions, can be implemented by means of a small pump systems, driven by the patient for dispensing receipts therapeutic agent into the patient. Pharmaceutical composition containing bespecifically single-chain antibodies according to the invention directed against and generated against independent from the environment of CD3 epitopes, can be entered using the pumping systems. These pumping systems are well known in the art and are typically designed for periodic replacement of the cartridge containing an injectable infusion of therapeutic agent. When you replace the cartridge in such a pump system may be provided with a temporary interruption in all other circumstances�Ah continuous flow of therapeutic agent into the patient. And in this case, the phase of introduction before replacing the cartridge and the introduction process after replacement of the cartridge are still considered within an understanding of the pharmaceutical means and methods according to the invention together to form one continuous introduction of such therapeutic agent.

Permanent or continuous introduction of these bespecifically single-chain antibodies according to the invention that are directed against and generated against independent from the environment of CD3 epitopes, can be intravenous or subcutaneous using the device for delivery of liquid or a small pumping system, comprising a feed mechanism for feeding the fluid from the reservoir, and a trigger mechanism for actuating the feeding mechanism. A pump system for the subcutaneous administration can include a needle or cannula for piercing the patient's skin and delivering the right composition to the patient. These pumping systems can be directly fixed on the skin or attached to skin of the patient independently from the vein, artery or blood vessel, thereby providing a direct contact between the pump system and the patient's skin. The pumping system can be attached to the skin of the patient during the time period from 24 hours up to several days. The pumping system may be a system that is small, �eservoir for small volumes. As a non-limiting example, the volume of the tank suitable for pharmaceutical compositions intended for administration, may be from 0.1 to 50 ml.

Continuous administration may be transdermal using a patch placed on the skin and replaced at certain intervals of time. Specialist in the art is aware of the systems applying patch for drug delivery that are suitable for this task. It should be noted that transdermal introduction is particularly suitable as a coherent introduction, since the first replacement of the used patch may preferably be carried out simultaneously with the substitution of a new, second patch, for example on the surface of the skin that are in the vicinity of the first used the patch and just before you remove the first used the patch. Problems with thread interruption or severe cell damage does not occur.

The composition of the present invention containing, in particular, bespecifically single-chain antibody directed against and generated against independent from the environment of CD3 epitopes, can optionally contain a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in this field �Ethniki and include solutions, for example with phosphate buffered saline, water, emulsions such as emulsions of oil/water, various types of wetting agents, sterile solutions, liposomes, etc. Compositions comprising such carriers can be manufactured by well known conventional methods. Compositions may contain carbohydrates, buffers, amino acids and/or surfactants. Carbohydrates can be nevosstanovlenie sugar, preferably trehalose, sucrose, octasulphate, sorbitol or xylitol. Such compositions can be used for continuous infusions, which may be intravenous or subcutaneous with and/or without the use of pumping systems. Amino acids may represent a charged amino acid, preferably lysine, lysine acetate, arginine, glutamate and/or histidine. Surfactants may constitute detergents, preferably with a molecular weight of more than 1.2 kDa, and/or easy polyester, preferably with molecular weight greater than 3 kDa. Non-limiting examples of preferred surfactants are Tween 20, Tween 40, Tween 60, Tween 80 or Tween 85. Non-limiting examples of preferred polyether are PEG 3000, PEG 3350, PEG 4000 or PEG 5000. The buffer system used in the present invention may have preferred�Stateline pH value of 5-9, and they can contain citrate, succinate, phosphate, histidine and acetate. The compositions of the present invention can be administered to the subject at a suitable dose can be determined, for example, as a result of research with application of increasing doses by injecting increasing doses of the molecules described here especifismo single-chain antibody of the invention exhibiting cross-species specificity against human primates, which is not a chimpanzee, for example macaques. As mentioned above, described herein is a molecule especifismo single-chain antibody of the invention exhibiting cross-species specificity, can be preferably used in identical form in preclinical testing on primates, which is not a chimpanzee, and in the form of the drug in humans. These compositions can also be administered in combination with other protein and non-protein drugs. These medicines can be administered simultaneously with the composition comprising a molecule described herein especifismo single-chain antibody of the invention exhibiting cross-species specificity, or separately before or after the introduction of the specified polypeptide in accordance with defined time intervals and doses. The dosage will be to identify the attending physician on the clinical indicators. As the Glo�but in medicine, dosages for any one patient depend on many factors, including the size of the patient's body, the body surface of the patient, the patient's age, the particular compound that is intended for insertion, the patient's sex, time and route of administration, General health of the patient and other medications are injected together. Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions and emulsions. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters, such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered environment. Parenteral diluents include sodium chloride solution, dextrose in ringer's solution, dextrose and sodium chloride, ringer solution with lactose, or fixed oils. Intravenous diluents include liquid and nutritious fillers, electrolytes fillers (for example on the basis of dextrose in ringer's solution) and the like. May also be present preservatives and other additives, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like. In addition, the composition of the present invention m�can contain protein carriers, such as, for example, serum albumin or immunoglobulin, preferably of human origin. It is envisaged that a composition according to the invention may contain, in addition to the molecule described here especifismo single-chain antibodies according to the invention, the additional biologically active agents depending on the intended application of the composition. Such agents might be drugs acting on the gastrointestinal system, drugs acting as cytotoxic, drugs, preventing hyperuricemia, medicines that suppress the immune response (e.g. corticosteroids), drugs, modulating the inflammatory response, drugs that act on the circulatory system, and/or agents such as cytokines, known in this field.

The biological activity of the pharmaceutical compositions as defined herein, may be determined, for example, cytotoxicity assays, as described in the examples below, in WO 99/54440, or in Schlereth et al. Cancer Immunol. Immunother., 20 (2005), 1-12. Used in this description, the terms "effectiveness" or "efficiency in vivo" refers to the response to therapy a pharmaceutical composition according to the invention, which is defined with required�Niemi, for example, standardized criteria for response assessment NCI (national cancer Institute, USA). The success or effectiveness of therapy in vivo using the pharmaceutical compositions according to the invention relates to the effectiveness of the composition in relation to its purpose, i.e. to the ability of the composition to cause the desired effect, i.e. the depletion of abnormal cells, such as tumor cells. Efficacy in vivo can be controlled by approved standard methods of determining relevant clinical entities, including, without limitation, the determination of the number of leukocytes, differential blood count, sorting fluorescence-activated cells, the bone marrow aspiration. In addition, there can be used methods for determination of clinical chemistry parameters specific to various diseases, and other approved standard methods. In addition, can be used computed tomography, x-ray, nuclear magnetic resonance imaging (e.g., for response assessment based on the criteria of the National cancer Institute of the USA [Cheson BD, Horning SJ, Coiffier B, Shipp MA, Fisher RI, Connors JM, Lister TA, Vose J, Grillo-Lopez A, Hagenbeek A, Cabanillas F, Klippensten D, Hiddemann W, Castellino R, Harris NL, Armitage JO, Carter W, Hoppe R, Canellos GP. Report of an international workshop to standardize response criteria for non-Hodgkin's lymphomas. NCI Sponsored International Working roup.J. Clin. Oncol., 1999 Apr; 17(4): 1244]), positron emission tomographic scanning, the determination of the number of leukocytes, differential blood count, sorting fluorescence-activated cells, aspiration bone marrow biopsy/histology of lymph nodes and identify the different lymphoma-specific clinical chemistry parameters (e.g. lactate dehydrogenase) and other approved standard methods.

Another big problem in the development of such drugs, as pharmaceutical composition according to the invention, is the predicted modulation of pharmacokinetic properties. For this purpose, define the pharmacokinetic profile of the drug candidate, i.e. the profile of pharmacokinetic parameters, which affect the ability of a particular drug to treat this condition. The pharmacokinetic parameters of the drug that affect the drug's ability to treat a specific disease state, include, but are not limited to, the elimination half-life, volume of distribution, metabolism "first passage" through the liver and the degree of binding to serum. The effectiveness of this medicinal agent can influence each of the above-mentioned parameters.

"Half-life" means�no time for which 50% of the administered drug is excreted as a result of biological processes, such as metabolism, excretion, etc.

Under the "first pass metabolism through the liver" means the ability of the drug to be metabolized after the first contact with the liver, i.e. during its first passage through the liver.

"Volume of distribution" means the degree of retention of the drug in all the various compartments of the body such as, for example, the intracellular and extracellular spaces of tissues and organs, etc., and distribution of the drug in these compartments.

"The degree of binding to serum" refers to the tendency of the drug to interact or communicate with serum proteins such as albumin, resulting in reduction or loss of biological activity of the drug.

Pharmacokinetic parameters also include bioavailability, the latent period (Tlag), Tmax, the rate of absorption, the start time and/or Cmax for a given amount of input of the drug.

"Bioavailability" means the amount of drug in the blood compartment.

"Latent period" means the time delay between administration of the drug and its detection and the ability measured�I in the blood or plasma.

"Tmax" is the time after which the maximum concentration of the drug in the blood, and "Cmax" is the maximum concentration in the blood, resulting from the use of this drug. For a period of time to achieve a concentration of the drug in the blood or tissues, which is required for the biological effect of this medicinal product, is influenced by all the parameters. Pharmacokinetic parameters bespecifically single-chain antibodies exhibiting cross-species specificity, which can be determined in preclinical animal tests, on primates, which are not chimpanzees, listed above, are also given, for example in the publication Schlereth et al. Cancer Immunol. Immunother., 20 (2005), 1-12.

Used in this description, the term "toxicity" refers to the toxic effects of the drug, manifested in adverse events or serious adverse events. These side effects can be treated to a complete intolerance of the drug and/or lack of local tolerability after administration. Toxicity may also include teratogenic or carcinogenic effects caused by this drug.

Used in this description, the terms "safety", "safety in vivo �whether "tolerance" determine the administration of the drug without inducing severe adverse events after the administration (local tolerance) and for longer periods of drug use funds. "Security", "safety in vivo" or "tolerance" can be assessed, for example, at regular intervals during treatment and subsequent follow-up period. Measurements include clinical assessment, such as manifestations of a number of authorities, and screening laboratory abnormalities. In accordance with the standards of the NCI-CTC and/or MedDRA is possible to carry out a clinical assessment and record/encode the deviation with respect to normal values. The existence of bodies can include criteria such as Allergy/immunology blood/bone marrow cardiac arrhythmia, coagulation and the like, which are described in Common terminology criteria for adverse events (Common Terminology Criteria for adverse events) v3.0 (CTCAE). Laboratory parameters that may be tested include, for example, haematological, clinical chemical, coagulation profile, and urinalysis, and examination of other body fluids such as serum, plasma, lymph or cerebrospinal fluid, cerebrospinal fluid, etc. Thus, the safety can be evaluated, for example, by medical examination, the use of visualization techniques (i.e. ultrasound, x-ray examination, CT-scan (computed tomography), magnetic resonance imaging (MRI)), other measurements used�using technical means (i.e. an electrocardiogram), vitals condition of the body, by measuring laboratory parameters and registration of adverse events. For example, unfavorable phenomena in primates that are not chimpanzees, in applications and methods of the invention can be investigated histopathological and/or histochemical methods.

Used in this description, the term "effective and non-toxic dose" refers to tolerated dose especifismo single-chain antibodies as defined herein, which is high enough to cause depletion of abnormal cells, elimination of a tumor, reduction of tumor size or stabilization of disease without or essentially without major toxic effects. Such effective and non-toxic doses can be determined, for example, is described in this field of research with increasing doses, and it must be below the dose that causes severe adverse side effects (limiting toxicity (DLT)).

The above terms are also referenced, for example, in Preclinical safety evaluation of biotechnology-derived pharmaceuticals (Preclinical evaluation of the safety of biotechnology-derived pharmaceuticals) S6; ICH Harmonised Tripartite Guideline; ICH Steering Committee meeting on July 16, 1997.

In addition, this invent�s relates to pharmaceutical compositions, containing molecule especifismo single-chain antibodies according to the invention or obtained by the method according to the invention to prevent, treat or reduce the symptoms of cancer or autoimmune diseases.

Preferably, the molecules PSCA×CD3 especifismo single-chain antibodies specified cancer is a prostate cancer, bladder cancer or pancreatic cancer.

Preferably, the molecule CD19×CD3 especifismo single-chain antibodies specified cancer is a b-cell malignancy such as non-Hodgkin's lymphoma, mediated by b-cells in autoimmune diseases or the depletion of b-cells.

Preferably, the molecule c-MET×CD3 especifismo single-chain antibodies specified cancer is a carcinoma, sarcoma, glioblastoma/astrocytoma, melanoma, mesothelioma, Wilms tumor or a hematopoietic malignancy such as leukemia, lymphoma or multiple myeloma.

Preferably, the molecules endothelin×CD3 especifismo single-chain antibodies specified cancer is a carcinoma (breast, kidney, lung, colorectal, colon, pancreas mesothelioma), sarcoma or neuroectodermal tumor (melanoma, glioma, neuroblastoma).

Preferably, d�I molecule EpCAM×CD3 especifismo single-chain antibodies specified cancer is an epithelial cancer or a minimal residual cancer.

Preferably, the molecules of FAPα×CD3 especifismo single-chain antibodies specified cancer is an epithelial cancer.

Preferably, the molecule IGF-1R×CD3 especifismo single-chain antibodies specified cancer is a cancer of bone or soft tissue (e.g. Ewing sarcoma), breast cancer, liver cancer, lung cancer, cancer in the head and neck, colorectal cancer, prostate cancer, leiomyosarcoma, cervical and endometrial cancer, ovarian cancer, prostate cancer and pancreatic cancer. Alternatively, the molecule IGF-1R×CD3 especifismo single-chain antibody/polypeptide as defined herein above, is also used in the prevention, treatment or reduction of symptoms of autoimmune diseases, preferably psoriasis.

Preferably, the specified pharmaceutical composition further comprises suitable carriers, stabilizers and/or excipient.

In another aspect the invention relates to the use of molecules especifismo single-chain antibody/polypeptide as defined herein above, or obtained by the method as defined herein above, for the manufacture of pharmaceutical compositions for the prevention, treatment or reduction of symptoms.

Preferably, UK�related disease is a cancer or autoimmune diseases.

More preferably, the molecules PSCA×CD3 especifismo single-chain antibody/polypeptide as defined herein above specified cancer is a prostate cancer, bladder cancer or pancreatic cancer.

More preferably, the molecules CD19×CD3 especifismo single-chain antibody/polypeptide as defined herein above specified cancer is a b-cell malignancy such as non-Hodgkin's lymphoma, mediated by b-cells in autoimmune diseases or the depletion of b-cells.

More preferably, the molecule c-MET×CD3 especifismo single-chain antibody/polypeptide as defined herein above specified cancer is a carcinoma, sarcoma, glioblastoma/astrocytoma, melanoma, mesothelioma, Wilms tumor or a hematopoietic malignancy such as leukemia, lymphoma or multiple myeloma.

More preferably, the molecules endothelin×CD3 especifismo single-chain antibody/polypeptide as defined herein above specified cancer is a carcinoma (breast, kidney, lung, colorectal, colon, pancreas mesothelioma), sarcoma or neuroectodermal tumor (melanoma, glioma, neuroblastoma).

More p�edocfile, for molecules EpCAM×CD3 especifismo single-chain antibody/polypeptide as defined herein above specified cancer is an epithelial cancer or a minimal residual cancer.

More preferably, the molecules of FAPα×CD3 especifismo single-chain antibody/polypeptide as defined herein above specified cancer is an epithelial cancer.

More preferably, the molecule IGF-1R×CD3 especifismo single-chain antibody/polypeptide as defined herein above specified cancer is a cancer of bone or soft tissue (e.g. Ewing sarcoma), breast cancer, liver cancer, lung cancer, cancer in the head and neck, colorectal cancer, prostate cancer, leiomyosarcoma, cervical and endometrial cancer, ovarian cancer, prostate cancer and pancreatic cancer. Alternatively, the molecule IGF-1R×CD3 especifismo single-chain antibody/polypeptide as defined herein above, is also used in the prevention, treatment or reduction of symptoms of autoimmune diseases, preferably psoriasis.

In another preferred embodiment of the use of molecules especifismo single-chain antibodies according to the invention mentioned pharmaceutical composition suitable for administration in combination � additional drug i.e. as part of combination therapy. In said combination therapy, the active agent may be included in the same pharmaceutical composition, comprising in a molecule especifismo single-chain antibodies according to the invention, or may be part of separate pharmaceutical compositions. In this latter case, the specified separate pharmaceutical composition suitable for administration before, simultaneously or after the introduction of the specified pharmaceutical composition containing molecule especifismo single-chain antibodies according to the invention. Additional medicament or pharmaceutical composition can be a non-proteinaceous compound or a proteinaceous compound. In the case where the additional drug is a protein compound, then preferably a protein compound capable of providing an activation signal for immune effector cells.

Preferably, these proteinaceous compound or non-proteinaceous compound may be administered simultaneously or asynchronously with the molecule especifismo single-chain antibodies according to the invention, the nucleic acid molecule as defined herein above, a vector as defined herein above, or the owner, as defined in this description� above.

In another aspect the invention relates to a method for the prevention, treatment or reduction of symptoms in a subject in need, comprising the stage of introducing an effective amount of the pharmaceutical composition of the invention. Preferably, the disease is a cancer or autoimmune diseases.

More preferably, the molecules PSCA×CD3 especifismo single-chain antibody/polypeptide as defined herein above specified cancer is a prostate cancer, bladder cancer or pancreatic cancer.

More preferably, the molecules CD19×CD3 especifismo single-chain antibody/polypeptide as defined herein above specified cancer is a b-cell malignancy such as non-Hodgkin's lymphoma, mediated by b-cells in autoimmune diseases or the depletion of b-cells.

More preferably, the molecule c-MET×CD3 especifismo single-chain antibody/polypeptide as defined herein above specified cancer is a carcinoma, sarcoma, glioblastoma/astrocytoma, melanoma, mesothelioma, Wilms tumor or a hematopoietic malignancy such as leukemia, lymphoma or multiple myeloma.

More preferably, for molecules� endothelin×CD3 especifismo single-chain antibody/polypeptide, as defined herein above specified cancer is a carcinoma (breast, kidney, lung, colorectal, colon, pancreas mesothelioma), sarcoma or neuroectodermal tumor (melanoma, glioma, neuroblastoma).

More preferably, the molecule EpCAM×CD3 especifismo single-chain antibody/polypeptide as defined herein above specified cancer is an epithelial cancer or a minimal residual cancer.

More preferably, the molecules of FAPα×CD3 especifismo single-chain antibody/polypeptide as defined herein above specified cancer is an epithelial cancer.

More preferably, the molecule IGF-1R×CD3 especifismo single-chain antibody/polypeptide as defined herein above specified cancer is a cancer of bone or soft tissue (e.g. Ewing sarcoma), breast cancer, liver cancer, lung cancer, cancer in the head and neck, colorectal cancer, prostate cancer, leiomyosarcoma, cervical and endometrial cancer, ovarian cancer, prostate cancer and pancreatic cancer. Alternatively, the molecule IGF-1R×CD3 especifismo single-chain antibody/polypeptide as defined herein above, is also used in warned�and, treating or reducing symptoms of autoimmune diseases, preferably psoriasis.

In another preferred embodiment of the method according to the invention mentioned pharmaceutical composition suitable for administration in combination with an additional drug, i.e. as part of combination therapy. In said combination therapy, the active agent may be included in the same pharmaceutical composition, which includes a molecule especifismo single-chain antibodies according to the invention, or may be part of separate pharmaceutical compositions. In this latter case, the specified separate pharmaceutical composition suitable for administration before, simultaneously or after the introduction of the specified pharmaceutical composition containing molecule especifismo single-chain antibodies according to the invention. Additional medicament or pharmaceutical composition can be a non-proteinaceous compound or a proteinaceous compound. In the case where the additional drug is a protein compound, then preferably a protein compound capable of providing an activation signal for immune effector cells.

Preferably, these proteinaceous compound or non-proteinaceous, the connection can be synchronous� or simultaneously with the molecule especifismo single-chain antibodies according to the invention, the nucleic acid molecule as defined herein above, a vector as defined herein above, or the owner, as defined in this description above.

Preferably, in the above-described method, the specified subject is a person.

In another aspect this invention relates to a set containing molecule especifismo single-chain antibodies according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention or the owner of the invention.

These and other embodiments are described and covered by the description and Examples of the present invention. Recombinant techniques and immunological methods are described, for example, in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 3rdedition 2001; Lefkovits; Immunology Methods Manual; The Comprehensive Sourcebook of Techniques; Academic Press, 1997; Golemis; Protein-Protein Interactions: A Molecular Cloning Manual; Cold Spring Laboratory Press, 2002. Further literature concerning any of the antibodies, methods, uses and compounds for use in accordance with the present invention, can be found in public libraries and databases, using, for example, electronic devices. For example, you can use a recognized database "Medline", available via the Internet, such as an address http://www.ncbi.nlm.nih.gov/PubMed/medline.html. Further databases and addresses, such as http://www.ncbi.nlm.ih.gov/ or listed on the home page of the service, EMBL (European Molecular Biology Laboratory) under the address http://www.embl.de/services/index.html, known to those skilled in the art and can also be accessed using, for example, http://www.google.com.

Graphics illustrate:

Fig.1

Merged design of the N-terminal amino acids 1-27 of CD3-Epsilon primates with heterologous soluble protein.

Fig.2

Fig.2 presents the average values of absorption for the samples in four repetitions, measured in ELISA (ELISA) that determines the presence of a construct consisting of the N-terminal amino acids 1-27 of the Mature CD3-Epsilon-chain of human rights, is attached to a hinge and Fc-gamma-plot of human lgG1 and C-terminal 6-histidinol label in the supernatant (SN) temporarily transfected 293 cells. In the first column labeled "27 AA huCD3E", shows the average absorption for construction, in the second column, labeled as a "nereli. SN", shows the average value for the supernatant of 293 cells transfected irrelevant design, as a negative control. A comparison of the values obtained for this design, with the values obtained for the negative control, clearly demonstrates the presence of recombinant constructs.

Fig.3

Fig.3 shows the average values of absorption for the samples in four repetitions, measured in ELISA analysis, determining the binding of anti-CD3-binding �of Alakul with interspecies specificity in the form of crude preparations of single-stranded antibodies, expressed in the periplasmic space, with the construct containing the N-terminal amino acids 1-27 of the Mature CD3-Epsilon-chain of human rights, is attached to a hinge and Fc-gamma-plot of human lgG1 and C-terminal His6-tag. In columns show, from left to right, the average absorption values for specificdate designated as A2J HLP, I2C HLP, E2M HLP, F70 HLP, G4H HLP, H2C HLP, E1L HLP, F12Q HLP, F6A HLP and NE HLP. In the right column marked as neg. counter." shows the average absorption for the preparation of single-stranded mouse antibodies against human CD3 as a negative control. A comparison of the values obtained for anti-CD3-specificdate, with the values obtained for the negative control (neg. contr.), has clearly demonstrated strong binding of anti-CD3-specificdate with the N-terminal amino acids 1-27 of the Mature CD3-Epsilon-chain of human rights.

Fig.4

Merged design of the N-terminal amino acids 1-27 of CD3-Epsilon primates with heterologous membrane-bound protein.

Fig.5

Combined histograms of different transfectants, tested in FACS analysis, determining the presence of recombinant transmembrane fusion proteins consisting of Arcam Javanese macaque and N-terminal amino acids 1-27 of CD3-Epsilon-chain of human, marmoset, Tamarin, squirrel monkey, and domestic swine, respectively. The combined histograms, left EmOC�AVO, top to bottom, results are presented for transfectants expressing constructs containing the 27-Mer, man, 27-Mer marmoset, 27-Mer Tamarin, 27-Mer squirrel monkeys and 27-Mer pigs, respectively. On each individual combined histogram thin line means the sample incubated with PBS (phosphate buffered saline) containing 2% FCS (fetal calf serum) instead of anti-Flag M2 antibody, as negative control, while the thick line indicates the sample is incubated with anti-Flag M2 antibody. Combined histogram for each design shows the binding of anti-Flag M2 antibodies with transfectants that clearly demonstrates the expression of recombinant constructs in transfectant.

Fig.6

Combined histograms of different transfectants, tested in FACS analysis, determining the binding of anti-CD3-binding molecules with cross-species specificity in the form of crude preparations of single-stranded antibodies, expressed in the periplasmic space, with N-terminal amino acids 1-27 of CD3-Epsilon-chain of human, marmoset, Tamarin and squirrel monkeys, respectively, merged with Arcam Javanese macaque.

Fig.6A:

The combined histograms, from left to right and top to bottom, the results for transfectants expressing the 1-27 CD3-Arsam containing 27-Mer human, PR�tested with the use of CD3-specific binding molecules marked NS HLP, F12Q HLP, e2m boasts HLP and G4H HLP, respectively.

Fig.6B:

The combined histograms, from left to right and top to bottom, the results for transfectants expressing the 1-27 CD3-Arsam containing 27-Mer marmoset, tested using CD3-specific binding molecules identified NS HLP, F12Q HLP, e2m boasts HLP and G4H HLP, respectively.

Fig.6C:

The combined histograms, from left to right and top to bottom, the results for transfectants expressing the 1-27 CD3-Arsam containing 27-Mer Tamarin, tested using CD3-specific binding molecules identified NS HLP, F12Q HLP, e2m boasts HLP and G4H HLP, respectively.

Fig.6D:

The combined histograms, from left to right and top to bottom, the results for transfectants expressing the 1-27 CD3-Arsam containing 27-Mer squirrel monkeys tested with the use of CD3-specific binding molecules identified NS HLP, F12Q HLP, E2M HLP and G4H HLP, respectively.

Fig.6E:

The combined histograms, from left to right and top to bottom, the results for transfectants expressing the 1-27 CD3-Arsam containing 27-Mer pigs tested using CD3-specific binding molecules identified NS HLP, F12Q HLP, E2M HLP and G4H HLP, respectively.

On a separate combined histograms thin there�Oia sample means, incubated with a preparation of murine single-chain antibodies against human CD3 as a negative control, and the thick line indicates the sample is incubated with the relevant specified anti-CD3-binding molecules. Taking into account the lack of binding to transfectants 27-the measure of a pig and the expression levels of the structures shown in Fig.5, combined histograms demonstrate a specific and strong binding of the tested anti-CD3-specificdate human bespecifically single-chain antibodies with full cross-species specificity to cells expressing recombinant transmembrane fusion proteins containing N-terminal amino acids 1-27 of CD3-Epsilon-chain of human, marmoset, Tamarin and squirrel monkeys, respectively, merged with Arcam Javanese macaque, and thus demonstrating cross-species specificity anti-CD3-binding molecules against many primates.

Fig.7

FACS analysis for determination of CD3-Epsilon person on transfected murine EL4 T-cells. Graphical analysis shows the combination of histograms. A thick line means transfetsirovannyh cells incubated with the antibody UCHT-1 against human CD3. The thin line represents cells incubated with mouse control lgG1 isotype. The binding of an antibody UCHT1 against CD3 clearly demonstrates Express� CD3-Epsilon-Henn person on the cell surface of the transfected murine EL4 T cells.

Fig.8

Linking with interspecies specificity anti-CD3 antibody with alanine mutants in the experimental alanine scanning. Columns on separate Figures, from left to right and top to bottom, represent the values of binding, calculated in arbitrary units on a logarithmic scale for transfectant wild-type (WT) and for all alanine mutants for the position 1-27. The values of binding is calculated using the following formula:

In this equation value_Sample means a value binding in arbitrary units, showing the degree of binding of specific anti-CD3 antibodies with a specific alanine-mutant, as shown in this Figure; Sample means the geometric mean fluorescence value obtained for a specific anti-CD3 antibodies, and analyzed on a specific alanine-scanning transfectant; neg_Contr. means the geometric mean fluorescence value obtained for a negative control, were analyzed for specific alanine mutant; UCHT-1 means the geometric mean fluorescence value obtained for the antibody UCHT-1, analyzed for specific alanine mutant; WT means the geometric mean fluorescence value obtained for a specific anti-CD3 antibodies, proanalizirovat�tion on transfectant wild type; x indicates the corresponding transfectant; specifies appropriate anti-CD3-antibody; and wt indicates that the corresponding transfectant refers to wild-type. The individual situation of alanine mutants marked with a single letter code amino acid in the wild type and number of provisions.

Fig.8A:

Fig.8A presents the results for anti-CD3-antibodies A2J HLP with interspecies specificity expressed in the form of chimeric IgG molecules. Reduced activity of binding is observed for mutations with substitution at the alanine at position 4 (asparagine), position 23 (threonine) and in regulation 25 (isoleucine). Complete loss of binding was observed for mutations with replacement by alanine at position 1 (glutamine), in regulation 2 (aspartate), in regulation 3 (glycine) and in regulation 5 (glutamate).

Fig.8B:

Fig.8B presents the results for anti-CD3-antibodies e2m boasts HLP with interspecies specificity expressed in the form of chimeric IgG molecules. Reduced activity of binding is observed for mutations with substitution at the alanine at position 4 (asparagine), position 23 (threonine) and in regulation 25 (isoleucine). Complete loss of binding was observed for mutations with replacement by alanine at position 1 (glutamine), in regulation 2 (aspartate), in regulation 3 (glycine) and in regulation 5 (glutamate).

Fig.8C:

Fig.8C presents the results for anti-CD3-antibodies NS HLP with mivida�specificity Oh, expressed in the form of chimeric IgG molecules. Reduced activity of binding is observed for mutations with substitution at the alanine at position 4 (asparagine). Complete loss of binding was observed for mutations to alanine glutamine in position 1 (glutamine), in regulation 2 (aspartate), in regulation 3 (glycine) and in regulation 5 (glutamate).

Fig.8D:

Fig.8D presents the results for anti-CD3-antibodies F12Q HLP with interspecies specificity tested in the form periplasmatic of a murine single-chain antibodies. Complete loss of binding was observed for mutations with replacement by alanine at position 1 (glutamine), in regulation 2 (aspartate), in regulation 3 (glycine) and in regulation 5 (glutamate).

Fig.9

FACS-analysis, determining the binding of anti-CD3-binding molecules NS HLP exhibiting cross-species specificity, human CD3, containing and not containing N-terminal His6-tag.

Combined histogram performed on cell lines EL4, transfetsirovannyh CD3-Epsilon-chain wild-type (left histogram) or CD3-Epsilon-chain of human, containing N-terminal His6-tag (right histogram), were tested in FACS analysis, determining the binding of the binding molecules NS HLP with interspecies specificity. Samples were incubated with the corresponding control isotype as a negative control (thin line), the antibody UCHT-1 �contrast human CD3 as a positive control (dotted line) and anti-CD3-antibody NS HLP with interspecies specificity in the form of chimeric IgG molecules (thick line).

Combined histograms demonstrate comparable binding of the antibody UCHT-1 with both transfectants compared to isotype control, demonstrating the expression of both recombinant constructs. Combined histograms also show the binding of anti-CD3-binding molecules NS HLP only with CD3 Epsilon-chain wild-type person, but not with His6-CD3-Epsilon-chain of human. These results demonstrate that the presence of a free N-Terminus is essential for binding of anti-CD3-binding molecules NS HLP with interspecies specificity.

Fig.10

FACS analysis of binding of these bespecifically single-stranded structures with cross-species specificity to the cells SNO, transfected by MCSP D3 the person with CD3+T cell line HPB-ALL human cells SNO, transfected by MCSP D3 Javanese macaque, and T-cell line 4119LnPx macaque. FACS-staining carried out as described in Example 10. The thick line represents cells incubated with the purified protein at a concentration of 2 μg/ml, which are then incubated with anti-his-antibody and PE-labeled detection antibody. Thin line on the histograms reflects the negative control: cells incubated only with anti-his-antibody and a detection antibody.

Fig.11

FACS analysis of binding of these with interspecies specificity bespecifically one�chain-like structures with cells SNO, transfected by MCSP D3 the person with CD3+T cell line HPB-ALL human cells SNO, transfected by MCSP D3 Javanese macaque, and T-cell line 4119LnPx macaque. FACS-staining carried out as described in Example 10. The thick line represents cells incubated with the purified protein at a concentration of 2 μg/ml, which are then incubated with anti-his-antibody and PE-labeled detection antibody. Thin line on the histograms reflects the negative control: cells incubated only with anti-his-antibody and a detection antibody.

Fig.12

FACS analysis of binding of these bespecifically single-stranded structures with cross-species specificity to the cells SNO, transfected by MCSP D3 the person with CD3+T cell line HPB-ALL human cells SNO, transfected by MCSP D3 Javanese macaque, and T-cell line 4119LnPx macaque. FACS-staining carried out as described in Example 10. The thick line represents cells incubated with the purified protein at a concentration of 2 μg/ml, which are then incubated with anti-his-antibody and PE-labeled detection antibody. Thin line on the histograms reflects the negative control: cells incubated only with anti-his-antibody and a detection antibody.

Fig.13

The cytotoxic activity induced specified MCSP-specific single-stranded� designs with cross-species specificity redirected to these cell lines target. A) stimulated CD4-/CD56 - RVMS person used as effector cells, and the cells SNO, transfetsirovannyh MCSP D3 man, used as target cells. ): T-cell line 4119LnPx macaque used as effector cells, and the cells SNO, transfetsirovannyh MCSP D3 Javanese macaque, used as target cells. The analysis is performed as described in Example 11.

Fig.14

The cytotoxic activity induced specified MCSP-specific single-stranded structures with interspecies specificity redirected to the specified cell line-target. A) and b) T-cell line 4119LnPx macaque used as effector cells, and the cells SNO, transfetsirovannyh MCSP D3 Javanese macaque, used as target cells. The analysis is performed as described in Example 11.

Fig.15

The cytotoxic activity induced specified MCSP-specific single-stranded structures with interspecies specificity redirected to the specified cell line-target. A) and b): stimulated CD4-/CD56 - RVMS person used as effector cells, and the cells SNO, transfetsirovannyh MCSP D3 man, used as a T cell target. The analysis is performed as described in Example 11.

Fig.16

Cytotoxic�quarter activity, induced specified MCSP-specific single-stranded structures with interspecies specificity redirected to the specified cell line-target. A) stimulated CD4-/CD56 - RVMS person used as effector cells, and the cells SNO, transfetsirovannyh MCSP D3 man, used as target cells. ): T-cell line 4119LnPx macaque used as effector cells, and the cells SNO, transfetsirovannyh MCSP D3 Javanese macaque, used as target cells. The analysis is performed as described in Example 11.

Fig.17

The cytotoxic activity induced specified MCSP-specific single-stranded structures with interspecies specificity redirected to the specified cell line-target. A) stimulated CD4-/CD56 - RVMS person used as effector cells, and the cells SNO, transfetsirovannyh MCSP D3 man, used as target cells. ): T-cell line 4119LnPx macaque used as effector cells, and the cells SNO, transfetsirovannyh MCSP D3 Javanese macaque, used as target cells. The analysis is performed as described in Example 11.

Fig.18

Stability in plasma MCSP and CD3 bespecifically single-chain antibodies with cross-species specificity tested by measuring the cytotoxic asset�spine, induced samples of these single-stranded constructs, incubated with 50% human plasma at 37°C and 4°C for 24 hours, respectively, or with the addition of 50% human plasma immediately prior to cytotoxicity testing, or without the addition of plasma. Cells SNO, transfetsirovannyh human MCSP, used as target cells, and stimulated CD4/CD56-RVMS person used as effector cells. Analysis bipolar as described in Example 12.

Fig.19

The initial reduction and recovery (i.e. redistribution) the absolute number of T cells (empty squares) in the peripheral blood of patients with B-NHL (b-cell non-Hodgkin's lymphoma) (number of patients 1, 7, 23, 30, 31 and 33 in Table 4), essentially had no circulating S-positive target b cells (filled triangles), during the initial phase of intravenous infusion of CD3-binding molecules CD19×CD3, recognizing traditional dependent on the environment epitope of CD3. The absolute number of cells is given in 1000 cells per microliter of blood. The first point on the chart shows the original amount immediately prior to the infusion. Dose of CD19×CD3 are shown in parenthesis next to the patient number.

Fig.20

(A) the Re-redistribution of T cells (empty squares) in a patient with B-NHL No. 19 (Table� 4), had no circulating CD19-positive target b cells (filled triangles) and who developed CNS symptoms during continuous intravenous infusion of CD19×CD3 in a dose of 5 μg/m2/24 hours for one day, followed by a sharp increase dose to 15 g/m2/24 h. the Absolute number of cells is given in 1000 cells per microliter of blood. The first point on the chart shows the original amount immediately prior to the infusion. After recovery, the number of circulating T-cells as a result of the first episode of redistribution initiated by treatment with 5 μg/m2/24 h, step-by-step dose increase from 5 to 15 g/m2/24 h initiated the second episode of the redistribution of T cells associated with the development of CNS symptoms, among which was dominated by confusion and disorientation.

(B) the Re-redistribution of T-cells in patients with B-NHL who developed CNS symptoms after repeated intravenous bolus infusion of CD19×CD3 at 1.5 μg/m2. The absolute number of cells is given in 1000 cells per microliter of blood. Infusion duration for each bolus injection ranged from 2 to 4 hours. Vertical arrows indicate the beginning of bolus infusion. The point on the chart at the initial time of each bolus of the introduction shows the number of T cells n�mediocre before bolus infusion. Each bolus infusion initiated the episode redistribution of T cells, followed by recovery of the number of T-cells until the next bolus infusion. Finally, the third episode of redistribution of T-cells was associated with the development of CNS-related symptoms in this patient.

Fig.21

A comprehensive picture of the redistribution of T cells (empty squares) in a patient with B-NHL No. 20 (table 4) without circulating CD19-positive b-cell targets (filled triangles) for linearly increasing (ramp) initiation of infusion of CD19×CD3, i.e. a smooth gradual increase in flow rate from almost zero to 15 μg/m2/24 h during the first 24 hours of treatment. The absolute number of cells is given in 1000 cells per microliter of blood. The first point on the chart shows the original amount immediately prior to the infusion. Dose of CD19×CD3 are shown in parentheses next to the patient number. T-cells, re-emerging in the circulating blood after the initial redistribution initiated by the first impact of the CD19×CD3, again disappear from the circulating blood, which is partly still induced higher levels of CD19×CD3 during the ramp-up phase.

Fig.22

The number of T - and b-cells during treatment with the use of a CD19×CD3 patients with B-NHL No. 13 (table 4), who had a significant number of circulating CD19-positive�x In-target cells (lymphoma) (filled triangles). The absolute number of cells is given in 1000 cells per microliter of blood. The first point on the chart shows the original amount immediately prior to the infusion. Dose of CD19×CD3 are shown in parentheses next to the patient number. T cells (empty squares) disappear completely from the bloodstream after the start of infusion of CD19×CD3 and not to reappear until circulating CD19-positive b cells (lymphoma) (filled triangles) will not be depleted in the peripheral blood.

Fig.23

Re redistribution of T cells (empty squares) in a patient with B-NHL No. 24 (table 4), which essentially had no circulating CD19-positive b-cell targets (shaded triangles) and developed CNS symptoms as a result of the initiation of the infusion of CD19×CD3 without the addition of HSA (human serum albumin) required for stabilization of the drug (upper panel). After the first restoration of circulating T cells at the end of the initial redistribution, the uneven flow of drugs, due to the lack of stabilizing HSA, initiated the second episode of the redistribution of T cells associated with the development of CNS symptoms, dominated by confusion and disorientation. When the same patient again began to enter the solution CD19×CD3 containing additive HS for stabilization of the drug, the patient was not observed to any re-redistribution of T cells (lower panel), as well as the development of any CNS symptoms. The absolute number of cells is given in 1000 cells per microliter of blood. The first point on the chart shows the original amount immediately prior to the infusion. Dose of CD19×CD3 are shown in parentheses next to the patient number.

Fig.24

Model the adhesion of T cells to endothelial cells induced by monovalent binding is dependent on the environment of CD3 epitopes. Monovalent interaction of traditional CD3-binding molecule with its dependent environment epitope on CD3-Epsilon may cause allosteric changes in the conformation of CD3 with subsequent recruitment Nck2 to the cytoplasmic domain of the CD3-Epsilon (Gil et al. (2002) Cell 109: 901). Because Nck2 is directly linked to integrins via PINCH and ILK (Legate et al. (2006) Nat. Rev. Mol. Cell Biol. 7: 20), recruitment Nck2 to the cytoplasmic domain of the CD3-Epsilon after allosteric changes in the conformation of CD3 linking traditional CD3-binding molecules (such as CD19×CD3 from Example 13) with its dependent environment epitope on CD3-Epsilon, can increase the adhesion of T cells to endothelial cells by temporary transition of integrins on the surface of T-cells in their more adhesive isoforms through the signal transmission from the inside �arujo.

Fig.25

The cytotoxic activity of the tested substances, CD33-AF5 VH-VL × 12C VH-VL used in the in vivo study in cynomolgus rhesus described in Example 14. Specific lysis of CD33-positive target cells was determined in a standard analysis release51chromium with increasing concentrations of CD33-AF5 VH-VL x I2C VH-VL. The analysis time was 18 hours. T-cell line 4119LnPx macaque was used as source of effector cells. Cells SNO, transfetsirovannyh CD33 Javanese macaque, served as target cells. The ratio of effector cells and target cells (E:T ratio) was 10:1. The concentration of CD33-AF5 VH-VL x I2C VH-VL required for premaxillae lysis of target cells (EC50), was calculated from the curve dose-response, and it was 2.7 ng/ml.

Fig.26

(A) Dose - and time-dependent depletion of CD33-positive monocytes in peripheral blood of cynomolgus macaques as a result of continuous intravenous infusion of CD33-AF5 VH-VL x I2C VH-VL as described in Example 14. The percentage relative to baseline (i.e. 100%) the absolute number of circulating CD33-positive monocytes after treatment duration indicated above the columns shown for each of the two Javanese macaques for each dose level. The dose level (i.e., the flow rate of the infusion) is indicated under each column. No depletion �insulinaemic CD33-positive monocytes were observed in animals 1 and 2, treated for 7 days in a dose of 30 μg/m2/24 h. In animals 3 and 4, which were treated within 7 days, a dose of 60 μg/m2/24 h, the number of circulating CD33-positive monocytes was reduced to 68% and 40% of baseline, respectively. At 240 μg/m2/24 h circulating CD33-positive monocytes were almost completely depleted in the peripheral blood after 3 days of treatment (animals 5 and 6). At 1000 μg/m2/24 h depletion of circulating CD33-positive monocytes in peripheral blood was completed already after 1 day of treatment (animals 7 and 8).

(B) the Course of change in the number of T cells and CD33-monocytes in the peripheral blood of two cynomolgus macaques during continuous infusion of CD33-AF5 VH-VL x I2C VH-VL in a period of 14 days at 120 μg/m2/24 h. the Absolute number of cells is given in 1000 cells per microliter of blood.

The first point on the chart shows the original amount immediately prior to the infusion. After the initial mobilization of CD33-monocytes within the first 12 hours after start of infusion of CD33-monocytes in the peripheral blood (filled triangles) are depleted by two-thirds (animal 10) and 50% (animal 9) relative to the respective initial amounts during the subsequent infusion. The number of circulating T cells (empty squares) demonstrates a limited initial reduction, which only� recovery of the amount already in the presence of circulating CD33-positive monocyte-derived target cells.

Fig.27

The cytotoxic activity of the tested substances, MCSP-G4 VH-VL × I2C VH-VL used in the in vivo study in cynomolgus rhesus described in Example 15. Specific lysis of MCSP-positive target cells was determined in a standard analysis release51chromium with increasing concentrations of MCSP-G4 VH-VL × I2C VH-VL. The analysis time was 18 hours. T-cell line 4119LnPx macaque was used as source of effector cells. Cells SNO, transfetsirovannyh MCSP Javanese macaque, served as target cells. The ratio of effector cells and target cells (E:T ratio) was 10:1. The concentration of MCSP-G4 VH-VL × I2C VH-VL required for premaxillae lysis of target cells (EC50), was calculated from the curve dose-response, and it was 1.9 ng/ml.

Fig.28

The lack of initial episodes of reduction and subsequent recovery of absolute number of T cells (i.e., redistribution) in peripheral blood of cynomolgus macaques during the initial phase of intravenous infusion of CD3 binding molecule MCSP-G4 VH-VL × I2C VH-VL that recognizes essentially independent from the environment epitope of CD3. The absolute number of cells is given in 1000 cells per microliter of blood. The first point on the chart shows the original amount immediately prior to the infusion. Dose of MCSP-G4 VH-VL × I2C VH-VL are shown in round brackets�x next to the animal room. In the known absence of MCSP-positive target cells in the circulating blood of cynomolgus macaques is not observed induction of redistribution of T cells (i.e., initial episode of reduction and subsequent recovery of absolute T-cells) due to indirect target cells cross-linking of CD3. In addition, induction of redistribution of T cells (i.e., initial episode of reduction and subsequent recovery of absolute T-cells) caused by a signal that T cells can be obtained through exceptional communication only with CD3-binding site, can be avoided by using such a CD3-binding molecules, as MCSP-G4 VH-VL x 12C VH-VL that recognizes essentially independent of the environment in the CD3 epitope.

Fig.29

FACS analysis of binding of these with interspecies specificity bespecifically single-stranded structures with cells SNO, transfected by human CD33, CD3+T cell line HPB-ALL human cells SNO, transfected by a macaque CD33 and RVMS macaque, respectively. FACS-staining performed as described in Example 16.4. Thick lines indicate cells incubated with purified bespecifically single-stranded structure at a concentration of 5 μg/ml or with cell culture supernatant of transfected cells expressing constructionspending antibodies with cross-species specificity. Shaded histograms represent negative controls. As a negative control, the supernatant nitrostilbene cells SNO. For each bespecifically single-stranded design with interspecies specificity combined histogram shows the specific binding of this design with CD33 human and macaque and human CD3 and macaque.

Fig.30

The diagrams show the results of chromium release, provide measuring cytotoxic activity induced specified CD33-specific single-stranded structures with interspecies specificity redirected to the specified cell line-target. Also indicated effector cells used. Analyses are performed as described in Example 16.5. The diagrams clearly demonstrate for each design shown a strong increase in the cytotoxic activity of effector cells and against macaque cells SNO, transfected CD33 human and macaque, respectively.

Fig.31

Monitoring cleaning bespecifically single-stranded molecules with interspecies specificity designated E292F3 HL × I2C HL, using SDS-PAGE (polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) and Western blotting. Samples of the eluate, the cell culture supernatant (SN) and about�internal column fractions (FT) were analyzed, as indicated. Protein marker (M) was used as reference masses. Intense protein zone with a molecular weight of from 50 to 60 kDa in SDS-PAGE demonstrates effective cleaning bespecifically single-stranded molecules with cross-species specificity to a very high degree of purity using one-step purification method described in Example 17.2. Western blotting with the definition histidin-label confirms the identity of the protein zones in the eluate as bespecifically single-stranded molecules with cross-species specificity. A weak signal for the sample flow-through fraction in this sensitive detection method further points to the almost complete capture bespecifically single-stranded molecules as a result of the implementation of this method of cleaning.

Fig.32

Monitoring cleaning bespecifically single-stranded molecules with interspecies specificity designated V207C12 HL × H2C HL, using SDS-PAGE and Western blotting. Samples of the eluate, the cell culture supernatant (SN) and the flow-through fraction of the column (FT) were analyzed as indicated. Protein marker (M) was used as reference masses. Intense protein zone with a molecular weight of from 50 to 60 kDa in SDS-PAGE demonstrates effective cleaning bespecifically single-stranded molecules with cross-species specificity to a very high degree of pureness� using one-step purification method, described in Example 17.2. Western blotting with the definition of the histidine-tag confirms the identity of the protein zones in the eluate as bespecifically single-stranded molecules with cross-species specificity. A weak signal for the sample flow-through fraction in this sensitive detection method further points to the almost complete capture bespecifically single-stranded molecules as a result of the implementation of this method of cleaning.

Fig.33

Monitoring cleaning bespecifically single-stranded molecules with interspecies specificity designated AF5HL×F12QHL, using SDS-PAGE and Western blotting. Samples of the eluate, the cell culture supernatant (SN) and the flow-through fraction of the column (FT) were analyzed as indicated. Protein marker (M) was used as reference masses. Intense protein zone with a molecular weight of from 50 to 60 kDa in SDS-PAGE demonstrates effective cleaning bespecifically single-stranded molecules with cross-species specificity to a very high degree of purity using one-step purification method described in Example 17.2. Western blotting with the definition of the histidine-tag confirms the identity of the protein zones in the eluate as bespecifically single-stranded molecules with cross-species specificity. The signal in the sample flow-through fraction in this sensitive method for the detection of Ob�sdaetsa saturation affinity column due to the high concentration bespecifically single-stranded molecules in the supernatant.

Fig.34

Standard curve for AF5HL×I2CHL in 50% macaque serum. The upper diagram shows the standard curve obtained in the analysis described in Example 18.2.

The lower graph shows the results for control samples (QC samples) AF5HL×I2CHL in 50% macaque serum. The degree of detection is above 90% for the QC sample with high and medium concentration and above 80% for the QC sample concentrations are low.

Thus, this analysis allows us to determine AF5HL×I2CHL in samples with serum in the range from 10 ng/ml to 200 ng/ml (before dilution).

Fig.35

Standard curve for MCSP-G4 HL × I2C HL in 50% macaque serum. The upper diagram shows the standard curve obtained in the analysis described in Example 18.2.

The lower graph shows the results for the QC samples MCSP-G4 ML × 12C HL in 50% macaque serum. The degree of detection is above 98% for the QC sample with high and medium concentration and above 85% for the QC sample concentrations are low.

Thus, the analysis allows to determine the MCSP-G4 ML × I2C HL in samples with serum in the range from 10 ng/ml to 200 ng/ml (before dilution).

Fig.36

FACS analysis of the binding of anti-Flag antibodies with cells SNO, transfected by N-terminal amino acids 1-27 of CD3-Epsilon these species, merged with Arcam Javanese macaque. FACS-staining contains�and, as described in Example 19.1. Thick lines indicate cells incubated with anti-Flag-antibody. Shaded histograms represent negative controls. As a negative control used PBS with 2% FCS. Histograms show strong and comparable binding of anti-Flag antibodies with all transfectants, indicating a strong and equal expression of the transfected constructs.

Fig.37

FACS analysis of binding constructs I2C lgG1 with Cho cells expressing N-terminal amino acids 1-27 of CD3-Epsilon these species, merged with Arcam Javanese macaque. FACS staining was performed as described in Example 19.3. Thick lines indicate cells incubated with 50 µl of cell culture supernatant of cells expressing the design of I2C lgG1. Shaded histograms represent negative control. Cells expressing N-terminal amino acids 1-27 of CD3-Epsilon pigs, merged with Arcam Javanese macaque, was used as a negative control. In comparison with negative control histograms clearly demonstrate the binding design I2C lgG1 with the N-terminal amino acids 1-27 of CD3-Epsilon person, marmoset, Tamarin and squirrel monkey.

Fig.38

FACS analysis of binding constructs I2C lgG1, as described in Example 19.2, with human CD3 with and without N-terminal His6-tag, as described in Examples 6.1 and 5.1 �sootvetstvenno. Thick lines indicate cells incubated with the antibody UCHT-1 against human CD3, Penta-His-antibody (Qiagen) and the culture supernatant of cells expressing the design of the 12C lgG1, respectively, as indicated. Shaded histograms represent cells incubated with irrelevant mouse lgG1 antibody as a negative control.

The top two combined histograms show comparable binding of the antibody UCHT-1 with both transfectants compared to isotype control, demonstrating the expression of both recombinant constructs. Central combined histograms show the binding of Penta-His-antibody with cells expressing His6-CD3-Epsilon chain (His6-CD3), but not with cells expressing CD3-Epsilon-chain wild-type (WT-CD3). Lower combined histograms show the binding design I2C lgG1 with human CD3-Epsilon-chain of the wild type, but not His6-CD3-Epsilon-chain of human. These results demonstrate that the presence of a free N-Terminus is essential for binding of anti-CD3-binding molecules with I2C inter-species specificity to CD3-Epsilon chain.

Fig.39

FACS analysis of binding of these with interspecies specificity bespecifically single-stranded structures with cells SNO, transfected by MCSP D3 the person with CD3+T cell line HPB-ALL human, with CL�worry-beads SSS transfected by MCSP D3 macaque, and T-cell line 4119LnPx macaque, respectively. FACS staining was performed as described in Example 10. Thick lines indicate cells incubated with purified bespecifically single-stranded structure at a concentration of 2 μg/ml or cell supernatant containing this bespecifically single-stranded structure, respectively. Shaded histograms represent negative controls. Supernatant retransfusion of Cho cells was used as negative control for binding to T-cell lines. Single-stranded structure with irrelevant target specificity was used as negative control for binding to Cho cells transfected by MCSP D3. Combined histogram for each bespecifically single-stranded design with interspecies specificity demonstrates the specific binding of this design with MCSP D3 human and macaque and human CD3 and macaque.

Fig.40

The cytotoxic activity induced specified MCSP D3-specific single-stranded structures with interspecies specificity redirected to the specified cell line-target. Also used effector cells and the ratio of effector cells to target cells as indicated. The analysis was performed as described in Example 11. �of diagramme clearly demonstrate the strong recruitment of cytotoxic activity with interspecies specificity under the influence of each design.

Fig.41

FACS analysis of binding of these with interspecies specificity bespecifically single-stranded structures with cells SNO, transfected by human CD33, CD3+T cell line HPB-ALL human cells SNO, transfected by a macaque CD33 and RVMS macaque, respectively. FACS staining was performed as described in Example 21.2. Thick lines indicate cells incubated with cell culture supernatant of transfected cells expressing constructs bespecifically antibodies with cross-species specificity. Shaded histograms represent negative controls. As a negative control, the supernatant nitrostilbene cells SNO. Combined histogram for each bespecifically single-stranded design with interspecies specificity demonstrates the specific binding of this design with CD33 human and macaque and human CD3 and macaque.

Fig.42

Diagrams present the results of chromium release, provide measuring cytotoxic activity induced specified CD33-specific single-stranded structures with interspecies specificity redirected to the specified cell line-target. Also indicated effector cells used. Analyses were performed as described in Example 21.. The diagrams clearly demonstrate for each construct the strong recruitment of cytotoxic activity of effector cells and against macaque cells SNO, transfected CD33 human and macaque, respectively.

Fig.43

The redistribution of T cells in chimpanzees with weekly intravenous bolus infusion of PBS/5% HSA and PBS/5% HSA plus the design of the single-stranded Arcam/CD3 especificacao antibody at doses of 1.6 to 2.0, 3.0 and 4.5 µg/kg. infusion Duration for each bolus injection was 2 hours. Vertical arrows indicate the beginning of bolus infusion. The point on the chart at the beginning of each bolus of the introduction shows the number of T-cells immediately before bolus infusion. Each bolus infusion design single-stranded EpCAM/CD3 especificacao antibody, which recognizes traditional dependent on the environment CD3-epitope triggers an episode of redistribution of T-cells with subsequent restoration of T-cells to their initial values before the next bolus infusion.

Fig.44

CD3-specific ELISA analysis of periplasmic preparations containing Flag-tagged scFv protein fragments from selected clones. Periplasmic preparations of soluble scFv protein fragments were added to plate wells for ELISA, which were covered by a soluble fused protein of human CD3-EP�Ilon (AA 1-27)-Fc (aa means amino acid) were additionally blocked with PBS 3% BSA. The determination was performed using monoclonal anti-Flag-Biotin-labeled antibody is then conjugated with streptavidin peroxidase. ELISA showed a solution of ABTS substrate (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate). The values of OD (optical density) (y axis) was measured at 405 nm using ELISA reader. The names of the clones represented on the x-axis.

Fig.45

ELISA analysis of periplasmic preparations containing Flag-tagged scFv protein fragments from selected clones. The same periplasmic preparations of soluble scFv protein fragments, and Fig.44 were added to the plate wells for ELISA, which were not covered with a soluble fused protein of human CD3-Epsilon (AA 1-27)-Fc, but were covered with human lgG1 (Sigma) and blocked with 3% BSA in PBS.

The determination was performed using monoclonal anti-Flag-Biotin-labeled antibody is then conjugated with streptavidin peroxidase. ELISA showed a solution of ABTS substrate. The OD values (y-axis) was measured at 405 nm using ELISA reader. The names of the clones represented on the x-axis.

Fig.46

Fig.46A-G: FACS analysis of binding of these with interspecies specificity bespecifically single-stranded structures with cells SNO, transfected by human PSCA, with CD3+T cell line HPB-ALL human cells SNO, transfected by a macaque PSCA, and T-cell line 4119LnPx macaque resp�respectively. FACS staining was performed as described in Example 24.5. Thick lines indicate cells incubated with cell culture supernatant of transfected cells expressing bespecifically single-stranded structure. Shaded histograms represent negative controls. Supernatant nitrostilbene cells was used as negative control. Combined histogram for each bespecifically single-stranded design with interspecies specificity demonstrates the specific binding of this design with PSCA human and macaque and human CD3 and macaque.

Fig.47

Fig.47A-C: the diagrams show the results of chromium release, provide measuring cytotoxic activity induced by the indicated single-stranded structures with interspecies specificity redirected to the specified cell line-target. Also indicated effector cells used. Analyses were performed as described in Example 24.6. The diagrams clearly demonstrate for each construct the strong recruitment of cytotoxic activity of effector cells of human and macaque against cells that are positive for the PSCA of human and macaque, respectively.

Fig.48

FACS analysis of binding of these bespecifically single-stranded structures with inter-service Spa�ifinally with cells SNO, PSCA transfected by the person with CD3+T cell line HPB-ALL human cells SNO, transfected by a macaque PSCA, and T-cell line 4119LnPx macaque, respectively. FACS staining was performed as described in Example 45.5. Thick lines indicate cells incubated with cell culture supernatant of transfected cells expressing constructs bespecifically single-chain antibodies. Shaded histograms represent negative controls. Supernatant nitrostilbene cells was used as negative control. Combined histograms for each bespecifically single-stranded design with interspecies specificity demonstrate the specific binding of this design with PSCA human and macaque and human CD3 and macaque.

Fig.49

The diagrams show the results of chromium release, provide measuring cytotoxic activity induced by the specified specific single-stranded structures with interspecies specificity redirected to the specified cell line-target. Also indicated effector cells used. Analyses were performed as described in Example 24.6. The diagrams clearly demonstrate for each construct the strong recruitment of cytotoxic activity of effector T-cells of human and macaque about�Yves target cells, positive against PSCA human and macaque, respectively.

Fig.50

FACS analysis of binding of these bespecifically single-stranded structures with cross-species specificity to the cells SNO, transfected by human CD19, CD3-positive T-cell line HPB-ALL human and with CD3-positive T-cell line 4119LnPx macaque. FACS staining was performed as described in Example 25.4. Thick lines on the histograms indicate cells incubated with cell culture supernatant, and then incubated with mouse anti-His-antibody, and then with PE-labeled anti-mouse Ig detection antibody. The histogram depicted by thin lines represent negative control, i.e., cells incubated only with anti-His-antibody and anti-mouse Ig detection antibody.

Fig.51

Cytotoxic T-cell activity, specified redirected bespecifically single-stranded structures with interspecies specificity against the indicated cell lines target. A) Stimulated CD4/CD56-depleted RVMS person used as effector T cells, and the cells SNO, transfetsirovannyh human CD19, used as target cells. In) T-cell line 4119LnPx macaque used as source of effector cells, and the cells SNO, transfetsirovannyh human CD19, use�Vanir as target cells. The analysis is performed as described in Example 25.5.

Fig.52

FACS analysis of binding of these bespecifically single-stranded structures with cross-species specificity to C-MET-positive cancer cell line human breast MDA-MB-231, with CD3+T cell line HPB-ALL human and CD3+T-cell line 4119LnPx macaque, respectively. FACS staining was performed as described in Example 26.5. Thick lines indicate cells incubated with cell culture supernatant of transfected cells expressing constructs bespecifically single-chain antibodies. Shaded histograms represent negative controls. Supernatant nitrostilbene cells was used as negative control. Combined histograms for each bespecifically single-stranded design with interspecies specificity demonstrates the specific binding of this design with C-MET and CD3 human and macaque.

Fig.53

The diagrams show the results of chromium release, provide measuring cytotoxic activity induced specified bespecifically single-stranded structures with interspecies specificity redirected to the specified cell line-target. Also indicated effector cells used. Analyses were performed as described in Example 6.6. The diagrams clearly demonstrate for each construct the strong recruitment of cytotoxic activity of effector cells against cells positive for the C-MET.

Fig.54

FACS analysis of binding of these bespecifically single-stranded structures with cross-species specificity to the cells SNO expressing estimates of a person as described in Example 27.1, with CD3+T cell line HPB-ALL human cells SNO expressing estimates macaque as described in Example 27.1, and T-cell line 4119LnPx macaque, respectively. FACS staining was performed as described in Example 27.2. Thick lines indicate cells incubated with cell culture supernatant of transfected cells expressing constructs bespecifically single-chain antibodies. Shaded histograms represent negative controls. Supernatant nitrostilbene cells was used as negative control. Combined histograms for each bespecifically single-stranded design with interspecies specificity demonstrates the specific binding of this design with estimates of human and macaque and human CD3 and macaque.

Fig.55

The diagrams show the results of chromium release, provide measuring cytotoxic activity induced specified Smet-SP�CityCasino single-stranded structures with cross-species specificity redirected to a specified cell line-target is created as described in Example 27.1. Also indicated effector cells used. Analyses were performed as described in Example 27.4. The diagrams clearly demonstrate for each construct the strong recruitment of cytotoxic activity of effector T cells macaque against target cells that are positive for the estimates macaque.

Fig.56

FACS analysis of binding of these scFv antibodies with cross-species specificity to the cells SNO expressing estimates of a person as described in Example 27.1, and with Cho cells expressing estimates macaque as described in Example 27.1, respectively. FACS staining was performed as described in Example 27.3. Thick lines indicate cells incubated with periplasmic preparations containing scFv antibodies. Shaded histograms represent negative controls. The buffer used for periplasmic preparations were used as negative control. Combined histograms for each scFv antibodies with cross-species specificity demonstrates the specific binding of this design with estimates of human and macaque.

Fig.57

FACS analysis of binding of scFv-fragment antibodies with cross-species specificity to the cells SNO, transfected by indocyanine human and Cho cells transfected by the end�selinam macaque. FACS staining was performed as described in Example 28.3. Thick lines indicate cells incubated with periplasmic preparations containing scFv-antibody fragment. Thin lines indicate negative controls. Nitrostilbene of Cho cells was used as negative control. Combined histograms demonstrate specific binding of scFv-antibody fragment with indocyanine human and macaque.

Fig.58

FACS analysis of binding of these bespecifically single-stranded structures with cross-species specificity to the cells SNO, transfected by human CD248, with CD3+T cell line HPB-ALL human cells SNO, transfected by CD248 macaque, and T-cell line 4119LnPx macaque, respectively. FACS staining was performed as described in Example 29.1. Thick lines indicate cells incubated with cell culture supernatant of transfected cells expressing constructs bespecifically single-chain antibodies. Thin lines indicate negative controls. Supernatant retransfusion of Cho cells was used as negative control. Combined histograms for each bespecifically single-stranded design with interspecies specificity demonstrates the specific binding of this design with CD248 human and macaque and human CD3 and mA�aka.

Fig.59

The diagrams show the results of chromium release, provide measuring cytotoxic activity induced specified CD248-specific single-stranded structures with interspecies specificity redirected to the specified cell line-target. Also indicated effector cells used. Analyses were performed as described in Example 29.1. The diagrams clearly demonstrate for each construct the strong recruitment of cytotoxic activity of effector T-cells of human and macaque against target cells, positive in respect of CD248 human and macaque, respectively.

Fig.60

FACS analysis of binding of these bespecifically single-stranded structures with cross-species specificity to the cells SNO, transfected by Ersam the person with CD3+T cell line HPB-ALL human T cell line 4119LnPx macaque. FACS staining was performed as described in Example 30.4. Thick lines indicate cells incubated with cell culture supernatant, and then incubated with anti-his-antibody and antibody for RE-detection. The histogram depicted by thin lines, demonstrates the negative control: cells incubated only with anti-his-antibody and the antibody for detection.

Fig.61

The cytotoxic activity induced by the specified� bespecifically single-stranded structures with cross-species specificity redirected to cell line-target. A) Stimulated CD4-/CD56 - RVMS person used as effector cells, and the cells SNO, transfetsirovannyh Arcam person, used as target cells. In) T-cell line 4119LnPx macaque used as effector cells, and the cells SNO, transfetsirovannyh Arcam person, used as target cells. The analysis was performed as described in Example 30.5.

Fig.62

The cytotoxic activity induced specified bespecifically single-stranded structures with interspecies specificity directed to cell lines target. A) Stimulated CD4-/CD56 - RVMS person used as effector cells, and the cells SNO, transfetsirovannyh Arcam person, used as target cells. In) T-cell line 4119LnPx macaque used as effector cells, and the cells SNO, transfetsirovannyh Arcam person, used as target cells. The analysis was performed as described in Example 30.5.

Fig.63

The cytotoxic activity induced specified bespecifically single-stranded structures with interspecies specificity directed to cell lines target. A) and b) Stimulated CD4-/CD56 - RVMS person used as effector cells, and the cells SNO, transpire�created Arcam person used as target cells. The analysis was performed as described in Example 30.5.

Fig.64 and 65

The cytotoxic activity induced specified bespecifically single-stranded structures with interspecies specificity directed to cell lines target. A) and b) Stimulated CD4-/CD56 - RVMS person used as effector cells, and the cells SNO, transfetsirovannyh Arcam person, used as target cells. The analysis was performed as described in Example 30.5.

Fig.66

The cytotoxic activity induced specified bespecifically single-stranded structures with interspecies specificity directed to cell lines target. A) Stimulated CD4-/CD56 - RVMS person used as effector cells, and the cells SNO, transfetsirovannyh Arcam person, used as target cells. In) T-cell line 4119LnPx macaque used as effector cells, and the cells SNO, transfetsirovannyh Arcam person, used as target cells. The analysis was performed as described in Example 30.5.

Fig.67

FACS analysis of binding of these bespecifically single-stranded structures with cross-species specificity to the cells SNO, transfected by Arcam human, mouse, or hybrid human-mouse. FACS staining was performed as described in Example 30.7. The bars represent the median fluorescence intensity of labeled structures to specified antigens Ersam.

Fig.68

In FACS-analysis of these constructs showed binding to CD3 and FAP-alpha in comparison with negative control. Demonstrated interspecies specificity bespecifically antibodies to antigens CD3 and FAP-alpha human and macaque, respectively. The analysis was performed as described in Example 31.

Fig.69

All created designs bespecifically single-chain antibodies with cross-species specificity demonstrated cytotoxic activity against FAP-alpha-positive target cells human induced stimulated CD4/CD56-depleted RVMS person, and against FAP-alpha-positive target cells macaque induced T-cell line 4119LnPx macaque. The analysis was performed as described in Example 31.

Fig.70

FACS analysis of binding of these bespecifically single-stranded structures with cross-species specificity to the cells SNO, transfected by FAP-alpha man, with CD3+T cell line HPB-ALL human cells SNO, transfected by FAP-alpha macaque, and T-cell line 4119LnPx macaque, respectively. FACS staining was performed as described in Example 32.1. Thick lines indicate cells incubated with cell culture supernatant of transfi�yovanny cells expressing design bespecifically single-chain antibodies with cross-species specificity. Thin lines indicate negative controls. Supernatant nitrostilbene cells was used as negative control. Combined histograms for each bespecifically single-stranded design with interspecies specificity demonstrates the specific binding of this design with FAP-alpha human and macaque and human CD3 and macaque.

Fig.71

The diagrams show the results of chromium release, which measure the cytotoxic activity induced by the specified FAP-alpha-specific single-stranded structures with interspecies specificity redirected to the specified cell line-target. Also indicated effector cells used. Analyses are performed as described in Example 32.1. The diagrams clearly demonstrate for each construct the strong recruitment of cytotoxic activity of effector T-cells of human and macaque against target cells that are positive for the FAP-alpha human and macaque, respectively.

Fig.72

FACS analysis of binding of these specific scFv antibodies with cross-species specificity to the cells SNO, transfected by FAP-alpha man, and with Cho cells transfected by FAP-alpha macaque, respectively. FACS-�krasivaya performed, as described in Example 32.2. Thick lines indicate cells incubated with periplasmic preparations containing scFv antibodies with cross-species specificity. Shaded histograms represent negative controls. The buffer used for periplasmic preparations were used as negative control. Combined histograms for each specific scFv antibodies with cross-species specificity to demonstrate the specific binding of this design with FAP-alpha human and macaque.

Fig.73

FACS analysis of binding of these bespecifically single-stranded structures with cross-species specificity to the cells SNO, transfected by IGF-1R person with CD3+T cell line HPB-ALL human cells SNO, transfected by IGF-1R macaque, and T-cell line 4119LnPx macaque, respectively. FACS staining was performed as described in Example 33.3. Thick lines indicate cells incubated with cell culture supernatant of transfected cells expressing constructs bespecifically single-chain antibodies with cross-species specificity. Shaded histograms represent negative controls. Cell culture medium was used as negative control. Combined histograms for each bespecifically single-stranded design serviceway specificity demonstrates the specific binding of this design with IGF-1R of human and macaque and human CD3 and macaque.

Fig.74

The diagrams show the results of chromium release, provide measuring cytotoxic activity induced specified bespecifically single-stranded structures with interspecies specificity redirected to the specified cell line-target. Which effector cells. Analyses were performed as described in Example 33.3. The diagrams clearly demonstrate for each construct the strong recruitment of cytotoxic activity of effector T-cells of human and macaque against target cells that are positive for the IGF-1R of human and macaque, respectively.

The present invention is further illustrated below by non-limiting examples, which give a better understanding of the present invention and its advantages.

EXAMPLES

1. Identification of sequences of CD3-Epsilon from blood samples of primates, non-human

For identification of CD3-Epsilon used blood samples following primates, non-human: Callithrix jacchus, Saguinus oedipus and Saimiris ciureus. For isolation of total cellular RNA prepared samples of fresh, treated with heparin whole blood in accordance with the manufacturer's Protocol (QIAamp RNA Blood Mini Kit, Qiagen). Performed transcription of extracted mRNA into cDNA in accordance with the published� protocols. Briefly, 10 µl of precipitated RNA were incubated with 1.2 μl of 10-fold hexanucleotide mixture (Roche) at 70°C for 10 minutes and kept on ice. Added to the reaction mixture containing 4 μl of 5 × Superscript buffer II, and 0.2 µl of 0.1 M dithiothreitol, 0,8 µl Superscript II (Invitrogen) and 1.2 µl of deoxyribonucleosides (25 μm) and 0.8 μl of RNase inhibitor (Roche) and 1.8 μl of water, not containing Tnkase and RNase (Roth). The reaction mixture was incubated at room temperature for 10 minutes followed by incubation at 42°C for 50 minutes and at 90°C for 5 minutes. The reaction mixture was cooled on ice, then added to 0.8 ál RNase H (1 unit/μl, Roche) and incubated for 20 minutes at 37°C.

cDNA first chain from each sample was subjected to 35 separate cycles of a polymerase chain reaction using DNA polymerase Taq (Sigma) and the following combination of primers designed on the basis of study data base: direct primer 5'-AGAGTTCTGGGCCTCTGC-3' (SEQ ID NO: 253); reverse primer 5'-CGGATGGGCTCATAGTCTG-3' (SEQ ID NO: 254). Amplified zone 550 base pairs (BP) was isolated from the gel (Gel Extraction Kit, Qiagen) and sequenced (Sequiserve, Vaterstetten/Germany, see the sequence listing).

CD3-Epsilon Callithrix jacchus

Nucleotides

Amino acids (SEQ ID NO: 3)

CD3-Epsilon Saguinus oedipus

Nucleotides

Amino acids (SEQ ID NO: 5)

CD3-Epsilon Saimiris ciureus

Nucleotides

Amino acids (SEQ ID NO: 7)

2. Creating fragments (scFv) single-chain antibodies with cross-species specificity, binding to N-terminal amino acids 1-27 of CD3-Epsilon person and different non-human primates non-chimpanzee

2.1. Immunization of mice with the use of the N-Terminus of CD3-Epsilon, has been isolated from its native CD3 environment through fusion with the heterologous soluble protein

Mice from F1 crosses of balb/c x C57black at the age of ten weeks were immunized fused protein CD3-Epsilon-Fc bearing a large part of the N-terminal amino acids 1-27 of the Mature CD3-Epsilon chain (1-27 CD3-Fc) of human and/or squirrel monkey. For this purpose, 40 mg fused protein 1-27 CD3-Fc together with 10 nmol of tioat-modified CpG-oligonucleotide (5'-tccatgacgttcctgatgct-3') (SEQ ID NO: 343) in 300 µl PBS per mouse were injected intraperitoneally. Mice received a booster immunization after 21, 42 and 63 may day in the same way. Ten days after the first booster immunization took blood samples, and the titer of antibodies in serum against the fused protein 1-27 CD3-Fc was tested by ELISA method. Additionally tested titer against CD3-positive T-cell lines HPBall method man flow� cytometry according to standard protocols. Serum titers from immunized animals were significantly higher than in unimmunized animals.

2.2. Creating immune scFv library mouse antibodies: construction of a combinatorial library of antibodies and phage display

Three days after the last injection, spleen cells of mice were collected to obtain total RNA according to standard protocols.

By RT-PCR (polymerase chain reaction with reverse transcription) RNA in the spleen of mice using VK and VH-specific primers designed a library of DNA fragments of variable regions of light chain (Kappa) (VK) and variable region of the heavy chain (VH) immunoglobulin (Ig) mice. cDNA was synthesized according to standard protocols.

Primers were designed in such a way as to create a site of recognition for the 5'Xhol and 3'BstEII for amplified V-fragments of the heavy chain and the site of recognition for 5'-Sacl and a 3'-Spel for amplified VK-DNA fragments.

For the PCR amplification of DNA fragments of VH each of the eight different primers specific to the 5'-VH-family (MVH1(GC)AG GTG CAG CTC GAG GAG TCA GGA CCT (SEQ ID NO: 344); MVH2 GAG GTC CAG CTC GAG CAG TCT GGA CCT (SEQ ID NO: 345); MVH3 CAG GTC CAA CTC GAG CAG CCT GGG GCT (SEQ ID NO: 346); MVH4 GAG GTT CAG CTC GAG CAG TCT GGG GCA (SEQ ID NO: 347); MVH5 GA(AG) GTG AAG CTC GAG GAG TCT GGA GGA (SEQ ID NO: 348); MVH6 GAG GTG AAG CTT CTC GAG TCT GGA GGT (SEQ ID NO: 349); MVH7 GAA GTG AAG CTC GAG GAG TCT GGG GGA (SEQ ID NO: 350); MVH8 GAG GTT CAG CTC GAG CAG TCT GGA GCT (SEQ ID NO: 351)),combined with one 3'-VH primer (3'MuVHBstEII tga gga gac ggt gac cgt ggt ccc ttg gcc cca g (SEQ ID NO: 352)); for the PCR amplification of fragments VK-circuit each of seven different primers specific to 5'-VK-family (MUVK1 CCA GTT CCG AGC TCG TTG TGA CTC AGG AAT CT (SEQ ID NO: 353); MUVK2 CCA GTT CCG AGC TCG TGT TGA CGC AGC CGC CC (SEQ ID NO: 354); MUVK3 CCA GTT CCG AGC TCG TGC TCA CCC AGT CTC CA (SEQ ID NO: 355); MUVK4 CCA GTT CCG AGC TCC AGA CCC TGA AGT CTC CA (SEQ ID NO: 356); MUVK5 CCA GAT GTG AGC TCG TGA TGA CCC AGA CTC CA (SEQ ID NO: 357); MUVK6 CCA GAT GTG AGC TCG TCA CCC TGA AGT CTC CA (SEQ ID NO: 358); MUVK7 CCA GTT CCG AGC TCG TGA TGA CAC AGT CTC CA (SEQ ID NO: 359)), combined with one 3'-VK-primer (3'MuVkHindIII/BsiW1 tgg tgc act agt cgt acg ttt gat etc aag ctt ggt ccc (SEQ ID NO: 360)).

For amplification using the following PCR program: denaturation at 94°C for 20 s; primer annealing at 52°C for 50 s and elongation of the primers at 72°C for 60 s; and 40 cycles, then a final elongation for 10 min at 72°C.

450 ng of the fragments of the light chain Kappa (split Sacl-Spel) was ligated using 1400 ng family pComb3H5Bhis (split Sacl-Spel; large fragment). Obtained a combinatorial library of antibodies is then subjected to a transformation in 300 µl electrocompetent cells of Escherichia coli XL1 Blue by electroporation (2.5 kV; cuvette with a gap of 0.2 cm; 25 UF; 200 Ohm, Biorad gene-pulser) obtaining as a result the library is larger than 107independent clones. After one hour the phenotypic expression of selected positive transformants by resistance to carbenicillin encoded vector pComb3H5BHis, 100 ml of liquid superbullen (SB) culture in tech�of the night. Then the cells were collected by centrifugation and plasmids were obtained using a commercially available kit to obtain plasmids (Qiagen).

2800 ng of this plasmid-DNA containing the VK-library (split Xhol-BstEII; large fragment), was ligated with 900 ng V-fragments of the heavy chain (split Xhol-BstEII) and again subjected to transformation into two aliquots of 300 µl electrocompetent cells E. coli XL1 Blue by electroporation (2.5 kV; cuvette with a gap of 0.2 cm; 25 μf, 200 Ω), to obtain libraries of VH-VK scFv (single chain variable fragment) General larger than 107independent clones.

After phenotype expression and slow adaptation to carbenicillin E. coli cells containing a library of antibodies, transferred into SB-carbenicillin(50 µg/ml) selective medium. Cells of E. coli containing a library of antibodies, and then was infected with an infectious dose of 1012particles of helper phage VCSM13 resulting in the production and secretion of filamentous M13 phage, where phage particle contains single stranded pComb3H5BHis-DNA encoding a murine scFv-fragment and displayed the corresponding scFv-protein in the form of a translational fusion with the shell III protein of phage. This pool of phages exhibiting a library of antibodies, are then used for selection of antigen-binding structures.

2.3. Selection of CD3-specific binding fragments on the basis of fagbag� display

Phage library carrying the cloned scFv-repertoire was harvested from the respective culture supernatant by precipitation with PEG8000/NaCl and centrifugation. Approximately 1011up to 1012scFv-phage particles were resuspended in 0.4 ml PBS/0,1% BSA and incubated with 105-107Jurkat cells (CD3-positive lineage T-cells) for 1 hour on ice under slow agitation. These Jurkat cells were grown in advance in RPMI medium enriched with fetal calf serum (FCS) (10%), glutamine and penicillin/streptomycin, collected by centrifugation, washed in PBS and were resuspended in PBS/1% FCS (containing sodium azide). scFv-bearing phages that did not bind specifically with Jurkat cells, were removed by carrying out up to five stages of washing with PBS/1% FCS (containing sodium azide). After washing the bound structure was suirable with cells by resuspension of cells in HCl-glycine buffer, pH 2.2, which is a 10 minute incubation followed by intensive mixing), and after neutralization with 2 M Tris solution, pH 12, the eluate was used for infection of a fresh uninfected culture of E. coli XL1 Blue (OD600 greater than 0.5). The E. coli culture containing E. coli cells subjected to successful transduction fahmideh copy, encoding a human scFv-fragment, were again subjected to selection for resistance to carbenicillin and �ATEM was infected with a helper phage VCMS 13, to begin the second round of exposure of antibodies and in vitro selection. Usually carried out a total of from 4 to 5 cycles of selection.

2.4. Screening CD3-specific binding fragments

Plasmid DNA corresponding to 4-5 rounds of panning, was isolated from cultures of E. coli, after selection. To obtain soluble scFv-protein, fragments of VH-VL-DNA is excised from the plasmid (Xhol-Spel). These fragments were cloned via the same restriction sites in the plasmid pComb3H5BFIag/His differing from the original pComb3H5BHis the fact that gene-expression design (e.g. scFv) includes a Flag-tag (TGD YKDDDDK) between the scFv and the His6-tag and the additional phage proteins removed. After ligation each pool (different rounds of panning) plasmid DNA was subjected to transformation into 100 μl competent to thermal shock E. coli TG1 or XLI blue and were plated on carbenicillin-LB-arap.Single colonies were selected and transferred in 100 µl LB-carb (50 µg/ml).

E. coli, transformed pComb3H5BHis containing a VL - and VH-segment, produce soluble scFv in significant amounts after the cut fragment of the gene III and induction using 1 mm IPTG (isopropylcyclopentadienyl). Thanks to a suitable signal sequence of scFv-chain is exported to the periplasm, where it folds into a functional conformation.

A single bacterial colony of E. coli TG1 were selected with cups for transformation for �of Holocene small amounts of periplasmic and grown in SB-medium (e.g. 10 ml), supplemented with 20 mm MgCl2and carbenicillin (50 μg/ml), and were resuspended in PBS (e.g. 1 ml) after harvesting. As a result of four cycles of freezing at -70°C and thawing at 37°With the outer membrane of the bacteria was destroyed by temperature shock and the soluble periplasmic proteins including the scFv, is released in the supernatant. After removal by centrifugation of intact cells and cell debris were collected supernatant containing scFv against human CD3, and used for further studies.

2.5. Identification of CD3-specific binding fragments

Linking the selected scFv was tested by flow cytometry on eukaryotic cells expressing on their surface heterologous protein, exhibiting at its N-end first 27 N-terminal amino acids of CD3-Epsilon.

As described in Example 4, the first amino acids 1-27 of the N-terminal sequence of the Mature CD3-Epsilon-chain T-cell receptor complex (amino acid sequence:

QDGNEEMGGITQTPYKVSISGTTVILT SEQ ID NO: 2) was subjected to fusion with the N-end transmembrane protein Arcam so that the N-end was located on the outer surface of the cell. In addition, between the N-terminal 1-27 sequence of CD3-Epsilon and consistency Arcam embed FLAG-epitope. This product of the merger of p�was saravali in the cells of the human embryonic kidney (HEK) cells and Chinese hamster ovary (Cho) cells.

Eukaryotic cell, exposing a large portion 27 N-terminal amino acids of the Mature CD3-Epsilon other Primate species were obtained in the same manner for Sainniri sciureus (N-terminal amino acid sequence of the CD3-Epsilon: QDGNEEIGDTTQNPYKVSISGTTVTLT SEQ ID NO: 8) for Callithrix jacchus (CD3-Epsilon N-terminal amino acid sequence: QDGNEEMGDTTQNPYKVSISGTTVTLT SEQ ID NO: 4) for Saguinus oedipus (CD3-Epsilon N-terminal amino acid sequence: QDGNEEMGDTTQNPYKVSISGTTVTLT SEQ ID NO: 6).

For analysis by flow cytometry of 2.5×105cells were incubated with 50 µl of the supernatant or with 5 μg/ml of the purified constructs in 50 μl PBS with 2% FCS. Binding of the constructs was determined using an anti-his-antibody (Penta-His-antibody not containing BSA, Qiagen GmbH, Hilden, FRG) at a concentration of 2 mg/ml in 50 μl PBS with 2% FCS. As the reagent for the second stage used conjugated with R-phycoerythrin, affinity purified F(ab')2-a fragment of the antibody goat against mouse IgG (specific to Fc-gamma fragment), diluted in the ratio 1:100 in 50 μl PBS with 2% FCS (Dianova, Hamburg, FRG). Samples were measured on a FACSscan (BD biosciences, Heidelberg, FRG).

Linking consistently confirmed by flow cytometry, as described in the previous paragraph, in primary T-cells of humans and different primates (e.g., Saimiri sciureus, Callithnx jacchus, Saguinus oedipus).

2.6. The creation of human/humanized equivalents of CD3-Epsilon-scFv not human

VH-region of murine anti-CD3 scFv was aligned relative to the amino acid sequences of antibody germline of the person. Choosing VH sequence of the antibody germline of the person who had the highest homology with the VH is not the person, and perform direct data alignment of two amino acid sequences. There were a number of frame balances for VH is not the person that are different from the frame of the VH regions ("different positions in the frame"). Some of these residues may participate in the binding of an antibody to its target and the activity of the antibody against its target.

To construct a library that contains the murine CDR and at each position of the frame differs from both options selected human VH sequences ("human" and maternal "murine" amino acid residue), synthesized degenerate oligonucleotides. In the composition of these oligonucleotides is in different positions human residue with a probability of 75% and murine residue with a probability of 25%. For a single human VH, for example, it was necessary to synthesize the six oligonucleotides that overlap in a terminal stretch of approximately 20 nucleotides. To do this, each second primer was an antisense primer. The restriction sites necessary for subsequent clone�of duplication within oligonucleotides, removed.

These primers may have a length of from 60 to 90 nucleotides, depending on the number of primers necessary to overlap across the V-sequence.

These primers, for example, six primers were mixed in equal amounts (e.g. 1 µl of each primer (stock solution of primers: 20-100 µm) with 20 µl PCR reaction mixture) was added to PCR mix consisting of PCR buffer, nucleotides and Taq polymerase. This mixture is incubated at 94°C for 3 minutes, at 65°C for 1 minute, 62°C for 1 min, 59°C for 1 min, at 56°C for 1 min, at 52°C for 1 minute, at 50°C for 1 minute and at 72°C for 10 minutes in a PCR cycler. Thereafter, the product was subjected to electrophoresis in agarose gel and the product size of from 200 to 400 was isolated from the gel according to standard methods.

This PCR product is then used as template for a standard PCR reaction using primers that introduce suitable for cloning the N-terminal and C-terminal restriction sites. The DNA fragment of the correct size (for VH approximately 350 nucleotides) was isolated by electrophoresis in agarose gel according to standard methods. In this way was sufficient amplified DNA fragment VH. This VH-fragment now was a pool of VH-fragments, each of which had different Koli�the degree of "human" and "mouse" relevant residues in different positions in the frame (pool humanized VH). A similar procedure was performed for VL-region of murine anti-CD3 scFv (pool of humanized VL).

The pool of humanized VH was then combined with the pool of humanized VL in the vector for phage display pComb3H5Bhis for the formation of a library of functional scFv from which after the display using filamentous phage were selected by anti-CD3-binding fragments, produced screened, identified and confirmed as described above for the parent not human (murine) anti-CD3 scFv. Single clones were then analyzed on the availability of suitable properties and amino acid sequence. The scFv amino acid sequence which has the highest homology to the V-segments of the germline of the person, are preferred, in particular those in which at least one among CDR CDR I and II of the VH and CDRs I and II VL-Kappa or CDR I and II VL-lambda exhibit more than 80% amino acid sequence identity with the closest corresponding CDR of all V-segments of the germline of the person. Anti-CD3 scFv was converted into recombinant bespecifically single-chain antibodies as described in the examples below, 9, 16, and 24.

3. The creation of a recombinant fused protein consisting of the N-terminal amino acids 1-27 of CD3-Epsilon-chain of human fused with the Fc region of lgG1 (1-27 CD3-Fc)

3.1. Cloning and expression -27 CD3-Fc

Encoding a sequence of N-terminal amino acids 1-27 of CD3-Epsilon-chain of human, fused to the hinge region and Fc-gamma-plot of human immunoglobulin lgG1, and consisting of 6 histidinemia label residues, was obtained by gene synthesis according to standard protocols (cDNA sequence and amino acid sequence of the recombinant fused protein represented in SEQ ID NO: 230 and 229). The fragment of gene synthesis was designed so that it contained the first website Kozak for eukaryotic expression of this design, then immunoglobulin leader peptide of 19 amino acids, then, in reading frame, encoding the sequence of the first 27 amino acids of the extracellular Mature plot of CD3-Epsilon-chain of human rights, then, in reading frame, encoding the sequence of the hinge region and Fc-gamma-phase of human lgG1, then, in reading frame, encoding a sequence consisting of 6 histidinemia label residues and a stop codon (Fig.1). The fragment of gene synthesis was also designed in such a way as to introduce restriction sites at the beginning and end of the cDNA encoding this protein. The introduced restriction sites, EcoRI at the 5'end and Sail at the 3'-end, was used in subsequent cloning procedures. The fragment of gene synthesis was cloned via EcoRI and Sail into a plasmid, oboznachenie�Yu pEF-DHFR (pEF-DHFR is described in Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025, and in Raum et al. Cancer Immunol Immunother 50 (2001) 141-150), following standard protocols. The plasmids with confirmed sequence was used for transfection in expressing the system of FreeStyle 293 (Invitrogen GmbH, Karlsruhe, Germany) according to the manufacturer's Protocol. After 3 days he collected the supernatants of cell cultures of transfectants and tested by ELISA for the presence of the recombinant construct. Specific to Fc-gamma fragment of human IgG antibody goat (obtained from Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK) diluted in PBS to a concentration of 5 μg/ml and was applied at 100 μl/well on a 96 well plate for ELISA MaxiSorp (Nunc GmbH & Co. KG, Wiesbaden, Germany) overnight at 4°C. the Wells were washed with PBS with 0.05% Tween 20 (PBS/Tween) and blocked with 3% BSA in PBS (bovine albumin, fraction V, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) for 60 minutes at room temperature (KG). After that, the wells are again washed with PBS/Tween and then incubated with cell culture supernatants for 60 minutes at KG. After washing, wells were incubated with anti-His6-antibody conjugated with peroxidase (Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany), diluted in the ratio 1:500 in PBS with 1% BSA, for 60 minutes at KG. Then the wells were washed with 200 μl PBS/Tween was added 100 μl of the substrate solution SIGMAFAST OPD (SIGMAFAST OPD [dihydrochloride o-phenylenediamine]) (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) in accordance with�tvii with the manufacturer's Protocol. The reaction was terminated by adding 100 µl of 1 M H2SO4. Color reaction was measured in a spectrophotometer for PowerWaveX microplate (BioTek Instruments, Inc., Winooski, Vermont, USA) at 490 nm with subtraction of the background absorption at 620 nm. As shown in Fig.2, the presence of construction compared to irrelevant supernatant imaginary travelround cells HEK 293, used as a negative control, were clearly defined.

3.2. Analysis of binding of single-chain antibodies with cross-species specificity with the 1-27 CD3-Fc

The binding of the crude drugs expressed in the periplasm single-chain antibodies with cross-species specificity, specific to CD3-Epsilon, 1-27 CD3-Fc were tested in the ELISA analysis. Goat antibody against human IgG specific for Fc-gamma fragment (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK), diluted in PBS to a concentration of 5 μg/ml and was applied at 100 μl/well on a 96 well plate for ELISA MaxiSorp (Nunc GmbH & Co. KG, Wiesbaden, Germany) overnight at 4°C. the Wells were washed with PBS with 0.05% Tween 20 (PBS/Tween) and blocked with PBS 3% BSA (bovine albumin, fraction V, Sigma-Aldrich Chemie GmbH, Taufkirchen Germany) for 60 minutes at KG. After that, the wells were washed with PBS/Tween and were incubated with the supernatants of cells expressing the design of the 1-27 CD3-Fc, for 60 minutes at KG. The wells were washed with PBS/Tween and were incubated with crude preparations expressed in Periplus�the single-chain antibodies with cross-species specificity as described above, for 60 minutes at room temperature. After washing with PBS/Tween, the wells were incubated with anti-Flag M2 antibody conjugated with peroxidase (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), diluted in the ratio 1:10000 in PBS with 1% BSA, for 60 minutes at KG. The wells were washed with PBS/Tween and incubated with 100 μl of the substrate solution SIGMAFAST OPD (OPD [dihydrochloride o-phenylenediamine]) (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) in accordance with the manufacturer's Protocol. Color reaction was stopped by adding 100 µl of 1 M H2SO4and performed measurement on the spectrophotometer for PowerWaveX microplate (BioTek Instruments, Inc., Winooski, Vermont, USA) at 490 nm with subtraction of the background absorption at 620 nm. Observed strong binding of human single-chain antibodies with cross-species specificity, specific to CD3-Epsilon, with the design of the 1-27 CD3-Fc compared to the murine anti-CD3 single-chain antibody (Fig.3).

4. The development of recombinant transmembrane fusion proteins consisting of the N-terminal amino acids 1-27 of CD3-Epsilon of various primates, non chimpanzees, merged with Arcam Javanese macaque (1-27CD3-EpCAM)

4.1. Cloning and expression of the 1-27 CD3-EpCAM

CD3-Epsilon was isolated from different primates, non-chimpanzee (marmoset, Tamarin, squirrel monkey), and pigs. Coding sequence of the N-terminal amino acids 1-27 of the Mature CD3-Epsilon-chain of human, marmoset (Calithhx jacchus), Oedipus Tamarin (Saguinus oedipus), squirrel monkeys (Saimiri sciureus) and domestic pig (Sus scrota; used as negative control), merged with M-end Flag-tagged Arcam Javanese macaque, was obtained by gene synthesis according to standard protocols (cDNA sequence and amino acid sequence of the recombinant fusion proteins represented in SEQ ID NO: 231-240). Fragments of gene synthesis was designed so that they contained the first BsrGI site to provide fusion in the correct reading frame with the coding sequence of the immunoglobulin leader peptide of 19 amino acids already present in the target expressing vector, then, in reading frame, encoding a sequence of N-terminal amino acids 1-27 of the extracellular Mature plot of CD3-Epsilon chains, then, in reading frame, encoding the sequence of the Flag-tag and then, in reading frame, encoding the Mature sequence of a transmembrane protein Arcam Javanese macaque (Fig.4). Fragments of gene synthesis was also designed in such a way as to introduce a restriction site at the end of the cDNA encoding this protein. The introduced restriction sites, BsrGI at the 5'end and Sail at the 3'-end, was used in subsequent cloning procedures. Fragments of gene synthesis is then cloned via BsrGI and Sail, following standard n�autokola, in the derived plasmid designated pEF DHFR (pEF-DHFR is described in Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025), which already contained the sequence encoding immunoglobulin leader peptide of 19 amino acids. Plasmids with confirmed sequence was used for temporary transfection of 293 cells NECK using a reagent MATra-A (IBA GmbH, Gottingen, Germany) and 12 μg of plasmid DNA for attached cells 293-HEK in bottles for cell culture capacity 175 ml according to the manufacturer's Protocol. After 3 days of culturing the cells transfectant tested against expression on the cell surface of recombinant transmembrane protein FACS analysis according to standard protocols. With this order of 2.5×105cells were incubated with anti-Flag M2 antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) at a concentration of 5 μg/ml in PBS with 2% FCS. Bound antibody was detected using conjugated with R-phycoerythrin, affinity purified F(ab')2-fragment of the antibody goat against mouse IgG specific to Fc-gamma fragment, diluted in the ratio 1:100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Samples were measured on a FACScalibur (BD biosciences, Heidelberg, Germany). The expression of Flag-tagged recombinant transmembrane fusion proteins consisting of Arcam Javanese macaque and N-terminal amino acids 1-27 of CD3-Epsilon-chain of human, marmoset,Tamarin, squirrel monkeys and pigs respectively defined in transfected cells (Fig.5).

4.2. The binding of single-chain anti-CD3 antibodies with cross-species specificity with the 1-27 CD3-EpCAM

The binding of the crude drugs expressed in the periplasm single-chain anti-CD3 antibodies with cross-species specificity to the N-terminal amino acids 1-27 of CD3-Epsilon-chain of human, marmoset, Tamarin and squirrel monkeys, respectively, merged with Arcam Javanese macaque, were tested in FACS analysis according to standard protocols. With this order of 2.5×105cells were incubated with crude preparations expressed in the periplasm single-chain anti-CD3 antibodies with cross-species specificity (getting carried out as described above and according to standard protocols) and murine single-chain antibody against human CD3 as a negative control. As secondary antibody used Penta-His-antibody (Qiagen GmbH, Hildesheim, Germany) at a concentration of 5 mg/ml in 50 μl PBS with 2% FCS. Binding of antibodies was determined using conjugated with R-phycoerythrin, affinity purified F(ab')2-fragment of the antibody goat against mouse IgG specific to Fc-gamma fragment, diluted in the ratio 1:100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Samples were measured on a FACScalibur (BD biosciences, Heidelberg, Germany). As shown in Fig.6 (A-�), observed binding of single-chain antibodies with transfectants expressing recombinant transmembrane fusion proteins consisting of the N-terminal amino acids 1-27 of CD3-Epsilon-chain of human, marmoset, Tamarin, or squirrel monkeys, merged with Arcam Javanese macaque. No binding single-chain antibodies with cross-species specificity with fused protein consisting of the 1-27 N-terminal amino acids of CD3-Epsilon chain of the pig, merged with Arcam Javanese macaque, which was used as a negative control, was observed. Was demonstrated interspecies specificity, single chain anti-CD3 antibodies against many primates. The signals obtained with anti-Flag M2 antibodies and single-chain antibodies with cross-species specificity were comparable, indicating high activity of binding single-chain antibodies with cross-species specificity to the N-terminal amino acids 1-27 of CD3-Epsilon.

5. Analysis of binding of single-chain anti-CD3 antibodies with cross-species specificity by alanine scanning of mouse cells transfected CD3-Epsilon-chain of human rights and its alanine mutants

5.1. Cloning and expression of the CD3-Epsilon person, wild-type

The encoding sequence of the CD3-Epsilon chain of human gene synthesis according to standard protocols (PEFC�dovalidate cDNA and amino acid sequence of the CD3-Epsilon chain of the person represented in SEQ ID NO: 242 and 241). The fragment of gene synthesis was designed so that it contained the website Kozak for eukaryotic expression of this design and the restriction sites at the beginning and at the end of the cDNA encoding the CD3-Epsilon person. The introduced restriction sites, EcoRI at the 5'end and Sail at the 3'-end, was used in subsequent cloning procedures. The fragment of gene synthesis is then cloned via EcoRI and Sail into a plasmid designated pEF-NEO, following standard protocols. pEF-NEO was obtained from pEF DHFR (Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025) by replacing DHFR cDNA in cDNA (gene) resistance to neomycin by traditional molecular cloning. The plasmids with confirmed sequence was used for transfection of murine T cell line EL4 (ATSS № TIB-39), cultured in RPMI medium with a stabilizing additive L-glutamine, supplemented with 10% FCS, 1% penicillin/streptomycin, 1% HEPES (N-2-hydroxyethyl-piperazine-N-2-econsultancy acid), 1% pyruvate, 1% nonessential amino acids (all Biochrom AG Berlin, Germany), at 37°C, 95% humidity and 7% CO2. Transfection was carried out using a reagent for transfection with SuperFect (Qiagen GmbH, Hilden, Germany) and 2 μg of plasmid DNA in accordance with the manufacturer's Protocol. After 24 hours cells were washed with PBS and again cultured in the above medium for culturing cells with 600 μg/ml G418 for selection (PAA Laboratories GmbH, asching, Austria). After 16-20 hours after transfection was observed the growth of resistant cells. After 7-14 days, the cells were tested against the expression of the CD3-Epsilon person FACS analysis according to standard protocols. of 2.5×105cells were incubated with the antibody UCHT-1 against human CD3 (BD biosciences, Heidelberg, Germany) at a concentration of 5 μg/ml in PBS with 2% FCS. Binding of antibodies was determined using conjugated with R-phycoerythrin, affinity purified F(ab')2-fragment of the antibody goat against mouse IgG (specific to Fc-gamma fragment), diluted in the ratio 1:100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Samples were measured on a FACSCalibur (BD biosciences, Heidelberg, Germany). Expression of human CD3-Epsilon chains in wild-type transfected EL4 cells shown in Fig.7.

5.2. Cloning and expression of single-chain anti-CD3 antibodies with cross-species specificity in the form of lgG1 antibodies

With the aim of creating improved tools for the identification of binding single-chain anti-CD3 antibodies with cross-species specificity NS HLP, A2J HLP and HLP, e2m boasts turned in lgG1 antibodies with murine and human lgG1 constant regions of the lambda. The cDNA sequence encoding the heavy and light chains corresponding lgG antibodies, obtained by gene synthesis according to standard protocols. Fragments of gene synthesis for each specificity designed in such a way that�s they contained the first website Kozak for eukaryotic expression of this design, then immunoglobulin leader peptide of 19 amino acids (SEQ ID NO: 244 and 243), then, in reading frame, encoding the sequence of the corresponding variable regions of the heavy chain or the corresponding variable region of light chain, then, in reading frame, encoding the sequence of the constant region of the heavy chain of murine lgG1 (SEQ ID NO: 246 and 245), or a sequence encoding a constant region light chain lambda (SEQ ID NO: 248 and 247), respectively. The restriction sites were introduced at the beginning and end of the cDNA encoding this protein. The restriction sites, EcoRI at the 5'end and Sail at the 3'-end, was used for subsequent cloning procedures. Fragments of gene synthesis was cloned via EcoRI and Sail into a plasmid designated pEF DHFR (Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025) for the heavy chain, and pEF ADA (pEF ADA is described in Raum et al., Cancer Immunol. Immunother., 50(3), (2001), 141-50) for designs light chain according to standard protocols. Plasmids with confirmed sequence was used to cotransfection relevant structures of light and heavy chains in expressing the system of FreeStyle 293 (Invitrogen GmbH, Karlsruhe, Germany) according to the manufacturer's Protocol. After 3 days of cell culture supernatants of transfectants collected and used for the experiment on alanine scanning.

5.3. Cloning expressia alanine mutants of CD3-Epsilon person for alanine scanning

27 cDNA fragments encoding the CD3-Epsilon chain of human rights, with the substitution of one codon in the sequence of wild-type CD3-Epsilon person at a codon encoding an alanine (GCC), for each amino acid from amino acids 1-27 of the extracellular domain of the Mature CD3-Epsilon-chain of human rights, respectively, were obtained by gene synthesis. With the exception of the replaced codon, the cDNA fragments were identical to the above-mentioned cDNA fragment of human wild-type CD3. Only one codon was replaced in each construct compared with the above-described cDNA fragment of human wild-type CD3. The restriction sites EcoRI and Sail were introduced into the cDNA fragments in position, identical design wild type. All scanned by alanine constructs cloned in pEF-NEO, and plasmids with confirmed sequence was transfusional in EL4 cells. Transfection and selection of transfectants was performed as described above. The result was a panel of murine constructions in which the first amino acid of CD3-Epsilon-chain of human rights, glutamine (Q, Gln) at position 1 was replaced with alanine. The last amino acid is replaced by alanine, was threonine (T, Thr) at position 27 of the Mature CD3-Epsilon-chain wild-type human. For each amino acid between 1 and glutamine threonine 27 respectively were obtained transfectant with the replacement of the wild-type amino acid by alanine

5.4. Experiment on alanine scanning

Chimeric lgG antibodies described in paragraph 5.2, and single-chain antibodies with cross-species specificity, specific to CD3-Epsilon, were tested in the experiment by alanine scanning. Antibody binding to cell lines EL4 transfected by mutant by alanine designs CD3-Epsilon person described in paragraph 5.3, was tested by FACS analysis according to standard protocols. of 2.5×105cells corresponding transfectants were incubated with 50 µl of cell culture supernatant containing lgG chimeric antibodies, or with 50 µl of the crude drugs expressed in the periplasm single-chain antibodies. For samples incubated with crude preparations expressed in the periplasm single-chain antibodies, anti-Flag M2 antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was used as secondary antibody at a concentration of 5 mg/ml in 50 μl PBS with 2% FCS. For samples incubated with lgG chimeric-antibody, the need in the secondary antibody was not. For all samples the binding of molecules of antibodies was determined using conjugated with R-phycoerythrin, affinity purified F(ab')2-fragment of the antibody goat against mouse IgG (specific to Fc-gamma fragment), diluted in the ratio 1:100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). �the best was measured on a FACSCalibur (BD biosciences, Heidelberg, Germany). Was determined by differential binding of lgG chimeric molecules or single-chain antibodies with cross-species specificity of cell lines EL4 transfected by alanine mutants of CD3-Epsilon person. As a negative control was used or isotype control, or untreated, the drug is expressed in the periplasm single-chain antibody of irrelevant specificity, respectively. The antibody UCHT-1 was used as a positive control, the expression level of alanine mutants of CD3-Epsilon person. Cell line EL4, transfetsirovannyh alanine mutants of the amino acid tyrosine at position 15, valine at position 17, isoleucine at position 19, valine at position 24, or leucine at position 26 of Mature CD3-Epsilon chains, not evaluated due to very low expression levels (data not shown). The binding of single-chain antibodies with cross-species specificity and single-chain antibodies in the format of chimeric IgG with cell lines EL4 transfected by alanine mutants of CD3-Epsilon person shown in Fig.8 (A-D) relative binding in arbitrary units with a geometric mean fluorescence values of the respective negative controls, deducted from all relevant geometric means of fluorescence for samples. For whom�anscii various levels of expression for all values of the samples for specific transfectant then divided by the geometric mean fluorescence of the antibody UCHT-1 for the corresponding transfectant. For comparison with the value of specificity for the sample of wild type, all the values of the corresponding specificdate for samples definitively divided on the value for sample wild-type, with the value for sample wild type was taken as 1 unit of the binding.

Used computing specifically shown in the following formula:

In this equation value_Sample means a value binding in arbitrary units representing the degree of binding of specific anti-CD3 antibodies with a specific alanine-mutant, as shown in Fig.8(A-D); Sample means the geometric mean fluorescence value obtained for a specific anti-CD3 antibodies, and analyzed on a specific alanine-scanning transfectant; neg_Contr. means the geometric mean fluorescence value obtained for a negative control, were analyzed for specific alanine mutant; UCHT-1 means the geometric mean fluorescence value obtained for the antibody UCHT-1, analyzed for specific alanine mutant; WT means the geometric mean fluorescence value obtained for a specific anti-CD3 antibodies, and analyzed on transfectant wild type; x specifies the appropriate transfectant; sets suitable�e anti-CD3-antibody, and wt indicates that the corresponding transfectant refers to wild-type.

As can be seen from Fig.8(A-D), lgG-antibody A2J HLP showed a pronounced loss of binding for the following amino acids: asparagine at position 4, threonine at position 23 and isoleucine at position 25 of Mature CD3-Epsilon chain. Complete loss of binding of IgG-antibodies A2J HLP was observed for the following amino acids: glutamine at position 1, the aspartate at position 2, glycine at position 3 and the glutamate at position 5 of the Mature CD3-Epsilon chain. lgG-antibody e2m boasts HLP showed a pronounced loss of binding for the following amino acids: asparagine at position 4, threonine at position 23 and isoleucine at position 25 of Mature CD3-Epsilon chain. lgG-antibody e2m boasts HLP showed complete loss of binding of the following amino acids: glutamine at position 1, the aspartate at position 2, glycine at position 3 and the glutamate at position 5 of the Mature CD3-Epsilon chain. lgG-antibody NS HLP demonstrated intermediate loss of binding to amino acid asparagine at position 4 of the Mature CD3-Epsilon chains, and it demonstrated a complete loss of binding for the following amino acids: glutamine at position 1, the aspartate at position 2, glycine at position 3 and the glutamate at position 5 of the Mature CD3-Epsilon chain. Single-chain antibody F12Q HLP demonstrated essentially complete loss of binding for�next amino acids: glutamine at position 1, aspartate at position 2, glycine at position 3 of the Mature CD3-Epsilon chains and a glutamate at position 5 of the Mature CD3-Epsilon chain.

6. Analysis of the binding of anti-CD3-binding molecules NS HLP with interspecies specificity with CD3-Epsilon-chain of human N-terminal His6-tag and without it, which was transfusional in murine T cell line EL4

6.1. Cloning and expression of the CD3-Epsilon chain of human N-terminal hexakustodianos tag (His6-tag)

The cDNA fragment encoding CD3-Epsilon-chain of human N-terminal His6-tag was obtained by gene synthesis. The fragment of gene synthesis was designed so that it contained the first website Kozak for eukaryotic expression of this design, then, in reading frame, immunoglobulin leader peptide of 19 amino acids, then, in reading frame, encoding the sequence of the His6-tag, then, in reading frame, encoding the sequence of the Mature CD3-Epsilon chain (cDNA sequence and amino acid sequence of this design is presented in SEQ ID NO: 256 and 255). The fragment of gene synthesis was also designed so that it contained the restriction sites at the beginning and at the end of the cDNA. The introduced restriction sites, EcoRI at the 5'end and Sail at the 3'-end, was used in subsequent cloning procedures. The fragment of gene synthesis is then cloned via EcoRI and Sail plasmid, designated pEF-NEO (as described above), following standard protocols. The plasmids with confirmed sequence was used for transfection of murine T cell line EL4. Transfection and selection of transfectants was performed as described above. After 34 days of culturing the cells transfectant used in the analysis described below.

6.2. Binding of anti-CD3-binding molecules NS HLP with interspecies specificity with CD3-Epsilon-chain of human N-terminal His6-tag and without it

Chimeric lgG-antibody NS HLP with binding specificity to CD3-Epsilon, were tested in binding to CD3-Epsilon person with N-terminal His6-tag and without it. The binding of this antibody with the cell lines EL4 transfected by His6-CD3-Epsilon person and the human CD3-Epsilon wild-type, respectively, was tested by FACS analysis according to standard protocols. of 2.5×105cells transfectants were incubated with 50 µl of cell culture supernatant containing lgG chimeric-antibody or 50 µl of appropriate control antibodies at a concentration of 5 μg/ml in PBS with 2% PCS. As a negative control used the appropriate isotype control, and as a positive control, expression of the construct were CD3-specific antibody UCHT-1, respectively. Binding of antibodies was determined using�ing conjugated with R-phycoerythrin, affinity purified F(ab')2-fragment of the antibody goat against mouse IgG (specific to Fc-gamma fragment), diluted in the ratio 1:100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Samples were measured on a FACSCalibur (BD biosciences, Heidelberg, Germany). Compared to the EL4 cell line, transfetsirovannyh human CD3-Epsilon wild type was determined apparent loss of binding of chimeric IgG antibody NS HLP with the specificity of binding to CD3-Epsilon person with N-terminal His6-tag. These results showed that a free N-end of the CD3-Epsilon is essential for the binding of specific anti-CD3 binding molecules NS HLP with interspecies specificity with Epsilon-chain of CD3 (Fig.9).

7. Cloning and expression of the C-terminal, transmembrane and truncated extracellular domains of human MCSP

The encoding sequence of the C-terminal, transmembrane and truncated extracellular domain of human MCSP (amino acids 1538-2322) was obtained by gene synthesis according to standard protocols (cDNA sequence and amino acid sequence of the recombinant construct for expression of the C-terminal, transmembrane and truncated extracellular domain of human MCSP (indicated by D3 man) is presented in SEQ ID NO: 250 and 249). The fragment of gene synthesis was designed so that it contained the first website Kozak� for eukaryotic expression of this design, then the encoding sequence of the immunoglobulin leader peptide of 19 amino acids, then, in reading frame, FLAG-tag, then, in reading frame, a sequence containing several restriction sites for cloning purposes and encoding an artificial linker of 9 amino acids (SRTRSGSQL), then, in reading frame, encoding the sequence of the C-terminal, transmembrane and truncated extracellular domain of human MCSP and a stop codon. The restriction sites were introduced at the beginning and end of the DNA fragment. The restriction sites, EcoRI at the 5'end and Sail at the 3'-end, was used in subsequent cloning procedures. The fragment was digested EcoRI and Sail and cloned into pEF-DHFR (pEF-DHFR is described in Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025), following standard protocols. The plasmids with confirmed sequence was used for transfection of cells CHO/dhfr- (ATCC no CRL 9096). Cells were cultured in medium RPMI 1640 with stabilizing additive glutamine, supplemented with 10% FCS, 1% penicillin/streptomycin (all purchased from Biochrom AG Berlin, Germany) and nucleosides from a stock solution of reagent purity "of cell cultures (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) to a final concentration of adenosine 10 μg/ml, deoxyadenosine 10 μg/ml and thymidine 10 μg/ml in an incubator at 37°C, 95% humidity and 7% CO2. Transfection was carried out using real�NTA for transfection with PolyFect (Qiagen GmbH, Hilden, Germany) and 5 µg of plasmid DNA in accordance with the manufacturer's Protocol. After culturing for 24 hours, the cells were washed with PBS and once again were cultured in RPMI 1640 with stabilizing additive glutamine and 1% penicillin/streptomycin. Thus, the cell culture medium did not contain nucleosides, so the selection was carried out on transfected cells. Approximately 14 days after transfection was observed the growth of resistant cells. After 7-14 days transfectant was tested by FACS analysis in relation to the expression of design. of 2.5×105cells were incubated with 50 μl anti-Flag-M2 antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), diluted to a concentration of 5 μg/ml in PBS with 2% FCS. Binding of antibodies was determined using conjugated with R-phycoerythrin, affinity purified P(ab')2-fragment antibodies goat against mouse IgG (specific to Fc-gamma fragment), diluted in the ratio 1:100 in PBS with 2% FCS (ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Samples were measured on a FACScalibur (BD biosciences, Heidelberg, Germany).

8. Cloning and expression of the C-terminal, transmembrane and truncated extracellular domains of macaque MCSP

The cDNA sequence C-terminal, transmembrane and truncated extracellular domains of macaque MCSP (indicated by D3 macaque) was obtained by conducting a series of three PCR on cDNA from macaque skin (catalogue number S-su-sun; BioCat GmbH, Heidelberg, Germany) in the following reaction conditions: 1 cycle at 94°C for 3 min, 40 cycles at 94°C for 0.5 min, 52°C for 0.5 min and 72°C for 1.75 min, then a final cycle at 72°C for 3 min, Using the following primers:

direct primer: 5'-GATCTGGTCTACACCATCGAGC-3' (SEQ ID NO: 361)

reverse primer: 5'-GGAGCTGCTGCTGGCTCAGTGAGG-3' (SEQ ID NO: 362)

direct primer: 5'-TTCCAGCTGAGCATGTCTGATGG-3' (SEQ ID NO: 363)

reverse primer: 5'-CGATCAGCATCTGGGCCCAGG-3' (SEQ ID NO: 364)

direct primer: 5'-GTGGAGCAGTTCACTCAGCAGGACC-3' (SEQ ID NO: 365)

reverse primer: 5'-GCCTTCACACCCAGTACTGGCC-3' (SEQ ID NO: 366).

As a result of these PCR received three overlapping fragments (A: 1-1329, In: 1229-2428 With: 1782-2547), which were isolated and sequenced according to standard protocols using the PCR primers, and thus received a portion of the cDNA sequence macaque MCSP size 2547 p. O. (cDNA sequence and amino acid sequence of this section of macaque MCSP is presented in SEQ ID NO: 252 and 251) from 74 p. O. above sequence encoding the C-terminal domain, to 121 p. O. below the stop codon. Another PCR, which was performed in the following reaction conditions: 1 cycle at 94°C for 3 min, 10 cycles at 94°C for 1 min, at 52°C for 1 min and at 72°C for 2.5 min, and a final cycle at 72°C for 3 min, was used for the fusion PCR products of the above reactions A and B. Used� the following primers:

direct primer: 5'-tcccgtacgagatctggatcccaattggatggcggactcgtgctgttctcacacagagg-3' (SEQ ID NO: 367)

reverse primer: 5'-agtgggtcgactcacacccagtactggccattcttaagggcaggg-3' (SEQ ID NO: 368).

The primers for this PCR were designed in such a way as to introduce restriction sites at the beginning and end of the cDNA fragment encoding the C-terminal, transmembrane and truncated extracellular domains of macaque MCSP. The introduced restriction sites, Mfel at the 5'end and Sail at the 3'-end, was used in subsequent cloning procedures. The PCR fragment then was cloned using Mfel and Sail into the Bluescript plasmid containing the EcoRI fragment/Mfel aforementioned plasmids pEF-DHFR (pEF-DHFR is described in Raum et al. Cancer Immunol. Immunother. 50 (2001) 141-150), by replacing the C-terminal, transmembrane and truncated extracellular domains of human MCSP. The fragment of gene synthesis contained the coding sequence of the immunoglobulin leader peptide and Flag-tags, and artificial linker (SRTRSGSQL), in reading frame with the 5'-end cDNA fragment encoding the C-terminal, transmembrane and truncated extracellular domains of macaque MCSP. This vector was used for transfection of cells CHO/dhfr- (ATCC no CRL 9096). Cells were cultured in RPMI 1640 with stabilizing additive glutamine, supplemented with 10% FCS, 1% penicillin/streptomycin (all from Biochrom AG Berlin, Germany) and nucleosides from a stock solution of reagent purity "of cell cultures (Sigma-Aldrich Chemie GmbH, Taufkirchen, German) to final concentration of adenosine 10 μg/ml, deoxyadenosine 10 μg/ml and thymidine 10 μg/ml, in an incubator at 37°C, 95% humidity and 7% CO2. Transfection was carried out using a reagent for transfection with PolyFect (Qiagen GmbH, Hilden, Germany) and 5 µg of plasmid DNA in accordance with the manufacturer's Protocol. After culturing for 24 hours, the cells were washed with PBS and once again were cultured in RPMI 1640 with stabilizing additive glutamine and 1% penicillin/streptomycin. Thus, the cell culture medium did not contain nucleosides, and therefore the selection was carried out on transfected cells. Approximately 14 days after transfection was observed the growth of resistant cells. After 7-14 days transfectant was tested by FACS analysis against recombinant expression constructs. of 2.5×105cells were incubated with 50 μl anti-Flag-M2 antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), diluted to a concentration of 5 μg/ml in PBS with 2% FCS. Binding of antibodies was determined using conjugated with R-phycoerythrin, affinity purified F(ab')2-fragment of the antibody goat against mouse IgG (specific to Fc-gamma fragment), diluted in the ratio 1:100 in PBS with 2% FCS (ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Samples were measured on a FACScalibur (BD biosciences, Heidelberg, Germany).

9. Establishment and characterization of MCSP and CD3 bespecifically single-stranded molecules with interspecific specifications�cestu

Molecules bespecifically single-chain antibodies, each of which contains a binding domain with cross-species specificity to CD3-Epsilon person and the primacy of non-chimpanzee, and binding domain with cross-species specificity to human MCSP and the primacy of non-chimpanzee, designed, as indicated in Table 1 below.

td align="left"> MCSP-G4 HLP × F12Q HL
Table 1
Formats MCSP and CD3 bespecifically single-chain antibodies with cross-species specificity
SEQ ID (u./Bel.)Formats of protein constructs (N → C)
190/189MCSP-G4 HL × H2C HL
192/191MCSP-G4 HL × F12Q HL
194/193MCSP-G4 HL × I2C HL
196/195MCSP-G4 HLP × F6A HLP
198/197MCSP-G4 HLP × H2C HLP
202/201MCSP-G4 HLP × G4H HLP
206/205MCSP-G4 HLP × E1L HLP
208/207MCSP-G4 HLP × E2M HLP
212/211
214/213MCSP-G4 HLP × I2C HL
216/215MCSP-D2 HL × H2C HL
218/217MCSP-D2 HL × F12Q HL
220/219MCSP-D2 HL × I2C HL
222/221MCSP-D2 HLP × H2C HLP
224/223MCSP-F9 HL × H2C HL
226/225MCSP-F9 HLP × H2C HLP
228/227MCSP-F9 HLP × G4H HLP
318/317MCSP-A9 HL × H2C HL
320/319MCSP-A9 HL × F12Q HL
322/321MCSP-A9 HL × I2C HL
324/323MCSP-C8 HL × I2C HL
328/327MCSP-B7 HL × I2C HL
326/325MCSP-B8 HL × I2C HL
330/329MCSP-G8 HL × I2C HL
332/331MCSP-D5 HL × I2C HL
334/333MCSP-F7 HL × I2C HL
336/335 MCSP-G5 HL × I2C HL
338/337MCSP-F8 HL × I2C HL
340/339MCSP-G10 HL × I2C HL

The aforementioned constructs containing the variable domains of the heavy chain (VH) and the variable domains of the light chain (VL) with cross-species specific for MCSP D3 human and macaque, and VH - and VL-domains cross-species specific for human CD3 and macaque, was obtained by gene synthesis. Fragments of gene synthesis was designed so that they contained the first website Kozak for eukaryotic expression of this design, then immunoglobulin leader peptide of 19 amino acids, then, in reading frame, encoding a sequence corresponding molecules especifismo single-chain antibodies, then, in reading frame, encoding the sequence of the histidine-tag and a stop codon. The fragment of gene synthesis was also designed in such a way as to introduce the suitable N - and C-terminal restriction sites. The fragment of gene synthesis was cloned via these restriction sites into a plasmid designated pEF-DHFR (pEF-DHFR is described in Raum et al. Cancer Immunol. Immunother. 50 (2001) 141-150), according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (2001)). These structures were transfusional temporarily in a stable or defective in DHFR �the tap hole of SSS (ATSS No. CRL 9096) by means of electroporation or alternative, temporarily transfusional in HEK 293 (kidney cells of the human embryo, ATSS number: CRL-1573) according to standard protocols.

Eukaryotic protein expression in defective in DHFR cells SSS was performed as described in Kaufmann R. J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the constructs was induced by adding increasing concentrations of methotrexate (MTX) to a final concentration of MTX 20 nm inclusive. After two passages of stationary culture cells were grown in roller bottles in liquid soy medium HyQ PF Cho without nucleosides (with 4.0 mm L-glutamine, 0.1% Pluronic F-68; HyClone) for 7 days, then collected. Cells were isolated by centrifugation, and the supernatant containing the expressed protein was stored at -20°C.

For chromatography system was used Akta® Explorer (GE Health Systems) and Unicorn software®. Affinity chromatography with immobilized metal ("IMAC") was performed using a Fractogel EMD chelate® (Merck) which was loaded ZnCb in accordance with the Protocol suggested by the manufacturer. The column was equilibrated with buffer A (20 mm sodium phosphate buffer pH 7.2; 0.1 M NaCl), and cell culture supernatant (500 ml) were applied to a column (10 ml) at a flow rate of 3 ml/min the Column was washed with buffer A to remove unbound sample. Bound protein was suirable using dogsleding� gradient of buffer B (20 mm sodium phosphate buffer pH 7.2; 0.1 M NaCl; 0.5 M imidazole) as follows:

stage 1: 20% buffer In 6 column volumes;

stage 2: 100% buffer In 6 column volumes.

- Eluted protein fractions from step 2 were pooled for further purification. All chemical reagents were of research degree of purity and were purchased from Sigma (Deisenhofen) or Merck (Darmstadt).

Gel filtration was performed on a preparative column (HiLoad 16/60 Superdex 200 (GE/Amersham), equilibrated Equi-buffer (25 mm citrate, 200 mm lysine; 5% glycerol; pH 7.2). - Eluted protein samples (flow rate 1 ml/min) were subjected to standard SDS-PAGE and Western blotting for detection. Before cleaning the column was calibrated to determine the molecular weight (the set of markers of molecular weight, Sigma MW-GF-200). Protein concentration was determined by OD 280 nm.

Purified protein bespecifically single-chain antibody, analyzed by the method of SDS-PAGE under reducing conditions using pre-filled 4-12%-tion of gels in bis-Tris buffer (Invitrogen). Preparation and application of samples was performed according to the manufacturer's Protocol. The molecular weight was determined using a protein standard MultiMark (Invitrogen). The gel was stained with colloidal Coomassie (Invitrogen Protocol). Determined by SDS-PAGE the purity of separated protein is over 95%.

Bespecifically single-chain antibody has a molecular mass� approximately 52 kDa in native conditions, as determined by gel filtration in phosphate-buffered saline (PBS). All constructs were purified according to this method.

Western blotting was carried out using membrane Optitran® BA-S83 in the apparatus for blotting Invitrogen in accordance with the Protocol provided by the manufacturer. For the detection of protein especificacao single-stranded antibodies used antibody against His-tag (Penta-His, Qiagen). The antibody goat against mouse Ig labeled with alkaline phosphatase (AP) (Sigma) was used as secondary antibody, a BCIP/NBT (Sigma) as substrate. Detected a single band at 52 kDa, corresponding purified bespecifically single-chain antibody.

Alternative, construction temporarily expressively defective in DHFR cells SNO. Briefly, 4×105cells per construct was cultured in 3 ml complete medium RPMI 1640 with stabilizing additive glutamine, supplemented with 10% fetal calf serum, 1% penicillin/streptomycin and nucleosides from a stock solution of reagent purity "of cell cultures (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) to a final concentration of adenosine 10 μg/ml, deoxyadenosine 10 μg/ml and thymidine 10 μg/ml, in an incubator at 37°C, 95% humidity and 7% CO2one day before the transfection. Transfection was carried out using Rea�yente for transfection Fugene 6 (Roche, No. 11815091001) in accordance with the manufacturer's Protocol. 94 μl of OptiMEM medium (Invitrogen) and 6 µl of Fugene 6 were mixed and incubated for 5 minutes at room temperature. After this was added 1.5 μg DNA per design, mixed and incubated for 15 minutes at room temperature. At the same time, defective in DHFR cells SSS were washed with 1x PBS and were resuspended in 1.5 ml complete medium RPMI 1640. Mixture for transfection was diluted with 600 μl of complete medium RPMI 1640 was added to the cells and incubated over night at 37°C, 95% humidity and 7% CO2. A day after the transfection incubated volume for each approach was increased to 5 ml by adding complete medium RPMI 1640. Supernatant was collected after 3 days of incubation.

10. Flow-cytometric analysis of binding of MCSP and CD3 bespecifically antibodies with cross-species specificity

For the purpose of testing the functionality of the designs bespecifically antibodies with cross-species specificity regarding the ability to contact MCSP D3 and CD3 human and macaque, respectively, was carried out by FACS-analysis. For this purpose, cells SNO, transfetsirovannyh MCSP D3 (as described in Example 7) and CD3-positive cell line HPB-ALL T cell human leukemia (DSMZ, Braunschweig, ACC483) was used to test the binding to human antigens. The ability to bind to macaque antigens those�preted using the created transfectant MCSP D3 macaque (described in Example 8) and T-cell lines 4119LnPx macaque (kindly provided by Professor Fickenscher, Hygiene Institute, Virology, ErIangen-Nuernberg; published in Knappe A, et al., and Fickenscher H., Blood 2000, 95, 3256-61). 200,000 cells of the respective cell lines were incubated for 30 min on ice with 50 μl of purified protein structures bespecifically antibodies with cross-species specificity (2 μg/ml) or cell culture supernatant of transfected cells expressing these constructs bespecifically antibodies with cross-species specificity. Cells were washed twice in PBS with 2% FCS and binding of this structure was determined using mouse anti-His-antibody (Penta-His-antibody; Qiagen; diluted in the ratio 1:20 in 50 μl PBS with 2% FCS). After washing, bound anti-His-antibody was determined using the Fc-gamma-specific antibody (Dianova), conjugated with phycoerythrin, diluted in the ratio 1:100 in PBS with 2% FCS. As a negative control for binding to T-cell lines used supernatant nitrostilbene cells SNO. Single-stranded structure with irrelevant target specificity was used as negative control binding to MCSP-D3 cells transfected by the SSS.

Flow cytometry was performed with apparatus FACS-Calibur, and to retrieve and analyze data using the software CellQuest (Becton Dickinson biosciences, Heidelberg). FACS-staining and measurement of the fluorescence intensity is�have been, as described in Current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-Interscience, 2002).

Bespecifically binding single-chain molecules listed above, which demonstrate cross-species specificity to MCSP D3 and interspecies specificity for human CD3 and macaque, explicitly defined, as shown in Fig.10, 11, 12, and 39. In the FACS analysis all constructs showed binding to CD3 and MCSP D3 compared to the corresponding negative controls. Was demonstrated interspecies specificity bespecifically antibodies to antigens CD3 and MCSP D3 human and macaque.

11. Biological activity of MCSP and CD3 bespecifically single-chain antibodies with cross-species specificity

Biological activity created bespecifically single-chain antibodies was analyzed by determining the release of chromium-51 (51Cr) in the analysis of cytotoxicity in vitro using MCSP D3-positive cell lines described in Examples 7 and 8. As effector cells used the stimulated CD4/CD56-depleted RVMS man, stimulated RVMS person or a T-cell line 4119LnPx macaque, as shown in the relevant drawings.

Getting stimulated CD4/CD56-depleted RVMS performed as follows: a Petri dish (diameter 145 mm; Greiner bio-one GmbH, Kremsmunster) was applied with a commercially available anti-CD3-SP�nificence antibody (e.g. OCT, Othoclone) to a final concentration of 1 μg/ml for 1 hour at 37°C. Unbound protein was removed in a single stage of leaching, using PBS. Fresh RWMS was isolated from peripheral blood (30-50 ml human blood) by centrifugation in ficoll gradient according to standard protocols. 3-5×107RWMS was added to pre-coated Petri dish in 120 ml of RPMI 1640 with stabilizing additive mixture glutamine/10% FCS/IL-2 20 u/ml (Proleukin, Chiron) and stimulated for 2 days. On the third day the cells were collected and washed once with RPMI 1640. Added IL-2 to a final concentration of 20 units/ml, and the cells were again cultured for one day in the same environment for culturing cells as described above. Through the depletion of CD4+T cells and CD56+NK-cells according to standard protocols implemented enrichment of CD8+cytotoxic T lymphocytes (CTL).

Target cells were washed twice with PBS and labeled to 11.1 MBq51Cr in a final volume of 100 μl RPMI with 50% FCS for 45 minutes at 37°C. After this, the labeled target cells were washed 3 times with 5 ml RPMI and then used in the analysis of cytotoxicity. The analysis was performed in 96-well plate in a total volume of 250 μl supplemented RPMI medium (as above) with the ratio (E:T of 10:1. Inflicted 1 µg/ml molecules especifismo single-chain antibodies with cross-species specificity and their 20 three-fold dilution. If used soup�mutant, containing molecules bespecifically single-chain antibodies with cross-species specificity, stabbed 21 of his two-fold dilution and 20 of its three-fold dilution in the analysis of cytotoxicity for macaque and human, respectively. The analysis time was 18 hours, and cytotoxicity was measured as relative values of released chromium in the supernatant, corresponding to the difference of maximum lysis (addition of Triton-X) and spontaneous lysis (without effector cells). All measurements were performed in four replicates. Radioactivity measurements of chromium in the supernatants was performed using a gamma counter Wizard 3" (Perkin Elmer Life Sciences GmbH, Koln, Germany). Analysis of experimental data was performed using Prism 4 for Windows (version 4.02, GraphPad Software Inc., San Diego, California, USA). Sigmoidal curves dose-response, typically have values of R2above of 0.90, as determined using this software. The values of EC50calculated using the analysis program, was used to compare the biological activity.

As shown in Fig.13-17 and 40, all created designs bespecifically single-chain antibodies with cross-species specificity demonstrated cytotoxic activity against target cells that are positive for the MCSP D3 human induced stimulated CD4/CD56-depleted� RVMS person or stimulated RVMS person and against target cells that are positive for the MCSP D3 macaque induced T-cell line 4119LnPx macaque.

12. Stability in plasma MCSP and CD3 bespecifically single-chain antibodies with cross-species specificity

The stability created bespecifically single-chain antibodies in human plasma was analyzed by incubation bespecifically single-chain antibodies in 50% human plasma at 37°C and 4°C for 24 hours and then test the biological activity. Biological activity was investigated in the analysis of cytotoxicity in vitro by determining the release of chromium-51 (51Cr) using MCSP-positive cell line Cho (expressing MCSP, cloned in accordance with Example 14 or 15) as a target and stimulated CD8-positive T-cells as effector cells.

The values of EC50calculated using the analysis program, which is described above, was used to compare the biological activity bespecifically single-chain antibodies, incubated with 50% human plasma for 24 hours at 37°C and 4°C respectively with bespecifically single-chain antibodies without the addition of plasma or mixed with the same amount of plasma directly before analysis.

As shown in Fig.18 and Table 2, biological �aktivnosti G4 H-L × I2C H-L, G4 H-L × H2C H-L and G4 H-L × F12Q H-L bespecifically antibodies was not significantly reduced compared to controls without addition of plasma or with the addition of plasma directly before testing the biological activity.

Table 2
Biological activity bespecifically antibodies without the addition of plasma and with the addition of plasma
DesignWithout plasmaWith plasmasPlasma 37°CPlasma 4°C
G4 H-L × I2C H-L300796902867
G4 H-L × H2C H-L49657523631449
G4 H-L × F12Q H-L49335815211040

13. Redistribution of circulating T cells in the absence of circulating target cells, initiated by the first impact of the CD3-binding molecules directed to traditional, that is hung�most from the environment, the epitopes of CD3, is a major risk factor of adverse events associated with the beginning of treatment

The redistribution of T cells in patients with b-cell non-Hodgkin lymphoma (B-NHL) after the start of treatment traditional CD3-binding molecules

Traditional S×CD3 binding molecule is a CD3-binding molecule of format bespecifically tandem scFv (Loffler (2000, Blood, Volume 95, Number 6) or WO 99/54440). It consists of two different binding sites aimed at (1) CD19 on the surface of normal and malignant b-cells and (2) CD3 on T-cells. As a result of cross-linking of CD3 on T cells with CD19 on b cells this design initiates redirected lysis of normal and malignant b-cells through cytotoxic activity of T cells. the CD3 epitope recognized by this traditional CD3-binding molecule localized on CD3-Epsilon chains, where he adopts the proper conformation only when immersed in the rest of the Epsilon-chain and held in proper position by means of heterodimerization Epsilon-chain or gamma-or Delta-chain of CD3. The interaction of this highly dependent on environment epitope with conventional CD3 binding molecule (see, for example, Loffler (2000, Blood, Volume 95, Number 6) or WO 99/54440), even when it is exclusively� monovalent manner and without any cross-linking, may induce an allosteric change in the conformation of CD3, leading to the exposure of hidden in other cases, Proline-rich site in the cytoplasmic domain of the CD3-Epsilon chain. Being exposed Proline-rich phase may stimulate signal transduction molecule Nck2, which is able to run further intramolecular signals. Although this is not sufficient for full T-cell activation, which necessarily requires cross-linking of several of CD3 molecules on the surface of T cells, for example by cross-linking multiple anti-CD3 molecules associated with several molecules on CD3 T-cell with multiple CD19 molecules on the surface of b cells, exclusively monovalent interaction of traditional CD3-binding molecules with dependent environment epitope on CD3-Epsilon still does not remain inert in respect of T cells in terms of signal transmission. Without any connection with theory, traditional monovalent CD3 binding molecules known in the art) can induce some T-cell response during infusion of their people, even in cases when the circulating target cells is not available for cross-linking of CD3. Important T-cell response to intravenous infusion of traditional monovalent CD19×CD3 binding molecule pats�customers with B-NHL, essentially having no circulating CD19-positive b-cells, is the redistribution of T cells after treatment. It was found that in phase I clinical trials of this T-cell reaction takes place during the initial phase of intravenous infusion of CD19×CD3 binding molecules to all individuals without circulating CD19-positive target b cells, essentially regardless of the dose CD19×CD3 binding molecules (Fig.19). However, it was found that a sharp increase in the impact of CD19×CD3 binding molecules will initiate virtually the same reaction redistribution of T cells from these patients, and the initial impact on T cells CD19×CD3-binding molecules at the beginning of treatment (Fig.20A), and even the gradual increase in the influence of CD19×CD3 binding molecules may still influence the redistribution of circulating T cells (Fig.21). It was also found that this is essentially not dependent on dose, T-cell redistribution reaction in the absence of circulating target cells, initiated by traditional CD3-binding molecules, such CD19×CD3 binding molecule (described, for example, in WO 99/54440), 100% of the treated individuals is a major risk factor of adverse events associated with the beginning of treatment.

In accordance with the study Protocol for phase I, open plan Liv� multicenter trials on patients (interpatient) with increasing doses were selected patients with recurrent histologically confirmed slow-growing b-cell non-Hodgkin lymphoma (B-NHL), including cell lymphoma mantle zone. The research Protocol was approved by independent ethics commissions of all participating centers and sent for notification to the competent regulatory authorities.

For inclusion in this study were required to have measurable disease (at least one pathological change of at least 1.5 cm), which is documented by CT scan (computed tomography). Patients received traditional CD19×CD3 binding molecule continuous intravenous infusion using a portable pump minisystem for four weeks at a constant flow rate (i.e., at a constant dose level). Patients were hospitalized for the first two weeks of treatment, after which they were discharged from the hospital, and they continued the treatment at home. Patients without apparent disease progression after four weeks were offered to continue treatment for the next four weeks. Up to this point have tested six different dose levels without reaching the maximum tolerated dose (MTD): 0,5, 1,5, 5, 15, 30 and 60 μg/m2/24 h. Each group consisted of three patients, if not observed any adverse events, as determined in accordance with the research Protocol as DLT (limiting toxicity). If noted one case of DLT among the first t�ex patients the group expanded to six patients, which, in the absence of the second case, DLT, allowed to continue to increase the dose. Accordingly, the dose levels without DLT in groups of 3 patients or one case of DLT in groups of 6 patients were considered as safe. Used in the study treatment was discontinued in all patients who developed DLT. For doses of 15 and 30 µg/m2/24 h during the first 24 h was investigated different modes of initiation of treatment in several additional groups: (1) step increase after 5 μg/m2/24 h during the first 24 h to a maintenance dose of 15 μg/m2/24 h (patients 15-step), (2) the continuous linear increase of flow velocity (ramp) virtually from zero to 15 or 30 μg/m2/24 h (group 15 patients-ramp 30-ramp) and (3) onset (treatment) maintenance dose (group of 15 patients-flat, 30-flat and 60-flat). All groups of patients with doses of 0.5, 1.5 and 5 μg/m2/24 h started treatment maintenance dose from the beginning (i.e., flat-initiation (initiation constant dose)).

The temporal variation in the absolute number of b - and T-cells in peripheral blood was determined by four-color FACS analysis as follows:

Collect blood samples and standard analysis

In patients of groups 15-ramp, 15-flat, 30-ramp, 30-flat and 60-flat took blood samples (6 ml) prior to infusion and through 0,75, 2, 6, 12, 24,30, 48 hours after the start of infusion of CD19×CD3 binding molecules (as described in WO 99/54440), and on the 8th, 15th, 17th, 22nd, 24th, 29th, 36th, 43rd, 50th, 57th day of treatment and 4 weeks after the infusion of traditional CD19×CD3 binding molecules, using containing EDTA (ethylenediaminetetraacetic acid) tubes Vacutainer™ (Becton Dickinson), who was delivered for analysis at 4°C. In control patients 15-step took blood samples (6 ml) prior to infusion and after 6, 24, 30, 48 hours after the start of infusion of CD19×CD3-binding molecules, as well as on the 8th, 15th, 22nd, 29th, 36th, 43rd, 50th, 57th day of treatment and 4 weeks after the infusion of traditional CD19×CD3-binding molecules. For dose levels of 0.5, 1.5 and 5 μg/m2/24 h took blood samples (6 ml) prior to infusion and after 6, 24, 48 hours after the start of infusion of CD19×CD3-binding molecules, as well as on the 8th, 15th, 22nd, 29th, 36th, 43rd, 50th, 57th day of treatment and 4 weeks after the infusion of traditional CD19×CD3-binding molecules. In some cases, for practical reasons, there have been small variations of these time points. FACS analysis of lymphocyte subpopulations was performed within a time period 24-48 h after collection of blood samples. The absolute number of subpopulations of leukocytes in blood samples was determined by differential blood test for CoulterCounter™ (Coulter).

The allocation RVMS sample

The allocation RVMS (mononuclear�x cells in peripheral blood) was carried out in accordance with an adapted Protocol selection in a gradient of Ficoll™. The blood was transferred at room temperature in tubes Leucosep™ (Greiner) with a capacity of 10 ml, pre-filled 3 ml Biocoll™ (Biochrom). Centrifugation was performed in an oscillatory rotor for 15 min at 1700 × g and 22°C without reducing speed. RVMS layer Biocoll™ was separated, washed once with a buffer for FACS (PBS/2% FBS [fetal serum cows; Biochrom]), centrifuged and were resuspended in FACS buffer for. Centrifugation during all stages of leaching was carried out in an oscillatory rotor for 4 min at 800 × g and 4°C. If necessary, lysis of erythrocytes was performed by incubation allocated RVMS in 3 ml of buffer for lysis of erythrocytes (8,29 g NH4Cl; 1.00 g of the JISC3; 0,037 g EDTA; 1.0 l of H2Obidest(double-distilled), pH 7.5) for 5 min at room temperature followed by washing stage in the buffer for FACS.

Staining RVMS antibodies with a fluorescent tag molecules against the cell surface

Monoclonal antibodies were obtained from Invitrogen (1catalogue number MHCD1301,2catalogue number MHCD1401), Dako (5catalogue number S) or Becton Dickinson (3catalogue number 555516,4catalogue number 345766), and they were used in accordance with the manufacturer's recommendations. From 5×105to 1×106cells were stained using the following combination of antibodies: anti-CD131/anti-CD142/sup> (FITC (fluoresceinisothiocyanate)) × anti-CD563(RE (phycoerythrine)) × anti-CD34(PerCP (pyridiniumyl)) × anti-CD195(ASC (allophycocyanin)). The cells were precipitated in 96-well multilateration tablets with V-shaped wells (Greiner), and the supernatant was removed. The cell sediments were resuspended in a total volume of 100 µl, containing specific antibodies diluted in FACS buffer for. Incubation was performed in the dark for 30 min at 4°C. afterwards the samples were washed twice with buffer for FACS, and cell sediments were resuspended in FACS buffer for analysis by flow cytometry.

Flow-cytometric determination of stained lymphocytes FACS-analysis

The data collection was performed using a 4-color FACSCalibur™ BD (Becton Dickinson). For each measurement took 1×104cells of certain subpopulations of lymphocytes. To get the percentage of lymphocyte subpopulations and to classify intensities of expression of cell surface molecules, carried out statistical analysis using the program CellQuest Pro™ (Becton Dickinson). Then the percentage of subpopulations of lymphocytes relative to total lymphocytes (i.e., b cells plus T cells plus NK cells, with the exception of myeloid cells by CD13/14-staining), as identified by FACS analysis, was correlated with the number of limfotsitov�, the resulting differential blood analysis, to calculate the absolute number of T cells (CD3+, CD56-, CD13/14-) and b cells (CD19+, CD13/14-).

The redistribution of T cells during the initial phase of treatment traditional CD19×CD3 binding molecule (e.g., described in WO 99/54440) in all patients, are essentially no circulating CD19-positive b-cells at the beginning of treatment, is shown in Fig.19. For comparison, Fig.22 shows representative example of redistribution of T cells during the initial phase of treatment, CD19×CD3 binding molecule in a patient with a significant number of circulating CD19-positive b cells.

In both cases (i.e., when essentially no, or when there are many circulating b-cells) the number of circulating T cells decreases rapidly after the start of treatment. However, in the absence of circulating b-cells T-cells tend very early to return to the circulating blood, while the return of T-cells circulating in the blood of those patients, which contained a significant number of circulating b-cells at the beginning of treatment, is usually delayed as long as these circulating B-cells are depleted. Thus, the distribution pattern of T cells mainly differ in the kinetics of the return of T-TC�the current in the circulating blood.

The assessment of effectiveness based on the CT scan performed on the results of comparative Central radiology (central radiology reference) after 4 weeks of treatment, and for patients additionally receiving treatment within 4 weeks, after 8 weeks of treatment plus, in all cases, four weeks after the end of treatment. The disappearance and/or normalize the size of all known pathological changes (including increased spleen), as well as the purification of bone marrow from lympany cells in cases of infiltration of the bone marrow was taken for complete response (CR). A reduction of at least 50% from baseline of the sum of products of two of the greatest diameters (SPD) of each predefined target pathological changes took over partial response (PR); a reduction by at least 25% was considered as a minimal response (MR). Progressive disease (PD) was defined as not less than 50% increase in SPD from baseline. Deviations SPD from baseline in the range from +50% to -25% considered as stable disease (SD).

Demographic data of patients who received dose and clinical results for 34 patients are summarized in Table 3. Clinical antitumor activity of CD19×CD3 binding molecules is clearly demonstrated dose-dependent manner: persistent �lomenie circulating CD19-positive B(lympany) cells in peripheral blood was observed from the dose of 5 μg/m 2/24 h At doses of 15 μg/m2/24 h and 30 μg/m2/24 h was recorded first objective clinical response (PR and CR), as well as cases of partial and complete elimination of b-lymphoma cells from infiltrated bone marrow. Finally, at the dose of 60 μg/m2/24 h, the level of response increased to 100% (PR and CR), and purification of bone marrow b-cell lymphomas are complete in all evaluated cases.

CD19×CD3 binding molecule is well tolerated by most patients. The most common adverse events with severity levels 1-4 in 34 patients, regardless of reason, are summarized in Table 4. Adverse effects associated with CD19×CD3 binding molecule, were usually temporary and completely reversible. In particular, there were 2 patients (patients No. 19 and No. 24 in Table 3) essentially no circulating CD19-positive b-cells whose treatment was discontinued prematurely due to adverse CNS effects (main symptoms: confusion and disorientation associated with the re-redistribution of T cells during the initial phase infusion of CD19×CD3 binding molecule.

One of these patients (No. 19) was in a group of 15 step. He received a CD19×CD3 binding molecule at a dose of 5 μg/m2/24 h during the first 24 h, followed by a sharp increase to a maintenance dose of 15 μg/m 2/24 h Corresponding to the pattern of redistribution of T cells shows that the number of circulating T cells rapidly decreased at the beginning of infusion at a dose of 5 μg/m2/24 h with a subsequent early re-appearance of T cells in the circulating blood essentially no circulating CD19-positive b cells. As a consequence, the number of peripheral T cells fully restored, when the dose of CD19×CD3 binding molecules increased after 24 h with 5 to 15 g/m2/24 h. dose Therefore step would initiate the second episode of the redistribution of T cells, as shown in Fig.20A. This is a re-redistribution of T cells has been associated with side effects on CNS (main symptoms: confusion and disorientation) in this patient, what was the reason of the termination of the infusion. The relationship between repeated redistribution of T-cells and such is unfavorable in respect of the Central nervous system phenomena was also observed in preliminary phase I clinical trials in patients with B-NHL treated with CD19×CD3 binding molecule (e.g., described in WO 99/54440) in the form of repeated bolus infusion over 2-4 hours each, then usually followed with a 2-h daily period of time without treatment (Fig.20V). Each single bolus infusion initiated one episode of redistribution of T cells, is rapid �the reduction of the number of circulating T cells and restoring T-cells before the next bolus infusion. Overall, in adverse CNS effects associated with the repeated redistribution of T cells was observed in 5 of 21 patients. Fig.20B shows representative example of one patient involved in clinical trials using bolus infusion who developed CNS symptoms after the third episode of redistribution of T cells. Usually patients with adverse CNS events in clinical trials using bolus infusion are also low number of circulating b cells.

The second patient (No. 24) from clinical trials with the use of continuous infusion, treatment was discontinued prematurely due to adverse CNS effects (main symptoms: confusion and disorientation associated with the re-redistribution of T cells during the initial phase infusion of CD19×CD3 binding molecules, was in a group of 15-flat. By mistake, this patient received an infusion of CD19×CD3 binding molecules without the addition of HSA, which is required for stabilization of the drug. The resulting uneven flow of the drug was initiated by recurrent episodes of redistribution of T cells, instead of only one (Fig.23A), after which a decision was made about the need for infusions due to the development of CNS symptoms. One�about when the same patient again began to enter correct solution CD19×CD3 binding molecule, contains additional HSA for stabilising drugs (eg as described in WO 99/54440), no re-redistribution of T cells was not observed, and the patient has no CNS symptoms did not develop (Fig.23V). Since this patient also had essentially no circulating b cells, circulating T cells could react with the rapid kinetics of redistribution even with subtle changes in the effects of medicines that were observed. Adverse CNS effects associated with the redistribution of T-cells in patients that do not have essentially no circulating target cells, can be explained by a temporary increase in the adhesion of T cells to endothelial cells with subsequent strong simultaneous adhesion of circulating T cells to the blood vessel walls with subsequent decrease in the number of T cells in the circulating blood, which was observed. Mass simultaneous attachment of T cells to the walls of the blood vessels may cause increased permeability of the endothelium and activation of endothelial cells. The consequence of increased permeability of the endothelium is a shift of fluid from intravascular compartment to interstitial tissue compartments, including the interstitial space of the CNS. Activation of endothelial cells way� labeling of T-cells may have procoagulation effects (Monaco et al. J. Leukoc. Biol. 71 (2002) 659-668) with possible abnormalities in blood flow (including cerebral blood flow), in particular in relation to the capillary microcirculation. Thus, in adverse CNS effects associated with the redistribution of T-cells in patients, which do not have circulating target cells, may be the result of capillary leak and/or abnormal capillary microcirculation due to adhesion of T cells to endothelial cells. Endothelial stress caused by one episode of redistribution of T cells, tolerated by most patients, whereas enhanced endothelial stress by the repeated redistribution of T cells, is often the cause of adverse CNS effects. More than one episode of redistribution of T cells may create less risk only in patients with very low numbers of circulating T cells. However, the limited endothelial stress caused by one episode of redistribution of T-cells, may also cause adverse CNS effects in rare cases of hypersensitivity to such phenomena that were observed in 1 of 21 patients in clinical trials with bolus infusion of CD19×CD3 binding molecule.

Without any connection with theory, a temporary increase in T-cell adhesion to andot�Lialina cells in patients essentially not having a circulating target cells, may explain how T-cell response to monovalent interaction of traditional CD3-binding molecules, such as CD19×CD3 binding molecule (see, e.g., WO 99/54440), with its dependent environment epitope on CD3-Epsilon leading to the allosteric change in the conformation of CD3 with subsequent recruitment Nck2 to the cytoplasmic domain of the CD3-Epsilon, as described above. Because Nck2 is directly linked to integrins via PINCH and ILK (Fig.28), recruitment Nck2 to the cytoplasmic domain of the CD3-Epsilon after allosteric changes in the conformation of CD3 linking traditional CD3-binding molecules, such as CD19×CD3 binding molecule, with its dependent environment epitope on CD3-Epsilon, can increase the adhesion of T cells to endothelial cells by temporary transition of integrins on the surface of T-cells in their more adhesive isoforms as a result of signaling from the inside out.

Table 3
Patient demographics and clinical results
GroupThe patientAge/genderDisease (Klas�according Ann Arbor) The dose level [mg/m2/day]Purification of bone marrowBest response* (CR duration in months or weeks)
1171/mIC (immunocytoma), With Binet0,0005noSD
267/WMCL (b-cell lymphoma mantle zone), stage IV/A/E0,0005n.d.PD
367/mCLL (chronic lymphocytic leukemia), stage IV/B/E0,0005n.d.MR
2469/mMCL stage IV/B0,0015n.i.SD
549/mMCL stage IV/A/S0,0015 n.d.SD
671/mMCL stage IV/B/E0,0015n.i.PD
777/mMCL stage IV/B/E/S0,0015n.i.SD
865/mCLL, stage IV/B/E/S0,0015n.d.PD
975/mFL (follicular lymphoma),0,0015n.i.SD
Stage II/B
31058/mMCL, stage III/B/S0,005n.i. PD
1168/WFL, stage IV/B0,005n.d.SD
1265/mMCL, stage III/A/E0,005n.i.SD
4a1360/mSLL, stage IV/B/S0,015FullPR
1473/mMCL, stage II/A/E0,015n.i.SD
1544/mFL, stage IV/B/E/S0,015PartialPR
1661/mFL, stage IV/A/S0,015FullCR (7 m�S.)
1767/mMZL (marginal zone lymphoma), stage IV/B/S0,015n.i.n.e.
1864/mFL, stage IV/A/E0,015n.i.PD
1975/mMCL, stage III/A0,015n.i.n.e.
2065/WFL; stage III/A0,015n.i.SD
2160/mMCL stage IV/A/E0,015noSD
2267/WFL, stage IV/B0,015Full2367/mDLBCL (diffuse large B-cell lymphoma), stage I II/B0,015n.i.n.e.
2465/WFL, stage III/A0,015n.d.SD
2574/WWD (Whipple's), stage IV/B0,015PartialSD
52667/mMCL, stage0,03FullSD
IV/A
2748/mFL, stage III/A0,03n.i. PD
2858/mMCL, stage III/A0,03n.i.CR (10 months+)
2945/fMCL stage IV/B0,03PartialPD
3059/mMZL, stage III/A0,03n.i.n.e.
3143/mFL, stage III/A0,03n.i.MR
63272/mMCL stage IV/A0,06FullPR
3355/mMCL stage IV/B0,06FullCR (4 months+)
3452/mFL, stage IV/A0,06n.i.CRb(1 week+)
*Centrally confirmed complete (CR) and partial (PR) response according to Cheson criteria bold; MR - minimal response (≥25 to <50%); SD - stable disease; PD - progressive disease; in parentheses is given the period of time from the first documented confirmation response; + means existing at the moment the response.
aGroup 4 was increased with the aim of the study three different modes of initiation of treatment.
bPR after 8 weeks of treatment, which was passed in CR after additional 4-week cycle of treatment with the same dose after 7 weeks without treatment.
n.e.: not evaluated, because the treatment period was less than 7 days.
n.d.: not determined (with infiltration, but the second biopsy after treatment, did not do).
n.i.: without infiltration at the beginning of treatment.

4 (11,8)
Table 4
The frequency of adverse events observed during the treatment
The adverse events occurring in at least 3 patients (N=34)Grade 1-4 N (%)The degree 3-4 N (%)
Hyperthermia22 (64,7)2 (5,9)
Leukopenia21 cases (61.8)11 (32,4)
Lymphopenia21 cases (61.8)21 cases (61.8)
Coagulopathy (increased number of D-dimers)16 (47,1)6 (17,6)
Enzyme abnormality (AP, LDH, CRP)16 (47,1)10 (29,4)
Violation of liver function (ALT, AST, GGT)16 (47,1)1 (2,9)
Anaemia13 (38,2)5 (14,7)
Chills13 (38,2)0 (0,0)
Headache12 (35,3)1 (2,9)
Hypokalemia12 (35,3)2 5,9)
Thrombocytopenia12 (35,3)6 (17,6)
The increase in mass12 (35,3)0 (0,0)
Hyperglycemia11 (32,4)2 (5,9)
Neutropenia11 (32,4)8 (23,5)
Hematuria10 (29,4)0 (0,0)
Peripheral edema10 (29,4)2 (5,9)
Anorexia9 (26,5)1 (2,9)
Diarrhea9 (26,5)0 (0,0)
Weight9 (26,5)0 (0,0)
Fatigue8 (23,5)1 (2,9)
Proteinuria8 (23,5)0 (0,0)
Hypocalcemia7 (20,6)2 (5,9)
impaired function of pancreatic enzymes 7 (20,6)0 (0,0)
Cough6 (17,6)0 (0,0)
Dyspnoea6 (17,6)0 (0,0)
Back pain5 (14,7)0 (0,0)
Pain at the site of catheter insertion5 (14,7)0 (0,0)
Hyperbilirubinemia5 (14,7)2 (5,9)
Hypoalbuminemia5 (14,7)0 (0,0)
Hypogammaglobulinemia5 (14,7)1 (2,9)
Hypoproteinemia5 (14,7)0 (0,0)
Pleural effusion5 (14,7)1 (2,9)
Vomiting5 (14,7)0 (0,0)
Asthenia4 (11,8)1 (2,9)
State of confused consciousness0 (0,0)
Constipation4 (11,8)0 (0,0)
Dizziness4 (11,8)0 (0,0)
Hypertension4 (11,8)0 (0,0)
Hyponatremia4 (11,8)2 (5,9)
Dry mucous membranes4 (11,8)0 (0,0)
Muscle spasms4 (11,8)0 (0,0)
Nausea4 (11,8)0 (0,0)
Night sweats4 (11,8)0 (0,0)
Abdominal pain3 (8,8)1 (2,9)
Ascites3 (8,8)0 (0,0)
Hypercoagulability3 (8,8)0 (0,0)
Hyperhidrosis3 (8,8)Hypoglobulinemia3 (8,8)0 (0,0)
Insomnia3 (8,8)0 (0,0)
Disorder liver3 (8,8)1 (2,9)
Nasopharyngitis3 (8,8)0 (0,0)
Itching3 (8,8)0 (0,0)

Used abbreviations: AE - adverse phenomenon; AP - alkaline phosphatase; LDH - lactate dehydrogenase; CRP - C-reactive protein; ALT-abanindranath; AST - aspartatamino; GGT - gamma-glutamyl transferase; data AE with an additional cycle of treatment of the patient 34 is still enabled.

As explained above, conventional CD3 binding molecule (described, for example, in WO 99/54440) capable of contact-dependent environment epitope, even functional, lead to the undesired effect of redistribution of T cells from patients calling in adverse CNS effects. In contrast, the binding molecules of the present invention, contacting independent from the environment of the N-terminal amino acids 1-27 of CD3-Epsilon chains, do not cause t�fir effects of redistribution of T cells. Consequently, the CD3-binding molecules of the invention are associated with a better safety profile compared with conventional CD3 binding molecules.

14. Bespecifically CD3-binding molecule of the invention, the inducing mediated T-cell lysis of target cells by recognition of surface antigen of the target, Deplete antigen-positive target cells in vivo

Bespecifically CD3-binding molecule of the invention that recognizes CD33 as an antigen target, drains CD33-positive circulating monocytes in the peripheral blood of cynomolgus macaques

The design of CD33-AF5 VH-VL × I2C VH-VL (amino acid sequence: SEQ ID NO: 267) was produced by expression in Cho cells using nucleotide sequence encoding SEQ ID NO: 268. Coding sequence (1) N-terminal leader peptide of the heavy chain of immunoglobulin containing a start codon embedded in a consensus Kozak sequence, and (2) the C-terminal His6-tag followed by a stop codon were both attached in frame readout, to the nucleotide sequence of SEQ ID NO: 268, and then the resulting DNA fragment obtained by gene synthesis, inserted in the multiple cloning site expressing vector pEF-DHFR (Raum et al. Cancer Immunol. Immunother. 50 (2001) 141-150). Stable transfection defective in DHFR �notches SSS selection of DHFR-positive transfectants secreting CD3-binding molecule CD33-AF5 VH-VL × I2C VH-VL in the culture supernatant and gene amplification with methotrexate to enhance expression levels was performed as described in Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025. Analytical SEC-profile of CD33-AF5 VH-VL x I2C VH-VL for use in cynomolgus macaques showed that the test substance is almost entirely comprised of monomer. The effectiveness of the test substances was measured in the analysis of cytotoxicity, as described in Example 16.5, using cells SNO, transfetsirovannyh CD33 cynomolgus macaque, as target cells and T-cell line 4119LnPx macaque as a source of effector cells (Fig.25). It was determined that the concentration of CD33-AF5 VH-VL × 12C VH-VL required for premaxillae lysis of target cells (EC50) effector T-cells, was 2.7 ng/ml.

Young (aged approximately 3 years) adult cynomolgus macaques (Masasa fascicularis) was administered by continuous intravenous infusion of CD3-binding molecule CD33-AF5 VH-VL x 12C VH-VL at different flow rates (i.e. at different dose levels for the study of the depletion of circulating CD33-positive monocytes in the peripheral blood. This situation is equivalent to the traditional treatment CD3-binding molecule CD19×CD3 (specific to CD19 on b cells and CD3 on T-cell�) those patients with B-NHL, have circulating CD19-positive target b cells (see, e.g., WO 99/54440). Depletion of circulating CD19-positive target b cells In peripheral blood was consistent with the equivalent overall clinical efficacy of traditional CD3-binding molecules (CD19×CD3, proposed in WO 99/54440) in patients with CD19-positive b-cell malignani, similar B-NHL. Similarly, depletion of circulating CD33-positive monocytes in the peripheral blood is considered as an adequate equivalent overall clinical efficacy of CD33-directed bespecifically CD3-binding molecules according to the invention, such as CD33-AF5 VH-VL x I2C VH-VL in patients with CD33-positive myeloid malignani, such kbkaml (acute myeloid leukemia).

Continuous infusion was carried out using a rotary mechanism as follows: the Monkeys through the femoral vein into a hollow tail vein venous catheter. The advance the catheter subcutaneously to the back shoulder area and brought to the surface of the body near the rear of the blade. Then the tube is passed through the corset and protective spring. The corset is fastened around the animal, and through the catheter tube is attached to the infusion pump.

Solution for injection (1.25 M lysine and 0.1% Tween 80, pH 7) without test substance was administered by continuous infusion speed�and 48 ml/24 h for 7 days before treatment, so animals are accustomed to the conditions of infusion. Treatment was started by adding the test substance CD33-AF5 VH-VL x I2C VH-VL in a solution for injection in an amount necessary for each individual tested dose level (i.e., the flow rate CD33-AF5 VH-VL x 12C VH-VL). Infusion reservoir was changed every day during all phases of addiction and treatment. The planned duration of treatment was 7 days, except for the dose level of 120 mg/m2/24 h, during which the animals were treated for 14 days.

The course of change in time of the absolute numbers of circulating T cells and CD33-positive monocytes was determined by 4 - or 3-color FACS analysis, respectively.

Collect blood samples and standard analysis

Blood samples (1 ml) was obtained before the start of continuous infusion and through 0,75, 2, 6, 12, 24, 30, 48, 72 hours after the start of continuous infusion MCSP-G4 VH-VL x I2C VH-VL as well as after 7 and 14 days (and after 9 days at the dose level of 120 mg/m2/24 h) treatment using EDTA-containing tubes Vacutainer™ (Becton Dickinson) which were shipped for analysis at 4°C. In some cases, for practical reasons, there have been small variations of these time points. FACS analysis of lymphocyte subpopulations was performed within a time period 24-48 h after collection of blood samples. The absolute number of subpopulations of leukocytes in blood samples was determined� differential blood count was normal veterinary laboratory,

The allocation RVMS sample

RVMS (mononuclear cells of peripheral blood) were allocated in accordance with the Protocol described above in Example 13, with the adjustments used in the volumes.

Staining RVMS antibodies with a fluorescent tag molecules against the cell surface

Monoclonal antibodies capable of reacting with antigens Javanese macaque, were obtained from Becton Dickinson (1catalogue number 345784,2No. no directory 556647,3catalogue number 552851,6catalogue number 557710), Beckman Coulter (4catalogue number IM2470) and Miltenyi (5catalogue number 130-091-732) and used in accordance with the manufacturer's recommendations. From 5×105to 1×106cells were stained using the following combination of antibodies: anti-CD141(FITC) × anti-CD562(RE) × anti-CD33 (PerCP) × anti-CD194(APC) and anti-CD141(FITC) × anti-CD335(RE) × anti-CD166(Alexa Fluor 647™). An extra stage was performed as described in Example 13 above.

Flow-cytometric determination of stained lymphocytes FACS-analysis

The data collection was carried out using 4-color BD FACSCalibur™ (Becton Dickinson). For each measurement took 1×104cells of certain subpopulations of lymphocytes. To get the percentage of lymphocyte subpopulations and to classify intensities of expression of cellular molecules� surface, carried out statistical analysis using the program CellQuest Pro™ (Becton Dickinson). Then the percentage of subpopulations of lymphocytes relative to total lymphocytes (i.e., b cells plus T cells plus MK-cells, with the exception of myeloid cells, CD13/14-staining) according to the FACS correlated with the number of lymphocytes resulting from the differential blood test to count the absolute number of t cells (CD3+, CD56-, CD13/14-). The absolute number of CD33-positive monocytes was calculated by multiplying the number of monocytes obtained by the differential blood analysis, the corresponding relationship CD33-positive monocytes (CD33+, CD14+to all of the monocytes (CD14+) according to the FACS.

The percentage compared to baseline (i.e. 100%) absolute number of circulating CD33-positive monocytes at the end of treatment with the use of CD33-AF5 VH-VL x I2C VH-VL in 4 groups of 2 Javanese macaques with increasing doses within the group from 30 to 60 and from 240 to 1000 μg/m2/24 h is shown in Fig.26A.

As shown in Fig.26A, a continuous intravenous infusion of CD33-AF5 VH-VL × I2C VH-VL induces depletion of circulating CD33-positive monocytes dose-dependent manner. Despite the fact that at 30 μg/m2/24 h still was not observed nick�whom significant depletion of circulating CD33-positive monocytes, the first change in the direction of decreasing the number of CD33-positive monocytes was noticeable at 60 μg/m2/24 hours after 7 days of treatment. At 240 μg/m2/24 h circulating CD33-positive monocytes were almost completely depleted in the peripheral blood after 3 days of treatment. This was achieved faster at 1000 μg/m2/24 h, when the depletion of circulating CD33-positive monocytes in peripheral blood was completed already after 1 day of treatment.

These data were confirmed by the results shown in Fig.26B, showing the depletion of circulating CD33-positive monocytes by two-thirds and 50 per cent compared to the corresponding baseline levels in two cynomolgus macaques treated by continuous infusion of CD33-AF5 VH-VL × 12C VH-VL at the dose of 120 µg/m2/24 h for 14 days.

This result clearly indicates the clinical efficacy of the CD3-binding molecules of the invention in General, and bespecifically CD33-directed CD3-binding molecules according to the invention for the treatment of CD33-positive malignum, such as AML, in particular. In addition, the redistribution of T cells during the initial phase of treatment with the use of CD33-AF5 VH-VL × I2C VH-VL in the presence of circulating target cells (i.e., CD33-positive monocytes) was less pronounced than the redistribution of T cells during the initial phase Leche�Oia with traditional CD19×CD3-designs, as described in WO 99/54440 for patients with B-NHL with a significant number of circulating target cells (i.e., CD19-positive b-cells), as shown in Fig.22. Although T cells disappear from the blood after the start of infusion of CD19×CD3 and not to reappear until circulating CD19-positive target b cells will not be depleted in the peripheral blood (Fig.22), the initial disappearance of circulating T cells is not complete after the start of infusion of CD33-AF5 VH-VL × I2C VH-VL, and the number of T cells will still recover, while there are circulating CD33-positive target cells (Fig.(26B). This is proof that the CD3-binding molecule of the invention (directed against and generated against epitope CD3ε(Epsilon) chain of human and Primate, non chimpanzees, and represents a part or a fragment or a full-sized amino acid sequence represented in SEQ ID NO: 2, 4, 6 or 8), due to the recognition independent from the environment of the CD3 epitope showed a more favourable profile of redistribution of T cells than conventional CD3 binding molecules that recognize dependent environment the CD3 epitope, such binding molecules, proposed in WO 99/54440.

15. CD3-binding molecules according to the invention is aimed at essentially independent of the environment apito�s CD3, due to the induced less redistribution of circulating T cells in the absence of circulating target cells reduces the risk of adverse events associated with the beginning of treatment

Reduced redistribution of T cells in cynomolgus macaques after the start of treatment representative CD3-binding molecule with cross-species specificity according to the invention

The design of MCSP-G4 VH-VL × 12C VH-VL (amino acid sequence: SEQ ID NO: 193) was produced by expression in Cho cells using nucleotide sequence encoding SEQ ID NO: 194. Coding sequence (1) N-terminal leader peptide of the heavy chain of immunoglobulin containing a start codon embedded in a consensus Kozak sequence, and (2) the C-terminal His6-tag followed by a stop codon were both attached in frame readout, to the nucleotide sequence of SEQ ID NO: 194, then inserted the resulting DNA fragment obtained by gene synthesis into the multiple cloning site expressing vector pEF-DHFR (Raum et al. Cancer Immunol. Immunother. 50 (2001) 141-150). Stable transfection defective in DHFR cells SNO, selection of DHFR-positive transfectants secreting CD3 binding molecule MCSP-G4 VH-VL x I2C VH-VL in the culture supernatant and gene amplification with methotrexate to enhance expression levels was performed�, as described in Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025. The test substance for the treatment of cynomolgus macaques had produced in 200-liter fermenter. For protein purification from collecting used affinity chromatography IMAC is aimed at the C-terminal His6-tag on MCSP-G4 VH-VL x I2C VH-VL, with subsequent pressure preparative chromatography (SEC). The overall yield of the final, not containing endotoxin, the test substance was 40 mg of the Test substance consisted of 70% of monomers, 30% of dimers and contained a small amount of impurity multimers of higher order. The effectiveness of the test substances was measured in the analysis of cytotoxicity, as described in Example 11, using cells SNO, transfetsirovannyh MCSP cynomolgus macaque, as target cells and T-cell line 4119LnPx macaque as a source of effector cells (Fig.27). It was determined that the concentration of MCSP-G4 VH-VL × I2C VH-VL required for premaxillae lysis of target cells (EC50) effector T-cells, is 1.9 ng/ml.

Young (aged approximately 3 years) adult cynomolgus macaques (Masasa fascicularis) was administered continuous intravenous infusion of CD3 binding molecule MCSP-G4 VH-VL × I2C VH-VL at different flow velocities (i.e., at different dose levels) to study the redistribution of circulating T cells after initiation of treatment in the absence of� circulating target cells. Despite the fact that the CD3-binding molecule MCSP-G4 VH-VL × I2C VH-VL can recognize as MCSP Javanese macaque, and CD3 Javanese macaque, there was no circulating blood cells expressing MCSP. Therefore, the only form of interaction that is possible in the circulating blood, is binding CD3-specific "shoulder" MCSP-G4 VH-VL × I2C VH-VL CD3 on T-cells. This situation is equivalent to the traditional treatment CD3-binding molecule (CD19×CD3 binding molecule specific to CD19 on b cells and CD3 on T cells) in patients with B-NHL who have no circulating CD19-positive b-cells-targets, as described in Example 13.

Continuous infusion was carried out using a rotary mechanism as follows: the Monkeys through the femoral vein into a hollow tail vein venous catheter. The advance the catheter subcutaneously to the back shoulder area and brought to the surface of the body near the rear of the blade. Then the tube is passed inside of the corset and protective spring. The corset is fastened around the animal, and the catheter attached to the infusion pump via a tube.

Solution for injection (1.25 M lysine and 0.1% Tween 80, pH 7) without test substance was administered by infusion continuously at a speed of 48 ml/24 h for 7 days before treatment that the animals are accustomed to the conditions of infusion. Treatment was started by doba�ing a test substance MCSP-G4 VH-VL × I2C VH-VL in a solution for injection in a quantity required for each individual tested dose level (i.e., the flow rate MCSP-G4 VH-VL × I2C VH-VL). The reservoir for the infusion was changed every day during all phases of addiction and treatment. The duration of treatment was 7 days.

The course of change in time of the absolute numbers of T cells in peripheral blood was determined by 4-color FACS analysis as follows:

Collect blood samples and standard analysis

Blood samples (1 ml) was obtained before the start of continuous infusion and through 0,75, 2, 6, 12, 24, 30, 48, 72 hours after the start of continuous infusion MCSP-G4 VH-VL x I2C VH-VL as well as after 7 days of treatment using EDTA-containing tubes Vacutainer™ (Becton Dickinson) which were shipped for analysis at 4°C. In some cases, for practical reasons, there have been small variations of these time points. FACS analysis of lymphocyte subpopulations was performed within a time period 24-48 h after collection of blood samples. The absolute number of subpopulations of leukocytes in blood samples was determined by differential blood count was normal veterinary laboratories.

The allocation RVMS sample

The allocation RVMS carried out in accordance with the Protocol described in Example 13 above, with the adjustments used in the volumes.

Staining RVMS antibodies with a fluorescent tag molecules against the cell surface

Monoclinic�tional antibodies, able to react with antigens Javanese macaque, were obtained from Becton Dickinson (1catalogue number 345784,2catalogue number 556647,3catalogue number 552851) and Beckman Coulter (4catalogue number IM2470) and used in accordance with the manufacturer's recommendations. From 5×105to 1×106cells were stained using the following combination of antibodies: anti-CD141(FITC) × anti-CD562(RE) × anti-CD33 (PerCP) × anti-CD194(ASC). An extra stage was performed as described in Example 13 above.

Flow-cytometric determination of stained lymphocytes FACS-analysis

The data collection was performed using a 4-color BD FACSCalibur™ (Becton Dickinson). For each measurement took 1×104cells of certain subpopulations of lymphocytes. To get the percentage of lymphocyte subpopulations and to classify intensities of expression of cell surface molecules, carried out statistical analysis using the program CellQuest Pro™ (Becton Dickinson). Then the percentage of subpopulations of lymphocytes relative to total lymphocytes (i.e., b cells plus T cells plus NK cells, with the exception of myeloid cells, CD13/14-staining) according to the FACS analysis correlated with the lymphocyte count obtained as a result of differential blood test to count the absolute number of T cells (CD3+ , CD56-, CD13/14-).

The redistribution of T cells during the initial phase of treatment with the use of MCSP-G4 VH-VL x I2C VH-VL in cynomolgus rhesus at dose levels of 60, 240 and 1000 μg/m2/24 h is shown in Fig.28. These animals never showed any signs of redistribution of T cells during the initial phase of treatment, i.e. the number of T-cells rather increased than decreased after the start of treatment. Taking into account that the redistribution of T cells constantly observed in 100% of patients without circulating target cells after the start of treatment traditional CD3-binding molecule (e.g. design CD19×CD3, as described in WO 99/54440) against the dependent environment of the CD3 epitope, it was demonstrated that significantly less redistribution of T cells in the absence of circulating target cells after the start of treatment can be observed when applying the CD3-binding molecule of the invention, directed and developed against an epitope of CD3-Epsilon-chain of human rights and the primacy of non-chimpanzee as defined any amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, or its fragment. This clearly differs from the case of CD3-binding molecules directed against dependent environment of the CD3 epitope, similar to the designs described in WO 99/54440. Binding molecules against independent from the environment epic�POV, as they suggested (among other things) to any of SEQ ID NO: 2, 4, 6 or 8 (or fragments of these sequences), provide significantly less (harmful and unwanted) redistribution of T cells. Because the redistribution of T cells during the initial phase of treatment with CD3 binding molecules is a major risk factor in adverse CNS events, CD3-binding molecules proposed in the present description and is able to recognize an independent environment from the CD3 epitope, have a significant advantage compared with CD3 binding molecules known in the art and directed against dependent environment of CD3 epitopes. Indeed, none of Javanese macaques, who were treated with MCSP-G4 VH-VL × I2C VH-VL, showed no signs of CNS symptoms.

According to the present invention proposed independent from the environment of the CD3 epitope, and it corresponds to the first 27 N-terminal amino acids of CD3-Epsilon or fragments of this stretch of 27 amino acids. This is independent of the environment epitope is removed from its native environment in the CD3 complex and subjected to fusion with a heterologous amino acid sequences without losing its structural integrity. Anti-CD3-binding molecules, as proposed according to the invention and created (and directed) against an independent otobrajenie epitope of CD3-Epsilon, provide unexpected clinical improvement in relation to the redistribution of T cells and, consequently, a more favorable safety profile. Without any connection with theory, since the CD3 epitope does not depend on the environment, making it an Autonomous self-sufficient subdomain without much impact on the rest of the CD3-complex, CD3-binding molecules, as proposed according to the invention, to a lesser extent, induce allosteric changes in the conformation of CD3 than conventional CD3 binding molecules (analogous to the molecules proposed in WO 99/54440) that recognize dependent environment epitopes of CD3, such molecules proposed in WO 99/54440. As a consequence (again, without communication with theory), induction of intracellular recruitment of NcK2 under the action of the CD3-binding molecules, as proposed according to the present invention, there is also decreased, leading to less pronounced transition of T-cell integrins in their isoforms and lesser adhesion of T cells to endothelial cells. Preferably, the preparations CD3-binding molecules of the invention (directed against and generated against independent from the environment epitope, as defined in this description) consist essentially of the monomer molecules. These monomer molecules are even more effective (than dimeric or multimeric molecules) to cancel pererasprede�t of T cells and, consequently, the risk of adverse effects on CNS phenomena during the initial phase of treatment.

16. Establishment and characterization of CD33 and CD3 bespecifically single-stranded molecules with cross-species specificity

16.1. Receipt of CHO cells expressing human CD33

The encoding sequence of human CD33, which is published in GenBank (access number NM_001772), was obtained by gene synthesis according to standard protocols. The fragment of gene synthesis was designed so that it contained the first website Kozak for eukaryotic expression of this design, then immunoglobulin leader peptide of 19 amino acids, then, in reading frame, encoding the Mature sequence of the protein CD33 person, then, in reading frame, encoding the sequence of the dipeptide serine-glycine, histidine6-tag and a stop codon (the cDNA sequence and amino acid sequence of this design is presented in SEQ ID NO: 305 and 306). The fragment of gene synthesis was also designed in such a way as to introduce restriction sites at the beginning and end of the fragment. The introduced restriction sites, EcoRI at the 5'end and Sail at the 3'-end, was used in subsequent cloning procedures. The fragment of gene synthesis was cloned via EcoRI and Sail into a plasmid designated pEF-DHFR (pEF-DHFR is described in Raum et al. Cancer Immunol. Immunother. 50 (2001) 141-10), following the standard protocols. The above procedures were performed according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (2001)). A clone with a confirmed nucleotide sequence was transfusional defective in DHFR cells SSS for eukaryotic expression of this design. Eukaryotic protein expression in defective in DHFR cells SSS was performed as described in Kaufmann R. J. (1990) Methods Enzymol. 185, 537-566. Gene amplification constructs was induced by increasing concentrations of methotrexate (MTX) to a final concentration of 20 nm MTX, inclusive.

16.2. Obtaining cells SNO expressing CD33 extracellular domain macaque

The cDNA sequence macaque CD33 received a series of 3 PCR on cDNA from macaque bone marrow according to standard protocols. Used the following reaction conditions: 1 cycle at 94°C for 3 min, then 35 cycles at 94°C for 1 min, 53°C for 1 minute and at 72°C for 2 min, then a final cycle at 72°C for 3 minutes and the following primers:

1. direct primer:

5'-gaggaattcaccatgccgctgctgctactgctgcccctgctgtgggcaggggccctggctatgg-3' (SEQ ID NO: 369)

reverse primer: 5'-gatttgtaactgtatttggtacttcc-3' (SEQ ID NO: 370)

2. direct primer: 5'-attccgcctccttggggatcc-3' (SEQ ID NO: 371) reverse primer: 5'-gcataggagacattgagctggatgg-3' (SEQ ID NO: 372)

3. direct primer: 5'-gcaccaacctgacctgtcagg-' (SEQ ID NO: 373) reverse primer: 5'-agtgggtcgactcactgggtcctgacctctgagtattcg-3' (SEQ ID NO: 374).

As a result of these PCR received three overlapping fragments, which were isolated and sequenced according to standard protocols using the PCR primers, and thus received a portion of the cDNA sequence macaque CD33 from the second nucleotide in the codon +2 to the third nucleotide in the codon +340 Mature protein. To create a construct for expression of macaque CD33 cDNA fragment was obtained by gene synthesis according to standard protocols (cDNA sequence and amino acid sequence of this design is presented in SEQ ID NO: 307 and 308). In this design sequence encoding macaque CD33 from amino acids +3 to +340 Mature CD33 protein was subjected to fusion with the coding sequence of human CD33, replacing encoding the human sequence, con