Bispecific binding agents with interspecific specificity

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a polypeptide containing two binding fragments presented by antibodies; the first of them binds to CD3e(epsilon) chain epitope of a human or a primate, other than a chimpanzee, particularly Callithrix jacchus, Saguinus oedipus and Saimiri sciureus; the second one - to EGFR, Her2/neu or IgE of a human or a primate, other than a chimpanzee, with the above CD3e epitope containing the amino acid sequence Gln-Asp-Gly-Asn-Glu. There are also disclosed a coding sequence of the nucleic acid, a vector, a host cell and a method for preparing the above peptide, as well as a pharmaceutical composition and using the polypeptide in preventing, treating or relieving a proliferative disease, a malignant disease or an immunological disorder.

EFFECT: invention provides the clinical improvement of T-cell redistribution and the enhanced safety profile.

17 cl, 8 tbl, 26 dwg, 26 ex

 

The present invention relates to a polypeptide containing the first binding domain able to contact the CD3 epitope (from the English. cluster of differentiation - cluster of differentiation) (Epsilon) of the person and the primacy of non-chimpanzee, and a second binding domain capable of contact with EGFR (the receptor for epidermal growth factor), Her2/neu (member of the family of receptors for epidermal growth factor) or IgE (immunoglobulin E) human and/or Primate, not a chimpanzee, as well as to a method for producing the above-mentioned polypeptide. In addition, the invention relates to nucleic acids coding for the polypeptide, vectors containing the data nucleic acids, and cells of the host containing the vector. In another aspect according to the invention proposed pharmaceutical composition containing the above-mentioned polypeptide, and the proposed medical use of this polypeptide.

T-cell recognition is mediated clonotypes distributed alpha-beta and gamma-Delta T-cell receptors (TcR) that interact with the peptide-loaded molecules (complex) peptide-MHC (major histocompatibility complex) (rmns) (Davis & Bjorkman, Nature 334 (1988), 395-402). Antigen-specific chain TcR do not contain domains of signaling, but instead are associated with conservative mnogozaryadnymi complexo the 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 directly related with the handset signal transmission, remains a fundamental question of T-cell biology (Alarcon, above; Davis, Ceil 110 (2002), 285-287). It seems obvious that in a stable T-cell responses involved contact with the receptor oligomerization TcR 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 the signal through the TcR occurs in the absence of these events and may involve ligand-induced conformational change in CP3-Epsilon (Alarcon, above, Davis (2002), above, 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 CD3 Epsilon-gamma-heterodimer, CD3 Epsilon-Delta of heterodimer and CD3 Zeta-Zeta of glycosilated (Call, above). In various studies found that the presence of CD3 molecules is important for the proper expression 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). On the basis of the allowed patterns of fragments ectodomain mouse CD3 Epsilon-gamma-heterodimer shown that both the Epsilon-gamma-subunit represents a C2-like C2-set Ig domains, which interact with each other with the formation of an unusual dimeric configuration type "side-to-side" (Sun, Cell 105 (2001), 913-923). Although enriched in cysteine residues of the "stem" (stalk), seems to play an important role in the launch of the dimerization CD3 (Su, above, Borroto, J. Biol. Chem. 273 (1998), 12807-12816), through interaction of the extracellular domains of the CD3-Epsilon and CD3-gamma is sufficient for the complex formation of these proteins with TcR-beta (Manolios, Eur. J. Immunol. 24 (1994), 84-92, Manolios & Li, Immunol. Cell Biol. 73 (1995), 532 to 536). Although this point is still controversial, the predominant stoichiometry TcR, most likely contains one alpha-beta TcR, one CD3 Epsilon-gamma heterodimer, one CD3 Epsilon-Delta heterodimer and one CD3 Zeta-Zeta-glycosilated (Call, above). The Central role of CD3 Epsilon-gamma-heterodimer human immune response is understandable in connection with the recent establishment of the crystal structure of this complex is associated with a therapeutic antibody OKT (Kjer-Nielsen, PNAS, 101 (2004), 7675-7680).

Many therapeutic strategies modulate T-cell immunity by targeted impact on TcR signaling, in particular monoclonal antibodies (mAb) against human CD3, which are widely used in the clinic in the schemes of immunosuppressive funds. CD3-specific murine mAb ACT was the first mAb approved for use in humans (Sgro, Toxicology 105 (1995), 23-29), and it is widely the use in the clinic as an immunosuppressive agent in transplantation (Chatenoud, Clin. Transplant. 7 (1993), 422-430, Chatenoud, Nat. Rev. Immunol. 3 (2003), 123-132, Kumar, Transplant. Proc. 30 (1998), 1351-1352), type 1 diabetes (Chatenoud (2003), above) and psoriasis (Utset, J. Rheumatol. 29 (2002), 1907-1913). Moreover, anti-SR mAb can induce partial T-cell signaling and clonal tolerance (Smith, J. Exp. Med. 185 (1997), 1413-1422). OCT been described in the literature as a potent T-cell mitogen (Van Wauve, J. Immunol. 124 (1980), 2708-18), as well as a powerful T-cell killer (Wong, Transplantation 50 (1990), 683-9). OCT shows the time-dependent change both of these activities: after an early activation of T cells, leading to cytokine release and subsequent introduction ACT blocks all known T-cell function. Thanks to this later to block T-cell function for AKT was shown such wide application as immunosuppressant in treatment regimens to reduce or even eliminate the rejection of tissue allograft.

OCT draws the rejection of tissue allograft most likely by blocking the function of T cells, which play 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-recognizing structure of T-cells (TCR) and is important for signal transduction. Numerous studies concerned how the I of the subunits of the TCR/CD3 associated with OCT. However, there are some indications specificity ACT in respect of the Epsilon-subunit complex of the TCR/CD3 (Tunnacliffe, Int. Immunol. 1 (1989), 546-50; Kjer-Nielsen, PNAS 101 (2004), 7675-7680). According to other sources it is shown that for binding ACT with complex TCR/CD3 need the presence of other subunits of this complex (Salmeron, J. Immunol. 147 (1991), 3047-52).

Other known antibodies with the specificity of the CD3 molecule are listed in Tunnacliffe, Int. Immunol. 1 (1989), 546-50. As noted above, such CD3-specific antibodies are able to induce different T-cell responses, such as 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, "Limphociting Typing II” 1 (1986), 223). That is, depending on the experimental conditions CD3-specific monoclonal antibody can either inhibit or 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 Immunol. 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 many antibodies against CD3, described in the art, according to reports, recognize CD3-Epsilon-subunit of CD3-complex, most of them are in fact associated with conformational epitopes and, therefore, from the onset CD3-Epsilon only in the native environment TCR. Conformational epitopes are characterized by the presence of two or more individual amino acid residues that are spaced apart in the primary sequence, but converge on the surface of the molecule, when the polypeptide is folded in the native protein/antigen (Sela (1969) Science 166, 1365 and Laver (1990) Cell 61, 553-6). Conformational epitopes that bind with antibodies against CD3-Epsilon described in this technical field can be divided into two groups. In the main group these epitopes are formed by two subunits of CD3, such as CD3-Epsilon chain and CD3-gamma or CD3-Delta circuit. For example, several studies found that the most widely used monoclonal antibodies against CD3-Epsilon, namely OCT, WT31, UCHT1, 7D6 and Leu-4, was associated with the cells, transfitsirovannykh one CD3-Epsilon chain. However, these antibodies stained cells, transfetsirovannyh dual combination of CD3-Epsilon plus or CD3-gamma, or CD3-Delta (Tunnacliffe, above; Law, Int. Immunol. 14 (2002), 389-400; Salmeron, J. Immunol. 147 (1991), 3047-52; Coulie, Eur. J. Immunol. 21 (1991), 1703-9). In the second less numerous group conformational epitope formed in the CD3-Epsilon the subunit. A member of this group is, for example, mAb ARA 1/1, which is raised against denatured CD3-Epsilon chain (Risueno, Blood 106 (2005), 601-8). In total, the majority of antibodies against CD3-Epsilon described in the art will recognize Informatsionnye epitopes, located on two or more subunits of CD3. Individual amino acid residues that form the three-dimensional structure of these epitopes, can be localized either on the CD3-Epsilon the subunit, or CD3-Epsilon the subunit and other subunits of CD3, such as CD3-CD3 gamma or Delta.

Another problem associated with antibodies against CD3, is that antibodies against CD3, as it was discovered, are species-specific. Ahth-CD3 monoclonal antibody, which is usually the case for any other monoclonal antibodies, operated by highly specific recognition of their target molecules. They recognize only one site, or epitope on a target molecule CD3. For example, one of the most widely used and best characterized monoclonal antibodies specific for CD3 complex, is OCT-3. This antibody interacts with CD3 chimpanzees, 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). Ahth-CD3 monoclonal antibody UCHT-1 also interacts with CD3 from chimpanzees, but not CD3 from macaque (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, is directed is on macaque CD3 (Uda et al., J. Med. Primatol. 30 (2001), 141-147). Interesting fact that peripheral lymphocytes from approximately 12% of Javanese macukow do not interact with a monoclonal antibody against CD3 macaque-rhesus (FN-18) due to polymorphism of the CD3 antigen of Makarov. Uda et al described the replacement of two amino acids in the sequence CD3 Javanese of Makarov, resulting in no interaction with antibodies FN-18, compared with CD3 other animals that interact with antibodies FN-18 (Uda et al., J Med. Primatol. 32 (2003), 105-10; Uda et al., J. Med. Primatol. 33 (2004), 34-7).

In addition, this selective, i.e., species specificity, characterized monoclonal antibodies against CD3 and their fragments, is a significant obstacle to their development as therapeutic agents for the treatment of human diseases. In order to get permission to sell any new drug candidate must pass rigorous testing. This testing can be divided into preclinical and clinical phases: while the latter additionally includes well-known clinical phase I, II and III, conducted on patients people preceding carried out on animals. The purpose of preclinical testing is evidence that the drug candidate has the desired activity and, most importantly, the beach is safe. Only after the establishment of animal safety and possible efficacy of a drug candidate in preclinical testing of the drug candidate will receive the approval of the relevant regulatory authority to conduct clinical testing on humans. Drug candidates can be tested for safety on animals the following three ways: (1) on the relevant form, i.e. the form in which the drug candidate can recognize ontologicheskie antigens, (2) transgenic animal containing human antigens, and (3) by using the simulator of the medicinal product candidate, which can communicate with ontologicheskie antigens present in 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. Constraints simulator drug candidate, are in a different composition of substances compared to real drug candidate, and often animals used are rodents, with the limitation that discussed above. Thus, doclines the data obtained in rodents, have limited predictive ability in relation to drug candidate. The approach to selection of security testing is to use relevant species, preferably a lower Primate. Currently, the limitation relating to the CD3-binding molecules suitable for therapeutic intervention in humans and described in the art, is that relevant species are higher primates, particularly chimpanzees. Chimpanzees are an endangered species, and due to their human-like nature of the use of such animals to test the safety of the drug is banned in Europe and very limited elsewhere.

The present invention relates to a polypeptide containing the first binding domain, preferably a human, capable of contacting epitope CD3ε(Epsilon) chain of human and Primate, not a chimpanzee, and a second binding domain capable of contact with EGFR, Her2/neu or IgE of a human and/or Primate, not a chimpanzee, and the specified epitope is a part of the amino acid sequence that is a member of the group consisting of SEQ ID NO: 2, 4, 6 or 8. Sequence shown in SEQ ID NO: 2, 4, 6 and 8, and fragments thereof are independent of the environment (context independnt) CD3 epitopes.

The advantage of the present invention is to offer polypeptide containing the binding domain, preferably human, demonstrating cross-species specificity against CD3ε(Epsilon) chain of human and Primate, not a chimpanzee, which can be used for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains, preferably humans, primates, and, in identical form, as a medicinal product in humans. In preclinical animal studies and in clinical studies in humans can be used the same molecule. This results in a very comparable results and significant prognostic ability of animal studies compared with species-specific molecules simulator. In the present invention unexpectedly was identified by N-terminal containing amino acid residues 1-27, polypeptide fragment of the extracellular domain of CD3-Epsilon, which in contrast to all other known epitopes of the CD3-Epsilon described in this technical field retains its three-dimensional structural integrity when removed from its native environment in the CD3 complex (and merging with the heterologous amino acid sequence, such as Arcam (a molecule of edge the AI epithelial cells) or Fc region of immunoglobulin).

The phrase "independence from the environment" epitope of CD3 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", as used in this application with respect to the CD3 epitope, means that the binding is considered in this application is binding molecules/antibody molecules according to the invention does not change or modification of conformation, sequence or structure, environmental antigenic determinant or epitope. In contrast, the CD3 epitope recognized by conventional CD3 binding molecule (e.g., described in WO 99/54440 or WO 04/106380), localized in the C-terminal region of CD3 Epsilon-chain relative to the N-terminal 1-27 amino acids independent of the environment epitope, and he accepts the proper conformation only when immersed in the rest of Epsilon-chain and held in proper position by means of heterodimerization Epsilon-chain or gamma-, or Delta-chain of CD3.

Anti-CD3 binding molecules as part bespecifically binding molecule, which is proposed in this application is created (and directed) against independent from the environment epitope of CD3, provide unexpected clinical improvement for the redistribution of T cells and, therefore, breatheasy security profile. Without communication with theory believe that because the CD3 epitope does not depend on the environment, forming an Autonomous self-sufficient subdomain without much impact on the rest of the CD3 complex, CD3-binding molecules, as proposed in this application, to a lesser extent induce an allosteric change in the conformation CD3 than conventional CD3 binding molecules (similar molecules, as proposed in WO 99/54440) that recognize dependent environment CD3 epitopes.

Independence from the environment epitope of CD3 CD3 binding molecules according to the invention as part bespecifically binding molecules associated with less redistribution of T cells during the initial phase of treatment, CD3 binding molecules according to the invention, resulting in getting the best safety profile of CD3 binding molecules according to the invention compared with conventional CD3 binding molecules known in the art that recognize dependent environment CD3 epitopes. In particular, since the redistribution of T cells during the initial phase of treatment, CD3 binding molecules is a major risk factor for adverse in relation to CNS events, CD3-binding molecules according to the invention by recognition rather independent from the environment than dependent environment CD3 epitope have considerable Ave the property in terms of safety compared to the CD3-binding molecules, known in the art. Patients with these adverse in relation to Central nervous system events associated with the redistribution of T cells during the initial phase of treatment, conventional CD3 binding molecules, usually suffer from confusion and disorientation, in some cases also from 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. Usually the patient has difficulties with concentration, and the process of thinking is not only vague and unclear, but often much slower. Patients with an unfavorable relation to Central nervous system events associated with the redistribution of T cells during the initial phase of treatment, conventional CD3 binding molecules may also suffer from memory loss. Often confusion leads to loss of ability to distinguish between people and/or places, or to call time and date. Feelings of disorientation are usually in confusion, and impaired decision making. Adverse in relation to Central nervous system events associated with a redistribution of T cells during the initial phase of treatment, conventional CD3 binding molecules may also include confused speech and/or difficulty in finding words. This disorder can be the t worsen as expressiveness, and speech perception, and the process of reading and writing. In addition to urinary incontinence, vertigo and dizziness can also accompany unfavourable in respect of Central nervous system events associated with a redistribution of T cells during the initial phase of treatment, conventional CD3 binding molecules, in some patients.

Maintaining the three-dimensional structure within the mentioned 27-amino acid N-terminal polypeptide fragment of CD3 Epsilon can be used to create a binding domain, preferably humans, which are associated with the N-terminal polypeptide fragment of CD3 Epsilon in vitro and native CD3 complex (CD3-Epsilon-subunit of CD3 complex on T cells in vivo with the same affinity binding. These data are strictly indicate that the N-terminal fragment, as described in this invention, forms a tertiary conformation, which is similar to its structure, normally existing in vivo. It was a very sensitive test of 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 was replaced by alanine (alanine scanning) to test the sensitivity of amino acids 1-27 of the N-terminal polypeptide fragment of CD3 Epsilon to minor drug is the making. CD3-specific antibody molecules as part bespecifically binding molecules according to the invention is used to test the binding alanine mutants of amino acids 1-27 of the N-terminal polypeptide fragment of CD3 Epsilon (see attached Example 5). Individual replacement of the first five amino acid residues in the same N-terminal end of the fragment and two amino acids at positions 23 and 25 additional amino acids 1-27 of the N-terminal polypeptide fragment of CD3 Epsilon were critical for binding molecules are antibodies. Replacement of amino acid residues in positions 1 to 5, containing the remains of Q (glutamine in position 1), D (aspartic acid in position 2), G (glycine at position 3), N (asparagine at position 4) and E (glutamic acid at position 5), alanine abolished the binding of the binding molecules, preferably human, according to the invention with the specified fragment. While at least some binding molecules, preferably human, according to the invention, two amino acid residue at the C-end of the above-mentioned fragment of T (threonine at position 23) and I (isoleucine at position 25) reduced the binding energy of the binding molecules, preferably human, according to the invention.

Suddenly discovered that the selected thus binding molecules, preferably human, not only recognize the N-end is howling fragment of CD3 Epsilon of man, but also the corresponding homologous fragments CD3-Epsilon various primates, including monkeys of the New world (gruntovye, ordinary igranka (Cailithrix jacchus); Oedipus Tamarin (Saguinus oedipus), common squirrel monkey (Saimiri sciureus) and Old world monkeys (long-tailed macaques (Masasa fascicularis), also known as cynomolgus macaques; or Massa mulatta, also known as rhesus). Thus was established the specificity CD3 binding molecules according to the invention in many primates (multi-primate specificity). Further analyses of the sequences confirmed that man and apes have a common area with vysokomaslichnoy sequence at the N end of the extracellular domain of CD3 Epsilon.

In the present invention have found that you can create binding molecules, preferably human, specific for CD3 Epsilon, in preclinical animal testing, and clinical studies in humans and even in human therapy can be used the same molecule. This is due to the unexpected identification of binding molecules, preferably human, which in addition to binding to the CD3-Epsilon person (and likely due to genetic similarities with the equivalent of a chimpanzee), have also been associated with homologues of these antigens primates that are not chimpanzees, including obeshannogo Light and Old world monkeys. As shown in the following Examples, these CD3-Epsilon-specific binding molecules, preferably human, can be integrated in bespecifically single-chain antibodies with the aim of creating therapeutic agents against various diseases, including, but not limited to, cancer or immunological disorder. Thus, a need in the construction of the CD3-Epsilon-binding domain simulator or especifismo single-chain antibodies, including his own, to test phylogenetically distant (from the people), no. As a result, in preclinical animal testing can be used the same molecule, which is designed to introduce people in clinical testing, and to permit the sale and introduction as therapeutic drugs. The ability to use the same molecule for preclinical testing on animals, as with the later introduction people actually eliminates or at least greatly reduces the danger that the data obtained in preclinical animal testing, will have limited applicability in the case of man. Briefly, obtaining preclinical safety data animal using the same molecules that f is effectively will be to introduce people largely guarantees the applicability of these data for the scenario suitable for people. Conversely, in traditional approaches using molecules imitators, these molecules simulators must be tailored at the molecular level to the animal test system used for preclinical safety assessment. Thus, the molecule that is to be used in the treatment of people, in fact, differs in sequence and also, probably, on the structure of the molecule simulator used in preclinical testing, pharmacokinetic parameters and/or biological activity, resulting in the data obtained in preclinical animal testing, have limited applicability/suitability in the case of people. The use of molecules of simulators requires the design, obtaining, purification and characterization of a completely new design. This leads to additional cost and development time required for obtaining such molecules. In General, simulators should be developed separately in addition to real drug that will be used in the treatment of people, therefore, should be two lines of development for the two molecules. Therefore, the main advantage of linking molecules person or structures on the Nove antibodies demonstrating cross-species specificity described in this invention, is that the same molecule can be used for therapy in humans and in preclinical animal testing.

Preferably, the polypeptide according to the invention, the first binding domain capable of contact with the epitope of CD3-Epsilon chain of human and Primate, not a chimpanzee, had a human origin.

In addition, due to the human origin of the binding molecules of the person according to the invention, the development of an immune reaction against these binding molecules are eliminated as much as possible after the introduction of these binding molecules to patients-people.

Another main advantage of the CD3-Epsilon-specific binding molecules, preferably human, as part bespecifically binding molecules according to the invention is used for preclinical testing of various primates. The behavior of the drug candidate in animals in the ideal case, is a measure of the expected behavior of the drug candidate after the introduction of the people. In the end, the data obtained on the basis of such pre-clinical testing, therefore, will typically have a high predictive power in the case of the of the ne. However, as became clear from the tragic results of a recent clinical trial, phase I, TGN1412 (CD28 monoclonal antibody), a drug candidate can act on species of primates and humans: despite the fact that in pre-clinical testing of the specified antibodies are not observed or no observed only limited adverse effects in animal studies, conducted with cynomolgus macaques, 6 patients-people developed multi-organ failure after administration of the indicated antibodies (Lancet 368 (2006), 2206-7). The results of these undesirable adverse events suggest that the restriction of preclinical testing, only one species (primates) may be insufficient. The fact that the molecules according to the invention specifically bind to the CD3-Epsilon person, contact number of CD3 monkeys of the New world and the Old world, can help to overcome problems encountered in the above-mentioned case. In line with this, according to the present invention proposed means and methods to minimize species differences in the effects during the development and testing of drugs for human therapy.

In addition, when using CD3-Epsilon-binding domain, preferably human, with interspecies specificity as part bespecifically bonding mo is ecoli according to the invention is no longer necessary to adapt the test animal to the drug candidate, intended for the introduction of the people, as, for example, when creating transgenic animals. CD3-Epsilon-specific binding molecules (or the containing bespecifically single-chain antibodies, preferably human, demonstrating cross-species specificity according to the uses and methods according to the invention can be directly used in preclinical testing in primates, which are not chimpanzees, without any genetic manipulation of animals. As is well known to specialists in this field techniques, approaches, in which the test animal will adapt to the drug candidate, always bear the risk that the results obtained in preclinical safety testing will be less representative and to have less predictive value for humans due to the modification of the animal. For example, the transgenic animal proteins encoded by transgenes are often sverkhekspressiya in a high degree. Thus, the data obtained for the biological activity of antibodies against this protein antigen, may be limited in their predictive value for people who have this protein is expressed at a much lower, more physiological levels.

Another advantage of applications CD3-Epsilon-specific link is their molecules (or the containing bespecifically single-chain antibodies), preferably humans, demonstrating cross-species specificity, is the fact that chimpanzees as endangered species of animals, exclude from ongoing animal testing. Chimpanzees are the closest relatives of people and have recently been grouped in the family of hominids on the basis of data from genome sequencing (Wildman et al., PNAS 100 (2003), 7181). Therefore, in General it is believed that the data obtained with the use of chimpanzees, have a high predictive value for humans. However, because of their status as endangered species, the number of chimpanzees, which can be used for medical experiments is very limited. Therefore, as indicated above, the provision of chimpanzees to test on animals is a costly and ethical problem. Application of CD3-Epsilon-specific binding molecules, preferably human, according to the invention (or the containing bespecifically single-chain antibodies) takes as an ethical objections, and financial difficulties in the process of preclinical testing without damaging the quality, i.e. the applicability of the obtained animal test results. In light of this, the use of CD3-Epsilon-specific binding molecules (or the containing bespecifically single-chain antibodies, preferably human, who has a reasonable alternative research on chimpanzees.

Another advantage CD3-Epsilon-specific binding molecules, preferably human, according to the invention (or the containing bespecifically single-chain antibodies) is the possibility of selecting multiple blood samples when used as part of the preclinical testing on animals, for example during pharmacokinetic studies in animals. Repeated sampling of blood can be obtained much easier with the use of the primacy of non-chimpanzee than using the lower animals, such as mice. The selection of multiple blood samples enables continuous testing of blood parameters to determine the biological effects induced binding molecule (or bespecifically single-chain antibody), preferably human, according to the invention. In addition, the selection of multiple blood samples allows the researcher to assess the pharmacokinetic profile of a binding molecule (or especifismo single-chain antibodies, preferably human, as defined in this application. Besides the possible side effects that may be induced by the specified binding molecule (or bespecifically single-stranded antirelay), preferably human, which is reflected in the blood parameters that can be measured in different samples of blood, otobrannyh during the introduction of the indicated antibodies. This allows you to determine possible toxicity profile of binding molecules (or especifismo single-chain antibodies, preferably human, as defined in this application.

The advantages of linking molecules (or bespecifically single-chain antibodies, preferably human, as defined in this application, demonstrating cross-species specificity, briefly can be summarized as set forth below.

First, a binding molecule (or bespecifically single-chain antibodies, preferably human, as defined in this application, used in preclinical testing, the same that are used in the treatment of people. Thus, it is no longer necessary in the development of two independent molecules, which may differ in their pharmacokinetic properties and biological activity. A huge advantage is that, for example, the pharmacokinetic results can be more directly transferred to the human environment (human setting) and apply to it than, for example, in the traditional approaches with the use of simulators.

Secondly, the use of binding molecules (or bespecifically single-chain antibodies, preferably human, as defined in this application, for the manufacture of therapeutic agents for humans is less than the costly and time-consuming process, than approaches with the use of simulators.

Thirdly, a binding molecule (or bespecifically single-chain antibodies, preferably human, as defined in this application can be used for preclinical testing not only on a single species of primates, but also on the number of other Primate species, thus limiting the risk of possible species differences between primates and man.

Fourthly, chimpanzees as endangered species of animals, are excluded from the test.

Fifth, to provide intensive pharmacokinetic studies it is possible to select multiple blood samples.

Sixth, due to the fact that binding molecules, preferably human, according to a preferred embodiment of the invention are of human origin, development, immune response against these binding molecules is minimized when administered to patients-people. Induction of an immune response with antibodies specific to the drug candidate derived from species that are not human, such as a mouse, leading to the development of human artemisinin antibodies (HAMA) against therapeutic molecules of murine origin, is excluded.

The term "protein" is well known in the art and describes a biological connection. Proteins contain one or more am nekisnotnice chains (polypeptides), in which amino acids linked together by peptide bonds. The term "polypeptide" as used in this application, describes a group of molecules which comprise more than 30 amino acids. In accordance with the invention, this group of polypeptides contains "proteins", provided that proteins consist of a single polypeptide. In addition, in accordance with the definition of the term "polypeptide" refers to fragments of proteins, provided that these fragments are composed of more than 30 amino acids. The polypeptides can also form multimer, such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide molecule. Polypeptide molecules forming such dimers, trimers, and so on, may be the same or not the same. The corresponding higher order structures such multimeric indicated, therefore, the Homo - or heterodimer, Homo - or heterotrimeric etc. Example of heteropolymer is a molecule antibodies, 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 glycosylation, AC is melirovanie, phosphorylation and the like. Such modifications are well known in the art.

As used in this application, the "human" and "person" refers to the species Homo sapiens. As stated on the medical applications of data structures described in this invention, the patients-people should be treated the same molecule.

The term "human origin", as it is used in the context of the molecules according to the invention, describes molecules derived from human libraries or having a structure/sequence corresponding to the human equivalent. Accordingly, the proteins having the amino acid sequence corresponding to the same sequence in humans, for example, the antibody fragment having the amino acid sequence in the frame corresponding to the sequences of the germline of the person, understood as molecules having a human origin.

As used in this application, the term "primacy, not a chimpanzee or APE, not sin." or its grammatical variants belong to any Primate that is different from chimpanzees, i.e. different from an animal belonging to the genus Pan, and including the species Pan paniscus (pygmy chimps, bonobos) and Pan troglodytes (common chimpanzee), also known Anthropopithecus troglodytes (common chimpanzee) or Simia satyrus (orangutan). The term "primacy", "view primates", "primates" or grammatical variants indicate the detachment of placental mammals, divided into two suborder prosimians and anthropoids and including man, apes, monkeys and lemurs. Specifically, the term "Primate" as used in this application contains the suborder Strepsirrhini (Micronesia monkeys) (paleobotany non-tarsier (non-tarsier prosimians)), including the infraorder Lemuriformes (glamourouslesya) (comprised of the superfamily Cheirogaleoidea (dwarf lemurs) and Lemuroidea (Lemuria)), the infraorder Chiromyiformes (reconocieran) (which includes the family Daubentoniidae (reconozco)) and the infraorder Lohsiformes (lorianne) (includes family Lorisidae (laravie) and Galagidae (ganagobie)). The term "Primate" as used in this application also includes the suborder Haplorrhini (shanasia monkeys), including the infraorder Tarsiiformes (Dolgopyatova) (which includes the family Tarsiidae (Dolgopyatova)), the infraorder Simiiformes (aleshanobleni) (includes Platyrrhini (new world monkeys or apes of the New world, and Catarrhini (old world monkeys), including Cercopithecidea (maryshopanne) or Old world monkeys)).

In the framework of the invention species of primates that are not chimpanzees, can be understood as representing the lemur, dolgopyat, Gibbon, igranka (owned by the monkeys of the New the Council of the family Cebidae (cacahuete)) or Old world monkey (belongs to the superfamily Cercopithecoidea (abacadabra)).

As used in this application, the term "Old world monkey" includes any monkey falling in the superfamily Cercopithecoidea, which itself is divided into families: Cercopithecinae (martyshkova monkeys), which are found mainly in Africa, but include other kind of Makarov found in Asia and North Africa; and Colobinae (Tonkolili), which include the majority occurring in Asia childbirth, as well as the African representatives calstatela monkeys.

Specifically, within the subfamily Cercopithecinae preferred Primate, not a chimpanzee, it can come from the tribe Martynovich (Cercopithecini), within the genus Allenopithecus (swamp monkey Allen, Allenopithecus nigrovihdis); within the genus Miopithecus (monkey dwarf talapoin, Miopithecus talapoin; Gabon, talapoin, Miopithecus ogouensis); within the genus Erythrocebus (monkey ordinary hussars, Erythrocebus patas); within the genus Chlorocebus (green monkey, Chlorocebus sabaceus; monkey green grivet, Chlorocebus aethiops; monkey chornobylska vervet, Chlorocebus djamdjamensis; monkey antalosky, Chlorocebus tantalus; verveda, Chlorocebus pygerythrus; malbrouck, Chlorocebus cynosuros); or within the genus Cercopithecus (dryas monkey or monkey sologaistoa, Cercopithecus dryas; Diana monkey, Cercopithecus diana; monkey rolovacie, Cercopithecus roloway; most belonoha monkey, Cercopithecus nictitans; blue monkey, Cercopithecus mitis; the silver monkey, Cercopithecus dogetti; the Golden monkey, Cercopithecus kandti; monkey six, Cercopithecus albogularis; Mona monkey, Cercopithecus mona; monkey Mona Campbell, Cercopithecus campbelli; monkey Mona Lowe, Cercopithecus lowei; monkey crested Mona, Cercopithecus pogonias; monkey Mona wolf, Cercopithecus wolfi; monkey Mona dent, Cercopithecus denti; monkey small belonoha, Cercopithecus petaurista; monkey rock, Cercopithecus erythrogaster, monkey of Sclater, Cercopithecus sclateri; red monkey, Cercopithecus erythrotis; monkey globaliza [bearded], Cercopithecus cephus; red monkey, Cercopithecus ascanius; monkey L Hosta, Cercopithecus Ihoesti; monkey Preuss, Cercopithecus preussi; monkey solncenosca, Cercopithecus solatus; monkey cofinality, Cercopithecus hamlyni; monkey de Brass, Cercopithecus neglectus).

An alternative, preferred Primate, not a chimpanzee, also within the subfamily Cercopithecinae, but within the tribe Avianova (Papionini), may come from the family of Makarov (Massa) (Barbary APE, Masasa sylvanus; macaques linkworthy, Masasa silenus; macaques South swingvote or beruk (Beruk), Masasa nemesthna; macaques North swingvote, Masasa leonina; macaques paryski or Bokkoi, Masasa pagensis; macaques to sibera, Masasa siberu; macaques Moore, Masasa maura; macaques ground, Masasa ochreata; macaques Tonkin, Masasa tonkeana; macaques Geka, Massa hecki; macaques gerontology, Masasa nigriscens; macaque Celebes crested or black monkey, Masasa nigra; cynomolgus macaques or monkeys-having, or the Mac is to the long-tailed or Kera (Kera), Massa fascicularis; Arctic monkeys or macaques bear, Masasa arctoides; rhesus, Masasa mulatta; macaques formanski mountain, Masasa cyclopis; Japanese macaques, Masasa fuscata; macaques Ceylon, Masasa sinica; Indian macaques, Masasa radiata; tailless macaques, Masasa sylvanmus; Assamese macaques, Masasa assamensis; Tibetan macaques or macaques Milne-Edwards, Masasa thibetana; macaques arunachali or "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; mangabey chernokogyi, Lophocebus aterrimus; mangabey Odenbach, Lophocebus opdenboschi; mangabey highland, Lophocebus kipunji); within the genus Papio (baboon galadril, Papio hamadryas; the Sphinx, or Guinea baboon, Papio papio; the Anubis baboon, Papio anubis; the baboon baboon, or yellow 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; mangabey common, Cercocebus torquatus; mangabey prompt, Cercocebus agilis; mangabey zolotodobychi, Cercocebus chrysogaster, mangabey river (Tang), Cercocebus galeritus; mangabey Sanya, Cercocebus sanjei); or within the genus Mandrillus (mandrill, Mandrillus sphinx; drill, Mandrillus leucophaeus).

Most preferred is Masasa fascicularis (also known as cynomolgus macaques, or having (Cynomolgus monkey) and so designated in the Examples as "Javanese" and Masasa mulatta (rhesus designated as "RH").

Within the subfamily Colobinae preferred Primate, not a chimpanzee, it can come from the African group within the genus Colobus (colobus black, Colobus satanas; Angolan colobus, Colobus angolensis; colobus Royal, Colobus polykomos; Ursine colobus, Colobus vellerosus; black and white guereza, Colobus guereza); within the genus Piliocolobus (Western red colobus, Piliocolobus badius; Piliocolobus badius badius; Piliocolobus badius temminckii; Piliocolobus badius waldronae; colobus of 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 Primate, not a chimpanzee, an alternative may be from the group of leaf monkeys (tanatarov), within the genus Semnopithecus (grey langur Nepali, Semnopithecus schistaceus; grey langur Kashmir, Semnopithecus ajax; grey langur taraski, Semnopithecus hector, langur Severianin grey, Semnopithecus entellus; grey langur ceroscopy, Semnopithecus hpoleucos; langur Generalny grey, Semnopithecus dussumieri; langur grey pubescent, Semnopithecus priam); within the group So vetulus of the genus Trachypithecus (langur purpuralis, Trachypithecus vetulus; Nilgiri langur, Trachypithecus johnii); within the group of T. cristatus genus Trachypithecus (langur Javanese, Trachypithecus auratus; conatel silver, or silver langur, Trachypithecus cristatus; langur Indochina, Trachypithecus germaini; langur tenasserim, Trachypithecus barbel); within the group of T. obscurus of the genus Trachypithecus (langur smoky, or spectacle, Trachypithecus obscurus; langur Feira, Trachypithecus phayrei); within group I. pileatus species of Trachypithecus (langur saposhnikovia, Trachypithecus pileatus; langur, Shortridge, Trachypithecus shortridgei; the Golden langur, or trahepatic gay, Trachypithecus geei); within the group of T. francoisi of the genus Trachypithecus (Francois langur, Trachypithecus francoisi; langur hatinhensis, Trachypithecus hatinhensis; the white-headed langur, Trachypithecus poliocephalus; langur lauriski, Trachypithecus laotum; langur of Delacour, Trachypithecus delacouri; langur black Indochina, Trachypithecus ebenus); or within the genus Presbytis (surely Sumatran, Presbytis melalophos; conatel striped, Presbytis femoralis; conatel karawacki, Presbytis chrysomelas; conatel belovedly, Presbytis of; conatel white-fronted, Presbytis frontata; conatel Javanese, Presbytis comata; conatel Thomas, Presbytis thomasi; thekotel of house, Presbytis hosei; conatel chestnut, Presbytis rubicunda; langur metawiki or Joja, Presbytis potenziani; conatel natonski, Presbytis natunae).

Within the subfamily Colobinae, prepact the strong primacy, not a chimpanzee, an alternative may be from the group nosed (odd-nosed) monkeys, within the genus Pygathrix (pygathrix krasnoprudny, Pygathrix nemaeus; pygathrix chernobilsky, Pygathrix nigripes; pygathrix seroiusly, Pygathrix cinerea); within the genus Rhinopithecus (monkey cornea Golden, Rhinopithecus roxellana; monkey cornea black, Rhinopithecus bieti; monkey cornea grey, Rhinopithecus brelichi; langur Tonkin snub, Rhinopithecus avunculus); within the genus Nasalis (Nosach ordinary, Nasalis larvatus); or within the genus Simias (langur swingvote, Simias concolor).

As used in this application, the term "igranka, or marmosets" means any New world monkey of the genus Callithrix, for example, belong to Maroseika the Atlantic coast of the subgenus Callithrix (so!) (igranka ordinary, or marmosets, Callithrix (Callithrix) jacchus; marmosets cernoia, Callithrix (Callithrix) penicillata; marmoset Species (Wied''s known as marmoset), Callithrix (Callithrix) kuhlii; marmosets bald, Callithrix (Callithrix) geoffroyi; igranka citrine, Callithrix (Callithrix) flaviceps; igranka beloha, Callithrix (Callithrix) aurita); belonging to Maroseika the Amazonian forests of the subgenus Mico (silk marmoset) (igranka Rio-Akari, Callithrix (Mico) acariensis; igranka Manicore, Callithrix (Mico) manicorensis; igranka silver, Callithrix (Mico) argentata; igranka white, Callithrix (Mico) leucippe; igranka Emilia (Emilia''s known as marmoset), Callithrix (Mico) emiliae; marmosets chernositona, Callithrix (Mico) nigriceps; igranka Mark (Marca''s known as marmoset), Callithrix (Mico) marcai; Marmo is etka mule, Callithrix (Mico) melanura; igranka sea, Callithrix (Mico) humeralifera; marmosets Maués, Callithrix (Mico) mauesi; marmosets gold (gold-and-white known as marmoset), Callithrix (Mico) chrysoleuca; marmosets Gershkovich (Hershkovitz''s known as marmoset), Callithrix (Mico) intermedia; marmosets Satere, Callithrix (Mico) saterei); marmosets Roosmalens' Dwarf belonging to the subgenus Callibella (Callithrix (Callibella) humilis); or dwarf igranka belonging to the subgenus Cebuella (Callithrix (Cebuella) pygmaea).

Other genera of New world monkeys include tamarins (or Zagainov) of the genus Saguinus (including group S. oedipus, group S. midas, the group of S. nigricollis group S. mystax, the group of S. bicolor and group S. inustus) and squirrel monkeys of the genus Saimiri (e.g., Saimiri sciureus, Saimiri oerstedii, Saimiri ustus, Saimiri boliviensis, Saimiri vanzolini).

The term "binding domain" refers in accordance with the present invention, the domain of the polypeptide, which specifically binds to/interacts with the given(output) target(output) 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 a specific group of antigens, for example the same antigen in different species. It is also clear that the binding/interaction defines "specific recognition". The term "specificeski discriminating means in accordance with this invention, what molecule antibodies able to specifically interact and/or bind at least two, preferably at least three, more preferably at least four amino acids of the antigen, for example antigen CD3 person specified in the application. Such linking can be illustrated with specificity in accordance with the principle of lock and key". Thus, specific motifs in the amino acid sequence of the binding domain and the antigen are associated with each other due to their primary, secondary, or tertiary structure, as well as due to secondary modifications of this structure. Specific interaction of this site of interaction with the antigen with its specific antigen can also lead simply to bind the specified site to the antigen. In addition, specific interaction of this site of interaction with the antigen with its specific antigen alternative may cause the initiation signal, for example, due to the induction of changes in the conformation of the antigen, oligomerization antigen, etc. Preferred example of a binding domain in accordance with the present invention is an antibody. Binding domain can be a monoclonal or polyclonal antibody or be derived from monoclonal or police the national antibodies.

The term "antibody" includes derivatives or their functional fragments, which still retain the binding specificity. Methods of obtaining antibodies are well known in the art and 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" includes immunoglobulin (Ig) different classes (i.e., IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgG1, IgG2, and so on). These antibodies can be used, for example, thus, affinity purification and immunolocalization polypeptide or fused protein according to the invention, as well as for monitoring the presence and quantity of such polypeptides, for example, in cultures of recombinant prokaryotic or eukaryotic cells or organisms.

The definition of the term "antibody" also includes such embodiments, as chimeric, single-chain, and humanized antibodies, and antibody fragments, like, inter alia, Fab-fragments. Fragments or derivatives of antibodies in addition contain fragments F(ab')2, Fv, scFv (single-chain Fv) or single domain antibodies, antibodies, comprising a single variable domain or immunoglobulin single variable domain that contains only one variable domain, which can be a VH or VL, which is specificeski contact with the antigen or epitope independently of other V regions or domains; see, for example, Harlow and Lane (1988) and (1999), above. Such immunoglobulin single variable domain includes not only the selected polypeptide single variable domain of the antibody, but also larger polypeptides that contain one or more monomers of the polypeptide sequence of a single variable domain of the antibody.

In the art there are known various methods that can be used to obtain such antibodies and/or fragments. So, these derivative (antibodies) can be obtained with the use of peptidomimetics. Further, techniques described for obtaining single-chain antibodies (see, inter alia, U.S. patent 4946778), can be adapted to obtain a single-chain antibody that has specificity to the selected time polypeptide(s). In addition, for the expression of humanized antibodies specific to the polypeptides and fused proteins according to this invention, it is possible to use transgenic animals. To obtain monoclonal antibodies, you can use any technique which provides antibodies produced by cultures of stable cell lines. Examples of such techniques include the hybridoma technique (Köhler and Milstein, Nature, 256 (1975), 495-497), tianwu method, the hybridoma technique using In-human cells (Kozbor, Immunology Today 4 (1983), 72) and the EBV-hybridoma IU is the odik (EBV - the virus of Epstein-Barr) for producing human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96). Surface plasmon resonance, which is used in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of the target polypeptide, such as CD3-Epsilon (Schier, Human Antibodies Hybridomas, 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). In the context of the present invention also provides that the term "antibody" includes or antibody-based test design, which can be expressed in the host, as described in this invention below, for example, or antibody-based test design, which can be transliterowany and/or transpulmonary using, among other things, viral or plasmid vectors.

The term "specific interaction" as it is used in accordance with the present invention, means that the binding(s)th (domain) molecule has no cross-reactivity or has no substantial cross-reactivity with a polypeptide that has a similar structure to that of the polypeptide that forms a link linking molecule, and which could be expressed by the same cells as representing the interest of the polypeptide. Cross reactivity panel investigated the binding molecules can be tested for example, by assessing the binding of the specified panel binding molecules under standard conditions (see, for example, 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 a specific antigen include the specificity of the ligand to its receptor. This definition, in particular, includes the interaction of ligands, which induce the signal after binding to its specific receptor. An example of the specified interaction, which, in particular, is also covered by the specified definition, is the interaction of antigenic determinants (epitopes) with a binding domain (antigennegative site) antibodies.

The term "cross-species specificity" or "species specificity" as used in this application means the binding of the binding domain described in this invention, with the same molecule-target in humans and primates, are not chimpanzees. Thus, "cross-species specificity" or "species specificity" should be understood as cross-species reactivity of the same molecule X, expressed in different forms, and not the molecule that differs from X. Interspecies specificity of monoclonal antibodies that recognize, for example, CD3-Epsilon person, assigned and CD3-Epsilon primacy, not a chimpanzee, for example, CD3-Epsilon macaque, can be determined, for example, using FACS analysis (cell sorters with activation of fluorescence (fluorescence-activated cell sorter)). FACS analysis is performed so that an appropriate monoclonal antibody is tested for binding to human cells and Primate, not a chimpanzee, for example macaque cells expressing these antigens CD3-Epsilon person and the primacy of non-chimpanzee, respectively. The corresponding analysis is shown in the following examples.

As used in this application, the term "CD3-Epsilon" means a molecule that is expressed as part of the T-cell receptor, and has the meaning usually ascribed to him in the prior art. As for man, he covers individual independently or a combination of all known subunits of CD3, such as CD3 Epsilon, CD3-Delta, CD3-gamma, CD3-Zeta, CD3-alpha and CD3-beta. CD3 antigens primates that are not chimpanzees, which are referred to in this application are, for example, CD3 of Massa fascicularis and CD3 of Massa mulatta. As for Massa fascicularis, it covers the CD3-Epsilon FN-18-negative and CD3-Epsilon FN-18-positive, CD3 gamma and CD3 Delta. As for Massa mulatta, it covers CD3 Epsilon, CD3 gamma and CD3 Delta. Preferably, the specified CD3, as used in this Declaration, the ke, is 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 person listed in GenBank under the number of access NM_000732.

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

The chain of the CD3-Epsilon "FN-18-positive" Massa fascicularis (i.e., CD3-Epsilon recognized by a monoclonal antibody FN-18) are 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 Massa fascicularis listed in GenBank under the number of access AV.

Nucleic acid sequence and amino acid sequence corresponding CD3-Epsilon, gamma, and Delta homologues of Massa mulatta can be identified and selected using recombinant techniques described in the art (Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 3rd edition, 2001). This applies, with necessary changes, to the CD3-Epsilon, gamma and Delta homologues of other primates, are not chimpanzees, which are defined in this application. Identification of the amino acid sequence of the common marmoset (Callithrix jacchus), common squirrel monkey (Saimiri sciureus) and marmoset Oedipus (Saguinus oedipus) OPI the Ana in the accompanying examples. Amino acid sequence of the extracellular domain of CD3-Epsilon Callithrix jacchus presented in SEQ ID NO: 3, the same sequence Saguinus oedipus is presented in SEQ ID NO: 5, and the same sequence Saimiri sciureus presented in SEQ ID NO: 7.

In accordance with the above, the term "epitope" determines the antigenic determinant that specifically binds to/identified a binding molecule as defined above. Binding domains or molecules can specifically bind/interact with conformational or continuous epitopes that are unique to the target structure, for example with CD3-Epsilon chain of human and primates, are not chimpanzees. The hallmark of conformational or continuous epitopes is to polypeptide antigens, the presence of two or more individual amino acid residues that are spaced apart in the primary sequence, but converge on the surface of the molecule, when the polypeptide is folded in the native protein/antigen (Sela, (1969) Science 166, 1365 and Laver, (1990) Cell, 61, 553-6). Two or more individual amino acid residues that contribute to this epitope are present in separate areas of one or more than one polypeptide chain. These balances are converging on the surface of the molecule, when the polypeptide(s) chain(s) is minimized(are) in the three-dimensional structure with education is receiving 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 the Epsilon-chain of CD3, may be in the correct conformation, only if it is immersed in the remainder of Epsilon-chain and held in proper position by means of heterodimerization Epsilon-chain or gamma-, or Delta-chain of CD3. And Vice versa, independent of the environment of the CD3 epitope, as proposed in this application, refers to the N-terminal containing amino acid residues 1-27, polypeptide, or functional fragment of CD3 Epsilon. This N-terminal containing amino acid residues 1-27, polypeptide or functional fragment retains its three-dimensional structural integrity and correct conformation when it is removed from native environment in the CD3-complex. Independence from the environment N-terminal containing amino acid residues 1-27, polypeptide or functional fragment, which is part of the extracellular domain of CD3-Epsilon, and is, thus, epitope, which is completely different from the epitope of CD3-Epsilon described in WO 2004/106380 due sosososo obtain human binding molecules. In the specified method used separately expressed recombinant CD3-Epsilon. The conformation of this separately expressed recombinant CD3-Epsilon differed from the conformation adapted to its natural form, i.e. the form in which 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 used separately expressed recombinant protein CD3-Epsilon, from the library identify antibodies specific to that antigen, despite the fact that this library does not contain antibodies with specificity to native antigens/autoantigens. This is because the separately expressed recombinant protein CD3-Epsilon does not exist in vivo; it is not autoantigens. Therefore, a subpopulation of b cells expressing antibodies specific to this protein, were not depleted in vivo; library of antibodies constructed from these b cells will contain the genetic material for antibodies specific to this separately expressed recombinant protein CD3-Epsilon.

However, because independent of the environment N-terminal containing amino acid residues 1-27, polypeptide or its function is optional fragment represents the epitope which is folded in its native form, binding domains of the present invention can not be identified by methods based on the approach described in WO 04/106380. So you can confirm in the tests that binding molecules, described in WO 04/106380 not able to contact the N-terminal amino acid residues 1-27 Epsilon-chain of CD3. Consequently, the traditional anti-CD3 binding molecules or molecules of antibodies against CD3 (for example, described in WO 99/54440) contact Epsilon-chain of CD3 in a position that is closer to the end than those that are independent of the environment N-terminal containing amino acid residues 1-27, polypeptide or functional fragment, proposed in this application. Molecules antibodies ACT and UCHT-1 of the prior art have specificity in relation to the Epsilon-subunit of the TCR/CD3 complex between amino acid residues 35 and 85 and, accordingly, the epitope of these antibodies is also located closer to the C-end. In addition, UCHT-1 binds to the Epsilon-chain of CD3 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 molecules of the prior art are not associated with a specific in this application is independent from the environment N-terminal containing amino acid residues 1-27, epitope is (or functional fragment) and not directed against him.

To create a binding domain, preferably a human, is contained in the polypeptide according to the invention, for example in bespecifically single-chain antibody as defined in this application can be used, for example, monoclonal antibodies that bind to the CD3-Epsilon both human and Primate, non chimpanzees (e.g., CD3-Epsilon macaque).

In the preferred embodiment of the polypeptide according to the invention the primacy of non-chimpanzee, is the Old world monkey. In a more preferred embodiment of this polypeptide by Old world monkey is the monkey of the genus Papio (baboon) or kind of Makarov. Most preferably, the monkey kind of Makarov is Assamese macaques (Masasa assamensis), the Barbary macaque (Masasa sylvanus), Indian macaques, or bonnet (Masasa radiata), macaques ground (Masasa ochreata), macaque Celebes crested (Masasa nigra), macaques formanski mountain (Masasa cyclopis), snow monkey or Japanese macaque (Masasa fuscata), cynomolgus macaques, or monkeys-having, or long-tailed macaques, or cynomolgus macaques (Masasa fascicularis), linkworthy macaques (Masasa silenus), macaques South swingvote (Masasa nemestrina), rhesus (Masasa mulatta), Tibetan macaques Arctic (Masasa thibetana), macaques Tonkin (Masasa tonkeana), macaques Ceylon (Masasa sinica), short-tailed macaques, or the red-faced monkeys, or mA the AK bear (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 polypeptide according to the invention, the primacy of non-chimpanzee, is a New world monkey. In a more preferred embodiment of this polypeptide New world monkey is the monkey of the genus Callithrix (gruntovye), genus Saguinus (Tamarina or sagouine) or genus Saimiri (squirrel monkeys). Most preferably, the monkey of the genus Callithrix is Callithrix jacchus (igranka common), the monkey of the genus Saguinus is Saguinus oedipus (Oedipus Tamarin), and the monkey of the genus Saimiri is Saimiri sciureus (monkey common squirrel).

As described in this invention above, the polypeptide according to the invention is bound by the first binding domain epitope CD3ε(Epsilon) chain of human and Primate, not a chimpanzee, and this epitope is a part of the amino acid sequence of a member group, consisting of 27 amino acid residues as shown in SEQ ID NO: 2, 4, 6, or 8.

In accordance with the present invention for a polypeptide according to the invention it is preferable that the specified epitope was a part of the amino acid sequence containing 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, , 7, 6 or 5 amino acids.

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

Within the present invention, a "functional fragment N-terminal 1-27 amino acid residue" means that the specified functional fragment is still an independent environment epitope which retains its three-dimensional structural integrity when it is removed from its native environment in the CD3 complex (and merge with heterologous amino acid sequence, such as Arcam or Fc-region of an immunoglobulin, for example, as shown in Example 3.1). The preservation of three-dimensional structures within a 27 amino acid N-terminal polypeptide, or functional fragment, CD3-Epsilon can be used to create binding domains that bind to N-terminal polypeptide fragment of CD3 Epsilon in vitro and native CD3 complex (CD3-Epsilon-subunit of CD3 complex on T cells in vivo with the same affinity binding. In the present invention, a "functional fragment N-terminal 1-27 amino acid residue" means that the CD3-binding molecules, as proposed in this application can still associate with such functional fragments, and it does not depend on the environment. Specialist in the art OS is edalen about the methods of epitope mapping to determine, what amino acid residues of the epitope recognized by such anti-CD3 binding molecules (for example, alanine scanning or pep-spot analysis).

In the preferred embodiment of the invention the polypeptide according to the invention contains a first binding domain capable of contacting the epitope CD3ε-chain of human and Primate, not a chimpanzee, and a second binding domain able to bind to the antigen on the cell surface.

The term "cell surface antigen", as used in this application, 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 is still available in its tertiary form outside the cells. Non-limiting examples of cell surface molecules, which is localized in the plasma membrane, is a transmembrane protein that contains, in its tertiary conformation, the field of hydrophilicity and hydrophobicity. At least one hydrophobic region makes possible the introduction or insertion of cell surface molecules in the hydrophobic plasma membrane of the cell, whereas the hydrophilic region are located on both sides of the plasma membrane to the cytoplasm and extracellular space, meet the but. Non-limiting examples of cell surface molecules that are located on the plasma membrane are proteins that are modified by cysteine residue in order to carry Palmitoyl group, proteins, modified by C-terminal cysteine residue, in order to carry varnishlog group, or proteins that are modified on the C-end, in order to carry glycosyl-phosphatidylinositol ("GPI") anchor. These groups allow covalent attach 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 include EGFR, EGFRvlll (EGFR variant, type III), MCSP, carbonic anhydrase IX (CAIX), CD30, CD33, Her2/neu, IgE, CD44v6 (CD44 variant type 6) and IIA-1. In addition, the examples for the respective antibodies on the cell surface contain antigens specific to the disease, i.e. cancer, autoimmune diseases or infectious diseases, including viral infections. Accordingly, the term "cell surface antigens", obviously, includes viral proteins, such as native, not subjected to the processing of viral proteins exposed on the surface of infected cells (described additional area for envelope proteins of the virus hepati the while, With HIV-1 (human immunodeficiency virus)).

One of the protective functions of cytotoxic T cells is the destruction of virus-infected cells, therefore unique property bespecifically binding molecules according to the invention to activate and redirect cytotoxic T cells regardless of their autochthonous specificity has a strong influence on a wide range of chronic viral infections. For most of these infections removal persistence infected cells is the only chance to recover. Currently being developed adoptive therapy on the basis of T-cells against chronic infections CMV (cytomegalovirus) and EBV (Rooney, C. M., et al., Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr-virus-related lymphoproliferation. Lancet, 1995. 345 (8941): p.9-13; Walter, E. A., et al., Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T cells clones from the donor. N. Engl. J. Med., 1995. 333 (16): p.1038-44).

Chronic infection with hepatitis b (HBV) is undoubtedly one of the most interesting and noteworthy readings. All over the world from 350 to 400 million people are infected with HBV. Modern treatment of chronic HBV hepatitis based on the use of γ-interferon and nucleoside or nucleotide analogues, long-term therapy with significant side effects, such as induction of a sudden exacerbation of hepatitis, fever, myalgias, is trombocytopenia and depression. Despite the fact that currently there are more than 4 of the approved schemes therapeutic treatment, is rarely achieved by removal of the virus. Persisting inflammation in chronic hepatitis leads to liver cirrhosis and hepatocellular carcinoma more than 25% of patients. In addition, up to 40% of patients with chronic hepatitis b die because of the serious complications that cause 0,6-1,0 million deaths per year worldwide.

HBV is the prototype of hepadnaviruses, is an enveloped virus, the genome of which relaksirano ring (RK) form transcribed back into RNA-pregenome. After infection kDNK is imported to the nucleus of hepatocytes, where it is converted to covalently closed circular DNA (tzcdnk), which contains four overlapping reading frames. He serves as a transcriptional matrix for pregenome RNA and three subgenomic RNA. RNA pregenome functions as mRNA for translation of viral crustal protein and the viral polymerase. Infected cells continuously produce a surface protein of HBV (HBsAg) with tzcdnk, even when HBV replication stops. HBsAg consists of a small surface (S) protein with a very small share of medium and large (L) surface proteins. As HBV S and L are targeted to the membrane of the endoplasmic reticulum (ER), where untransported in membrane vesicles through the TRANS-Golgi organelles to the plasma membrane (Gorelick, F. S. and C. Shugrue, Exiting the endoplasmic reticulum. Mol. Cell Endocrinol., 2001. 177 (1-2): p.13-8). S and L proteins are constantly expressed on the surface of HBV-replicating hepatocytes, as shown recently (Chu, C. M. and Y. F. Liaw, Membrane staining for hepatitis In surface antigen on hepatocytes: a sensitive and specific marker of active viral replication in hepatitis B. J. Clin. Pathol., 1995, 48(5): p.470-3).

Prototypes of viruses that exhibit envelope proteins on the cell surface, are hepatitis b virus (HBV), hepatitis C virus (HCV) and HIV-1, which represent a heavy burden of diseases in General. For HIV-1 virus recently shown that T-cells, engineered chimeric design TCR with Fv-antibody directed on envelope protein gp120, can destroy HIV-1-infected target cells (Masiero, S., et al., T-cell engineering by a chimeric T-cell receptor with fntibody-type specificity for the HIV-1 gp120. Gene Ther., 2005, 12 (4): p.299-310). Owned hepadnavirus hepatitis b virus (HBV) expresses the complex envelope protein (HBsAg), which is produced continuously from episomal tzcdnk, even when HBV replication is reduced.

The expression in the form of intact S and L HBV proteins on the cell surface makes them accessible to antibodies that are a hallmark of seroconversion, when patients recover from the acute phase of infection and is replaced circulating in the blood of HBsAg to anti-HBs. If seroconversion does not occur, up to 30% hepatocyte who continue to Express the S protein of HBV after highly active antiviral therapy of long duration. Thus, in addition to T-lymphocytes that specifically recognize versions of intracellular HBV peptides and presented by MHC molecules on the cell surface, other forms of T-cell interactions may focus on the intact surface protein, such as S and L antigens available in the outer cell membrane. Using single-stranded fragments of antibodies that recognize small (S) and large (L) envelope proteins of hepatitis b virus, artificial T-cell receptors, allowing you to direct the transplanted T cells to infected hepatocytes and after antigen-contact activation of these T cells to secrete cytokines and destroy infected hepatocytes.

A limitation to this approach is that (1) there is a need for manipulation of T-cells in vitro, (2) used for transfer of T-cell receptors retroviruses can lead to insertional mutagenesis in T-cells, and (3) after such transfer of T-cell cytotoxic response cannot be limited.

To overcome these limitations can be created bespecifically single-stranded molecule of the antibody containing the first domain with binding specificity to the antigen CD3-Epsilon person and the primacy of non-chimpanzee (as proposed in this application in the context of this image is to be placed), and a second domain with binding specificity with envelope proteins of HBV or HCV infected hepatocytes, and they are included in the scope of this invention.

According to the present invention also preferably, the second binding domain was associated with the cell surface antigen of human and/or originating from the primacy of non-chimpanzee, copies of cell surface antigens of human, selected from EGFR, Her2/neu or IgE.

For the formation of the second binding domain of the polypeptide according to the invention, for example bespecifically single-chain antibodies as defined in this application can be used monoclonal antibodies to bind to the appropriate cell surface antigens of human and/or Primate, not a chimpanzee. The corresponding binding domains for especifismo polypeptide as defined in this application may be obtained, for example, monoclonal antibodies with cross-species specificity in accordance with the recombinant methods described in the art. Monoclonal antibody binding to the cell surface antigen of a man and a homologue of the indicated cell surface antigen of the primacy of non chimpanzees, can be tested in FACS-analyses outlined above. Specifications the sheets in the art it is obvious, what antibodies with cross-species specificity can also be created hybridoma methods described in the literature (Milstein and Köhler, Nature, 256 (1975), 495-7). For example, mice can alternately to immunize CD33 human and Primate, not a chimpanzee. From these mice by hybridoma technology produce hybridoma cells producing antibodies with cross-species specificity, and analyzed by FACS, as described above. Creation and analysis bespecifically polypeptides, such as bespecifically single-chain antibodies, demonstrating cross-species specificity, as described in this invention, shown in the following examples. Advantages bespecifically single-chain antibodies, demonstrating cross-species specificity, include the points listed below.

As for the polypeptide according to the invention, it is particularly preferable that the first binding domain capable of contacting the epitope CD3ε-chain of human and Primate, not a chimpanzee, contained VL-region containing CDR-L1, CDR-L2 and CDR-L3 selected from:

(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-L, as represented in SEQ ID NO: 155.

Variable regions, i.e., the variable light chain (L or VL) and variable heavy chain ("H" or "VH") is understood in the art as providing binding domain antibodies. These variable regions contain hypervariable sites.

The term "hypervariable area" (CDR) is well known in the art to determine the antigenic specificity of the antibodies. The term "CDR-L" or "L CDR" refers to the CDRs in VL, while the term "CDR-H" or "H CDR" refers to the CDRs in VH.

In an alternative preferred embodiment of this polypeptide according to the invention, the first binding domain capable of contacting the epitope D3ε-chain of human and Primate, not a chimpanzee, contains a VH-region containing CDR-H1, CDR-H2 and CDR-H3 selected from:

(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, and CDR-H3 as shown in SEQ ID NO: 32;

(b) 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;

(d) 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;

(e) CDR-H1 as presented is prohibited 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;

(C) CDR-H1 as shown 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

(C) 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.

Also preferably, the first binding domain capable of contacting the epitope CD3ε-chain of human and Primate, not a chimpanzee, contained 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.

Alternative preferably, the first binding domain capable of contacting the epitope CD3ε-chain of human and Primate, not a chimpanzee, contained 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 or 181.

More preferably, the polypeptide according to the invention is characterized by the first binding domain capable of contacting the epitope D3ε-chain of human and Primate, not a chimpanzee, which contains a VL region and a VH region selected from the group consisting of:

(a) VL-is blasti, as represented in SEQ ID NO: 17 or 21, and a VH-region, which is presented in SEQ ID NO: 15 or 19;

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

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

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

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

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

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

(and) 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

(K) 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 polypeptide according to the invention a pair of VH and VL regions-regions are in the format of single-chain antibodies (scFv). VH - and VL-region arranged in the following order: VH-VL or VL-VH. Preferably, the VH-region was located at the N-end linker sequence. VL-region is located at the C-end of the linker consistently the tee.

The preferred embodiment described above, the polypeptide according to the invention is characterized by the first binding domain capable of contacting the epitope CD3ε-chain of human and Primate, 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 invention also relates to above-described polypeptide, where the second binding domain binds to a cell surface antigen, preferably representing a tumor antigen.

The term "tumor antigen" as used in this application, you can understand how those antigens that are presented on the tumor cell. These antigens can be presented on the cell surface through the extracellular area, which is often combined with the transmembrane and cytoplasmic portion of the molecule. These antigens can sometimes be presented only by tumor cells and never normal cells. Tumor antigens can be expressed only on tumor cells, or may represent a tumor-specific mutation in comparison with normal cells. In this case they are called tumor-specific antigens. More common are the antigens that are presented tumor cell and normal cells, and they are called tumor-associated antigens. These tumor-associated antigens can sverkhekspressiya compared with normal cells or are available for binding of antibodies in tumor cells due to the less compact structure of the tumor tissue compared to normal tissue. Non-limiting examples of tumor antigens as used in this application is EGFR (Liu, Br. J. Cancer, 82/12 (2000), 1991-1999; Bonner, Semin. Radiat. Oncol., 12 (2002), 11-20; Kiyota, Oncology, 63/1 (2002), 92-98; Kuan, Brain Tumor Pathol., 17/2 (2000), 71-78).

EGFR (also known as c-erb1 or HER1) belongs to the erbB-family receptors tyrosinekinase. When activated by binding of a ligand of the EGF-family growth factors EGFR forms homodimer or heterodimer with the second EGFR or another member of the erbB family of receptors, respectively, triggering a signaling cascade through the mitogen-activated protein kinases and other transcription factors, leading to proliferation, differentiation and repair (Olayioye, EMBO J. 19 (2000), 3159-67). EGFR sverkhekspressiya in many cancer types of cancer, including colorectal cancer, breast cancer, lung and head and neck (Mendelsohn, J. Clin. Oncol., 21 (2003), 2787-99; Mendelsohn, J. Clin. Oncol., 20 (18, Suppl.) (2002), 1S-13S; Prewett, Clin. Cancer Res., 8 (2002), 994-1003). Overexpression and/or mutation of EGFR in malignant cells causes constitutive activation of the kinase act is Vesti, that leads to proliferation, angiogenesis, invasion, metastasis and inhibition of apoptosis (Mendelsohn (2003, above; Ciardiello, Clin. Cancer Res., 7 (2001), 2958-70; Perez-Soler, Oncologist, 9 (2004), 58-67). It is shown that monoclonal antibodies directed at the extracellular ligand-binding domain or the intracellular tyrosinekinase EGFR signaling cascade, effective as anticancer target (Laskin, Cancer Treat. Review, 30 (2004), 1-17). For example, humanitariannet monoclonal antibody to EGFR cetuximab (Erbitux), which competitively inhibits the extracellular domain of EGFR in order to inhibit ligand activation of the receptor, was approved by the Department for quality control of food, drugs and cosmetics (FDA) in 2004 for the treatment of metastatic colon cancer in combination with a topoisomerase inhibitor with irinotecan.

In the preferred embodiment of the invention the polypeptide is a molecule especifismo single-chain antibodies.

Described above in this invention the problems related to the design of molecules of models for preclinical studies also worsen if the drug candidate is bespecifically antibody, for example bespecifically single-chain antibody. So, bespecifically antibody requires that both the recognized antigen possessed of Merv the annual specificity for this type of animal, to enable security testing on this animal.

As mentioned in the application above, according to the present invention proposed a polypeptide containing the first binding domain capable of contacting the epitope CD3ε-chain of human and Primate, not a chimpanzee, and a second binding domain capable of contact with a cell surface antigen selected from EGFR, Her2/neu or IgE, and the second binding domain is preferably associated with the cell surface antigen of human and/or Primate, not a chimpanzee. The advantage bespecifically single-chain antibody molecules as drug candidates that meets the requirements for preferred polypeptide according to the invention lies in the use of such molecules in pre-clinical testing in animals and in clinical studies and even for therapy in humans. In the preferred embodiment bespecifically single-chain antibodies with cross-species specificity according to the invention, the second binding domain capable of contact with a cell surface antigen, is of human origin. In bespecifically molecule with interspecies specificity according to the invention binding domain able to bind epitope Epsilon-chain of CD3 person and the primacy, not awlays is gosia chimpanzees, located in the order VH-VL or VL-VH N-end or end bespecifically molecules. Examples bespecifically molecules with interspecies specificity according to the invention with a different arrangement of the VH-and VL-circuit in the first and second binding domain are described in the accompanying examples.

As used in this application, "bespecifically single-chain antibody" refers to a single polypeptide chain containing two binding domain. Each binding domain contains one variable region of the heavy chain antibody (VH-region"), and the VH-region of the first binding domain specifically binds to a molecule CD3ε, and VH-region of the second binding domain specifically binds to a cell surface antigen, as defined in more detail below. Two binding domain may be linked to each other through a short polypeptide spacer. Non-limiting examples of polypeptide spacer is Gly-Gly-Gly-Gly-Ser (G-G-G-S) and its repetitions. Each binding domain may additionally contain one variable region of the light chain of the antibody (VL-region") and the VH region and the VL-region within each of the first and second binding domains are linked to each other via a polypeptide linker, for example, of the type described and claimed in EP 623679 B1, but in any case having a length sufficient th is would allow the VH region and the VL-region of the first binding domain and a VH region and the VL-region of the second binding domain to form a pair with each other so so that being together, they were able to specifically contact the respective first and second molecules.

According to a preferred embodiment of the present invention described above, the molecule especifismo single-chain antibody contains a group of the following sequences, such as CDR H1, CDR H2, CDR, NC, CDR L1, CDR L2 and CDR L3 in the second binding domain selected from SEQ ID NO: 441-446, SEQ ID NO: 453-458, SEQ ID NO: 463-468, SEQ ID NO: 481-486.

Especially preferred embodiment of the invention relates to the polypeptide described above, where the molecule especifismo single-chain antibody contains a sequence chosen from:

(a) amino acid sequence as shown in any of SEQ ID NO: 389, 391, 393, 395, 397, 399, 409, 411, 413, 415, 417, 419, 429, 431, 433, 435, 437, 439, 447, 449, 451, 469, 471, 473, 475, 477, 479, 495, 497, 499, 501, 503 and 505; and

(b) amino acid sequence encoded by the nucleic acid sequence as represented in any of SEQ ID NO: 390, 392, 394, 396, 398, 400, 410, 412, 414, 416, 418, 420, 430, 432, 434, 436, 438, 440, 448, 450, 452, 470, 472, 474, 476, 478, 480, 496, 498, 500, 502, 504 and 506.

In the preferred embodiment of the invention bespecifically single-chain antibodies have cross-species specificity to the CD3-Epsilon and to tumor antigen, raspoznavaniya their second binding domain.

In an alternative embodiment according to the present izaberete is s presented with a sequence of nucleic acids, encoding the above polypeptide according to the invention.

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

Many suitable vectors known to experts in molecular biology, the choice of which will depend on the desired function, and include plasmids, Comedy, viruses, bacteriophages and other vectors used in genetic engineering. To construct various plasmids and vectors can be used by methods well known to experts in the art; see, for example, the techniques described in Sambrook et al. (above) and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N. Y. (1989), (1994). Alternatively, polynucleotides and vectors according to 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 expression of a particular polypeptide, the relevant sequences can be transferred in expressing vectors. Typical cloning vectors include pBluescript SK, pGEM, pUC9, pBR322 and pGBT9. Typical expressing vectors include pTRE, pCAL-n-EK, pESP-1, ROSAT.

Preferably, this vector contains a nucleic acid sequence, which is subauriculatum sequence, functionally associated with the specified sequence of the nucleic acid defined in this application.

The term "regulatory sequence" refers to DNA sequences which are necessary to affect the expression of coding sequences to which they legirovanyh. The nature of such regulatory sequences differs depending on the host body. In prokaryotic regulatory sequences generally include promoter, the binding site of the ribosome and terminators. In eukaryotes in General regulatory sequences include promoters, terminators and, in some instances, enhancers, transactivator or transcription factors. The term "regulatory sequence" is intended to include 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, which describes thus the components are in the relationship, allowing them to function properly. Regulatory sequence, "functionally linked" to the coding sequence, Legerova in such a way that expression of the coding sequence on schaut in terms compatible with the regulatory sequences. In the case when the regulatory sequence is a promoter, expert obviously, it is preferable to use double-stranded nucleic acid.

Thus, the said vector is preferably expressing vector. "Expressing vector" is a structure which can be used for transforming the selected host and provides the expression of the coding sequence in the selected host. Expressing vectors can represent, for example, cloning vectors, binary vectors, or integrating vectors. The expression involves transcription of the nucleic acid molecule preferably in broadcast (region) of the mRNA. Regulatory elements ensuring expression in prokaryotes and/or eukaryotic cells are well known to specialists in this field of technology. In the case of eukaryotic cells, and they contain norm promoters, ensuring initiation of transcription, and possibly poly-A signals ensuring termination of transcription and stabilization of the transcript. Possible regulatory elements allowing expression in prokaryotic cells-hosts include, for example, the promoter PL, lac, trp or tac in E. coli, and examples for regulatory elem is now, allowing expression in eukaryotic cells, the owners, are the promoter AOH or GAL1 in yeast or promoters based on the CMV, SV40 (simian virus 40), RSV (rous sarcoma virus), CMV-enhancer, SV40-enhancer or glubinoy introns in mammals and other animals.

In addition to the elements that are 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 site tk-poly-A, below this polynucleotide. In addition, depending on expressing system to the coding sequence described nucleic acid sequence can be added to a leader sequence capable of directing the polypeptide to a cellular compartment or secrete it into the environment, and they are well known in the art; see also the accompanying Examples. Leader(s) sequence(s) of Sebire(s)in appropriate phase with sequences of initiation and termination of translation, and preferably, a leader sequence was capable of directing secretion of translated protein or its part in periplasmatic space or the extracellular environment. It is possible that the heterologous sequence can encode a protein, including the N-to zeway identifying peptide, providing desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product; see above. In this context, in the art suitable known expressing vectors, such as Okayama-Berg cDNA-expressing vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, 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, when expressing regulatory sequence will be a eukaryotic promoter systems in vectors capable of transforming transfinitely eukaryotic cell hosts, but you can also use the regulatory sequences of prokaryotic hosts. After the vector will be incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and, if desired, then can follow the collection and purification of the polypeptide according to the invention; see, for example, the attached Examples.

Alternative expressing system that can be used for expression of the protein active against cell cycle is the system of insects. In one of these systems as vectors for expression of foreign genes in cells of Spodoptera frugiperda, or l the escarpment Trichoplusia used the nuclear polyhedrosis virus of Autographa californica NPV (AcNPV). The coding sequence of the described nucleic acid molecules can be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the specified coding sequence will inactivate polyhedrin 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 expressed protein of the invention (Smith, J. Virol. 46 (1983), 584; Engelhard, Proc. Nat. Acad. Sci. USA, 91 (1994), 3224-3227).

Additional regulatory elements may include transcriptional and translational enhancers. Preferably, the above-described vectors according to the invention contain breeding and/or comparative marker (scorable marker).

Breeding marker genes useful for selection of transformed cells and, for example, plant tissue and plants, well known to experts in the art and give, 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 is resistant to hygromycin (Marsh, Gene, 32 (1984), 481-485). Describes the advanced breeding genes, namely trpB, which allows cells to utilize indole instead of tryptophan; hisD, which allows cells to utilize gastinel instead of histidine (Hartman, Proc. Natl. Acad. Sci. USA, 85 (1988), 8047); mannose-6-fortisalberta, which allows cells to utilize mannose (WO 94/20627); and ODC (ornithindecarboxilase), which provides resistance to the inhibitor interdiscursivity 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 comparative markers are also known to experts in the art and are commercially available. Preferably, when the specified marker is the gene encoding the luciferase (Giacomin, PI. Sci., 116 (1996), 59-72; Scikantha, J. Bact, 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett., 389 (1996), 44-47) or (3-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 above-mentioned vector.

As described above, the nucleic acid molecule may be used by itself or as part of a vector for expression of the polypeptide according to the invention in cells, for example, for cleaning and for genotype the political tasks. The nucleic acid molecule or vectors containing a sequence and DNA encoding(s) of any of the above described polypeptide according to the invention is introduced into cells, which, in turn, produce the desired polypeptide. Gene therapy based on the introduction of therapeutic genes into cells using methods ex vivo or in vivo, is one of the most important applications of gene transfer. Suitable vectors, methods or systems for delivery of genes for gene therapy in vitro or in vivo are described in the literature and known to the specialist in the art; see, for example, 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 Then, 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. Mentioned molecules of nucleic acids and vectors can be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell. Preferably, this cell was an embryonic cell lines, embryonic cell or an egg or descended from them, most preferably the specified cell is provided which allows a stem cell. An example of an embryonic stem cell can be, among other things, a stem cell, which is described in Nagy, Proc. Natl. Acad. Sci. USA, 90 (1993), 8424-8428.

In addition, according to the invention proposed by the owner, subject to transformation or transfection vector according to the invention. The specified host can be obtained by introducing the above-described vector according to the invention or the above-described nucleic acid molecules according to the invention into the host. The presence of at least one vector or at least one molecule of nucleic acid in the host may mediate the expression of the gene encoding the above-described construction of single-chain antibodies.

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

The host can be any prokaryotic or eukaryotic cell.

The term "prokaryotic" is meant to include all bacteria which can be transformed or transliterowany molecules of DNA or RNA to protein expression according to the invention. Prokaryotic hosts may include gram-negative and gram-positive bacteria, such as E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. The term "eukaryotic" is intended to include yeast cells, the higher the x the plants, insects and preferably mammals. Depending on the host used in the procedure of recombinant obtain protein encoded by polynucleotides of the present invention may be glycosylated or may be deglycosylation. Especially it is preferable to use a plasmid or virus containing the coding sequence of the polypeptide according to the invention, genetically fused with N-terminal FLAG-tag and/or C-terminal His-tag. Preferably, the length of the specified FLAG-tag is approximately 4-8 amino acids, most preferably 8 amino acids. The above polynucleotide can be used for transformation or transfection of a host using any of the methods generally recognized among experts of ordinary skill in the art. In addition, methods of obtaining merged, functionally related genes and their expression, for example in mammalian cells and bacteria, are well known in the art (Sambrook, supra).

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

In particular, it is envisaged that the specified host can be a mammalian cell. Particularly, preferred cell hosts include cells Cho (Chinese hamster ovary), COS cells (fibroblasts Africa is a Julian green monkey), line myeloma cells of type SP2/0 or NS/0. As illustrated in the accompanying examples, as owners particularly preferred cells SNO.

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

Thus, in the following embodiment of the present invention relates to a method for producing a polypeptide according to the invention, comprising culturing the host of the invention under conditions conducive to the expression of this polypeptide according to the invention, and extraction of the resulting polypeptide from the culture.

Transformed hosts can be grown in fermenters and cultivated in accordance with techniques known in the art, to achieve optimal cell growth. Then the polypeptide according to the invention can be isolated from the growth medium, cellular lysates, or cellular membrane fractions. Isolation and purification, for example, expressed by microbial polypeptides according to the invention can be carried out by any conventional methods, such as, for example, preparative chromatographic separations and immunological separations, as, for example, separation involving the use of monoclonal or polyclonal antibodies directed against, for example, the label (tg) of the polypeptide of the invention or as described in the accompanying examples.

The cultivation conditions of the host, allowing the expression known in the art as system-dependent host and expressing the system/expressing vector used in this way. The parameters that must be modified to meet the conditions conducive to the expression of the recombinant polypeptide, are known in the art. Thus, suitable conditions can be determined by an expert in the field of technology in the absence of additional inventive contribution.

After conducting the expression of the polypeptide according to the invention can be purified according to standard in the art treatments, including the deposition of ammonium sulfate, affinity columns, column chromatography, electrophoresis gel, and the like; see Scopes, "Protein Purification", Springer-Verlag, N. Y. (1982). For pharmaceutical applications preferred essentially pure polypeptide with a homogeneity of at least about 90-95%, and most preferred with homogeneity 98-99% or more. Being purified, partially or to homogeneity, if desired, the polypeptide according to the invention can then be used for therapeutic purposes (including in vitro) or to design and perform analytical procedures. In addition, in the accompanying examples described examples m the methods of extraction of the polypeptide according to the invention from the culture.

In addition, according to this invention proposed a composition comprising a polypeptide according to the invention or the polypeptide obtained by the method described above. Preferably, the composition is a pharmaceutical composition.

In accordance with the invention, the term "pharmaceutical composition" refers to compositions for administration to a patient, preferably a patient man. Specific preferred pharmaceutical composition of this invention contains binding molecules directed against and created against independent from the environment CD3 epitopes. Preferably, the pharmaceutical composition contains a suitable composition of carriers, stabilizers and/or excipients. In the preferred embodiment the pharmaceutical composition comprises a composition for parenteral, transdermal, intraluminal, intraarterial, intrathecal and/or intranasal 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. Introduction of suitable compositions may be effected in various ways, for example, by intravenous, intraperitoneal, subcutaneous, intramuscular, local, or intradermal injection. In particular, according to the present invention proposed a continuous introduction of a suitable composition. As a non-limiting example, continuous, i.e. continuous introduction can be realized by means of a small pump, which is driven by the patient for dispensing receipt of therapeutic agent into the patient. A pharmaceutical composition comprising a binding molecule directed against and created against independent from the environment CD3 epitopes according to the invention, can be entered using these pumping systems. Such pumping systems in General are known in the art and are typically designed for periodic replacement of cartridges containing entered by the infusion of therapeutic agent. When replacing the cartridge in such a pumping system may occur suspension in other cases, a continuous flow of therapeutic agent into the patient. In this case, the phase of introduction before replacing the cartridge and the introduction process after replacing the cartridge will be considered together constituting the "continuous introduction of such a therapeutic agent in accordance with the meaning of the pharmaceutical means and methods according to the invention.

Permanent or continuous introduction of these binding molecules directed against and created against independent from the environment CD3 epitopes according to this invention, can be intravenous or subcutaneous with what ispolzovaniem device for delivery of liquid or small pumping system, including the mechanism of fluid supply for supplying fluid from the reservoir and triggering mechanism for actuating the drive mechanism. Pumping system for subcutaneous administration may include a needle or cannula for piercing the skin of a patient and deliver the right composition into the patient. These pumping systems can be directly fixed on the skin or attached to the skin of the patient in isolation from the vein, artery or blood vessel, thereby providing a direct contact between the pumping system and the patient's skin. The pumping system can be attached to the patient's skin from 24 hours to several days inclusive. The pumping system can be small in size with a reservoir for small amounts. As a non-limiting example, the volume of the tank to a suitable pharmaceutical composition, which must be entered, can be from 0.1 to 50 ml.

Continuous introduction may be using transdermal patch placed on the skin and replaced after a certain period of time. Specialist in the art is aware of the systems using the patch for the delivery of drugs that are suitable for this task. It should be noted that the transdermal introduction is particularly suitable as a non-interruptible injection, pascalc the replacement first used the patch preferably can be carried out simultaneously replacing them with new, the second patch, for example, on the surface of the skin, located in the immediate vicinity of the first used the patch and just before the destruction of 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, the binding molecules directed against and created against independent from the environment CD3 epitopes, can optionally contain a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include solutions such as phosphate buffered saline solutions, water, emulsions such as emulsions of oil/water, various types of wetting agents, sterile solutions, liposomes, etc. Compositions containing such carriers can be manufactured by well known conventional methods. The composition may contain carbohydrates, buffer solutions, amino acids and/or surfactants. Carbohydrates can be nereguliruemyi sugar, preferably trehalose, sucrose, octasulphate, sorbitol or xylitol. Such compositions can be used for continuous infusions, which can be intravenous or subcutaneous with and/or without use of the pump is item. Amino acids may constitute a charged amino acid, preferably lysine, lysine acetate, arginine, glutamate and/or histidine. Surfactants can be a detergent, preferably with molecular weight of more than 1.2 kDa, and/or easy polyester, preferably with a molecular mass of more than 3 kDa. Non-limiting examples of preferred detergent is Tween 20, Tween 40, Tween 60, Tween 80 or Tween 85. Non-limiting examples of preferred polyether is PEG (polyethylene glycol) 3000, PEG 3350, PEG 4000 or PEG 5000. The preferred pH value of the buffer systems used in the present invention, may be 5-9, and they may contain citrate, succinate, phosphate, histidine and acetate. The compositions of the present invention it is possible to introduce the subject at a suitable dose, which may be determined, for example, as a result of studies using increasing doses by injection of increasing doses of the polypeptide according to the invention, showing cross-species specificity described in this invention in relation to primates, not a chimpanzee, for example, Makarov. As mentioned above, the polypeptide according to the invention, showing cross-species specificity described in this invention, can be preferably used in the same form in polinices the Ohm testing on primates, not a chimpanzee, and in the form of a medicinal product in humans. These compositions can also be entered in combination with other protein and non-protein drugs. These drugs can be administered simultaneously with a composition comprising a polypeptide according to the invention, which is defined in this application, or separately before or after the introduction of the specified polypeptide in accordance with defined time intervals and doses. The dosage regimen will be determined by the attending physician and clinical indicators. As is well known in the fields of medicine, dosages for any one patient depends on many factors, including the size of the patient, the surface area, age, the particular input connection, sex, time and route of administration, General health and other drugs proposed for joint injection. Compositions for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters, such as etiloleat. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered environment. Parenteral times avieli include sodium chloride solution, dextrose in ringer's solution, dextrose and sodium chloride, ringer's solution with lactose or non-volatile oils. Intravenous diluents include liquid and nutritious fillers, electrolytes fillers (for example, on the basis of dextrose in ringer's solution) and the like. Can also contain 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 may contain protein carriers, such as, for example, serum albumin or immunoglobulin, preferably of human origin. It is envisaged that the composition according to the invention may contain, in addition to the polypeptide according to the invention is defined in this application, additional biologically active agents depending on the intended use of the composition. Such agents may be drugs acting on the gastrointestinal system, drugs acting as cytotoxic tools, medicines, warning hyperuricemia, medicines that suppress the immune response (e.g., corticosteroids), medicines, modulating the inflammatory response, medicines acting on the circulatory system, and/is whether such agents, as cytokines known in the art.

The biological activity of the pharmaceutical composition defined in this application may be determined, for example, on the basis of the analysis of cytotoxicity, as described in the following examples, in WO 99/54440, or Schlereth and others (Cancer Immunol. Immunother., 20 (2005), 1-12). "Efficiency" or "effectiveness in vivo", as used in this application refers to the response to therapy a pharmaceutical composition according to the invention, obtained by using, for example, standardized criteria for evaluation of response NCl (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 for its intended 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 in accordance with the approved standard methods of determining appropriate forms of diseases including, but not limited to, the determination of the number of leukocytes, differential blood count, sort method fluorescently-activated cells, the bone marrow aspiration. In addition to this can be used in methods for the determination of clinical chemistry parameters, specification is cnyh for various diseases, and other approved standard methods. In addition, can be used computed tomography, x-ray, nuclear magnetic resonance imaging (eg, for evaluation of the response based on the criteria of the National cancer Institute 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 Group. J. Clin. Oncol., 1999 Apr; 17(4): 1244]), positron emission tomographic scanning, identifying leukocyte count, differential blood count, sort fluorescently-activated cells, aspiration bone marrow biopsy/histology of lymph nodes and identify the different lymphoma-specific clinical-chemical parameters (e.g., lactate dehydrogenase) and other approved standard methods.

Another major problem in the development of drugs such as pharmaceutical composition according to the invention, the predicted modulation of pharmacokinetic properties. For this purpose, define the pharmacokinetic profile of the drug candidate, i.e. pharmacokinetic profile options that affect the ability of a particular drug to treat a given condition. Pharmacokinetic Parametrierung means, affect the ability of drugs to treat a specific disease state, include, but are not limited to: half-life, volume of distribution, metabolism first pass through the liver and the degree of binding to serum. On the efficiency of a given medicinal agent can influence each of the above parameters.

"Half-life" means the time at which 50% of the injected drug is excreted through biological processes such as metabolism, excretion and so on

Under the "first pass metabolism through the liver" understand 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 medicinal product in all the various compartments of the body, such as, for example, intracellular and extracellular spaces of tissues and organs and so on, and distribution of medicines in these compartments.

"The degree of binding to serum" refers to the ability of drugs to interact or communicate with serum proteins such as albumin, resulting in reduction or loss of biological activity of the medicinal product

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

"Bioavailability" refers to the amount of the drug in the blood compartment.

"Latent period" means the time lag between the introduction of the drug and its detection and measurement in blood or plasma.

"Tmax" is the time after which the maximum concentration of drug in the blood, and "Cmax" is the maximum concentration in the blood, resulting from the use of this medicinal product. For the period of time to achieve a concentration of the medicine in the blood or tissues, which is necessary for its biological effect is influenced by all parameters. Pharmacokinetic parameters bespecifically single-chain antibodies, the preferred embodiment of the polypeptide according to the invention, showing cross-species specificity, which can be determined in preclinical testing on animals-primates, non-chimpanzee listed above are, for example, in the publication Schlereth and others (Cancer Immunol. Immunother., 20 (2005), 1-12).

The term "toxicity" as used in this application, tositsa to the toxic effects of the medicinal product, expressed in adverse events or severe adverse events. These side effects could be attributed to lack of tolerability of the drug in whole and/or in the absence of local tolerance after injection. Toxicity could also include teratogenic or carcinogenic effects caused by this drug.

The term "security", "security " in vivo" or "tolerance", as used in this application determines the administration of a medicinal product without inducing serious adverse events immediately after the introduction (local tolerability) and for a longer period of drug administration. "Security", "security " in vivo" or "tolerance" can be estimated, for example, at regular intervals during treatment and follow-up. Measurements include clinical assessment, for example, organ manifestations and screening laboratory abnormalities. In accordance with the standards of NCI-CTC and/or MedDRA can be clinical evaluation and registered/encoded deviation with respect to the normal data. Organ manifestations may include criteria such as Allergy/immunology blood/bone marrow, cardiac arrhythmia, folding, and the like are described, for example, a Common terminology is riteria for adverse events (Common Terminology Criteria for adverse events) v3.0 (CTCAE). Laboratory parameters that may be tested include, for example, Hematology, clinical chemistry, coagulation profile and urine analysis and examination of other body fluids such as serum, plasma, lymph or cerebrospinal fluid, cerebrospinal fluid, and the like. Thus, security can be assessed, for example, by medical examination, the use of imaging techniques (i.e., ultrasound, x-rays, CT-scan, magnetic resonance imaging (MRI)), other dimensions with the use of technical devices (i.e., electrocardiogram), key indicators of the condition of the body, through the measurement of laboratory parameters and registration of adverse events. For example, adverse events in primates, not chimpanzees, applications and methods according to the invention can be investigated histopathological and/or histochemical methods.

The term "effective and non-toxic dose" as used in this application refers to the tolerance dose especifismo single-chain antibodies as defined in this application, which is high enough to cause depletion of abnormal cells, elimination of a tumor, reducing tumor or stabilization of the disease without or essentially without major toxic effects. So the e effective and non-toxic dose can be determined, for example, in studies of the action of increasing doses described in this technical field, and it must be lower than the doses that induce severe adverse side effects (doselimiting toxicity (dose limiting toxicity, DLT)).

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

In addition, the invention relates to pharmaceutical compositions containing the polypeptide according to this invention (i.e., a polypeptide containing at least one binding domain capable of contacting the epitope Epsilon-chain of CD3 person and the primacy of non-chimpanzee, and this epitope is a part of the amino acid sequence that is a member of the group consisting of SEQ ID NO: 2, 4, 6 or 8 according to this invention, or produced in accordance with the method according to the invention) to prevent, cure or ameliorate disease selected from a proliferative disease, tumor diseases or immunological disorders. Preferably, the specified pharmaceutical composition further comprises a suitable composition of carriers, stabilizers and/or excipients.

Another aspect of the invention is tositsa to the use of the polypeptide, which is defined in the application above or obtained according to the method specified in the application above, for the manufacture of pharmaceutical compositions for the prevention, cure or ameliorate disease. Preferably, the disease is a proliferative disease, a tumorous disease, or an immunological disorder. Also preferably, when the specified tumor disease is a malignant disease, preferably cancer.

In another preferred embodiment of the use of this polypeptide according to the invention specified in the pharmaceutical composition is suitable for administration in combination with an additional drug, i.e., as part of combination therapy. In the specified combination therapy active agent may be included in the same pharmaceutical composition, and the polypeptide according to the invention, or may be included in a separate pharmaceutical composition. In this latter case, the specified individual a pharmaceutical composition suitable for application to introducing, simultaneously with or after the introduction of the introduction of specified pharmaceutical compositions containing the polypeptide according to the invention. Additional drug or pharmaceutical composition can be a non-protein connection of the giving or protein connection. In that case, if the additional drug is a protein compound, it is preferable that this protein connection could provide an activation signal for immune effector cells.

Preferably, the specified protein compound or a non-protein compound can be administered simultaneously or asynchronously with the polypeptide according to the invention, the nucleic acid molecule as defined in this application above, a vector as defined in this application above, or the owner, as defined in this application above.

Another aspect of the invention relates to a method for prevention, cure or ameliorate disease in a subject in need thereof, comprising the stage of introducing an effective amount of the pharmaceutical composition according to the invention. Preferably, the disease is a proliferative disease, a tumorous disease, or an immunological disorder. Even more preferably, the specified tumor disease is a malignant disease, preferably cancer.

In another preferred embodiment of the method according to the invention specified in the pharmaceutical composition is suitable for administration in combination with an additional drug, i.e., as part of combination therapy. In the specified combined t is rapie active agent, may be included in the same pharmaceutical composition, and the polypeptide according to the invention, or may be included in a separate pharmaceutical composition. In this latter case, the specified individual a pharmaceutical composition suitable for application to introducing, simultaneously with or after the introduction of the introduction of specified pharmaceutical compositions containing the polypeptide according to the invention. Additional drug or pharmaceutical composition can be a non-protein compound or protein connection. In that case, if the additional drug is a protein compound, it is preferable that this protein connection could provide an activation signal to the effector cells of the immune system.

Preferably, the specified protein compound or a non-protein compound can be administered simultaneously or asynchronously with the polypeptide according to the invention, the nucleic acid molecule as defined in this application above, a vector as defined in this application above, or the owner, as defined in this application above.

Preferably, for the above-described method according to the invention, the specified subject is people.

The following aspect of the invention relates to a kit containing the polypeptide according to the invention, the molecule nuclei the OIC acid according to the invention, the vector according to the invention or of the owner according to the invention.

The present invention is additionally characterized by the following list of items.

Paragraph 1. A method for the identification of (a) polypeptide(s) that contain(s) binding domain with cross-species specificity, the ability to communicate with the epitope of CD3-Epsilon (CD3ε) of the person and the primacy of non-chimpanzee, which includes stages:

(a) bringing into contact of the polypeptide(s) with the N-terminal fragment of the extracellular domain of CD3ε, consisting of a maximum of 27 amino acids containing an amino acid sequence Gln-Asp-Gly-Asn-Glu-Glu-Met-Gly (SEQ ID NO: 381) or Gln-Asp-Gly-Asn-Glu-Glu-lle-Gly (SEQ ID NO: 382), fixed by means of its C-end on the solid phase;

(b) elution of bound(s) of the polypeptide(s) with the specified fragment; and

(C) isolation of the polypeptide(s) of the eluate (b).

Preferably, the polypeptide(s) identified by(e) the above-mentioned method according to the invention, had(and) of human origin.

This "way of identifying (a) polypeptide(s)" is understood as a way of separating one or more different polypeptides with the same specificity to 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: 381) or Gln-Asp-Gly-Asn-Glu-Glu-lle-Gly (SEQ ID NO: 382), from a variety of polypeptides candidate as well as the method of purification of poly is eptide from the solution. A non-limiting embodiment of the method of selection of one or more different polypeptides with the same specificity to the fragment of the extracellular domain of CD3ε include methods of selection of antigen-specific binding structures, for example, panning methods commonly used for hybridoma screening, screening temporarily/stably transfected clones of eukaryotic host cells or in the methods of phage display. A non-limiting example of the method of purification of the polypeptide from a solution is, for example, cleaning recombinante expressed polypeptide from the culture supernatant or preparation of such a culture.

As mentioned above, the fragment used in the method according to the invention, is an N-terminal fragment of the extracellular domain of CD3ε molecule primates. Amino acid sequence of the extracellular domain of CD3ε molecule different primates is presented in SEQ ID NO: 1, 3, 5 and 7. Two forms of the N-terminal of the octamer is presented in SEQ ID NO: 381, and 382. Preferably, this N-end was available without restrictions for binding polypeptides, which must be identified according to the method according to the invention. The term "available without restriction" in the context of the invention is understood as no additional motives, such as His-tag. The interference of such a His-tag with svyazyvayuscyego, identified by the method according to the invention described in the attached Example 6.

According to the method according to the invention the specified portion is fixed via its C-end on the solid phase. Specialist in the art easily and without any inventive difficulties will choose a suitable solid 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 granules, polystyrene granules, dextranase granules, tablets (cultural tablets or advance tablets), as well as chips, are known, for example, Biacore®. The choice of means and methods of fixation/immobilization fragment on a given solid substrate depends on the choice of the solid substrate. The most commonly used method of fixation/immobilization is binding through the N-hydroxysuccinimidyl (NHS) ester. Underlying this binding chemistry, as well as alternative methods of fixation/immobilization well-known specialist in the art, for example, 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 dextranase pellets (Sigma) or activated polymethacrylate. These reagents can also be used in the periodic approach. In addition, periodic approach can be used dextranase pellets containing iron oxide (e.g., available from Miltenyi). These granules can be used in combination with a magnet to separate the pellets from the solution. The polypeptides can be immobilized on the Biacore chip (for example, chips SM) by using NHS-activated carboxymethylcysteine. The following examples of appropriate solid substrate are advance tablets with reactive amino groups (for example, tablets, Nunc Immobilizer™).

According to the method according to the invention the specified fragment of the extracellular domain of CD3-Epsilon can be associated with the solid substrate directly or through a stretch of amino acids, which may be a linker or other protein/polypeptide group. Alternatively, the extracellular domain of CD3-Epsilon can be connected directly through one or more than one adapting molecule.

Tools and methods for evaluation of peptide bound to the immobilized epitope are well known in the art. The same is true for cues identifiable(s) of the polypeptide(s) of the eluate.

In accordance the invention, the method of selection from a variety of polypeptides candidates of one or more different polypeptides with the same specificity to the fragment of the extracellular domain CD3e, containing at its N-end amino acid sequence Gln-Asp-Gly-Asn-Glu-Glu-X-Gly, may include one or more stages following the selection of antigen-specific structures.

CD3ε-specific binding molecules may be selected from the repertoire of antibodies. Library of phage display can be constructed on the basis of standard procedures, which are 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 the antibodies in the library of antibodies can be scFv, but in the General case can also be Fab-fragment, or even a fragment of single-domain antibodies. For allocating fragments of antibodies can be used naive libraries of fragments of antibodies. For selection of binding structures with the lowest possible immunogenicity in their subsequent therapeutic use of preferred may be libraries of fragments of human antibodies for direct selection of fragments of human antibodies. In some cases, they can form the basis for a synthetic libraries of antibodies (Knappik et al. J. Mol. Biol., 2000, 296: 57). The appropriate format can be Fab, scFv (as described below) or a domain antibody (dAb, which are reviewed in Holt et al., Trends Biotechnol., 2003, 21: 484 ff).

In the art it is also known that in many cases there is no source of human them is unnuh antibodies useful against the target antigen. Therefore, a given target antigen is subjected to immunization of the animal, and from animal tissues, such as spleen or RVMS (mononuclear cells peripheral blood), allocate the appropriate library of antibodies. N-terminal fragment can be biotinylated or covalently associated with a protein type KLH (hemocyanin lymph snails) or bovine serum albumin (BSA). According to the General approaches used for immunization of rodents. Some repertoires of immune antibodies of human origin can be especially beneficial for other reasons, for example, due to the presence of single-domain antibodies (VHH), originating from the 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 corresponding format of the library of antibodies can be Fab, scFv (as described below) or single domain antibodies (VHH).

In one of the possible approaches mouse F1 at the age of 10 weeks from crossing balb/c x 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 Mature CD3ε-chain. Alternatively, mice can be immunized fused protein 1-27 CD3-Epsilon-MS (corresponding to the approach described in the accompanying Example 2). After busterna the(s) immunization(s) can be selected samples of blood and serum titers of antibodies against CD3-positive T cells can be tested, for example, in FACS-analysis. Usually serum titers significantly higher in immunized than in unimmunized animals.

Immunized animals may become the basis for the creation of immune libraries of antibodies. Examples of such libraries include the libraries of phage display. Such libraries in General can be based on the standard methods, which are described, for example, in "Phage Display: A Laboratory Manual"; Ed. Barbas, Burton, Scott & Silverman; Cold Spring Harbor Laboratory Press, 2001.

In addition, human antibodies can be humanitarian by phage display in the establishment of libraries more variable antibodies, which can then be enriched in relation linking agents (binders) in the selection process.

In the approach using phage display any of the pools of phage, which exhibits a library of antibodies, forms the basis for selection of the linking structures using the corresponding antigen as a target molecule. The Central stage on which secrete antigen-specific antigen-related phages, designated as panning. Due to the exposure of fragments of antibodies on the surface of phages, this General method is called rahovym display. One of the preferred methods of selection is the use of small proteins, such as domain N2 filamentous phage, trance is azione fused with the N-end scFv, exhibited by this phage. Another display method known in the art, which can be used to identify binding structures is the method of ribosomal display (an overview of which is given 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 from the fused protein 1-27 CD3ε-Fc phage library that carries the cloned scFv-repertoire can be obtained from the corresponding culture supernatants using PEG (polyethylene glycol). scFv-containing phage particles can be preincubator merged with immobilized protein CD3ε-Fc. Immobilized protein CD3ε-Fc can be applied in the form of a coating on a solid phase. Linking patterns can be suirvey, and this eluate can be used to infect a fresh portion uninfected bacterial hosts. Bacterial hosts, subject to the successful transduction fahmideh copy that encodes a human scFv-fragment, can again be subjected to selection for resistance to carbenicillin and then infected with, for example, helper phage VCMS 13 to start the second round of exposure of antibodies (antibody display and selection in vitro. In the normal conduct a total of from 4 to 5 cycles of selection.

Linking the selected binding is x structures can be tested on CD3-positive Jurkat cells, the HPBall cells, RVS or transfected eukaryotic cells that carry N-terminal CD3ε-sequence, merged with the exposed surface Arcam using flow-cytometrics analysis (see attached Example 4).

Paragraph 2. The method according to claim 1 wherein the polypeptide(s) contains(at) identified binding domain as the first binding domain and a second binding domain capable of contact with a cell surface antigen.

To create a second binding domain polypeptide identified by the method according to the invention, for example bespecifically single-chain antibodies as defined in this application can be used monoclonal antibodies to bind to the appropriate cell surface antigens of human and Primate, not a chimpanzee. The corresponding binding domains for especifismo polypeptide as defined in this application may be obtained, for example, monoclonal antibodies with cross-species specificity using recombinant methods described in the art. Monoclonal antibody binding to the cell surface antigen of a man and a homologue of the indicated cell surface antigen of the primacy of non chimpanzees, can be tested in FACS-anal is Zech, above. In addition, to generate antibodies with cross-species specificity can be used hybridoma technique described in the literature (Milstein and Kohler, Nature 256 (1975), 495-7). For example, mice can alternately to immunize CD33 human and Primate, not a chimpanzee. From these mice by hybridoma technology produce hybridoma cells producing antibodies with cross-species specificity, and analyzed by FACS as above. Creation and analysis bespecifically polypeptides, such as bespecifically single-chain antibodies, demonstrating cross-species specificity, which are described in the present invention, shown in the following Examples. Advantages bespecifically single-chain antibodies, demonstrating cross-species specificity, include the points listed below.

Item 3. The method according to paragraph 2, where the second binding domain binds to a cell surface antigen of human and/or Primate, not a chimp.

Item 4. The method according to any of paragraphs 1 to 3, where the first binding domain is an antibody.

Paragraph 5. The method according to item 4, wherein the antibody is a single-chain antibody.

Item 6. The method according to any of paragraphs 2-5, where the second binding domain is an antibody.

Item 7. The method according to any of items 1-6, g the e fragment of the extracellular domain of CD3ε consists of one or more fragments of the polypeptide, having an amino acid sequence selected from any of the sequences shown in SEQ ID NO: 2, 4, 6, or 8.

Item 8. The method according to paragraph 7, where the length of the specified fragment is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 amino acid residues.

Item 9. The method according to any of items 1-8, where the authentication method is a method of screening a variety of polypeptides containing a binding domain with cross-species specificity, the ability to communicate with epitope CD3ε human and Primate, not a chimp.

Paragraph 10. The method according to any of items 1-8, where the authentication method is a method of purification/separation of the polypeptide containing the binding domain with cross-species specificity, the ability to communicate with epitope CD3ε human and Primate, not a chimp.

Paragraph 11. The use of the N-terminal fragment of the extracellular domain of CD3ε, consisting of a maximum of 27 amino acids containing an amino acid sequence Gln-Asp-Gly-Asn-Glu-Glu-Met-Gly (SEQ ID NO: 381) or Gln-Asp-Gly-Asn-Glu-Glu-lle-Gly (SEQ ID NO: 382) to create a binding domain with cross-species specificity.

In accordance with the use according to the invention is preferably created binding domain with interspecies specificity was of human origin.

Item 12. Application under paragraph 11, which connects the omen with interspecies specificity is an antibody.

Paragraph 13. Application under paragraph 12, where the antibody is a single-chain antibody.

Paragraph 14. Use paragraphs 12-13, where the antibody is bespecifically antibody.

These and other embodiments are described and included in 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, 3rd edition, 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. Additional literature concerning any of the antibodies, methods, applications and connections used in accordance with the present invention, it is possible to extract from public libraries and databases, using, for example, electronic device. For example, you can use a shared database "Medline", available via the Internet, for example, the url http://www.ncbi.nlm.nih.gov/PubMed/medline.html. Additional databases and addresses, such as http://www.ncbi.nlm.nih.gov/ or listed on the home page, service EMBL (European Molecular Biology Laboratory) under the address http://www.embl.de/services/index.html known to the specialist in the art and can also be obtained using, for example, http://www.google.com.

Graphic materials (see below).

Fig.1

Fused construction of N-terminal amino acids 1-27 CD3-Epsilon PR the mats with heterologous soluble protein.

Fig.2

In Fig. shows the average absorbance values repeated in quadruplicate samples measured in the ELISA (enzyme-linked immunosorbent assay) determining the presence of a construct consisting of the N-terminal amino acids 1-27 Mature Epsilon-chain of human CD3, merged with the hinge and Fc-gamma-section of human IgG1 and a C-terminal 6-his-tag label in the supernatant (SN) temporarily transfected 293 cells. In the first column, labeled "27 AA huCD3E", shows the average absorbance value for construction, in the second column, marked as "nedelev. SN", shows the average value for the supernatant of 293 cells, transfected with irrelevant design as a negative control. A comparison of values obtained for this design, with the values obtained for the negative control, clearly demonstrates the presence of the recombinant construct.

Fig.3

In Fig. shows the average absorbance values repeated in quadruplicate samples measured in ELISA analysis, determining the binding of anti-CD3 binding molecules with cross-species specificity in the form of crude preparations of single-chain antibodies expressed in periplasmatic space, with a design containing N-terminal amino acids 1-27 Mature Epsilon-chain of human CD3, merged with the hinge and Fc-gamma-what Astok human IgG1 and C-terminal His6-tag. The columns show, from left to right, the average absorbance values for specificdate designated as A2J HLP, I2C HLP, EM HLP, F70 HLP, G4H HLP, NS HLP, E1L HLP, F12Q HLP, F6A HLP and NE HLP. In the rightmost column, labeled "neg. CTR.", shows the average absorbance value for the preparation of single-chain mouse antibodies against human CD3 as a negative control. A comparison of values obtained for anti-CD3-specificdate, with values obtained for the negative control (neg. CTR.), clearly demonstrates the strong binding of anti-CD3-specificdate N-terminal amino acids 1-27 Mature Epsilon-chain of human CD3.

Fig.4

Fused construction of N-terminal amino acids 1-27 CD3-Epsilon primates with heterologous membrane-bound protein.

Fig.5

Overlapping histograms of different transfectants tested in FACS analysis, determining the presence of recombinant transmembrane fused protein consisting of Arcam cynomolgus macaque and N-terminal amino acids 1-27 Epsilon-chain of CD3 human, marmoset, Tamarin, squirrel monkey and domestic pigs, respectively. Overlapping histograms, from left to right and top to bottom, show the results for transfectants expressing constructs containing the 27-Mer man, 27-Mer marmoset, 27-Mer Tamarin, 27-Mer squirrel monkeys and 27-Mer pigs, respectively. To separate the switch is of Ivani thin line represents the sample incubated with PBS (phosphate buffered saline) containing 2% FCS (fetal calf serum instead of anti-Flag M2 antibody as a negative control, and the bold line shows the sample incubated with anti-Flag M2 antibody. For each design the overlap of the histograms shows the binding of anti-Flag M2 antibodies with transfectants that clearly demonstrates the expression of recombinant constructs in the transfectants.

Fig.6

Overlapping 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-chain antibodies expressed in periplasmatic space, with N-terminal amino acids 1-27 Epsilon-chain of CD3 human, marmoset, Tamarin and squirrel monkeys, respectively, merged with Arcam cynomolgus macaque.

Fig.6A

Overlapping histograms, from left to right and top to bottom, show the results for transfectants expressing the 1-27 CD3-EpCAM containing 27-Mer man, tested using the CD3 specific binding molecules, designated as NS HLP, F12Q HLP, EM HLP and G4H HLP, respectively.

Fig.6V

Overlapping histograms, from left to right and top to bottom, show the results for transfectants expressing the 1-27 CD3-EpCAM, soda is containing 27-Mer marmoset, tested using the CD3 specific binding molecules, designated as NS HLP, F12Q HLP, EM HLP and G4H HLP, respectively.

Fig.6S

Overlapping histograms, from left to right and top to bottom, show the results for transfectants expressing the 1-27 CD3-EpCAM containing 27-Mer Tamarin, tested using the CD3 specific binding molecules, designated as NS HLP, F12Q HLP, EM HLP and G4H HLP, respectively.

Fig.6D

Overlapping histograms, from left to right and top to bottom, show the results for transfectants expressing the 1-27 CD3-EpCAM containing 27-Mer squirrel monkeys tested using the CD3 specific binding molecules, designated as NS HLP, F12Q HLP, EM HLP and G4H HLP, respectively.

Fig.6E

In histograms, from left to right and top to bottom, shows the results of transfectants expressing the 1-27 CD3-EpCAM containing 27-Mer pigs tested using the CD3 specific binding molecules, designated as NS HLP, F12Q HLP, EM HLP and G4H HLP, respectively.

For individual interleaved thin line represents the sample incubated with drug-stranded mouse antibodies against human CD3 as a negative control, and the bold line shows the sample incubated with the appropriate specified anti-CD3 binding molecules of the mi. Given the lack of binding to the transfectants 27-measure pigs and levels of expression of the structures shown in Fig.5, overlapping 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 fused proteins containing N-terminal amino acids 1-27 Epsilon-chain of CD3 human, marmoset, Tamarin and squirrel monkeys, respectively, merged with Arcam cynomolgus macaque, and thus exhibit species specificity of anti-CD3 binding molecules for many primates.

Fig.7

FACS-analysis for opredeleniya CD3-Epsilon person in transfected murine EL4 T cells. Graphical analysis shows the overlap between the histograms. Bold line shows transfetsirovannyh cells incubated with the antibody UCHT-1 against human CD3. The thin line represents cells incubated with mouse control IgG1 isotype. The binding of an antibody UCHT1 against CD3 clearly demonstrates the expression of Epsilon-chain of human CD3 on the cell surface of transfected murine EL4 T cells.

Fig.8

Binding of anti-CD3 antibodies with cross-species specificity with alanine mutants in the experiment alanine functional is. Columns in a separate graphic materials, from left to right and top to bottom, show the values of binding calculated in arbitrary units on a logarithmic scale for transfectant wild-type (WT) and for all alanine mutants for position 1-27. The values of binding calculated using the following formula:

In this equation value_Sample means the amount of binding in arbitrary units representing the degree of binding of specific anti-CD3-antibodies with specific alanine mutant, as shown in this Fig., Sample mean geometric average value of the fluorescence obtained for the specific anti-CD3 antibodies, and analyzed for specific alanine-scanning transfectant, neg_Contr. mean geometric average value of the fluorescence obtained for the negative control, were analyzed for specific alanine mutant, UCHT-1 means the average geometric size of the fluorescence obtained for antibody UCHT-1, were analyzed for specific alanine mutant, WT denotes the average geometric size of the fluorescence obtained for the specific anti-CD3 antibodies, and analyzed on transfectant wild-type, x indicates the corresponding transfectant, indicates the corresponding anti-CD3 antibody, a wt, transfectant refers to the wild type. Individual provisions of the alanine mutants labeled using the one-letter code of amino acids of the wild type and position.

Fig.8A

In Fig. showing results for anti-CD3-antibodies A2J HLP with interspecies specificity expressed in the form of the chimeric molecule of IgG. Reduced activity of binding observed for mutations and replaced with alanine in position 4 (aspartic acid, at position 23 (threonine) and in regulation 25 (isoleucine). Complete loss of binding was observed for mutations and replaced with alanine in position 1 (glutamine), in position 2 (aspartate), position 3 (glycine) and position 5 (glutamate).

Fig.8B

In Fig.showing results for anti-CD3-antibodies EM HLP with interspecies specificity expressed in the form of the chimeric molecule of IgG. Reduced activity of binding observed for mutations and replaced with alanine in position 4 (aspartic acid, at position 23 (threonine) and in regulation 25 (isoleucine). Complete loss of binding was observed for mutations and replaced with alanine in position 1 (glutamine), in position 2 (aspartate), position 3 (glycine) and position 5 (glutamate).

Fig.8S

In Fig.showing results for anti-CD3-antibodies NS HLP with interspecies specificity expressed in the form of the chimeric molecule of IgG. Reduced activity of binding observed for evolution is th and replaced with alanine in position 4 (asparagine). Complete loss of binding was observed for mutations to alanine glutamine in position 1 (glutamine), in position 2 (aspartate), position 3 (glycine) and position 5 (glutamate).

Fig.8D

shows the results for anti-CD3-antibodies F12Q HLP with interspecies specificity tested in the form of periplasmatic expressed single-chain antibodies. Complete loss of binding was observed for mutations and replaced with alanine in position 1 (glutamine), in position 2 (aspartate), position 3 (glycine) and position 5 (glutamate).

Fig.9

FACS-analysis, which determines the binding of anti-CD3 binding molecules NS HLP with interspecies specificity with human CD3, containing and not containing N-terminal His6-tag.

Overlapping histograms carried out on cell lines EL4, transtitional CD3 Epsilon-chain wild-type human (left histogram) or CD3 Epsilon-chain of man, containing N-terminal His6-tag (right histogram), tested in FACS analysis, determining the binding of the binding molecules NS HLP with interspecies specificity. Samples incubated with control of the relevant isotype as a negative control (thin line), antibody UCHT-1 against human CD3 as a positive control (dashed line) and anti-CD3-antibody NS HLP with interspecies specificity in the form of the chimeric molecule IgG (bold line)

Overlapping histograms show comparable binding antibody UCHT-1 with both transfectants compared with ezotericheskim control, demonstrating the expression of both recombinant constructions. Overlapping histograms 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 man. These results demonstrate that the presence of free N-Terminus is essential for the binding of anti-CD3 binding molecules NS HLP with interspecies specificity.

Fig.10

Saturating the binding of EGFR-21-63 LH x NS on CD3-positive RVMS of a person for the purpose of determining the value of KD CD3-binding cells using FACS analysis. The analysis is carried out, as described in Example 7.

Fig.11

FACS analysis of binding of these bespecifically single-stranded structures with cross-species specificity to cells SNO, transfitsirovannykh EGFR person, with CD3+ T-cell line human HPB-ALL cells SNO, transfitsirovannykh EGFR cynomolgus macaque, and with T-cell line macaque 4119 LnPx. FACS-staining carried out as described in Example 12. Bold line represents cells incubated with 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 shown in detail the t negative control: cells, incubated only with anti-his-antibody and detecting antibody.

Fig.12

The cytotoxic activity induced by these EGFR-specific single-stranded structures with cross-species specificity, redirected to the indicated target cell lines. A) and b): stimulated CD4/CD56-RVMS person used as effector cells, Cho, transfetsirovannyh human EGFR as target cells. The analysis is carried out, as described in Example 13.

Fig.13

The cytotoxic activity induced by these EGFR-specific single-stranded structures with cross-species specificity, redirected to the indicated target cell lines. A) and b): T-macaque cell line 4119 LnPx used as effector cells, Cho, transfetsirovannyh EGFR cynomolgus macaque, as target cells. The analysis is carried out, as described in Example 13.

Fig.14

FACS analysis of binding of these bespecifically single-stranded structures with cross-species specificity to cells SNO, transfitsirovannykh MCSP D3 of the person with CD3+T-cell line human HPB-ALL cells SNO, transfitsirovannykh MCSP D3 cynomolgus macaque, and with T-cell line macaque 4119 LnPx. FACS staining was performed as described in Example 17. Bold line represents cells incubated with purified Belko is at a concentration of 2 μg/ml, which then incubated with anti-his-antibody and PE-labeled detection antibody. Thin line on the histogram reflects the negative control: cells incubated only with anti-his-antibody and detecting antibody.

Fig.15

FACS analysis of binding of these bespecifically single-stranded structures with cross-species specificity to cells SNO, transfitsirovannykh MCSP D3 of the person with CD3+ T-cell line human HPB-ALL cells SNO, transfitsirovannykh MCSP D3 cynomolgus macaque, and with T-cell line macaque 4119 LnPx. FACS staining was performed as described in Example 17. Bold line represents cells incubated with 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 histogram reflects the negative control: cells incubated only with anti-his-antibody and detecting antibody.

Fig.16

FACS analysis of binding of these bespecifically single-stranded structures with cross-species specificity to cells SNO, transfitsirovannykh MCSP D3 of the person with CD3+T-cell line human HPB-ALL cells SNO, transfitsirovannykh MCSP D3 cynomolgus macaque, and with T-cell line macaque 4119 LnPx. FACS staining was performed as described in Example 17. Bold line represents cells, inkubirovanii is with purified protein at a concentration of 2 μg/ml, which then incubated with anti-his-antibody and PE-labeled detection antibody. Thin line on the histogram reflects the negative control: cells incubated only with anti-his-antibody and detecting antibody.

Fig.17

Cytotoxic activity induced specified MCSP-specific single-stranded structures with cross-species specificity, redirected to the indicated target cell lines. : Stimulated CD4/CD56-RVMS person used as effector cells, Cho, transfetsirovannyh MCSP D3 man, as target cells. ): T-macaque cell line 4119 LnPx used as effector cells, Cho, transfetsirovannyh MCSP D3 cynomolgus macaque, as target cells. The analysis is carried out, as described in Example 18.

Fig.18

Cytotoxic activity induced specified MCSP-specific single-stranded structures with cross-species specificity, redirected to the indicated target cell lines. A) and b): T-macaque cell line 4119 LnPx used as effector cells, Cho, transfetsirovannyh MCSP D3 cynomolgus macaque, as target cells. The analysis is carried out, as described in Example 18.

Fig.19

Cytotoxic activity induced specified MCSP-specific single-stranded what instrukciyami with interspecies specificity, redirected to the indicated target cell lines.

A) and b): stimulated CD4/CD56-RVMS person used as effector cells, Cho, transfetsirovannyh MCSP D3 man, as target cells. The analysis is carried out, as described in Example 18.

Fig.20

Cytotoxic activity induced specified MCSP-specific single-stranded structures with cross-species specificity, redirected to the indicated target cell lines. : Stimulated CD4/CD56-RVMS person used as effector cells, Cho, transfetsirovannyh MCSP D3 man, as target cells. ): T-macaque cell line 4119 LnPx used as effector cells, Cho, transfetsirovannyh MCSP D3 cynomolgus macaque, as target cells. The analysis is carried out, as described in Example 18.

Fig.21

Cytotoxic activity induced specified MCSP-specific single-stranded structures with cross-species specificity, redirected to the indicated target cell lines. : Stimulated CD4/CD56-RVMS person used as effector cells, Cho, transfetsirovannyh MCSP D3 man, as target cells. ): T-macaque cell line 4119 LnPx used as effector cells, Cho, transfetsirovannyh MCSP D3 Javanese mA is aka as target cells. The analysis is carried out, as described in Example 18.

Fig.22

Stability in plasma MCSP and CD3-bespecifically single-chain antibodies with cross-species specificity tested by measuring the cytotoxic activity induced samples of these single-stranded structures, inkubiruemykh in the presence of 50% human plasma at 37°C and 4°C for 24 hours, respectively, or with the addition of 50% human plasma directly before testing cytotoxicity or without the addition of plasma. Cells SNO, transfetsirovannyh MCSP person, used as a target cell lines and stimulated CD4/CD56-RVMS person used as effector cells. The analysis is carried out, as described in Example 19.

Fig.23

FACS analysis of binding of these bespecifically single-stranded structures with cross-species specificity to cells SNO, transfitsirovannykh HER2 person, with CD3+ T-cell line human HPB-ALL cells SNO, transfitsirovannykh HER2 macaque T cell line macaque 4119 LnPx, respectively. FACS-staining carried out as described in Example 23.4. The bold lines represent cells incubated with purified bespecifically-stranded structure at a concentration of 2 µg/ml Thin lines represent negative controls. As a negative is the control used PBS with 2% FCS. For each bespecifically-stranded structure with interspecies specificity overlap of the histograms shows the specific binding of this design with HER2 human and macaque and CD3 human and macaque.

Fig.24

The graphs depict the results of chromium release, which measure the cytotoxic activity induced specified NER-specific single-stranded structures with cross-species specificity, redirected to the indicated target cell lines. Also specify the effector cells. The analyses carried out as described in Example 23.5. The chart shows clearly shown for each design strong increase of the cytotoxic activity of effector cells of human and macaque cells against SNO, HER2 transfected human and macaque, respectively.

Fig.25

CD3-specific ELISA analysis periplasmatic preparations containing Flag-labeled protein scFv-fragments of the selected clones. Periplasmic preparations of soluble scFv protein fragments were added to wells for ELISA, which were coated with soluble fused protein [CD3 Epsilon (AA 1-27) man]-Fc (AA denotes an amino acid) and were further blocked with PBS 3% BSA. Opredelenie was performed using monoclonal anti-Flag-Biotin-labeled antibody, the ATEM conjugated with horseradish peroxidase streptavidin. ELISA showed a solution of ABTS substrate (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate). Size OD (optical density) (y axis) was measured at 405 nm using ELISA reader. The names of the clones represented on the axis X.

Fig.26

ELISA analysis periplasmatic preparations containing Flag-labeled protein scFv-fragments of the selected clones. The same periplasmic preparations of soluble scFv protein fragments, as in Fig.25, was added to the wells for ELISA, which were not covered soluble fused protein [CD3 Epsilon (AA 1-27) man]-Fc, but were covered with human IgG1 (Sigma) and blocked in 3% BSA in PBS.

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

The present invention is additionally described by the following non-limiting illustrative examples that provide a better understanding of the present invention and many of its advantages.

EXAMPLES

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

To identify CD3-Epsilon used blood samples following primates, non-human: Callithrix jacchus, Sagunus oedipus and Saimiris ciureus. For isolation of total cellular RNA was prepared fresh samples treated with heparin whole blood in accordance with the manufacturer's Protocol (QIAamp RNA Blood Mini Kit, Qiagen). Carried out the transcription of extracted mRNA into cDNA in accordance with published protocols. Briefly, 10 µl of the precipitated RNA was incubated with 1.2 μl of 10-fold hexanucleotide mixture (Roche) at 70°C for 10 minutes and kept on ice. Was added to the reaction mixture containing 4 μl of 5 × superscript buffer II, 0.2 µl 0.1 M dithiothreitol, and 0.8 μl of 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 Dinkas 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 left to cool on ice, then added to 0.8 ál of RNase H (1 unit/μl, Roche) and incubated for 20 minutes at 37°C.

cDNA first circuit of each sample were subjected to 35 separate cycles of polymerase chain reaction using DNA polymerase Tag (Sigma) and the following combination of primers designed based on the study database: direct primer 5'-AGAGTTCTGGGCCTCTGC-3' (SEQ ID NO: 377); reverse primer 5'-CGGATGGGCTCATAGTCTG-3' (SEQ ID NO: 378). Amplificatoare zone 550 p. O. was isolated from the gel (Gel Extraction Kit, Qiagen) and sequenced (Sequierve, Vaterstetten/Germany, see sequence listing).

CD3-Epsilon Callithrix iacchus

Nucleotides

CAGGACGGTAATGAAGAAATGGGTGATACTACACAGAACCCATATAAAGTTTCCATCTCAGGAACCACAGTAACACTGACATGCCCTCGGTATGATGGACATGAAATAAAATGGCTCGTAAATAGTCAAAACAAAGAAGGTCATGAGGACCACCTGTTACTGGAGGACTTTTCGGAAATGGAGCAAAGTGGTTATTATGCCTGCCTCTCCAAAGAGACTCCCGCAGAAGAGGCGAGCCATTATCTCTACCTGAAGGCAAGAGTGTGTGAGAACTGCGTGGAGGTGGAT

Amino acids

QDGNEEMGDTTQNPYKVSISGTTVTLTCPRYDGHEIKWLVNSQNKEGHEDHLLLEDFSEMEQSGYYACLSKETPAEEASHYLYLKARVCENCVEVD

CD3-Epsilon Saguinus oedipus

Nucleotides

CAGGACGGTAATGAAGAAATGGGTGATACTACACAGAACCCATATAAAGTTTCCATCTCAGGAACCACAGTAACACTGACATGCCCTCGGTATGATGGACATGAAATAAAATGGCTTGTAAATAGTCAAAACAAAGAAGGTCATGAGGACCACCTGTTACTGGAGGATTTTTCGGAAATGGAGCAAAGTGGTTATTATGCCTGCCTCTCCAAAGAGACTCCCGCAGAAGAGGCGAGCCATTATCTCTACCTGAAGGCAAGAGTGTGTGAGAACTGCGTGGAGGTGGAT

Amino acids

QDGNEEMGDTTQNPYKVSISGTTVTLTCPRYDGHEIKWLVNSQNKEGHEDHLLLEDFSEMEQSGYYACLSKETPAEEASHYLYLKARVCENCVEVD

CD3-Epsilon Saimiri sciureus

Nucleotides

CAGGACGGTAATGAAGAGATTGGTGATACTACCCAGAACCCATATAAAGTTTCCATCTCAGGAACCACAGTAACACTGACATGCCCTCGGTATGATGGACAGGAAATAAAATGGCTCGTAAATGATCAAAACAAAGAAGGTCATGAGGACCACCTGTTACTGGAAGATTTTTCAGAAATGGAACAAAGTGGTTATTATGCCTGCCTCTCCAAAGAGACCCCCACAGAAGAGGCGAGCCATTATCTCTACCTGAAGGCAAGAGTGTGT GAGAACTGCGTGGAGGTGGAT

Amino acids

QDGNEEIGDTTQNPYKVSISGTTVTLTCPRYDGQEIKWLVNDQNKEGHEDHLLLEDFSEMEQSGYYACLSKETPTEEASHYLYLKARVCENCVEVD

2. The creation of single-stranded fragments of antibodies (scFv) with interspecies specificity, binding to the N-terminal amino acids 1-27 CD3-Epsilon person and various primates, non-chimpanzee

2.1. Immunization of mice using N-end of the CD3-Epsilon, isolated from its native CD3 environment by merging with heterologous soluble protein

Mice F1 from crosses balb/c x C57black at the age of ten weeks immunizer the Wali fused protein CD3-Epsilon-Fc, bearing a large part of the N-terminal amino acids (1-27) Mature Epsilon-chain of CD3 (1-27 CD3-Fc) of human and/or common squirrel monkeys. For this 40 µg fused protein 1-27 CD3-Fc with 10 nmol of tiout-modified CpG-oligonucleotide (5'-tccatgacgttcctgatgct-3') in 300 μl PBS per mouse was administered by intraperitoneal injection. Mice received a booster immunization after 21, 42 and 63 may day in the same way. Ten days after the first booster immunization was collected the blood samples and the antibody titer in the serum against the fused protein 1-27 CD3-FC was tested with ELISA. Additionally tested titer against CD3-positive T-cell line human HPBall using flow cytometry according to standard protocols. Serum titers were significantly higher in immunized than in unimmunized animals.

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

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

Using 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 region of the light chain (to the PAP) (VK) and variable regions of the heavy chain (VH) of immunoglobulins (Ig) mouse. cDNA was synthesized according to standard protocols.

The primers were designed in such a way as to create a website recognition for the 5'Xhol and 3'-BstEII for amplified V-fragments of the heavy chain and site recognition for the 5'Sacl and 3'-Spel for amplified VK-DNA fragments. For 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; MVH2 GAG GTC CAG CTC GAG CAG TST GGA CCT; MVH3 CAG GTC CAA CTC GAG CAG CCT GGG GCT; MVH4 GAG GTT CAG CTC GAG CAG TCT GGG GCA; MVH5 GA(AG) GTG AAG CTC GAG GAG TCT GGA GGA; MVH6 GAG GTG AAG CTT CTC GAG TCT GGA GGT; MVH7 GAA GTG AAG CTC GAG GAG TCT GGG GGA; MVH8 GAG GTT CAG CTC GAG CAG TCT GGA GCT) was combined with one of the 3'VH primer (3'MuVHBstEII tga gga gac ggt gac cgt ggt ccc ttg gcc cca g); for PCR amplification of fragments VK-circuit each of the seven different primers specific to the 5'-VK-family (MUVK1 CCA GTT CCG AGC TCG TTG TGA CTC AGG AAT ARTICLE; MUVK2 CCA GTT CCG AGC TCG TGT TGA CGC AGC CGC SS; MUVK3 CCA GTT CCG AGC TCG TGC TCA VCC AGT CTC CA; MUVK4 CCA GTT CCG AGC TCC AGA TGA CCC AGT CTC CA; MUVK5 CCA GAT GTG AGC TCG TGA TGA CCC AGA CTC CA; MUVK6 CCA GAT GTG AGC TCG TCA TGA CCC AGT CTC CA; MUVK7 CCA GTT CCG AGC TCG TGA TGA CAC AGT CTC CA) combined with a single 3'-VK-primer (3'MuVkHindIII/BsiW1 tgg tgc act agt cgt acg ttt gat ctc aag ctt ggt ccc).

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

450 ng of fragments of the light chain of the cap is a (split Sacl-Spel) ligated with 1400 ng family pComb3H5Bhis (split Sacl-Spel; a large fragment). The obtained combinatorial biblioteque antibodies were then subjected to 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 μf; 200 Ohm, Biorad gene-pulser), receiving in the library larger than 107 independent clones. After one hour the phenotypic expression of selected positive transformants for resistance to carbenicillin, encoded by the vector pComb3H5BHis, 100 ml of liquid SB(super broth) culture during the night. Then the cells were collected by centrifugation and plasmid was obtained using a commercially available kit to obtain plasmid (Qiagen).

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

After phenotypic expression and slow adaptation to carbenicillin cells of E. coli containing a library of antibodies, transferred to the SB-carbenicillin(50 μg/ml)-selective environment. Cells of E. coli containing a library of antibodies, and then infected infectio the Noah dose, equal to 1012 particles of helper phage VCSM13 resulting in the production and secretion of filamentous M13 phage, where the phage particle contains a single-stranded pComb3H5BHis-flHK encoding murine scFv-fragment, and exhibited the corresponding scFv-protein in the form of a translational fusion with the envelope protein III of phage. This pool of phages exhibiting a library of antibodies, and then used for breeding antigenspecific structures.

2.3. Selection of CD3-specific binding agents on the basis of phage display

Phage library that carries the cloned scFv-repertoire, received from the respective culture supernatant by precipitation with PEG8000/NaCl and centrifugation. Approximately 1011up to 1012scFv-phage particles resuspendable in 0.4 ml of PBS/0,1% BSA and incubated with 105-107 cells Jurkat (CD3-positive T line is a human cell) for 1 hour on ice with weak stirring. These Jurkat cells were grown in advance in RPMI medium enriched with fetal calf serum (10%), glutamine and penicillin/streptomycin were collected by centrifugation, washed in PBS and resuspendable in PBS/1% FCS (containing sodium azide). scFv-bearing phages that were not specifically associated with Jurkat cells were removed through the five stages of washing using PBS/1% FCS (containing sodium azide). After washing, the bound peroxidase patterns which were lurvely with cells by resuspendable cells in HCl-glycine buffer pH of 2.2 (10 min incubation followed by intensive mixing) and after neutralization with 2 M Tris pH 12 the eluate was used to infect fresh uninfected culture of E. coli XL1 Blue (OD600 greater than 0.5). A culture of E. coli containing E. coli cells subjected successful transduction fahmideh copy that encodes a human scFv-fragment, was again subjected to selection for resistance to carbenicillin and then infected helper phage VCMS 13 to start the second round of exposure antibodies and selection in vitro. Usually carried a total of from 4 to 5 cycles of selection.

2.4. Screening CD3-specific binding agents

Plasmid DNA corresponding to 4-5 cycles of panning, was isolated from cultures of E. coli after breeding. To obtain soluble scFv protein, fragments of the VH-VL-DNA was cut out from the plasmid (Xhol-Spel). These fragments were cloned via the same restriction sites in plasmid pComb3H5BFlag/His, different from the original pComb3H5BHis those that expressed design (e.g., scFv) includes a Flag-tag (TGD YKDDDDK) between the scFv and the His6-tag and additional phage proteins deleterows. After ligating each pool (various rounds of panning) plasmid DNA was subjected to transform 100 μl of competent to thermal shock E. coli TG1 or XLI blue and sown on carbenicillin-LB-agar. A single colony was transferred by periclymenum in 100 µl LB-carb (50 µg/ml).

E. coli transformed pComb3H5BHis containing VL - and VH-segment, produce soluble scFv in significant amounts after cutting the fragm is the gene III and induction with 1 mm IPTG (isopropylcyclopentadienyl). Thanks a suitable signal sequences of scFv-chain is exported to periplasm, where she collapsed in a functional conformation.

Single bacterial colonies of E. coli TG1 with cups for transformation were selected to receive small quantities of periplasm and were grown in SB medium (e.g., 10 ml), supplemented with 20 mm MgCl2and carbenicillin (50 μg/ml), and resuspendable in PBS (e.g., 1 ml) after collection. As a result of four cycles of freezing at -70°C. and thawing at 37°C outer membrane of the bacteria was destroyed under the action of temperature shock and soluble periplasmatic proteins, including scFv, was 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 agents

Linking the selected scFv was tested by flow cytometry on eukaryotic cells expressing on their surface a heterologous protein, which at its N end of the 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 Epsilon-chain of CD3 T-cell receptor complex (amino acid consistently is th: QDGNEEMGGITQTPYKVSISGTTVILT) were fused to the N-end transmembrane protein Arcam thus, to N-end was located on the outer cell surface. In addition, FLAG-epitope was inserted between the N-terminal 1-27 CD3-Epsilon sequence and the sequence Arcam. This product merge expressed in the cells of the human embryo kidney (NECK) and cells of the Chinese hamster ovary (Cho).

Eukaryotic cells, exposing a large part of the 27 N-terminal amino acids of Mature CD3-Epsilon other Primate species, was obtained in the same way for Saimiri sciureus (common squirrel monkey) (CD3-Epsilon N-terminal amino acid sequence: QDGNEEIGDTTQNPYKVSISGTTVTLT), Callithrix jacchus (CD3-Epsilon N-terminal amino acid sequence: QPGNEEMGPTTQNPYKVSISGTTVTLT) and Saguinus oedipus (CD3-Epsilon N-terminal amino acid sequence: QPGNEEMGPTTQNPYKVSISGTTVTLT).

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

The binding is always confirmed by flow cytometry, as described in the preceding paragraph, on primary T-cells of human and other primates (e.g., common squirrel monkey (Saimiri sciureus), common marmoset (Callithrix jacchus); Oedipus Tamarin (Saguinus oedipus)).

2.6. The creation of human/humanized equivalents of scFv specific for the CD3-Epsilon type, not a man

Carried out the alignment of the VH-region of murine anti-CD3 scFv relative to the amino acid sequence of antibody germline person. Chose the VH sequence of the antibody germline person, which had the highest homology with VH species that are not human, and implemented a direct alignment of these two aminokisloty sequences. There were a number of residues frame for VH type, not a person, which differed from the frame regions VH person ("the various provisions in the frame"). Some of these residues may be involved in the binding of an antibody to its target and activity of the antibody for its target.

To construct the library, which contains the murine CDR and in which each frame position differs from both options selected human VH sequences (human and maternal murine amino acid residue), Sintesi the Wali degenerate oligonucleotides. The composition of these oligonucleotides is in different positions "human" remains with a probability of 75% and a mouse with a probability of 25%. For a single human VH, for example, it was necessary to synthesize six of these oligonucleotides that overlap the tail section of approximately 20 nucleotides. For this purpose, each second primer is represented antisense primer. The restriction sites required for subsequent cloning the oligonucleotides were removed.

These primers can have a length of from 60 to 90 nucleotides, depending on the number of primers required for closing across the V-sequence.

These primers, for example, six primers were mixed in equal amounts (for example, 1 μl of each primer (concentrated solution primer: 20-100 μm) with 20 ál PCR reaction) was added to the PCR mixture 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, 56°C for 1 min, 52°C for 1 minute, at 50°C for 1 minute and at 72°C for 10 minutes in a PCR-collere. After that, the product was subjected to agarose gel electrophoresis and the product size from 200 to 400, was isolated from the gel according to standard methods.

This PCR product is then used as the Mat is Itza for a standard PCR reaction using primers introducing suitable for cloning of N-terminal and C-terminal restriction sites. The DNA fragment of the correct size (for VH approximately 350 nucleotides) were isolated using agarose gel electrophoresis according to standard methods. This way was amplified sufficient VH-DNA fragment. This VH fragment was now a pool of VH fragments, each of which had different amounts of "human" and "mouse" relevant residues in different positions in the frame (pool humanized VH). The same procedure was carried out for the VL-region of murine anti-CD3 scFv (pool humanized VL).

Pool humanized VH 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 display using filamentous phage selected anti-CD3-binding agents, made screening, identified and confirmed as described above for the parent non-human (mouse) anti-CD3 scFv. Single clones were then analyzed for the presence of suitable properties and amino acid sequence. The scFv, amino acid sequence which has the highest homology with V-segments in the germline of the person, are preferred, in particular those in which at least one CDR among CDR I and I VH and CDR 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 in the germline of the person. Ahth-CD3 scFv was converted into recombinant bespecifically single-chain antibodies, as described in the following Examples 10 and 16 and further characterized.

3. The creation of a recombinant fused protein consisting of the N-terminal amino acids 1-27 Epsilon-chain of human CD3, fused with the Fc region of IgG1 (1-27 CD3-Fc)

3.1. Cloning and expression of the 1-27 CD3-Fc

The coding sequence of the 1-27 N-terminal amino acid Epsilon-chain of human CD3, merged with the hinge region and Fc-gamma-section of human immunoglobulin IgG1, and also consisting of a 6 his-tag remnants of label, received gene synthesis gene synthesis according to standard protocols (cDNA sequence and amino acid sequence of the recombinant fused protein represented in SEQ ID NO: 350 and 349). The fragment of gene synthesis was designed so that it contained the first site 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 site Mature Epsilon-chain of CD3 person, then, in reading frame, encoding the sequence of the hinge region and Fc-gamma-phase of human IgG1, then, in reading frame, encoding a sequence consisting of 6 his-tag residues of the label and the top-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. Introduced restriction sites, EcoRI at the 5'-end and Sall on the 3'-end, used in subsequent cloning procedures. A fragment of the gene fusion was cloned via EcoRI and Sall in a plasmid designated pEF-DHFR (pEF-DHFR is described in Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025), following standard protocols. Plasmid 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 collected supernatant cell cultures of transfectants and tested using ELISA for the presence of recombinant constructs. Specific to Fc-gamma fragment of human IgG antibody goat (obtained from Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK) was diluted in PBS to a concentration of 5 μg/ml and they were coated (100 μl per well) to the wells of 96-hole tablet for ELISA MaxiSorp (Nunc GmbH & Co. KG, Wiesbaden, Germany) over night at 4°C. the Wells were washed in PBS with 0.05% of Tween 20 (PBS/Tween) and blocked 3% BSA in PBS (bovine albumin, fraction V, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) for 60 minutes at room temperature (RT). After that, the wells were again washed in PBS/Tween and then incubated with supernatant cell culture for 60 minutes at RT. After washing Luna which was carried out by incubation with anti-His6-antibody conjugated to peroxidase (Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany), diluted 1:500 in PBS with 1% BSA for 60 minutes at RT. Then the wells were washed with 200 μl PBS/Tween was added 100 μl of substrate solution SIGMAFAST OPD (SIGMAFAST OPD [dihydrochloride o-phenylenediamine]) (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) according to the manufacturer's Protocol. The reaction was stopped by adding 100 μl of 1 M H2SO4. The intensity of the resulting color was measured on the spectrophotometer for PowerWaveX microplate (BioTek Instruments, Inc., Winooski, Vermont, USA) at 490 nm with subtraction of the background absorbance at 620 nm. As shown in Fig.2, the presence of design in comparison with irrelevant supernatant putatively translated (mock-transfected) cells SOME 293, used as a negative control, defined.

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

Binding of the crude drugs expressed in periplasm single-chain antibodies with cross-species specificity, specific for CD3 Epsilon, 1-27 CD3-Fc was tested in ELISA analysis. Goat antibody against human IgG specific to Fc-gamma fragment (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK), diluted in PBS to a concentration of 5 μg/ml and was applied in the form of a coating on a 100 µl per well of 96-well plate for ELISA MaxiSorp (Nunc GmbH & Co. KG, Wiesbaden, Germany) over night at 4°C. the Wells prom is Wali 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 RT. After that, the wells were washed in PBS/Tween, and incubated with supernatants cells expressing design 1-27 CD3-FC, for 60 minutes at RT. The wells were washed in PBS/Tween, and incubated with crude preparations expressed in periplasm single-chain antibodies with cross-species specificity, as described above, for 60 minutes at room temperature. After washing PBS/Tween, the wells were incubated with peroxidase conjugated with anti-Flag M2 antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), diluted 1:10000 in PBS with 1% BSA for 60 minutes at RT. The wells were washed in PBS/Tween, and incubated with 100 µl of substrate solution SIGMAFAST OPD (OPD [dihydrochloride o-phenylenediamine]) (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) according to the manufacturer's Protocol. The color development was stopped with 100 ál of 1 M H2SO4and was measured on the spectrophotometer for PowerWaveX microplate (BioTek Instruments, Inc., Winooski, Vermont, USA) at 490 nm with subtraction of the background absorbance at 620 nm. Observed strong binding of human single-chain antibodies with cross-species specificity, specific for CD3 Epsilon, design 1-27 CD3-fc compared to the murine anti-CD3 single-chain antibody (Fig.3).

4. The development of recombinant transmembrane fused protein of N-terminal amino acids 1-27 CD3-Epsilon various primates, n is which is the chimpanzee, merged with Arcam cynomolgus macaque (1-27 CD3-Arsam)

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

CD3-Epsilon was isolated from various primates, non-chimpanzee (marmoset, Tamarin, squirrel monkey, and pig. The sequence encoding the N-terminal amino acids 1-27 Mature Epsilon-chain of human CD3, common marmoset (Callithrix jacchus), Oedipus Tamarin (Saguinus oedipus), common squirrel monkey (Saimiri sciureus) and domestic pigs (Sus scrota; used as negative control), fused with the N-end of the Flag-labeled Arcam cynomolgus macaque, 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: 351-360). Fragments of gene synthesis designed so that they contained the first BsrGI site that allows you to merge in the correct reading frame with the coding sequence of immunoglobulin leader peptide of 19 amino acids already present in the target expressing vector, then, in reading frame, encoding the sequence of the 1-27 N-terminal amino acids of the extracellular site Mature Epsilon chain of CD3, then, in reading frame, encoding the sequence of the Flag-tag and then, in reading frame, the coding sequence of the Mature transmembrane protein Arcam Yavan is anyone 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. Introduced restriction sites, BsrGI at the 5'-end and Sall on the 3'-end, was used in the subsequent cloning procedures. Fragments of the gene fusion was then cloned via BsrGI and Sall, following standard protocols, 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 coding sequence of the immunoglobulin leader peptide of 19 amino acids. Plasmids with confirmed sequence used for temporary transfection of cells SOME 293 using reagent MATra-A (IBA GmbH, Gottingen, Germany) and 12 μg of plasmid DNA for attached cells SOME 293 175 ml bottles for cell culture in accordance with the Protocol of the manufacturer. After 3 days of culturing cells, the transfectants were tested against the expression on cell surfaces recombinant transmembrane protein using FACS analysis according to standard protocols. This purpose 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 peroxidase antibody was determined using conjugated with R-phycoerythrin, affinity purified F(ab')2-fragment antibodies goat about the Yves mouse IgG (specific to Fc-gamma fragment), diluted 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 Flag-labeled recombinant transmembrane fused protein consisting of Arcam cynomolgus macaque and N-terminal amino acids 1-27 Epsilon-chain of CD3 human, marmoset, Tamarin, squirrel monkeys and pigs, respectively, defined in transfected cells (Fig.5).

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

Binding of the crude drugs expressed in periplasm single-chain anti-CD3 antibodies with cross-species specificity to N-terminal amino acids 1-27 of the Epsilon chain of CD3 human, marmoset, Tamarin and squirrel monkeys, respectively, merged with the EP ITSELF cynomolgus macaque, were tested using FACS analysis according to standard protocols. This purpose of 2.5×105cells were incubated with crude preparations expressed in periplasm single-chain anti-CD3 antibodies with cross-species specificity (getting carried out, as described above, and according to standard protocols) and single-chain murine antibody against human CD3 as a negative control. As secondary antibodies used Penta-His antibody (Qiagen GmbH, Hildesheim, Germany) at a concentration of 5 μg/ml in 50 μl PBS with 2% FCS. Binding of antibodies was determined and the use conjugated with R-phycoerythrin, affinity purified F(ab')2-fragment antibodies goat against mouse IgG (specific to Fc-gamma fragment), diluted 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-E), observed the binding of single-chain antibodies with transfectants expressing recombinant transmembrane fused protein consisting of the 1-27 N-terminal amino acids of CD3-Epsilon human, marmoset, Tamarin, or squirrel monkeys, merged with Arcam cynomolgus macaque. No binding of single-chain antibodies with cross-species specificity with fused protein consisting of the 1-27 N-terminal amino acids of CD3-Epsilon pigs, merged with Arcam cynomolgus macaque, which was used as a negative control, were not observed. Was shown interspecies specificity of single-stranded anti-CD3 antibodies against many primates. The signals obtained with anti-Flag M2 antibody and single-chain antibodies with cross-species specificity were comparable, indicating a strong binding activity of single-chain antibodies with cross-species specificity to N-terminal amino acids 1-27 CD3-Epsilon.

5. The analysis of binding single-chain anti-CD3 antibodies with cross-species specificity by means alanine scanning of mouse cells transfected with the Epsilon-chain of human CD3 is a and its alanine mutants

5.1. Cloning and expression of the human CD3-Epsilon-chain wild-type

The coding sequence of Epsilon-chain of human CD3 were obtained by gene synthesis according to standard protocols (cDNA sequence and amino acid sequence of Epsilon-chain of human CD3 presented in SEQ ID NO: 362 and 361). The fragment of gene synthesis was designed so that it contains the website Kozak for eukaryotic expression of this design and the restriction sites at the beginning and at the end of the cDNA coding CD3-Epsilon person. Introduced restriction sites, EcoRI at the 5'-end and Sall on the 3'-end, was used in the subsequent cloning procedures. A fragment of the gene fusion was then cloned via EcoRI and Sall in a plasmid designated pEF-NEO, following the standard protocols. pEF derived from NEO pEF DHFR (Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025) the replacement DHFR cDNA in cDNA (gene) resistance to neomycin using traditional molecular cloning. Plasmid with confirmed sequence was used for transfection of murine T-cell line EL4 (ATSS №TIB-39), cultured in RPMI with 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. T is inspectio was performed using reagent for transfection with SuperFect (Qiagen GmbH, Hilden, Germany) and 2 μg of plasmid DNA in accordance with the Protocol of the manufacturer. After 24 hours, cells were washed in PBS and again cultured in the above medium for culturing cells with 600 µg/ml G418 for selection (PAA Laboratories GmbH, Pasching, Austria). After 16-20 days after transfection observed the growth of resistant cells. After 7-14 days the cells were tested against the expression of CD3-Epsilon person using FACS analysis according to standard protocols. a 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. Antibody binding was determined using conjugated with R-phycoerythrin, affinity purified F(ab')2-fragment antibodies goat against mouse IgG (specific to Fc-gamma fragment), diluted 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 the human CD3-Epsilon-chain 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 IgG1 antibodies

To provide improved means of determining the binding of single-chain anti-CD3 antibodies with cross-species specificity NS HLP, A2J HLP and AM HLP turned in IgG1-antibodies containing murine IgG1 and constant region of human lambda. Sequence is lnasty cDNA, encoding the heavy and light chains corresponding IgG antibodies, were obtained by gene synthesis according to standard protocols. Fragments of gene synthesis for each specificity designed so that they contained the first site Kozak for eukaryotic expression of this design, then immunoglobulin leader peptide of 19 amino acids (SEQ ID NO: 364 and 363), then, in reading frame, encoding the sequence of the corresponding variable regions of the heavy chain or the corresponding variable region of the light chain, then, in reading frame, encoding the sequence of the constant region of the heavy chain of murine IgG1 (SEQ ID NO: 366 and 365) or the coding sequence of the constant region of the light chain lambda human (SEQ ID N0: 368 and 367), 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 Sall on the 3'-end, was used for subsequent cloning procedures. Fragments of the gene fusion was cloned via EcoRI and Sall in a plasmid designated pEF DHFR (Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025) for construction of heavy chain and pEF ADA (pEF ADA is described in Raum et al., Cancer Immunol. Immunother., 50(3), (2001), 141-50) for construction of light chain, according to standard protocols. Plasmids with confirmed sequences used for cotransfection suitable the structures of the light and heavy chains in expressing the system of Freestyle 293 (Invitrogen GmbH, Karlsruhe, Germany) according to the manufacturer's Protocol. After 3 days supernatant cell cultures of transfectants were collected and used for the experiment by alanine scanning.

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

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

5.4. Experiment by alanine scanning

Chimeric IgG antibodies, as described in 2), and single-chain antibodies with cross-species specificity, specific for CD3 Epsilon, tested in the experiment by alanine scanning. Antibody binding to cell lines EL4, transfitsirovannykh mutated to alanine designs CD3-Epsilon person, as described in 3), were tested using FACS analysis according to standard protocols. a 2.5×105cells corresponding transfectants were incubated with 50 μl of cell culture supernatant containing the chimeric IgG-antibodies, or with 50 µl of the crude drugs expressed in periplasm single-chain antibodies. For samples incubated with crude preparations expressed in periplasm single-chain antibodies, anti-Flag M2 antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was used as secondary antibody at a concentration of 5 μg/ml in 50 μl PBS with 2% FCS. For samples incubated with chimeric IgG-antibodies that are needed in the secondary antibody was not. For all samples linking mole is str antibodies was determined using conjugated with R-phycoerythrin, affinity purified F(ab')2-fragment antibodies goat against mouse IgG (specific to Fc-gamma fragment), diluted 1:100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Samples were measured on a FACSCalibur (BP biosciences, Heidelberg, Germany). Was determined by differential binding of chimeric IgG-molecules or single-chain antibodies with cross-species specificity to cell lines EL4, transfitsirovannykh alanine mutants of CD3-Epsilon person. As a negative control was used or izotopicheskii control or untreated drug expressed in periplasm single-chain antibodies of irrelevant specificity, respectively. 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 Mature Epsilon-chain of CD3, were not estimated due to the very low levels of expression (data not shown). Binding of single-chain antibodies with cross-species specificity and single-chain antibodies in the format of chimeric IgG from cell lines EL4, transfitsirovannykh alanine mutants of CD3-Epsilon person, shown in Fig.8 (A-D) relative binding in a conventional unit of the middle geometric quantities fluorescence of the corresponding negative controls, deducted from all relevant geometric mean fluorescence for samples. To compensate for different levels of expression of all values for samples for specific transfectant then divided by the average geometric size fluorescence antibody UCHT-1 for the corresponding transfectant. In order to compare with the value of specificity for sample wild-type all the values of the corresponding specificdate for samples completely divided on the value for sample wild type, with the value for sample wild type was taken as 1 unit binding.

Used the calculations presented in detail by the following formula:

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

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

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

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

The cDNA fragment encoding the human CD3-Epsilon-chain with N-terminal His6-tag was obtained by gene synthesis. The fragment of gene synthesis was designed so that it contained the first site Kozak for eukaryotic expression of this construction, then, in reading frame, immunoglobulin leader peptide of 19 amino acids, then, in reading frame, encoding a sequence of His6-tag, then, in reading frame encoding the Mature sequence Epsilon-chain of CD3 (cDNA sequence and amino acid sequence of this design is presented in SEQ ID NO: 380 and 379). The fragment of gene synthesis was also designed so the m way to contain restriction sites at the beginning and at the end of the cDNA. Introduced restriction sites, EcoRI at the 5'-end and Sall on the 3'-end, was used in the subsequent cloning procedures. A fragment of the gene fusion was then cloned via EcoRI and Sall in a plasmid designated pEF-NEO (as described above), following standard protocols. Plasmid 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 cell culturing transfectants used in the following analysis.

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

Chimeric IgG-antibody NS HLP with binding specificity specific for the CD3-Epsilon, tested against binding CD3-Epsilon person with N-terminal His6-tag and without it. Binding of this antibody from cell line EL4, transfitsirovannykh His6-CD3-Epsilon and human CD3-Epsilon wild-type, respectively, were tested using FACS analysis according to standard protocols. 2,j cell transfectants were incubated with 50 μl of cell culture supernatant containing the chimeric IgG-antibody or 50 ál of the appropriate control antibodies at a concentration of 5 μg/ml in PBS with 2% FCS as a negative control used corresponds izotopicheskii control, and as a positive control, expression constructs Biacore. Unbound protein was removed by vigorous washing with subsequent blocking of the remaining unreacted NHS-activated carboxylate by adding a solution of ethanolamine. The successful accession of the protein was confirmed by a higher value of the signal measured in units response Units) compared to the signal before joining. The cell comparison was prepared in the same way as described, but without the addition of protein solution.

Purified bespecifically antibody EGFR-21-63 LH × NS HLP vigorously were dialyzed against HBS-EP buffer (Biacore, Uppsala, Sweden) in the cell for initializa Slide-A-Lyzer® (Pierce, Rockford II, USA). The protein concentration after dialysis was determined by absorption at 280 nm, obtaining a concentration of 43 ág/ml.

The protein solution was transferred into a 96-well plate and serially diluted with buffer HBS-EP at a ratio of 1:1 to 10 subsequent holes.

Measurement method of surface plasmon resonance was performed by separately depositing samples in all 11 holes. Between the measurement flow cell regenerates acetate buffer to release the bound protein.

Signals binding molecules bespecifically antibodies were obtained by subtracting the signal of the cell comparison of the signal measured cells, conjugated with protein 1-27 CD3-Fc. Curves is Sociali and dissociation was measured in units of the response was recorded. The binding constants were calculated using the software Biacore® to fit curves based on the model of Langmuir.

The calculated value of the binding constant KD for the first five concentrations was 1.52×10-7M

7.2. Determination of the binding constant CD3 in the measurement using FACS

To test the affinity of molecules especifismo antibodies with cross-species specificity in relation to the strength of binding to native human CD3 carried out additional analysis saturating binding using FACS. Selected molecule especifismo antibody EGFR-21-63 LH × NS HLP used to establish the number of dilutions by a factor of 1:1.5 and the initial concentration 63,3 µg/ml was Used CD3-specific antibody UCHT-1, respectively. Antibody binding was determined using conjugated with R-phycoerythrin, affinity purified F(ab')2 fragment antibodies goat against mouse IgG (specific to Fc-gamma fragment), diluted 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 with the EL4 cell line, transtitional human CD3-Epsilon wild-type was determined by the apparent loss of binding of chimeric IgG with binding specificity - NS HLP with CD3-Epsilon person with N-terminal His6-tag. These results showed that a free N-con the CD3-Epsilon is essential for the binding of specific anti-CD3 binding molecules NS HLP with interspecies specificity with Epsilon-chain of human CD3 (Fig.9).

7. Determination of the binding constant KD especifismo single-chain antibodies with cross-species specificity to EGFR primates and CD3 primates (EGFR LH × NS HLP), with the merged protein 1-27 CD3-Fc measured using surface plasmon resonance, in comparison with the binding CD3-expressing RVS measured using a cell sorting device with activation of fluorescence (FACS)

7.1. The measurement method of surface plasmon resonance

To determine the affinity of binding especifismo single-chain antibody EGFR-21-63 LH × NS HLP with full cross-species specificity with amino acids 1-27 of the N-end Epsilon-chain of human CD3 was performed by the measurement method of surface plasmon resonance with recombinant fused protein consisting of the N-terminal amino acids 1-27 Mature Epsilon-chain of human CD3, fused with the Fc region of human IgG1 (1-27 CD3-Fc). To do this, the system Biacore 2000® (Biacore, Uppsala, Sweden) were placed carboxymethyl-dextranase Biacore chip SM (Biacore, Uppsala, Sweden). One flow cell (flow cell) was activated with a solution of the hydrochloride of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide/N-hydroxysuccinimide in accordance with standard procedures. Then was added a solution of fused protein 1-27 CD3-Fc, which led to the formation of a stable covalent bond between the protein and dextranomer layer of the chip. Each of the molecules bespar the specific antibodies were incubated in various concentrations from 1.25×10 5RVMS person for 1 hour at 4°C, followed by two stages of washing PBS at 4°C. Determination of bound peroxidase molecules especifismo antibodies was performed using Penta-His antibodies (Qiagen GmbH, Hildesheim, Germany) at a concentration of 5 μg/ml in 50 μl PBS with 2% FCS. After incubation for 45 minutes at 4°C and two stages of leaching linking Penta-His antibody was determined using conjugated with R-phycoerythrin, affinity purified F(ab')2-fragment antibodies goat against mouse IgG (specific to Fc-gamma fragment), diluted 1:100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Flow cytometry was carried out on the apparatus FACS-Canto II, for data collection and analysis used software FACS Diva (Becton Dickinson biosciences, Heidelberg). FACS-staining and measurement of fluorescence intensity was performed as described in Current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-lnterscience, 2002). Build the graph of the obtained average values of fluorescence intensity on the concentration of molecules used especifismo antibodies and analyzed using biomathematical software Prism analysis one-way linking one side of the binding analysis) (hyperbole). Using software to calculate the relevant value of KD, describing the binding of the ligand (molecule of bispecific the mini-antibodies) receptor (CD3-positive sbraccia RVMS) in accordance with the law of the masses. The underlying formula has the following form: Y=Bmax×X/(Kd+X), where Bmax is the maximum binding. KD is the concentration of ligand required to reach premaxillae binding. FACS staining was performed in two replicates, the values of R2were more of 0.95.

The results definitions premaxillae binding to molecules especifismo antibody EGFR-21-63 LH × NS HLP was achieved at a concentration of 8472 ng/ml, which corresponds to 154 nm (1,54×10-7M) if the value of the molecular weight of 55,000 daltons (Fig.10).

Thus it is proved that the affinity of EGFR-21-63 LH × NS HLP to N-terminal amino acids 1-27 Epsilon-chain of human CD3, isolated from its native CD3 environment, equal affinity EGFR-21-63 LH × NS HLP to native CD3 in intact T cells.

8. Obtaining cells SNO, transfected with human EGFR

Cell line, positive attitude EGFR human A431 (epidermoid carcinoma cells, CRL-1555, American type culture collection, Rockville, MD) was used to obtain total RNA, which was isolated in accordance with the instructions of the kit (Qiagen, RNeasy Mini Kit, Hilden, Germany). The obtained RNA was used to synthesize cDNA by polymerase chain reaction with reverse transcription with random primers. To clone the full sequence of the EGFR antigen people the century used the following oligonucleotides:

5' EGFR AG Xbal 5'-GGTCTAGAGCATGCGACCCTCCGGGACGGCCGGG-3';

3' EGFR AG Sall 5'-TTTTAAGTCGACTCATGCTCCAATAAATTCACTGCT-3'.

The coding sequence amplified by PCR (denaturation at 94°C for 5 min, annealing at 58°C for 1 min, elongation at 72°C for 2 min for the first cycle; denaturation at 94°C for 1 min, annealing at 58°C for 1 min, elongation at 72°C for 2 min for 30 cycles; final elongation at 72°C for 5 min). The PCR product was then digested Xbal and Sall, ligated into appropriately cleaved expressing vector pEF-DHFR (Raum et al., Cancer Immunol. Immunother. 2001; 50: 141-150) and subjected to transformation into E. coli. The above procedure was carried out according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (2001)). The clone with confirmed nucleotide sequence (SEQ ID 370, amino acid sequence SEQ ID 369) was transfusional in defective in DHFR cells SNO for eukaryotic expression of this design. The expression of eukaryotic protein defective in DHFR cells SNO was carried out as described in Kaufmann R. J. (1990) Methods Enzymol. 185, 537-566. Gene amplification structure induced by increasing concentrations of methotrexate (MTX) to a final concentration of 20 nm MTX inclusive.

9. Obtaining cells SNO expressing the extracellular domain of cynomolgus EGFR m is Kaka

The cDNA sequence of the extracellular domain of EGFR cynomolgus macaque received in the result of a series of two PCR on cDNA from the colon cynomolgus macaque (catalog No. S-su-SU; obtained from BioCat GmbH, Heidelberg, Germany), using the following reaction conditions: 1 cycle at 94°C for 3 minutes, then 35 cycles at 94°C for 1 min, 53°C for 1 minute and at 72°C for 2 minutes, then a final cycle at 72°C for 3 minutes. Used the following primers:

1) direct primer: 5'-CGCTCTGCCCGGCGAGTCGGGC-3',

reverse primer: 5'-CCGTCTTCCTCCATCTCATAGC-3';

2) direct primer: 5'-ACATCCGGAGGTGACAGATCACGGCTCGTGC-3,

reverse primer: 5'-CAGGATATCCGAACGATGTGGCGCCTTCGC-3'.

As a result of these PCR received two overlapping fragments (A: 1-869, In: 848-1923), which were isolated and sequenced according to standard protocols, using PCR primers, and was thus given a sequence region EGFR cDNA cynomolgus macaque size 1923 p. O. third nucleotide in the codon +1 of the Mature protein to the 21st codon transmembrane domain. To create constructs for expression of EGFR cynomolgus macaque, the 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: 372 and 371). In this construct, the coding sequence of cynomolgus EGFR poppy is from amino acids +2 to +641 Mature protein EGFR included in the coding sequence of human EGFR, replacing the coding sequence of amino acids from +2 to +641. The fragment of gene synthesis was also designed so that it contains the website Kozak for eukaryotic expression of this design and the restriction sites at the beginning and at the end of the cDNA that encodes essentially of the extracellular domain of EGFR cynomolgus macaque, fused with the transmembrane and intracellular domains of EGFR person. In addition, introduced a conservative mutation in the position corresponding to the amino acid 627 (4th amino acid transmembrane domain), substituting valine for leucine, to create a restriction site (Sphl) for cloning. Introduced restriction sites, Xbal at the 5'-end and Sall on the 3'-end, was used in the subsequent cloning procedures. A fragment of the gene fusion was then cloned via Xbal and Sall in a plasmid designated pEF-DHFR (pEF-DHFR is described in Mack et al. Proc. Natl. Acad. Sci. USA, 92 (1995) 7021-7025). The clone with the confirmed sequence of this plasmid was used for transfection of cells CHO/dhfr-, as described above.

10. The creation of EGFR and CD3-bespecifically single-stranded molecules with cross-species specificity

10.1. Cloning of binding molecules with cross-species specificity

Typically, molecules bespecifically single-chain antibodies, each of which contains a domain with binding specificity, demonstrating cross-species specificity to CD3-Epsilon of man and of the primacy, not a chimpanzee, as well as a domain with binding specificity, demonstrating cross-species specificity to EGFR human and Primate, not a chimpanzee, was designed, as shown in the following Table 1.

Table 1
Formats molecules with anti-CD3 and anti-EGFR bespecifically single-chain antibodies with cross-species specificity
SEQ ID (n./Bel.)The formats of protein constructs (N→C)
294/293EGFR-21-63 LH × NS HL
296/295EGFR-21-63 LH × NS HLP
302/301EGFR-21-63 LH × A2J HLP
298/297EGFR-21-63 LH × H1E HLP
306/305EGFR-21-63 LH × E2M HLP
308/307EGFR-21-63 LH × F70 HLP
390/389EGFR1 HL × I2C HL
392/391EGFR1 LH × I2C HL
394/393EGFR1 HL × F12Q HL
396/395EGFR1 LH × F12Q HL
398/397EGFR1 HL × H2C HL
400/399EGFR1 LH × H2C HL
448/447EGFR1 HL × H2C HL
450/449EGFR1 HL × F12Q LH
452/451EGFR1 HL × I2C HL
410/409EGFR2 HL × I2C HL
412/411EGFR2 LH × I2C HL
414/413EGFR2 HL × F12Q HL
416/415EGFR2 LH × F12Q HL
418/417EGFR2 HL × H2C HL
420/419EGFR2 LH × H2C HL

The above constructs containing the variable domains of the light chain (L) and the variable domains of the heavy chain (H) cross-species specificity to EGFR human and cynomolgus macaque, was obtained by gene synthesis. Fragments of gene C is thesis was designed so to contain the first site Kozak for eukaryotic expression of this design, then immunoglobulin leader peptide of 19 amino acids, then, in reading frame, encoding a sequence corresponding molecule especifismo single-chain antibodies, then, in reading frame, encoding a sequence consisting of 6 his-tag residues tag and a stop codon. The fragment of gene synthesis was also designed in such a way as to enter the appropriate N - and C-terminal restriction sites. A fragment of the gene fusion was cloned, using these restriction sites in the 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)). The clone with confirmed nucleotide sequence was transfusional in defective dihydrotetrazolo (DHFR) cells Chinese hamster ovary (Cho) for eukaryotic expression of the construct.

These structures were transfusional stable or temporarily defective in DHFR cells Cho (ATSS No. CRL 9096) via electroporation or, alternatively, temporarily transfusional in SOME 293 (embryonic cells of the human kidney, ATS number: CRL-1573) according to standard protocols.

10.2. Expression and purification of molecules bespecifically od is azeotech antibodies

Molecules bespecifically single-chain antibodies expressed in cells of the Chinese hamster ovary (Cho). The expression of eukaryotic protein defective in DHFR cells SNO was carried out as described in Kaufmann R. J. (1990) Methods Enzymol. 185, 537-566. Gene amplification structures induced by increasing the final concentration of MTX up to 20 nm inclusive. After two passages stationary culture cells were grown in roller bottles in liquid soy medium HyQ PF Cho, not containing nucleosides (with 4.0 mm L-glutamine, 0.1% Pluronic F-68; HyClone), within 7 days before collection. Cells were isolated by centrifugation and the supernatant containing the expressed protein was stored at -20°C. alternatively, a structure temporarily expressed in the cells of SOME 293. Transfection was performed using 293fectin reagent (Invitrogen, No. 12347-019) in accordance with the Protocol of the manufacturer.

For chromatography used the system Akta® Explorer (GE Health Systems) and software Unicorn®. Affinity chromatography with immobilized metal ("IMAC") was performed using Fractogel EMD chelate® (Merck), which were loaded with ZnCl2 in accordance with the Protocol suggested by the manufacturer. The column was balanced with buffer A (20 mm sodium phosphate buffer pH 7,2; 0.1 M NaCl) and cell culture supernatant (500 ml) was applied to a column (10 ml) at a flow rate of 3 ml/min the Column was washed with buffer is m a to remove unbound sample. Bound peroxidase protein was suirable using a two-step gradient of buffer B (20 mm sodium phosphate buffer pH 7,2; 0.1 M NaCl; 0.5 M imidazole) in accordance with the following Protocol:

stage 1: a 20% buffer In 6 column volumes;

stage 2: 100% buffer In 6 volumes of the column.

Erwerbende protein fractions from stage 2 were combined for further purification. All chemical reagents were of a purity suitable for research, and were purchased from Sigma (Deisenhofen) or Merck (Darmstadt).

Gel filtration was performed on preparative column (HiLoad 16/60 Superdex 200 (GE/Amersham), balanced Equi-buffer (25 mm citrate, 200 mm lysine; 5% glycerol; pH of 7.2). Erwerbende protein samples (flow rate 1 ml/min) and then were subjected to standard SDS-PAGE (polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate) and Western-blotting for detection. Before cleaning the column was calibrated to determine the molecular weight (set of markers of molecular weight, Sigma MW-GF-200). The protein concentration was determined using OD at 280 nm.

Purified protein bespecifically single-chain antibody, were analyzed in SDS-PAGE in reducing conditions and pre-filled 4-12% bear bis-Tris gels (Invitrogen). Preparation and application of samples was carried out according to the Protocol suggested by the manufacturer. The molecular weight was determined using Belk is a new standard MultiMark (Invitrogen). The gel was stained with colloidal Coomassie (Invitrogen Protocol). The purity of the selected protein was more than 95% as determined by SDS-PAGE.

Bespecifically single-chain antibody has a molecular weight of approximately 52 kDa in native conditions, as determined by gel filtration in PBS. All constructs were purified according to this method.

Western blotting was carried out using the membrane Optitran® BA-S83 in apparatus for blotting Invitrogen according to the Protocol suggested by the manufacturer. Used antibodies were directed against the His-tag (Penta-His, Qiagen) and antibodies goat against mouse Ig labeled with alkaline phosphatase (AP) (Sigma), and BCIP/NBT (5-bromo-4-chloro-3-indolylacetic)/nitrotetrazolium blue) (Sigma) as substrate. Identified a single band at 52 kDa, which corresponded cleaned bespecifically single-chain antibody.

11. Determination of the binding constant KD bespecifically single-chain antibodies, with full cross-species specificity, with the merged protein 1-27 CD3-Fc measured using surface plasmon resonance

To determine affinely binding molecules especifismo single-chain antibodies with cross-species specificity to EGFR primates and CD3 primates with amino acids 1-27 of the N-end of the Mature Epsilon-chain of human CD3 carried out measurements by means of surface plasmon resonance with recombinantly fused protein, consisting of N-terminal amino acids 1-27 Epsilon-chain of human CD3, fused with the Fc region of human IgG1 (1-27 CD3-Fc). To do this, the system Biacore 2000® (Biacore, Uppsala, Sweden) was established carboxymethyl-dextranase SM chip Biacore (Biacore, Uppsala, Sweden). The flow cell was activated with a solution of the hydrochloride of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide/N-hydroxysuccinimide in accordance with standard procedures. Then was added a solution of fused protein 1-27 CD3-Fc, which led to the formation of a stable covalent bond between the protein and dextranomer layer of the Biacore chip. Unbound protein was removed by vigorous washing with subsequent blocking of the remaining unreacted NHS-activated carboxylate by adding a solution of ethanolamine. The successful accession of the protein was confirmed by a higher value of the signal, measured in units of the response, in comparison with the signal before joining. The cell comparison was prepared in the same way as described, but without the addition of protein solution.

The concentration of purified bespecifically single-chain antibodies listed below were adjusted to 5 μg/ml of buffer HBS-EP (Biacore, Uppsala, Sweden) and transferred to 96-well plate, each in a volume of 150 μl.

Measurement method of surface plasmon resonance was performed for all samples and flow cells were regenerated between measured is s acetate buffer to release the bound protein (all according to standard protocols).

The signals linking bespecifically single-chain antibodies were obtained by subtracting the signal of the cell comparison of the signal measured cells, conjugated with protein 1-27 CD3-Fc.

Curves of Association and dissociation were measured in units of the response was recorded. The binding constants were calculated using the software Biacore® to fit curves based on the model of Langmuir. The calculated values of affinely to test bespecifically single-stranded molecules with a full cross-species specificity to N-terminal amino acids 1-27 CD3-Epsilon person is given in the form of KD values below and lie in the range of 2.54×10-6M to 2.49×10-7M "LH" refers to the variable domains in the order VL-VH. "HL" refers to the variable domains in the order VH-VL. G4H, F70, A2J, E1L, EM, NE F6A and treat various CD3-binding molecules with cross-species specificity.

Molecule especifismo antibodiesKD (M)
EGFR LH × F70 HLP1,01×10-6
EGFR LH × A2J HLP2,49×10-7
EGFR LH × E2M HLPthe 2.46×10-6

EGFR LH × H1E HLP2.54×10-6

12. Flow-cytometrics the analysis of binding EGFR and CD3-bespecifically antibodies with cross-species specificity

To test the functionality of the structures bespecifically antibodies with cross-species specificity in terms of their ability to communicate with EGFR and CD3 human and cynomolgus macaque, respectively, was carried out by FACS-analysis. For this, cells SNO, transfetsirovannyh human EGFR, as described in Example 8, and CD3-positive cell line HPB-ALL T-cell human leukemia (DSMZ, Braunschweig, ASS) was used to test binding to antigens of human rights. The ability to bind to antigens cynomolgus macaque was tested with the created transfectant with EGFR cynomolgus macaque described in Example 9, and T-macaque cell line 4119 LnPx (courtesy of Prof. Fickenscher, Hygiene Institute, Virology, Erlangen-Nuernberg; published in Knappe A, et al., and Fickenscher H., Blood 2000, 95, 3256-61). 200,000 cells of the respective cell populations were incubated for 30 min on ice with 50 μl of purified protein structures bespecifically antibodies with cross-species specificity (2 μg/ml). An alternative, used the supernatant of the cell culture with temporarily produced proteins. Cells are washed twice in PBS and linking this structure was determined using mouse is th Penta-His antibody (Qiagen; diluted 1:20 in 50 μl PBS with 2% FCS). After washing, the bound peroxidase anti-His antibodies were determined using an Fc-gamma-specific antibody (Dianova) conjugated with phycoerythrin, diluted 1:100 in PBS with 2% FCS. As a negative control used fresh culture medium.

Flow cytometry was carried out on the apparatus FACS-Calibur; for data collection and analysis used the software CellQuest (Becton Dickinson biosciences, Heidelberg). FACS-staining and measurement of fluorescence intensity was performed as described in Current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-Interscience, 2002).

Linking the ability of some bespecifically single-stranded molecules that are specific for EGFR and having inter-species specific for human CD3 and of the primacy of non-chimpanzee, was clearly defined, as shown in Fig.11. In FACS analysis all constructs showed binding to CD3 and EGFR compared with the culture medium and the first and second detection antibody as a negative control. Was demonstrated interspecific specificity especifismo antibodies to CD3 and EGFR human and cynomolgus macaque.

13. The biological activity of EGFR and CD3-bespecifically single-chain antibodies with cross-species specificity

Biological activity created bespecifically single-stranded anti who ate were analyzed for the release of chromium 51 ( 51Cr) in the analysis of cytotoxicity in vitro, using EGFR-positive cell lines described in Examples 8 and 9. As effector cells used stimulated CD8-positive T-cells or T-macaque cell line 4119 LnPx, respectively.

Getting stimulated CD8+T cells was carried out as follows.

On a Petri dish (145 mm diameter; Greiner) pre-inflicted commercially available anti-CD3 specific antibody at a final concentration of 1 μg/ml for 1 hour at 37°C. Unbound protein was removed per stage of washing using PBS. Fresh RWMS was isolated from peripheral blood (30-50 ml of blood) by centrifugation in a gradient of ficoll according to standard protocols. 3-5×107RVMS added to pre-coated Petri dish in 120 ml of RPMI 1640/10% FCS/IL-2 (interleukin-2) 20 units/ml (Proleukin, Chiron) and stimulated for 2 days. On the third day the cells were collected, washed once with RPMI 1640. Added IL-2 to a final concentration of 20 units/ml, and were again cultured for one day. CD8+cytotoxic T lymphocytes (CTL) were isolated by depletion of CD4+T cells and CD56+NK-cells.

Target cells were twice washed in PBS and were marked to 11.1 MBq51Cr in the 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 the analysis of cytotoxicity. The analysis was carried out in 96-well-plate in a total volume of 250 μl supplemented RPMI medium (as above) with a ratio of E:T of 10:1. Put 1 μg/ml of molecules especifismo single-chain antibodies with cross-species specificity and their 20 three-fold dilutions. Alternatively, the cell culture supernatant temporarily proteins produced serially diluted 1:2 in stages. The analysis time is 18 hours, and cytotoxicity was measured in relative terms chromium released into the supernatant related to the difference of maximum lysis (adding Triton-X) and spontaneous lysis (without effector cells). All measurements were carried out in four replications. Measurement of the activity of chromium in supernatant was performed using a gamma counter Wizard 3" (Perkin Elmer Life Sciences GmbH, Köln, 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-effect usually had a value of R2more than 0.90 as determined using the software. Size EU50calculated by the program analysis was used to compare the biological activity.

As shown in Fig.12 and 13, all designs bespecifically single-chain antibodies with cross-species specificity was demonstrated cytotoxic asset is ity against target cells, positive EGFR human induced CD8+human cells, and target cells, positive for the EGFR cynomolgus macaque, induced T-cell line macaque 4119 LnPx. Bespecifically single-chain antibody with a different target specificity was used as negative control.

14. Cloning and expression of the C-terminal, transmembrane and shortened extracellular domains of human MCSP

The coding sequence of the C-terminal, transmembrane and a 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 recombinant constructs for the expression of C-terminal, transmembrane and a truncated extracellular domain of human MCSP (labeled D3 man) is presented in SEQ ID NO: 374 and 373). The fragment of gene synthesis was designed so that it contained the first site Kozak for eukaryotic expression of this design, then coding sequence of the immunoglobulin leader peptide of 19 amino acids, then, in reading frame, FLAG-tag, then, in reading frame, a sequence containing multiple restriction sites for cloning purposes and encoding an artificial linker of 9 amino acids (SRTRSGSQL) then in reading frame encoding the sequence C-terminal, transmembrane and a truncated extracellular domain of human MCSP and a stop codon. The restriction sites were introduced at the beginning and at the end of this DNA fragment. The restriction sites, EcoRI at the 5'-end and Sall on the 3'-end, was used in the subsequent cloning procedures. The fragment was digested EcoRI and Sall 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. Plasmid with confirmed sequence was used for transfection of cells CHO/dhfr - ATSC 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 concentrated solution of the reagent purity "cell cultures" (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) to a final concentration of adenosine, 10 μg/ml deoxyadenosine 10 μg/ml thymidine, 10 μg/ml, in an incubator at 37°C, 95% humidity and 7% CO2. Transfection was performed using reagent for transfection PolyFect (Qiagen GmbH, Hilden, Germany) and 5 μg of plasmid DNA in accordance with the Protocol of the manufacturer. After culturing for 24 hours, the cells were washed once with PBS and again cultured in RPMI 1640 with stabilizing additive glutamine and 1% penicillin/streptomycin. Thus, the cell the culture medium did not contain nucleosides, and so the selection was performed on transfected cells. Approximately 14 days after the implementation of transfection was observed the growth of resistant cells. After 7-14 days the transfectants were tested against the expression constructs using FACS analysis. a 2.5×105cells were incubated with 50 µl of 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 antibodies goat against mouse IgG (specific to Fc-gamma fragment), diluted 1:100 in PBS with 2% FCS (ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Samples were measured on a FACScalibur (BD biosciences, Heidelberg, Germany).

15. Cloning and expression of the C-terminal, transmembrane and shortened extracellular domains macaque MCSP

The sequence of the DNA C-terminal, transmembrane and shortened extracellular domains macaque MCSP (labeled D3 macaque) received a series of three PCR on cDNA from skin macaque (catalog No. S-su-SU; BioCat GmbH, Heidelberg, Germany) using 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 a 1.75 min, then a final cycle at 72°C for 3 min Used the following primers:

direct primer: 5'-GATCTGGTCTACACCATCGAGC3';

reverse primer: 5'-GGAGCTGCTGCTGGCTCAGTGAGG-3';

direct primer: 5'-TTCCAGCTGAGCATGTCTGATGG-3';

reverse primer: 5'-CGATCAGCATCTGGGCCCAGG-3';

direct primer: 5'-GTGGAGCAGTTCACTCAGCAGGACC-3';

reverse primer: 5'-GCCTTCACACCCAGTACTGGCC-3'.

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 these PCR primers, and was thus given a sequence region cDNA macaque MCSP size 2547 p. O. (cDNA sequence and amino acid sequence of this area of the macaque MCSP presented in SEQ ID NO: 376 and 375) 74 p. O. above sequence that encodes a C-terminal domain, to 121 p. O. below the stop codon. Another PCR using the following reaction conditions: 1 cycle at 94°C for 3 min, 10 cycles at 94°C for 1 min, 52°C for 1 min and 72°C for 2.5 min, and a final cycle at 72°C for 3 min, was used for fusion PCR products a and b of the above-mentioned reactions. Used the following primers:

direct primer: 5'-tcccgtacgagatctggatcccaattggatggcggactcgtgctgttctcacacagagg-3'; reverse primer: 5'-agtgggtcgactcacacccagtactggccattcttaagggcaggg-3'.

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 C-terminal, transmembrane and short vnekletochnye macaque MCSP. Introduced restriction sites, Mfel at the 5'-end and Sall on the 3'-end, was used in the subsequent cloning procedures. The PCR fragment is then cloned via Mfel and Sall in the Bluescript plasmid containing the fragment EcoRI/Mfel the above-mentioned 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 shortened extracellular domains of human MCSP. The fragment of gene synthesis contained the coding sequences of immunoglobulin leader peptide and Flag-tag, as well as artificial linker (SRTRSGSQL), in reading frame with the 5'-end of the cDNA fragment encoding C-terminal, transmembrane and shortened extracellular domains macaque MCSP. This vector was used for transfection of cells CHO/dhfr - ATSC 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 concentrated solution of the reagent purity "cell cultures" (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) to a final concentration of adenosine, 10 μg/ml deoxyadenosine 10 μg/ml thymidine, 10 μg/ml, in an incubator at 37°C, 95% humidity and 7% CO2. Transfection was performed using reagent for transfection PolyFect (Qiagen GmbH, Hilden, Germany) and 5 μg of plasmid DNA in accordance with the Protocol of the manufacturer. After culturing for 4 hours, the cells were washed once with PBS and again 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 performed on transfected cells. Approximately 14 days after the implementation of transfection was observed the growth of resistant cells. After 7-14 days the transfectants were tested against the recombinant expression constructs using FACS analysis. a 2.5×105cells were incubated with 50 µl of 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 antibodies goat against mouse IgG (specific to Fc-gamma fragment), diluted 1:100 in PBS with 2% FCS (ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Samples were measured on a FACScalibur (BD biosciences, Heidelberg, Germany).

16. Creation and characterization of MCSP and CD3-bespecifically single-stranded molecules with cross-species specificity

Molecules bespecifically single-chain antibodies, each of which contains a binding domain with interspecies specificity for CD3-Epsilon person and the primacy of non-chimpanzee, and binding domain with cross-species specificity to human MCSP and the primacy, not a chimpanzee, was designed, as shown in the following Table 2.

Table 2
Formats MCSP and CD3-bespecifically single-chain antibodies with cross-species specificity
SEQ ID (n./Bel.)The formats of protein constructs (N→C)
310/309MCSP-G4 HL × NS HL
312/311MCSP-G4 HL × F12Q HL
314/313MCSP-G4 HL × I2C HL
316/315MCSP-G4 HLP × F6A HLP
318/317MCSP-G4 HLP × NS HLP
322/321MCSP-G4 HLP × G4H HLP
326/325MCSP-G4 HLP × E1L HLP
328/327MCSP-G4 HLP × EM HLP
332/331MCSP-G4 HLP × F12Q HL
334/333MCSP-G4 HLP × I2C HL
336/335MCSP-D2 HL × NS HL
338/337MCSP-D2 HL × F12Q HL
340/339MCSP-D2 HL × I2C HL
342/341MCSP-D2 HLP × NS HLP
344/343MCSP-F9 HL × NS HL
346/345MCSP-F9 HLP × NS HLP
348/347MCSP-F9 HLP × G4H HLP

The above constructs containing the variable domains of the heavy chain (VH) and the variable domains of the light chain (VL) of interspecific specific for MCSP D3 human and macaque, and VH and VL domains with inter-species specific for human CD3 and macaque, was obtained by gene synthesis. Fragments of gene synthesis designed so that they contained the first site Kozak for eukaryotic expression of this design, then immunoglobulin leader peptide of 19 amino acids, then, in reading frame, encoding a sequence corresponding molecule especifismo single-chain antibodies, then, in reading frame encoding the histidine sequence6-tag and a stop codon. The fragment of gene synthesis was also designed in such a way as to enter the appropriate N - and C-terminal restriction sites. A fragment of the gene fusion was cloned, using these restriction sites in the 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 cabelino or temporarily defective in DHFR cells Cho (ATSS No. CRL 9096) via electroporation or alternatively, temporarily transfusional in SOME 293 (embryonic cells of the human kidney, ATS number: CRL-1573) according to standard protocols.

The expression of eukaryotic protein defective in DHFR cells SNO was carried out as described in Kaufmann R. J. (1990) Methods Enzymol. 185, 537-566. Gene amplification structures induced by adding increasing concentrations of methotrexate (MTX) to a final concentration of 20 nm MTX inclusive. After two passages 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) within 7 days before collection. Cells were isolated by centrifugation and the supernatant containing the expressed protein was stored at -20°C.

For chromatography used the system Äkta® Explorer (GE Health Systems) and software Unicorn®. Affinity chromatography with immobilized metal ("IMAC") was performed using Fractogel EMD chelate® (Merck), which were loaded with ZnCl2in accordance with the Protocol suggested by the manufacturer. The column was balanced with buffer A (20 mm sodium phosphate buffer pH 7,2; 0.1 M NaCl), and cell culture supernatant (500 ml) was applied to a column (10 ml) at a flow rate of 3 ml/min the Column was washed with buffer to remove unbound sample. Bound peroxidase protein was suirable using ducted logo gradient of buffer B (20 mm sodium phosphate buffer pH 7,2; 0.1 M NaCl; 0.5 M imidazole) in accordance with the following Protocol:

stage 1: a 20% buffer In 6 column volumes;

stage 2: 100% buffer In 6 volumes of the column.

Erwerbende protein fractions from stage 2 were combined for further purification. All chemical reagents were of a purity suitable for research, and were purchased from Sigma (Deisenhofen) or Merck (Darmstadt).

Gel filtration was performed on preparative column (HiLoad 16/60 Superdex 200 (GE/Amersham), balanced Equi-buffer (25 mm citrate, 200 mm lysine; 5% glycerol; pH of 7.2). Erwerbende 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 (set of markers of molecular weight, Sigma MW-GF-200). The protein concentration was determined using OD at 280 nm.

Purified protein bespecifically single-chain antibody, were analyzed using SDS-PAGE in reducing conditions and pre-filled 4-12% gels were in bis-Tris buffer (Invitrogen). Preparation and application of samples was carried out according to the Protocol suggested by the manufacturer. The molecular weight was determined using a protein standard MultiMark (Invitrogen). The gel was stained with colloidal Coomassie (Invitrogen Protocol). The purity of the selected protein was more than 95% as determined by SDS-PAGE.

Bespecifically odnosa the targeted antibody has a molecular weight of 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 the membrane Optitran® BA-S83 in apparatus for blotting Invitrogen according to the Protocol suggested by the manufacturer. To determine protein especifismo single-chain antibodies used antibody against His-tag (Penta-His, Qiagen). Antibody goat against mouse Ig labeled with alkaline phosphatase (AP) (Sigma) was used as secondary antibody, a BClP/NBT (Sigma) as substrate. Identified a single band at 52 kDa, which corresponded cleaned bespecifically single-chain antibody.

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

17. Flow-cytometrics the analysis of binding MCSP and CD3-bespecifically antibodies with cross-species specificity

To test the functionality of the structures bespecifically antibodies with cross-species specificity in terms of their ability to contact MCSP D3 and CD3 human and macaque, respectively, was carried out by FACS-analysis. For this, cells SNO, transfetsirovannyh MCSP D3 person (as described in Example 14), and CD3-positive cell line HPB-ALL T-cell human leukemia (DSMZ, Braunschweig, ASS) was used to test binding to antigens of human rights. The ability to bind to antigens macaque TES is Aravali using the created transfectant with MCSP D3 macaque (described in Example 15) and T-macaque cell line 4119 LnPx (courtesy of Prof. Fickenscher, Hygiene Institute, Virology, Erlangen-Nuernberg; published in Knappe A, et al., and Fickenscher H., Blood 2000, 95, 3256-61). 200,000 cells of the respective cell populations 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 are washed twice in PBS with 2% FCS and linking this structure was determined using mouse anti-His antibody (Penta-His antibody; Qiagen; diluted 1:20 in 50 μl PBS with 2% FCS). After washing, the bound peroxidase anti-His antibodies were determined using an Fc-gamma-specific antibody (Dianova) conjugated with phycoerythrin, diluted 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 a negative control binding to MCSP-D3-transfitsirovannykh cells SNO.

Flow cytometry was carried out on the apparatus FACS-Calibur; for data collection and analysis used the software CellQuest (Becton Dickinson biosciences, Heidelberg). FACS-staining and measurement of fluorescence intensity was performed as opisanoj Current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-Interscience, 2002).

Bespecifically binding single-stranded molecules above that demonstrate cross-species specific for MCSP D3 and cross-species specific for human CD3 and macaque, clearly defined, as shown in Fig.14, 15, 16. In FACS analysis all constructs showed binding to CD3 and MCSP D3 compared with negative controls. Was demonstrated interspecific specificity bespecifically antibodies to antigens CD3 and MCSP D3 human and macaque.

18. The biological activity of MCSP and CD3-bespecifically single-chain antibodies with cross-species specificity

As shown in Fig.17-21, all designs bespecifically single-chain antibodies with cross-species specificity was demonstrated cytotoxic activity against MCSP-positive target cells human induced CD8+ human cells, and MCSP-positive target cells cynomolgus macaque, induced T-cell line macaque 4119 LnPx. Bespecifically single-chain antibody with a different target specificity is used as a negative control.

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

The stability created bespecifically single-chain antibodies in plasma was analyzed by incubation of the do Bispo is specific 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 the release of chromium 51 (51Cr) using MCSP-positive cell line Cho (expressing MCSP, cloned in accordance with Example 14 or 15) as the target, and stimulated CD8-positive T cells as effector cells.

Size EU50calculated using program analysis, which is described above, is used to compare the biological activity bespecifically single-chain antibodies, inkubiruemykh 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 immediately prior to analysis.

As shown in Fig.22 and in Table 3, the biological activity bespecifically antibodies G4 H-L × I2C H-L, G4 H-L × NS H-L and G4 H-L × F12Q H-L was slightly reduced compared to controls without the addition of plasma or with the addition of plasma directly before testing biological activity.

Table 3
Biological activity bespecifically antibodies without adding or adding plasma
Design Without plasmaWith plasmasPlasma 37°CPlasma 4°C
G4 H-L × I2C H-L300796902867
G4 H-L × H2C H-L49657523631449
G4 H-L × F12Q H-L49335815211040

20. Creating bespecifically single-stranded molecules with interspecies specificity against EGFR and CD3 person

Molecules bespecifically single-chain antibodies with binding domain, demonstrating cross-species specificity to CD3-Epsilon human and cynomolgus macaque, and binding domain, demonstrating cross-species specific for human EGFR, was designed, as shown in the following Table 4.

Table 4
Formats EGFR and CD3-bespecifically single-chain antibodies with cross-species specificity
SEQ ID (n./Bel.)/td> The formats of protein constructs (N→C)
390/389EGFR H-L × I2C HL
392/391EGFR L-H × I2C HL
394/393EGFR H-L × F12Q HL
396/395EGFR L-H × F12Q HL
398/397EGFR H-L × H2C HL
400/399EGFR L-H × H2C HL
448/447EGFR HL × H2C HL
450/449EGFR HL × F12Q LH
452/451EGFR HL×I2C HL

The above constructs containing the variable domains of the light chain (L) and the variable domains of the heavy chain (H) cross-species specificity to EGFR human and cynomolgus macaque, was obtained by gene synthesis. Fragments of gene synthesis designed so that they contained the first site Kozak for eukaryotic expression of this design, then immunoglobulin leader peptide of 19 amino acids, then, in reading frame, encoding a sequence corresponding molecule especifismo single-chain antibodies, then, in reading frame, coding sequence is alnost consisting of 6 his-tag residues tag and a stop codon.

The fragment of gene synthesis was also designed in such a way as to enter the appropriate N - and C-terminal restriction sites. A fragment of the gene fusion was cloned, using these restriction sites in the 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)). The data structure was stable transfusional in defective in DHFR cells Cho (ATSS No. CRL 9096), and had received and was purified as described in Example 10.

21. Creating bespecifically single-stranded molecules with interspecies specificity against EGFR and CD3 person

Molecules bespecifically single-chain antibodies with binding domain, demonstrating cross-species specificity to CD3-Epsilon human and cynomolgus macaque, and binding domain, demonstrating cross-species specific for human EGFR, was designed, as shown in the following Table 5.

EGFR H-L×I2C HL
Table 5
Formats EGFR and CD3-bespecifically single-chain antibodies with cross-species specificity
SEQ ID (n./Bel.)The formats of protein constructs (N→C)
410/4099
412/411EGFR L-H×I2C HL
414/413EGFR H-L×F12Q HL
416/415EGFR L-H × F12Q HL
418/417EGFR H-L × H2C HL
420/419EGFR L-H × H2C HL

The above constructs containing the variable domains of the light chain (L) and the variable domains of the heavy chain (H) cross-species specificity to EGFR human and cynomolgus macaque, was obtained by gene synthesis. Fragments of gene synthesis designed so that they contained the first site Kozak for eukaryotic expression of this design, then immunoglobulin leader peptide of 19 amino acids, then, in reading frame, encoding a sequence corresponding molecule especifismo single-chain antibodies, then, in reading frame, encoding a sequence consisting of 6 his-tag residues tag and a stop codon.

The fragment of gene synthesis was also designed in such a way as to enter the appropriate N - and C-terminal restriction sites. A fragment of the gene fusion was cloned, using these restriction sites in the plasmid designated pEF-DHFR (pEF-DHFR is described in Raum et al. Cancer Immunol. Immunother. 50 (2001) 141-150), coz the ACLs standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (2001)). The data structure was stable transfusional in defective in DHFR cells Cho (ATSS No. CRL 9096), and had received and was purified as described in Example 10.

22. Creating bespecifically single-stranded molecules with interspecies specificity against Her2/neu and human CD3

Molecules bespecifically single-chain antibodies with binding domain, demonstrating cross-species specificity to CD3-Epsilon human and cynomolgus macaque, and binding domain, demonstrating cross-species specificity to Her2/neu human designed, as shown in the following Table 6.

Table 6
Formats Her2/neu - and CD3-bespecifically single-chain antibodies with cross-species specificity
SEQ ID (n./Bel.)The formats of protein constructs (N→C)
430/439HER2/neu VH-VL × I2C VH VL
432/431HER2/neu VL-VH × I2C VH VL
434/433HER2/neu VH-VL × F12Q VH VL
436/435HER2/neu VL-VH × F12Q VH VL
438/437HER2/neu VH-VL × H2C VH VL
440/439HER2/neu VL-VH × H2C VH VL

The above constructs containing the variable domains of the light chain (L) and the variable domains of the heavy chain (H) cross-species specificity to Her2/neu human and cynomolgus macaque, was obtained by gene synthesis. Fragments of gene synthesis designed so that they contained the first site Kozak for eukaryotic expression of this design, then immunoglobulin leader peptide of 19 amino acids, then, in reading frame, encoding a sequence corresponding molecule especifismo single-chain antibodies, then, in reading frame, encoding a sequence consisting of 6 his-tag residues tag and a stop codon.

The fragment of gene synthesis was also designed in such a way as to enter the appropriate N - and C-terminal restriction sites. A fragment of the gene fusion was cloned, using these restriction sites in the 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)). The data structure was stable transfusional in defective in DHFR cells Cho (ATSS No. CRL 9096), and had received and was purified as described in Example 0.

23.1. Obtaining cells SNO expressing human HER2

The coding sequence of the human HER2, which is published in GenBank (access number H), get gene synthesis according to standard protocols. A fragment of the gene fusion construct so that it contained the coding sequence of the protein HER2 person, including its leader peptide (cDNA sequence and amino acid sequence of this design is presented in SEQ ID NO: 459 and 460). A fragment of the gene fusion construct also in such a way as to introduce restriction sites at the beginning and end of a segment. Introduced restriction sites, Xbal at the 5'-end and Sall on the 3'-end, used in subsequent cloning procedures. The fragment of gene synthesis clone using Xbal and Sall in plasmid designated pEFDHFR (pEFDHFR described in Raum et al. Cancer Immunol. Immunother. 50 (2001) 141-150), following standard protocols. The above procedures are conducted according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (2001)). The clone with confirmed nucleotide sequence used for transfection defective in DHFR cells SNO for eukaryotic expression of this design. The expression of eukaryotic protein defective in DHFR cells SNO carried out, as described in Kaufmann R. J. (1990) Methods Enzymol. 185, 537-566. Gene is th amplification design induce by increasing concentrations of methotrexate (MTX) to a final concentration of 20 nm MTX inclusive.

23.2. Obtaining cells SNO expressing the extracellular domain Neg macaque

The above-described coding sequence Neg person modify to include amino acids 123-1038 protein Neg macaque, as published in GenBank (access number HR). The coding sequence of this chimeric protein get gene synthesis according to standard protocols (cDNA sequence and amino acid sequence of this design is presented in SEQ ID NO: 461 and 462). A fragment of the gene fusion construct also so that it contains the website Kozak for eukaryotic expression of the construct and restriction sites at the beginning and at the end of the fragment. Introduced restriction sites, Xbal at the 5'-end and Sall on the 3'-end, used in subsequent cloning procedures. The fragment of gene synthesis then clone using Xbal and Sall in plasmid designated pEFDHFR (pEFDHFR described in Raum et al. Cancer Immunol. Immunother. 50 (2001) 141-150). The clone with the confirmed sequence of this plasmid used for transfection of cells CHO/dhfr-, as described above.

23.3. The creation of HER2 and CD3-bespecifically single-stranded molecules with cross-species specificity

3.1. Cloning of binding molecules with cross-species specificity

Typically, molecules bespecifically single-chain antibodies, each of which contains a domain with specificity what was mentioned link, demonstrating cross-species specificity to CD3-Epsilon human and macaque, as well as a domain with binding specificity, demonstrating cross-species specificity to HER2 human and macaque, design, as shown in the following Table 7.

Table 7
Formats molecules with anti-CD3 and anti-HER2 bespecifically single-chain antibodies with cross-species specificity
SEQ ID (n./Bel.)The formats of protein constructs (N→C)
432/431Her2 LH × I2C HL
436/435Her2 LH × F12Q HL
440/439Her2 LH × NS HL
430/429Her2 HL × I2C HL
434/433Her2 HL × F12Q HL
438/437Neg HL × NS HL
480/479I2C HL × Her2 LH
478/477F12Q HL×Her2 LH
476/475NS HL × Her2 LH

The above mentioned structures containing variable house is by the light chain (L) and the variable domains of the heavy chain (H) cross-species specificity to HER2 human and macaque, and CD3-specific VH - and VL-combination with inter-species specific for human CD3 and macaque, get gene synthesis. Fragments of gene synthesis design so that they contained the first site Kozak for eukaryotic expression of this design, then immunoglobulin leader peptide of 19 amino acids, then, in reading frame, encoding a sequence corresponding molecule especifismo single-chain antibodies, then, in reading frame, encoding a sequence consisting of 6 his-tag residues tag and a stop codon. A fragment of the gene fusion construct also thus to introduce suitable restriction sites at the beginning and end of a segment. Introduced restriction sites used in subsequent cloning procedures. The fragment of gene synthesis clone using these restriction sites in the plasmid, designated pEFDHFR (pEFDHFR described in Raum et al. Cancer Immunol. Immunother. 50 (2001) 141-150), following standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (2001)). The clone with confirmed nucleotide sequence transferout in defective in DHFR cells SNO for eukaryotic expression of this design. The expression of eukaryotic protein defective in DHFR cells SNO carried out, as described in Kaufmann R. J. (1990) Methods Enzymol. 185, 537-566. the military amplification design induce by increasing concentrations of methotrexate (MTX) to a final concentration of 20 nm MTX inclusive.

3.2. Expression and purification of molecules bespecifically single-chain antibodies

Molecules bespecifically Express a single-chain antibodies in the cells of the Chinese hamster ovary (Cho). The expression of eukaryotic protein defective in DHFR cells SNO carried out, as described in Kaufmann R. J. (1990) Methods Enzymol. 185, 537-566. Gene amplification structures induce by adding increasing concentrations of MTX to a final concentration of 20 nm MTX inclusive. After two passages of the stationary cell culture cells 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) within 7 days before collection. Cells release by centrifugation and the supernatant containing the expressed protein is stored at -80°C. the Transfection is carried out using 293fectin reagent (Invitrogen, No. 12347-019) in accordance with the Protocol of the manufacturer.

For chromatography using the system Äkta® Explorer (GE Health Systems) and software Unicorn®. Affinity chromatography with immobilized metal ("IMAC"), make use of Fractogel EMD chelate® (Merck), which load ZnCb in accordance with the Protocol suggested by the manufacturer. Column offset buffer A (20 mm sodium phosphate buffer pH 7,2; 0.1 M NaCl) and column (10 ml) is applied to the cell culture supernatant (500 ml) at a speed of Patoka ml/min The column is washed with buffer to remove unbound sample. Bound peroxidase protein elute using a two-step gradient of buffer B (20 mm sodium phosphate buffer pH 7,2; 0.1 M NaCl; 0.5 M imidazole) in accordance with the following Protocol:

stage 1: a 20% buffer In 6 column volumes;

stage 2: 100% buffer In 6 volumes of the column.

Erwerbende protein fractions from stage 2 unite for further purification. All chemicals have a degree of purity suitable for research, and purchased from Sigma (Deisenhofen) or Merck (Darmstadt).

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

Purified protein bespecifically single-chain antibody, and analyzed using SDS-PAGE in reducing conditions and pre-filled 4-12% gels were in bis-Tris buffer (Invitrogen). Preparation and application of samples is carried out in accordance with the Protocol suggested by the manufacturer. The molecular weight determined using protein article shall ndart MultiMark (Invitrogen). Gel paint colloidal Coomassie (Invitrogen Protocol). The purity of the selected protein is greater than 95% as determined by SDS-PAGE.

Bespecifically single-chain antibody has a molecular weight of approximately 52 kDa in native conditions, as determined by gel filtration in PBS. All designs are cleaned in accordance with this method.

Western blotting performed using the membrane Optitran® BA-S83 in apparatus for blotting Invitrogen according to the Protocol suggested by the manufacturer. To determine protein especifismo single-chain antibodies using antibody against His-tag (Penta-His, Qiagen). Antibody goat against mouse Ig labeled with alkaline phosphatase (AP) (Sigma), used as a secondary antibody, a BCIP/NBT (Sigma) as substrate. Define a single band at 52 kDa, which corresponds to the purified bespecifically single-chain antibody.

23.4. Flow-cytometrics analysis of the binding of HER2 and CD3-bespecifically single-chain antibodies with cross-species specificity

To test the functionality of the structures bespecifically antibodies with cross-species specificity in terms of their ability to communicate with HER2 and CD3 human and macaque, respectively, carried out FACS analysis. For this, cells SNO, transfetsirovannyh HER2 person as described in Example 23.1, and CD3-positive cell line HPB-ALL T-cell leukemia is a ne (DSMZ, Braunschweig, ACC483) is used to test binding to antigens of human rights. The ability to contact the macaque antigens tested using the generated transfectant with HER2 macaque described in Example 23.2, and T-macaque cell line 4119 LnPx (courtesy of Prof. Fickenscher, Hygiene Institute, Virology, Erlangen-Nuernberg; published in Knappe A, et al., and Fickenscher H., Blood 2000, 95, 3256-61). 200,000 cells of the respective cell lines incubated for 30 min on ice with 50 μl of purified protein structures bespecifically antibodies with cross-species specificity (2 μg/ml). Cells are washed twice in PBS with 2% FCS and linking this design determined using mouse anti-His antibody (Penta-His antibody; Qiagen; diluted 1:20 in 50 μl PBS with 2% FCS). After washing, the bound peroxidase anti-His-antibody determined with the use of Fc-gamma-specific antibody (Dianova) conjugated with phycoerythrin, diluted 1:100 in PBS with 2% FCS. PBS with 2% FCS is used as a negative control binding to T-cell lines with HER2-transfitsirovannykh cells SNO.

Flow cytometry is performed on the device FACS-Calibur; for data collection and analysis used the software CellQuest (Becton Dickinson biosciences, Heidelberg). FACS-staining and measuring the intensity of fluorescence is carried out, as described in Current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-Interscince, 2002).

Bespecifically binding single-stranded molecules above that demonstrate cross-species specificity to HER2 and cross-species specific for human CD3 and of the primacy of non-chimpanzee, clearly defined, as shown in Fig.23. In FACS-analysis of all designs demonstrate binding to CD3 and HER2 when compared to the respective negative controls. Demonstrated cross-species specificity bespecifically antibodies to antigens CD3 and HER2 human and macaque.

23.5. The biological activity of HER2 and CD3-bespecifically single-chain antibodies with cross-species specificity

Biological activity created bespecifically single-chain antibodies analyze the release of chromium 51 (51Cr) in the analysis of cytotoxicity in vitro, using R2-positive cell lines described in the Examples 23.1 and 23.2. As effector cells using stimulated CD4/CD56-depleted RVMS person or T-macaque cell line 4119 LnPx, as shown in the corresponding graphic materials.

Getting stimulated CD4/CD56-depleted RVMS carried out as follows.

On a Petri dish (diameter 85 mm; Nunc) applied a commercially available anti-CD3 specific antibody (e.g., OCT, Othoclone) at a final concentration of 1 μg/ml for 1 hour at 37°C. Unbound protein removed per stud is Yu washing using PBS. Fresh VRMS isolated from the peripheral blood (30-50 ml of blood) by centrifugation in a gradient of ficoll according to standard protocols. 3-5×107RVMS added to pre-coated Petri dish in 50 ml of RPMI 1640 with stabilizing additive glutamine/10% FCS/IL-2 20 unit/ml (Proleukin, Chiron) and stimulated for 2 days. On the third day the cells are harvested and once washed with RPMI 1640. Add IL-2 to a final concentration of 20 units/ml and the cells are again cultured for one day in the same environment for culturing cells, as mentioned above. By depletion of CD4+T cells and CD56+NK cells according to standard protocols, carry out the enrichment of CD8+cytotoxic T lymphocytes (CTL).

Target cells washed twice by PBS and mortality of 11.1 MBq51Cr in the final volume of 100 μl RPMI with 50% FCS for 45 minutes at 37°C. After this, the labeled target cells are washed 3 times with 5 ml RPMI and then used in the analysis of cytotoxicity. The analysis is performed in 96-well-plate in a total volume of 250 μl supplemented RPMI medium (as above) with the ratio of E:T of 1:1 or 10:1, which is specified in the relevant graphic materials. Put 1 μg/ml of molecules especifismo single-chain antibodies with cross-species specificity and 15-21 their fivefold dilutions. The analysis time is 18 hours, and cytotoxicity measured in relative terms, visualaid the frame of chromium in the supernatant, corresponding to the difference between the maximum lysis (adding Triton-X) and spontaneous lysis (without effector cells). All measurements carried out in four replications. Measurement of radioactivity chrome supernatant carried out using a gamma counter Wizard 3" (Perkin Elmer Life Sciences GmbH, Koln, Germany). Analysis of experimental data carried out using Prism 4 for Windows (version 4.02, GraphPad Software Inc., San Diego, California, USA). Sigmoidal curves dose-effect typically have a value of R2above 0,90, as determined using this software. Size EU50designed with the help of this program analysis is used to compare the biological activity.

As shown in Fig.24, designs bespecifically single-chain antibodies with cross-species specificity demonstrate cytotoxic activity against target cells, positive for the HER2 human induced stimulated CD4/CD56-depleted RVMS person, and against target cells, positive for the HER2 macaque induced T-cell line macaque 4119 LnPx.

Example 24. Cloning and expression of the membrane-bound form of IgE human and macaque

Murine cell line J558L (obtained from Interlab Project, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy, ECACC 88032902), a variant of the myeloma cell line with spontaneous perspiration is it heavy chain, which synthesizes and secretes a light chain lambda, used to complementaly membrane-bound variant of the heavy chain of IgE human and macaque, respectively. To create such structures synthetic molecule was obtained by gene synthesis according to standard protocols (nucleotide sequence are shown in SEQ ID NO: 507 and 508). In these constructs the coding sequence of Epsilon-chain of human and macaque were subjected to fusion with the transmembrane region of the human IgE, respectively. Built-in function in specificity VH-chain directed against the hapten ((4-hydroxy-3-nitro-phenyl)acetyl) (NP). The fragment of gene synthesis was also designed so that it contains the website Kozak for eukaryotic expression of this design, immunoglobulin leader peptide and the restriction sites at the beginning and at the end of the DNA. Introduced restriction sites, EcoRI at the 5'-end and Sall on the 3'-end, was used during the stage of cloning in expressing plasmid, designated pEFDHFR. After confirmation of the sequence (macaques: HM: Epsilon-With-pane Ig macaque-rhesus (Masasa mulatta), mRNA; man: NC_000014: chromosome 14 of Homo sapiens, the full sequence, the national center for biotechnology information, http://www.ncbi.nlm.nih.gov/entrez these plasmids were used for transfection of cells CHO/dhfr-, as described above, the Expression of eukaryotic protein defective in DHFR cells SNO exercise, as described in Kaufmann R. J. (1990) Methods Enzymol. 185, 537-566. Gene amplification design induce by increasing concentrations of methotrexate (MTX) to a final concentration of 20 nm MTX inclusive.

Example 25. Creating IgE - and CD3-bespecifically single-stranded molecules with cross-species specificity

Typically, molecules bespecifically single-chain antibodies, each of which contains a domain with binding specificity with the CD3 antigen of human and cynomolgus macaque, as well as a domain with binding specificity with IgE-antigen of human and macaque, was designed, as shown in the following Table 8.

506/505
Table 8
Formats molecules with anti-CD3 and anti-IgE bespecifically single-chain antibodies with cross-species specificity
SEQ ID (n./Bel.)The formats of protein constructs (N→C)
496/495IgE HL × NS HL
498/497IgE HL × F12Q HL
500/499IgE HL × I2C HL
502/501IgE LH × H2C HL
504/503IgE LH × F12Q HL
IgE LH × I2C HL

The above constructs containing the variable domains of the light chain (L) and the variable domains of the heavy chain (H) with interspecies specificity for IgE human and macaque and CD3-specific VH - and VL-combination with inter-species specific for human CD3 and macaque, get gene synthesis. Fragments of gene synthesis design so that they contained the first site Kozak for eukaryotic expression of this design, then immunoglobulin leader peptide of 19 amino acids, then, in reading frame, encoding a sequence corresponding molecule especifismo single-chain antibodies, then, in reading frame, encoding a sequence consisting of 6 his-tag residues tag and a stop codon. A fragment of the gene fusion construct also thus to introduce suitable restriction sites at the beginning and end of a segment. Introduced restriction sites used in subsequent cloning procedures. The fragment of gene synthesis clone using these restriction sites in the plasmid, designated pEFDHFR, following the standard protocols. The clone with confirmed nucleotide sequence transferout in defective in DHFR cells SNO for eukaryotic expression of this design. The expression of eukaryotics the CSOs protein defective in DHFR cells SNO exercise, as described in Kaufmann R. J. (1990) Methods Enzymol. 185, 537-566. Gene amplification design induce by increasing concentrations of methotrexate (MTX) to a final concentration of 20 nm MTX inclusive. Alternative designs are used for temporary transfection defective in DHFR cells SNO according to standard protocols.

To assess the ability to communicate with human IgE was carried out experiments on binding using FACS for cell line J558L, transtitional IgE person. Cross-species specificity to cells positive macaque IgE was tested using J558L cells, transfetsirovannyh macaque IgE. The same changes in cell lines used in the cytotoxicity assays conducted with IgE - and CD3-bespecifically single-chain antibodies with cross-species specificity. Except for this analysis was carried out as described in Examples 4 and 5.

As is shown in Fig.23 created IgE - and CD3-bespecifically single-chain antibodies with cross-species specificity was demonstrated by the binding of antigens to both human and cynomolgus macaque, which confirmed their full cross-species specificity.

As shown in Fig.24, all designs bespecifically single-chain antibodies with cross-species specificity was demonstrated cytotoxic activity against target cells, put the lnyh against human IgE, induced CD8+human cells, and target cells positive macaque IgE induced T-cell line macaque 4119 LnPx. As a negative control was used irrelevant bespecifically single-chain antibody.

Example 26. Specific binding of scFv clone-N-end of the CD3-Epsilon person

26.1. Bacterial expression of scFv constructs in E. coli XL1 Blue

As mentioned above, cells of E. coli XL1 Blue, transformed pComb3H5Bhis/Flag containing the VL and VH segments, produce soluble scFv in sufficient quantities after cutting a fragment of the gene III and induction with 1 mm IPTG. scFv-chain is exported to periplasm, where she collapsed in a functional conformation.

For this experiment were selected following scFv clones:

1) scFv4-10, 3-106, 3-114, 3-148, 4-48, 3-190 and 3-271, which are described in WO 2004/106380,

2) scFv clones NS, F12Q and I2C-linking anti-CD3-Epsilon person described in this invention.

To obtain periplasmatic drugs bacterial cells transformed by the corresponding scFv-containing plasmids, allowing periplasmatic expression were grown in SB-medium, supplemented with 20 mm MgCl2and carbenicillin (50 μg/ml), and resuspendable in PBS after collection. As a result of four cycles of freezing at -70°C. and thawing at 37°C outer membrane of the bacteria which was destroyed under the action of osmotic shock and soluble periplasmatic proteins, including scFv, was 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. These crude supernatant containing scFv, hereinafter will be referred to as periplasmatic drugs (PPP).

26.2. Binding of scFv with fused protein [CD3 Epsilon (AA 1-27) man]-Fc

Experiments using ELISA was performed by coating the wells of 96-well plastic plates (Nunc, maxisorb) fused protein [CD3 Epsilon (AA 1-27) man]-Fc usually at 4°C over night. The antigen solution for coating was then removed, the wells were washed once with PBS/0,05% Tween 20 and then blocked PBS/3% BSA for at least one hour. After removal of the blocking solution in the wells was added PPP and control solutions and usually incubated for one hour at room temperature. The wells were then washed three times with PBS/0,05% Tween 20. The definition of scFv, contacting the immobilized antigen was carried out using labeled with Biotin antibody against FLAG-tag (anti Flag M2-Bio, Sigma, usually at a final concentration of 1 μg/ml PBS), and was determined using peroxidase labeled streptavidin (Dianova, 1 μg/ml PBS). The signal was developed by adding ABTS substrate solution and was measured at a wavelength of 405 nm. Nonspecific tie is of the tested samples with a blocking agent and/or plot IgG1 person of the fused protein [CD3 Epsilon (AA 1-27) man]-Fc was investigated by carrying out an identical analysis with the same reagents and the same timing tablets for ELISA, which were covered with human IgG1 (Sigma). As a negative control was used PBS.

As shown in Fig.25, scFv NS, F12Q and I2C demonstrate strong binding signals on fused protein [CD3 Epsilon (AA 1-27) man]-Fc. scFv person 3-106, 3-114, 3-148, 3-190, 3-271, 4-10 and 4-48 (described in WO 2004/106380) do not show any significant binding above the level of the negative control.

To exclude the possibility that the positive binding of scFv NS, F12Q and I2C with holes, covered with a fused protein [CD3 Epsilon (AA 1-27) man]-Fc, can be due to the binding with BSA (used as a blocking agent) and/or Fc-gamma-plot IgG1 person of the fused protein [CD3 Epsilon (AA 1-27) man]-Fc, were simultaneously implemented a second ELISA experiment. In this second ELISA experiment, all parameters were identical to the parameters in the first ELISA experiment, except that in the second ELISA experiment instead of the fused protein [CD3 Epsilon (AA 1-27) man]-Fc was applied human IgG1 (Sigma). As shown in Fig.26, none of the tested scFv did not show any significant binding to BSA and/or human IgG1, exceeding the background level.

Taken together, these results allow to conclude that the scFv 4-10, 3-271, 3-148, 3-190, 4-48, 3-106 and 3-114 not associated specifically with (AA 1-27)-area CD3-Epsilon person the century, while scFv NS, F12Q and I2C clearly demonstrate specific binding to the N-terminal 27 amino acids of CD3-Epsilon person.

1. The polypeptide containing the first binding domain that is an antibody that can specifically bind to epitope D3ε-chain of human andCallithrix jacchus, Saguinus oedipusorSaimirí sciureuswhere specified epitope is a part of the amino acid sequence that is a member of the group consisting of SEQ ID NO: 2, 4, 6 or 8, and a second binding domain that is an antibody that can specifically bind to EGFR (the receptor for epidermal growth factor), Her2/neu (member of the family of receptors for epidermal growth factor) or IgE (immunoglobulin E) human and/or Primate, non chimpanzees, where the first binding domain contains:
CDR (hypervariable site)-L1 selected from the group consisting of SEQ ID NO: 27, 117 and 153;
CDR-L2 is selected from the group consisting of SEQ ID NO: 28, 118 and 154;
CDR-L3 selected from the group consisting of SEQ ID NO: 29, 119 and 155;
CDR-H1 is selected from the group consisting of SEQ ID NO: 12, 30, 48, 66, 84, 102, 120, 138, 156 and 174;
CDR-H2, selected from the group consisting of SEQ ID NO: 13, 31, 49, 67, 85, 103, 121, 139, 157 and 175; and
CDR-H3 selected from the group consisting of SEQ ID NO: 14, 32, 50, 68, 86, 104, 122, 140, 158 and 176.

2. The polypeptide under item 1, where the specified epitope is a part of the amino acid sequence, entering the it group, consisting of SEQ ID NO: 2, 4, 6 or 8, and contains at least the amino acid sequence Gln-Asp-Gly-Asn-Glu.

3. The polypeptide under item 1, where the first binding domain contains a VL-region (variable region light chain), containing CDR-L1, CDR-L2 and CDR-L3 selected from:
(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.

4. The polypeptide under item 1, where the first binding domain contains a VH-region (variable region of the heavy chain), containing CDR-H1, CDR-H2 and CDR-H3 selected from:
(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, and CDR-H3 as shown in SEQ ID NO: 32;
(b) 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;
(d) 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;
(e) CDR-H1 as shown in SEQ ID NO: 102, CDR-H2 as shown in SEQ ID NO: 103, and CDR-H3 as presented is about 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;
(C) CDR-H1 as shown 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
(C) 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.

5. The polypeptide under item 1, where the first binding domain 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.

6. The polypeptide under item 1, where the first binding domain 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 or 181.

7. The polypeptide under item 1, where the first binding domain contains a VL region and a VH region selected from the group consisting of:
(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) a VL-region as shown in SEQ ID NO: 35 or 39, and a VH-region as shown in SEQ ID NO: 33 or 37;
(b) VL-region as shown in SEQ ID NO: 53 or 57, and a VH-region as shown in SEQ ID NO: 51 or 55;
(g) VL-region as shown in SEQ ID NO: 71 or 75, and a VH-region as shown in SEQ ID NO: 69 or 73;
(d) VL-region as shown in SEQ ID NO: 89 or 3, and VH-region as shown in SEQ ID NO: 87 or 91;
(f) a VL-region as shown in SEQ ID NO: 107 or 111, and a VH-region as shown in SEQ ID NO: 105 or 109;
(W) VL-region as shown in SEQ ID NO: 125 or 129, and a VH-region as shown in SEQ ID NO: 123 or 127;
(C) VL-region as shown in SEQ ID NO: 143 or 147, and a VH-region as shown in SEQ ID NO: 141 or 145;
(and) 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
(K) VL-region as shown in SEQ ID NO: 179 or 183, and a VH-region as shown in SEQ ID NO: 177 or 181.

8. The polypeptide under item 7, where the first binding domain contains an 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.

9. The polypeptide according to any one of paragraphs.1-8, where the specified polypeptide is a molecule especifismo single-chain antibodies.

10. The polypeptide under item 9, where the molecule especifismo single-chain antibody contains a group of the following sequences, such as CDR H1, CDR H2, CDR H3, CDR L1, CDR L2 and CDR L3 in the second binding domain selected from SEQ ID NO: 441-446, SEQ ID NO: 453-458, SEQ ID NO: 463-468, SEQ ID NO: 481-486.

11. The polypeptide under item 9, where the molecule especifismo single-chain antibody contains a sequence chosen from:
(a) amino acid sequence as shown in any of SEQ ID NO: 389, 39, 393, 395, 397, 399, 409, 411, 413, 415, 417, 419, 429, 431, 433, 435, 437, 439, 447, 449, 451, 469, 471, 473, 475, 477, 479, 495, 497, 499, 501, 503 and 505; and
(b) amino acid sequence encoded by the nucleic acid sequence as represented in any of SEQ ID NO: 390, 392, 394, 396, 398, 400, 410, 412, 414, 416, 418, 420, 430, 432, 434, 436, 438, 440, 448, 450, 452, 470, 472, 474, 476, 478, 480, 496, 498, 500, 502, 504 and 506.

12. The sequence of a nucleic acid encoding a polypeptide as defined in any of paragraphs.1-11.

13. A vector containing the nucleic acid sequence as defined in paragraph 12, to obtain a polypeptide as defined in any of paragraphs.1-11.

14. A host cell transformed or transfusiona vector defined in paragraph 13, to obtain a polypeptide as defined in any of paragraphs.1-11.

15. The method of producing the polypeptide according to any one of paragraphs.1-11, comprising culturing the host cell defined in paragraph 14, under conditions promoting expression of the specified polypeptide, and removing the specified derived polypeptide from the culture.

16. Pharmaceutical composition comprising an effective amount of the polypeptide according to any one of paragraphs.1-11 or obtained according to the method according to p. 15, to prevent, cure or ameliorate disease selected from a proliferative disease, neoplastic diseases or immunological disorders.

17. The use of the polypeptide according to any of the PP.1-11 or obtained according to the method according to p. 15 in the prevention, treatment or attenuation of a disease selected from a proliferative disease, neoplastic diseases or immunological disorders.



 

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35 cl, 15 tbl, 5 ex, 4 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a recovered human integrin α5β1 monoclonal antibody. The antibody is characterised by the fact that it contains 6 CDR, 3 CDR from a light chain and 3 CDR from a heavy chain. A nucleic acid (NA) coding the antibody according to the invention, an expression vector containing a NA molecule, a host cell containing the vector, and a method for preparing the antibody on the basis of the cell are described. There are disclosed: a composition and a method for growth inhibition of the tumour cells expressing human integrin α5β1 on the basis of the antibody. What is described is a version of the method for growth inhibition of the tumour cells expressing human integrin α5β1 using the composition.

EFFECT: invention provides the new antibodies with high (approximately nm, as measured by FACS) binding affinity for human integrin α5β1 that can find application in medicine in therapy of the tumours mediated by integrin α5β1 expression.

13 cl, 36 dwg, 3 tbl, 11 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, in particular, to method of producing amino acids derived from aspartate with application of recombinant microorganism, possessing at least the following properties in comparison with initial microorganism: (a) increased activity of glucose-6-phosphatedehydrogenase, (b) increased activity of fructose-1,6-biphosphatase, (c) weaker activity of isocitrate dehydrogenase, (d) increased activity of diaminopimelate dehydrogenase and (e) increased activity of aspartate kinase, where microorganism represents Corynebacterium.

EFFECT: invention makes it possible to produce amino acids of aspartate family, in particular L-lysine with high degree of efficiency.

15 cl, 35 dwg, 20 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents a combined recombinant protein of the formula: S-L-R, including SR10, SR13, SR15, SdR10, SdR13 or SdR15, which specifically recognises melanoma cells, where S - streptavidin monomer, L - linker having amino-acid sequence Ser-Arg-Asp-Asp-Asp-Asp-Lys containing a restriction site with enteropeptidase and marked as "d", or amino-acid sequence Ser-Arg-Ala-Gly-Ala,R - melanoma-addressing oligopeptide representing R10 having amino-acid sequence Asp-Gly-Ala-Arg-Tyr-Cys-Arg-Gly-Asp-Cys-Phe-Asp-Gly, or R13 having amino-acid sequence Leu-Ser-Gly-Cys-Arg-Gly-Asp-Cys-Phe-Glu-Glu, or R15 having amino-acid sequence Asp-Gly-Phe-Pro-Gly-Cys-Arg-Gly-Asp-Cys-Ser-Gln-Glu. This invention also describes recombinant plasmid DNAs pSR and pSdR for expression of the specified combined proteins, bacterial strains Escherichia coli MG1655/pSR and MG1655/pSdR, producers of the specified combined proteins and a producing method of melanoma-addressing oligopeptide R from combined recombinant proteins SdR10, SdR13 or SdR15.

EFFECT: invention allows producing combined proteins that provide selective and effective binding to receptors on the surface of melanoma cells and can be used in diagnostics and therapy of cancer of a human being.

9 cl, 7 dwg, 5 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes a constructed plasmid for expression in a cell of a Chinese hamster, in the following sequence, which mainly contains the following elements: pUC plasmid replication beginning region; an open reading frame (ORF) of beta-lactamase providing immunity to ampicillin; procaryotic gene promoter bla; a section of terminal repetition of Epstein-Barr virus of a human being; a functional gene promoter of elongation factor 1 alpha of the Chinese hamster, 5' non-translated region of this gene and a non-transcribed region flanking this gene, coding Kozak sequence for cap-dependent initiation of translation; ORF of the gene of subunit 1 of complex of 2,3-epoxyreductase of vitamin K (VKORC1) of the Chinese hamster with stop codon; a functional terminator and a signal of polyadenilation of the gene of elongation factor 1 alpha of the Chinese hamster, 3' non-translated region of this gene and a non-transcribed region flanking this gene; a promoter of early genes of virus SV40; gene of immunity to a selective agent; and a polyadenilation signal and terminator of virus SV40. Cells of the Chinese hamster - producer of protein with Gla-domain are transformed by the obtained plasmid, which are used in a method of recombinant obtainment of proteins with Gla-domain.

EFFECT: invention allows increasing productivity of the above cells owing to increasing activity of native VKORC1.

12 cl, 12 dwg, 2 tbl, 8 ex

FIELD: biotechnology.

SUBSTANCE: bispecific antibody is proposed, that binds to both the blood coagulation factor IX/activated blood coagulation factor IX and with the blood coagulation factor X, and functionally replaces the function of blood coagulation factor VIII. The nucleic acid is considered, encoding the antibody of the invention, a vector, a cell and a method of producing the antibody, and also a pharmaceutical composition and a kit for use in the method of preventing and/or treating bleeding or diseases associated with or caused by bleeding.

EFFECT: invention may find further application in the treatment of diseases associated with impaired blood clotting.

16 cl, 2 ex, 6 dwg

FIELD: chemistry.

SUBSTANCE: claimed is bispecific antibody, which is bound with both blood coagulation factor IX/activated blood coagulation factor IX and with blood coagulation factor X and functionally replaced function of blood coagulation factor VIII. Described are nucleic acid, coding antibody by invention, vector, cell and method of obtaining antibody, as well as pharmaceutical composition and set for application in method of prevention and/or treatment of bleeding or diseases, associated with or induced by bleeding.

EFFECT: invention can be applied in therapy of diseases, associated with blood coagulation disorders.

16 cl, 2 ex, 6 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology and biotechnology. There are presented variants of antagonist antibodies binding to the interleukin-7 receptor (IL-7R). There are described: variants of nucleic acids coding the antibodies; a host cell recombinant producing the antibody; a pharmaceutical composition inhibiting the human IL-7R function, and methods of treating and/or preventing: an autoimmune disease specified in the group of type 1 diabetes, lupus, multiple sclerosis, rheumatoid arthritis; type 2 diabetes or graft-versus-host disease based on using the antibody.

EFFECT: invention provides the antagonist anti-IL-7R antibodies that can find application in medicine.

17 cl, 15 dwg, 8 tbl, 13 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to molecular biology, in particular to short interfering RNA (siRNA), and can be used in antitumor therapy. On the basis of genome analysis the sequences of siRNA against the human gene HIF1A with SEQ ID NO:1-2 are designed, siRNA against the human gene HSP8A with SEQ ID NO:3-4, siRNA against the human gene APEX1 with SEQ ID NO:5-6, and siRNA against the human gene CCND3 with SEQ ID NO:7-8, associated with cell proliferation of human pancreatic adenocarcinoma. Using the obtained siRNA, including as part of a lentiviral vector, results in suppression of cell proliferation of human pancreatic adenocarcinoma and tumor destruction. The invention enables to achieve the level of suppression of proliferation up to 65% using siRNA to genes HIF1A and HSP8A associated with stress and responsible for the major proliferative (CCND3) and reparative (APEX-1) ways of cell division.

EFFECT: improving level of suppression of proliferation.

3 cl, 10 dwg, 2 ex

FIELD: biotechnology.

SUBSTANCE: expression cassette, the recombinant vector, the seed and the plant are disclosed, comprising the said isolated nucleic acid molecule, as well as the host cell and the plant comprising the said expression cassette. The invention also relates to the use of the said fusion protein in the insecticidal composition and the method of control of European corn worm, and also the method for its preparation. Also the method of production of a transgenic plant is disclosed, which is resistant to European corn worm, with use of the said fusion protein.

EFFECT: invention enables to obtain plants resistant to European corn worm.

39 cl, 8 dwg, 9 tbl, 45 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented group of inventions refers to biotechnology, and concerns a DLK1-Fc fused protein and using it for the metastases inhibition, a polynucleotide coding such a protein, an expression vector containing the polynucleotide, a host cell producing the above fused protein, a method for producing the fused protein by culturing the above host cell, a composition containing the above fused protein, and a method for the metastases inhibition. The characterised fused protein contains a DLK1 extracellular soluble domain consisting of the amino acid sequence SEQ ID NO:4 and Fc domain of a human antibody.

EFFECT: group of inventions can be used for preparing a therapeutic agent for reduction of cancer cell migration and the metastases inhibition.

11 cl, 36 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the field of biotechnology. Claimed is a recombinant plasmid pCHBH for an expression of a sequence, coding procarboxypeptidase B in Pichia pastoris, which has a size of10466 t.b.p. and consists of XhoI/EcoRI -fragment of DNA of vector pP1C9K with a size of 9246 t.b.p. and XhoI/EcoRI -fragment of DNA with a size of 1220 t.b.p., including a region, coding the signal peptide of α-factor of Saccharomyces cerevisiae,anda synthetic gene of human procarboxypeptidase B with a nucleotide sequence, corresponding to the sequence, represented on fig. 2. In addition, a recombinant strain of Pichia pastoris GS115CPBH - producent of human procarboxypeptidase, obtained as a result of the parent strain transformation by the said plasmid, is described.

EFFECT: invention makes it possible to obtain human procarboxypeptidase and a corresponding to it active form in an increased quantity, constituting not less than 9,0 units/ml in comparison with the prototype.

2 cl, 4 dwg, 4 ex

FIELD: biotechnology.

SUBSTANCE: bispecific antibody is proposed, that binds to both the blood coagulation factor IX/activated blood coagulation factor IX and with the blood coagulation factor X, and functionally replaces the function of blood coagulation factor VIII. The nucleic acid is considered, encoding the antibody of the invention, a vector, a cell and a method of producing the antibody, and also a pharmaceutical composition and a kit for use in the method of preventing and/or treating bleeding or diseases associated with or caused by bleeding.

EFFECT: invention may find further application in the treatment of diseases associated with impaired blood clotting.

16 cl, 2 ex, 6 dwg

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