Human antibodies to receptor of epidermal growth factor

FIELD: medicine.

SUBSTANCE: there is claimed isolated human antibody or its fragment, which binds to human EGFR. Antibody contains corresponding CDR areas of light and heavy chain. Its conjugate with anti-neoplastic means or marker is described. Also described are: coding nucleic acid, expression vector, recombinant cell-host for obtaining antibodies and method of inhibiting growth of tumor, expressing EGFR on the basis of antibody.

EFFECT: application of invention provides antibodies with affinity comparable or higher, than in IMC-C225, which neutralises EGFR activation, what can be applied in medicine for treatment of tumours.

36 cl, 14 dwg, 6 tbl, 13 ex

 

The SCOPE of the INVENTION

The invention relates to monoclonal antibodies that are specific to the receptor for epidermal growth factor (epidermal growth factor receptor, EGFR). These antibodies can be used, inter alia, in the treatment of neoplastic hyperproliferative diseases and disorders.

The LEVEL of TECHNOLOGY

While normal cells multiply by carefully controlled activation of tyrosine kinase receptor growth factor (receptor tyrosine kinases, RTKs) and their respective ligands, cancer cells also multiply by activating receptors growth factor, but with the loss of a reliable control of normal reproduction. Loss of control can be caused by many reasons, such as overexpression of growth factors and/or receptors, and Autonomous activation of biochemical pathways regulated by growth factors. Some examples of RTKs involved in oncogenesis, are the receptors for epidermal growth factor (EGFR), platelet-derived growth factor (platelet-derived growth factor, DERIVED), insulin-like growth factor (insulin-like growth factor, IGFR), nerve growth factor (nerve growth factor, NGFR) and fibroblast growth factor (fibroblast growth factor, FGF). The binding of these growth factors to their receptors on the cell surface leads to the activation of the receptor that initiates and modifies the transmission path of the signal and leads to ketocholesterol and differentiation.

Members of the group of receptors for epidermal growth factor (epidermal growth factor, EGF) are especially important tyrosine kinase receptors growth factor associated with the oncogenesis of epidermal cells. The first discovered member of the group of EGF receptor EGFR was expressed on many types of tumor cells. It was found that EGFR is involved in regulation of growth and division of cancer cells, repair and survival, angiogenesis, invasion and metastasis of tumors.

EGFR is a membrane glycoprotein with a molecular weight of 170 kD, with extracellular binding ligand domain, a transmembrane portion, and a cytoplasmic protein tyrosine kinase domain. Examples of ligands that stimulate EGFR include epidermal growth factor (EGF), transforming growth factor-a(transforming growth factor-a, TGF-a), heparinase growth factor (heparin-binding growth factor, HBGF), betacellulin and Cripto - 1 factor. Binding of specific ligands leads to autophosphorylation EGFR receptor, activating the cytoplasmic tyrosine kinase domain and initiation of multiple signal transduction pathways that regulate survival and tumor growth. EGFR also affects production in the tumor some other angiogenic factors, such KaKVEGF factor and basic fibroblastic growth factor (basic fibroblastic growth factor, bFGF).

It is believed that the growth factors that activate GFR, participate in tumor angiogenesis. Angiogenesis, indicating the formation of capillaries from existing blood vessels in embryonic and adult organisms, known as a key element in the process of tumor growth, survival and metastasian. It was reported that stimulation of tumor cells through EGFR leads to increased expression of angiogenic factors: vascular endothelial growth factor (VEGF), interleukin-8 (IL-8) and the base fibroblast growth factor (bFGF), which may cause activation of vascular endothelial cells associated with the tumor. Stimulation of vascular endothelial cells associated with the tumor, is also possible via the activation of their own EGF receptor factors produced by the tumor, such as TGF-α and EGF.

It was reported that many human tumors Express or sverkhekspressiya EGFR. The expression of EGFR correlates with poor prognosis, decreased survival and/or increased metastasis. Because of its involvement in oncogenesis, EGFR is a special object of anticancer therapy. These therapies mainly include or monoclonal antibodies that block the binding of ligand to the extracellular domain of the receptor, or a synthetic tyrosine kinase inhibitor, acting directly on the intracellular part and predotvraschauschii signal transmission.

For example, Mab Cetuximab (ERBITUX®) is a recombinant human-mouse hybrid monoclonal antibody that specifically binds the extracellular domain of human EGFR. Cetuximab is an EGFR antagonist that blocks the binding of ligand to EGFR, preventing activation of the receptor and inhibiting the growth of tumor cells expressing EGFR. Cetuximab has proven itself to use in conjunction with irinotecan, or in his absence, in the treatment of patients with metastatic colorectal cancer, accompanied by the production of receptors for epidermal growth factor, which was immune to irinotecanbased chemotherapy, or to which it could not be applied. Cetuximab is also effective in the treatment of psoriasis.

Description of the INVENTION

This invention is a monoclonal antibody or fragments thereof, specific for EGFR, preferably to the extracellular part of EGFR containing from one to six sections that define complementarity (complementarity determining regions, CDRs)selected from the following group: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14. Antibodies are preferably human. Even more preferably, antibodies of the present invention or their fragments contain SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6. Alternatively, but also preferably, the antibodies of this from the retene or their fragments contain SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14. The preferred way antibodies of the present invention or their fragments contain the variable region of the heavy chain SEQ ID NO:8 and/or the variable region of the light chain of SEQ ID NO:16. Such antibodies or fragments thereof of this invention have a variety of properties, including the ability to neutralize EGFR and prevent binding of the EGFR ligand to its receptor.

Also, this invention is selected polynucleotide encoding described antibodies or fragments thereof, and expression vectors containing these chains polynucleotides made as functioning with expressed sequence. Also in this invention presents a recombinant cell host containing an expression vector or its products, where expressed above antibodies or their products. Also presents methods for producing antibodies or their fragments in the culture of these cells under conditions enabling expression of antibodies or their fragments. Antibodies or fragments thereof can be purified from cells or cell environment.

Also, this invention is methods of influence on the growth of tumors in mammals, including the introduction of a mammal effective amounts of the described antibodies. Described antibodies can be combined with antibodies that bind other RTKs. These methods is s may also include the introduction of a mammal an anti-neoplastic funds or processing, including, for example, chemotherapeutic drugs and/or irradiation. In some cases, tumor growth is inhibited. A preferred way, the treatment leads to regression of the tumor.

This invention also presents methods of treatment in mammals of hyperproliferative diseases of non-oncological nature, such as psoriasis, including the introduction of a mammal effective amounts of the described antibodies.

BRIEF DESCRIPTION of DRAWINGS

On figa and 1B shows the expression vectors for cloning of immunoglobulin genes pDEC and pEE12.1L. On figs depicts the final vector plasmid pGS-11F8, containing one fully human anti-EGFR antibody.

Figure 2 shows the profile of splitting restrictase pGS-11F8. Label DNA size marked on the DNA marker in kilobase.

Figure 3 depicts the in vitro binding IMC-C11F8 and IMC-C225 with EGFR as measured by ELISA.

Figure 4 shows the results of in vitro compete IMC-11F8 and IMC-C225 labeled with125I EGF with EGFR binding.

Figure 5 demonstrates the effect of IMC-11F8 and IMC-C225 on EGFR phosphorylation in cells Whrs. Was used a control antibody IMC-1C11.

6 shows the inhibition of phosphorylation of EGFR using IMC-11F8 and IMC-C225 in A431 cells.

7 shows Western Blot analysis of EGFR phosphorylation in the presence of unstimulated control cells (Doro is ka 1), EGF (lane 2), IMC-C225 (lane 3), IMC-11F8 (lane 4), and a control antibody (lane 5). On figa using anti-phosphotyrosine antibodies shown phosphorylated EGFR and figv shows the total EGFR in stimulated cells.

On Fig shows inhibition of EGF-stimulated EGFR phosphorylation various concentrations of IMC-11F8. Figa depicts Western Blot analysis of anti-phosphotyrosine EGFR antibodies in unstimulated control cells (lane 1), stimulated cells treated with no antibody IMC-11F8 (lane 2), 15 µg/ml (lane 3), 3 µg/ml (lane 4) and 0.6 μg/ml (lane 3) IMC-11F8. On FIGU shows the total EGFR.

Fig.9 shows the inhibition of proliferation DiFi cells using IMC-11F8, IMC-C225 and control antibody and IMC-S assessment MTT method.

Figure 10 shows specific lysis51Cr-labeled DiFi cells treated with IMC-11F8 or IMC-C225 (ERBITUX™).

11 shows the growth of A431 tumor cells in mice, treated with IMC-11F8 or IMC-C225 (Cetuximab). For control of tumor growth was used untreated animals.

Fig shows the growth of tumor cells Whrs in mice, treated with IMC-11F8 or IMC-C225 (Cetuximab). For control of tumor growth was used untreated animals.

On Fig shows immunohistochemical staining xenotransplantion of naked mouse cells che is bacescu tumor, treated with saline or IMC-11F8. Sections a and b depict the A431 xenograft of naked mice treated with Salina (A) or IMC-11F8 (IN). Sections C and D depict the xenograft Whrs of naked mice treated with Salina (s) or IMC-11F8 (D). Sections E and F depict Ki-67 staining of xenograft Whrs of naked mice treated with Salina (E) or IMC-11F8 (F).

Fig demonstrates inhibition xenotransplantion in Nude mouse human colorectal carcinoma using IMC-11F8 in combination with CPT-11. Naked mice bearing xenografts of human colorectal tumor GEO (graph A), DLD-1 (graph B) or HT-29 (graph) were processed by intraperitoneally injection Salina or IMC-11F8 twice weekly 0.3 mg or 1.0 mg per injection, in pure form or in mixture with CPT-11 in the amount of 100 mg/kg once a week. Tumor size was measured twice a week. Data represent mean ±SE of measurements tumors in ten animals from each group. Graph D represents the regression of the tumor as a result of treatment with IMC-11F8 in pure form or in mixture with CPT-11. Each group consists of 10 infected tumor animals.

DETAILED description of the INVENTION

This invention is monoclonal antibodies and fragments thereof that are specific for EGFR, as well as dedicated or ocimene the polynucleotide sequence, encoding the antibody. Antibodies according to this invention are preferably human, and can be used for the treatment of neoplastic diseases, including solid and not solid tumor, as well as for treatment of hyperproliferative disorders.

Natural antibodies consist of two identical heavy chains and two identical light chains, each light chain is covalently attached to a heavy chain by using an intermediate disulfide bond, with multiple disulfide bonds connect, in turn, two heavy chains with one another. Individual circuits can be formed in domains characterized by similar size (110-125 amino acids) and structures, but with different functions. Light chain may contain one variable domain (VLand/or one constant domain (CL). Heavy chain can also contain one variable domain (VHand/or, depending on the class or isotype of antibody, three or four constant domains (CH1, CH2, CH3 and CH4). People are isotypes IgA, IgD, IgE, IgG and IgM, and IgA and IgG are further divided into subclasses or subtypes (lgA1-2and lgG1-4).

In General, the variable domains show considerable diversity in amino acid sequence from one antibody to another, especially in the area of the tee where the site of binding antigens. In each of the VLand VHthere are three zones, called hypervariable sites or sites that define complementarity (complementarity-determining regions, CDRs), while they are accompanied by a less variable regions, which are called frame variable regions (variable framework regions).

Part of the antibody consisting of domains VLand VHdenoted by Fv (fragment variable) and is the region of binding antigens. Fv, single chain (single chain Fv, scFv) is an antibody fragment containing the VLdomain and VHdomain on the same polypeptide chain, while N is the end of one domain and the other end connected domain movable linker (see, for example, U.S. patent No. 4 946 778 (Ladner et al.); WO 88/09344 (Huston et al.). WO 92/01047 (McCafferty et al.) describes the display of scFv fragments on the surface of soluble recombinant genetic imaging methods, such as bacteriophages.

Peptide linkers used to obtain single-chain antibodies, can be flexible peptides selected in such a way as to ensure the correct three-dimensional folding of the VLand VHdomains. The linker consists, in General, from 10 to 50 amino acid residues. Preferably, the linker consists of 10-30 amino acid residues. Even more preferred linker consisting of 12 to 30 amino acid residues. The most preferred one is by the linker, consisting of 15-25 amino acid residues. An example of such a peptide linker comprises (Gly-Gly-Gly-Gly-Ser)3(SEQ ID NO:19).

The single-chain antibodies lacking some or all of the constant domains of the whole antibody from which they originate. Therefore, they are able to circumvent some of the problems associated with the use of whole antibodies. For example, single-chain antibodies generally free from some undesirable interactions between the constant parts of the heavy chains with other biological objects. In addition, single-chain antibodies have significantly smaller than whole antibodies, and may have a greater ability to permeability than whole antibodies, which allows single-chain antibodies to detect and join the target antigen-binding regions more effectively. Moreover, the relatively small size of single-chain antibodies make less likely the occurrence of unwanted immune reaction in the recipient, compared to whole antibodies.

Several single-chain antibodies, each single chain which has one of the VHand one of the VLthe domain covalently linked to the first peptide linker can be covalently linked using one or more peptide linkers with the formation of multivalent single-chain antibodies, the cat is who can be monospecificity or multispecificity. Each circuit multivalent single-chain antibody contains a fragment of the variable region of the light chain and a fragment of the variable region of the heavy chain, and using the peptide linker is associated with at least one other circuit. The peptide linker comprises at least fifteen amino acid residues. The maximum number of amino acid residues is about one hundred.

Two single-chain antibodies may be combined with the formation of dimeric antibodies, also known as a bivalent dimer. Dimeric antibodies consist of two chains and two connecting areas and can be monospecificity or bispecific. Each chain dimeric antibody contains VHthe domain connected with VLdomain. The domains are connected via linkers, which are short enough to prevent mating between domains of the same chain, and thus, the pairing of complementary domains on different circuits, creating two antigen-binding region.

Three single-chain antibodies may be combined with the formation of the trimeric antibodies, also known as trivalent trimers. The trimeric antibodies constructed in such a way that the amino acid end of the VHand VLdomains directly linked to the carboxyl end of the VHand VLdomains, i.e. without in the sparkling linker sequences. The trimeric antibody has three Fv fragments with polypeptides arranged in a cyclical manner, "head to tail". A possible conformation of trimeric antibody is a planar conformation, in which three areas of binding are located in the same plane at angles of 120° to one another. The trimeric antibodies can be monospecificity, bespecifically and trapezitinae.

The term Fab (Fragment, antigen binding) refers to fragments of antibodies consisting of VHVLVHand CH1 domain. Those that are formed by splitting papain, simply Fab and do not contain the hinged section in the heavy chain. The splitting of pepsin, a lot of different Fab fragments containing the hinge area in the heavy chain. Those divalent fragments, which contain megapode disulfide bond, denoted F(ab')2whereas monovalent Fab' are formed in the case, if disulfide bonds are not preserved. F(ab')2the fragments have a greater avidity for antigen than the monovalent Fab fragments.

Fc (Fragment crystallization) is the designation for the portion or fragment of an antibody, which contains paired constant domains of the heavy chain. For example, IgG, Fc containsH2and CH3the domains. Fc antibodies IgA and IgM additionally containsH4domain. Fc has to Fc cocktail recipes. what specific binding, by the activation of complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity (antibody-dependent cellular-cytoxicity, ADCC). For such antibodies of IgA and IgM, which are complexes of several IgG-like protein, to complex formation requires constant Fc domains.

Finally, the hinge region separates the Fab and Fc portions of antibodies, providing mobility Fab-domains relative to each other and relative to the Fc, as well as with multiple disulfide bonds to covalent bonds linking the two heavy chains.

Thus, the antibodies according to this invention include, but are not limited to, the following: natural antibodies, bivalent fragments such as (Fab')2, monovalent fragments such as Fab, single-chain antibodies, single-chain Fv (scFv), single domain antibodies, multivalent single-chain antibodies, dimeric antibodies of the trimeric antibodies, and the like that have been specifically bind to the antigens.

Antibodies and their fragments according to this invention are specific for EGFR. Under the specificity of the antibody refers to specific recognition antibody to a specific epitope of the antigen. Antibodies and their fragments according to this invention can be, for example, monospecificity or bispecific. Bispecific antibodies (BsAbs) are antibodies that have DV is two different antigen - binding specificnosti or areas. In the case where the antibody has more than one specificity, recognizable epitopes can relate to one antigen or more than one antigen. Thus, this invention is bispecific antibodies or fragments thereof that bind to two different antigens, and at least one specificity for EGFR.

The specificity of these antibodies or their fragments to EGFR can be determined based on affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with the antibody (Krf), measures the strength of binding between an antigenic determinant and area of binding antigen. The avidity is a measure of the strength of binding of an antibody to its antigen. The avidity due to an affinity between the epitope and the binding region of the antigen to the antibody, and the valency of the antibody, which is defined as the number of regions of the antigen binding to a specific epitope. Antibodies usually bind with a dissociation constant (Kd) of the order of 10-5up to 10-11l/mol. Any less than 10-4l/mol, generally denoting the nonspecific binding. The smaller the value of Kdthe more strength of binding between an antigenic determinant and area of binding antigen.

As the decrees of the W "antibody" and "antibody fragments" comprise modifications which retain the specificity of EGF receptor. Such modifications include, but are not ogranichivayutsya this, the connection with the effector molecule such as a chemotherapeutic agent (such as cisplatin, Taxol, doxorubicin) or a cytotoxin (e.g., protein or non-protein organic chemotherapeutic agent). Antibodies may be modified by conjugation with detectivesyme reporter molecules. Also included are antibodies with changes affecting non-svyazyvaniye characteristics such as half-life (e.g., tahilramani).

Protein and non-protein funds can be combined with antibodies using methods known in this field. Connection methods include direct connection, connection via covalently attached linkers and specific United pair members (e.g., avidin-Biotin). Such methods include, for example, described in Greenfield et al., Cancer Research 50, 6600-6607 (1990) for attachment of doxorubicin and described in Arnon et al., Adv. Exp.Med. Biol. 303, 79-90 (1991) and in Kiseleve et al., Mol. Biol. (USSR)25, 508 to 514 (1991) for attaching the platinum compounds.

Equivalents of antibodies or their fragments according to this invention also include polypeptides with amino acid sequences in large is largely equivalent to the amino acid sequences of the variable or hypervariable sites full of anti-EGFR antibodies included in this invention. Under substantially equivalent amino acid sequence " means a sequence with at least 70%, preferably at least 80%, and more preferably at least 90%homology, by definition, search FASTA method in accordance with Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85, 2444-8 (1988)), including sequences that are at least 70%, preferably at least 80%, more preferably at least 90% identical.

Such antibodies will have the same or similar binding ligand blocking and receptor-neutralizing activity against the antibodies of the present invention, including SEQ ID NO:8 and 16, especially in the case of conservative substitutions of amino acids. Conservative substitution of amino acids is defined as the change in amino acid composition by replacing one or more amino acids in the peptide, polypeptide or protein, or fragments. It can be replaced by amino acids with similar overall characteristics (e.g., acidity, basicity, aromaticity, size, positive or negative charge, polarity, Polarnet) so that these changes do not cause significant changes of the respective peptide, polypeptide or protein characteristics (e.g., charge, isoelectric the political point, the affinity, avidity, conformation, solubility or activity. Typical conservative substitutions are selected from the groups of amino acids, which include, but are not limited to, the following:

(1) hydrophobic: methionine (M), alanine (A), valine (V), leucine (L), isoleucine (I);

(2) hydrophilic: cysteine (C), serine (S), threonine (T), aspargine (N), glutamine (Q);

(3) acidic: spartanova acid (D), glutamic acid (E);

(4) alkaline: histidine (H), lysine (K), arginine (R);

(5) aromatic: phenylalanine (F), tyrosine (Y) and tryptophan (W);

(6) residues that influence chain orientation: gly, pro.

Antibodies according to this invention further include such that their binding characteristics have been improved by direct mutation, methods of development of affinity phage display or mixing circuit. The affinity and specificity can be modified or improved by mutation sites CDR and selecting regions of binding antigens with desired characteristics (see, for example, Yang et al., J. Mol. Biol., 254: 392-403 (1995)). CDR mutate in different ways. One way is to randomize individual residues or combinations of residues so that the population is initially identical to the regions of the antigen binding all twenty amino acids are found in certain places. Otherwise, mutations are introduced in the set of CDR OST Dow method of error-prone PCR (see, for example, Hawkins et. al., J. Mol. Biol., 226: 889-896 (1992)). For example, vectors phage display containing the genes for the variable regions of the heavy and light chains, can proliferate in mutant strains of E. coli (see, e.g., Low et al., J. Mol. Biol., 250: 359-368 (1996)). These methods mutagenesis illustrate only some of those methods, which are known to experts in this field.

Each domain antibody according to this invention can be complete domain immunoglobulin (e.g., variable or constant domain of the heavy or light chain), or may be a functional equivalent or a mutant or derivative of the natural domain, or a synthetic domain, generated, for example, in vitro using a method, such as what is described in WO 93/11236 (Griffiths et al.). For example, it is possible to connect together the domains corresponding to the variable domains of antibodies that are missing at least one amino acid. An important characteristic feature of the antibodies is the existence of binding antigen. The terms of the fragment of the variable region of the heavy chain and a fragment of the variable region of the light chain should not be so construed as to exclude options that do not have a significant impact on specificity.

Antibodies according to this invention or fragments thereof is a human antibody having one, two, three, four LMA and/or six areas, determining complementarity (CDR)selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14.

Preferably, antibodies (or fragments thereof) according to this invention have lots CDR from the group of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6. Alternatively, and preferably, these antibodies or fragments thereof, have lots CDR from the group of SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14. Amino acid sequence plots CDR listed in Table 1.

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TABLE 1
Heavy chain
CDR1SGDYYWSSEQIDN0:2
CDR2YIYYSGSTDYNPSLKSSEQIDN0:4
CDR3VSIFGVGTFDYSEQIDN0:6
Light chain
CDR1RASQSVSSYLASEQIDN0:10
CDR2DASNRATSEQIDN0:12
CDR3HQYGSTPLTSEQIDN0:14

In another embodiment of the invention, these antibodies or fragments thereof can have a variable region heavy chain SEQ ID N0:8 and/or variable region of the light chain of SEQ ID N0:16. IMC-11F8 is particularly preferred antibody according to this invention. This antibody has a human VHand VLareas of the structure (framework regions, FWs), as well as areas CDR. Variable domain VHIMC-11F8 (SEQ ID N0:8) has three parts CDR (SEQ ID NO:2, 4 and 6) and four FW and VLdomain (SEQ ID NO:16) has three parts CDR (SEQ ID NO:10, 12 and 14) and four FW.

Preferably, antibodies or fragments thereof according to this invention neutralize EGFR. The binding of the ligand, for example, EGF or TGF-a, to the outside, the extracellular domain of EGFR stimulates dimerization of the receptor, autophosphorylation EGFR, activation of the internal receptor cytoplasmic tyrosine-kinases the domain and the initiation of multiple signal transduction pathways and development, involved in the regulation of DNA synthesis (gene activation), as well as the development of cell cycle or cell division. Also preferably, the anti-EGFR antibodies (or fragments thereof) of the present invention are specific for the extracellular region of EGFR. These antibodies or fragments thereof additionally, preferably, prevent binding of the EGFR ligand with its receptor. In this embodiment of the invention, the antibodies according to this invention, or fragments thereof, bind EGFR at least as much as the natural EGFR ligands (EGF and TFG-α).

Neutralization includes EGFR inhibition, diminution, inactivation and/or terminate one or more activities associated with signal transmission. Thus, neutralization of EGFR results in many effects, including inhibition, diminution, inactivation and/or interruption of growth (proliferation and differentiation), angiogenesis (recovery, invasion and metastasis of blood vessels), as well as cellular activity and metastasis (cell adhesion and invasiveness).

One of the measures by neutralizing EGFR is the inhibition of tyrosine kinase activity of the receptor. Tyrosine kinase inhibition can be determined using well-known methods, for example, by measuring the level of autophosphorylation recombinant kinase prescriptions is ora, and/or phosphorylation of natural or synthetic substrates. Thus, analyses of phosphorylation are useful in the determination of neutralizing antibodies in the context of the present invention. Phosphorylation can be detected, for example, by using antibodies specific to phosphotyrosine, ELISA or Western Blot analysis. Some of the tests for the tyrosine kinase activity described in Panek et al., I. Pharmacol. Exp.Thera. 283:1433-44 (1997) and Batley et al., life Sci.62: 143-50 (1998).

In addition, methods of detecting expression of the protein can be used to determine EGFR-neutralizing, if these proteins or protein activity, or activated state, regulated tyrosine kinase activity of EGFR. These methods include immunohistochemistry (IHC) to determine protein expression, fluorescent in situ hybridization (FISH) to determine gene amplification, competitive radioligand binding analysis techniques blots on a solid support, such as Northern blot and southern blot (Northern and Southern blots), polymerase chain reaction with reverse transcription (reverse transcriptase polymerase chain reaction, RT-PCR) and ELISA. Look, for example, Grandis et al., Cancer, 78:1284-92 (1996); Shimizu et al., Japan J. Cancer Res., 85:567-71 (1994); Sauteretal., Am. J. Path., 148:1047-53 (1996); Collins, Glia, 15:289-96(1995); Radinsky et al., Clin. Cancer Res., 1:19-31 (1995); Petrides et al., Cancer Res., 50:3934-39 (1990); Hofmiann et al., Anticancer Res., 17:4419-26 (1997); Wikstrand et al., Cancer Res., 55:3140-48 (1995).

In vivo assays can also be used to determine the neutralizing EGFR. For example, tyrosine kinase inhibition of the receptor can be observed using mitogenic analysis using cells stimulated receptor ligand in the presence and absence of inhibitor. For example, A431 cells (American Type Culture Collection (ATSS), Rockville, MD), stimulated by EGF, can be used for analysis of EGFR inhibition. Another method includes examining, on inhibition of growth of expressing EGFR tumor cells, using, for example, human tumor cells embedded in mice. See, for example, U.S. patent No. 6 365 157 (Rockwell et al.).

This invention is not limited to any particular mechanism of neutralization of EGFR. Anti-EGFR antibodies according to this invention can outer contact surface receptor cells EGF, block ligand binding (e.g., EGF or TGF-a) and the corresponding signal transmission is taking place with the participation of related receptor tyrosine kinases, as well as to inhibit the phosphorylation of EGFR and other next protein in the cascade signal. Receptore-or antibody-based test complex can also be absorbed and destroyed, which leads to a weakening of the activity of the receptor on the cell surface. The activity of matrix metalloproteinases, which are involved in invasion and metastasis of puhalovich cells, can also be weakened by the antibodies according to this invention. Moreover, antibodies to Danna invention can contribute to the inhibition of the production of the growth factor and angiogenesis.

Antibody fragments can be obtained by dividing the whole antibody or by expression of DNA that encodes a fragment. Possible methods of obtaining fragments of the antibodies described in Lamoyi et al., J. Immunol. Methods, 56: 235-243 (1983) and Parham, I. Immunol. 131:2895-2902 (1983). Such fragments may contain one or both of the Fab fragment of the F(ab')2the fragment. Such fragments may also contain single-stranded fragments of variable regions of antibodies, such as scFv, a dimer antibodies or other antibody fragments. Methods of producing such functional equivalents are disclosed in PCT application WO 93/21319, European patent application No. EP 239400; PCT application WO 89/09622; European patent application EP 338745 and European patent application EP 332424.

Preferred cells-hosts for transformation of vectors and expression of receptor antagonists are mammalian cells, such as cells COS-7 cells Chinese hamster ovary (Chinese hamster ovary, Cho), and colonies of cells of lymphoid origin, such as cell lymphoma, myeloma (e.g., NSO) or hybridoma. Alternatively, it may be other eukaryotic recipients, such as yeast.

If want what Ino to Express the gene construct in yeast, a suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7. Stinchcomb et al. Nature, 282: 39 (1979); Kingsman et al., Gene, 7:141 (1979). Trpl gene provides a selection marker for mutaciones yeast strain unable to grow in tryptophan, for example, ATS No. 44076 or RER-1. Jones, Genetics, 85:12 (1977). Damage trpl in the genome of the yeast host cell, thus, provides a convenient environment to determine a transform with growth in the absence of tryptophan. Similarly, yeast strains with the lack of Leu2 (ATS 20, 622 or 38, 626) are complemented by known plasmids bearing Leu2 gene.

Transformed cell hosts are cultivated using methods known in this field, in a liquid medium containing assimilated sources of carbon (hydrocarbons, such as glucose or lactose), nitrogen (amino acids, peptides, proteins or products of their degradation, such as gelatin, ammonium salt and the like), and inorganic salts (sulfates, phosphates and/or carbonates of sodium, potassium, magnesium and calcium). The environment also contains, for example, substances that promote growth, such as micronutrients, such as iron, zinc, manganese and the like).

As described in the examples below, highly specific anti-EGFR antibodies, in accordance with this invention, can be isolated from the phage library is on display, composed of genes of variable regions of human light and heavy chains. For example, the variable domain according to this invention can be obtained from lymphocyte peripheral blood, which contains the rearranged variable gene region. Alternatively, part of the variable domain, such as CDR and FW plots can be obtained from various human sequences. More than 90% of the obtained clones after three repetitions of selection specific for EGFR. The affinity for binding to EGFR for the studied Fab is in the nanomolar range, which is the same high value as for several bivalent anti-EGFR monoclonal antibodies generated by hybridoma technology.

Antibodies and antibody fragments according to this invention can be obtained, for example, from natural antibodies, or libraries of phage display Fab or scFv. Whereas, that for a single-domain antibodies from antibodies containing VHand VLdomains can be desirable, certain substitutions of amino acids outside areas CDR, to improve binding, espressi or solubility. For example, it may be desirable to modify amino acid residues, which otherwise would be hidden at VH-VLinteraction.

Additionally, antibodies and antibody fragments Dunn is th invention can be obtained by a standard hybrid technology (Harlow & Lane, ed., Antibodies: A Laboratory Manual, Cold Spring Harbor, 211-213 (1998), included by reference) using transgenic mice (e.g., mouse KM from Medarex, San Jose, Calif.), which produce gamma heavy and Kappa light chain of human immunoglobulin. The preferred way, a significant portion of the human genome that encodes the antibody is introduced into the genome of the mouse and compensates for the lack of production of endogenous mouse antibodies. Such mice can be subcutaneously injected (subcutaneously, s.c.) immunized using KDR (VEGFR-2) in complete Freund's adjuvant.

Protein is used to identify EGFR-associated antibodies according to this invention, is primarily EGFR and, more predominantly, the extracellular domain of EGFR. The extracellular domain of EGFR can be free or bound to another molecule.

The invention also provides isolated polynucleotide encoding antibodies or fragments thereof, as described earlier. The invention includes a nucleic acid having a sequence encoding one, two, three, four, five or all six plots CDR. Table 2 shows the nucleic acid sequences.

DNA encoding a human antibody can be obtained by recombination of DNA that encodes the constant and variable regions of a person other than teaching Dow CDR, what is happening is essentially or exclusively from the corresponding human antibody and DNA encoding the areas CDR derived from human (SEQ ID NO:1, 3, and 5 for plots CDR variable domain of the heavy chain and SEQ ID NO:9, 11 and 13 for areas CDR variable domain of the light chain).

Suitable sources of DNA that encode fragments of antibodies include any cell, such as hybridoma and spleen cells, which Express full-length antibodies. Fragments can be used by themselves, as equivalents of the antibodies, or can be recombined equivalents, as described above. Deletion and recombination of DNA, as described in this section, can be produced by known methods such as described in the above publications, taking into account the equivalence of antibodies, and/or other standard techniques of DNA recombination, such as described below. Another source of DNA can serve as a single-chain antibodies derived from phage libraries displays known in the field of ways.

In addition, this invention represents the expression vector containing the above-described nucleotide sequences that are functionally related expressed sequence, promoter and enhancer sequence. Have developed a variety of expression vector is for the efficient synthesis of polypeptides antibodies in prokaryotes, for example, the bacterial and eukaryotic systems, including, but not limited to, yeast and systems mammalian cell cultures. The vectors of this invention can contain segments of chromosomal, non-chromosomal and synthetic DNA sequences.

Can be used any suitable expression vector. For example, prokaryotic cloning vectors, including plasmids from E. coli, such as co1EI, pCRI, pBR322, RMV, pUC, pKSM, and RP4. Prokaryotic vectors include derivatives of DNA phages, such as M13 and other DNA single-stranded phage filament. An example of a suitable yeast vector is a plasmid 2µ. Suitable vectors for expression in mammalian cells include the well-known derivatives of SV40, adenovirus, a DNA sequence derived from retroviruses, and Shuttle vectors derived from combinations of functional vectors mammals, such as described above, as well as functional plasmids and DNA phages.

In this area there are other eukaryotic expression vectors (e.g., P.J. Southern and P. Berg, J. Mol. Appl. Genet, 1, 327-341 (1982); Subramani et al., Mol. Cell. Biol., 1: 854-864 (1981); Kaufmann and Sharp, "Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene," J. Mol. Biol. 159, 601-621 (1982); Kaufmann and Sharp, Mol. Cell. Biol. 159, 601-664 (1982); Scahill et al., "Expression And Characterization of the Product of a Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells," Pro. Nat'1 Acad. Sci. USA 80, 4654-659 (1983); Urlaub and Chasin, Proc. Nat'1 Acad. Sci. USA, 77, 4216-4220 (1980)a.

The expression vectors according to this invention contain at least one sequence control expression, which is functionally connected with the expressed DNA sequence or fragment. The control sequence is introduced into a vector for the control and regulation of expression of the cloned DNA sequence. Examples of suitable sequences for controlling expression are the lac system, the trp system, the tac system, the trc system, major operator and promoter sites of the lambda phage, a control plot of the protein shell fd, the glycolytic promoters of yeast, e.g., the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid fosfotazy, e.g., Pho5, the promoters drogowych alpha-factor mating, and promoters derived from the polynomials, adenovirus, retrovirus and monkey virus, for example, the early and late promoters or SV40, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses, or combinations thereof.

This invention is also a recombinant cell host containing the above-described expression vectors. Antibodies according to this invention can be expressed in cell lines other than a hybrid. Nukleinovokisly, containing sequences encoding the polypeptides according to the invention can be used to transform suitable host cells of mammals.

Cell lines of particular interest, are selected on the basis of a high level of expression, constitutive expression of interest protein and minimal contamination with proteins of the host. Lines of mammalian cells, available as hosts for expression are well known in this area and include many immortalized cell line, such as cells of the ovary of the Chinese hamster (Chinese Hamster Ovary, Cho), kidney cells, baby hamster (Baby Hamster Kidney, KSS) and many others. Additional suitable eukaryotic cells include yeast and other fungi. Suitable prokaryotic hosts include, for example, E. coli, such as E. coli SG-936, E. coli HB 101, E. coli W3110, E. coli XI776, E. coli H, E. coli DHI, and E. coli MRC1, Pseudomonas, Bacillus, such as Bacillus subtilis and Streptomyces.

Presents the recombinant cell host may be used to produce antibodies or fragments by culturing these cells under conditions that ensure the expression of the antibody or its fragments, and purification of the antibody or its fragments from the host cell or medium surrounding the cell host. Targeting expressed antibody or fragment secretion in R is combinant cells hosts can facilitate the introduction of a signal or secretory sequence, encodes a leader peptide (see Shokri et al., Appl Environ Biotechnol. 60(6):654-64 (2003), Nielsen et al., Prot. Eng. 10:1-6 (1997) and von Heinje et al., Nucl. Acids Res. 14:4683-690 (1986)) at the 5'end of the gene of interest that encodes the antibody. These elements secretory leader peptide can be derived from both prokaryotic and eukaryotic sequences. Accordingly appropriately used secretory leader peptides, which are amino acids attached to the N-terminal end of the polypeptide, in order to guide the movement of the polypeptide outside cytosole host cell and secrete into the environment.

Antibodies according to this invention can be fused to additional amino acid residues. This amino acid residue may be a peptide tag, it is possible to facilitate the selection. We also look at other amino acid residues to the direction of antibodies to specific organs and tissues.

In another embodiment of the invention, the antibody according to this invention is produced by expression of nucleic acid encoding the antibody in a transgenic animal, such that the antibody is expressed and can be extracted. For example, the antibody can be expressed Dane-specific way that promotes the recovery and purification. In one of the embodiments of the invention, the antibody in question is in the invention is expressed in the mammary gland for secretion during lactation. Examples of transgenic animals include, but are not limited to, mice, goats and rabbits.

This invention is also a method for treatment of tumors in mammals by introduction of a mammal an effective amount of the above-described antibodies. Suitable for the treatment of tumors, in accordance with this invention are preferably those that Express EGFR. Despite the fact that diseases and conditions that can be treated or prevented presents methods do not mean to be bound to any particular mechanism, these include, for example, those in which tumor growth or pathogenic angiogenesis is stimulated by paracrine and/or autocrine loop of EGFR. This means that tumors expressing EGFR, characteristic sensitive to the presence of EGFR in their environment and future products can be stimulated by EGF and/or TGF-ct autocrine stimulyatorom cycle. The treatment of such tumors in accordance with this invention includes the complete or partial inhibition of tumor growth. It is noteworthy that in certain embodiments, inhibition includes advanced regression of tumors.

Expression of EGFR was observed in many human tumors both in vitro and in vivo, and the levels of expression of EGFR vary widely depending on the type of tumor. EGFR exp is esteroide at different levels on the cell surface of a significant part of human tumors, such as colorectal, head and neck (epidermoid), pancreatic, lung, tumour of the breast and renal carcinoma, and glioblastoma. For some types of tumors, the expression of EGFR is very common (for example, from 35% to 70% of cases of ovarian cancer and approximately from 25% to 77% of cases of colorectal cancer). High levels of EGFR expression can be achieved in correlation with the production of receptor ligands (e.g., EGF and TGF-α). Expression of EGFR is also corallium with increased resistance to certain chemotherapeutic drugs and radiotherapy. The expression of EGFR can also serve as a predictive factor for certain types of tumors, because it is associated with reduced survival and poor prognosis and/or increased risk of metastasis. In addition, increased expression of EGFR is present in many types of tumors.

The tumor to be treated, include primary and metastatic tumors and refractory tumors. Refractory tumors include tumors that do not respond or are resistant to treatment separately chemotherapeutics, separately antibodies, separately radiation techniques, or combinations thereof. To intractable tumors are those that inhibited the processing of ways, but receiver who are within five years, sometimes for ten or more years after stopping treatment.

Tumors that can be treated with antibodies of this invention include those that revascularization, or insignificant vascularized and vascularized tumors. Examples of solid tumors that may be susceptible to treatment include carcinomas of the breast, lung carcinoma, colorectal carcinoma, carcinoma, pancreatic cancer, glioma and lymphoma. Some examples of such tumors include epidermoid tumors, squamous tumors, such as head and neck tumors, colorectal tumors, prostate tumors, breast tumors, lung tumors, including small cell and non-small cell lung tumors, pancreatic tumors, thyroid tumors, ovarian tumors, and liver tumors. Other examples include Kaposi's sarcoma, neoplasm CNS neuroblastoma, capillary hemangioblastomas, meningiomas and cerebral metastases, melanoma, gastrointestinal and renal carcinomas and sarcomas, rhabdomyosarcoma, glioblastoma, preferably polymorphic glioblastoma and leiomyosarcoma.

In another aspect of the present invention, the anti-EGFR antibodies inhibit angiogenesis associated with tumors. EGFR stimulation of vascular endothelium is associated with vascularization of the tumor. Usually vascular endo is the men stimulated by paracrine manner, through, for example, EGF and/or TGF-α from other sources (e.g., tumor cells).

In accordance with this human anti-EGFR antibody is effective for the treatment of subjects with vascularized tumors or neoplasma or angiogenic diseases. Such tumors and neoplasma include, for example, malignant tumors and neoplasm, such as blastoma, carcinoma and sarcoma, as well as vysokokontsentrirovannye tumors and neoplasm. Cancers that can be treated by the methods provided in this invention include, for example, brain cancer, genitourinario tract, lymphatic system, stomach, kidney, colon, larynx, lung and bone. Non-limiting examples further include epidermoid tumors, squamous tumors, such as head and neck tumors, colorectal tumors, prostate tumors, breast tumors, lung tumors, including lung adenocarcinoma and small cell and non-small-cell lung tumors, pancreatic tumors, thyroid tumors, ovarian tumors, and liver tumors. The method is also used for the treatment of vascularized skin cancers, including squamous cell carcinoma, basal cell carcinoma, and skin cancers that can be treated by suppressing the growth of malignant the x keratinocytes. Other cancers that can be treated include Kaposi's sarcoma, neoplasm CNS (neuroblastoma, capillary hemangioblastomas, meningiomas and cerebral metastases), melanoma, gastrointestinal and renal carcinomas and sarcomas, rhabdomyosarcoma, glioblastoma, including polymorphic glioblastoma and leiomyosarcoma.

This invention also provides a method of treating non-cancer hyperproliferative diseases in mammals, including the introduction of a mammal an effective amount of an antibody according to this invention. Here we have in mind that "hyperproliferative disease" is defined as a condition caused by excessive growth of cancer cells, which Express some of EGFR family receptors. Excess cells are generated in the result of hyperproliferative diseases, Express EGFR at a normal level, or can sverkhekspressiya EGFR.

Types of hyperproliferative disorders that can be treated in accordance with this invention are any of hyperproliferative disorders, which are stimulated EGFR ligands or mutations such ligands. Examples of hyperproliferative diseases include psoriasis, senile keratoses and sabranie the keratoses, warts, keloid scars and eczema. Also included are the guy who ecoliterate disease, caused by viral infections, such as viral infection papillomavirus. Psoriasis, for example, may occur in different forms and with varying degrees of severity. Different types of psoriasis are manifested in the form of purulent vesicles (pustular psoriasis), multiple necrotic exclusion skin (erythrodermic psoriasis), teardrop-shaped points (drip psoriasis) and smooth inflammatory lesions (inverse psoriasis). This invention addresses the treatment of all types of psoriasis (e.g., psoriasis vulgaris, psoriasis pustulosa, psoriasis erythrodermica, psoriasis arthropathica, parapsoriasis, palmoplantar pustulosis).

Within the methods of the present invention, a therapeutically effective amount of an antibody according to the invention is administered to a mammal in case of need. The term "introduced" is used here as denoting the provision of antibodies according to this invention to a mammal by any method that provides the desired result. They can be introduced, for example, intravenously or intramuscularly. Although human antibodies according to this invention are particularly suitable for administration to humans, they can also be entered and other mammals. The term "mammals" in this context means including (not limited to humans, laboratory animals and Pets and farm animals. "Therapeutics and effective amount" means such amount of the antibody of the present invention, which, being introduced to a mammal, effectively lead to the desired effect, such as inhibition of kinase activity or the inhibition of tumor growth.

Identification of such diseases is determined by the abilities and knowledge of specialists in this field. For example, human individuals suffering from clinically significant manifestations of neoplastic or angiogenic diseases or who are at risk of manifestation of clinically significant symptoms, are good candidates for data entry EGFR antibodies. Clinical practitioner who is a specialist in this area, could readily determine, for example, results of clinical tests, physical studies, and medical and family history, whether the individual is a candidate for such treatment.

Data anti-EGFR antibodies can be administered for therapeutic treatment to a patient suffering from a tumor or pathological condition associated with angiogenesis, in amounts sufficient to prevent, inhibit or reduce tumor progression or pathological condition. Progression includes, for example, the growth, invasiveness, metastasis and/or recurrence of a tumor or pathological state. Amount sufficient to achieve this purpose, is defined as therape is automatically effective dose. Amounts effective for this purpose will depend on the severity of the disease and the General state of their own immune system of the patient. The dosing schedule will also be different depending on the stage of the disease and the patient's condition and usually varies from one loading dose or continuous infusion to several injections per day (e.g., every 4-6 hours), or in accordance with the instructions of the attending physician and the patient. However, it should be noted that this invention is not limited to any particular dosage.

A mixture of EGFR antagonists, for example, monoclonal antibodies, is a particularly effective method of treatment for inhibiting the growth of tumor cells. The mixture can include not-or antibody-based test EGFR antagonists in the amount of 2, 3 or 4 receptor antagonists to 6, 8 or 10.

In the embodiment of the invention, the anti-EGFR antibodies can be administered in combination with one or more anti-neoplastic agents. For examples of combination therapy, see, for example, U.S. Patent No. 6,217,866 (Schlessinger et al.) (anti-EGFR antibodies in combination with anti-neoplastic agents); WO 99/60023 (Waksal et al.) (anti-EGFR antibodies in combination with radiation). Can be any suitable anti-neoplastic agents such as chemotherapeutic agents, irradiation, or their combination. Anti-neoplas what practical means can be an alkylating agent or an anti-metabolite. Examples of alkylating funds include, but are not limited to, the following: cisplatin, cyclophosphamide, melphalan, and dacarbazine. Examples of anti-metabolites include, but are not limited to, the following: doxorubicin, daunorubicin, paclitaxel, irinotecan (CPT-11) and topotecan. If the anti-neoplastic agent is radiation, the radiation source can be external (Nesmelova radiation therapy, external beam radiation therapy - EBRT)and internal (brachytherapy, brachytherapy - W) relative to the patient. Dosage appointed anti-neoplastic funds is determined by many factors, including, for example, the type tool, type and severity of the tumor to be treated and the route of administration tools. However, it should be emphasized that the invention is not limited to any particular dosage.

In the treatment of hyperproliferative diseases, antibodies of the present invention described above techniques can be combined with the introduction of any traditional remedies. For example, when the hyperproliferative disease is psoriasis, there are a large number of available General funds and local action. General funds for psoriasis include methotrexate, and oral retinoids, such as acitretin, etretinate and isotretinoin. Other common drugs for psoriasis includes the t hydroxyurea, NSAIDS, sulfasalazin and 6-tioguanin. Antibiotics and antimicrobial drugs can be used to prevent infections that can cause exacerbation and worsening of psoriasis. Local remedies for psoriasis include anthralin, calcipotriene, coal tar, corticosteroids, retinoids, keratolytic, tazarotene. Steroids topical applications are one of the most common therapies prescribed for light and medium psoriasis. Steroids topical application is applied to the surface of the skin, but some of them are introduced in the area of psoriatic lesions by injection.

The treatment of hyperproliferative diseases further includes the introduction of anti-EGFR antibodies in conjunction with phototherapy. Phototherapy involves the processing of light of any wavelength, which reduces the symptoms of hyperproliferative diseases, as well as the photo-activation of the chemotherapeutic agents (photochemotherapy). For further discussion on the treatment of hyperproliferative disorders watch WO 02/11677 (Teufel et al.) (treatment of hyperproliferative disorders using receptor antagonist of epidermal growth factor).

Anti-EGFR antibodies according to this invention can be entered together with EGFR antagonists and/or antagonists of other RTK, such as antibodies, blocking RTK ligands or neutralizing RTK in other ways. Ligands include EGF, for example, EGF, TGF-α amphiregulin, heparin-binding EGF (HB-EGF) and betacellulin. EGF and TGF-α are considered to be the main endogenous ligands, causing stimulation through EGFR, although it has been shown that TGF-α is a stronger promoter of angiogenesis. Accordingly, EGFR antagonists include antibodies that bind such ligands and thereby block the binding of EGFR and activation.

Another example of this RTK is a VEGFR. In the embodiment of the present invention, the anti-EGFR antibody is used in combination with a VEGFR antagonist. In one embodiment of the present invention, the anti-EGFR antibody is used in combination with a receptor antagonist that specific binds to VEGFR-2/KDR receptor (PCT/US92/01300, filedFeb. 20,1992; Terman et al., Oncogene 6; 1677-1683 (1991)). In another embodiment of the present invention, the anti-EGFR antibody is used in combination with a receptor antagonist that specific binds to the receptor VEGFR-1/Flt-l (Shibuya M. et al., Oncogene 5, 519-524 (1990)). Especially preferred antigen-binding proteins that bind the extracellular domains of VEGFR-1 or VEGFR-2 and block the binding of ligands (VEGF or P1GF) and/or neutralize activation by VEGF or PIGF. For example, Mab IMC-1121 binds soluble and produced at the cell surface KDR. Mab IMC-1121 contains VHand VLdomains obtained is passed from human libraries phage display Fab (see WO 03/075840). In another example, ScFv 6.12 binds soluble and produced at the cell surface Flt-1. ScFv 6.12 contains VHand VLdomains of murine monoclonal antibodies b 6.12. The cell line of hybridoma producing b 6.12. deposited under ATSS number of MOUTH-3344.

Another example of this RTK is the receptor for insulin-like growth factor (insulin-like growth factor receptor, IGFR). In some types of tumor cells inhibition of EGFR function may be compensated by increased signaling pathways other receptors growth factor, in particular the stimulation of IGFR. Further, inhibition of signality IGFR increases the sensitivity of tumor cells to specific therapeutic agents. The stimulation of EGFR and IGFR leads to the phosphorylation of molecules of the General cascade of signal transmission, including Akt and R/42, although in varying degrees. Accordingly, in the embodiment of the present invention, the antagonist IGFR (for example, the antibody that binds IGF or IGFR and neutralize receptor) is introduced together with an antibody according to this invention, blocking, thus, the second entry in the General cascade of signaling pathways (e.g., inhibition of Akt activation and/or R/42). Example of human antibodies specific to IGFR, serves as IMC-A12 (see WO 2005/016970).

Other examples of receptors growth factor involved in oncogenesis include re atory platelet-derived growth factor (platelet-derived growth factor, PDGF), nerve growth factor (NGF) and fibroblast growth factor (FGF).

Anti-EGFR antibodies can also be entered with the intracellular RTK antagonists, which inhibit the activity of the RTK or elements associated signaling cascades that are involved in tumor growth or associated with tumor angiogenesis. Intracellular RTK antagonists are mostly small molecules. Some examples of small molecules include organic compounds, ORGANOMETALLIC compounds, salts of organic and ORGANOMETALLIC compounds and inorganic compounds. The atoms in small molecules are joined by covalent and ionic bonds, the first typical small organic compounds such as small molecule tyrosine kinase inhibitors, and the latter is typical of small molecules to inorganic substances. The arrangement of the atoms in the small organic molecule may be a chain, for example, carbon-carbon chain or carbon-heteroatomic chain, or to include cycles of carbon atoms, for example, benzene or polycyclic system, or a combination of carbon and heteroatoms, such as heterocycles, such as pyrimidine or hinzelin. Although small molecules can have any molecular weight, they generally include molecules that could be considered biologicalchemical, if their molecular weight would not be less D. Small molecules include compounds occurring in nature, such as hormones, neurotransmitters, nucleotides, amino acids, sugars, lipids and their derivatives, and received traditional organic synthesis, biosynthesis or their combinations. Cm. for example, Ganesan, DrugDoscov. Todayl(1): 47-55 (Jan. 2002); Lou, Drug Discov. Today, 6(24): 1288-1294 (Dec. 2001).

More preferably, small molecules for use as intracellular RTK antagonists, in accordance with the present invention, are intracellular EGFR antagonists that compete with ATP binding to the intracellular site of EGFR binding having a kinase domain, or a protein involved in transmission of the activation of EGFR. Examples of such signal transduction pathways are RAS-mitogen-activated protein kinase (ras-mitogen activated protein kinase, MARK) path, phosphatidylinositol-3 kinase (phosphatidylinosital-3 kinase, (P13K)-Akt)pathway, the stress-activated protein kinase (stress-activated protein kinase, SAPK) path and the path of the transmitter signal and activators of transcription (signal transducers and activators of reduced, STAT). He limiting examples of proteins involved in such ways (and which can join to small molecule EGFR antagonists in accordance with this invention, serve GRB-2, SOS, Ras, Raf, MEK, MARK, and matrix metalloproteinases (MMPs).

About them examples of EGFR antagonist small molecule is IRESSA™ (ZD1939), which is a derivative of hinazolina which works as an ATP-mimetic to inhibit EGFR. Watch U.S. Patent No. 5,616,582 (Zeneca Limited); WO 96/33980 (Zeneca Limited) at p.4; see also Rowinsky et al, 5 Abstract presented at the 37th Annual Meeting of ASCO, San Francisco, CA, 12-15 May 2001; Anido et al., Abstract 1712 presented at the 37th Annual Meeting of ASCO, San Francisco, CA, 12-15 May 2001. Another example of an EGFR antagonist small molecule is TARCEVA™ (OSI-774), which represents a 4-(substituted phenylamino) hinzelin derived [6,7-bis(2-methoxy-ethoxy)-hinzelin-4-yl]-(3-ethinyl-phenyl)amine hydrochloride] EGFR-inhibitor. Watch WO 96/30347 (Pfizer Inc.) on, for example, page 2, line 12 to page 4, line 34 and page 19, lines 14-17. Cm. also Moyeref al, Cancer Res., 57:4838-48 (1997); Pollack et al, J. Pharmacol, 291: 739-8 (1999). TARCEVA™ can operate through inhibition of phosphorylation of EGFR and its cascading PI3/AJkt and MAP (mitogen activated protein) kinase of signal transduction pathways, leading to the cessation of the cell cycle through P27. Watch Hidalgo et al, Abstract 281 presented at the 37th Annual Meeting of ASCO, San Francisco, CA, 12-15 May 2001.

Reported other small molecules, inhibiting EGFR, many of them through the tyrosine kinase domain of EGFR. Some examples of these EGFR-antagonist small molecule is described in WO 91/116051, WO 96/30347, WO 96/33980, WO 97/27199 (Zeneca Limited). WO 97/30034 (Zeneca Limited), WO 97/42187 (Zeneca Limited), WO 97/49688 (Pfizer Inc.), WO 98/33798 (Warner Lambert Company), WO 00/18761 (American Cyanamid Company) and WO 00/31048 (Warner Lambert Company). Examples of pacificnew EGFR-antagonist small molecules include CI 033 (Pfizer), which is an inhibitor of tyrosine kinases, hinzelin (N-[4-(3-chloro-4-fluoro-phenylamino)-7-(3-morpholine-4-yl-propoxy) hinzelin-6-yl]-acrylamide), particularly EGFR and described in WO 00/31048 on page 8, lines 22-6; PKI 66 (Novartis), which is pyrrolopyrimidine inhibitor of EGFR and which is described in WO 97/27199 on p.10-12; GW2016 (GlaxoSmithKline), which is an inhibitor of EGFR and HER2; EQU (Wyeth), which reportedly inhibits the growth of tumor cells, sverkhekspressiya EGFR or HER2 in vitro and in vivo; AG-1478 (Tryphostin), which is hinazolinam with Malm size of the molecule, which inhibits signaling from EGFR and erbB-2; AG-1478 (Sugen), which is bisubstrate inhibitor, which also inhibits protein kinase W2; PD 153035 (Parke-Davis), which reportedly inhibits EGFR kinase activity and tumor growth, induces apoptosis of cells in culture and increases the cytotoxicity of cytotoxic chemotherapeutic agents; SPM-924 (Schwarz Pharma), which is a tyrosine kinase inhibitor used for the treatment of prostate cancer; CF-546,989 (OSI Pharmaceuticals), which, reportedly, is an inhibitor of angiogenesis and is used for the treatment of solid tumors; ADL-681, which is an EGFR kinase inhibitor and used to treat cancer; PD 158780, which is pyridopyrimidines and which reportedly inhibits tumor growth in xenografts A umasa; CP-358,774, which is hinazolinam and which reportedly inhibits autophosphorylation in the HN5 xenografts in mice; ZD1839, which is hinazolinam and which, reportedly, has antitumor activity in murine xenotransplantation models, including cancer of the female external genital organs, NSCLC, prostate, colorectal, and ovarian, CGP A, which is pyrrolopyrimidine, which reportedly inhibits the growth of EGFR-positive xenografts in mice; PD 165557 (Pfizer); CGP54211 and CGP53353 (Novartis), which are diaminopyrimidine. Natural tyrosine kinase inhibitors of EGFR include genistein, herbimycin And, quercetin and abstain.

Other small molecules that are reported to inhibit EGFR and, thus, fall within the scope of this invention are tricyclic compounds, such as compounds described in U.S. patent No. 5679683; derivatives hintline, such as described in U.S. patent No. 5616582; and indole compounds, such as described in U.S. patent No. 5196446.

In another embodiment, the EGFR antagonist may be administered in combination with one or more adjuvants, such as, for example, cytokines (such as IL-10 and IL-13) or other immune stimulators, such as, chemokine, tumor-specific antigens and peptides. Look, for example, Larrivee et al., above. However, it should be taken in the Iman, introduction only EGFR antibodies is sufficient to therapeutically effective prevention, inhibition or reduction of the progression of a tumor.

In combination therapy, the anti-EGFR antibody is introduced before, during or after the start of therapy other means, as well as in any combination, for example, before and during, before and after, during and after, or before, during, and after the start of therapy anti-neoplastic agent. For example, anti-EGFR antibody can be administered between 1 and 30 days, preferably 3 and 20 days, more preferably between 5 and 12 days before the start of radiation therapy. In a preferred embodiment of the present invention, the chemotherapy given together or, more preferably, followed by antibody therapy.

In the framework of this invention, any available method or path can be used for the introduction of anti-EGFR antibodies according to this invention, and optional for joint introduction of anti-neoplastic funds and/or antagonists of other receptors. Reception modes anti-neoplastic funds, in accordance with this invention include any mode that is considered most appropriate for the treatment of the neoplastic condition of the patient. Various malignancy may require use of a specific antitumor antibodies and specific the STI-neoplastic funds that is determined for each patient individually. Methods purpose include, for example, oral, intravenous, intraperitoneal, subcutaneous or intramuscular administration. Prescribed dose of antagonist depends on many factors, including, for example, the type of antagonist, type and severity of the tumor to be treated and the route of administration of the antagonist. However, it should be noted that this invention is not limited to any particular method or by injection.

It should be noted that the anti-EGFR antibody according to this invention may be in the form of a conjugate that is bound by a specific receptor and exerts toxic, lethal action, followed by internalization of the complex ligand-toxin.

Conjugate the antibody-drug/small molecule can be formed directly or via a linker, peptide or non-peptide.

In another aspect of the present invention, the anti-EGFR antibody according to this invention can be chemically or biosynthetic associated with one or more anti-neoplastic or anti-angiogenic agent.

The invention further includes an anti-EGFR antibodies with attached fragments in the role of target or label. The target serves as the first associate member of the pair. Anti-neoplastic agents, such as anywhereelse with the second member of the pair and thus is m, oriented towards the area where the anti-EGFR antibody. A common example of such a binding pair is avidin and Biotin. In a preferred embodiment, Biotin is conjugated with anti-EGFR antibody and thus is a target for anti-neoplastic funds or other substances, conjugated with Avidya or streptavidin. Alternatively, Biotin or other similar substance attached to the anti-EGFR antibody according to this invention and used as a label, for example in a diagnostic system, in which the designated tool, which generates the signal, conjugated with Avidya or streptavidin.

It is assumed that anti-EGFR antibodies according to this invention, in the case of application in relation to the mammal for the prevention or treatment that are introduced in the form of a composition additionally comprising a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, for example, one or more of the following: water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, and combinations thereof. Pharmaceutically acceptable carriers may further comprise some amount of auxiliary substances such as uvlazhniaushii or emulsifying means, preservatives or buffers, which enhance the shelf life or effectiveness of the awn related proteins. Injectable compositions, as is well known in this field can be selected in such a way as to provide rapid, continuous or delayed release of the active ingredient after administration to the mammal.

The invention also includes kits for inhibiting tumor growth and/or associated with tumor angiogenesis comprising a therapeutically effective amount of a human anti-EGFR antibodies. The kit may also contain any suitable antagonist, for example, another growth factor receptor involved in tumorigenesis or angiogenesis (e.g., VEGFR-l/Flt-1, VEGFR-2, DERIVED, IGFR, NGFR, FGFR, etc. as described above). Alternatively, or in addition, the kits of this invention may also contain anti-neoplastic agent. Examples of suitable anti-neoplastic means, in the context of this invention have been described above. Kits according to this invention can also contain Adjumani, examples were described above.

In addition, within this invention, also included is the use of these antibodies in vivo and in vitro in research and diagnostic methods known in this field. Diagnostic methods include kits containing the antibodies according to this invention.

Accordingly, the data receptor antagonists, thus, mo is ut be used in vivo and in vitro research, diagnostic, prophylactic and therapeutic methods known in this field. Of course, is understandable and expected that experts in this field can be changes in the elements of this invention, and it is implied that such changes are included in the scope of this invention.

Increased activation of EGFR sometimes associated with conditions that are treated in accordance with this invention. The high concentration of ligand, EGFR amplification, increased transcription of the receptor or mutations that cause unregulated receptor signal transmission, can be a cause of increased EGFR activation. Amplification of the gene encoding EGFR, also causes an increase in the number of ligands that bind EGFR, which can further stimulate the proliferation of cells. EGFR can sverkhekspressiya in the absence of gene amplification, presumably by mutations that increase the EGFR transcription, mRNA translation or stability of the protein. Mutants of EGFR have been identified in gliomas, non-small cell lung carcinomas, carcinomas of the ovary and prostate, with constitutively active tyrosine kinase, which is likely to be significant for high-level activity of EGFR than overexpression of EGFR in these types of cancer. Look, for example, Pedersen et al., Ann. Oncol., 12(6):745-60 (001). (Mutation EGFR third type is called in different cases EGFRvlll, de2-7 EGFR or AEGFR - no part of the extracellular binding site of the ligand encoded by exons 2-7.); see also Wikstrand et al., Cancer Res., 55:3140-3148 (1995).

EXAMPLES

The following examples serve to further illustrate the invention, but should not be construed as any limitation of the scope of the invention. Detailed descriptions of standard methods such as those used to create vectors and plasmids, the introduction of genes encoding polypeptides into such vectors and plasmids, the introduction of plasmids into host cells, as well as the production and study of genes and gene products can be obtained from a number of publications, including Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Laboratory Press (198 9). Everything mentioned here references are included throughout the volume.

Example 1 - Allocation of human anti-EGFR antibodies

In General, human antibodies isolated from the bacteriophage library of natural human Fab obtained from Dyax, Cambridge, MA, method biologiste (multiple affine selection) on the activity of soluble human EGFR isolated from tumour cells, it produces. Bacteriophobia library natural Fab, containing the variable regions of the heavy and light chains of the antibodies produced by the human who their cells, (peripheral lymphocytes), composed of natural, non-immunized human cells basophilia spleen of a patient with gastric carcinoma, using amplification reactions in the primary PCR using forward and reverse primers, specific for the V gene, and cloning individual VHand VLgenes in separate vectors (WO 00/70023).

The original Fab library was grown to log phase, treated with phage-assistant MC and amplified overnight in the environment 2YTAK (2YT with 100 μg/ml ampicillin and 50 μg/ml kanamycin) at 30°C. the Phage preparation was precipitated in 4% PEG/0,5M NaCl and re-resuspendable in 3% fat-free milk/PBS to block nonspecific binding.

Approximately 1×1012pfu pre-blocked phage were incubated with 106cells A431, sverkhekspressiya EGFR, 1 ml of a clean environment DMEM at 4°C for 1 h, after which cells were washed 15 times with PBS. Bound phages were recovered by incubation at room temperature for 30 minutes in 1 ml of PBS containing 0.5 mg/ml MS-S. Retrieved phages were incubated in 10 ml under half of the log-phase TG1 cells at 37°C for 30 minutes stationary and within 30 minutes with shaking. The infected TG1 cells were pelletierine and placed on several large 2YTAG tablets and were incubated over night at 30°is. All grown up on the tablet colonies were erased in 3-5 ml 2YTA environment, mixed with glycerol (final concentration 10%), liquation and stored at - 70°C. For the next round of selection, 100 μl of phage strain was added to 25 ml 2YTAG medium and grown to half of the log phase. The culture was treated with phage-assistant MC, amplified, precipitated and used for breeding as described above.

Individual clones TG1 allocated at each level of selection, were randomly selected and grown at 37°C in 96-well tablet and processed using phage-assistant MC as described above. Preparation of phage was blocked with 1/6 volume of 18% of a mixture of milk/PBS at room temperature for 1 hour and added to 96-well Maxi-sorp tablet (Nunc)coated with recombinant EGFR (1 μg/ml, 100 μl). After incubation at room temperature for 1 h, the plate was washed three times with PBST and incubated with mouse anti-M13-phage conjugate-HRP (Amersham Pharmacia Biotech, Piscataway, NJ). The plate was washed five times, added peroxidase TMB substrate (KPL, Gaithersburg, MD) and using a tablet reader (Molecular Devices, Sunnyvale, CA) was defined absorption at 450 nm.

Identified clones were further tested for blocking the binding of EGF. To differentiate unique clones used method of fingerprinting DNA clones Representative clones from each alternative cleavage were selected and subjected to DNA sequencing.

Example 2 Expression and purification of soluble Fab fragments

Plasmids containing genes encoding 11F8 Fab, was used for transformation of non-suppressing E. coli host NW. Expression of Fab fragments in NV stimulated by culturing cells in 2YTA medium containing 1 mm isopropyl-1-thio-β-D-galactopyranoside (IPTG, Sigma) at 30°C. Perepleteny extract pf cells was prepared by repeated resuspending cellular tablets in 25 mmol Tris (pH 7.5)containing 20% (weight/volume) sucrose, 200 mmol NaCl, ImM EDTA, and 1 mmol PMSF, followed by incubation at 4°C With careful shaking for 1 h After centrifugation the soluble Fab protein was purified from the supernatant affinity chromatography using a Protein G column, following the manufacturer's instructions (Amersham Pharmacia Biotech).

Example 3 - Construction of human anti-EGFR IgGl antibody

Human anti-EGFR Fab was transformed into a full human IgGI. The selected Fab candidate was selected from natural human Fab-ragovoy library due to its high affinity and activity by blocking ligand human EFGR (ErbB). The DNA sequence of genes 11F8 Fab encoding the variable parts of the lungs (SEQ ID NO:15) and heavy chains were obtained (SEQ ID NO:7) by PCR amplification and cloned in the expression vector containing the constant domain of a human is th lgG1, using sexpressions system glutamine synthase from Lonza Biologies, Inc.

The PCR amplification was performed in two stages using a set Expand PCR (Boehringer Mannheim, Inc.) in accordance with the manufacturer's instructions, and examples, are given in table 3.

In General, PCR products of heavy and light chains were amplified using 25 ng C11F8 Fab plasmid DNA as a model and a pair of forward and reverse primers heavy (C11F8HF and C11F8HR) and lung (C11F8LF and C11F8LR) chains in 50 μl Expand Buffer System #3 in the following circumstances cycles, see Table 4:

Table 4
1 cycle94°C2 minutes
5 cycles94°C20 seconds
48°C2 minutes
68°C20 seconds
20 cycles94°C20 seconds
65°C60 seconds
68°C2 minutes
1 cycle65°C5 minutes

The resulting PCR products are added to the sequence of the 57 base pairs to the 5'-end of the 19 amino acid signal sequence of the gene of murine heavy chain immunoglobulin (MGWSCHLFLVATATGVHS, SEQ ID NO:25), which provides efficient education and secretion of immunoglobulin. For efficient initiation of translation of genes in mammalian cells, is added to the consensus sequence "Kozak" (J.Mol. Biol 196:947) by amplification of heavy and light chains in the secondary PCR reaction, using the direct primer OSEF in combination with CH11F8HR or C11F8LR respectively. This PCR product also provides the location of cleavage by restriction enzyme 5' Hind III cloning the amplified product into a suitable expression vector.

Purified in agarose gel Hind 111-Nhe I fragment of the heavy chain was cloned into managed the CMV promoter vector pDFc (figa) to create a continuous stretch of cDNA encoding the variable and constant region DNA sequences. Hind Ill-Xba I fragment of the light chain was cloned in the second run CMV promoter vector p12.1L (pigv). The resulting structure contains a single intron that separates the variable light and Kappa constant participation is key, which effectively visiplate of the resulting RNA transcript. Recombinant plasmids were transformed into competent E. coli, plasmid and selected strains were subjected to screening for the subject of the transient co-expression of heavy and light chains in COS cells.

Example 4 Expression of human anti-EGFR IgGI antibodies

For stable transfection of a single plasmid vector was generated by cloning Not1-Sal1 fragment from the expression cassette, containing the heavy chain under the CMV promoter in the vector p12.1L containing light chain. The resulting plasmid vector pGS-11F8 was restriction mapped (figs). Restriction analysis shown in figure 2.

Recombinant cell line used for production of monoclonal antibody 11F8, comes from " non-secretory cell line murine myeloma NS0 (according to Barnes et al., Cytotechnology 32:109 (2000)). Cell line NS0 was obtained from Lonza Biologies, Inc. (Slough, Berkshire, UK).

Cell line NSO myeloma was transliterowany the plasmid pGS-11F8 by electroporation using the BioRad Gene Pulser II, at a voltage of 250 V with a capacity of 400 μf and the time constant of 9.0 msec. Elektrooborudovanie cells were re-resuspension in DMEM (JRH Biosciences, Inc., Lenexa, KS)containing 10% detalizirovannoi fetal calf serum, dFCS (HyClone, Logan, UT) and 2 mmole glutamine (InVitrogen/Life Technologies, Paisley, PA). 50 m is l re resuspending cells were seeded on 96-hole plate with a density of 5000-10000 cells per well. Glutamine synthetase (GS)-no3HTHBHbie the transfectants were selected by adding DMEM medium that does not contain glutamine containing 10% dFCS with additional 1×GS (JRH Biosciences, Inc.) twenty-four hours post-transfection. Cells were cultured for 2-4 weeks at 37°C With 5% CO2to ensure the growth and development of colonies before screening for clones expressing the antibody.

Clones expressing anti-EGFR antibody were identified by screening using ELISA-based analysis of conjugate anti-human Fc (gamma) and horseradish peroxidase ordinary, detection was carried out with A450 nm. Positive clones were propagated and re-checked after 3-5 days of cultivation period. The most positive (production of antibodies at 25 µl/mg or more) were propagated for further analysis. Based on the production of antibodies in 24 9 µg/ml, clone number 34 was selected for sublimirovanny with limited dilution and rechecking. Clone 34-5 was chosen based on a constant level of production that meets or exceeds the level of the original cell line (production series 310 µg/ml, injection of 0.75-0.8 g/l). Clone 34-5-3 was selected after the second stage of sublimirovanny and according to the results of the analysis of clone 34-5-3 produces the antibody at a high level (the production of a series of 324 mg/ml, injection of 1.0-1.2 g/l). In the following the x examples were produced further study of the characteristics of this clone.

Example 5 - In Vitro antibody binding to EGFR

Antibodies were scanned using ELISA analysis on a firm surface when compared IMC-11F8 and IMC-C225 in binding characteristics. Defenestration microtitration plate was coated overnight with EGFR concentration 1 μg/ml in carbonate buffer at 4°C. the plate was blocked with saline phosphate buffer (phosphate buffered saline, PBS) supplemented with 10% newborn calf serum for one hour at 37°C. Different amounts of IMC-11F8 or IMC-C225 was added to the tablet and were incubated at room temperature for another 60 minutes followed by washing with PBS. Next was added conjugate mouse anti-human Fc with horseradish peroxidase (horse radish buffer, HRP) and incubated for additional 60 minutes at room temperature followed by intensive washing with PBS. The tablet then incubated with HRP substrate for 30 sec - 2 min, and the reaction was stopped with 0.1 M H2SO4. The tablet was read using ELISA analysis with OD450 nm.

Figure 3 shows the binding of IMC-11F8 and IMC-C225 antibody to EGFR. As IMC-11F8, and IMC-C225 find comparable binding to EGFR.

Example 6 - the Kinetics of binding of anti-EGFR antibodies

The kinetics of sesivany IMC-11F8 and IMC-C225 IgG antibodies and their Fab fragments was measured using a BIAcore sensor (Pharmacia Biosensor). Hybrid baie is OK EGFR-AP was immobilized on a sensor chip and injected soluble IMC-11F8 and IMC-C225 antibody at concentrations from 1.5 nm to 100 nm. Sensorgram read at each concentration and analyzed using BIA Evaluation 2.0, a program for determining the rate constants of the process, toonand koff. The affinity constant, Kd, was calculated from the ratio of these constants and koff/kon.

The kinetics of binding of anti-EGFR antibodies of the present invention is illustrated in Table 5. As you can see, both IgG antibodies have similar kinetics of binding to EGFR.

5,4±1,0
TABLE 5
AntibodyFormattoon(105M-1c-1)koff(10-4c-1)kd(nm)
IMC-11F8Fab22,9±9,936,7±8,51,78±0,5
IMC-11F8IgG18,6±7,75,8±2,20,32±0,06
IMC-C225Fab23,1±4,811,7±3,40,53±0,17
IMC-C225IgG21,3±7,30,3±0,2
Results represent mean ± statistical error of at least three measurements

Example 7 - Specificity of antibodies to EGFR

Shvatyvaemost antibodies to EGFR was estimated using125I-EGF competitive analysis. Cells NT were sown with a concentration of 2×104cells per well in 24-hole tablet COSTAR™ (Fisher Scientific, U.S.A.) in the environment McCoy''s 5A with the addition of 1.5 mmol L-glutamine, 10% CS and antibiotics at 37°C. Then the cell monolayer was incubated at room temperature for 1 h with different concentrations of unlabeled EGF, 11F8 or IMC-C225, which were mixed with different quantities125I-labeled EGF. Cells were washed with cold PBS and measured cellular radioactivity using a gamma counter-radiation.

Figure 4 shows the inhibition of binding125I-EGF with EGFR in cells NT. At concentrations between 10 and 100 nmol, IMC-11F8 effective as IMC-C225 in the inhibition of binding125I-EGF with EGFR in cells NT. Both antibodies are more successful in linking than EGF, which is a natural ligand of EGFR. Similar results were obtained for inhibition of the binding125I-EGF with EGFR in A431 cells.

Example 8 Activation of EGFR

In General, the analysis of the activation of kinase receptor (kinase receptor activation assay, KIRA assay) wire, and using cells Whrs or A431. Initially, cells were grown to 90% coverage by a monolayer in DME with the addition of up to 4 mmol L-glutamine and content of 1.5 g/l sodium bicarbonate and 4.5 g/l glucose, 10% CS at 37°C. Before the experiment the cells were placed for 24 h in DME containing 0.5% CS. To evaluate the effect of antibody, IMC-11F8, IMC-C225 and IMC-1 C11 on the activation of EGFR induced with EGF, various concentrations of the antibody pre-bound at room temperature for 30 minutes, followed by stimulation with EGF concentration of 8 ng/ml for an additional 15 minutes. After stimulation, cell monolayers were washed in ice-cold PBS containing 1 mmol of orthovanadate sodium. Cells were literally in lyse buffer (20 mmol Tric-HCl, pH 7.4, 1% Triton X-100, 137 mmol NaCl, 10% glycerol, 10 mmol EDTA, 2 mmol of orthovanadate sodium, 100 mmol NaF, 100 mmol of sodium pyrophosphate, 5 mmol PEFABLOC®SC (Boehringer Mannheim Biochemicals, Indianapolis, IN), 100 μg of Aprotinin and 100 μg/ml leupeptin) and centrifuged at 14000 g for 10 minutes. Cleared cell lysate was added to the wells of 96-well plates coated with polyclonal anti-EGFR antibodies. Tablets washed to remove nonspecific binding proteins and determined the level of EGFR phosphorylation by the addition of anti-phosphotyrosine antibodies. After extensive washing, the amount of bound anti-phosphotyrosine antibodies was measured with the help of ELISA analysis with OD450 nm.

P the results indicate a significant reduction in EGFR phosphorylation antibody IMC-11F8 as in cells Whrs (figure 5), and A431 (6) compared to the control antibody IMC-1C11.

Inhibition of EGF-stimulated EGFR phosphorylation was further evaluated by analyzing in Western Band immunoprecipitating EGFR. The A431 cells were pre-bound with antibodies followed by stimulation with EGF, as described above. Was used a control antibody that binds to EGFR, but did not inhibit phosphorylation of EGFR. Protein (EGFR) was immunoprecipitated purified from lysates using polyclonal anti-EGFR antibody and then pellet Protein A Sepharose. The pellets were then washed once with 0.2% of Triton X-100, 10 mmol Tris-HCl, pH 8.0, 150 mmol NaCl, 2 mm EDTA (buffer A), twice with buffer a containing 500 mm NaCl and twice with Tris-HCl, pH 8.0. The dried granules were mixed with 30 μl of 2×SDS loading buffer, boiled and caused the supernatant by SDS-PAGE. After separation of proteins by electrophoresis, protein zone was transferred to nitrocellulose filters for analysis in Western Band. Filters were blocked overnight blocking buffer, 50 mmol Tris-HCl, pH 7.4, 150 mmol NaCl (TBS)containing 5% bovine serum albumin and 10% skim milk powder. To determine the phosphorylated receptor, the blots were treated with anti-phosphotyrosine antibody in blocking buffer for 1 hour at room temperature. Then b is the notes were intensively washed with a 0.5×TBS containing 0.1% Tween-20 (TBS-T) and preincubator with goat anti-mouse Ig conjugated with HRP (Amersham, Little Chalfont, U.K.). The blots were washed with TBS and incubated for 1 minute with chemiluminescent reagent (ECL, Amersham, Little Chalfont, U.K.). Anti-phosphotyrosine reacting with phosphorylated proteins were detected by exposure to a high-performance film for the detection of luminescence (Hyperfilm-ECL, Amersham, Little Chalfont, U.K.) for from 0.5 to 10 minutes.

The analysis in Western Band on figa shows that IMC-11F8, as well as IMC-C225, inhibits the phosphorylation of EGFR. Neither EGF-antibody or cells treated with a control antibody that does not inhibit EGFR completely. Figv shows that the EGFR synthesis is not inhibited by adding antibodies to the cells. Fig shows that phosphorylation of EGFR inhibited IMC-11F8. More than 70% inhibition was observed for the three tumor cell cultures of different origin (A431,×RHS, HT-29) with the lowest evaluated concentrations of antibodies (0.8 nmol).

It was also tested the effect of IMC-11F8 on one of the main signaling molecules of EGFR cascade, MAP kinase R/R42 cable line. IMC-11F8 blocked the phosphorylation R/42 MAP kinase, following EGF stimulation, cells×RHS and HT-29 with effectiveness depending on the dosage (Fig).

Example 9 Inhibition of cell proliferation

Analysis of cell proliferation MTT was performed colorimetric as a result of the recovery of yellow tetrazole, MTT (3-(4, 5-dimethylthiazole-2)-2, 5-phenyltetrazol the rd bromide) metabolically active cell to the intracellular purple formisano product, which can be dissolved and quantified by spectrophotometric means. In General, the DiFi cells were cultured overnight in DMEM-10% CS. Antibody, IMC-11F8, IMC-C225 or IMC-1 C11, was added to each of three wells and incubated for an additional 72 hours at 37°C With 5% CO2. To measure the growth of cells in each well was added an aliquot of 20 µl tetrazolium dye and cells were incubated for 3 h at 37°C. When the purple precipitate was clearly visible under the microscope, the cells were literally by adding 100 μl of detergent. The absorbance formisano product was measured at OD570 nm as a measure of cell proliferation.

As shown in Fig.9, in contrast to the control antibody IMC-1C11, IMC-11F8 is as an effective inhibitor of cell proliferation, as IMC-C225.

Example 10 - Antibody-dependent cellular cytotoxic (Antibody-Dependent Cellular Cytotoxicity, ADCC) activity

One of the methods to estimate the mortality of the cells is the analysis of antibody-dependent cellular cytotoxicity (ADCC), which is commonly used radioisotope51Cr. Target cells labeled with51Cr, mixed with antibody and estimated level of mortality for the release of51Cr. Usually about 3×106DiFi cells suspended in 0.5 µl of culture medium and added to 0.5 mCi of Na51CrO4. A mixture of incubi is ovali for 1 h at 37°C With periodic shaking. The cells are then washed three times with cold culture medium. Then labeled cells suspended in 100 μl of culture medium containing different concentrations of anti-EGFR antibody (IMC-11F8 or IMC-C225), and incubated for 30 minutes at 4°C. After that, cells are washed three times culture medium using centrifugation. After addition of rabbit complement the treated cells are again incubated for 1 h at 37°C. Then added 50 μl of cold environment and ofcentrifugal. Separated supernatant liquid was measured using a gamma counter radioactivity, the highlighted cells in this fluid. The maximum allocation of radioactivity was registered in the case of adding to the investigated cells with 1% Triton X. the Percent of cytotoxicity was calculated as CPT highlighted in the course of the experiment minus CPT background, multiplied by 100, divided by CPT maximum allocation minus CPT background.

Figure 10 shows the mortality of the cells through IMC-11F8 and IMC-C225 (or ERBITUX™) when you activate antibody-dependent cellular cytotoxicity, or ADCC activity.

Example 11 Inhibition of growth of tumor cells in mice in vivo

Antitumor studies in vivo studies were performed to determine the ability of IMC-11F8 to block the growth of tumor cells xenotransplantation model. Devoid of the thymus gland of a mouse (nu/nu; Charles River Lab, Wilmington, MA) p is dkone were injected in the flank of 1-2 million cells A431 or×RS-3. Anti-EGFR antibody (IMC-11F8 and IMC-C225) or the control antibody was administered intraperitoneally injected in the amount of 1 mg per dose or 0.3 mg per dose three times per week. Tumor size was measured at least three times a week using calipers and calculated tumor volume (see, for example Baselga et al., J Natl. Cancer Inst. (1993) 85:1327-1333).

11 shows anti-tumor activity of IMC-11F8 on A431 xenotransplantation model. At a dose of 1 mg (11, right panel), IMC-11F8 effective as IMC-C225 (CETUXIMAB) in suppression or inhibition of tumor growth compared to control animals. When lower doses up to 0.3 mg, progression of tumor growth was slowed. Similarly, Fig shows the effect of IMC-11F8 and IMC-C225 in the second tumor models (xenograft×RS-3). The kinetics of tumor growth Whrs similar to the observed in the case of tumor models A431. At the dose level of 1.0 mg per mouse per injection, in the case of IMC-11F8 was observed in 6 cases, regression of tumors from 8 animals from A431 and 5 cases, regression of tumors from 8 mice In×RS-3.

Immunohistochemical staining as A431, and×PC-3 xenograft showed that treatment with IMC-11F8 significantly lowers the density of tumor cells and increases the area of non-cellular necrotic residues inside the tumor (Fig). Further, IMC-11F8 lowers the percentage of Ki-67-positive cells around the tumor phase is, that means a reduction of cell proliferation in tumors (Fig).

Example 12 - IMC-11F8 combination therapy

Naked mice with xenotransplantion human colorectal tumor GEO, DLD-1, HT-29, approximately 200-300 mm3, worked interperitoneal injection of IMC-11F8 twice weekly 0.3 mg or 1.0 mg per injection, in pure form or in combination with irinotecan (CPT-11) in the dose of 100 mg/kg once a week. Measurement of the tumor was performed once a week.

Treatment with IMC-11F8 in the amount of 0.3 mg or 1.0 mg per mouse per injection had a significant inhibitory effect on the growth of all three colorectal xenografts (GEO, DLD-1 and HT-29; figa-C). With the introduction of cambiale with CPT-11 mouse with GEO-xenograft, IMC-11F8 significantly increased the inhibitory effect on tumor growth compared with CPT-11 in its pure form (figa, p<0.01 for both doses of IMC-11F8). Moreover, while CPT-11 in its pure form did not cause regression of the tumor in this model, in 4 out of 10 and 9 out of 10 cases, regression of the tumor was achieved with the use of CPT-11 in combination with IMC-11F8 in the amount of 0.3 mg or 1.0 mg per mouse per injection, respectively (p=0.004 and p<0.0001, respectively). Such combined antitumor effects were observed in two other xenografts, DLD-1 (pigv) and HT-29 (figs) with equivalent statistics the practical significance of tumor regression in the group with high dose of antibody (1.0 mg). Fig.14D illustrates a significant increase in the number of tumor regressions in the case of a combination of CPT-11 with IMC-11F8 on these three models xenotransplantion colorectal carcinoma.

Example 13 - Pharmakinetics IMC-11F8

Pharmakinetic IMC-11F8 studied in cynomolgus monkeys (cynomolgus monkeys) and compared with pharmakinetics IMC-C225. Single dose for pharmokinetics research 20.5 mg/kg125I-labeled IMC-11F8 and MS-S separately injected monkeys, intravenous and blood sample to determine the level of antibody remaining in the plasma of the animal. Table 6 shows pharmakinetics comparison of IMC-11F8 and IMC-C225 in cynomolgus macaques.

Table 6
IMC-11F8IMC-C225
Cmax(mg/l)12131161
Tmax(h)0,750,117
T1/2(h)116117
AUC (mg×h/l)11540097871
CI (ml/h)0,7360,636

Is understandable and expected that a specialist in this field can be produced modifications of the described principles of the present invention, and it is implied that such modifications are included within the scope of this invention.

1. Isolated human antibody or antibody fragment binding to the EGFR and containing the complementarity determining the sites of SEQ ID NO:2 at CDRH1; SEQ ID NO:4 at CDRH2; SEQ ID NO:6 at CDRH3; SEQ ID NO:10 at CDRL1; SEQ ID NO:12 for CDRL2 and SEQ ID NO:14 at CDRL3.

2. The antibody or antibody fragment according to claim 1, containing SEQ ID NO:8.

3. The antibody or antibody fragment according to claim 1, containing SEQ ID NO:16.

4. The antibody or antibody fragment according to claim 1, containing SEQ ID NO:8 and SEQ ID NO:16.

5. The antibody or antibody fragment according to claims 1-4, selectively bind to the EGFR.

6. The antibody or antibody fragment according to claims 1-4, inhibiting EGFR binding with a ligand of EGFR.

7. The antibody or antibody fragment according to claims 1-4, EGFR neutralizing.

8. The antibody or antibody fragment according to claims 1-4, selected from the group consisting of single-chain antibody, Fab, single-chain Fv dimer antibodies and trimeric antibodies.

9. Conjugate antibodies described in claims 1 to 4, or a functional fragment with anti-neoplastic agent or a marker for specific binding to its receptor EGFR.

10. Isolated polynucleotide encoding the antibody or frag the UNT antibody and containing the nucleotide sequence of SEQ ID NO:1 for CDRH1; SEQ ID NO:3 at CDRH2; SEQ ID NO:5 for CDRH3; SEQ ID NO:9 at CDRL1; SEQ ID NO:11 at CDRL2; and SEQ ID NO:13 at CDRL3.

11. Isolated polynucleotide of claim 10, containing SEQ ID NO:7.

12. Isolated polynucleotide of claim 10, containing SEQ ID NO:15.

13. The expression vector containing polynucleotide on p-12.

14. Recombinant a host cell containing the expression vector according to item 13.

15. Recombinant cell host according to 14, which produces a polypeptide containing SEQ ID NO:8, and the polypeptide containing SEQ ID NO:16.

16. Recombinant cell host according to 14, which produces a polypeptide containing SEQ ID NO:8 and SEQ ID NO:16.

17. Method of inhibiting the growth of tumors expressing EGFR, in a mammal, comprising introducing a therapeutically effective amount of the antibody according to claims 1-8.

18. The method according to 17, in which the tumor sverkhekspressiya EGFR.

19. The method according to 17, in which the tumor is a primary cancer.

20. The method according to 17, in which the tumor is a metastatic cancer.

21. The method according to 17, in which the tumor is resistant cancer.

22. The method according to 17, in which the tumor is vascularizing cancer.

23. The method according to 17, in which the tumor is selected from the group consisting of colorectal cancer, cancer of the head or neck, cancer of the pancreas, cancer of the lung, cancer of the milk the gland, renal carcinoma and glioblastomas.

24. The method according to 17, in which the antibody or antibody fragment is introduced in combination with anti-neoplastic agent.

25. The method according to paragraph 24, in which the anti-neoplastic agent is a chemotherapeutic agent.

26. The method according to paragraph 24, in which the anti-neoplastic agent is irinotecan (CPT-11).

27. The method according to paragraph 24, in which the anti-neoplastic agent is a radiation.

28. The method according to 17, in which the antibody or antibody fragment is administered with an EGFR antagonist.

29. The method according to p in which EGFR antagonist is intracellular EGFR antagonist.

30. The method according to 17, further comprising introducing a therapeutically effective amount of an antagonist of the receptor for vascular endothelial factor (vascular endothelial factor receptor, VEGFR).

31. The method according to 17, comprising introducing a therapeutically effective amount of an antagonist of the receptor for insulin-like growth factor (insulin like growth factor receptor, IGFR).

32. The method of treatment of hyperproliferative diseases caused by excessive growth of cancer cells expressing EGFR comprising introducing a therapeutically effective amount of the antibody according to claims 1-8.

33. The method according to p in which the hyperproliferative disease is psoriasis.

34. The method according to p, in which the antibody or fragment of the anti-Christ. ate is administered in combination with local or systemic agent for psoriasis.

35. The method according to p, in which the antibody or antibody fragment is administered in combination with a corticosteroid.

36. The method according to p, in which the antibody or antibody fragment is administered in combination with a retinoid.



 

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7 cl, 7 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to immunology and biotechnology. The invention describes a nucleotide sequence which codes the V9 nanoantibody and its expression vector with extra epitope(s) on the C-end for detection and extraction and a signal peptide on the N-end. The invention discloses a method of obtaining the V9 nanoantibody, a method of inhibiting proliferation of endothelial cells using the V9 nanoantibody, as well as use of the V9 nanoantibody for qualitative and quantitative determination of VEGF in a sample. Use of the invention provides high-affinity neutralising monovalent single-strand nanoantibodies which are more resistant to external factors (temperature, pH) and cheaper to produce compared to conventional VEGF antibodies, which can be useful in medicine for treating and diagnosing diseases associated with regulation of the activity of the vascular endothelial growth factor (VEGF).

EFFECT: invention discloses a monomer single-strand V9 nanoantibody which can bind and inhibit the human vascular endothelial growth factor.

7 cl, 7 dwg, 7 ex

FIELD: medicine.

SUBSTANCE: plasmid contains a DNA fragment encoding laccase C1 of ligninolytic fungus Trametes hirsuta or a DNA fragment hybridised with SEQ ID N0:1 in tough conditions, under control of promoter functioning in this cell. The resulting cell is a producer of laccase C1. The invention also refers to method of laccase obtaining using to the indicated cell.

EFFECT: obtaining laccase with high catalytic activity.

6 cl, 3 dwg, 1 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to biotechnology and immunology. An antibody against angiopoietin-2 is proposed. Versions of the antibody are disclosed, which are produced by hybridome ATCC PTA-7258, ATCC PTA-7259, ATCC PTA-7260. The corresponding coding nucleic acid and expression vector are disclosed. A host cell which produces the antibody based on the said vector is described. The disclosed antibodies have Kd of the order of 10-10-10-12 M, for the antibody 3.19.3 (from ATCC PTA-7260) IC50=99 nM. The said antibody properties can be used in treating human tumours.

EFFECT: design of a method of treating pathological angiogenesis based on an antibody and use of the antibody to prepare a medicinal agent for treating pathological angiogenesis.

33 cl, 18 dwg, 18 tbl, 24 ex

FIELD: medicine.

SUBSTANCE: trans-sialydase enzyme has been recovered from a unicell Trypanosoma congolense. Trans-sialydase is characterised by one of the following amivo acid sequences: SEQ ID NO:2, SEQ ID NO:4 or a sequence being 75% identical with one of said sequences.

EFFECT: extended range of enzymes with trans-sialydase activity.

6 cl, 3 dwg, 1 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to anti-M-CSF-specific antibodies based on RX1 or originating from RX1, and which more than 785% compete with monoclonal antibodies RX1, MC1 and/or MC3 for bonding with M-CSF (macrophagal colony-stimulating factor). The non-mouse antibody is two-stranded, contains a certain amino acid sequence (given in the formula of invention and list of sequences) and retains high affinity towards M-CSF. The invention discloses an isolated nucleic acid which codes the said antibody, an expression vector, a host cell and a method of producing the anti-M-CSF-antibody using a host cell or hybridome, particularly ATCC PTA-6263 or ATCC PTA-6264 hybridome. The invention describes a pharmaceutical composition containing said antibodies, sets containing pharmaceutical compositions and methods of preventing and treating osteoporosis in a person suffering from an osteolytic disease.

EFFECT: disclosed antibodies can inhibit osteoclast differentiation, which facilitates their use as highly effective preparations for treating osteolysis, cancer with metastases and osteoporosis associated with cancer metastases.

131 cl, 44 dwg, 12 tbl, 16 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to immunology and biotechnology. The invention discloses a monomer single-strand V93 nanoantibody which can bind and inhibit the human vascular endothelial growth factor. The invention describes a nucleotide sequence which codes the V93 nanoantibody and its expression vector with extra epitope(s) on the C-end for detection and extraction and a signal peptide on the N-end. The invention discloses a method of obtaining the V93 nanoantibody, a method of inhibiting proliferation of endothelial cells using the V93 nanoantibody, as well as use of the V93 nanoantibody for qualitative and quantitative determination of VEGF in a sample.

EFFECT: use of the invention provides high-affinity neutralising monovalent single-strand nanoantibodies which are more resistant to external factors (temperature, pH) and cheaper to produce compared to conventional VEGF antibodies, which can be useful in medicine for treating and diagnosing diseases associated with regulation of the activity of the vascular endothelial growth factor (VEGF).

7 cl, 7 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to immunology and biotechnology. The invention describes a nucleotide sequence which codes the V9 nanoantibody and its expression vector with extra epitope(s) on the C-end for detection and extraction and a signal peptide on the N-end. The invention discloses a method of obtaining the V9 nanoantibody, a method of inhibiting proliferation of endothelial cells using the V9 nanoantibody, as well as use of the V9 nanoantibody for qualitative and quantitative determination of VEGF in a sample. Use of the invention provides high-affinity neutralising monovalent single-strand nanoantibodies which are more resistant to external factors (temperature, pH) and cheaper to produce compared to conventional VEGF antibodies, which can be useful in medicine for treating and diagnosing diseases associated with regulation of the activity of the vascular endothelial growth factor (VEGF).

EFFECT: invention discloses a monomer single-strand V9 nanoantibody which can bind and inhibit the human vascular endothelial growth factor.

7 cl, 7 dwg, 7 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to biotechnology and immunology. An antibody against angiopoietin-2 is proposed. Versions of the antibody are disclosed, which are produced by hybridome ATCC PTA-7258, ATCC PTA-7259, ATCC PTA-7260. The corresponding coding nucleic acid and expression vector are disclosed. A host cell which produces the antibody based on the said vector is described. The disclosed antibodies have Kd of the order of 10-10-10-12 M, for the antibody 3.19.3 (from ATCC PTA-7260) IC50=99 nM. The said antibody properties can be used in treating human tumours.

EFFECT: design of a method of treating pathological angiogenesis based on an antibody and use of the antibody to prepare a medicinal agent for treating pathological angiogenesis.

33 cl, 18 dwg, 18 tbl, 24 ex

FIELD: medicine.

SUBSTANCE: method facilitates linkage of sequences, coding immunoglobulin variable regions, T-cells receptors or B-cells receptors. Method is instrument of higher effectivity for making sequence data libraries. Capability of multiple RT-PCR with chain extension by interruption with employment of matrix, derived from single cell, provides highly effective creation of sister pairs libraries.

EFFECT: method is effective for linkage of two or few nucleotide sequences, coding domens or subunits of heteromeric protein as a result of single reaction performance.

51 cl, 25 dwg, 27 tbl, 14 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of human IL-13 antibodies, including based on CDR antibody BAK278D6. There is described a based composition, and also isolated nucleic acid, a host cell for preparing antibodies and versions of the method for preparing antibodies. There is disclosed application of antibodies for preparing a drug and a composition for treating various diseases mediated by IL-13 activity. Application of the invention provides antibodies neutralising IL-13.

EFFECT: applicable in medicine for preparing a vaccine.

52 cl, 32 dwg, 7 tbl, 29 ex

FIELD: chemistry.

SUBSTANCE: invention relates to immunology and biotechnology. Described are versions of the humanised antibody CD45RO/RB which carry a light and a heavy strand. Versions of the following are disclosed: isolated polynucleotide, coding antibody, expression vector containing a polynucleotide and host cells containing the expression vector. Described also is use of the antibody to treat and/or prevent various diseases, including as a component of a pharmaceutical composition.

EFFECT: invention provides antibodies identified as CD45RO and CD45RB, which can find use in medicine.

9 cl, 14 dwg, 2 tbl, 13 ex

FIELD: chemistry.

SUBSTANCE: invention relates to humanised anti-TGF-beta-antibody which is linked to TGF-beta. The humanised antibody has a variable domain VH which contains residues of the hypervariable region (non-human), which are contained in the human domain VH which includes a modified framework region (FR) (amino acid and nucleotide sequences are given in the list of sequences). The humanised antibody can contain residues of the complementarity determining region (CDR) of the variable domain of the light strand VL. The invention also relates to a composition for treating TGF-beta mediated disorders, e.g. malignant tumours, nucleic acid, coding monoclonal antibody, and a method of obtaining the latter using host cells. The invention provides a method of treating and detecting TGF-beta in a sample from the body using the disclosed antibody, as well as to a product which contains the humanised antibody and directions for use for treating TGF-beta mediated disorders.

EFFECT: invention enables control of TGF-beta molecules, which can prevent possible changes in antibodies, enables preparation of high-affinity humanised antibodies which act as TGF-beta antagonists.

57 cl, 45 dwg, 4 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: there is offered a monoclonal antibody specific to human interleukine-4 (hIL-4) containing two domains with the related CDR1-3 region. There are described versions thereof that contain specified CDR, polynucleotide coding said antibody. There are described an expression vector and a host-cell for preparing the antibody to human interleukine-4 (hIL-4). There are opened: application of the antibody for preparing a pharmaceutical agent for treating the diseases mediated by interleukine-4 and/or IgE. There is discovered the pharmaceutical composition for treating the diseases mediated by interleukine-4 and/or IgE is opened.

EFFECT: application of the invention ensured the high-affinity neutralised monoclonal antibodies to human interleukine-4.

14 cl, 1 tbl, 6 ex

FIELD: pharmacology.

SUBSTANCE: present invention refers to immunology and biotechnology. There are antibody-antagonist to CD40 with their variable areas derived from an antibody produced of hybridoma 4D11 (FERM BP-7758). The constant areas of antibodies are derived from human IgG4 with mutations S228P and L235E. There are described related coding polynucleotides and the based expression vector. There is disclosed host-cell containing said vector. There is described method for preparing monoclonal antibody and application thereof in the pharmaceutical composition.

EFFECT: application of the invention provides reduced ADCC and CDC activity that can find application in therapy of autoimmune diseases and graft rejection.

10 cl, 26 dwg, 2 tbl, 22 ex

FIELD: medicine.

SUBSTANCE: method of producing an antigen-binding VH domain wherein an elongated CDR3 loop similar to a camel loop is absent, involves transformation of a mammal cell by a heavy-chain VH heterolocus. The locus contains a gene coding a variable area, at least containing one segment of VH gene, one segment of D gene not being camel's, one segment of J gene not being camel's, one invariable heavy chain region provided that any gene coding the invariable regions does not code a Sn1 functional domain. The segments of V, D and J genes are capable to recombination and formation of the VDJ coding sequence. The transformed cell is capable to express a heavy chain antibody only containing an antigen-binding VH domain and the invariable constant effector region without the functional domain Sn1. The cell is used for producing a transgene animal to be immunised with an antigen of concern. Further the cells or tissues expressing specific antibodies to the heavy chain antigen of concern are recovered; nucleic acid coding the VH domain of the specific heavy chain antibody only is recovered; and the specified antigen-binding VH domain is expressed. The specified VH domains can be used for production of fused proteins or binding complexes of monovalent, bivalent or polyvalent polypeptide.

EFFECT: invention allows producing antigen-specific human antibodies of any class, exhibiting high affinity.

11 cl, 26 dwg, 8 ex

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