Binding proteins specific to insulin-like growth factors, and using them

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a completely human monoclonal antibody, which binds insulin-like growth factor-II (IGF-II) and has a cross responsiveness to IGF-I, as well as its antigen-binding fragment. There are disclosed a nucleic acid molecule coding an antibody according to the invention, a vector and a host cell for the expression of the antibody according the invention. There are described a pharmaceutical composition, as well as conjugates for treating and diagnosing malignant tumour, using the antibody according to the invention in preparing the therapeutic agent and a method for determining IGF-II and IGF-I levels in a patient's sample.

EFFECT: present invention can find further application in cancer therapy.

16 cl, 27 ex, 18 tbl

 

CROSS-REFERENCE TO RELATED APPLICATIONS

In this application claimed priority under 35 U. S. C. §119, of provisional application U.S. serial number U60/750,085, filed December 13, 2005; provisional application U.S. serial number 60/750,772, filed December 14, 2005; provisional application U.S. serial number 60/774,747, filed February 17, 2005; and provisional application U.S. serial number 60/808,183, filed may 24, 2006, each of which is fully incorporated into this application by reference.

Background of the INVENTION

Field of the invention the Invention relates to binding proteins that bind insulin-like growth factor - 2 (IGF-II) with cross-reactivity to insulin-like growth factor - 1 (IGF-I) and to the use of such binding proteins. More specifically, the invention relates to monoclonal antibodies directed to IGF-II with cross-reactivity to IGF-I and to the use of such antibodies. Aspects of this invention also relate to hybridomas or other lines that Express such antibodies.

Description of the prior art

Insulin-like growth factors IGF-I and IGF-II are small polypeptides that are involved in the regulation of cell proliferation, survival, differentiation and transformation. IGFs in various ways affect the way the OS�Aries, interaction with the specific receptor on the cell surface, the receptor for IGF-I (IGF-IR) and activation of various intracellular signaling cascades. IGFs circulate in the serum, mostly binding to IGF-binding proteins (IGFBP-1-6). The interaction of IGFs with IGF-IR is regulated by IGFBPs, a IGFs can bind only to IGF-IR after being released from IGFBPs (mainly by proteolysis of IGFBPs). IGF-I can also bind the hybrid receptor consisting of IGF-IR and insulin receptor (IR). It has been shown that IGF-II binds the "A" isoform of the insulin receptor.

Malignant transformation include the imbalance of various processes such as cell growth, differentiation, apoptosis and transformation. IGF-I and IGF-II were involved in the pathophysiology of a wide range of conditions, and it is believed that they are involved in mutagenesis, due to their mitogenic and antiapoptotic properties, receptor mediated IGF-IR. LeRoith and Roberts, Cancer Lett. 195: 127-137 (2003).

IGF-I was opened like growth factor, which is produced by the liver under the regulation of growth hormone mucosa, and originally called somatomedin - C. Salmon et al., J. Lab. Clin. Med. 49: 825-826 (1957). As IGF-I and IGF-II expedious everywhere and act as endocrine, paracrine and autocrine growth factors, through their interaction with IGF-IR, TRANS-membrane tyrosine-kinase to�I structurally and functionally belongs to the insulin receptor (IR). IGF-I functions primarily through the activation of IGF-IR, where IGF-II can act through IGF-IR or via the isoforms IR-A. LeRoith and Roberts, Cancer Lett. 195: 127-137 (2003). Additionally, the interaction of IGF-I and IGF-II with IGF-binding proteins can influence the half life and bioavailability of IGFs, as well as their direct interaction with receptors in similar cases. Rajaram et al., Endocr. Rev. 18: 801-831 (1997).

IGF-I exerts a prolonged effect on cell proliferation, differentiation, and apoptosis. Experiments with cultured osteosarcoma and breast cancer suggested that IGF-I is a protein mitogen and exerts its mitogenic action by increasing DNA synthesis and by stimulating the expression of zaklinivalo D1, accelerating progression of the cell cycle from G1to S phase. Furlanetto et al., Mol. Endocrinol. 8: 510-517 (1994); Dufourny et al., J. Biol. Chem. 272: 311663-31171 (1997). Suppression of expression of D1 in cancer cells of the pancreas abolishes the mitogenic action of IGF-I. Kornmann et al., J. Clin. Invest. 101: 344-352 (1998). In addition to stimulation of cell cycle progression, IGF-I also inhibits apoptosis. It has been shown that IGF-I stimulates the expression of Bl proteins and inhibits expression of Bax, increasing the relative amount of heterodimer Bcl/Bax, blocking, thus, the path apoptosis. Minshall et al., J. Immunol. 159: 1225-1232 (1997); Parrizas et al, Endocrinology 38: 1355-1358 (1997); Wang et al., Endocrinology 139: 1354-1360 (1998).

Similarly, IGF-I, IGF-II also has mutagenic and antiapilepticescoy action and regulates cell proliferation and differentiation. Compared to IGF-I in serum by high circulating concentrations of IGF-II. The high content of IGF-II in serum was found in patients with bowel cancer, with a trend of increasing concentration with the progression of the disease. Renehan et al., Br. J. Cancer 83:1344-1350. In addition, the majority of primary tumors and transformed cell lines redundantly Express IGF-II and RNA and protein. Werner and LeRoith Adv. Cancer Res. 68:183-223 (1996). Overexpression of IGF-II colon cancer is associated with aggressive phenotype, loss of fingerprints (loss of allele-specific expression) IGF-II gene may be important in colon carcinogenesis. Michell et al., Br. J. Cancer 76:60-66 (1997); Takano et al., Oncology 59: 210-216 (2000). Cancer cells, which have a strong tendency to the formation of metastasis are the expression levels of IGF-four times greater than the expression in those cells that have a weak tendency to form metastases Guerra et al., Int. J. Cancer 65: 812-820 (1996).

Scientific and clinical studies have highlighted the role of IGF family members in the development, maintenance and progression of cancer. It has been shown that many cancer cells have an over expression of h-IGF-IR and/or IGF ligands. For example, IGF-I and IGF-II are potent mitogens wide range�and cancer cell lines, including sarcoma, leukemia, and cancer of the prostate, breast, lung, colon, stomach, esophagus, liver, pancreas, kidney, thyroid, brain, ovarian, and uterine cancer. Macaulay et al., Br. J. Cancer 65: 311-320 (1992); Oku et al., Antisepsis Res. 11: 1591-1595 (1991); LeRoith et al., Ann. Intern. Med. 122:54-59 (1995); Yaginuma et al., Oncology 54: 502-507 (1997); Singh et al., Endocrinology 137: 1764-1774 (1996); Frostad et al., Eur. J. Haematol 62: 191-198 (1999). When IGF-I was injected into malignant cancer cells of the esophagus, they became resistant to apoptosis induced by cytokines. Remacle-Bonnet et al., Cancer Res. 60: 2007-2017 (2000).

The role of IGFs in cancer development is also confirmed by epidemiological studies in which it was shown that high levels of circulating IGF-I and low levels of IGFBP-3 are associated with an increased risk of developing several kinds of cancer (prostate, breast, colon and lung). Mantzoros et al., Br. J. Cancer 76: 1115-1118 (1997); Hankinson et al., Lancet 351: 1393-1396 (1998); Ma et al., J. Natl. Cancer Inst. 91: 620-625 (1999); Karasik et al., J. Clin. Endocrinol Metab. 78: 271-276 (1994). These results suggest that IGF-I and IGF-II act as a potent mitogenic and anti-apoptotic signals, and their overexpression is correlated with poorer prognosis for patients with several types of cancer.

Using knockout mice, several studies have further established the role of IGFs in the growth of tumors. With the technology development of tissue-specific, conditional� gene deletions, was developed murine model of hepatic IGF-I deficiency (LID). Liver-specific deletion of the gene igfl has abolished the expression of IGF-I mRNA and caused a strong decrease in levels of circulating IGF-I. Yakar et al., Proc. Natl. Acad. Sci. USA 96: 7324-7329 (1999). When mammalian tumor was induced in mice LID, reducing levels of circulating IGF-1 led to a significant decrease in cancer development, growth and metastasis formation, while increased levels of circulating IGF-1 is associated with increased tumor growth. Wu et al., Cancer Res. 63: 4384-4388 (2003).

In several articles it was reported that the inhibition of IGF-IR expression and/or signal leads to inhibition of growth of tumors, both in vitro and in vivo. It was also shown that the inhibition of IGF signaling increases the sensitivity of tumor cells to chemotherapeutic agents. We developed a variety of strategies (antisense oligonucleotides, soluble receptors, peptide inhibitors, dominant negative receptor mutants, small molecules inhibitors of kinase activity and anti-hIGF-IR antibody) for inhibiting IGF-IR transmission signal in tumor cells. One approach was to defeat kinase activity of IGF-IR small molecule inhibitors. Were recently identified two compounds as small molecule kinase inhibitors that are able �elective to inhibit IGF-IR. Garcia-Echeverria et al., Cancer Cell 5: 231-239 (2004); Mitsiades et al., Cancer Cell 5: 221-230 (2004). Inhibition of kinase activity of IGF-IR did not allow IGF-I-mediated survival and the formation of colonies in soft agar cancer cells MCF-7 human breast cancer. Garcia-Echeverria et al., Cancer Cell 5: 231-239 (2004). When administered kinase inhibitor of IGF-IR mice with tumor xenograft, signaling IGF-IR in tumor xenotransplantation ingibirovalos, and growth caused by IGF-IR fibrosarcoma decreased significantly. Garcia-Echeverria et al., Cancer Cell 5: 231-239 (2004). A similar effect was observed in hematological malignancies, particularly multiple myeloma. In cells of multiple myeloma (a disease of Rusticola-Calera, a small molecule IGF-IR kinase inhibitor, was shown to >16-higher power (in relation to IGF-IR, compared with the insulin receptor, and showed a similar efficacy in inhibiting growth and survival of cells. Mitsiades et al., Cancer Cell 5: 221-230 (2004). Similar compounds were intraperitoneally injected to the mice and inhibited the growth of multiple myeloma cells and increased the survival of mice Mitsiades et al., Cancer Cell 5: 221-230 (2004). In combination with other chemotherapeutic agents in sub-therapeutic doses, the inhibition of kinase activity of IGF-IR synergy has reduced the weight of tumors Mitsiades et al., Cancer Cell 5: 221-230 (2004).

Another approach to inhibit �eredeti signals IGF was to develop an effective neutralizing antibodies, directed against the receptor for IGF-IR. Various groups have developed antibodies to IGF-IR to the inhibition of stimulated receptors IGF-I autophosphorylate, inducing receptor internalization and degradation and reduce the proliferation and survival of various human cancer cell lines. Hailey et al., Mol Cancer Ther. 1: 1349-1353 (2002); Maloney et al., Cancer Res. 63: 5073-5083 (2003); Benini et al., Clin. Cancer Res. 7: 1790-1797 (2001); Burtrum et al., Cancer Res. 63: 8912-8921 (2003). In addition, xenotransplantation tumor models, blockade of IGF-IR resulted in a significant inhibition of growth of mammary tumors, kidney and pancreas in vivo. Burtrum et al., Cancer Res. 63: 8912-8921 (2003); Maloney et al., Cancer Res. 63: 5073-5083 (2003). The experiments used a chimeric humanized antibody IGF-IR led to a similar result, by inhibiting the growth of cancerous breast cells in vitro and in tumor xenograft. Sachdev et al., Cancer Res. 63: 627-635 (2003). Other humanized IGF-IR antibody blocked caused by IGF-I phosphorylation thyroid and inhibition of tumor growth in the breast and small lung cells and in vivo. Cohen et al., Clin. Cancer Res. 11: 2063-2073 (2005); Goetsch et al., Int. J. Cancer 113: 316-328 (2005).

Increased IGF-I levels were also associated with several non-cancerous pathological conditions, including acromegaly and gigantism (Barkan, Cleveland Clin. J. Med. 65: 343, 347-349, 1998), at the time�I like abnormal functioning of the receptor for IGF-I/IGF-II were involved in psoriasis (Wraight et al., Nat. Biotech. 18: 521-526, 2000), atherosclerosis and smooth muscle restenosis of blood vessels after angioplasty (Bayes-Genis et al., Circ. Res. 86: 125-130, 2000). Increased IGF-I levels were involved in diabetes or complications associated with diabetes, such as microvascular proliferation (Smith et al., Nat. Med. 5: 1390-1395, 1999).

Antibodies to IGF-I and IGF-II are described in the prior art. See, for example, Goya et al., Cancer Res. 64: 6252-6258 (2004); Miyamoto et al., Clin. Cancer Res. 11: 3494-3502 (2005). Additionally, see WO 05/18671, WO 05/28515 and WO 03/93317.

BRIEF description of the INVENTION

Embodiments of this invention relate to binding proteins that bind specific insulin-like growth factors and reduce the growth of tumors. In one embodiment, the binding proteins are fully human monoclonal antibodies, or binding fragments that bind specific insulin-like growth factors and reduce the growth of tumors. The mechanisms for achieving this may include and are not limited to either inhibition of binding of IGF-I/II and its receptor IGF-IR inhibition caused by IGF-I/II signaling IGF-IR, or increased clearance of IGF-I/II, thus reducing the effective concentration of IGF-I/II.

Thus, some embodiments provide a fully dedicated human-specific binding protein, which preferentially binds insulinpump�th growth factor-II (IGF-II) and has cross-reactivity to insulin-like growth factor I (IGF-I), neutralizes the activity of IGF-I and IGF-II. In some aspects, the binding protein binds IGF-II with epinasty, which is at least 2.5 times greater than its affinity towards the IGF-I. In other aspects, the binding protein that binds IGF-II with epinasty, which is at least 3, at least 4, at least 5, at least 7, at least 10, at least 50, at least 60, at least 100 or at least 150 times greater than its affinity towards the IGF-I.

In some embodiments, specific binding protein has an EC50not more than 15 nm for inhibiting IGF-I-dependent phosphorylation of IGF-I receptor in NIH3T3 cells, which ectopiceski Express IGF-1R. In some aspects, the specific binding protein has an EC50not more than 15 nm and not more than 10 nm, or not more than 8 nm for inhibiting IGF-I-dependent phosphorylation of IGF-I receptor in NIH3T3 cells, which ectopiceski, Express IGF-1R.

In some embodiments, specific binding protein has an EC50not more than 5 nm and not more than 4 nm, or not more than 3 nm for inhibiting IGF-I-dependent phosphorylation of IGF-I receptor in NIH3T3 cells, which ectopiceski Express IGF-1R.

In other embodiments, specific binding protein inhibits more than 70% of IGF-II dependent proliferation of NIH3T3 cells that Express recombinant hIGF-IR values when EC50/sub> not exceeding 25 nm, less than 20 nm, no greater than 15 nm, or not greater than 10 nm.

In other embodiments, specific binding protein inhibits more than 70% of IGF-I dependent proliferation of NIH3T3 cells that Express recombinant hIGF-IR values when EC50not exceeding 40 nm, no greater than 30 nm, no greater than 25 nm.

In some embodiments, specific binding protein competes for binding with a monoclonal antibody, which contains a sequence of variable heavy chain, which is selected from the group consisting of SEQ ID no: 2, SEQ ID no: 6, SEQ ID no: 10, SEQ ID no: 14 and SEQ ID no: 18, the sequence of the variable light chain, which is selected from the group consisting of SEQ ID no: 4, SEQ ID no; 8, SEQ ID no: 12 and SEQ ID no: 16.

One of the embodiments of the present invention is a fully human antibody that binds IGF-I with Kd, which has a value of less than 500 picomolar (PM). More preferably, the antibody binds with Kd, which has a value of less than 450 picomole (PM). More preferably, the antibody binds with Kd, which has a value of less than 410 picomole (PM). More preferably, the antibody binds with Kd, which has a value less than 350 PM. Even more preferably, the antibody binds with Kd, which has a value of less than 300 PM. Measure epinasty and/or AB�gnosti can be performed using a BIACORE ®as described in this application.

Another embodiment of this invention is a fully human antibody that binds IGF-II with Kd, which has a value of less than 175 picomole (PM). More preferably, the antibody binds with Kd, which has a value less than 100 picomolar (PM). More preferably, the antibody binds with Kd, which has a value of less than 50 picomolar (PM). More preferably, the antibody binds with Kd, which has a value of less than 5 picomoles (PM). Even more preferably, the antibody binds with Kd, which has a value of less than 2 PM.

In some embodiments, specific binding protein is a fully human monoclonal antibody or binding fragment is a fully human monoclonal antibody. Binding fragments include Fab, Fab(or F(ab')2and Fv.

One of the embodiments of this invention include fully human monoclonal antibodies 7.251.3 (the access number of ATSS MOUTH-7422), 7.34.1 (the access number of ATSS MOUTH-7423), and 7.159.2 (the access number of ATSS MOUTH-7424) that specifically bind IGF-I/II, as discussed more below.

In some embodiments, specific binding protein that binds insulin-like growth factor-II (IGF-II) and has Perek�Jan reactivity to insulin-like growth factor-I (IGF-I), or its binding fragment may include a heavy chain polypeptide having the sequence of SEQ ID no: 6 and a light chain polypeptide having the sequence of SEQ ID no: 8.

Specific binding protein can include a polypeptide heavy chain having the sequence of SEQ ID no: 10 and a light chain polypeptide having the sequence of SEQ ID no: 12.

The specific binding protein according to the invention can include a heavy chain polypeptide having the sequence of SEQ ID no: 14 and a light chain polypeptide having the sequence of SEQ ID no: 16.

In some embodiments, specific binding protein may be a mixture with a pharmaceutically acceptable carrier.

Another embodiment includes the selected nucleic acid molecule encoding any of the specific binding proteins described in this application, the vector with the selected nucleic acid molecule encoding the specific binding proteins, or the host cell transformed with any of such nucleic acid molecules and vectors.

In some embodiments, specific binding protein that binds insulin-like growth factor-II (IGF-II) and having cross-reactivity to insulin-like growth factor-I (IGF-I), or its binding fragment does not bind with�elficiency IGF-II or IGF-I proteins, if these proteins are associated with the binding proteins of the insulin growth factor.

Additional embodiments include methods for determining levels of insulin-like growth factor-II (IGF-II) and insulin-like growth factor I (IGF-I) in the patient sample. These methods can include providing a sample of a patient; contacting the sample with a specific binding protein that binds insulin-like growth factor-II (IGF-II) and having cross-reactivity to insulin-like growth factor-I (IGF-I), or its binding fragment; and determining the level of IGF-I and IGF-II in this sample. In some aspects, the disruption of the patient's blood.

Additional embodiments include methods of treating malignant tumors in mammals. These methods may include the selection of a mammal requiring the treatment of malignant tumors; and the introduction to the mammal a therapeutically effective dose of a specific binding protein that binds insulin-like growth factor-II (IGF-II) and has cross-reactivity to insulin-like growth factor-I (IGF-I), or their binding fragments. In some aspects of animal is man. In some aspects, the binding protein is a fully human monoclonal antibody, and it is chosen from the group consisting of mAb 7.251.3 (n�measures access ATSS MOUTH-7422), mAb 7.34.1 (the access number of ATSS MOUTH-7423), and mAb 7.159.2 (the access number of ATSS MOUTH-7424).

Diseases amenable to treatment, may include melanoma, non-small cell lung cancer, glioma, hepatocellular cancer, tumor thyroid, stomach cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer, pancreatic cancer and squamous cell carcinoma.

Additional embodiments include methods of treating diseases that are dependent on growth factor in mammals. These methods involve the selection of a mammal requiring the treatment of the disease; and the introduction of the specified mammal therapeutically effective dose of a specific binding protein that binds insulin-like growth factor-II (IGF-II) and has cross-reactivity to insulin-like growth factor-I (IGF-I), or their binding fragments. In some aspects, the mammal can be a human. In some aspects, the binding protein is a fully human monoclonal antibody, and it is chosen from the group consisting of mAb 7.251.3 (the access number of ATSS MOUTH-7422), mAb 7.34.1 (the access number of ATSS MOUTH-7423), and mAb 7.159.2 (the access number of ATSS MOUTH-7424).

Treatable diseases that are dependent on the growth factor may include osteoporosis, diabetes, and heart�but the cardiovascular disease. Other treatable disease state include acromegaly and gigantism, psoriasis, atherosclerosis and smooth muscle restenosis of blood vessels, and diabetes.

Additional embodiments include a conjugate consisting of a fully human monoclonal antibody that binds to insulin-like growth factor-II (IGF-II) and has cross-reactivity to insulin-like growth factor-I (IGF-I), or its binding fragment, and a therapeutic agent. In some aspects, therapeutic agent can be a toxin, a radioactive isotope, or a pharmaceutical composition.

In other embodiments, the invention provides fully human monoclonal antibody, or binding fragment that bind insulin-like growth factor-II (IGF-II) and have cross-reactivity to insulin-like growth factor-I (IGF-I), and contain hypervariable section 1 (CDR1) of the heavy chain having the amino acid sequence of "Ser Tyr Tyr Trp Ser" (SEQ ID no: 21); hypervariable segment 2 (CDR2) of the heavy chain, having the amino acid sequence of "Tight Phe Phe Tyr Ser Gly Tyr Thr Asn Tyr Asn Pro Ser Leu Lys Ser" (SEQ ID no: 22); and the hypervariable section 3 (CDR3) of the heavy chain having the amino acid sequence Ile Thr Gly Thr Thr Lys Gly Gly Met Asp Val" (SEQ ID no: 23).

Further embodiments include fully human�ical monoclonal antibodies, or binding fragment containing the hypervariable area 1 (CDR1) of the light chain having the amino acid sequence of "Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His" (SEQ ID no: 24). The antibodies in this application can also include the hypervariable segment 2 (CDR2) a light chain having the amino acid sequence of "Gly Asn Asn Asn Arg Pro Ser" (SEQ ID no: 25); and the hypervariable section 3 (CDR3) light chain having the amino acid sequence of "Gin Ser Phe Asp Ser Ser Leu Ser Gly Ser Val" (SEQ ID no: 26).

In other embodiments, the invention provides fully human monoclonal antibody, or binding fragment that bind insulin-like growth factor-II (IGF-II) and have cross-reactivity to insulin-like growth factor-I (IGF-I), and contain hypervariable section 1 (CDR1) of the heavy chain having the amino acid sequence of "Ser Tyr Tyr Trp Ser" (SEQ ID no: 27); hypervariable segment 2 (CDR2) of the heavy chain, having the amino acid sequence of "Tyr Phe Phe Tyr Ser Gly Tyr Thr Asn Tyr Asn Pro Ser Leu Lys Ser" (SEQ ID no: 28); and the hypervariable section 3 (CDR3) of the heavy chain having the amino acid sequence Ile Thr Gly Thr Thr Lys Gly Gly Met Asp Val" (SEQ ID no: 29).

Further embodiments include fully human monoclonal antibody, or binding fragment, containing the hypervariable area 1 (CDR1) of the light chain having the amino acid PEFC�dovalidate "Thr Gly Arg Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His" (SEQ ID no: 30); hypervariable segment 2 (CDR2) a light chain having the amino acid sequence of "Gly Asn Ser Asn Arg Pro Ser" (SEQ ID no: 31); and the hypervariable section 3 (CDR3) light chain having the amino acid sequence of "Gln Ser Tyr Asp Ser Ser Leu Ser Gly Ser Val" (SEQ ID no: 32).

In other embodiments, the invention provides fully human monoclonal antibody, or binding fragment that bind insulin-like growth factor-II (IGF-II) and have cross-reactivity to insulin-like growth factor-I (IGF-I), and contain hypervariable section 1 (CDR1) of the heavy chain having the amino acid sequence of "Ser Tyr Asp Ile Asn" (SEQ ID no: 33); hypervariable segment 2 (CDR2) of the heavy chain, having the amino acid sequence of "Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe Gln Gly" (SEQ ID no: 34); and the hypervariable section 3 (CDR3) of the heavy chain having the amino acid sequence of "Asp Pro Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val" (SEQ ID no: 35).

Further embodiments include fully human monoclonal antibody, or binding fragment, containing the hypervariable area 1 (CDR1) of the light chain having the amino acid sequence of "Ser Gly Ser Ser Ser Asn Ile Glu Asn Asn His Val Ser" (SEQ ID no: 36); hypervariable segment 2 (CDR2) a light chain having the amino acid sequence of "Asp Asn Asn Lys Arg Pro Ser" (SEQ ID no: 37); and the hypervariable section 3 (CDR3) light chain�and, having the amino acid sequence of "Glu Thr Trp Asp Thr Ser Leu Ser Ala Gly Arg Val" (SEQ ID no: 38).

In other embodiments, the invention provides fully human monoclonal antibodies, their bkb-binding fragment that bind insulin-like growth factor-II (IGF-II) and have cross-reactivity to insulin-like growth factor-I (IGF-I), and contain hypervariable section 1 (CDR1) of the heavy chain having the amino acid sequence of "Ser Ser Ser Tyr Tyr Trp Gly" (SEQ ID no: 81); hypervariable segment 2 (CDR2) of the heavy chain, having the amino acid sequence of "Gly Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser" (SEQ ID no: 82); and the hypervariable section 3 (CDR3) of the heavy chain having the amino acid sequence of "Gln Arg Gly His Ser Ser Gly Trp Trp Tyr Phe Asp Leu" (SEQ ID no: 83).

Further embodiments include fully human monoclonal antibody, or binding fragment, containing the hypervariable area 1 (CDR1) of the light chain having the amino acid sequence of "Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Ala" (SEQ ID no: 84); hypervariable segment 2 (CDR2) a light chain having the amino acid sequence of "Ala Ala Ser Ser Leu Gln Ser" (SEQ ID no: 85); and the hypervariable section 3 (CDR3) light chain, having the amino acid sequence of "Gln Gln Ala Asn Asn Phe Pro Phe Thr" (SEQ ID no: 86).

In other embodiments, the invention provides fully human monoc�national antibodies, or binding fragment that bind insulin-like growth factor-II (IGF-II) and have cross-reactivity to insulin-like growth factor-I (IGF-I), and contain hypervariable section 1 (CDR1) of the heavy chain having the amino acid sequence of "Ser Ser Ser Asn Tyr Trp Gly" (SEQ ID no: 87); hypervariable segment 2 (CDR2) of the heavy chain having the amino acid sequence of "Gly Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Arg Ser" (SEQ ID no: 88); and the hypervariable section 3 (CDR3) of the heavy chain having the amino acid sequence of "Gln Arg Gly His Ser Ser Gly Trp Trp Tyr Phe Asp Leu" (SEQ ID no: 89).

Further embodiments include fully human monoclonal antibody, or binding fragment, containing the hypervariable area 1 (CDR1) of the light chain having the amino acid sequence of "Arg Ala Ser Arg Gly Ile Ser Ser Trp Leu Ala" (SEQ ID no: 90); hypervariable segment 2 (CDR2) a light chain having the amino acid sequence of "Thr Ala Ser Ser Leu Gln Ser" (SEQ ID no: 91); and the hypervariable section 3 (CDR3) light chain, having the amino acid sequence of "Gln Gln Ala Asn Ser Phe Pro Phe Thr" (SEQ ID no: 92).

Some embodiments provide the use of specific binding proteins described in this application, to obtain drugs for the treatment of malignant tumors. In some aspects, the specific binding protein can performance�perform a fully human monoclonal antibody. In certain aspects, the binding protein is a mAb 7.251.3 (the access number of ATSS MOUTH-7422) or mAb 7.34.1 (the access number of ATSS MOUTH-7423) or mAb 7.159.2 (the access number of ATSS MOUTH-7424). In some aspects, a drug approved for use in combination with a second antineoplastic agent which is selected from the group consisting of antibody, chemotherapeutic agent or radioactive means. In some aspects, a drug approved for use in combination or after traditional surgery, stem cell transplantation bone marrow or transplantation of peripheral stem cells.

A malignant tumor can be, for example, melanoma, non-small cell lung cancer, glioma, hepatocellular cancer, tumor thyroid, stomach cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer, pancreatic cancer and squamous cell carcinoma.

Other embodiments provide the use of specific binding proteins described in this application, to obtain drugs for the treatment of diseases dependent growth factor. In some aspects, the specific binding protein is a fully human�mechanical monoclonal antibody and may be selected from the group consisting of mAb 7.251.3 (the access number of ATSS MOUTH-7422), mAb 7.34.1 (the access number of ATSS MOUTH-7423), and mAb 7.159.2 (the access number of ATSS MOUTH-7424).

Disease-specific growth factor can be, for example, osteoporosis, diabetes and cardiovascular disease.

Advantageously, the antibody contains the amino acid sequence of the heavy chain having the hypervariable segment (CDR) with one or more sequences given in Table 11. For example, the antibody may contain the amino acid sequence of a heavy chain having CDR1, CDR2, or CDR3 of one or more of the sequences shown in Table 11, or a combination of both. Note that the person skilled in the art can easily make the definitions of CDR. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

Embodiments in accordance with the invention described in this application relate to monoclonal antibodies that bind IGF-I/II and affect the function of IGF-I/II. Other embodiments relate to fully human anti-IGF-I/II antibodies and obtaining anti-IGF-I/II antibodies with desirable properties from the point of view of a therapeutic perspective, including high epinasty binding to IGF-I/II, the ability to neutralize IGF-I/II in vitro and in vivo, and the ability to inhibition of cell proliferation induced IGF-I/II.

DETAILED DESCRIPTION�

Embodiments in accordance with the invention described in this application, are binding proteins that specifically bind IGF-II and have cross-reactivity to IGF-I (in this application called "IGFI/II"). In some embodiments, the binding proteins are antibodies or their binding fragments, and bind IGF-II and have cross-reactivity to IGF-I and ingibiruet binding of these proteins with their receptor, IGF-IR. Other embodiments in accordance with the present invention include fully human neutralizing anti-IGF-I/II antibodies, and antibody drugs, therapeutically useful and bind both insulin-like growth factors. Such drugs are anti-IGF-I/II antibodies, predominantly, have desirable therapeutic properties, including strong epinasty binding of IGF-I/II, the ability to neutralize IGF-I/II in vitro, and the ability to inhibition caused by IGF-I/II cell proliferation in vivo.

Embodiments in accordance with this invention also include selected binding fragments of the anti-IGF-I/II antibodies. Mostly, binding fragments derived from a fully human anti-IGF-I/II antibodies. Illustrative fragments include Fv, Fab(or other well-known fragments of antibodies, as described in more detail below. Embodiments in accordance with the present invention also� include cells, that Express fully human antibodies to IGF-I/II. Examples of cells include hybridomas, or recombinante created cells, such as cells hamster ovary (Cho) cells, which produce antibodies to IGF-I/II.

In addition, embodiments in accordance with the present invention include methods of using these antibodies to treat diseases. Anti-IGF-I/II antibodies useful for the prevention of IGF-I/II is mediated by IGF-I/II signaling, inhibiting, thus, cell proliferation. The mechanism of action of this inhibition of IGF-I/II due to the binding of its receptor, IGF-IR, inhibiting the IGF-I/II-induced IGF-IR signal transmission, or increased clearance of IGF-I/II, leading to decreased effective concentration of IGF-I/II for binding to IGF-IR. Diseases amenable to treatment through this mechanism of inhibition include, without limitation listed, neoplastic diseases such as melanoma, non-small cell lung cancer, glioma, hepatocellular cancer, gynecological tumors, head and neck cancer, esophageal cancer, glioblastoma, and various types of cancer and tumors of the thyroid gland, stomach, prostate, breast, ovarian, bladder, lung, uterus, kidney, colon and pancreas, salivary gland and colon and rectum.

Other embodiments in accordance with the present invention on�companion diagnostic tests for the specific determination of the quantity of IGF-I/II in the biological sample. Analytic resources can include anti-IGF-I/II antibodies along with the necessary labels for labels such antibodies. These diagnostic assays are useful for screening of diseases associated with the growth factor, including but not limited to shows such neoplastic diseases such as melanoma, non-small cell lung cancer, glioma, hepatocellular cancer, gynecological tumors, head and neck cancer, esophageal cancer, glioblastoma, and carcinoma of the thyroid, stomach, prostate, breast, ovarian, bladder, lung, uterus, kidney, colon, and pancreas, salivary gland, and colorectum. Other non-neoplastic conditions can be acromegaly and gigantism, psoriasis, osteoporosis, atherosclerosis and smooth muscle restenosis of blood vessels, and diabetes.

Additional embodiments, features, etc., relative to anti-IGF-I/II antibodies are further described in detail below.

Sequence listing

Embodiments in accordance with the present invention include the specific anti-IGF-I/II antibodies listed below in Table 1. In this table it is reported the identification number of each anti-IGF-I/II antibodies, along with the SEQ ID number of the corresponding genes of light and heavy chains. In addition, the germline sequence of lines each derivative formed�Oh and the heavy chain are also shown in Table 1 below.

Each antibody was given an ID number, which includes two or three numbers, separated by one or two decimal points. In such cases, get several clones of a single antibody. Although the clones contain the same nukleinovokisly and the amino acid sequence of the parent sequence, they may also be listed separately, and the number of the clone shown by the number to the right of the second decimal point. Thus, for example, nukleinovokisly and the amino acid sequence of the antibody 7.159.2 identical to the sequences of the antibody 7.159.1.

As can be seen by comparing the sequences in the sequence listing, SEQ ID nos: 1-20 differ from SEQ ID nos: 39-58 as SEQ ID nos: 39-58 includes netransliruemye signal peptide and the constant region sequenced for each heavy and light chain.

TABLE 1
mAb ID no:SequenceSEQ ID no:
7.158.1The nucleotide sequence encoding the variable region of the heavy chain1
Amino acid posledovatelno�ü, encoding the variable region of the heavy chain2
The nucleotide sequence that encodes a variable region light chain3
Amino acid sequence encoding the variable region light chain4

7.159.2The nucleotide sequence encoding the variable region of the heavy chain5
Amino acid sequence encoding the variable region of the heavy chain6
The nucleotide sequence that encodes a variable region light chain7
Amino acid sequence encoding the variable region light chain8
7.34.1The nucleotide sequence encoding the variable region of the heavy chain9
Amino acid sequence encoding the variable region of the heavy chain 10
The nucleotide sequence that encodes a variable region light chain11
Amino acid sequence encoding the variable region light chain12
7.251.3The nucleotide sequence encoding the variable region of the heavy chain13
Amino acid sequence encoding the variable region of the heavy chain14
The nucleotide sequence that encodes a variable region light chain15
Amino acid sequence encoding the variable region light chain16
7.234.1The nucleotide sequence encoding the variable region of the heavy chain17
Amino acid sequence encoding the variable region of the heavy chain18
The nucleotide sequence encoding the variable region l�gcoi chain 19
Amino acid sequence encoding the variable region light chain20
7.158.1The nucleotide sequence encoding the variable region of the heavy chain39
Amino acid sequence encoding the variable region of the heavy chain40
The nucleotide sequence that encodes a variable region light chain41
Amino acid sequence encoding the variable region light chain42
7.159.2The nucleotide sequence encoding the variable region of the heavy chain43
Amino acid sequence encoding the variable region of the heavy chain44
The nucleotide sequence that encodes a variable region light chain45
Amino acid sequence, encoding�the decisive variable region light chain 46

7.34.1The nucleotide sequence encoding the variable region of the heavy chain47
Amino acid sequence encoding the variable region of the heavy chain48
The nucleotide sequence that encodes a variable region light chain49
Amino acid sequence encoding the variable region light chain50
7.251.3The nucleotide sequence encoding the variable region of the heavy chain51
Amino acid sequence encoding the variable region of the heavy chain52
The nucleotide sequence that encodes a variable region light chain53
Amino acid sequence encoding the variable region light chain54
7.234.1The nucleotide sequence encoding the variable region of the heavy chain55
Amino acid sequence encoding the variable region of the heavy chain56
The nucleotide sequence that encodes a variable region light chain57
Amino acid sequence encoding the variable region light chain58
Germ line (7.158.1)The nucleotide sequence encoding the variable region of the heavy chain59
Amino acid sequence encoding the variable region of the heavy chain60
The nucleotide sequence that encodes a variable region light chain61
Amino acid sequence encoding the variable region light chain62
Germ line (7.159.1)The nucleotide sequence�, encoding the variable region of the heavy chain63
Amino acid sequence encoding the variable region of the heavy chain64
The nucleotide sequence that encodes a variable region light chain65
Amino acid sequence encoding the variable region light chain66
Germ line (7.34.1)The nucleotide sequence encoding the variable region of the heavy chain67
Amino acid sequence encoding the variable region of the heavy chain68
The nucleotide sequence that encodes a variable region light chain69
Amino acid sequence encoding the variable region light chain70

Germ line (7.251.3)Nucleotide after�outermost, encoding the variable region of the heavy chain71
Amino acid sequence encoding the variable region of the heavy chain72
The nucleotide sequence that encodes a variable region light chain73
Amino acid sequence encoding the variable region light chain74

Definition

If not defined otherwise, technical and scientific terms used in this application, will have the meanings generally understood by the experts in this field. In addition, unless the context otherwise requires, the singular will include the plural, and the plural only. In General, the nomenclature and methods used in connection with cell and tissue cultures, molecular biology and chemistry of proteins and oligo - and polynucleotides and hybridization, described in this application are well known and widely used in this field.

For recombinant DNA, oligonucleotide synthesis and tissue culture and transformation using standard methods (e.g., electroporation and lipofection). The enzymatic reaction and m�Toda purification were performed according to the manufacturer's instructions or generally accepted practices, known in the art, or as described in this application. The above methods and techniques, in General, carried out in accordance with conventional methods, well known in the art and as described in various General and more specific references that are cited in this application, which are discussed in the description to it. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)), which is incorporated into this application by reference. Nomenclature and laboratory methods that were used in connection with analytical chemistry, synthetic organic chemistry, medical and pharmaceutical chemistry described in this application are well known and are widely used in the field. For chemical syntheses, chemical analyses, pharmaceutical preparation, composition and delivery and treatment of patients used the standard methods.

As used in accordance with this description, the following terms, unless otherwise indicated, shall be construed as having the following values.

The term "IGF-I" refers to a molecule insulin-like growth factor-I, and the term "IGF-II" refers to a molecule insulin-like factor-II. The term "IGF-I/II" refers to both molecules insulin-like growth factor s-I and-II, refers to the preferred binding of IGF-II and have Perek�Jan reactivity to IGF-I. Thus, the antibody that binds IGF-I/II, will be, preferably, to bind IGF-II, but can cross-react with IGF-I, binding of IGF-II with afinety greater than the affinity towards the IGF-I. for Example, the antibody may bind IGF-II with epinasty, 2.5 times higher than the affinity towards the IGF-I. In some embodiments, the antibody may bind IGF-II with epinasty, which is at least 5, at least 10, at least 25, at least 50 or at least about 150 times greater than the affinity towards the IGF-I.

The term "neutralizing" when used in relation to an antibody refers to the ability of the antibody to destroy, or greatly reduce, the activity of the antigen target. Accordingly, a "neutralizing" anti-IGF-I/II antibody capable of the destruction of or significant decrease in the activity of IGF-I/II. Neutralizing IGF-I/II antibody may, for example, act by blocking the binding of IGF-I/II with its receptor IGF-IR. By blocking the binding of IGF-IR-mediated signal transmission largely or completely destroyed. Ideally, a neutralizing antibody to IGF-I/II inhibits cell proliferation.

The term "selected polynucleotide", as used in this application to denote the polynucleotide that has been isolated from its natural source. Such polynucleotides may be of genomic, cDNA or synthetic. �edelenyi polynucleotides mostly not associated with all or part of polynucleotide with which they are associated in nature. The selected polynucleotide may be functionally linked to another polynucleotide to which it is not associated in nature. In addition, the selected polynucleotides, preferably, does not occur in nature as part of a larger sequence.

The term "selected polynucleotide", as indicated in this application, means a protein isolated from a natural source. Such proteins may be derived from genomic DNA, cDNA, recombinant DNA, recombinant RNA, or be of synthetic origin or represent some combination of them, where by virtue of its origin or source of a selected protein" (1) is not associated with proteins found in nature, (2) does not contain other proteins derived from the same source, i.e. not containing murine proteins, (3) is expressed by cells from different species, or (4) does not occur in nature.

The term "polypeptide" is used in this application as a generic concept to refer to the natural protein, fragments or analogs of a polypeptide sequence. Therefore, a natural protein, fragments and analogs are species of the polypeptide genus. Preferred polypeptides in accordance with this invention contain human MOLEKULYaRNAYa heavy chain and human Kappa immunoglobulin molecule light chain, as well as antibody molecules formed by combinations containing molecules of the immunoglobulin heavy chains and molecules of the immunoglobulin light chain, such as Kappa or lambda immunoglobulin molecule light chain, and Vice versa, as well as fragments and analogs. Preferred polypeptides in accordance with this invention, can also contain only molecules of the immunoglobulin heavy chain and fragments thereof and analogues.

The term which occurs in nature", as used in this application, as applied to objects, refers to the fact that the object could appear in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a natural source and has not been intentionally modified by man in the laboratory or otherwise, is one that occurs in nature.

The term "functionally linked", as used in this application, refers to the provisions for the features described in such a way that their relative position allows them to function as intended. For example, a control sequence that is "functionally linked" to a coding sequence is connected in such a way that expression of the coding sequence is achieved under conditions with�big with the control sequences.

The term "polynucleotide" as used in this application, means a polymeric form of nucleotides having at least 10 bases in length, of ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide, or hetero-duplexes of RNA and DNA. The term includes single and double stranded forms of DNA.

The term "oligonucleotide", as used in this application, includes naturally occurring and modified nucleotides linked together by relationships that occur or do not occur in nature. Oligonucleotides are a polynucleotide subset, in General consisting of 200 or fewer bases. Advantageously, the oligonucleotides having a length of 10-60 bases and most predominantly 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases. Oligonucleotides are usually single stranded, for probes; although oligonucleotides may be double stranded, e.g. for use in the construction of a gene mutant. The oligonucleotides can be semantic or antimuslim oligonucleotides.

The term "nucleotides, which occur in nature", as used in this application, includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" as used in this application, includes nucleotides with modified or substituted sugar groups, etc. T�pmin "oligonucleotide communication", as used in this application, includes oligonucleotide, such as phosphorothioate, phosphorodithioate, phosphorescent, phosphorodithioate, postranslational, phosphoramidate, phosphoroamidite, etc., for example, LaPlanche et al. Nucl. Acids Res. 14: 9081 (1986); Stec et al. J. Am. Chem. Soc. 106: 6077 (1984); Stein et al. Nucl. Acids Res. 16: 3209 (1988); Zon et al. Anti On Drug Design 6:539 (1991); Zon et al. Onuzouywieomuds and Analogues: A Practical Approach, pp.87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al. U.S. patent No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90: 543 (1990), descriptions of which are included in this application by reference. The oligonucleotide can include a label for detection, optional.

The term "selectively hybridizing" as used in this application, means-detectable and selectively bind. Polynucleotides, oligonucleotides and fragments thereof selectively hybridized in strands of nucleic acids under hybridization and washing that minimize significant number of detected binding to nonspecific nucleic acids. Very stringent conditions can be used to achieve selective hybridization conditions as known in the art and is discussed in this application. In General, nukleinovokisly homology between the polynucleotides, oligonucleotides, or fragments of antibodies and nucleic acid, which is considered, is at least 80%, and more typically, priemuschestvenno greater homology, which is at least, 85%, 90%, 95%, 99% and 100%.

Two amino acid sequences are "homologous" if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% amino acid identical if two sequences are linearized for the maximum match. To maximize the overlap of allowed gaps (in one of the sequences that are the same); the preferred length of the gap is 5 or less, more preferably 2 or less. Alternative and mainly, two protein sequences (or polypeptide sequences derived from them of length at least about 30 amino acids) are homologous, meaning of term used in this application, if the score of the alignment is more than 5 (in standard deviation units) using the program ALIGN with the mutation matrix data and gap penalties, amounting to 6 or more. Cm. Dayhoff, M. O., in Atlas of Protein Sequence and Structure, pp.101-110 (Volume 5, National Biomedical Research Foundation (1972)) and Supplement 2 to this volume, pp.1-10. Two sequences or parts thereof are more preferably homologous if their amino acids have an identity of 50% or more under optimal alignment using the ALIGN program. It should be appreciated that there may be times�ary region of homology in two antologicheskih sequences. For example, the functional sites of orthology mice and humans may have a higher degree of homology than non-functional regions.

The term "corresponds" is used in this application to denote the homology (i.e. identity, not simply evolutionary relationships) polynucleotide sequence of all or a portion of the reference polynucleotide sequence or polynucleotide sequence identical to a reference polypeptide sequence.

On the contrary, the term "complementary to" is used in this application to mean that the complementary sequence is homologous to all or a portion of the reference polynucleotide sequence. As an illustration, the nucleotide sequence "TATAC" corresponds to a reference sequence "TATAC" and is complementary to the reference sequence "GTATA".

The following terms are used to describe the relationship of sequences of two or more polynucleotide or amino acid sequences: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity." "Reference sequence" is a defined sequence used on the basis of comparison �of posledovatelnostei. The reference sequence may be a subset of a larger sequence, for example, the segment of full-size cDNA or gene sequence given in the sequence listing, or may contain a full-sized DNA or gene sequence. In General, the reference sequence contains at least 18 nucleotides or 6 amino acids, often at least 24 nucleotides or 8 amino acids, often at least 48 nucleotides or 16 amino acids. Since each of the two polynucleotides or amino acid sequences may (1) contain a sequence (i.e., a portion of the complete polynucleotide or amino acid sequence) that is similar in the two molecules, and (2) may further comprise a sequence which differs in the two polynucleotides or amino acid sequence, comparison of sequences between two (or more) molecules are typically performed by comparing sequences of the two molecules in the "comparison window" to identify and compare local regions of sequence identity. A "comparison window", as used in this application, refers to a conceptual segment of at least approximately 18 adjacent nucleotide positions, or about 6 amino acids, where the polynucleotide sequence or �aminokislotnoi sequence compared to a reference sequence, at least 18 neighbouring nucleotide, or 6 amino acid sequences and where the portion of the polynucleotide sequence in the comparison window may include additions, deletions, substitutions, etc. (i.e., gaps) of 20 percent or less compared to the reference sequence (which does not include additions or deletions) for optimal alignment of two sequences. Optimal alignment of sequences for aligning in the window of comparison may be conducted using the algorithm of the local homology of Smith and Waterman Adv. Appl. Math. 2:482 (1981), using the algorithm of the homology alignment Needleman and Wunsch J. Mol. Biol. 48: 443 (1970), by finding the method similarity Pearson and Lipman Proc. Natl. Acad. Sci. (U. S. A.) 85: 2444 (1988), by computerized implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0 (Genetics Computer Group, 575 Science Dr., Madison, Wis.), GENEWORKS™, or software MACVECTOR®), or choose by checking, or the best alignment (i.e. resulting in the highest percentage homology in the window of comparison), which can be obtained by applying different methods.

The term "sequence identity" means that two polynucleotide or amino acid sequences are identical (i.e., that the basis of the nucleotide-nucleotide or residue-residue) in the window of comparison. The term "percent identity pic�of egovernance" is calculated by comparing two optimally aligned sequences in the comparison window, to determine the number of provisions that are identical nukleinovokisly base (e.g. A, T, C, G, U, or I) or amino acid residues in the two sequences to get the number of matched positions, dividing the number of matched positions on total number of positions in the comparison window (i.e. the window size) and multiplying by 100 to obtain a percent identity of the sequences. The terms "substantial identity" as used in this application, means a characteristic of a polynucleotide or amino acid sequence, where the polynucleotide or amino acid contains a sequence that has at least 85 percent sequence identity, preferably at least 90-95% sequence identity, more preferably at least 99% sequence identity, compared to the reference sequence in the comparison window of at least 18 nucleotide (6 amino acid) positions, frequently in a window of at least 24-48 nucleotide (8-16 amino acid) positions, where the percentage of sequence identity is calculated by comparing the reference sequence to the sequence which may include deletions or additions, comprises a total of 20 percent or less from the reference sequence�eljnosti in the window of comparison. The reference sequence may be a subset of a larger sequence.

As used in this application, the twenty-traditional amino acids and their abbreviations are traditional uses. Cm. Immunology - A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)) included in this application by reference. Stereoisomers (e.g., D-amino acids) of the twenty-traditional amino acids, unnatural amino acids, such as α-, α-disubstituted amino acids, N-alkyl amino acids, laktonovye acid, and other unconventional amino acids may also be suitable components for polypeptides in accordance with the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetylized, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine and other similar amino - and aminokisloty (for example, 4-hydroxyproline). When referring to the polypeptides used in this application, the left-hand direction is the N-terminal direction and the right - hand direction is the carboxy-terminal direction, in accordance with standard usage.

Similarly, unless otherwise indicated, the left end of the single-stranded polynucleotide sequences is the 5(end; levot�side direction of the double-stranded polynucleotide sequence is called 5(direction. The direction 5'-3(adding the resulting RNA transcripts is called the direction of transcription; the field sequence of the DNA strand having the same sequence as the RNA and which are 5'-5(the ends of the RNA transcript are referred to as "upper sequences"; sequences pane of the DNA strand having the same sequence as the RNA and which are 3'-3(the ends of the RNA transcript are referred to as "lower sequences".

As applied to polypeptides, the term "substantially identical" means that the peptide sequences, when optimally aligned, for example, using the programs GAP or BESTFIT using the weight of the gaps by default, have at least 80 percent sequence identity, preferably at least 90% sequence identity, more preferably at least 95 percent sequence identity, and most predominantly, at least 99 percent sequence identity. Mainly, the provisions of residues that are not identical differ by conservative amino acid substitutions. Conservative amino acid substitution refers to the interchangeability of residues with similar side chains. For example, a group of amino acids having aliphatic Bokova� chain represents glycine, alanine, valine, leucine and isoleucine; a group of amino acids having alifaticheskii-hydroxyl side chains is a serine and threonine; a group of amino acids having amide-containing side chains, represents asparagine and glutamine; a group of amino acids having aromatic side chains is a phenylalanine, tyrosine and tryptophan; a group of amino acids having basic side chains is a lysine, arginine and histidine; a group of amino acids having sulfur-containing side chains, represents cysteine and methionine. Preferred substitution groups of conservative amino acids are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutama-barkanova, and asparagine-glutamine.

As discussed in this application, the minimum variation of the amino acid sequences of antibodies or immunoglobulin molecules are considered as such, which are described in the present invention, provided that the variations of the amino acid sequence maintain at least 75%, more preferably at least, 80%, 90%, 95%, and most prevalently, 99% identity to the sequences of antibodies or immunoglobulin molecules described in this application. In particular, we consider a conservative amino acid substitution. Conservati�governmental substitutions are that occur in the family of amino acids with similar side chains. Genetically encoded amino acids, in General, are divided into families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred families are: serine and threonine are alifaticheskii-hydroxy family; asparagine and glutamine are an amide-containing family; alanine, valine, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan and tyrosine are an aromatic family. For example, it is reasonable to expect that the selected substitution of leucine for isoleucine or valine, aspartate for glutamate, threonine for serine, or a similar replacement of an amino acid at a structurally related amino acid will not have much impact on the function of binding or properties of the resulting molecule, especially if the substituting amino acid is not involved in wireframe website. Could the substitution of amino acids lead to the formation of functional peptide can be easily determined by analyzing the specific activity of the derived polypeptide. Analyses in detail the OPI�Ana in this application. Fragments or analogs of antibodies or immunoglobulin molecules can be readily obtained by the skilled in the art. The predominant amino - and carboxy-ends of the fragments or analogues are located near boundaries of functional domains. Structural and functional domains can be identified by comparing data of the nucleotide and/or amino acid sequences that are present in public or private databases of sequences. Advantageously, the use of computerized methods for comparison to identify fragments of sequences or predicted conformations of the protein domains that occur in other proteins of known structure and/or function. Known methods of identification of protein sequence covered by the known three-dimensional structure. Bowie et al. Science 253: 164 (1991). Therefore, these examples demonstrate that the person skilled in the art may recognize fragments of the sequences and structural conformations that may be used to define structural and functional domains in accordance with the antibodies described in this application.

Predominant amino acid substitutions are those in which: (1) reduced sensitivity to proteolysis, (2) reduced sensitivity to oxidation, (3) change�is epinasty binding for the formation of protein complexes, (4) changes epinasty binding, and (5) are formed or modify other physicochemical or functional properties of such analogs. Analogs can include various mutant sequences that differ from the peptide sequences found in nature. For example, single or multiple amino acid substitution (mainly conservative amino acid substitution) can be made in the sequences that occur in nature (mainly the portion of the polypeptide outside the domain (domain)) form intermolecular contacts. Conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., amino acid substitution should not be directed at the destruction of helix present in the parent sequence, or the breaking of other types of secondary structures that characterize the parent sequence). Examples of recognized technical level of secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et at. Nature 354: 105 (1991), each of which is included in this application by reference.

The term "polypeptide fragment" as used in this application, refers to a polypeptide�, having N-terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence identical to the corresponding provisions of the sequences found in nature, obtained, for example, from full-size cDNA sequence. The fragments typically have a length of at least 5, 6, 8 or 10 amino acids, preferably at least 14 amino acids, more preferably at least 20 amino acids, usually at least 50 amino acids, even more preferably at least 70 amino acids. The term "analog" as used in this application, refers to the polypeptides, which consist of a segment of at least 25 amino acids that has substantial identity with a part of the obtained amino acid sequence and which have at least one of the following properties: (1) the specific binding of IGF-I/II, in a suitable binding conditions, (2) ability to block appropriate IGF-I/II binding, or (3) the ability to inhibit the activity of IGF-I/II. Typically, polypeptide analogs contain conservative amino acid substitution (or addition or deletion) in relation to the sequence that occurs in nature. Counterparts, typically have a minimum length of at least 20 amino acids, preferably at least 50 amino acids or more, and can often have the same length, h�about and a full-sized polypeptide, found in nature.

Peptide analogs are traditionally used in the pharmaceutical industry as non-peptide drugs with properties analogous to the properties of the template peptides. These types of non-peptide compounds are termed "peptidomimetic" or "peptide mimetics". Fauchere, J. Adv. Drug Res. 15: 29 (1986); Veber and Freidinger TINS p.392 (1985); and Evans et al. J. Med. Chem. 30: 1229 (1987), which is included in this application by reference. Such connections often develop with the help of computer molecular modeling. Peptidomimetics that are structurally similar to therapeutically useful peptides may be useful to obtain an equivalent therapeutic or prophylactic effect. In General, peptidomimetics, structurally similar to the reference polypeptide (i.e. a polypeptide that has a biochemical property or pharmacological activity), such as human antibody, but have one or more peptide linkages optionally replaced by a linkage, which is selected from the group consisting of: --CH2NH--, --CH2S--, --CH2-CH2--, -CH=CH-- (CIS and TRANS), --COCH2--, --CH(OH)CH2- and-CH2SO--, by methods well known in the art. Systematic substitution of one or more contiguous sequence of amino acids to D-amino acid of the same type (e.g., D-lysine instead of L-lysine) can be used DL� more stable peptides. In addition, restricted peptides containing a continuous sequence or a substantially identical variation continuous sequence, can be obtained by methods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61: 387 (1992), included in this application by reference); for example, by adding internal cysteine residues capable of forming intramolecular sulfide bridges, cyclessa peptide.

As used in this application, the term "antibody" refers to a polypeptide or group of polypeptides, consisting of at least one binding domain, formed by folding of polypeptide chains having three-dimensional linking of the distance, the shape of the inner surfaces and the charge distribution which is complementary to the characteristics of the antigenic determinants of the antigen. The antibody typically has a tetrameric form, contains two identical pairs of polypeptide chains, each pair has one "easy" and one "heavy" chain. The variable regions of each light/heavy chain form a binding site of the antibody.

"Binding fragments of antibodies are produced by recombinant DNA techniques or enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv, and single chain antibodies. An antibody other than a "bispecific" and�and "bifunctional" antibody interpret it as such, in which all binding sites are identical. An antibody substantially inhibits adhesion of a receptor to protivorechathie, if an excess of antibody reduces the quantity of receptor that is associated with protivorechivogo, at least approximately, 20%, 40%, 60% or 80%, or more traditionally, more than about 85% (in accordance with measurement results of the competitive analysis of binding in vitro).

As used in this application, the "binding protein" or "specifically binding the protein are proteins that specifically bind the molecule-target. Antibodies and binding fragments of the antibodies are binding proteins.

The term "epitope" includes any protein determinant capable of specifically bind immunoglobulin or receptor of T cells. Epitope determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and can, but not always, have specific three dimensional structural characteristics, as well as specific charging characteristics. They say that the antibody specifically binds an antigen when the dissociation constant is ≤1 µm, preferably ≤100 nm and most predominantly ≤10 nm.

The term "agent" used in this application to refer to chemical compounds, mixtures of chemical compounds, biologists�certification macromolecule, or extract derived from biomaterial.

"Active" or "activity" in relation to IGF-I/II polypeptide refers to a piece of IGF-I/II polypeptide having a biological or immunological activity of natural IGF-I/II polypeptide. "Biological", as used in this application, refers to a biological function, which is the result of natural activity of IGF-I/II polypeptide. The predominant biological activity of IGF-I/II includes, for example, IGF-I/II-induced cell proliferation.

"Mammal" as used in this application, refers to any animal that is considered to be mammals. Advantageously, the mammal is man.

Digestie antibodies by enzyme, papain, results in two identical antigen-binding fragments, known also as "Fab" fragments, and a "Fc" fragment, having no antigen-binding activity but having the ability to crystallization. Digestie antibodies by enzyme, pepsin, results in the formation of F(ab')2the fragment in which two branches antibodies remain linked and comprise two-antigen binding sites. The fragment F(ab')2has the ability to cross-link the antigen.

"Fv", as used in this application, refers to the minimum fragment of an antibody that retains both antigen-recognizing and ant�gene-binding sites.

"Fab," as used in this application, refers to a fragment of the antibody that contains the constant domain of the light chain and CH1 domain of the heavy chain.

The term "mAb" refers to a monoclonal antibody.

"Liposome" as used in this application, refers to a small vesicle that may be used for drug delivery, and may include IGF-I/II polypeptides according to the invention or antibodies to such an IGF-I/II polypeptide in mammals.

"Label" or "labeled", as used in this application, refers to the addition of apparently detected group to the polypeptide, for example, radioactive labels, fluorescent labels, enzyme labels, chemiluminescent labels or biodieselnow group. Radioactive isotopes or radionuclides may include a3N,14C,15N,35S,90Y99Cu111In125I,131L fluorescent labels may include rhodamine, the lanthanides, phosphorus or FITC and enzymatic labels may include horseradish peroxidase, β-galactosidase, luciferase, and alkaline phosphatase.

The term "pharmaceutical agent or drug" as used in this application, refers to a chemical compound or composition capable of causing the desired therapeutic effect when properly administered to a patient. Other chemical terms in this application use, as this is customary in this field, as shown in The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)), (included in this application by reference).

As used in this application, "substantially pure" means a dominant presence kinds of objects (i.e. with a molar perspective, a larger presence than any other individual species in the composition), and, preferably, a substantially purified fraction is a composition where the types of objects comprise at least about 50% (molar point of view) of all macromolecular species present. In General, a substantially pure composition will contain more than approximately 80% of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95% and 99%. Most advantageously, the object species is purified to essential homogeneity (no contamination can be detected in the composition by conventional detection methods), and the composition consists essentially of a single macromolecular species.

The term "patient" includes human and veterinary facilities.

Human antibodies and humanization of antibodies

The human antibodies are absent some of the problems associated with antibodies that are available from murine or rat variable and/or constant regions. The presence of such obtained from mice or rats proteins �can lead to rapid clearance of the antibodies or the immune response of the patient to the antibody. Avoid the use of antibodies derived from mice or rats, can be derived fully human antibody by introduction of functional loci of human antibodies in rodent, other mammal or animal so that the mammal or the animal had produced fully human antibodies.

One way of obtaining fully human antibodies is the use of strains of XenoMouse mice®designed to contain less than 1000 kb-sized germline lines configured fragments of the locus, the human heavy chain and Kappa locus light chain. Cm. Mendez et al. Nature Genetics 15: 146-156 (1997) and Green and Jakobovits J. Exp. Med. 188: 483-495 (1998). The XenoMouse strains®available from Abgenix, Inc. (Fremont, CA).

Obtaining strains of XenoMouse mice®is discussed further in United States patent application Nos. 07/466,008, filed January 12, 1990, 07/610,515, filed November 8, 1990, 07/919,297, filed July 24, 1992, 07/922,649, filed July 30, 1992, 08/031,801, filed March 15, 1993, 08/112,848, filed August 27, 1993, 08/234,145, filed April 28, 1994, 08/376,279, filed January 20, 1995, 08/430, 938, filed April 27, 1995, 08/464,584, filed June 5, 1995, 08/464,582, filed June 5, 1995, 08/463,191, filed June 5, 1995, 08/462,837, filed June 5, 1995, 08/486,853, filed June 5, 1995, 1995, 08/486,857, filed June 5, 1995, 08/486,859, filed June 5, 1995, 08/462,513, filed yuna 1995 08/724,752, filed October 2, 1996, 08/759,620, filed December 3, 1996, publications of the U.S. 2003/0093820, filed November 30, 2001, and U.S. patents№№6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 and in Japanese patents Nos. 3068180 B2, 3068506 B2, and 3068507 B2. Cm. also European patent number EP 0463151 B1, issued June 12, 1996, international patent application no WO 94/02602, published February 3, 1994, international patent application no WO 96/34096, published October 31, 1996, WO 98/24893, published June 11, 1998, WO 00/76310 published December 21, 2000. Descriptions of each of the aforementioned patents, applications and references is included in its entirety in this application by reference.

In an alternative approach, others, including GenPharm International, Inc., used "miniatury" approach. In this approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Therefore, one or more VHgenes, one or more DHgenes, one or more JHgenes, a mu constant region, and usually a second constant region (mainly gamma constant region) to form a construct for insertion into an animal. This patent is described in U.S. patent No. 5,545,807, issued Surani et al., and U.S. patents№№5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458, each of which issued to Lonberg and Kay, U.S. patent No. 5,591,669 and 6,023.010 issued to Krimpenfort and Berns, U.S. patents Nos. 5,612,205, 5,721,367 and 5,789,215 you�data Berns et al., and U.S. patent No. 5,643,763 issued to Choi and Dunn, and United States patent application GenPharm International, serial No. 07/574,748 filed August 29, 1990, 07/575,962, filed August 31, 1990, 07/810,279 filed 17 December 1991, 07/853,408, filed March 18, 1992, 07/904,068 filed 23 June 1992, 07/990,860, filed December 16, 1992, 08/053,131, filed April 26, 1993, 08/096,762, filed July 22, 1993, 08/155,301 filed 18 November 1993, 08/161,739, filed December 3, 1993, 08/165,699, filed December 10, 1993, 08/209,741, filed March 9, 1994, the description of which is included in this application by reference. Cm. also European patent No. 0546073 B1, international patent application Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, WO 98/24884 and U.S. patent No. 5,981,175, the descriptions of which are fully incorporated into this application by reference. Cm. also Taylor et al., 1992, Chen et al., 1993, Tuaillon et al., 1993, Choi et al., 1993, Lonberg et al., (1994), Taylor et al., (1994), and Tuaillon et al., (1995), Fishwild et al., (1996), the disclosures of which are fully incorporated in this application by reference.

Kirin also demonstrated obtaining human antibodies from mice in which, through microcell fusion, were introduced in large parts of chromosomes or whole chromosomes. Cm. European patent application No. 773288 and 843961, the descriptions of which are included in this application by reference. Additionally, there were obtained km™mouse obtained by crossing mice Kirin TC minilogue Medarex mice (Huma). These mice have the human transhumanity IgH Kirin mice and the Kappa chain transgene of the Genpharm mice (Ishida et al., Cloning Stem Cells(2002), 4: 91-102).

Human antibodies can also be obtained by in vitro methods. Acceptable examples include, without limitation listed, phage display (CAT, Morphosys, Dyax, Biosite/Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed) ribosomal display (CAT), yeast display, etc.

Antibodies

Antibodies, as described in this application were generated using XenoMouse technology®, described below. Such mice are then capable of producing human immunoglobulin molecules and antibodies, deficient production of murine immunoglobulin molecules and antibodies. Technologies used for this are described in the patents, applications and references described in the section "Background of invention" section of this application. In particular, however, the preferred embodiment of transgenic production of mice and antibodies described in the patent application U.S. serial number No. 08/759,620, filed December 3, 1996 and international patent application Nos. WO 98/24893, published June 11, 1998 and WO 00/76310 published December 21, 2000, the descriptions of which are included in this application by reference. Cm. also Mendez et al., Nature Genetics 15: 146-156 (1997), descriptions of which are included in this application by reference.

Through the use of such technology were p�derived fully human monoclonal antibodies to many antigens. Mainly, lines of mice XenoMouse® immunized antigen, which is treated (for example, IGF-I/II), lymphatic cells (such as b-cells) are recovered from the Hyper-immunized mice, and the recovered lymphocytes are fused with a cell line of myeloid type to gain immortal hybrid cell lines. These hybrid cell lines have been screened and selected to identify cell lines that produced antibodies specific to the antigen that is being considered. In this application are provided methods of producing multiple hybrid cell lines that produce antibodies specific to IGF-I/II. Additionally, in this application are provided with the characteristics of the antibodies produced by such cell lines, including the analysis of nucleotide and amino acid sequences of the heavy and light chains of such antibodies.

Alternatively, instead of merging with myeloma cells to obtain hybridomas, cells can be analyzed directly. For example, CD19+B cells can be isolated from hyperimmune XenoMouse mice®and left for proliferation and differentiation into antibody-secretory plasma cells. Antibodies from the cell supernatants were then subjected to screening using ELISA for reactivity against IG-I/II to the immunogen. The supernatants might also be subjected to screening for immunoreactivity against fragments of IGF-I/II for an additional application to map the different antibodies for binding to domains of functional interest IGF-I/II. Antibodies may also be subjected to screening for other related human chemokines and against rats, mice and nonhuman primates, such as cynomolgus monkeys, orthologic to IGF-I/II, the last to determine the cross-reactivity species. In the cells from the wells containing antibodies, which are considered, can be immortality variety of ways, including merging to obtain a hybrid individual or joint holes, or by EBV infection or transfection of known genes immortalization, and subsequent placement in an acceptable environment. Alternative, single plasma cells secreting antibodies with the desired properties, then was extracted with the use of IGF-I/II-specific hemolytic analysis of belascoaran (Babcook et al., Proc. Natl. Acad. Sci. USA 93: 7843-48 (1996)). Targets of lysis are mainly red blood cells of sheep (SRBCs) coated with the antigen IGF-I/II.

In the presence of the culture of B-cells containing plasma cells secreting the immunoglobulin, which is considered, and complement, the formation of plaques indicates Oper�devanny-specific IGF-I/II lysis of red blood cells of sheep, surrounded by plasma cells, which are considered. Individual antigen-specific plasma cell in the center of the plaque can be selected, and the genetic information that encodes the specificity of the antibodies isolated from single cells of the plasma. Using reverse transcription followed by PCR (RT-PCR), can be cloned DNA that encodes the variable regions of the heavy and light chains of the antibody. Such cloned DNA can then be inserted in a suitable expression vector, preferably, a vector cassette such as rdnk, more preferably such as rdnk a vector containing the constant domains of the heavy and light chains of immunoglobulin. The generated vector can then be transfection in the host cell, for example, NECK cells, Cho cells, and cultured in a traditional nutrient medium, modified appropriately to induce transcription, selection of transformants, or amplification of genes encoding the desired sequences.

In General, antibodies that have been produced merged by the hybridomas were of the heavy chain of human IgG2 with fully human Kappa or lambda light chains. The antibodies described in this application, have a human IgG4 heavy chain and IgG2 heavy chain. Antibodies can also be antibodies other human isotypes, including� IgG1. Antibodies have a high epinasty, typically, have a Kd from about 10-6to around 10-10M or below, the measurements of the solid-phase or liquid-phase methods. Antibodies having a KD of at least 10-11M are preferred to inhibit the activity of IGF-MI.

As will be appreciated, the anti-IGF-I/II antibodies can be expressed in cell lines that are different from hybrid cell lines. Sequences encoding particular antibodies can be used to transform an acceptable host cells of mammals. Transformation can be carried out by any known methods of introducing polynucleotides into a host, including, for example, packaging the polynucleotide in a virus (or viral vector) and transuterine the host with the virus (or vector) or by transfection methods known in the art as shown in U.S. patent No. 4,399,216, 4,912,040, 4,740,461 and 4,959,455 (these patents are incorporated in this application by reference). The transformation procedure used here, depends on the host that will be transformed. Ways of introduction of heterological polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, deposition of calcium phosphate, polybrene-mediated transfection, protoplast merger, �electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

Mammalian cell lines that are present as hosts for the expression, well known in the art and include many immortalisant cell lines available in the American type culture collection (of ATS), including but not limited to shows, cages hamster ovary (Cho) cells, HeLa cells, kidney cells baby hamster (BHK) cells, monkey kidney (COS) cells, human hepatocellular cancer (e.g., Hep G2), epithelial cells, human kidney 293 and other cell lines. Particularly advantageous cell lines are selected by identifying cell lines with high expression levels, which produce antibodies with significant IGF-I/II binding properties.

Anti-IGF-I/II antibodies useful for the detection of IGF-I/II in the samples of patients and, accordingly, useful for the diagnosis of disease States, as described in this application. Furthermore, based on their ability to significantly neutralize the activity of IGF-I/II (as shown in the Examples below), anti-IGF-I/II antibodies have therapeutic effects for the treatment of symptoms and conditions that result in expression of IGF-I/II. In specific embodiments, the antibodies and methods described in this application relate to the treatment� symptoms the resulting cell proliferation induced IGF-I/II. Additional embodiments include the use of antibodies and the techniques described in this application, for the treatment of diseases, including neoplastic diseases, such as melanoma, non-small cell lung cancer, glioma, hepatocellular cancer, tumor thyroid, stomach cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer, gynecological tumors, head and neck cancer, esophageal cancer and pancreatic cancer. Other non-neoplastic painful conditions can be acromegaly and gigantism, psoriasis, osteoporosis, atherosclerosis and smooth muscle restenosis of blood vessels, and diabetes.

Therapeutic introduction and composition

Embodiments in accordance with the present invention include sterile pharmaceutical compositions of anti-IGF-I/II antibodies, useful for the treatment of diseases. Such compositions can inhibit the binding of IGF-I/II with its receptor IGF-IR, providing, thus, effectively treating pathological conditions where, for example, abnormally elevated serum or tissue IGF-I/II. Anti-IGF-I/II antibodies, predominantly, have a corresponding epinasty for possible neutralization of IGF-I/II, and, mostly, have the adequate�capital duration of action, to reduce dosage for humans. Long period of exposure will reduce the frequency of injections by the use of alternative routes of parenteral administration such as subcutaneous or intramuscular.

Sterile compositions can be created, for example, by filtration through sterile filtration membranes, prior to or after lyophilizate and recovery of the antibody. Usually the body is stored in the lyophilized form or in solution. Therapeutic compositions of antibodies, in General, placed in a container with a sterile access port, for example, a container or vial with intravenous solution with an adapter that allows you to extract composition, in which the tube is permeable to the needle for subcutaneous injections.

The route of administration of the antibody corresponds to known methods, for example intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intrathecal, inhalation introduction or infusion, or the introduction or injection into the affected tissue, or by sustained release systems as noted below. Antibody, preferably administered continuously by infusion or bolus injection.

An effective amount of the antibody that will be entered therapeutically, depends on your goals of treatment, route of administration, and �condition of the patient. Accordingly, priority is that the doctor has titrated the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. Typically, the doctor will inject the antibody until a dosage to have the desired effect. The success of treatment is easy to estimate using traditional methods of analysis, or methods of analysis described below in this application.

Antibodies, as described in this application can be obtained in a mixture with a pharmaceutically acceptable carrier. This therapeutic composition may be administered intravenously or through the nose or through the lungs, primarily as liquid or powder aerosol (lyophilized). The composition may also be introduced parenterally or subcutaneously, as desired. With systemic administration, therapeutic composition should be sterile, does not contain pyrogen free and be in a parenterally acceptable solution with acceptable pH, isotonicity and stability. These conditions known to specialists in this field. Briefly, dosage formulations of the compounds described in this application, prepared for storage or administration by mixing the compound desired degree of purity with physiologically acceptable carriers, excipients or stabilizers. These materials are not toxic to patients at the dosages koncentracija, they are used, and these buffers can contain as TRIS HCl, phosphate, citrate, acetate and other organic salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) peptides such as polyalanine, proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamina acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating drugs, such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium and/or nonionic surfactants such as TWEEN, PLURONICS or polyethyleneglycol.

Sterile compositions for injection can be formulated according to conventional pharmaceutical procedures, as described in Remington: The Science and Practice of Pharmacy (20thed, Lippincott Williams &Wilkens Publishers (2003)). For example, desirable can be a solution or suspension of the active substance in the basis, such as water or natural vegetable oil, such as sesame, coconut or cotton, or synthetic fatty bases, such as ethyloleate, etc., Buffers, preservatives, antioxidants, etc. may be included in accordance with FA�matemticas procedures.

Acceptable examples of sustained release preparations include semi-impermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) as described by Langer et al., J. Biomed Mater. Res. (1981), 15: 167-277 and Langer, Chem. Tech., (1982) 12: 98-105, or poly(vinyl alcohol)), polylactic acid called PLA (Pat. U.S. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, (1983) 22: 547-556), non-biodegradable ethylene-vinyl acetate (Langer et al., supra), degradable copolymers of lactic and glycolic acids, such as LUPRON Depot™ (microspheres for injection, consisting of a copolymer of lactic and glycolic acid and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid (EP 133,988).

While such polymers as ethylene-vinyl acetate, lactic acid and glycolic acid, are capable of releasing molecules for over 100 days, certain hydrogels release proteins for shorter time. When encapsulated proteins remain in the body for a longer period of time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. For with�abilitati protein can be a rational strategy depending on the mechanism. For example, if you discovered that the mechanism of aggregation is the formation of intramolecular S-S linkages through disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilization from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

The sustained release composition also contain preparations of antibody crystals suspended in an acceptable composition, is able to maintain the crystals in suspension. These drugs when administered subcutaneously or intraperitoneally can also cause a slow release effect. Other compositions also contain the captured antibodies. Liposomes containing such antibodies, prepared by methods known from: U.S. patent No. DE 3,218,121; Epstein et al., Proc. Natl Acad. Sci. USA (1985) 82: 3688-3692; Hwang et al., Proc. Natl Acad. Sci. USA (1980) 77: 4030-4034; EP 52,322; EP 36,676; EP 88,046; EP 143,949; 142,641; Japanese patent application 83-118008; U.S. patents №№4,485,045 and 4,544,545; and EP 102,324.

The dosage of these compositions of antibodies for a given patient will be determined by attending physician, considering various factors known to modify the effects of drugs including severity and type of disease, body weight, sex, diet, time and route of administration, other pharmaceutical means�and relevant clinical factors. Therapeutically effective dosages may be determined by methods in vitro or in vivo.

An effective amount of the antibodies described in this application, for therapeutic use will depend on, for example, from therapeutic objectives, the route of administration and patient status. Accordingly, for the doctor will be the predominant titration of dosage and modify the route of administration, is required to obtain the optimal therapeutic effect. A typical daily dose can be in the range from approximately 0.001 mg/kg to 100 mg/kg or more, depending on the listed factors. Typically, the doctor will inject therapeutic antibody until a dosage is reached that achieves the desired effect. The success of treatment is easy to estimate using traditional methods of analysis or as described in this application.

It will be appreciated that the introduction of therapeutic facilities in accordance with the compositions and methods of this application will be implemented with acceptable carriers, fillers and other agents included in the formulation to provide improved transfer, delivery, tolerance, and so on. These compositions include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids (cationic or anionic) containing a base (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, emulsion�AI type "oil in water" and "water in oil", emulsions carbowax (polyethylene glycols with different molecular weight), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate for treatment and therapy in accordance with this invention, provided that the active ingredient in the composition is not activated composition, and the composition is physiologically compatible and portable with this route of administration. Cm. also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. Toxicol. Pharmacol. 32(2): 210-8 (2000), Wang W. "Lyophilization and development of solid protein pharmaceuticals." Int. J. Pharm. 203 (1-2): 1-60 (2000), Charman WN "Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts." J Pharm Sci.89(8): 967-78 (2000), Powell et al. "Compendium of excipients for parenteral formulations" PDA J Pharm Sci Technol. 52: 238-311 (1998) and references there for more information concerning compositions, fillers and carriers, known to chemists-pharmacists.

Development and creation of other drugs

In accordance with this invention and based on the activity of antibodies, prepared and characterized in this application in relation to IGF-I/II, development of other therapeutic modalities is facilitated and described by specialists in this field. Such modalities include, without limitation, advanced drugs based on antibodies, such as bespecifically antibodies, immunotoxin, radioactively labeled tools and �dinnie domains of antibodies V, the antibody-like binding agents based on other fields than the field V, and the creation of peptide drugs, gene therapy, especially intrathalamic, antisense therapy and small molecules.

In connection with the establishment of advanced therapy with antibodies, where the fixation of complement is desirable, possible independence from complement killing of cells by using, for example, bespecifically antibodies, immunotoxins or radioactive labels.

For example, can be obtained bespecifically antibodies that contain (i) two antibodies, one specific to the IGF-I/II, and another antibody specific to another molecule, which konjugierten together, (ii) a single antibody that has one chain specific to IGF-I/II and which chain specific to a second molecule, or (iii) single-stranded antibody, specificity both in relation to IGF-I/II, and in relation to another molecule. Such bespecifically antibodies can be obtained using techniques which are well known in the art; for example, in connection with (i) and (ii) see e.g., Fanger et al. Immunol Methods 4: 72-81 (1994) and Wright and Harris, supra. And in connection with (iii) see e.g., Traunecker et al. Int. J. Cancer (Suppl.) 7: 51-52 (1992). In each case, the second specificity can be obtained when needed. For example, the second specificity can be obtained for receptorpositive heavy chain, including, without limitation, CD16 or CD64 (see e.g., Deo et al., 18: 127 (1997)) or CD89 (see, e.g., Valerius et al. Blood 90: 4485-4492 (1997)).

Antibodies can also be modified to act as immunotoxins, using methods known in the art. See, for example, Vitetta Immunol Today 14: 252 (1993). Cm. also U.S. patent No. 5,194,594. In connection with the receipt of radioactively labeled antibodies, such modified antibodies can also be easily obtained using techniques that are well known in the art. See, for example, Junghans et al. in Cancer Chemotherapy and Biotherapy 655-686 (2d edition, Chafner and Longo, eds., Lippincott Raven (1996)). Cm. also U.S. patent№№4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471 and 5,697,902. Each of immunotoxins and radioactively labeled molecules, likely to kill cells expressing the desired multidimensional enzyme subunit oligomerization domains. In some embodiments, is provided a pharmaceutical composition comprising an effective amount of the antibody in combination with a pharmaceutically acceptable carrier or diluent.

In some embodiments, the anti-IGF-I/II antibody is associated with an agent (e.g., a radioactive isotope, a pharmaceutical composition or a toxin). Mostly, such antibodies can be used to treat diseases, such diseases may be considered to cells that Express�tion of IGF-I/II or excessively expressing IGF-I/II. For example, described that the drugs have pharmaceutical properties and are selected from the group which consists of antineoplastic, alkylating, of antimetabolitnyh, antiangiogenic, apofaticheski, alkaloid, COX-2, and antibiotic agents and combinations thereof. The medication is selected from the group consisting of nitrogen mustard, ethyleneimine derivatives, alkyl sulphonates, nitrosomonas, triazino, analogs of folic acid, anthracycline, tcsanow, inhibitors SOH-2, pyrimidine analogs, purine analogs, antimetabolites, antibiotics, enzymes, epipodophyllotoxins, platinum coordination complexes, Vinca alkaloids, substituted ureas, derivatives of methylhydrazine, the suppressor activity of the adrenal cortex, antagonists, endostatin, tokalov, camptothecine, oxiplatin, doxorubicinol and their analogues, and combinations thereof.

Examples of toxins include gelonin, exotoxin Pseudomonas (RE), RE, RE, diphtheria toxin, ricin, abrin, alpha-toxin, saporin, ribonuclease (RNase), DNase I, the toxin of staph-A, an antiviral protein, Phytolacca American, and their derivatives, combinations and modifications.

Examples radiometric isotopes include gamma generators, positron-generators and x-ray generators, which can be used for localization and/or therapy, and beta-and alpha generators-generators, �have a quiet can be used for therapy. Radiometrie isotopes described above as useful for diagnostics, prognostics and transportation, are also useful for treatment. Unlimited examples of anti-cancer or protivodiabeticheskih agents include anthracyclines such as doxorubicin (adriamycin), daunorubicin (daunomycin), idarubicin, demoralizing, carminomycin, epirubicin, zorubicin, and morpholino and substituted derivatives, combinations and modifications. Illustrative pharmaceutical agents include CIS-platinum, Taxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin, idarubicin, fludarabine, chlorambucil, interferon alpha, hydroxyurea, temozolomide, thalidomide, bleomycin, and derivatives, combinations and modifications. Mainly, cancer or protivoanemicakimi agents are doxorubicin, morpholino-doxorubicin or morpholinomethyl.

As will be appreciated by the person skilled in the art, the above-mentioned embodiment, while the affinity values can be important, other factors may be as or more important depending on the specific functions of the antibody. For example, to immunotoxin (toxin associated with the antibody) the antibody binding with the target can be useful; however, in some embodiments, the desired goal is internationalises�I of the toxin into the cell. As such, antibodies with a high percentage of internalization may be desirable in these situations. Therefore, in one embodiment, the described antibodies with high efficiency in the internalization. High efficiency of internalization can be measured in percentage of internalized antibodies and can range from low values to values of 100%. For example, in various embodiments, 0,1-5, 5-10, 10-20, 20-30, 30-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-99 and 99-100% can be high efficiency. As will be appreciated by the person skilled in the art, the desired effectiveness may vary in different embodiments, depending on, for example, the associated agent, the amount of antibody that can be introduced into the region, side effects of a complex of antibody-agent types (e.g., cancer) and the severity of the disease that will be treated.

In other embodiments, the antibodies described in this application, provide a test kit to detect the expression of IGF-I/II in tissues or mammalian cells for screening of diseases or disorders associated with changes in expression of IGF-I/II. The kit contains an antibody that binds IGF-I/II and is intended for indicating the reaction of the antibody with the antigen, if present.

In some embodiments, are provided with the finished product containing container which contains a composition of an anti-IGF-I/II EN�of icela and packaging or label stating the composition may be used for the treatment of a disease mediated by expression of IGF-I/II. Mainly, mammals, and more preferably, people get anti-IGF-I/II antibody.

Combination

Anti-IGF-I/II antibodies listed previously in this application, can be used to treat by themselves or may contain, optionally, a compound in accordance with this invention, conventional surgery or radiotherapy or chemotherapy. Such chemotherapy may include one or more of such categories of anti-tumor agents:

(i) antiproliferative/antineoplastic drugs and combinations thereof, which are used in medical Oncology, such as alkylating agents (e.g., cisplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulfan and nitrocefin); antimetabolites (for example antifolates like torpedinidae, such as 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinose and hydroxyurea; antitumor antibiotics (for example, anthracycline-like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example Vinca alkaloids, like vincristine, vinblastine, vindesine and vinorelbine, and such taxaide, like Taxol and Taxotere); and �the topoisomerase inhibitors (for example, epipodophyllotoxins, such as etoposide and teniposide, amsacrine, topotecan and camptothecin);

(ii) cytostatic agents such as antiestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene and idoxifene), regulators of the estrogen receptor (for example fulvestrant), antiandrogens (for example, bikalutamid, flutamide, nilutamid and ciproteron acetate), LHRH antagonists or LHRH agonists (for example, goserelin, leuprorelin and buserelin), progestogen (for example, megestrol acetate), aromatase inhibitors (e.g. anastrozole, letrozole, Forasol and exemestane) and inhibitors of 5α-reductase such as finasteride;

(iii) agents which inhibit invasion of cancer cells (for example, such inhibitors metalloprotease as marimastat inhibitors activator receptor function urokinase plasminogen);

(iv) inhibitors of the function of growth factor, such inhibitors include antibodies, growth factor, receptor antibodies, growth factor (e.g., anti-erbb2 antibody trastuzumab [Herceptin™] and the anti-erbb1 antibody cetuximab), farnesyl, transferase inhibitors, MEK inhibitors, tyrosine kinase inhibitors and inhibitors of serine/trionychidae, for example, inhibitors of the family of epidermal growth factor (for example, such inhibitors of EGFR family tyrosine kinases, asN-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropan)hinzelin-4-amine (�gefitinib, AZD1839),N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)hinzelin-4-amine (erlotinib, OSI-774) and 6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropan)hinzelin-4-amine (CI 1033)), for example inhibitors of the platelet-family of growth factors and, for example, inhibitors hepatocytes family growth factor;

(v) antiangiogenic agents that can inhibit the influence of the factor vascular endothelial growth (for example, anti-antibody growth factor of endothelial cells of blood vessels bevacizumab [Avastin™], compounds such are described in international patent applications WO 97/22596, WO 97/30035, WO 97/32856 and WO 98/13354) and compounds acting through other mechanisms (e.g. linomide, inhibitors of integrin αvβ3 function, angiostatin and inhibitors of the action of angiopoetines, such as angiopoietin 1 and angiopoietin 2);

(vi) the agents of vascular lesions, such as combretastatin A4 and compounds described in international patent applications WO 99/02166, WO 00/40529, WO 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213;

(vii) antisense therapies, for example, directed to the above targets, such as ISIS 2503, anti-ras antisense;

(viii) gene therapy approaches, including for example approaches to replace these aberrant genes, as aberrant p53 or aberrant BRCA1 or BRCA2, these approaches GDEPT (gene-directed enzyme Pro-drug therapy) approaches used�Itanium cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase the tolerability of the patient chemotherapy or radiotherapy, such as gene therapy multilocational resistance;

(ix) immunotherapeutic approaches, including for example ex-vivo and in-vivo approaches to enhance the immunogenicity of tumor cells of the patient, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte macrofamily factor stimulation of the colon, approaches to reduce the fatigue of T cells, approaches using transfecting immune cells such as cytokine-transfecting dendritic cells, approaches using cytokine-transfecting tumor cell lines and approaches using anti-idiotypic antibodies;

(x) inhibitors of cell cycles, including for example CDK inhibitors (eg, flavopiridol) and other inhibitors of checkpoint of the cell cycle (e.g., checkpoint kinase); inhibitors of Aurora kinase and other kinases involved in the regulation of mitosis and cytokinesis (for example, mitotic kinesins); and inhibitors of the histone deacetylase;

(xi) endothelin antagonists, including antagonists of endothelin a antagonists, endothelin B antagonists of endothelin A and B; for example ZD4054 and ZD1611 (WO 96 40681), atrasentan and YM598; and

(xii) of biotherapeutic healing�e approaches for example, using peptides or proteins (such as antibodies or soluble external design of receptor domains) with the sequencing of the receptor ligands, block ligand binding to receptor or decrease signaling receptor (e.g. due to enhanced receptor degradation or lowered expression levels).

Such Conjoint treatment may be achieved by simultaneous, sequential or separate dosing of the individual components of treatment. Such combination products contain compounds in accordance with this invention in the range of dosages described above and the other pharmaceutically active agents in the approved range of dosages.

EXAMPLES

The following examples, including experiments and obtained the results given for illustration only and should not be construed as limiting theories and teachings, described in this application.

EXAMPLE 1

Immunization and titration

Immunization

As the antigens used recombinant human IGF-I and IGF-II obtained from R&D Systems, Inc. (Minneapolis, MN Cat. No. 291-292 G1 and-G2, respectively). Monoclonal antibodies to IGF-I/II was developed by sequential immunization of XenoMouse mice®(XenoMouse strains XMG2 and XMG4 (3C-1 strain), Abgenix, Inc. Fremont, CA). XenoMouse animals were immunized� through the introduction of all injections in the pads. Total volume of each injection was 50 μl per mouse, 25 µl for each pad of paws. In each group were immunized ten (10) mice. Each injection was 10 μg IGF-I or IGF-II on the mouse itself or in combination with the antigen Keyhole Limpet Hemocyanin (KLH) as a carrier, as detailed in Table 2. The first injection was carried out in phosphate-buffered saline (PBS), modified by Dulbecco (DPBS) and mixed 1:1 vol. with the adjuvant Titermax Gold Adjuvant (SIGMA Cat. #T2684, party #K). Then injected just 8-11 additional boost injections within 27-38 days, mixed with 25 μg of Adju-Phos (aluminum phosphate gel, catalog number 1452-250, batch #8937, HCI Biosector) and 10 μg CpG (15 µl adjuvant ImmunEasy Mouse Adjuvant, catalog number 303101; lot of #11553042; Qiagen) on the mouse, with final boost injection of 10 μg of antigen in non-pyrogen DPBS, without adjuvant. For combination immunization (immunized animals as IGF-I and IGF-II), the second antigen was administered in the last two (2) boost-injection.

TABLE 2.
BRIEF DESCRIPTION IMMUNIZATION
Group immunizationThe initial immunogenThe final immunogenKLH conjugated The isotype of the mouseGroup merge
1IGF-1IGF-1-IgG2-λ1
3IGF-1IGF-1-IgG4-λ1
5IGF-1IGF-1+IgG2-λ1
7IGF-1IGF-1+IgG4-λ1
2IGF-2IGF-2-IgG2-λ2
4IGF-2IGF-2-IgG4-λ2
6IGF-2IGF-2+IgG2-λ 2
8IGF-2IGF-2+IgG4-λ2
9IGF-1IGF-2-IgG2-λ3
11IGF-1IGF-2-IgG4-λ3
13IGF-1IGF-2+IgG2-λ3
15IGF-1IGF-2+IgG4-λ3
10IGF-2IGF-1-IgG2-λ4
12IGF-2IGF-1-IgG4-λ4
14/td> IGF-2IGF-1+IgG2-λ4
16IGF-2IGF-1+IgG4-λ4

EXAMPLE 2

RECOVERY of LYMPHOCYTES, the SECRETION of b-CELLS, MERGERS AND the FORMATION of HYBRID

Immunized mice were immortalitate by displacement of the cervical vertebrae and drained lymph nodes from each group were collected and accumulated. The lymphoid cells dissociatively by grinding in the medium Needle, modified Dulbecco (DMEM) to extract cells from the tissues, and the cells suspended in DMEM. Cells were counted and 0.9 ml of DMEM, 100 million lymphocytes were added cellular tablets for re-suspension, gently but completely. Use 100 ál of CD90+ magnetic particles per 100 million cells, the cells were labeled by incubation of cells with magnetic particles at 4°C for 15 minutes. A suspension of magnetically-labeled cells that contained up to 108positive cells (or up to 2×109cells), were immersed in the LS+ column and washed her DMEM. All spilled fluid was collected as D90-negative fraction (expected that most of these cle�are OK B cells).

The fusion was performed by mixing washed enriched In cells obtained as described above, and non-secretory myeloma cells P3X63Ag8.653 acquired from ATS, cat. # CRL 1580 (Kearney et al., J. Immunol. 123, 1979, 1548-1550) at a ratio of 1:1. The cell mixture gently was pelletized by centrifugation at 800×g. After complete removal of the supernatant, the cells were treated with 2-4 ml of a solution of pronase (CalBiochem, cat. # 53702; 0.5 mg/ml in PBS) not more than 2 minutes. Then 3-5 ml of fetal bovine serum (FBS) was added to stop enzyme activity and the suspension was adjusted to a total volume of 40 ml with a solution to electroline cells (ECFS, 0.3 M sucrose, Sigma, cat. # S7903, 0.1 mm magnesium acetate, Sigma, cat. # M, 0.1 mm calcium acetate, Sigma, cat. # S). Supernatant was removed after centrifugation, and the cells re-suspended in 40 ml of ECFS. This stage washing was repeated and the cells again re-suspended in ECFS to a concentration of 2×106cells/ml.

Electroline cells were counted using a generator merge (model ECM, Genetronic, Inc., San Diego, CA). The size of the used camera the merger was 2.0 ml, using such settings devices:

the clearing conditions: voltage: 50 V, time: 50 sec;

the destruction of the membrane at: voltage: 3000 V, time: 30 msec;

retention time after the merger: 3 sec.

After ECF cell susp�nsii carefully removed from the chamber of the merger under sterile conditions and transferred into a sterile tube, containing the same amount of hybrid culture medium (DMEM, JRH Biosciences), 15% FBS (Hyclone), with the addition of L-glutamine, pen/strep, OPI (oxaloacetate, pyruvate, cow insulin) (all obtained from Sigma) and IL-6 (Boehringer Mannheim). Cells were incubated for 15-30 minutes at 37°C, and then centrifuged at 400×g (1000 rpm) for five minutes. Cells gently re-suspended in a small volume of hybridization breeding environment (hybrid the culture medium with 0.5 x (Sigma, cat. # A)) and the volume was adjusted by adding appropriate quantities of hybridization breeding environment, based on the final tablets at 5×106B cells only on the plate of 96 wells and 200 μl per tablet. The cells are gently mixed and with a pipette was added to the plates of 96 wells and left to grow. On a 7 or 10 day half of the medium was removed and cells re-added hybridization breeding environment.

EXAMPLE 3

The POSSIBLE SELECTION of ANTIBODIES USING ELISA

After 14 days of culturing the hybrid supernatants were subjected to screening for IGF-I/II-specific monoclonal antibodies. Tablets ELISA (Fisher, cat. no.12-565-136) were coated with 50 µl/well of human IGF-I or IGF-II (2 μg/ml) covering the buffer (0.1 M carbonate buffer, pH 9,6, NaHCO38.4 g/l), then incubated at 4°C overnight. After incubation the plates were washed with washing buffer (0.05% of Tween 0 in PBS) 3 times. Added 200 μl/well blocking buffer (0.5% of BSA, 0,1% Tween 20, and 0.01% thimerosal in 1x PBS) and the plates were incubated at room temperature for 1 hour. After incubation the plates were washed with washing buffer three times. Added 50 μl/well of hybrid supernatants and positive and negative controls and the plates were incubated at room temperature for 2 hours.

After incubation tablets three times washed with wash buffer. 100 μl/well goat anti-huIgGFc-HRP (Caltag, cat. No. H10507) for detection antibody was added, and the plates were incubated at room temperature for 1 hour. At the second screening positive controls in the first screening were subjected to screening in two stages, one for the detection of human IgG (heavy chain) and the rest for the detection of human Ig Kappa light chain (goat anti-hIg Kappa-HRP (Southern Biotechnology, cat. No. 2060-05) to demonstrate fully human composition as the IgG and Ig Kappa. After incubation tablets three times washed with wash buffer. 100 μl/well of TMB (BioFX Lab. cat. No. TMSK-0100-01) was added and left tablets for approximately 10 minutes (until the wells of the negative control has clearly started to show color). Was then added 50 μl/well of stop solution (TMB stop solution (BioFX Lab. cat. no.STPR-0100-01)) and read tablets on tablet ELISA reader at 450 nm. As shown in table 3, all�about 1,233 wells contained antibodies to IGF-I and-II.

All antibodies that bound to the ELISA analysis were subjected to a quantitative analysis of the binding of insulin using ELISA to exclude those that cross-react with insulin. Tablets ELISA (Fisher, Cat. No. 12-565-136) were coated with 50 ál/well of recombinant human insulin (concentration: 1 µg/ml; Sigma, catalog # 12643) in covering buffer (0.1 M carbonate buffer, pH 9,6, NaHCO38.4 g/l), then incubated at 4°C overnight. As detailed in Table 3, only 1,122 antibodies from 1233 source cross-react with insulin.

TABLE 3.
BRIEF DESCRIPTION SCREENING
Screening confirmation
Merge#Murine strainThe immunogenThe goal with the determination of hGThe purpose of the definition hK+hLIGF-I(+) & IGF-II(+)IGF-I(+) & IGF-II(+) and human insulin (-)
1G2 KLIGF-I-KLHIGF-II IGF-I3628
2G4 KLIGF-I-KLHIGF-IIIGF-I6555
3G2 KLIGF-II or IGF-II-KLHIGF-IIGF-II168150
4G4 KLIGF-II or IGF-II-KLHIGF-IIGF-II197194
5G2 KLIGF-I/-II-KLHIGF-IIIGF-II5450
6G4 KLIGF-I/-II-KLHIGF-IIIGF-II10186
7G2 KLIGF-II/-I-KLHIGF-IIIGF-I 294271
8G4 KLIGF-II/-I or IGF-II/-I-KLHIGF-IIIGF-II318288
Only F1-F4466427
Only F5-F8767695
Total1,2331,122

Finally, antibodies, selected through a quantitative screening, were tested using ELISA to confirm binding proteins IGF-I and IGF-II. Just identified 683 hybrid lines having cross-reactivity to murine IGF-I/II. Accordingly, these hybrid lines expressively antibodies that bound human IGF-I, human IGF-II, murine IGF-I and murine IGF-II, but not linked human insulin.

EXAMPLE 4

INHIBITION of IGF-I And IGF-II BINDING to IGF-IR

The aim of this study was to conduct a screening 683 anti-IGF-I/II human IgG2 and IgG4 antibodies on stage hybrid of the supernatant at PR�dmet neutralizing activity, which was determined by inhibition of IGF-I and IGF-II receptor binding of IGF-IR. Therefore, the analysis of the binding of the receptor/ligand was performed with NIH3T3 cells that excessively expressively human IGF-IR receptor, as described below.

Briefly, multisyringe filter plates (0.65 μm MultiScreen 96-well PVDF, Millipore, Cat. No. MADV N0B 10), blocked with blocking buffer (PBS containing 10% BSA with 0.02% NaN3) at 200 μl/well overnight at 4°C.-labeled IGF (Amersham Life Sciences cat. No. IM172 (IGF-I) or IM238 (IGF-II)) at 100 m Ci ml and 50 nm were diluted to appropriate concentration (70 PM final IGF-I and 200 PM final for IGF-II) in binding buffer (PBS containing 2% BSA with 0.02% NaN3). Filter plate covered with blocking buffer, were washed once with 200 μl PBS and 50 μl of anti-IGF-I/II Ab of the supernatant (diluted in binding buffer to a final 25% of final volume) were pre-incubated with 25 μl of IGF in the tablet MultiScreen for 30-60 minutes on ice. Subconfluent NIH3T3 mouse fibroblasts stably expressing hIGF-IR (received from AstraZeneca) were collected with trypsin and re-suspended in cold binding buffer with 6×106/ml and 25 μl of cells was added to the tablet for a two-hour incubation in ice. The tablet was washed four times with 200 μl cold PBS and dried overnight. Added twenty-five μl/well of scintillant (cocktail SuperMix, Wallac/Perkin Elmer cat. No. 200-439) and read the tablet with the help of a reader Microbeta Trilux (Wallac).

For each screening tablets used such a control: the absence of antibodies (all linked IGF), the control neutralizing anti-IGF-I (#05-172, Upstate) or anti-IGF-II (#MAB, R&D Systems) mAbs at 50 ál/ml (non-specific background) and 0.075-0.5 ál/ml (approximate values / EC50 neutralizing antibodies), and the isotope-matched control human IgG2 (RK 16.3.1, Abgenix, party #360-154) or IgG4 (108.2.1, Abgenix, batch #718-53A) mAbs at a concentration of 0.5 ál/ml (approximate values / EC50 neutralizing antibodies). Additional titration control neutralizing antibodies and the isotope-matched control human antibody was added in one tablet for one screening analysis (1/10 serial dilution of 50 μg/ml (the 333.3 nm)). All controls with or without antibodies were prepared in binding buffer with the addition of anti-KLH human IgG2 or IgG4 depleted supernatant at 25% of the final volume.

The percentage of inhibition was determined as follows:

% Inhibition=([(Mean CPM total number associated125I-IGF)-(Mean CPM associated125I-IGF in the presence of antibody)]/[(Mean CPM total number associated125I-IGF)-(Mean CPM associated125I-IGF in the presence of an excess of control neutralizing antibodies∗)])×100

* the non-specific background was determined as CPM of cells with an excess of control neutralizing anti-IGF Ab (50 mcgml, the 333.3 nm), which was found, was equivalent to the excess of cold IGF (less than or equal to 10% of total CPM)

Screening of anti-IGF-I/II of the supernatant was split into isotopes due to the presence of radioactively labeled ligand tissues. As shown in Table 4, the supernatants from anti-IGF-I/II antibodies with the IgG2 isotype (total 293) were first subjected to screening for Radiometrie IGF-I. the Limit of 40% inhibition was used for the first screening (i.e., were selected hybrid line, inhibiting at 40% and above) were selected and 111 hits for sequential screening for IGF-II. 111 hits were found only 91 lines that inhibit the binding of IGF-II to its receptor at 50% of the limit. Only 71 were selected ultimate hits by selection of the supernatants, which neutralize 50% the activity of IGF-I and IGF-II.

Was conducted an initial screening of all supernatants expressing IgG4 isotypes (390) in respect of radioactively labeled IGF-II, and 232 being hit with a limit of inhibition of 50% were subjected to sequential screening in relation to IGF-I. Total 90 lines were capable of inhibiting the binding of IGF-I with its receptor at the limit of 50%. After the combination of hits for IgG2 (71) and IgG4 (90), was obtained only 161 line, which inhibited IGF-I and IGF-II by 50% or more.

In conclusion, from 683 source supernatants 343 (111 IgG2 232 and IgG4, 50,2) were selected from the first screening with IGF-I or IGF-II. They received a total of 161 ultimate hits (23,6% from baseline), capable of blocking the binding of IGF-I and IGF-II to IGF-IR with the General criterion of the limit, which is 50% inhibition.

TABLE 4.
BRIEF description of the EXHAUSTION of the SUPERNATANT of the ANTI-IGF-I/II (50% LIMIT)
ESN activity
Merge #Murine strainThe immunogenhIGF-II chG+/hK+or hG+/hL+R/L analysis of the binding of IGF-IIGF-I+%R/L analysis of the binding of IGF-II to IGF-I+IGF-I/II+%R/L analysis of the binding of IGF-IIIGF-II+%R/L analysis of the binding of IGF-I on IGF-II+Total
1G2 KLIGF-I-KLH141285,71 7,1
2G4 KLIGF-I-KLH20420,0420,0
3G2 KLIGF-II or IGF-II-KLH491326,548,2
4G4 KLIGF-II or IGF-II-KLH1148675,43631,6
5G2 KLIGF-I/-II-KLH321134,4721,9
6G4 KLIGF-I/-II-KLH4721of 44.7817,0
7G2 KLIGF-II/-I-KLH1987537,95929,8
8G4 KLIGF-II/-I or IGF-II/-I-KLH20912157,94220,1
683111
The limit of 40%
16,37110,423234,09013,2
gG2 IgG4

EXAMPLE 5

Highly SENSITIVE AND LIMITED ANTIGENIC ANTIGENIC ELISA

To determine the relative Finasta among 161 hybrid lines selected in Example 4, and the concentration of antibody in the supernatants of each line, were held highly sensitive antigen (S) and limited antigen (LA) ELISA tests. In quantitative analysis, high concentrations of antigen and incubation period on all night and limited the influence of epinasty antibodies, allowing quantification of relative amount of antigen-specific antibody present in each sample. Low concentrations of antigen in the LA analysis limited the influence of the concentration of antibodies and led to the classification of antibodies based on their relative affinity.

Highly sensitive quantitative analysis of antigenic

Tablets ELISA covered a relatively large amount of antigen IGF-I or IGF-II (R&D Systems, Inc., Minneapolis, MN Cat. No. 291-292 G1 and-G2, respectively) at 500 ng/ml (67 nM). Containing hybrid antibodies, the supernatants were titrated in a range of dilution 1:50 - 1:12200. Control of known IGF-specific antibodies (R&D Systems, Inc., Minneapolis, MN Cat. No. MAB291 and MAV, respectively) were used to determine the linear range of the analysis. Data in the linear range was used to obtain relative concentrations of IGF-specific antibodies in each titrated sample. Limited antigenic analysis

Mikrotechnologie plates were plated at low concentrations of antigens. Fifty microlites (50 ml) IGF-I or IGF-II 64, 32, 16, 8, 4, and 2 ng/ml (covered in the range from 8.5 nm to be 0.26 nm) in 1% skim milk/1X PBS pH of 7.4/0.5% azide was added to each well. The plates were incubated for 30 minutes.

The plates were washed four times (4X) water and 50 µl of hybrid supernatant containing test antibodies diluted 1:25 in 1% skim milk/IX PBS pH 7.4/0.5% of the azide was added to the wells. Tablets tightly wrapped with plastic wrap or parafilm and incubated overnight with shaking at room temperature.

The following day, all plates were washed five times (5X) and to each well was added 50 ml of goat anti-human IgG Fc HRP polyclonal antibody at a concentration of 0.5 μg/ml in 1% milk, IX PBS pH of 7.4. The plates were incubated for 1 hour at room temperature.

The plates were washed at least five times (5X tap water). Fifty microliters (50) μl of the HPR substrate TMB was added to each well and the plates were incubated in tech�of 30 minutes. The reaction of the HRP-TMB was stopped by adding 50 µl of 1M phosphoric acid to each well. The optical density (absorption) at 450nm was measured for each well the plates.

Data analysis

The values of optical density of the test antibodies were averaged and counted their range. Antibodies with higher signal and acceptable low standard deviations were selected as the antibodies with the highest affinity towards the antigen, compared to the reference antibody.

Then carried out the analysis for the top selection of antibodies based on neutralization (Example 4), efficiency (low concentration of antibodies according to the results ON ELISA and high performance ligand binding), epinasty (LA ELISA), or by all three criteria. The results of this analysis was compiled a list of 25 antibodies. A separate analysis based on the mean % inhibition of IGF-I and-II binding and epinasty for IGF-I and IGF-II resulted in a second list of 25 antibodies. Sixteen antibodies coincided for both lists, the result was obtained the final list of 40 antibodies. The results of LA and for those 40 antibodies are shown in Table 5. These 40 lines were selected for cloning and 33 of them were successfully cloned.

TABLE 5.
RES�the OBJECTIVES HYPERSENSITIVE AND LIMITED ANTIGENIC ELISA FOR TOP 40 ANTIBODIES
IGF1IGF2IGF1 LAIGF2 LAIGF1 LAIGF2 LA
IDAverage (µg/ml) HA1The standard deviation HA1Average (µg/ml) HA2The standard deviation HA2IGF1 ng/m lIGF2 ng/m lIGF1 ng/m lIGF2 ng/m l
4.1211,160,163,56to 1.340,560,53to 1.140,90
4.1411,990,221,990,210,570,791,25of 1.52
4.1424,700,213,65 0,310,781,00of 1.761,78
4.1431,740,172,030,410,600,991,20of 1.91
4.251,260,231,480,360,710,811,31a 1.54
4.697,171,166,500,530,800,801,50a 1.54
4.901,150,123,680,770,580,481,040,89
7.118of 10.341,26 of 10.321,900,810,851,561,44
7.12313,43,212,02,81,01,71,66of 2.58
7.127to 7.280,446,591,551,191,372,292,40
7.1304,320,323,51to 1.340,641,171,361,98
7.14612,040,989,630,60,20,860,291,58
7.1589,29 0,497,10,561,711,423,002,46
7.159formed 16.531,8341,10,56the 1.650,982,47
7.1604,90,55,10,21,72,23,143,30
7.175is 8.460,496,211,220,130,340,140,62
7.20211,941,98with 15.241,721,111,682,282,69
7.212 11,301,9010,861,260,970,932,22a 1.54
7.21510,112,0510,941,391,011,092,251,93
7.234,300,263,990,290,551,221,402,17
7.2344,71,43,10,40,71,4to 1.792,44
7.25130,411,930,171,091,021,311,53
7.252the 8.250,515,551,681,221,232,532,09
7.268a 7.580,425,070,921,471,373,062,42
7.2912,51,2423,534,70,180,390,270,49
7.313,182,128,830,580,811,282,071,96
7.3412,541,9914,673,050,211,070,44 1,84
7.41of 3.690,194,970,81,021,532,212,32
7.5614,62,021,73,71,31,42,382,46
7.58drain rate is 17.520,0127,546,220,211,150,47of 1.82
7.66of 6.020,816.18 of0,710,490,971,421,53
7.77to 8.420,18the 7.251,120,640,46 1,501,00
7.85one-22.670,68of 23.630,930,10,330,160,51
7.997,90,25,91,60,91,01,87the 1.65

IGF1IGF2IGF1 LAIGF2 LAIGF1 LAIGF2 LA
IDAverage (µg/ml) HA1The standard deviation HA1Average (µg/ml) HA2The standard deviation HA2IGF1 2 ng/m lIGF2 2 ng/m lIGF1 4 ng/m lIGF2 4 ng/m l
8.119 1,260,000,770,162,370,793,771,36
8.1415,960,504,120,611,800,613,021,08
8.1464,030,452,550,740,971,062,131,98
8.2874,80,12,80,82,21,63,802,91
8.82,000,171,450,251,720,773,131,46
3,150,192,360,240,92of 1.35of 1.762,18

EXAMPLE 6

ANTIBODY BINDING TO IGF-I And IGF-II ASSOCIATED WITH IGFBP-3

IGF-I and-II that circulate in the serum, mainly associated with IGF-binding proteins (IGFBPs). Our goal was to identify antibodies that do not recognize IGFs in complex with IGFBPs, avoid deletions in vivo anti-IGF antibodies. The following format analysis was developed to characterize antibodies that recognize IGF-I or IGF-II if these growth factors are associated in a complex with IGFBP-3. Specifically, this analysis examined the ability of IGF in complexes with IGF/aHTH-IGF antibody to bind IGFBP-3.

Antibody-mediated blocking of IGF capture IGFBP-3

Was developed by the analysis, wherein the pre-formed complexes between IGF-I or IGF-II and IGF-specific antibodies described in the examples above. The ability of these complexes to bind IGFBP-3 was checked using the techniques of analysis AlphaScreen (PerkinElmer). In the tablet with 384 wells, 10 ál of 1:20 diluted hybrid of supernatants were mixed with 10 μl of 3 nm biotinylating IGF-I or IGF-II and incubated at room temperature within 2 hours. Added coated AlphaScreen streptavidin donor pellets and IGFBP-3-United AlphaScreen acceptor bead (10 ál of the mixture to 1/60 of final dilution of hybrid supernatants) and continued incubation for another hour. Then, the samples were read on a tablet reader Packard Fusion.

Three commercially available anti-IGF monoclonal antibody M23 (Cell Sciences), 05-172 (Upstate) and MAB (R&D Systems) showed different ability to inhibit binding of IGF to IGFBP with IC50 values in the range of low ng/ml to 100 ng/ml. the Inhibition of binding of IGF-I with IGFBP-3 was observed in IgG external mouse and human IgG to 10 μg/ml, causing the assumption that anti-IGF-I effect is specific. Commercially available monoclonal antibodies 5-166 (Upstate) and MAB (R&D) showed a significant difference in epinasty for inhibiting the interactions of IGF-II/IGFBP-3. These experiments showed that anti-IGF mAbs can block the binding of IGF to IGFBP-3, providing analysis that can be used to screen purified antibodies hybrid lines. The next stage is to assess the impact of expendable hybrid environment on the analytical signal.

Serial dilutions hybrid environment and anti-KLH hybrid consumable supernatants were tested in the analytical system. When breeding a hybrid supernatants of 1:10 with the prior incubation of IGFI/II the final dilution in the analysis was 1:60), almost no influence of environment on the results of the analysis. Based on these data, the hybrid supernatants were diluted with pre-incubation of IGF, providing preferential 1/60 dilution final dilution in the analysis.

Six hundred eighty three spent supernatants of positive relative to IGF-I and IGF-II binding were tested for their ability to inhibit the binding of IGF to IGFBP-3. Inhibition of over 50% for IGF-I and over 60% for IGF-II was used as the limiting criterion. The final results of the screening using the limits shown in Table 6.

TABLE 6
The NUMBER of POSITIVE HITS IDENTIFIED BY SCREENING
IGF-IIGF-IIIGF-I/II
SampleInhibition - >>50%>60%
376(1-4 tablets)485119
307(plates 5-8)397832
683Total8712951

In the competitive analysis using IGFBP analysis AlphaScreen identified 87 samples that inhibit the binding of IGF-I with IGFBP-3 and 129 samples, which inhibits the binding of IGF-II, IGFBP-3 among 683 tested supernatants. Fifty-one sample showed double competition of IGF-I and IGF-II. However, a more careful reconstruction of the function or behavior of antibodies in vivo, where IGF and IGFBP complexation can be accomplished to a greater extent, were carried out additional tests as described in Example 8.

EXAMPLE 7

DEFINITION of EPINASTY ANTI-IGF-I And IGF-II ANTIBODIES USING BIACORE ANALYSIS (SCREENING LOW RESOLUTION)

Screening low-resolution 34 of the purified monoclonal antibodies

Surface plasmon resonance (SPR), or Biacore, used to measure epinasty antibodies to the antigen. For this he received a surface goat anti-human antibodies with high density over CM 5 Biacore chip using standard amine combinations. All mAbs were diluted to approximately 20 μl/ml in HBS-P rolling puff�e, containing 100 μl/ml BSA. Each mAb was captured on a separate surface, the contact time was 30 seconds at 10 μl/min and washed for 5 minutes to stabilize the background lines mAb.

IGF-I was administered by injection 335,3 nm in all surfaces at 23°C for 120 seconds, followed by 5 min dissociation using a flow rate of 100 ál/min. the Samples were prepared in HBS-P rolling buffer described above. The surface was regenerated after each cycle of capture/injection with one 15-second pulse of 146 mm phosphoric acid (pH of 1.5). Similar cycles of capture/injection was repeated for each antibody with 114,7 nm IGF-II. Drift-free data binding for 34 mAbs were obtained by subtracting the signal from a control flow cell and subtracting the background drift of the line buffer, injected before each injection of antigen. Data globally coincided with a 1:1 interaction model using the CLAMP to determine the kinetics of binding (David G. Myszka and Thomas Morton (1998) "CLAMP©: a biosensor kinetic data analysis program", TIBS 23, 149-150). The mass transfer coefficient used when fitting the data. Kinetic analysis showed the binding of IGF-I and IGF-II at 25°C, are shown in Table 7 below. mAbs were ranked depending on epinasty: high to low.

TABLE 7.
IGF-I And IGF-II SCREENING HIGH-RESOLUTION BIACORE 34 MONOCLONAL ANTIBODIES
IGF-IIIGF-I
Sampleka(M-1c-1)kd(C-1)KD(PM)ka(M-1c-1)kd(C-1)KD(PM)
7.159.2a 3.5×1061,0×10-52,94,3×1069,3×10-4216,0
8.86.1of 6.1×106of 2.4×10-439,3the 3.4×106of 1.3×10-23823,0
4.25.19,3×1064,1×10-444.1 kHz5,1×106of 6.9×10-31353,0
7.234.2 of 6.4×1062.9 x 10-4of 45.3of 6.7×106of 2.2×10-3328,0
7.160.2a 4.6×106of 2.5×10-454,3of 5.6×1063,3×10-3589,0
7.146.3a 3.2×106of 1.8×10-456,2of 3.8×106of 8.7×10-32289,0
7.34.13,0×106of 1.8×10-460,0of 5.2×106a 3.2×10-3615,0
7.123.1of 4.0×106the 3.4×10-485,04,3×1069,0×10-32093,0
7.202.3of 1.8×106of 1.7×104 94,4of 1.2×106of 5.4×10-34500,0
4.141.1of 4.9×1064,7×10-495,9***
7.215.2a 3.2×1063,3×10-4103,03,6×106of 2.6×10-27222,0
8.287.2of 2.4×106of 2.5×10-4104,0of 7.7×106of 1.3×10-31688,0
8.146.2of 7.7×106of 8.0×10-4104,0of 2.4×106of 1.3×10-25417,0
4.143.2of 1.1×107of 1.2×10-3109,0of 6.4×106 of 1.9×10-22969,0
7.251.3a 3.5×1064,3×10-4123,0a 4.6×1064,3×10-3935,0
7.99.1of 6.9×106of 9.9×10-4143,06,0×106a 4.8×10-3800,0

IGF-IIIGF-I
Sampleka(M-1c-1)kd(C-1)KD(PM)ka(M-1c-1)kd(C-1)KD(PM)
4.142.25,3×106of 8.5×10-4160,0of 8.5×106of 1.9×10-2 2235,0
7.41.3a 3.2×106of 5.5×10-4172,0of 5.2×1062.9 x 10-3558,0
7.56.33,3×1066,0×10-4182,0a 4.8×106of 3.1×10-3646,0
7.127.14,1×106of 7.6×10-4185,0of 4.9×106a 3.5×10-3714,0
8.8.3of 4.0×1067,8×10-4195,0of 3.1 X 1068,2×10-4264,0
7.158.2the 3.4×106of 6.7×10-4197,04.4 X 106of 2.2×10-3500,0
7.23.3the 6.5×10-4197,04,1×106of 5.8×10-31415,0
7.252.1a 3.2×106the 6.5×10-4203,02.3 x 106of 2.1×10-3913,0
7.66.13,6×106of 7.7×10-4214,0of 2.0×106of 2.5×10-31250,0
7.130.14,2×1061,0×10-3238,0a 4.6×106of 1.8×10-23913,0
4.90.2of 4.9×106of 1.2×10-3245,0***
7.3.3a 3.2×1068, ×10-4 275,04,1×1063.9 x 10-3951,0
7.118.1a 4.6×106of 1.5×10-3326,0of 5.6×1065,1×10-3911,0
7.212.14,2×106of 1.6×10-3381,0of 3.1×1066,0×10-21935,0
7.175.2of 1.3×106of 7.4×10-4569,0of 1.8×106of 2.0×10-211111,0
4.121.1of 5.5×106*a 3.2×10-3*582,0*of 1.5×106of 2.1×10-31400,0
7.85.2of 1.9×106of 1.3×10-3684,0of 2.2×106 2.9 x 10-213182,0
7.58.38,9×106of 7.9×10-3888,07,2×106a 3.2×10-24444,0

Data of binding of IGF-I

Most mAbs are quite well matched 1:1 model. MAbs 4.90.2 and 4.141.1 was characterized by extremely complex data. These mAbs are marked with an asterisk in Table 7 because of the significant kinetic constants cannot be set from the model fit 1:1. The last phase of dissociation is very slow because of these mAbs (at least 1×10-5with-1) that can make these two mAbs are useful as therapeutic compounds.

Data binding IGF-II

Most Most mAbs are quite well matched 1:1 model. The dissociation constant for mAb 7.159.2 was constant at 1×10-5with-1due to the lack of sufficient data on the decay for an adequate assessment of kd.

Biacore analyses of low resolution in this example is designed as a semi-qualitative classification approach. For more accurate information about the characteristic rate constants and offendeth individual mAbs, analyses were conducted Biacore high RA�solutions as described in Example 8.

EXAMPLE 8

DEFINITION of EPINASTY ANTI-IGF-I And IGF-II ANTIBODIES USING BIACORE ANALYSIS (SCREENING HIGH RESOLUTION)

Analysis of high-resolution Biacore was conducted to further measure epinasty antibodies to the antigen. Each mAbs 7.159.2, 7.234.2, 7.34.1, 7.251.3, and 7.160.2 caught, and each antigen IGF-I and IGF-II were injected in the concentration range. The obtained binding constants are shown in Table 8.

TABLE 8.
EPINASTY ANTI-IGF ANTIBODY, determined BY the BIACORE ANALYSIS of LOW AND HIGH RESOLUTION
mAbLow resolution KD(PM)High resolution KD(PM)
IGF-IIGF-IIIGF-IIGF-II
7.159.2216,02,9294,01,9
7.234.2328,0of 45.33760,0295,0
7.34.1615,060,0km 436.0164,0
421,0162,0
7.251.3935,0123,0452,047,4
7.160.2589,054,32800,0237,0

Thus, embodiments in accordance with the present invention can include an antibody that preferentially binds IGF-II, but cross-react with IGF-I, binding of IGF-II with epinasty, greater than the affinity towards the IGF-I. for Example, the antibody may bind IGF-II with epinasty, which is 2.5 times higher than the affinity towards the IGF-I. In some embodiments, the antibody may bind IGF-II with epinasty, in which at least 5, at least 10, at least 25, at least 50 or at least 150 times greater than the affinity towards the IGF-I.

Screening of pre-formed complexes of IGF-I/GFBP-3

Competitive analysis IGFBP described in Example 6, identified 87 samples that inhibit the binding of IGF-I with IGFBP-3 and 129 samples, which inhibits the binding of IGF-II, IGFBP-3 among 683 tested super�of atento. Fifty-one sample showed double competition of IGF-I and IGF-II. However, for a more thorough restoration of function or behavior of antibodies in vivo, where the complexation of IGF and IGFBP can be carried out more tests were performed Biacore selected antibodies.

The six selected antibodies was subjected to screening to determine whether they bind IGF-I Il IGF-II to IGFBP complex. All six selected mAbs(7.159.2, 7.146.3, 7.34.1, 7.251.3, 7.58.3 and other irrelevant control antibody amplitude-time characteristic-MA) covalently immobilizerpower with high surface area (5,400-12,800 EN) on two chips Biacore cm 5 using standard amine binding by use of the Biacore 2000. on each chip CM 5 activated and blocked one flow cell (no immobilized mAb) for use as a control surface.

Then IGF-I and IGFBP-3 were mixed with a buffered salt solution Hepes, pH of 7.4, 0.005% P-20, 100 µg/ml BSA (HBS-P), to obtain the final solution at 193 nm and 454 nm, respectively. IGF-II and IGFBP-3 were mixed together to obtain the final solution at 192 nm and 455 nm, respectively. In these conditions, IGF-I and IGF-II to 99.97% formed a complex with IGFBP-3. Under these conditions, equilibrium was reached within minutes. Solutions related to the complex of IGF-I/IGFBP-3 and IGF-II/IGFBP-3 was passed through various mAb surface at 40 μl/min at 23°C within t�e 180 seconds, followed by dissociation for 120 seconds. Free IGF-I and IGF-II are then passed through each surface at 193 nm and 192 nm, respectively, a IGFBP-3 was passed through each surface at 454 nm. The surface was regenerated during the 20-second pulse of 10 mm glycine, pH 2,0.

Sensogram were processed using Scrubber by subtracting the volumetric change of the index of refraction and any signal of nonspecific binding of the analyte to colorless surfaces from the surfaces and/with immobilized mAb. After correction subtractions colorless surfaces, sensogram again compared by subtracting the average sensogram buffer injections at a specific flow cell. This "double comparison" adjusted sensogram mAb binding in regard to any systematic errors present in a particular flow cell.

Formed a complex and free IGF-I/IGFBP-3 and IGF-II/IGFBP-3 was associated rather poorly with bound antibodies, a rough estimate of the binding unspecific binding interaction was ToD>1 μm for all six mAbs, including negative controls amplitude-time characteristic-MA (see Table 9). However, since the amplitude-time characteristic-MA binding of IGF-I/II was weak and indicated that nonspecific binding interaction occurred in all three analytes. Obviously, the complexes of IGF/IGFBP-3 was associated with all of these mAbs, a IGFBP-3 on SEB� - no. However, due to the fact that both IGF-I and IGF-II and IGBP is-3 by themselves specifically associated with these mAbs, when they are related to each other, this leads to even more "sticky" unspecific binding protein complex, because of the large binding signal for the complex. Complexes of IGF-I/II/IGFBP-3 and IGFBP-3 are strongly associated with the control surface, which also indicates the nonspecific character of these two proteins. However sensogram below is background binding is subtracted from the first reference point during the processing of the data, as described above.

The experiment suggests that although it has been previously shown, 51 sample inhibited the binding of IGF-I/II, IGFBP3 (Example 6), the antibodies may also bind to the complex of IGF/IGFBP in vitro.

TABLE 9.
BRIEF DESCRIPTION of BINDING TO IGF-I/IGFBP-3 And IGF-II/IGFBP-3 BINDING WITH SIX MABS
mAbThe complex of IGF-MGFBP-3Complex GF-II/IGFBP-3IGFBP-3IGF-I (or II)
7.159.2++++++
7.146.3+++++++
7.34.1+++++++
7.251.3+++++++++
7.58.3+++++++++
Amplitude-time characteristic-MA++++
+ weak binding compared with IGF-I or IGF-II with mAb
++ secondary binding compared with IGF-I or IGF-II with mAb
+++ strong binding compared with IGF-I or IGF-II binding to mAb
*These rankings do NOT INDICATE TODfor such interactions.

EXAMPLE 9

DEFINITION of EPINASTY ANTI-INSULIN ANTIBODIES USING BIACORE ANALYSIS (SCREENING LOW RESOLUTION)

The cross-reactivity of antibodies related�Yu to IGF-I/II additionally, by measuring epinasty mAbs against human insulin. IGF-I/II antibodies were immobilizovana on the chip CM 5 Biacore, insulin in solution was injected to determine the constants of Association and dissociation constants. Five mAbs, including 7.234.2, 7.34.1, 7.159.2, 7.160.2 and 7.251.3, were tested in this experiment. Insulin was diluted to 502 nm in the movable buffer and injected into all of the arresting surface.

At 502 nm insulin binding insulin with any of the mAbs was observed. These results suggest no obvious cross-reactivity to IGF-I/II mAbs and insulin.

EXAMPLE 10

ANTIBODY BINDING

Epitope binding was performed in order to determine which of the anti-IGF-I/II antibodies can cross-compete with each other, and thus may bind the same epitope GF-I/II. The linking process described in the patent application U.S. No. 20030175760, as well as in Jia et al., J. Immunol. Methods (2004), 288: 91-98. both of which are included in this application fully by reference. Briefly, pellets Luminex associated with murine anti-huIgG (Pharmingen #555784) following the Protocol binding proteins provided on the web site of Luminex. Pre-associated granules were obtained for the binding of initially unknown antibody in accordance with the following procedure, protecting the pellets from light. For each individual supernatant used separate test tubes. The required volume of the supernatants was calculated using shadowsforsale: (p+10)×50 μl (where n=total number of samples). This analysis used a concentration of 0.1 μl/ml. Granules gently stirred on a vortex mixer and diluted with in granules/ml.

Samples were incubated in a shaker in the dark at room temperature overnight.

Filter plate was pre-wet by adding 200 ál wash buffer per well, then the buffer was removed. 50 μl of each of the pellets was then added to each well filter plate. Samples were washed once by adding 100 μl/well wash buffer and remove it. Antigen and controls were added to the filter plate at 50 μl/well. The plate was covered, incubated in the dark for 1 hour and then washed samples were incubated in the dark for 1 hour in a shaker and samples were washed 3 times. Second unknown antibody was then added at 50 μl/well. The concentration of 0.1 μg/ml were used for primary antibodies. Then the plate incubated in the dark for 2 hours at room temperature in a shaker, and then the samples were washed 3 times. Added 50 μl/well biotinylated mouse anti-human IgG (Pharmingen #555785) diluted 1:500, and the samples were incubated in the dark for 1 hour with stirring at room temperature.

Samples were washed 3 times. 50 µl/well streptavidin-PE at a 1:1000 dilution was added, and samples were incubated in t�note for 15 minutes with shaking at room temperature. After two cycles of washing on a Luminex100, samples were washed 3 times. The content of each well is re-suspended in 80 ál of blocking buffer. The sample is gently stirred with repeated pipetting to re-suspend the pellets. Then, the samples were analyzed using a Luminex100. The results are presented below in Table 10.

td align="center"> 8.287.2
TABLE 10.
The position of the TOP 34 IGF-I/II ANTIBODY POSITIVE IN FUNCTIONAL ANALYSIS
IGF-IIGF-II
Position 1Position 2Position 3The absence of a provisionPosition 1Position 2Position 3The absence of a provision
7.3.37.58.37.175.27.215.27.3.37.158.2 7.175.27.215.2
7.23.38.287.27.85.27.127.18.146.24.90.2
7.66.14.90.27.99.17.252.14.141.1
7.56.34.141.17.123.18.86.17.85.2
7.160.27.146.37.212.17.251.3
7.41.37.34.17.234.27.159.2
4.121.17.159.27.130.17.146.3
8.146.27.251.37.118.17.34.1
7.252.1
7.123.17.58.3
7.212.17.66.1
7.234.27.41.3
7.99.17.56.3
7.127.17.160.2
4.25.17.202.3
8.8.38.8.3
7.158.24.25.1
7.202.37.23.3
7.130.14.142.2
8.86.14.143.2
4.142.24.121.1
7.118.1
4.143.2

EXAMPLE 11

STRUCTURAL ANALYSIS of ANTI-IGF-I/II ANTIBODIES

Variable regions of heavy chains and variable regions of light chains was sequentially to determine their DNA sequences. Detailed information about sequences of anti-IGF-I/II antibodies are provided in the list of nucleotide and amino acid sequences for each gamma and �APPA circuits. Variable regions of the heavy chains were analyzed to determine the VH family, the sequence D-region and the sequence of the J-region. Then the sequence was broadcast to determine the primary amino acid sequence and comparison of sequences of the VH, D and J-region germ lines for estimation of somatic hypermutations.

Alignment of the sequences of these antibodies with their genes embryonic lines shown in the tables below. Table 11 is a table comparing regions of heavy chain antibodies and regions of heavy chains to their cognate germ line. Table 12 is a table comparing the areas of the Kappa light chain of the antibody with a light chain regions to their cognate germ line. Mutations in the germ line is shown as a new amino acid.

Variable (V) regions of immunoglobulin chains are encoded by multiple segments of DNA germ line attached to a functional variable region (VHDJHor VKJKduring b-cell ontogeny. Molecular and genetic divergence of reactions antibodies to IGF-I/II were studied in detail. These analyses revealed several characteristics that are specific to anti-IGF-I/II antibodies.

Analysis of five individual antibodies specific to IGF-I/II resulted in about�the definition, that antibodies were obtained from three different VH genes embryonic lines, four of which belonged to the family VH4, and 2 antibodies were obtained from a segment of the VH4-39 gene. In Tables 11 and 12 shows the results of this analysis.

It should be appreciated that the amino acid sequence among affiliated clones collected from each of the hybridomas, are identical. For example, sequences of heavy and light chains of mAb 7.159.2 identical to the sequences shown in Tables 11 and 12 for mAb 7.159.1.

CDR1 heavy chain antibodies according to the invention have a sequence described in Table 11. CDR1, which are described in Table 11, fall under the definition of Kabat. Alternative, CDR1 can be defined using an alternative definition, thus to enable the last five residues of the sequence FR1. For example, antibodies 7.159.1 FR1 sequence is QVQLVQSGAEVKKPGASVKVSCKAS (SEQ ID no: 93), a CDR1 sequence is GYTFTSYDIN (SEQ ID no: 94); for antibody 7.158.1 FR1 sequence is QLQLQESGPGLVKPSETLSLTCTVS (SEQ ID no: 95), a CDR1 sequence is GGSIRSSSYYWG (SEQ ID no: 96); for antibody 7.234.1 FR1 sequence is QLQLQESGPGLVKPSETLSLTCTVS (SEQ ID no: 97) and the CDR1 sequence is GGSINSSSNYWG (SEQ ID no: 98); for antibody 7.34.1 FR1 sequence is QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID no: 99) and CDR1 consisten�eTelestia is GGSISSYYWS (SEQ ID no: 100); and for antibodies 7.251.3 FR1 sequence is QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID no: 101), a CDR1 sequence is GGSISSYYWS (SEQ ID no: 102).

Should also be appreciated that in cases where a specific antibody differs from the sequence of its corresponding germ line at the amino acid level, the sequence of the antibody can be mutated back to the germline sequence line. Such correlation of mutation can occur in one, two, three or more positions, or a combination of the mutated positions, using standard biological techniques. As a non-limiting example, Table 12 shows the sequence of the light chain of mAb 7.34.1 (SEQ ID no: 12), which differs from the corresponding germ line sequence (SEQ ID no: 80) by mutation of Pro to Ala (mutation 1) in the FR1 and through mutation of Phe to Leu (mutation 2) in the FR2 region. Thus, amino acid or nukleinovokisly sequence that encodes the light chain of mAb 7.34.1, can be modified with a mutation changes 1 to obtain germ line sequence at the site of mutation 1. Furthermore, amino acid or nukleinovokisly sequence that encodes the light chain of mAb 7.34.1, can be modified with a mutation changes 2 to obtain germ line sequence in place �utali 2. Furthermore, amino acid or nukleinovokisly sequence that encodes the light chain of mAb 7.34.1, can be modified with both mutations change 1 and 2 to obtain the sequence of the germ line in both places, mutations 1 and 2.

EXAMPLE 12

INHIBITION of IGF-I And IGF-II-INDUCED phosphorylation of hIGF-IR, WHICH EPITOPE EXPRESSED IN NIH3T3 CELLS

IGF ligands cause of their proliferation and function of apoptosis by activation of receptor tyrosine kinases in the receptor IGF-IR. To assess the ability of anti IGF-I/II antibodies inhibit IGF-induced phosphorylation of IGF-IR, NIH3T3 cells, which ectopiceski Express hIGF-IR, used in the following analysis.

NIH3T3 cells, which ectopiceski Express human IGF-IR were seeded in the plate of 96 wells at a density of 10,000 cells per well and incubated overnight in a "hungry" medium (1% FBS in a mixture with coal). The next day, growth media was removed, the wells gently washed two times with PBS and 100 μl of the medium containing no serum (0% FBS) was added to kill the cells. After 1-2 hours, 100 μl of the medium containing serum with 0.05% BSA containing IGF-I (10 ng/ml) or IGF-II (10 nm) pre-incubated for 60 minutes at 37°C with different concentrations of antibodies and then added to �years in a set of three. Stimulation was performed for 10 minutes at 37°C, then the medium was removed and 100 μl of 3.7% formaldehyde in PBS/3%BSA was added to each well and incubated at room temperature for 20 minutes. Then the cells were washed 2X in PBS and each well was added 100 μl permeable buffer (0,1% Triton-X in 3% BSA/PBS). The mixture was incubated at room temperature for 10 minutes, removed and 100 μl of 0.6% hydrogen peroxide in PBS/3% BSA was added to inactivate any endogenous peroxidase activity. After 20 minutes of incubation at room temperature cells were washed 3X with PBS/0,1% Tween-20 and blocked by adding 100 μl of 10% FBS in PBS/0,1% Tween-20 at room temperature for 1 hour. Then the blocking buffer was removed and 50 ál of anti-phospho-IGFIR antibody at 1 ug/ml (cat. #44-804, BioSource) was added to each well in 10% FBS/PBS-T. After 2 hours incubation at room temperature, cells were washed 3X with soaking in PBST for 5 minutes each time before rinsing. After washings with 50 μl/well goat anti-rabbit IgGFc-HRP secondary antibody was diluted 1:250 in blocking buffer and added to each well. After 1 hour incubation at room temperature cells were washed 3X for 5 minutes with PBST as previously described, and dried. Was then added 50 μl of the reagent ECL (DuoLux) and immediately reads RLU (relative light units).

Screening was conducted�n for 32 (thirty two) lines of antibodies, and for each antigen was performed by two independent analysis. Results for the top ten of the antibodies are shown in Table 13 below.

TABLE 13.
BRIEF description of the INHIBITION of IGF-DEPENDENT IGF-IR PHOSPHORYLATION IN NIH3T3 CELLS
IGF-I pTYR results (n=2)IGF-II pTYR results (n=2)
% Max. activationEC50 (nm)*% Max. activation/ EC50 (nm)*
mAb IDAverageThe average deviationAverageThe average deviationAverageThe average deviationAverageThe average deviation
7.15 9.216,5%6,6%7,40,828,6% 5,4%3,10,2
7.34. 114,2%1,8%9,40,821,5%3,9%2,50,1
7.14 6.319,5%3,9%19,05,7the 23.6%2,1%3,60,2
7.25 1.316,9%5,4%14,50,715,9%1,5%3,00,9
7.23 4.221,1%3,0%24,31,021,1%0,1%7,7od
7.16 0.233,6%6,4%22,9od 21,5%0,6%4,70,2
7.15 8.222,7%0,9%28,30,533,7%2,4%11,33,2
7.56. 331,3%2,2%25,14,321,2%0,2%6,30,5
7.11 8.1to 24.1%5,2%of 40.82,621,8%3,2%13,95,3
7.41. 333,1%6,1%of 47.17,029,5%4,4%3,54,0
* These tests were conducted in antigen-limiting conditions, obtaining mAb KD d�I GF-I and IGF-II.

EXAMPLE 13

INHIBITION of IGF-I And IGF-II-INDUCED PROLIFERATION of NIH3T3 CELLS, TRANSFECTING WITH HIGF-IR

As discussed above, one of the criteria neutralization of IGF-I/II antibodies is the ability to inhibition of cell proliferation caused by IGF. To assess the ability of antibodies to inhibit induced IGF proliferation of human tumor cells of the pancreas Whrs, which Express endogenous hIGF-IR, used in the following analysis.

Cells Whrs that ectopiceski Express hIGF-IR, were seeded in a plate of 96 wells at a density of 5000 cells per well and were cultured overnight in standard culture medium (1% FBS in a mixture with coal). The next day, growth media was removed, the wells gently washed twice with medium containing no serum, and added 100 μl of the medium containing serum, for killing cells. 100 ál of "hungry" medium containing 15 ng/ml IGFI or 50 ng/ml of IGFII, pre-incubated for 30 minutes at 37°C with different concentrations of antibodies and added to the cells at double or triple the amount. After 20 hours of incubation the cells were exposed to pulses of BrdU for 2 hours and quantify the degree of incorporation (proliferation) using a kit for cell proliferation ELISA from Roche (Roche, cat. #1647229).

Screening was conducted for 32 lines anti�ate, and for each antigen was performed by two or three independent analysis. Results for top 10 antibodies are shown in Table 14 below.

0,6%
TABLE 14.
BRIEF description of the INHIBITION of IGF-DEPENDENT proliferation of NIH3T3/hIGF-IR CELLS
Analysis of cell proliferation IGF-IAnalysis of cell proliferation IGF-II
% inhibitionEC50 (nm)*% inhibitionEC50 (nm)*
mAb IDAverage (n=3)The average deviationAverage (n=3)The average deviationAverage (n=2)The average deviationEnvironments (n=2)The average deviation
7.15 9.277,0%9,6%24,15,999.3% of7,62,5
7.34. 172,6%5,6%23,48D73,6%11,8%16,30,4
7.14 6.365,3%5,5%37,24,582,0%6,9%15,93,4

7.25 1.372,4%15,3%38,94,379,2%7,8%22,03,7
7.23 4.267,3%6,9%40,64,662,1%17,2%24,32,4
7.16 0.262,8%the 5.7% 47,610,745,9%0,8%24,72,8
7.15 8.257,4%19,5%42,81,754,6%6,6%36,04,2
7.56. 350,2%7,8%65,731,948,0%10,9%38,37,5
7.11 8.159,4%14,5%1626,62714,568,3%0,8%49,93,8
7.41. 329,5%14,7%76,335,951,9%13,7%61,923,7
*These analyses would�and held in antigen-limiting conditions, obtaining mAb KD for IGF-I and IGF-II.

EXAMPLE 14

INHIBITION of IGF-I And IGF-II-INDUCED phosphorylation of hIGF-IR, WHICH is EXPRESSED IN CELLS of the HUMAN PANCREAS TUMOR Whrs

IGF-I/II trigger their proliferation and anti-apoptotic function by activation of the receptor tyrosine kinase IGF-IR. To assess the ability of antibodies to inhibit IGF-induced phosphorylation of IGF-IR, Whrs human tumor cells in the pancreas that Express endogenous hIGF-IR, used in the following analysis.

Cells Whrs were seeded in a plate of 96 wells with a density of 55 OOO cells per well and incubated overnight in normal growth media. The next day, growth media was removed, the wells gently washed twice with medium containing no serum, and 100 μl of the medium containing serum was added to kill the cells. After 24 hours the medium was removed and cells gently washed once with medium containing no serum. A medium without containing serum with 0.05% BSA containing IGF-I (20 ng/ml) or IGF-II (75 ng/ml) pre-incubated for 30 minutes at 37°C with different concentrations of antibody and 100 ál was then added to the cells in a set of three. The plates were incubated for 15 minutes at 37°C and then washed with cold PBS. 100 µl lisanova buffer added�whether to the wells and incubated tablet for 30 minutes at 4°C. Lysates centrifugal rotated at 2000 rpm for 10 minutes at 4°C, and the supernatants were collected. IGF-IR phosphorylation was quantitatively determined using Duoset human phospho-IGF-IR ELISA (R&D Systems, Cat. No. DYC1770).

The screening was held for ten lines, antibodies, and for each antigen was performed independent analysis. The results are shown in Table 15 below.

TABLE 15.
BRIEF description of the INHIBITION of IGF-DEPENDENT IGF-IR PHOSPHORYLATION
IGF-I pTYR results (n=2)IGF-II pTYR results (n=2)
n=1n=2n=1n=2
mAb IDMax.% inhibition (the 333.3 nm)/ EC50 (nm)Max.% inhibition (the 333.3 nm)/ EC50 (nm)Max.% inhibition (the 333.3 nm)/ EC50 (nm)*Max.% inhibition (133,3 nm)/ EC50 nm)
7.159.2100,03,3100,01,6100,01,691,21,7
7.34.1100,05,998,53,8100,02,089,71,9
7.146.396,416,194,210,7100,02,0of 87.92,0
7.251.395,77,595,25,3100,0N. D.91,32,6
7.234.297,35,191,52,998,51.,2,3
7.160.293,45,389,23,188,61,773,22,5
7.158.292,94,5of 89.43,692,4N. D.74.04.2
7.56.3to 84.9N. D.88,76,591,410,166,25,1
7.118.190,513,190,611,895,717,978,013,1
7.41.388,66,586,56,588,6 4,570,63,1
*333 nM for the last three 3 antibodies.
N. D.: Not determined

EXAMPLE 15

INHIBITION of IGF-I And IGF-II-INDUCED PROLIFERATION of HUMAN PANCREATIC TUMORS Whrs

As discussed above, one of the criteria of neutralizing IGF antibodies is the ability to inhibition of cell proliferation caused by IGF. To assess the ability of antibodies to inhibit induced IGF proliferation of human tumor cells of the pancreas Whrs, which Express endogenous hIGF-IR, used in the following analysis.

Cells Whrs were seeded in a plate of 96 wells at a density of 2000 cells per well and were cultured overnight in standard medium for cultivation. The next day, growth media was removed, the wells gently washed twice with medium containing no serum, and added 100 μl of the medium containing no serum, 10 µl/ml transferrin and 0.1% BSA (analytical environment) for killing of cells. After 24 hours the medium was removed, the cells are gently were washed once with serum and added 100 ál analytical medium containing 20 ng/ml IGF pre-incubated for 30 minutes at 37°C with various concentrations of antibodies were added to the cells in the d�oinam or triple the amount. The plates were incubated for 3 days and proliferation was quantified using reagent CellTiter-Glo (Promega).

The screening was held for ten lines, antibodies, and for each antigen was performed by two or three independent analysis. The results are shown in Table 16 below. On the basis of functional data below and the data of Example 14 were selected four of the best antibodies. Data analysis of cell proliferation caused by IGF-I, were excluded from the selection criteria due to the high observed variability analysis.

TABLE 16.
BRIEF description of the INHIBITION of IGF-DEPENDENT PROLIFERATION Whrs HUMAN PANCREATIC TUMOR CELL
IGF-II results in proliferation (n=2)
n=1n=2
MAb IDMax.% inhibition (the 333.3 nm)/ EC50 (nm)*Max.% inhibition (133,3 nm)/ EC50 (nm)
7.159.2120,0 0,8118,30,9
7.34.1117,00,5109,06,7
7.146.3128,73,0achieved sales of 119.57,3
7.251.3128,50,5to 105.34,4
7.234.2111,7N. D.200,72,6
7.160.279,71,1155,7N. D.
7.158.286,3with 0.0013of 148.3N. D.
7.56.387,0N. D.112,3102,0
7.118.1114,034,0137,054,7
7.41.3102,0N. D.73,0N. D.
*333 nm for the last 3 antibodies
N. D.: Not determined

EXAMPLE 16

The DEFINITION of CROSS-REACTIVITY TO MURINE IGF-I, IGF-II AND INSULIN

One of the objectives was to develop antibodies specific to IGF-I and IGF-II, but do not have cross-reactivity to insulin. To perform the following experiments on animals, antibodies were also to have a cross reactivity relative to a murine IGF-I/II, but not to mouse insulin. Accordingly, analyses were performed ELISA to determine the ability of selected antibodies cross-react with mouse and IGFs or insulin.

As shown in Table 17, five of the top ten antibodies were tested for cross reactivity with mouse or rat insulin using ELISA. The ELISA analyses showed that these antibodies had no cross-reactivity with mouse or rat insulin, compared with a negative control antibody RC.3.1, and in contrast to the positive control Anticriminal insulin antibodies.

0,58
TABLE 17
CROSS-REACTIVITY TO MOUSE INSULIN
OD 450 with different Ag
AntibodyMurine insulinRat insulinNo Ag
7.159.20,520,520,56
7.160.20,600,570,62
7.34.10,480,470,55
7.251.30,550,530,56
7.234.20,510,490,66
serum1,281,23to 1.34
anti rat insulin2,523,060,10
RC.3.10,580,62

EXAMPLE 17

The INHIBITION CAUSED by IGF-I And IGF-II MICE PHOSPHORYLATION of HUMAN IGF-IR, WHICH ECTOPICESKI EXPRESSED IN NIH3T3 CELLS

Monoclonal antibodies having cross-reactivity with mouse IGF-I and IGF-II were further tested to determine the degree of inhibition caused them IGF phosphorylation of IGF-IR. This analysis was performed as previously described for analysis using NIH3T3 cells, which ectopiceski Express the receptor hIGF-IR. The results are shown in Table 18.

NIH3T3 cells, which ectopiceski Express human IGF-IR were seeded in a plate of 96 wells at a density of 10,000 cells per well and incubated overnight in a "hungry" medium (1% FBS, decolorized with charcoal). The next day, growth media was removed, the wells gently twice washed with PBS and 100 μl of the medium containing no serum (0% FBS) was added to kill the cells. After 1-2 hours, 100 μl of the medium containing serum with 0.05% BSA containing murine IGF-I (10 nm) or IGF-II (10 nm) (R&D Systems, Inc., Minneapolis, MN Cat. no.791-792 MG and-MG, respectively), which were pre-incubated for 60 minutes at 37°C with various concentrations of antibodies were added to cells in a set of three. Stimulation was performed � for 10 minutes at 37°C, after stimulation, the medium was removed and 100 μl of 3.7% formaldehyde in PBS/3% BSA was added to each well and incubated at room temperature for 20 minutes. Then the cells were washed with 2X PBS and 100 µl permeable buffer (0,1% Triton-X in 3% BSA/PBS) was added to each well. It's all incubated at room temperature for 10 minutes, removed and 100 μl of 0.6% hydrogen peroxide in PBS/3% BSA was added to inactivate endogenous peroxidase activity. After 20 minutes incubation at room temperature, cells were washed 3X with PBS/0,1% Tween-20 and blocked by adding 100 μl of 10% FBS in PBS/0,1% Tween-20 at room temperature for 1 hour. Then the blocking solution was removed and 50 ál of anti-phospho-IGFIR antibody at 1 ug/ml (cat. #44-804, BioSource) was added to each well in 10% FBS/PBS-T. After 2 hours incubation at room temperature cells were washed 3X PBST, soaking for 5 minutes between each wash. After washings with 50 μl/well goat anticalcium IgGFc-HRP second antibody, diluted 1:250 in blocking buffer, was added to each well. After 1 hour incubation at room temperature cells were washed 3X for 5 minutes with PBST as described above, and dried. Was then added 50 μl of each ECL reagent (DuoLux) and immediately reads RLU.

TABLE 18.
INHIBITION of phosphorylation of hIGF-IR-INDUCED MURINE IGF
Murine IGF-I / EC50 (nm)Mouse IGF-II / EC50 (nm)
mAb IDn=1n=2n=1n=2
7.159.22,85,73,15,0
7.34.16,010,24,09,7
7.251.36,710,65,48,7
7.234.246,0the 36.1
7.160.2of 49.5225,2

EXAMPLE 18

The INHIBITION of growth of NIH3T3 CELLS, EXPRESSIONWHICH IGF-II And IGF-IR IN VIVO "NUDE" MICE

To assess the ability of antibodies to inhibit induced IGF-II proliferation�Oia in vivo, were conducted the following experiments.

"Naked" female mice aged 6-8 weeks (supplied by Charles River Laboratories, Wilmington, MA, USA) were implanted subcutaneously with 5x106 cells of clone 32 (NIH3T3 cells, ectopiceski redundantly expressing human IGF-I and human IGF-1R). Cells suspended in PBS vaccination in General the volume of 330 μl. Tumors were left to grow to 100-200 mm3before treatment with monoclonal antibodies 7.159.2, 7.34.1 and 7.251.3. IgG2 antibodies or isotype control antibodies suspended in PBS, were injected intraperitoneally in a randomized group consisting of 9 or 12 mice, twice a week for 4 weeks at 5 or 50 mg/kg, starting on Day 22. PBS was injected as control are the basis for further group, consisting of 11 mice weekly for 4 weeks, starting on Day 22. Tumor size and body weight were measured 2-3 times per week.

There was a significant inhibition of tumor growth, while none of the groups was not observed a significant reduction of body weight. Antibodies 7.159.2, 7.34.1 and 7.251.3 significantly inhibited the growth of tumors with clone 32 with 5 and 50 mg/kg/week.

EXAMPLE 19

The INHIBITION of growth of NIH3T3 CELLS EXPRESSING IGF-I And IGF-IR IN VIVO "NUDE" MICE

In the previous example, it was shown that the antibodies inhibit induced IGF-II proliferation in vivo. In order to evaluate the antibodies for their STRs�oblasti to inhibit induced IGF-I proliferation in vivo were conducted the following experiments.

Naked female mice (strain Alderley Park, obtained from strain Swiss bare mice, which is supplied by AstraZeneca) were implanted with 5×106viable P12 cells [NIH3T3 cells, ectopiceski redundantly expressing human IGF-I and human IGF-IR (Pietrzkowski et al., Cell Growth &Differentiation, 3, 199-205, 1992)] subcutaneously in the left side. Cells suspended in PBS in a total grafting volume of 0.1 ml. Two groups of animals (each n=10) dosed out twice a week, starting from the day of implantation NIH3T3 cells mAb 7.159.2 at 1.0 mg per mouse, or an equivalent volume basis PBS (0.3 ml) on the same schedule. All doses were given intraperitoneally (i.p.). Body weight of animals was measured every day and after the establishment of mass, measurement of tumors was performed twice a week using calipers. Volume of all measurable tumors was calculated on the basis of measurements of the compass, assuming an oval form.

Significant inhibition of tumor growth was observed when using mAb 7.159.2 after the intraperitoneal injection twice per week 1.0 mg of antibody/mouse. In one group of animals was not observed a significant loss of mass.

EXAMPLE 20

The INHIBITION of GROWTH of TUMOR CELLS IN PATIENTS-PEOPLE

A group of people suffering from cancer, who was diagnosed with pancreatic cancer, p�Osvaldo were divided into treatment groups. Each group of patients were treated with weekly intravenous injections of mAb 7.159.2, 7.34.1 or 7.251.3, described in this application. Each patient was given a dose of an effective amount of the antibody in the range of 50 mg/kg up to 2,250 mg/kg for 4-8 weeks. The control group received only standard chemotherapy

Periodically during and after treatment, the tumor mass was assessed using magnetic resonance imaging (MRI). It was found that patients who received weekly treatment with antibodies mAb 7.159.2, 7.34.1 or 7.251.3, showed a significant reduction in tumor size, compared with patients who do not receive such treatment. Some patients receiving treatment, the tumor was not found. On the contrary, in the control group, the size of tumors increased or did not change.

EXAMPLE 21

The INHIBITION of GROWTH of TUMOR CELLS IN PATIENTS-PEOPLE

If a male patient was diagnosed with a malignant tumor. The patient was treated through the introduction of weekly intravenous injections of mAb 7.159.2 for 8 weeks. Periodically during and after treatment, the tumor mass was assessed using magnetic resonance imaging (MRI). It was found a significant decrease in tumor size.

EXAMPLE 22

TREATMENT of ACROMEGALY IN PATIENTS-PEOPLE

In an adult male was prediagnostic�on acromegaly. The patient was treated through the introduction of twice-weekly intravenous injections of mAb 7.34.1 for 2 years. As a result, the patient had significant reduction in symptoms of acromegaly.

EXAMPLE 23

TREATMENT of PSORIASIS IN PATIENTS-PEOPLE

The adult woman was diagnosed with severe psoriasis. Patient was treated by introducing a twice weekly intravenous injections of mAb 7.251.3 for 3 weeks. As a result, the patient was observed a significant decrease in the symptoms of psoriasis.

EXAMPLE 24

TREATMENT of OSTEOPOROSIS IN PATIENTS-PEOPLE

In adult women diagnosed osteoporosis. Patient was treated by introducing a twice weekly intravenous injections of mAb 7.159.2 during the year. As a result this has led to a significant reduction of bone loss.

EXAMPLE 25

TREATMENT of ATHEROSCLEROSIS IN PATIENTS-PEOPLE

In an adult male was diagnosed atherosclerosis. The patient was treated through the introduction of twice-weekly intravenous injections of mAb 7.34.1 during the year. As a result, the patient had a reduction of symptoms of atherosclerosis, such as heart attack.

EXAMPLE 26

TREATMENT of RESTENOSIS IN PATIENTS-PEOPLE

Grown woman received angioplasty for the relief of blocked arteries. After the procedure, angioplasty patient was treated by injecting twice a week intravenously �of nycci mAb 7.251.3 during the year. As a result, the patient did not happen restenosis of the artery, which was treated.

EXAMPLE 27

DIABETES PATIENTS-PEOPLE

The adult woman was diagnosed with diabetes. Patient was treated by introducing a twice weekly intravenous injections of mAb 7.159.2 during the year. As a result, the symptoms of diabetes were reduced.

SEQUENCE

Sublimirovanny of hybridomas were sequenced to determine their primary structure, both at the level of nucleotide and amino acid levels, as genes for variable regions of heavy and light chains. Nucleotide and polypeptide sequences of the variable regions of monoclonal antibodies to IGF-I and IGF-II, as listed in Table 1 given in the sequence Listing.

INCORPORATION BY REFERENCE

All the references used in this application, including patents, patent applications, articles, books, etc., all cited in their references, to the extent in which they are entirely included in this application by reference.

EQUIVALENTS

The above written specifications are considered sufficient to enable a person skilled in the art could implement the invention. In the above Description and Examples have been described in detail preferred embodiments of the present invention and describes the best the regimes of mode - �latelyi. However, it will be appreciated that regardless of how detailed the foregoing, the present invention can be implemented in many ways and can be interpreted in accordance with the formula, which is attached, and any equivalents.

1. A fully human antibody or antigen-binding fragment that bind insulin-like growth factor-II (IGF-II), have cross-reactivity to insulin-like growth factor I (IGF-I) and neutralize the activity of IGF-I and IGF-II, where the specified antibody or its binding fragment includes a heavy chain polypeptide having the sequence of SEQ ID no: 14 and a light chain polypeptide having the sequence of SEQ ID no: 16.

2. The antibody or antigen-binding fragment according to claim 1, where the specified antibody is a fully human monoclonal antibody.

3. The antibody or antigen-binding fragment according to claim 1, where the specified antigen-binding fragment is an antigen-binding fragment of a fully human monoclonal antibody.

4. The antibody or antigen-binding fragment according to claim 3, where the specified antigen-binding fragment is selected from the group consisting of Fab, Fab' or F(ab')2and Fv.

5. The antibody or antigen-binding fragment according to any one of claims.1-4, where the specified antibody is a monoclonal� antibody 7.251.3 (produced by cell line which has the registration number of ATSS No. MOUTH-7422).

6. Composition for treating malignant tumors comprising a pharmaceutically acceptable carrier and an effective amount of the antibody or antigen-binding fragment according to any one of claims.1-5.

7. A nucleic acid molecule that encodes the antibody or antigen-binding fragment according to any one of claims.1-5.

8. Vector designed for expression of the antibody or antigen-binding fragment according to any one of claims.1-5 and contains the nucleic acid molecule according to claim 7.

9. A host for receiving the antibody or antigen-binding fragment according to claim 1 to 5, containing the vector of claim 8.

10. Human monoclonal antibody according to any one of claims.1-5, where the specified antibody does not bind specifically to IGF-II or IGF-I proteins, if these proteins are associated with insulin binding proteins growth factor.

11. The method of determining the level of insulin-like growth factor-II (IGF-II) and insulin-like growth factor I (IGF-I) in the patient sample, which includes:
contacting the said sample of the patient with the antibody or antigen-binding fragment according to any one of claims.1-5; and
determining the level of IGF-I and IGF-II in this sample.

12. The use of antibodies or antigen-binding fragment according to any one of claims.1 to 5 to obtain drugs for the treatment of SLAC�quality of the tumor.

13. The use according to claim 12, where the specified malignant tumor selected from the group consisting of melanoma, non-small cell lung cancer, glioma, hepatocellular cancer, thyroid tumor, gastric cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer, pancreatic cancer, and epidermoid carcinoma.

14. The use according to claim 12 or 13, where the specified drug is intended for use in combination with a second antineoplastic agent which is selected from the group consisting of antibody, chemotherapeutic agent or radioactive drugs.

15. Conjugate for the treatment of malignant tumors, comprising the antibody or antigen-binding fragment according to any one of claims.1-5 and a therapeutic agent.

16. The conjugate according to claim 15, where therapeutic agent is a toxin; a radioactive isotope or a pharmaceutical composition.



 

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FIELD: medicine, pharmaceutics.

SUBSTANCE: presented group of inventions concerns fused proteins, nucleic acids coding these proteins, an expressing cartridge providing nucleic acid expression, a vector comprising this cartridge, a diagnostic technique for in vitro borreliosis, a kit for this diagnostic technique, which use these proteins, as well as a vaccine composition for preventing borreliosis containing these proteins. The characterised fused proteins contain (i) at least one sequence of DbpA protein of the species Borrelia specified in B. afzelii, B. burgdorferi sensu stricto and B. garinii, and (ii) least one sequence of OspC protein of the species Borrelia specified in B. afzelii, B. burgdorferi sensu stricto and B. garinii.

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FIELD: chemistry.

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FIELD: medicine.

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43 cl, 98 dwg, 20 tbl, 26 ex

FIELD: medicine.

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11 cl, 20 dwg, 2 tbl, 2 ex

FIELD: medicine.

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3 tbl, 3 ex

FIELD: biotechnologies.

SUBSTANCE: invention can be used for determination of atherogenicity of immune complexes containing multiply modified low density lipoproteins (IC-MMLDLP). For this purpose basic calcium superphosphate of IC-MMLDLP is prepared from human blood serum by treatment with the buffer containing 10% solution of polyethyleneglycol with a molecular weight 3350 (PEG-3350), in the ratio 1:2.5, then it is incubate within 10 minutes at a room temperature. IC-MMLDLP aggregates are pelleted, dissolved in the buffer without PEG-3350, analysed for the content of cholesterol in immune complexes (ChIK) and the level of guinea pig complement binding (EBC) by precipitated immune complexes. IC-MMLDLP atherogenicity is calculated as SSK to HIK ratio. If the value is below 24 units a high blood atherogenicity because of the reduced complement activating IgG function in IC-MMLDLP is stated.

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1 tbl

FIELD: medicine.

SUBSTANCE: invention can be used for the instant assessment of atherogenicity of the immune complexes (IC) of human blood serum. Precipitated ICs from human blood serum are prepared by treating with a buffer containing 10% polyethylene glycol of molecular weight 3,350 (PEG-3350) in ratio 1:3.5, incubating for 10 min at room temperature. Aggregated ICs are deposited by centrifuging and dissolved in PEG-3350 free buffer; the immune complex cholesterol (ICC) is measured, and a guinea pig's complement fixation (CF) by the precipitated immune complexes is determined. The IC atherogenicity is calculated as CF/ICC relation, and if the derived value is less than 4 units, the high blood atherogenicity is stated.

EFFECT: higher assessment accuracy.

1 tbl

FIELD: biotechnology.

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5 dwg, 1 tbl, 5 ex

FIELD: medicine.

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2 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to oncohaematology, and can be used to monitor the effectiveness of the anti-tumour treatment of patients with multiple myeloma. For this purpose the concentration of free light chains of immunoglobulins is determined before and 24 hours after finishing the treatment course. In case of a 60% reduction of the concentration, the performed treatment is considered to be effective, in case the reduction of the concentration is 60% and lower - ineffective.

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5 ex

FIELD: medicine.

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33 cl, 21 dwg, 3 tbl

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and immunology. There are presented optimised genes of light and heavy chains of Infliximab, an anti-tumour necrosis factor alpha (TNF-alpha) antibody, as well as a cell line VKPM-N-131, and a method for antibody biosynthesis. Nucleotide sequences of the genes coding the light and heavy chains of Infliximab are optimised in order to provide the content of codones most specific for mammals; the G/C content is expected to make 50-60% of the total composition; the absence of expanded tracts of a degenerate composition and the absence of RNA secondary structures.

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4 cl, 3 dwg, 4 ex

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16 cl, 7 dwg, 13 tbl, 8 ex

FIELD: medicine.

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43 cl, 98 dwg, 20 tbl, 26 ex

FIELD: biotechnology.

SUBSTANCE: synthetic DNA is proposed, encoding human erythropoietin, having the sequence Seq ID No. 1, comprising its expression vector, the method of production of erythropoietin producer strain, and a strain of a Chinese hamster ovary cells - producer of recombinant human erythropoietin, deposited under the number RKKK(P) 761 D.

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5 cl, 1 tbl, 8 dwg, 4 ex

FIELD: medicine.

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23 cl, 74 dwg, 17 tbl, 33 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to antibodies including human antibodies and their antigen-binding portions, which specifically bind to CCR2, in particular to human CCR2, and can act as CCR2 inhibitors. Anti-CCR2 antibodies are those binding to first and/or second extra-cellular CCR2 loops. The present invention also refers to human anti-CCR2 antibodies and to their antigen-binding portions. The present invention refers to the recovered heavy and light chains of immunoglobulin initiated from human anti-CCR2 antibodies, and to nucleic acid molecules coding such immunoglobulins. The present invention also refers to methods for preparing human anti-CCR2 antibodies and their antigen-binding portions, to compositions containing such antibodies or their antigen-binding portions, and to methods for using antibodies and their antigen-binding portions, and compositions for diagnosing and treating.

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25 cl, 24 dwg, 8 tbl, 17 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. Presented are antibodies targeting integrin α2β1 containing humanised anti-integrin alpha-2 (α2) antibodies, as well as a method of treating by the integrin α2 antibodies. The humanised integrin α2 antibodies comprise a variable region of a light chain domain, a constant human light chain domain and a variable constant heavy chain domain of human IgG1, which exhibit the altered effector function. The variable constant heavy chain domain of human IgG1 comprises an S324N substitution. The invention can be used in medicine.

EFFECT: antibodies exhibit complement-dependent cytotoxicity, improved antibody-dependent cell-mediated cytotoxicity and improved CDC and ADCC.

33 cl, 3 dwg, 1 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology.

EFFECT: bispecific anti-human vascular endothelial growth factor VEGF and human angiopoietin-2 ANG-2 antibodies, methods for producing them, pharmaceutical compositions containing the above antibodies, and using them are described.

13 cl, 26 dwg, 15 tbl, 19 ex

Siglec-15 antibody // 2539790

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. What is described is a pharmaceutical composition used for treating and/or preventing pathological bone metabolism and containing this antibody. The invention can be used in medicine.

EFFECT: antibody and its functional fragment specifically recognising human Siglec-15 and possessing the osteoclast inhibitory activity are described.

73 cl, 57 dwg, 4 tbl, 33 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of immunology. Claimed is isolated antibody to ICOS protein of people with increased effector function. Also described are cell and method of obtaining antibody in accordance with claimed invention, pharmaceutical composition, method of treating autoimmune disease or disorder, transplant rejection and malignancy of human T-cells, as well as method of depletion of ICOS-expressing T-cells, method of destroying germ centre structure in secondary lymphoid organ of primates, methods of depleting B-cells of germ centre of secondary lymphoid organ and circulating B-cells, which have undergone class switching, in primates.

EFFECT: invention can be further applied in therapy of diseases, mediated by T-cells.

33 cl, 21 dwg, 3 tbl

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