HK1108391B

HK1108391B - Antibody formulation in histidine-acetate buffer

Info

Publication number: HK1108391B
Application number: HK08102619.9A
Authority: HK
Inventors: James D. Andya; Shiang C. Gwee; Jun Liu; Ye Shen
Original assignee: 健泰科生物技术公司
Priority date: 2004-10-20
Filing date: 2005-10-19
Publication date: 2012-05-25

Description

Antibody formulations in histidine-acetate buffer

This is a non-provisional application filed under 37CFR1.53(b), claiming priority from provisional application 60/620,413 filed on 10/20/2004, the contents of which are incorporated herein by reference.

Technical Field

The present invention relates to antibody formulations, including monoclonal antibodies formulated in histidine-acetate buffer, as well as formulations comprising antibodies that bind domain II of HER2 (e.g., Pertuzumab), and formulations comprising antibodies that bind DR5 (e.g., Apomab).

Background

In the last decade, advances in biotechnology have made it possible to produce a variety of proteins for pharmaceutical applications using recombinant DNA technology. The formulation of these proteins poses a particular problem because they are larger and more complex (i.e., have multiple functional groups in addition to a complex three-dimensional structure) than traditional organic and inorganic drugs. In order for a protein to retain biological activity, the formulation must maintain intact the conformational integrity of at least the core sequence of the protein's amino acids, while protecting the various functional groups of the protein from degradation. Degradation pathways for proteins may include chemical instability (i.e., any process involving modification of the protein by bond formation or cleavage, which results in a new chemical entity) or physical instability (i.e., a change in the higher order structure of the protein). Chemical instability can result from deamidation, racemization, hydrolysis, oxidation, beta elimination or disulfide exchange. Physical instability can result, for example, from denaturation, aggregation, precipitation or adsorption. The three most common protein degradation pathways are protein aggregation, deamidation and oxidation. Cleland et al Critical Reviews in therapeutics monitor Systems 10 (4): 307-377(1993).

Antibody formulations

Proteins for pharmaceutical applications include antibodies. An example of an antibody that may be used in therapy is an antibody that binds the HER2 antigen, such as Pertuzumab.

U.S. patent No. 6,339,142 describes a HER2 antibody composition comprising a mixture of an anti-HER 2 antibody and one or more acidic variants thereof, wherein the amount of acidic variants is less than about 25%. Trastuzumab is an exemplary HER2 antibody.

U.S. Pat. Nos. 6,267,958 and 6,685,940(Andya et al) describe lyophilized antibody formulations, including HER2 and IgE antibody formulations. WO97/04807 and US 2004/0197326A1(Fick et al) describe methods of treating allergic asthma with IgE antibodies. WO99/01556(Lowman et al) relates to IgE antibodies having asparagine (aspatyl) residues prone to isomerization, and improved variants thereof. US 2002/0045571(Liu et al) provides concentrated protein formulations with reduced viscosity, exemplified by humanized IgE antibody formulations, rhuMAb E25 and E26. WO 02/096457 and US2004/0170623(Arvinte et al) describe stable liquid formulations comprising the anti-IgE antibody E25. See also US 2004/0197324 a1(Liu and Shire) directed to high concentration anti-IgE formulations.

U.S. Pat. No. 6,171,586(Lam et al) describes stable aqueous antibody formulations. The F (ab') 2rhuMAb CD18 antibody was formulated in sodium acetate and histidine-HCl buffer. rhuM A preferred formulation for Ab CD18 is 10mM sodium acetate, 8% trehalose, 0.01% TWEEN20^TMpH5.0. Acetate (ph5.0) formulations of rhuMAb CD20 stored at 40 ℃ for one month showed greater stability than those samples formulated in histidine (ph5.0 or 6.0).

US 2003/0190316(Kakuta et al) relates to the formulated antibody hPM-1, which is a humanized IL-6 receptor antibody. The monomer loss was greatest in sodium citrate (pH6.7), followed by sodium phosphate (pH6.8), Tris-HCl (pH7.2), histidine-HCl (pH7.2) and glycine (pH7.6), in decreasing order. The effect of sodium phosphate (pH6.5), phosphate-His (pH6.0 or 6.5), His-HCl (pH6.5), and sodium phosphate (pH6.0) on hPM-1 stability was evaluated.

WO2004/071439(Burke et al) indicated that the degradation of polysorbate 80 in a formulation of natalizumab (a humanized monoclonal antibody against. alpha.4 integrin) resulted in an increase in impurities, apparently by an oxidation reaction involving metal ions and histidine. Thus, phosphate buffer was chosen.

WO2000/066160 (English corresponding to the text EP1174148A1) (Okada et al) relates to the formulation of humanized C4G1 antibodies that bind to the fibrinogen receptor of the human platelet membrane glycoprotein GPIIb/IIIa in a sodium phosphate or sodium citrate buffer.

WO2004/019861(Johnson et al) relates to CDP870, a pegylated anti-TNF α Fab fragment formulated at 200mg/ml in 50mM sodium acetate (pH5.5) and 125mM sodium chloride.

WO2004/004639(Nesta, P) relates to the formulation of huC242-DM1, a tumor-activated immunotoxin, in 50mM succinic acid buffer (pH6.0) and sucrose (5% w/v).

WO03/039485(Kaisheva et al) found that Daclizumab (a humanized IL-2 receptor antibody) had the highest stability in sodium succinate buffer pH6.0 and rapidly lost potency in histidine as the buffer was oxidized.

WO 2004/001007 relates to a CD80 monoclonal antibody in histidine HCl, sodium acetate or sodium citrate buffer.

U.S. patent No. 6,252,055 (relan, J.) relates to anti-CD 4 and anti-CD 23 antibodies formulated in maleate, succinate, sodium acetate or phosphate buffers, with phosphate being identified as the preferred buffer.

U.S. Pat. No. 5,608,038(Eibl et al) relates to highly concentrated polyclonal immunoglobulin preparations having immunoglobulins, glucose or sucrose, and sodium chloride.

WO03/015894(Oliver et al) relates to 100mg/mL25mM histidine-HCl, 1.6mM glycine, pH6.0, and lyophilized The aqueous preparation of (1), theAt the time of formulation (before lyophilization) 25mM histidine, 1.6mM glycine and 3% w/v mannitol pH6.0 were included.

US 2004/0191243A1(Chen et al) reports a preparation of the human IgG2 antibody ABX-IL 8.

US 2003/0113316a1(Kaisheva et al) relates to a lyophilized anti-IL 2 receptor antibody formulation.

HER2 antibody

The HER family of receptor tyrosine kinases is an important regulator of cell growth, differentiation and survival. The receptor family includes four distinct members, including epidermal growth factor receptor (EGFR, ErbB1, or HER1), HER2(ErbB2 or p 185)^neu) HER3(ErbB3) and HER4(ErbB4 or tyro 2).

EGFR encoded by the erbB1 gene is implicated in the etiology of human malignancies. In particular, increased EGFR expression has been observed in breast, bladder, lung, head, neck and stomach cancers as well as glioblastomas. Increased EGFR receptor expression is often associated with increased production of the EGFR ligand, transforming growth factor alpha (TGF- α), by the same tumor cells, which results in receptor activation through an autocrine stimulatory pathway. Baselga and Mendelsohn pharmac. ther.64: 127-154(1994). Monoclonal antibodies against EGFR or its ligands TGF-alpha and EGF have been evaluated as therapeutic agents in the treatment of these malignancies. See, e.g., Baselga and mendelsohn, supra; masui et al Cancer Research 44: 1002-1007 (1984); and Wu et al j.clin.invest.95: 1897-1905(1995).

Second member of the HER family, p185^neuOriginally identified as the product of the transforming gene from neuroblastoma in chemically treated rats. The activated form of the neu proto-oncogene is produced by point mutations (valine to glutamic acid) in the transmembrane region of the encoded protein. Amplification of the neu human homolog is observed in breast and ovarian cancers and is associated with poor prognosis (Slamon et al, Science, 235: 177-182 (1987); Slamon et al, Science, 244: 707-712 (1989); and U.S. Pat. No. 4,968,603). To date, no point mutations similar to those in Neu protooncogenes have been reported for human tumors. Overexpression of HER2 (often but not uniformly due to gene amplification) has also been observed in other cancers, including cancers of the stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas and bladder. See, King et al, Science, 229: 974 (1985); yokota et al, Lancet: 1: 765-; fukushige et al, Mol Cell biol, 6: 955- "958 (1986); guerin et al, Oncogene res, 3: 21-31 (1988); cohen et al, Oncogene, 4: 81-88 (1989); yonemura et al, Cancer res, 51: (1034 1991); borst et al, gynecol. oncol., 38: 364 (1990); weiner et al, Cancer res, 50: 421-; kern et al, Cancer res, 50: 5184 (1990); park et al, Cancer res, 49: 6605 (1989); zhau et al, mol. carcinog, 3: 254-; aasland et al br.j. cancer 57: 358-363 (1988); williams et al pathway 59: 46-52 (1991); and McCann et al, Cancer, 65: 88-92(1990). HER2 can be overexpressed in prostate Cancer (Gu et al Cancer Lett.99: 185-9 (1996); Ross et al hum.Pathol.28: 827-33 (1997); Ross et al Cancer 79: 2162-70 (1997); and Sadasivan et al J.Urol.150: 126-31 (1) 993))。

Anti-rat p185 has been described^neuAnd the human HER2 protein product. Drebin and colleagues cultivated anti-rat neu gene product, p185^neuThe antibody of (1). See, e.g., Drebin et al, cell 41: 695-706 (1985); myers et al, meth.enzyme.198: 277-290 (1991); and WO 94/22478. Drebin et al Oncogene 2: 273-277(1988) report with p185^neuThe mixture of antibodies reacting in two different regions brings about a synergistic antitumor effect on neu-transformed NIH-3T3 cells transplanted into nude mice. See also U.S. patent 5,824,311, published on month 10 and 20 of 1998.

Hudziak et al, mol.cell.biol.9 (3): 1165-1172(1989) describe the generation of a panel of HER2 antibodies, characterized by the human breast tumor cell line SK-BR-3. Relative cell proliferation of SK-BR-3 cells was determined by monolayer crystal violet staining after 72 hours exposure to the antibody. Using this assay, maximum inhibition was obtained with an antibody called 4D5 that inhibited cell proliferation by 56%. The other antibodies in this panel reduced cell proliferation to a lesser extent in this assay. It was further found that antibody 4D5 sensitizes breast tumor cell lines overexpressing HER2 to the cytotoxic effects of TNF-a. See also U.S. Pat. No. 5,677,171, published 10/14 in 1997. The HER2 antibody discussed in Hudziak et al is further characterized in the following literature. Fendly et al cancer research 50: 1550 and 1558 (1990); kotts et al in vitro 26 (3): 59A (1990); growth Regulation 1 of Sarup et al: 72-82 (1991); shepard et al j. clin. immunol.11 (3): 117-127 (1991); kumar et al mol.cell.biol.11 (2): 979-; lewis et al Cancer immunol.imother.37: 255-; pietras et al Oncogene 9: 1829-1838 (1994); vitetta et al Cancer Research 54: 5301-5309 (1994); sliwkowski et al j.biol.chem.269 (20): 14661-14665 (1994); scott et al j.biol.chem.266: 14300-5 (1991); d' souza et al proc.natl.acad.sci.91: 7202-; lewis et al Cancer research 56: 1457-1465 (1996); and Schaefer et al Oncogene 15: 1385-1394(1997).

Recombinant humanized forms of murine HER2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2, Trastuzumab orU.S. Pat. No. 5,821,337) is clinically effective in patients with breast cancer that overexpresses HER2 who have previously received extensive anti-cancer therapy (Baselga et al, j.clin.oncol.14: 737-744(1996)). Trastuzumab received marketing approval from the food and drug administration at 25.9.1998 for the treatment of patients with metastatic breast cancer whose tumors overexpress HER2 protein.

In Tagliabue et al int.j. cancer 47: 933 937 (1991); McKenzie et al Oncogene 4: 543 and 548 (1989); maier et al Cancer res.51: 5361-5369 (1991); bacillus et al Molecular Carcinogenesis 3: 350-362 (1990); stancovski et al PNAS (USA) 88: 8691 and 8695 (1991); bacillus et al Cancer Research 52: 2580 + 2589 (1992); xu et al int.j. cancer 53: 401-408 (1993); WO 94/00136; kasprzyk et al Cancer Research 52: 2771-2776 (1992); hancock et al Cancer Res.51: 4575-4580 (1991); shawver et al Cancer Res.54: 1367-; artemiga et al Cancer res.54: 3758-3765 (1994); harteth et al j.biol.chem.267: 15160- > 15167 (1992); U.S. Pat. nos. 5,783,186; and Klaper et al Oncogene 14: 2099-2109(1997) describe other antibodies to HER2 with various properties.

Homology screening has identified two other HER receptor family members; HER3 (U.S. Pat. Nos. 5,183,884 and 5,480,968 and Kraus et al PNAS (USA) 86: 9193-. Both of these receptors exhibit enhanced expression in at least some breast cancer cell lines.

Various combinations of HER receptors are commonly found in cells, and heterodimerization is thought to enhance the diversity of cellular responses to a variety of HER ligands (ear et al Breast cancer research and Treatment 35: 115-132 (1995)). EGFR binds to six different ligands; epidermal Growth Factor (EGF), transforming Growth factor alpha (TGF-. alpha.), amphiregulin (ampheregulin), heparin-binding epidermal Growth factor (HB-EGF), beta cell protein (betacellulin) and epithelial regulatory protein (epiregulin) (Groenen et al Growth Factors 11: 235-257 (1994)). The heregulin protein family resulting from single gene selective cleavage are ligands of HER3 and HER 4. The heregulin family includes the alpha, beta and gamma heregulins (Holmes et al, Science, 256: 1205-1210 (1992); U.S. Pat. No. 5,641,869; and Schaefer et al Oncogene 15: 1385-1394 (1997)); neu Differentiation Factors (NDFs), Glial Growth Factors (GGFs); acetylcholine Receptor Inducing Activity (ARIA); and sensory and motor neuron derived factor (SMDF). For a review see Groenen et al Growth Factors 11: 235-257 (1994); lemke, g.molec. & cell.neurosci.7: 247-: 51-85(1995). Three additional HER ligands have recently been identified; neuregulin-2 (NRG-2) reported to bind HER3 or HER4 (Chang et al Nature 387509-; neuregulin-3 binding to HER4 (Zhang et al PNAS (USA)94 (18): 9562-7 (1997)); and neuregulin-4 which binds HER 4(Harari et al Oncogene 18: 2681-89 (1999)). HB-EGF, beta cell protein and epithelial regulatory protein also bind HER 4.

Although EGF and TGF α do not bind HER2, EGF stimulates EGFR and HER2 to form a heterodimer, which activates EGFR and leads to transphosphorylation of HER2 in the heterodimer. Dimerization and/or transphosphorylation appear to activate HER2 tyrosine kinase. See, Earp et al, supra. Similarly, when HER3 is co-expressed with HER2, an active signaling complex is formed and anti-HER 2 antibodies are able to disrupt this complex (Sliwkowski et al, J.biol.chem., 269 (20): 14661-14665 (1994)). Furthermore, HER3, when co-expressed with HER2, increased the affinity for regulatory proteins (HRG) to a higher affinity state. For the HER2-HER3 protein complex see also Levi et al, Journal of Neuroscience 15: 1329-; morrissey et al, proc.natl.acad.sci.usa 92: 1431-1435 (1995); and Lewis et al, cancer res, 56: 1457-1465(1996). HER4, like HER3, forms an active signaling complex with HER2 (Carraway and Cantley, Cell 78: 5-8 (1994)).

To target the HER signaling pathway, rhuMAb2C4(Pertuzumab, OMNITARG) was used^TM) Developed as humanized antibodies that inhibit dimerization of HER2 with other HER receptors, thereby inhibiting ligand-driven phosphorylation and activation, as well as downstream activation of the RAS and AKT pathways. In a phase I trial of Pertuzumab as a single agent for treating solid tumors, three subjects with advanced ovarian cancer were treated with Pertuzumab. One patient had a sustained partial response, while the other subjects had stable disease for 15 weeks, Agus et al Proc Am Soc Clin Oncol 22: 192, Abstract 771 (2003).

DR5 antibody

Various ligands and receptors belonging to the Tumor Necrosis Factor (TNF) superfamily have been identified in the art. These ligands include tumor necrosis factor-alpha ("TNF-alpha"), tumor necrosis factor-beta ("TNF-beta" or "lymphotoxin-alpha"), lymphotoxin-beta ("LT-beta"), CD30 ligand, CD27 ligand, CD40 ligand, OX-40 ligand, 4-1BB ligand, LIGHT, Apo-1 ligand (also known as Fas ligand or CD95 ligand), Apo-2 ligand (also known as Apo2L or TRAIL), Apo-3 ligand (also known as TWEAK), APRIL, OPG ligand (also known as RANK ligand, ODF, or TRANCE), and TALL-1 (also known as BlyS, BAFF, or THANK) (see, for example, Ashkenazi, Nature Review, 2: 420-Bufonic 430 (2002); Ashkenazi and Xit, Science, 281: Ashkenazi 1305 (1998); Dihkenazi and Curxit, Currin, Op. 255, cell 11, cell 1308, 11, cell, 11, 75, 2000, 11, 75, 2000, 7, 3, 7, K, and D, D, 7: 750-; locksley et al, Cell, 104: 487 501 (2001); gruss and Dower, Blood, 85: 3378-3404 (1995); schmid et al, proc.natl.acad.sci., 83: 1881 (1986); dealtry et al, eur.j.immunol., 17: 689 (1987); pitti et al, j.biol.chem., 271: 12687-; wiley et al, Immunity, 3: 673-682 (1995); browning et al, Cell, 72: 847-; armitage et al Nature, 357: 80-82(1992), WO 97/01633 published 1997 on 16.1; WO 97/25428 published on 17.7.1997; marsters et al, curr. biol., 8: 525-528 (1998); chicaporthe et al, biol. chem., 272: 32401-32410 (1997); hahne et al, j.exp.med., 188: 1185-1190 (1998); WO98/28426, published on 2.7.1998; WO98/46751, published on 22/10/1998; WO/98/18921 published on 7/5/1998; moore et al, Science, 285: 260-263 (1999); shu et al, j.leukcyte biol., 65: 680 (1999); schneider et al, j.exp.med., 189: 1747 — 1756 (1999); mukhopadhyay et al, j.biol.chem., 274: 15978-15981(1999)).

The induction of various cellular responses mediated by these TNF family ligands is generally initiated by their binding to specific cellular receptors. Some, but not all TNF family ligands bind through cell surface "death receptors" and induce various biological activities to activate caspases, or enzymes that execute cell death or apoptosis pathways (Salvesen et al, cell, 91: 443-446 (1997)). Members of the TNF receptor superfamily identified to date include TNFR1, TNFR2, TACI, GITR, CD27, OX-40, CD30, CD40, HVEM, Fas (also referred to as Apo-1 or CD95), DR4 (also referred to as TRAIL-R1), DR5 (also referred to as Apo-2 or TRAIL-R2), DcR1, DcR2, Osteoprotegerin (OPG), RANK and Apo-3 (also referred to as DR3 or TRAMP), and the like.

Most of these TNF receptor family members share a structure typical of cell surface receptors, including extracellular, transmembrane and intracellular domains, while other members are found naturally as soluble proteins lacking transmembrane and intracellular domains. The extracellular portion of typical TNFRs contains an initiation NH sequence₂Repetitive amino acid sequence patterns of terminal multiple Cysteine Rich Domains (CRDs).

Ligands known as Apo-2L or TRAIL were identified a few years ago as members of the TNF family of cytokines (see, e.g., Wiley et al, Immunity, 3: 673-682 (1995); Pitti et al, J.biol.chem., 271: 12697-12690 (1996); WO 97/01633; WO 97/25428; U.S. Pat. No. 5,763,223, 6/9/1998; U.S. Pat. No. 6,284,236, 9/4/2001). Full-length native sequence human Apo2L/TRAIL polypeptide is a 281 amino acid long type II transmembrane protein. Some cells are capable of producing the polypeptide in its native soluble form by enzymatically cleaving the extracellular domain of the polypeptide (Mariani et al, J.cell.biol., 137: 221-229 (1997)). Soluble forms of Apo2L/TRAIL crystallization studies have shown a homotrimeric structure similar to that of TNF and other related proteins (Hymowitz et al, mol. cell, 4: 563-571 (1999); Cha et al, Immunity, 11: 253-261 (1999); Mongkolsapaya et al, Nature Structural Biology, 6: 1048 (1999); Hymowitz et al, Biochemistry, 39: 633-644 (2000)). However, Apo2L/TRAIL, unlike other TNF family members, was found to have unique structural features in which three cysteine residues (at position 230 of each subunit of the homotrimer) are coordinated together with a zinc atom, and zinc binding is important for trimer stability and biological activity (Hymowitz et al, supra; Bodmer et al, J.biol.chem., 275: 20632-one 20637 (2000)).

Apo2L/TRAIL has been reported in the literature to play a role in immune system regulation, including in autoimmune diseases such as rheumatoid arthritis (see, e.g., Thomas et al, J.Immunol., 161: 2195 & 2200 (1998); Johnsen et al, cytokine, 11: 664 & 672 (1999); Griffith et al, J.exp.Med., 189: 1343 & 1353 (1999); Song et al, J.exp.Med., 191: 1095 & 1103 (2000)).

Soluble forms of Apo2L/TRAIL have been reported to induce apoptosis in a number of Cancer cells, including tumors of the colon, lung, breast, prostate, bladder, kidney, ovary and brain, as well as melanoma, leukemia, and multiple myeloma (see, e.g., Wiley et al, supra; Pitti et al, supra; U.S. Pat. No. 6,030,945, 2/29/2000; U.S. Pat. No. 6,746,668, Rieger et al, FEBS Letters, 427: 124-. In vivo studies in murine tumor models further suggest that Apo2L/TRAIL, alone or in combination with chemotherapy or radiation therapy, is capable of exerting a substantial anti-tumor effect (see, e.g., Ashkenazi et al, supra; Walzcak et al, supra; Gliniak et al, Cancer Res., 59: 6153-6158 (1999); Chinnaiyan et al, supra; Roth et al, biochem. Biophys. Res. Comm., 265: 1999 (1999); PCT application US/00/15512; PCT application US/01/23691). In contrast to many types of cancer cells, most normal human cell types appear to be resistant to apoptosis induction by some recombinant form of Apo2L/TRAIL (Ashkenazi et al, supra; Walzcak et al, supra). Jo et al reported that polyhistidine-labeled soluble forms of Apo2L/TRAIL induce apoptosis in vitro in normally isolated human hepatocytes but not in non-human hepatocytes (Jo et al, Nature Med., 6: 564-567 (2000); see also, Nagata, Nature Med., 6: 502-503 (2000)). It is believed that the biochemical properties and biological activities of certain recombinant Apo2L/TRAIL preparations for diseased and normal cells may vary depending on, for example, the presence or absence of marker molecules, zinc content, and percent trimer content (see Lawrence et al, Nature Med., Letter to the Editor, 7: 383-385 (2001); Qin et al, Nature Med., Letter to the Editor, 7: 385-.

Apo2L/TRAIL has been found to bind to at least five different receptors. At least two of the Apo2L/TRAIL binding receptors contain a functional cytoplasmic death domain. One such receptor is termed "DR 4" (and/or as TR4 or TRAIL-R1) (Pan et al, Science, 276: 111-113 (1997); see also WO98/32856, published on 30.7.1998, WO99/37684, published on 29.7.1999, WO 00/73349, published on 7.12.7.2000, US6,433,147, published on 13.8.2002, US6,461,823, published on 8.10.8.2002, and US6,342,383, published on 29.1.2002).

Another such receptor for Apo2L/TRAIL is designated DR5 (or it is also designated Apo-2; TRAIL-R or TRAIL-R2, TR6, Tango-63, hAPO8, TRICK2 or KILLER) (see, for example, Sheridan et al, Science, 277: 818-821(1997), Pan et al, Science, 277: 815-818(1997), WO98/51793, 11.19.1998; WO98/41629, 24.1998; Scandaton et al, Curr.biol., 7: 693-696 (1997); Walczak et al, EMBO J., 16: 5386-5387(1997), Wu et al, Nature Genetics, 17: 141-143(1997), WO 98/98, 35, 1998: 35: 5386-5387(1997), WO 1998; WO 35: 1998; WO 2/35: 1998; 1998: 4610, 1998; WO 2/35: 1998; 1999, WO 2/11; 1999, 1998, WO 2, 1998, 35, 1999 2002/0072091, respectively; US 2002/0098550 published on 12/7/2001; US6,313,269 published at 12/6/2001; US2001/0010924, published on 8/2/2001; US 2003/01255540 published on 7/3/2003; US 2002/0160446, published at 31/10/2002, US2002/004878, published at 25/4/2002; US6,342,369 published in month 2 2002; US6,569,642 published on 27 th 5 th 2003, US6,072,047 published on 6 th 2000, US6,642,358 published on 4 th 11 th 2003; IS 6,743,625 published on 6/1/2004). Like DR4, DR5 is reported to contain a cytoplasmic death domain and to be capable of signaling apoptosis upon ligand binding (or via binding to a molecule, such as an agonist antibody, that mimics the activity of the ligand). The crystal structure of the complex formed between Apo-2L/TRAIL and DR5 is described in Hymowitz et al, Molecular Cell, 4: 563 and 571 (1999).

After ligand binding, both DR4 and DR5 are able to promote apoptosis by the death domain-containing linker molecule called FADD/Mort1, independently by recruiting and activating the apoptosis-initiating factor caspase-8 (Kischkel et al, Immunity, 12: 611-620 (2000); Sprick et al, Immunity, 12: 599-609 (2000); Bodmer et al, Nature Cell biol., 2: 241-243 (2000)).

Apo2L/TRAIL is reported to also bind to those receptors designated DcR1, DcR2 and OPG, which are believed to act as inhibitors, but not a signaling agent (see, e.g., DcR1 (also known as TRID, LIT or TRAIL-R3) (Pan et al, Science, 276: 111-113 (1997); sheridan et al, Science 277: 818-821 (1997); McFarlane et al, J.biol.chem., 272: 25417-; schneider et al, FEBS Letters, 416: 329- > 334 (1997); Degli-Esposti et al, J.Exp.Med., 186: 1165-1170 (1997); and Mongkolsapaya et al, J.Immunol., 160: 3-6 (1998); DcR2 (also known as TRUNDD or TRAIL-R4) (Marster et al, Curr. biol., 7: 1003-1006 (1997); pan et al, FEBS Letters, 424: 41-45 (1998); Degli-Esposti et al, Immunity, 7: 813-820(1997)), in contrast to DR4 and DR5, DcR1 and DcR2 receptors do not signal apoptosis.

Certain antibodies that bind DR4 and/or DR5 receptors have been reported in the literature. For example, anti-DR 4 antibodies directed against the DR4 receptor and having agonistic or apoptotic activity in certain mammalian cells are disclosed in, for example, WO 99/37684 at 29.7/1999; WO 00/73349, published on 12/7/2000; described in WO 03/066661 published on 8/14/2003. See also, for example, Griffith et al, j.immunol., 162: 2597-2605 (1999); chuntharapaai et al, j.immunol., 166: 4891 vs 4898 (2001); WO 02/097033, published on 12/2/2002; WO 03/042367, published on 5/22/2003; WO 03/038043, published on 8/5/2003; WO03/037913, 5/8/2003. Certain anti-DR 5 antibodies are likewise described, see, e.g., WO 98/51793, published at 11/8 of 1998; griffith et al, j.immunol., 162: 2597-2605 (1999); ichikawa et al, Nature med., 7: 954-960 (2001); hylander et al, "An Antibodyto DR5 (TRAIL-receiver 2) supresses the Growth of parent derived restricted specific turbines Growth in SCID Mouse", Abstract, 2d International Congress on Monoclonal antibodies in cancer, aug.29-Sept.1, 2002, Banff, Alberta, Canada; WO 03/038043, published on 8/5/2003; WO03/037913 published on 8/5/2003. In addition, certain antibodies have been described that have cross-reactivity to both DR4 and DR5 receptors (see, e.g., U.S. patent 6,252,050, 6/26, 2001).

Summary of The Invention

The present invention herein relates, in part, to the identification of histidine-acetate (histidine-acetate), ph5.5-6.5, as a particularly useful buffer for the formulation of monoclonal antibodies, particularly full length IgG1 antibodies that are susceptible to deamidation and/or aggregation. The formulation prevents degradation of the antibody product therein.

Thus, in a first aspect, the present invention relates to a stable pharmaceutical formulation comprising a monoclonal antibody in histidine-acetate buffer at ph 5.5-6.5. The monoclonal antibody preferably binds to an antigen selected from the group consisting of HER2, CD20, DR5, BR3, IgE and VEGF.

Furthermore, the present invention relates to a method of treating a disease or disorder in a subject comprising administering to the subject an effective amount of the formulation to treat the disease or disorder.

In another aspect, the present invention relates to a pharmaceutical formulation comprising: (a) a full length IgG1 antibody that is susceptible to deamidation or aggregation in an amount from about 10mg/mL to about 250 mg/mL; (b) histidine-acetate buffer (histidine-acetate buffer), ph 5.5-6.5; (c) a sugar selected from the group consisting of trehalose and sucrose in an amount from about 60mM to about 250 mM; and (d) polysorbate 20 in an amount of from about 0.01% to about 0.1%.

The invention also provides a method of reducing deamidation or aggregation of a therapeutic monoclonal antibody comprising formulating said antibody in histidine-acetate buffer, ph 5.5-6.5.

In yet another aspect, the invention relates to a pharmaceutical formulation comprising an antibody that binds to domain II of HER2 in histidine buffer at a pH of from about 5.5 to about 6.5, a sugar and a surfactant.

The present invention also relates to a pharmaceutical formulation comprising Pertuzumab in an amount from about 20mg/mL to about 40mg/mL, a histidine-acetate buffer, sucrose, and polysorbate 20, wherein the pH of the formulation is about 5.5 to about 6.5.

The invention also relates to a pharmaceutical formulation comprising a DR5 antibody in a histidine buffer at a pH of from about 5.5 to about 6.5, a sugar, and a surfactant.

In another aspect, the present invention relates to a pharmaceutical formulation comprising Apomab, histidine-acetate buffer, trehalose, and polysorbate 20 in an amount from about 10mg/mL to about 30mg/mL, wherein the pH of the formulation is about 5.5 to about 6.5.

In yet another aspect, the invention provides a method of treating cancer in a subject comprising administering to the subject an effective amount of a pharmaceutical formulation to treat cancer.

The invention also relates to a vial or stainless steel canister with a stopper pierceable by a syringe, said vial or canister containing said formulation, optionally in lyophilized form.

In addition, the present invention provides a method of preparing a pharmaceutical formulation comprising: (a) preparing a monoclonal antibody preparation; and (b) assessing the physical stability, chemical stability, or biological activity of the monoclonal antibody in the formulation.

Brief Description of Drawings

FIG. 1 depicts domains I-IV (SEQ ID NOS.19-22, respectively) of the extracellular domain of HER 2.

Fig. 2A and 2B depict an alignment of the following amino acid sequences: murine monoclonal antibody 2C4 is variable light (V)_L) (FIG. 2A) and variable weight (V)_H) (FIG. 2B) strand domains (SEQ ID Nos.1 and 2, respectively); humanized 2C4 version 574V_LAnd V_HDomains (SEQ ID Nos.3 and 4, respectively), and human V_LAnd V_HConsensus framework (hum. kappa.1, light kappa subunit I; humIII, heavy subunit III) (eachSEQ ID Nos.5 and 6). Asterisks identify the differences between humanized 2C4 version 574 and murine monoclonal antibody 2C4 or between humanized 2C4 version 574 and the human framework. Complementarity Determining Regions (CDRs) are in parentheses.

Figures 3A and 3B show the amino acid sequences of Pertuzumab light and heavy chains (SEQ id nos.15 and 16, respectively). CDRs are shown in bold. The calculated molecular weights of the light and heavy chains were 23,526.22Da and 49,216.56Da (reduced forms of cysteine). The sugar moiety is attached to Asn299 of the heavy chain.

FIGS. 4A and 4B show the amino acid sequences of the light and heavy chains of Pertuzumab, each including the complete amino-terminal signal peptide sequence (SEQ ID Nos.17 and 18, respectively).

Figure 5 illustrates binding of 2C4 at the heterodimer binding site of HER2, thereby preventing heterodimerization with activated EGFR or HER 3.

Figure 6 depicts HER2/HER3 conjugation to MAPK and Akt pathways.

Figure 7 compares the activity of Trastuzumab and Pertuzumab.

Figure 8 shows the stability of the Pertuzumab formulation by Ion Exchange (IEX) analysis.

Figure 9 shows the stability of the Pertuzumab formulation by Size Exclusion Chromatography (SEC) analysis.

Figure 10 reflects the physical stability of Pertuzumab in different formulations.

Figure 11 is from an agitation study (agitation study) of a liquid formulation of Pertuzumab.

Figure 12 is from other agitation studies of Pertuzumab liquid formulation.

Figure 13 is from a freeze-thaw study of Pertuzumab formulations.

FIGS. 14A and 14B show the amino acid sequences of Trastuzumab light chain (SEQ ID No.13) and heavy chain (SEQ ID No. 14).

Figures 15A and 15B depict the variant Pertuzumab light chain sequence (SEQ ID No.23) and the variant Pertuzumab heavy chain sequence (SEQ ID No. 24).

FIGS. 16A and 16B show oligosaccharide structures commonly observed in IgG antibodies.

FIGS. 17A and 17B show the sequences of the light and heavy chains of the specific anti-IgE antibodies E25, E26, HAE1 and Hu-901 (SEQ ID Nos. 37-44). In fig. 17A, the variable light chain domain ends at residue VEIK at residue 111. In fig. 17B, the variable heavy chain domain ends at residue VTVSS near residue 120.

FIG. 18A is a graph comparing the variable light domain (V) of each of murine 2H7(SEQ ID No.25), humanized 2H7V16 variant (SEQ ID No.26), and human kappa light chain subunit I (SEQ ID No.27)_L) Alignment of the amino acid sequences of (a). V of 2H7 and hu2H7V16_LThe CDRs of (A) are as follows: CDR1(SEQ ID No.57), CDR2(SEQ ID No.58), and CDR3(SEQ ID No. 59).

FIG. 18B is a graph comparing the variable heavy domain (V) of murine 2H7(SEQ ID No.28), the humanized 2H7V16 variant (SEQ ID No.29), and the human common sequence of heavy chain subunit III (SEQ ID No.30)_H) Alignment of the amino acid sequences of (a). V of 2H7 and hu2H7V16_HThe CDRs of (A) are as follows: CDR1(SEQ ID No.60), CDR2(SEQ ID No.61), and CDR3(SEQ ID No. 62).

In FIGS. 18A and 18B, CDR1, CDR2, and CDR3 in each chain are included in parentheses and flanked by framework regions denoted FR1-FR 4. 2H7 refers to murine 2H7 antibody. The asterisks between the two rows of the sequence indicate the positions that differ between the two sequences. Residues are numbered according to Kabat et al Sequences of Immunological Interest, 5th Ed.public Health Service, national institutes of Health, Bethesda, Md. (1991), and insertions are shown as a, b, c, d, and e.

FIG. 19 depicts the variable domain sequences of three different VEGF antibodies, which are SEQ ID Nos.31-36, respectively.

Fig. 20 shows the Size Exclusion Chromatography (SEC) elution profiles of the following Apomab samples: (a) controls and formulations prepared under conditions of (b) pH4.0, (c) pH5.0, (d) pH6.0 and (e) pH 7.0. The prepared samples were stored at 40 ℃ for 2 months prior to analysis.

Figure 21 depicts a pH rate curve for Apomab antibody monomer loss during storage. The monomer kinetics of SEC were monitored at 30 ℃ and 40 ℃ during storage and the first order rate constants were calculated.

Fig. 22 provides the Ion Exchange Chromatography (IEC) elution profile for Apomab samples as follows: (a) controls and formulations prepared under conditions of (b) pH4.0, (c) pH5.0, (d) pH6.0 and (e) pH 7.0. Formulated samples were stored at 40 ℃ for 2 months prior to analysis.

Figure 23 shows the pH rate curve of IEC main peak loss during storage. The main peak kinetics of the IEC were monitored at 30 ℃ and 40 ℃ during storage and the first order rate constants were calculated.

FIG. 24 shows the nucleotide sequence (SEQ ID No.45) of the human Apo-2 ligand cDNA and its derived amino acid sequence (SEQ ID No. 46). The "N" at nucleotide position 447 (in SEQ ID No.45) is used to indicate that the nucleotide base may be "T" or "G".

FIGS. 25A and 25B show the 411 amino acid sequences of the human DR5 receptor (SEQ ID No.47), and the encoding nucleotide sequence (SEQ ID No.48), disclosed in WO 98/51793 at 19/11/1998.

FIGS. 26A and 26B show the 440 amino acid sequence of the human DR5 receptor and the coding nucleotide sequence (SEQ ID No.50), which is also disclosed in WO98/35986 at 20.8.1998.

FIG. 27 shows the Apomab7.3 heavy chain amino acid sequence (SEQ ID No. 51).

FIG. 28 shows the Apomab7.3 light chain amino acid sequence (SEQ ID No. 52).

FIG. 29 shows an alignment of the amino acid sequences of the 16E2 heavy chain (SEQ ID No.53) and the Apomab7.3 heavy chain (SEQ ID No. 51).

FIG. 30 shows an alignment of the amino acid sequences of the 16E2 light chain (SEQ ID No.54) and Apomab7.3 light chain (SEQ ID No. 52).

FIGS. 31A and 31B depict the variable heavy chain amino acid sequence (FIG. 31A; SEQ ID No.55) and the variable light chain amino acid sequence (FIG. 31B; SEQ ID No.56) of Apomab7.3. CDR residues are identified in bold.

FIG. 32 shows an alignment of mature 2H7v16 and 2H7v511 light chains (SEQ ID Nos.63 and 64, respectively). The sequences are shown using the Kabat variable domain residue numbering method and the Eu constant domain residue numbering method.

FIG. 33 shows an alignment of mature 2H7v16 and 2H7v511 heavy chains (SEQ ID Nos.65 and 66, respectively). The sequences are shown using the Kabat variable domain residue numbering method and the Eu constant domain residue numbering method.

Detailed description of the preferred embodiments

I. Definition of

The term "pharmaceutical formulation" refers to a preparation which is in a form which allows the biological activity of the active ingredient to be effective, and which does not contain other components which are unacceptably toxic to the subject to which the formulation is to be administered. These formulations are sterile.

"sterile" preparations are sterilized (ascitic) or free of all living microorganisms and their spores.

As used herein, a "frozen" formulation is a formulation at a temperature below 0 ℃. Generally, a frozen formulation is not lyophilized, nor is it lyophilized before or after. Preferably, the frozen preparation comprises a frozen drug substance (in a stainless steel jar) or a frozen drug product (in the final vial configuration) for preservation.

A "stable" formulation is one in which the protein substantially retains its physical and/or chemical stability and/or biological activity after storage. Preferably, the formulation substantially retains its physical and chemical stability, as well as its biological activity, after storage. The shelf life is generally selected based on the desired shelf life of the formulation. Various analytical techniques for measuring Protein stability are known in the art and are described, for example, in Peptide and Protein Drug Delivery, 247-: a review is made in 29-90 (1993). Stability can be measured at a selected temperature for a selected time. Preferably, the formulation is stable at about 40 ℃ for at least about 2-4 weeks, and/or at about 5 ℃ and/or 15 ℃ for at least 3 months, and/or at about-20 ℃ for at least 3 months or at least 1 year. In addition, the formulation is preferably stable after freezing (to, e.g., -70 ℃) and thawing of the formulation, e.g., after 1, 2 or 3 cycles of freeze-thawing. Stability can be assessed qualitatively and/or quantitatively in a number of different ways, including assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); (ii) assessing charge heterogeneity by using cation exchange chromatography or capillary partition electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometry analysis; SDS-PAGE analysis to compare reduced and intact antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; assessing the biological activity or antigen binding function of the antibody; and so on. Instability may include any one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), cleavage/hydrolysis/fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extensions, C-terminal processing, glycosylation differences, and the like.

A "deamidated" monoclonal antibody herein is one in which one or more asparagine residues thereof have been derivatized, for example, to aspartic acid or iso-aspartic acid.

An antibody that is "susceptible to deamidation" is one that includes one or more residues that are found to be susceptible to deamidation.

An antibody that is "susceptible to aggregation" is one that has been found to aggregate with other antibody molecules, particularly after freezing and/or agitation.

An antibody that is "easily fragmented" is one that has been found to be cleaved into two or more fragments, for example at its hinge region.

"reducing deamidation, aggregation, or fragmentation" is intended to prevent or reduce the amount of deamidation, aggregation, or fragmentation relative to monoclonal antibodies formulated at a different pH or in a different buffer.

As used herein, "biological activity" of a monoclonal antibody refers to the ability of the antibody to bind to an antigen and result in a measurable biological response, which can be measured in vitro or in vivo. Such activity may be antagonistic (e.g. when the antibody is a HER2 antibody) or agonistic (e.g. when the antibody binds DR 5). For Pertuzumab, in one embodiment, biological activity refers to the ability of the formulated antibody to inhibit proliferation of the human breast cancer cell line MDA-MB-175-VII. When the antibody is Apomab, biological activity can refer, for example, to the ability of the formulated antibody to kill colon cancer, Colo205 cells.

By "isotonic" is meant that the formulation of interest has substantially the same osmotic pressure as human blood. An isotonic formulation will generally have an osmotic pressure of from about 250 to 350 mOsm. Isotonicity can be measured using, for example, a vapor pressure or freezing type osmometer.

As used herein, "buffer" refers to a buffered solution that is resistant to pH changes by the action of its acid-base conjugated components. The buffer of the invention preferably has a pH of from about 5.0 to about 7.0, preferably from about 5.5 to about 6.5, for example from about 5.8 to about 6.2, most preferably a pH of about 6.0. Examples of buffers that will control the pH in this range include acetate, succinate, gluconate, histidine, citrate, glycylglycine and other organic acid buffers. The preferred buffer herein is histidine buffer.

A "histidine buffer" is a buffer that includes histidine ions. Examples of histidine buffers include histidine chloride, histidine acetate, histidine phosphate, histidine sulfate. The preferred histidine buffer identified in the examples herein was found to be histidine acetate. In a preferred embodiment, the histidine acetate buffer is prepared by titration of L-histidine (free base, solid) with acetic acid (liquid). Preferably, the histidine buffer or histidine-acetate buffer is at pH5.5-6.5, preferably at pH 5.8-6.2.

"sugar" herein includes conventional Compositions (CH)₂O)_nAnd derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like. Examples of sugars herein include glucose, sucrose, trehalose (trehalose), lactose, fructose, maltose, dextran, glycerol, dextran, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, melibiose (mellibiose), melezitose (melezitose), raffinose (raffinose), mannotriose (manotriose), stachyose (stachyose), maltose, lactulose (lactulose), maltulose (maltulose), sorbitol (glucitol), maltitol, lactitol, iso-maltulose (maltulose), and the like. Preferred sugars herein are non-reducing disaccharides such as trehalose or sucrose.

Herein, "surfactant" refers to a surface active agent, preferably a nonionic surfactant. Examples of surfactants herein include polysorbates (e.g., polysorbate 20 and polysorbate 80); polyhydroxyalkylenes (poloxamers) (e.g., polyhydroxyalkylenes 188); triton; sodium Dodecyl Sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmityl Amidopropyl-, or isostearamidopropyl-betaine (e.g., lauramidopropyl); myristamidopropyl-, palmitoamidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl or sodium methyl oleyl taurate; and MONAQUAT^TMSeries (Mona Industries, inc., Paterson, New Jersey); polyethylene glycols, polypropylene glycols, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.); and so on. The preferred surfactant herein is polysorbate 20.

A "HER receptor" is a receptor protein tyrosine kinase that belongs to the HER receptor family, including the EGFR, HER2, HER3, and HER4 receptors, as well as other members of this family identified in the future. HER receptors will typically comprise an extracellular domain, which may bind a HER ligand; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxy-terminal signal domain with several tyrosine residues, which can be phosphorylated. Preferably the HER receptor is a native sequence human HER receptor.

The extracellular domain of HER2 comprises four domains, domain I (amino acid residues from about 1-195), domain II (amino acid residues from about 196-320), domain III (amino acid residues from about 321-488), and domain IV (amino acid residues from about 489-632) (residue numbering without signal peptide). See Garrett et al mol.cell.. 11: 495-505(2003), Cho et al Nature 421: 756-760(2003), Franklin et al human cancer cells 5: 317-: 1746-1750(1993). See also figure 1 herein.

The terms "ErbB 1", "HER 1", "epidermal growth factor receptor" and "EGFR" are used interchangeably herein and are as described, for example, in Carpenter et al ann.rev.biochem.56: 881 (1987) and is referred to as EGFR and includes naturally occurring mutant forms thereof (e.g., deletion mutant EGFR in Humphrey et al PNAS (USA) 87: 4207-4211 (1990)). erbB1 refers to the gene encoding the EGFR protein product.

The expressions "ErbB 2" and "HER 2" are used interchangeably herein and are referred to as human HER2 protein, for example, in Semba et al, pnas (usa) 82: 6497-: 230-. The term "erbB 2" refers to the gene encoding human erbB2 and "neu" refers to the gene encoding rat p185 neu. Preferred HER2 is native sequence human HER 2.

"ErbB 3" and "HER 3" refer, for example, to the ErbB3 and "HER 3" as described in U.S. Pat. Nos. 5,183,884 and 5,480,968, and Kraus et al PNAS (USA) 86: 9193-9197 (1989).

The terms "ErbB 4" and "HER 4" herein refer to ErbB 8926, for example, in european patent application No. 599,274; plowman et al, proc.natl.acad.sci.usa, 90: 1746 — 1750 (1993); and Plowman et al, Nature, 366: 473-475(1993), including isomers thereof, e.g. as disclosed in WO99/19488, published in 1999-04-22.

By "HER ligand" is meant a polypeptide that binds to and/or activates a HER receptor. Specific HER ligands of interest herein are native sequence human HER ligands such as Epidermal Growth Factor (EGF) (Savage et al, j.biol.chem.247: 7612-7621 (1972)); transforming growth factor alpha (TGF-. alpha.) (Marquardt et al, Science 223: 1079-1082 (1984)); amphiregulin is also known as Schwannoma (schwanoma) or keratinocyte autocrine growth factor (Shooyab et al Science 243: 1074-1076 (1989); Kimura et al Nature 348: 257-260 (1990); and Cook et al mol.cell.biol.11: 2547-2557 (1991)); beta cellulose (Shing et al, Science 259: 1604-1607 (1993); and Sasada et al, biochem. Biophys. Res. Commun.190: 1173 (1993)); heparin-binding epidermal growth factor (HB-EGF) (Higashiyama et al, Science 251: 936-939 (1991)); epithelial regulatory protein (epiregulin) (Toyoda et al, J.biol.chem.270: 7495-7500 (1995); and Komurasaki et al Oncogene 15: 2841-2848 (1997)); heregulin (see below); neuregulin-2 (NRG-2) (Carraway et al, Nature 387: 512-516 (1997)); neuregulin-3 (NRG-3) (Zhang et al, Proc. Natl. Acad. Sci.94: 9562-9567 (1997)); neuregulin-4 (NRG-4) (Harari et al Oncogene 18: 2681-89(1999)) or cripto (CR-1) (Kannan et al J.biol.chem.272 (6): 3330-3335 (1997)). HER ligands that bind EGFR include EGF, TGF- α, amphiregulin, β -cellulose, HB-EGF and epithelial regulatory proteins. HER ligands that bind HER3 include heregulin. HER ligands capable of binding to HER4 include beta cellulose, epithelial regulatory protein, HB-EGF, NRG-2, NRG-3, NRG-4 and heregulin.

As used herein, "heregulin" (HRG) refers to a protein encoded by a protein encoded: 312-318 (1993). Examples of heregulins include heregulin-alpha, heregulin-beta 1, heregulin-beta 2 and heregulin-beta 3(Holmes et al, Science, 256: 1205-1210 (1992); and U.S. Pat. No. 5,641,869); neu Differentiation Factor (NDF) (Peles et al cell 69: 205-216 (1992)); acetylcholine Receptor Inducing Activity (ARIA) (Falls et al Cell 72: 801. cona. 815 (1993));]glial Growth Factors (GGFs) (Marchionni et al, Nature, 362: 312-318 (1993)); sensory and motor neuron derived factor (SMDF) (Ho et al J.biol.chem.270: 14523-; gamma-heregulin (Schaefer et al Oncogene 15: 1385-1394 (1997)). The term includes biologically active fragments and/or amino acid sequence variants of native sequence HRG polypeptides, such as EGF-like domain fragments thereof (e.g., HRG β 1)_177-244)。

As used herein, a "HER dimer" is a non-covalently bound dimer comprising at least two different HER receptors. Such complexes may form when cells expressing two or more HER receptors are exposed to a HER ligand and may be isolated by immunoprecipitation and analyzed by SDS-PAGE, as described, for example, by Sliwkowski et al, j.biol.chem., 269 (20): 14661-. Examples of such HER dimers include EGFR-HER2, HER2-HER3, and HER3-HER4 heterodimers. In addition, the HER dimer may comprise two or more HER2 receptors that bind different HER receptors, such as HER3, HER4, or EGFR. Other proteins, such as cytokine receptor subunits (e.g., gp130) can be associated with the dimer.

A "heterodimer binding site" on HER2 refers to a region in the extracellular domain of HER2 that contacts, or contacts, a region in the extracellular domain of EGFR, HER3, or HER4 after dimer formation. This region is found in domain II of HER 2. Franklin et al Cancer Cell 5: 317-328(2004).

"HER activation" or "HER 2 activation" refers to the activation, or phosphorylation, of any one or more HER receptors, or HER2 receptors. In general, HER activation results in signal transduction (e.g., that initiated by the intracellular kinase domain of the HER receptor, which phosphorylates tyrosine residues in the HER receptor or substrate polypeptide). HER activation can be mediated by binding of a HER ligand to a HER dimer comprising the HER receptor of interest. HER ligand binding to the HER dimer may activate the kinase domain of one or more HER receptors in the dimer and thereby result in phosphorylation of tyrosine residues in one or more HER receptors, and/or phosphorylation of tyrosine residues in other substrate polypeptides, such as Akt or MAPK intracellular kinases.

The term "antibody" as used herein in the broadest sense specifically includes full length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two full length antibodies, and antibody fragments so long as they exhibit the desired biological activity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for variants that may occur during the production of the monoclonal antibody, which variants are typically present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, they are also advantageous in that they are not contaminated with other immunoglobulins. The modifier "monoclonal" refers to the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as limiting the production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the invention may be produced by a method described by Kohler et al, nature, 256: 495(1975) or can be prepared by recombinant DNA methods (e.g., U.S. Pat. No. 4,816,567). "monoclonal antibodies" can also be identified using Clackson et al, Nature, 352: 624-: 581-597(1991) from phage antibody libraries.

Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, provided that they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; Morrison et al, proceedings of the national academy of sciences USA 81: 6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising antigen binding sequences derived from non-human primate variable domains (e.g., Old World Monkey, ape, etc.) and human constant region sequences.

Antibody fragments "include a portion of a full-length antibody, preferably including the antigen-binding or variable regions thereof. Examples of antibody fragments include Fab, Fab ', F (ab') 2, and Fv fragments; a bivalent antibody; a linearized antibody; a single chain antibody molecule; and multispecific antibodies composed of antibody fragments.

A "full-length antibody" is an antibody comprising an antigen-binding variable region and a light chain constant domain (C) _L) And heavy chain constant domains s, C_H1，C_H2 and C_H3. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. Preferably, the full length antibody has one or more effector functions.

The term "major species antibody" herein refers to an antibody structure in a composition that is the predominant antibody molecule in the composition in terms of quantity. In one embodiment, the main species antibody is a HER2 antibody, such as an antibody that binds to domain II of HER2, an antibody that inhibits HER dimerization more effectively than Trastuzumab, and/or an antibody that binds to the heterodimeric binding site of HER 2. A preferred embodiment of the main component HER2 antibody herein is one which comprises the variable light and variable heavy amino acid sequences of SEQ ID nos.3 and 4, most preferably the light and heavy chain amino acid sequences of SEQ ID nos.15 and 16 (Pertuzumab).

An "amino acid sequence variant" antibody herein is an antibody having an amino acid sequence that differs from that of the main species of antibody. Generally, amino acid sequence variants will have at least about 70% homology with the main species of antibody, preferably they will have at least about 80%, more preferably at least about 90% homology with the main species of antibody. Amino acid sequence variants have substitutions, deletions, and/or additions at certain positions within or adjacent to the amino acid sequence of the main species of antibody. Examples of amino acid sequence variants herein include acidic variants (e.g., deamidated antibody variants), basic variants, antibodies having amino-terminal directed extensions (e.g., VHS-) on one or both of their light chains, antibodies having C-terminal lysine residues on one or both of their heavy chains, and the like, and include combinations of heavy and/or light chain amino acid sequence variations. A particular antibody variant of interest herein is an antibody comprising an amino-terminal directed extension on one or both of its light chains, optionally further comprising other amino acid sequences and/or glycosylation differences relative to the main species antibody.

A "therapeutic monoclonal antibody" is an antibody used to treat a human subject. Therapeutic monoclonal antibodies disclosed herein include: HER2 antibody for use in cancer and various non-malignant diseases or disorders; a CD20 or BR3 antibody for use in the treatment of a B cell malignancy, an autoimmune disease, transplant rejection, or suppression of an immune response against a foreign antigen; IgE antibodies for use in the treatment of IgE-mediated disorders; a DR5 or VEGF antibody for use in the treatment of cancer.

A "glycosylation variant" antibody herein is an antibody having one or more sugar moieties attached thereto that is different from the one or more sugar moieties attached to the main species of antibody. Examples of glycosylation variants herein include antibodies having the G1 or G2 oligosaccharide structure, but not the G0 oligosaccharide structure, attached to their Fc region, antibodies having one or two sugar moieties attached to one or two light chains thereof, antibodies having no sugar attached to one or two heavy chains of the antibody, and the like, as well as combinations of glycosylation variations.

When the antibody has an Fc region, an oligosaccharide structure such as that shown in figure 16 may be attached to one or both heavy chains of the antibody, for example at residue 299 (298, Eu numbering of residues). G0 is the predominant oligosaccharide structure for Pertuzumab, with lesser amounts of other oligosaccharide structures such as G0-F, G-1, Man5, Man6, G1-1, G1(1-6), G1(1-3) and G2 being found in Pertuzumab compositions.

Unless otherwise specified, "G1 oligosaccharide structures" herein include G-1, G1-1, G1(1-6) and G1(1-3) structures.

Herein, "amino-terminal leader extension" refers to one or more amino acid residues of an amino-terminal leader sequence, which are present at the amino terminus of any one or more heavy or light chains of an antibody. Exemplary amino-terminal guide extensions comprise or consist of three amino acid residues, VHS, present in one or both light chains of an antibody variant.

"homology" is defined as the percentage of amino acid sequence variant residues that are identical after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for performing the alignment are well known in the art. One such computer program is "Align 2" which was archived in its user file at the U.S. copyright office, washington, DC20559, 12/10, 1991.

Antibody "effector functions" refer to those biological activities attributable to the Fc region of an antibody (either the native sequence Fc region or the amino acid sequence variant Fc region). Examples of "effector functions" include C1q binding; complement-dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), and the like.

Full-length antibodies can be assigned to different "classes" based on the amino acid sequence of their heavy chain constant domains. There are five major full-length antibody classes: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA 2. The chain constant domains corresponding to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

"antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. The primary cells mediating ADCC, NK cells, express Fc γ R III only, whereas monocytes express Fc γ R I, Fc γ R II and Fc γ R III. Raveth and Kinet in immunological yearbook 9: FcR expression on hematopoietic cells is summarized in table 3 on pages 457-92(1991), 464. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, as described in U.S. patent No. 5,500,362 or 5,821,337. Useful effector cells in such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, ADCC activity of a molecule of interest may be assessed in vivo, for example in Clynes et al pnas (usa) 95: 652-.

A "human effector cell" is a leukocyte that expresses one or more fcrs and performs effector functions. Preferably, the cells express at least fcyr III and perform the function of an ADCC effector. For example, human leukocytes that mediate ADCC include human Peripheral Blood Mononuclear Cells (PBMCs), Natural Killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; among them, PBMC and NK cells are preferable. Effector cells may be isolated from their natural source, e.g., from blood and PBMCs as described herein.

The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Moreover, the preferred fcrs are receptors (gamma receptors) that bind IgG antibodies, including Fc γ R I, Fc γ R II and Fc γ R III subtypes, as well as allelic variants and alternatively spliced forms of these receptors. Fc γ R II receptors include Fc γ R IIA ("activating receptor") and Fc γ R IIB ("inhibiting receptor"), both of which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor Fc γ R IIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor Fc γ R IIB contains in its cytoplasmic domain an immunoreceptor tyrosine-based inhibitory motif (ITIM) (for review see Daeron, Ann. Rev.Immunol. 15: 203-234 (1997)). In ravatch and Kinet, annual immunology 9: 457-92 (1991); capel et al, immunization methods (immunological methods) 4: 25-34 (1994); and de Haas et al, journal of laboratory and clinical medicine (j.lab.clin.med.) 126: 330-41(1995) for FcR. Other fcrs, including those identified in the future, are encompassed by the term "FcR". The term also includes the neonatal receptor, FcRn, which is responsible for transporting maternal IgG to the fetus (Guyer et al, J Immunol 117: 587(1976) and Kim et al, J Immunol 24: 249 (1994)).

"complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) that forms a complex with a cognate antigen. To assess complement activation, one can use, for example, Gazzano-Santoro et al, J Immunol methods 202: 163(1996) CDC assays were performed.

"native antibodies" are typically heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, whereas the number of disulfide bonds varies among heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end thereof, followed by a plurality of constant domains. Each light chain end has a variable domain (VL) and the other end has a constant domain; the light chain constant domain is juxtaposed to the first constant domain of the heavy chain, and the variable domain of the light chain is juxtaposed to the variable domain of the heavy chain. It is believed that specific amino acids form an interface between the variable domains of the light and heavy chains.

The term "variable" means that certain portions of the variable regions of different antibodies differ widely in sequence and that these portions are useful in the binding and specificity of each particular antibody for its particular antigen. However, the distribution of variability is not uniform throughout the variable region of the antibody. It is concentrated in three segments called hypervariable regions of the light and heavy chain variable regions. The more highly conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native light and heavy chains each comprise four FRs, mostly in the beta-sheet configuration, connected by three hypervariable regions which form loops, sometimes partially, with the beta-sheet configuration. The hypervariable regions of each chain are linked in close proximity by FRs and form together with the hypervariable regions of the other chains the antigen-binding site of an antibody (see Kabat et al, sequences of proteins of immunological interest, 5 th edition, public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but exhibit various effector functions, such as participation of antibodies in antibody-dependent cell-mediated cytotoxicity (ADCC).

The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. Hypervariable regions typically contain amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 24-34(L1), 50-56(L2) and 89-97(L3) of the light chain variable region, residues 31-35(H1), 50-65(H2) and 95-102(H3) of the heavy chain variable region, Kabat et al, sequences of proteins of immunological interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), and/or residues of the "hypervariable loops" (e.g., residues 26-32(L1), 50-52(L2) and 91-96(L3) of the light chain variable region, residues 26-32(H1), 53-55(H2) and 96-101(H3) of the light chain variable region; Chia and Lesk, journal of molecular biology (1987) -. "framework region" or "FR" residues are variable region residues other than the hypervariable region residues defined herein.

Papain digestion of antibodies produces two identical antigen binding fragments, called "Fab" and one residual "Fc" fragment, each Fab fragment having a single antigen binding site, the name of Fc reflecting its ability to readily form crystals. Pepsin treatment produced an F (ab')₂A fragment which has two antigen binding sites and which is still capable of cross-linking with an antigen.

"Fv" is the smallest antibody fragment, containing the entire antigen recognition and antigen binding site. This region is a dimer of a heavy and a light chain variable region that are tightly and non-covalently joined. At V_H-V_LThe surface of the dimer, the three hypervariable regions of each variable region interact conformationally to define an antigen-binding site. The six hypervariable regions together confer antigen-binding properties to the antibody. However, even a single variable domain (or half of an Fv comprising only three antigen-specific hypervariable regions) can recognize and bind antigen with only a lower affinity than the entire binding site.

The Fab fragment also contains the light chain constant region and the first constant region of the heavy chain (CH 1). Fab' differs from Fab fragments in that the carboxy terminus of the heavy chain CH1 domain has been added to several residues, including one or more cysteines from the antibody hinge region. Fab '-SH is used herein to refer to a Fab' in which the cysteine residues of the constant domains have at least one free thiol group. F (ab') ₂Antibody fragments were originally produced as Fab' fragment pairs with hinge region cysteines between them. Other chemical linkages of antibody fragments are also known.

The "light chain" of an antibody from any species of vertebrate can be assigned to one of two completely different types, called kappa and lambda, based on their constant region amino acid sequences.

"Single chain Fv" or "scFv" antibody fragments include the V of an antibody_HAnd V_LDomains, which are present in a single polypeptide chainThe above. Preferably, the Fv polypeptide is at V_HAnd V_LThe domains also contain a polypeptide linker between them that enables the scFv to form the structure required for antigen binding. For reviews on scFv see Pluckthun in "pharmacology of monoclonal antibodies", Vol.113, eds Rosenburg and Moore, Springer-Verlag, New York, pp.269-315 (1994). HER2 antibody scFv fragments are described in WO 93/16185; U.S. patent nos. 5,571,894; and U.S. Pat. No. 5,587,458.

The term "bivalent antibodies (diabodies)" refers to small antibody fragments with two antigen binding sites, which fragments are in one polypeptide chain (V)_H-V_L) Has a heavy chain variable region (V) linked thereto_H) And a light chain variable region (V) _L). Two antigen binding sites are formed by using a very short linker that prevents pairing of the two domains on the same chain, forcing pairing with the complementary domains on the other chain. In EP 404, 097; WO 93/11161; and Hollinger et al, proceedings of the national academy of sciences USA, 90: 6444-.

A "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody comprising minimal sequences derived from non-human immunoglobulins. For the most part, humanized antibodies are human immunoglobulins (recipient) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species, such as mouse, rat, rabbit or non-human primate (donor) antibody), having the desired specificity, affinity, and capacity (capacity). In some cases, human immunoglobulin Framework Region (FR) residues are substituted with corresponding non-human residues. Furthermore, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications are intended to further refine antibody function. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally further comprises an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. Auspicious see Jones et al, Nature 321: 522-525 (1986); riechmann et al, Nature 332: 323-329 (1988); and Presta, curr. op. struct.biol.2: 593-596(1992).

Humanized HER2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 or TrastuzumabAs described in table 3 of U.S. patent 5,821,337, which is specifically incorporated herein by reference; humanized 520C9(WO93/21319) and humanized 2C4 antibodies described herein.

For purposes herein, "Trastuzumab", is "And "huMAb 4D 5-8" refers to antibodies comprising the light and heavy chain amino acid sequences in SEQ ID NOS.13 and 14, respectively.

Herein, "Pertuzumab," rhuMAb2C4, "and" omnitarget "refer to antibodies comprising the variable light and variable heavy amino acid sequences in SEQ ID nos.3 and 4, respectively. Wherein Pertuzumab is a full-length antibody, preferably it comprises the light and heavy chain amino acid sequences of SEQ ID nos.15 and 16, respectively.

A "naked antibody" is an antibody (as defined herein) that is not conjugated to a heterologous molecule, such as a cytotoxic moiety or a radioactive standard.

An "affinity-matured" antibody is an antibody that has one or more changes in one or more hypervariable regions that result in an increased affinity of the antibody for an antigen compared to a parent antibody that does not have those changes. Preferred affinity matured antibodies will have nanomolar or even picomolar affinity for the target antigen. Affinity-matured antibodies are produced by methods known in the art. Marks et al, Bio/Technology, 10: 779-783(1992) describes affinity maturation by rearrangement of VH and VL domains. Random mutation of CDR and/or framework residues is described by Barbas et al, proc.nat.acad.sci, USA, 91: 3809-3813 (1994); schier et al, Gene, 169: 147-; yelton et al, j.immunol., 155: 1994-2004 (1995); jackson et al, j.immunol., 154 (7): 3310-3319 (1995); and Hawkins et al, j.mol.biol., 226: 889-.

An "agonist antibody" is an antibody that binds to and activates a receptor. In general, the receptor activation capacity of an agonist antibody will be at least qualitatively similar (and may be substantially qualitatively similar) to the natural agonist ligand of the receptor. An example of an agonist antibody is an antibody that binds to a receptor in the TNF receptor superfamily, such as DR5, and which induces apoptosis in cells expressing a TNF receptor (e.g., DR 5). Assays for determining induction of apoptosis are described in WO98/51793 and WO99/37684, both of which are hereby incorporated by reference.

An "isolated" antibody is one that is identified and isolated and/or recovered from a component of its natural environment. Contaminant components of their natural environment are substances that would interfere with diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to a degree of purification of greater than 95% by weight, and most preferably greater than 99% by weight of the antibody as determined by the Lowry method, (2) to a degree sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by use of a rotating cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions and use of Coomassie blue or, preferably, silver stain. Isolated antibodies include antibodies in situ within recombinant cells, as at least one component of the antibody's natural environment is not present. In general, however, an isolated antibody is prepared by at least one purification step.

A HER2 antibody that "inhibits HER dimerization more effectively than Trastuzumab" is one that attenuates or eliminates HER dimers more effectively (e.g., at least about 2-fold more effectively) than Trastuzumab. Preferably, such antibodies are at least about as effective as murine monoclonal antibody 2C4, a Fab fragment of murine monoclonal antibody 2C4, Pertuzumab, and a Fab fragment of Pertuzumab for inhibiting HER2 dimerization. HER dimerization inhibition can be assessed by direct study of HER dimers, or by assessing HER activation, or downstream signals resulting from HER dimerization, and/or by assessing antibody-HER 2 binding sites, and the like. An assay for screening for antibodies that inhibit HER dimerization more effectively than Trastuzumab is described in Agus et al Cancer Cell 2: 127-137(2002) and WO01/00245(Adams et al). For example, inhibition of HER dimer formation can be assessed, for example (see, e.g., FIGS. 1A-B of Agus et al Cancer Cell 2: 127-137 (2002); and WO 01/00245); attenuation of HER ligand activation in HER dimer-expressing cells (e.g., FIGS. 2A-B of WO01/00245 and Agus et al Cancer Cell 2: 127-137 (2002)); repression of binding of HER ligands to cells expressing HER dimers (e.g., WO01/00245, and FIG. 2E of Agus et al Cancer Cell 2: 127-137 (2002)); inhibition of Cell growth of Cancer cells (e.g., MCF7, MDA-MD-134, ZR-75-1, MD-MB-175, T-47D cells) that express HER dimers in the presence (or absence) of HER ligands (e.g., WO01/00245 and FIGS. 3A-D of Agus et al Cancer Cell 2: 127-137 (2002)); inhibition of downstream signaling (e.g., inhibition of HRG-dependent AKT phosphorylation or inhibition of HRG-or TGF α -dependent MAPK phosphorylation) (see, e.g., WO01/00245, and FIGS. 2C-D of Agus et al Cancer Cell 2: 127-137 (2002)) to determine inhibition of HER dimerization. Whether an antibody inhibits HER dimerization can also be assessed by studying the binding site of antibody HER2, for example, by assessing the structure or model, such as crystal structure, of an antibody that binds HER2 (see, e.g., Franklin et al Cancer Cell 5: 317-.

The HER2 antibody may "inhibit HRG-dependent AKT phosphorylation" and/or "inhibit HRG-or TGF-dependent MAPK phosphorylation" more effectively (e.g., at least 2-fold more effectively) than Trastuzumab (see, e.g., Agus et al Cancer Cell 2: 127-137(2002) and WO 01/00245).

The HER2 antibody may "not inhibit HER2 external domain (ectomain) cleavage" (Molina et al Cancer Res.61: 4744-4749 (2001).

A HER2 antibody that "binds to the heterodimeric binding site of HER 2" binds to residues in domain II (optionally also binds to residues in other HER2 extracellular domains, such as domains I and III) and can sterically hinder, at least to some extent, the formation of HER2-EGFR, HER2-HER3, or HER2-HER4 heterodimers. Franklin et al Cancer Cell 5: 317-.

An antibody that "binds to domain II of HER 2" binds to residues in domain II and optionally binds to other domains of HER2, such as residues in domains I and III. Preferably the antibody binding domain II binds to the linkage between domains I, II and III of HER 2.

As used herein, "growth inhibitory agent" refers to a compound or composition that inhibits the growth of a cell, particularly a HER-expressing cancer cell, in vitro or in vivo. Thus, the growth inhibitory agent may be one that significantly reduces the percentage of HER expressing cells in S phase. Examples of growth inhibitory agents include agents that inhibit cell cycle progression (beyond S phase), such as agents that induce G1 arrest and M-phase arrest. Typical M-phase suppressants include vinca alkaloids (vincas) (vincristine and vinblastine), taxanes, and topological II inhibitors such as doxorubicin, epirubicin (epirubicin), daunorubicin (daunorubicin), etoposide (etoposide), and bleomycin. Those agents that block G1 also extend to S phase blockade, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine (dacarbazine), mechlorethamine (mechlororethamine), cisplatin, methotrexate, 5-fluorouracil, and cytarabine (ara-C). Further information can be found in The Molecular Basis of Cancer, Mendelsohnand Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and landinaneoplastic drugs" by Murakami et al (WB Saunders: Philadelphia, 1995), especially page 13.

Examples of "growth inhibitory" antibodies are those that bind to HER2 and inhibit the growth of HER2 cancer cells. A preferred growth inhibitory HER2 antibody inhibits the growth of SK-BR-3 breast tumor cells in cell culture by more than 20%, preferably more than 50% (e.g., from about 50% to about 100%) at an antibody concentration of about 0.5-30 μ g/ml, wherein the growth inhibition is determined after six days of exposure of the SK-BR-3 cells to the antibody (see U.S. Pat. No. 5,677,171 issued 10/14/1997). The SK-BR-3 cell growth inhibition assay is described in greater detail in this patent and below. A preferred growth inhibitory antibody is a humanized variant of murine monoclonal antibody 4D5, e.g., Trastuzumab.

An antibody that "induces apoptosis" refers to an antibody that induces programmed cell death as determined by binding of annexin (annexin) V, DNA fragmentation, cell shrinkage, endoplasmic reticulum swelling, cell fragmentation, and/or formation of a vacuole (known as an apoptotic body). The cell is typically one that expresses the antigen to which the antibody binds. Preferably the cell is a tumor cell. For example, Phosphatidylserine (PS) translocation can be measured by annexin binding; DNA fragmentation can be assessed by DNA gradients; whereas nuclear/chromatin condensation accompanied by DNA fragmentation can be assessed by any growth of hypodiploid cells. Preferably, the antibody that induces apoptosis is one that results in about 2-50 fold, preferably about 5-50 fold, and most preferably about 10-50 fold induction of annexin binding relative to untreated cells in an annexin binding assay that is performed using cells that express the antigen to which the antibody binds. Examples of antibodies that induce apoptosis are HER2 antibodies 7C2 and 7F3, and certain DR5 antibodies.

"epitope 2C 4" is the region in the extracellular domain of HER2 to which antibody 2C4 binds. To screen for Antibodies that bind the epitope of 2C4, a conventional cross-blocking assay can be performed, such as the assay described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988). Alternatively, epitope mapping can be performed to assess whether an antibody binds to the 2C4 epitope of HER 2. Epitope 2C4 comprises residues from domain II in the extracellular domain of HER 2. 2C4 and Pertuzumab bind the extracellular domain of HER2 at the junction of domains I, II and III. Franklin et al human cancer cell 5: 317-328(2004).

"epitope 4D 5" is the region in the extracellular domain of HER2 to which antibody 4D5(ATCC CRL10463) and Trastuzumab bind. This epitope is close to the transmembrane domain of HER2 and within domain IV of HER 2. To screen for Antibodies that bind the epitope of 4D5, a conventional cross-blocking assay can be performed, such as the assay described in Antibodies, available Manual, Cold Spring harbor laboratory, Ed harbor and David Lane (1988). Alternatively, epitope mapping can be performed to assess whether the antibody binds to the 4D5 epitope of HER2 (e.g., any one or more residues in the region from about residue 529 to about residue 625 of HER2, including residue 529 and residue 625).

"epitope 7C2/7F 3" is an amino-terminal region within domain I of the extracellular domain of HER2 to which 7C2 and/or 7F3 antibodies (both deposited under ATCC, see below) bind. To screen for Antibodies that bind the epitope 7C2/7F3, a conventional cross-blocking assay can be performed, such as the assay described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988). Alternatively, epitope mapping can be performed to confirm whether the antibody binds to the 7C2/7F3 epitope on HER2 (e.g., any one or more residues in the region from about residue 22 to about residue 53 of HER 2).

"treatment" refers to both therapeutic treatment and prophylactic measures. The person in need of treatment includes persons already suffering from the disease as well as persons to be protected from the disease. Thus, a patient to be treated herein may be diagnosed as having a disease or as likely predisposed to, or predisposed to, the disease.

The terms "cancer" and "cancerous" refer to or describe the physiological state of a mammal that is generally characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma, and islet cell carcinoma), mesothelioma, schwannomas (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, neck cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, scrotal cancer, esophageal cancer, tumors of the biliary tract, and cancers of the head and neck.

The term "effective amount" refers to an amount of a drug that is effective against the disease in a patient. When the disease is cancer, the effective amount of the drug may reduce the number of cancer cells; reducing tumor size; inhibit (i.e., delay and preferably stop to some extent) cancer cell infiltration into peripheral organs; inhibit (i.e., delay and preferably stop to some extent) tumor metastasis; inhibit tumor growth to some extent; and/or alleviate one or more symptoms associated with cancer to some extent. To the extent that the drug can prevent the growth of cancer cells and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. The effective amount may prolong progression free survival, result in a target response (including a partial response, PR, or complete response, CR), increase overall survival time, and/or ameliorate one or more symptoms of cancer.

A "HER 2 expressing cancer" is a cancer comprising cells having a HER2 protein present on their cell surface.

A cancer that "overexpresses" a HER receptor is one that has significantly higher levels of HER receptor, such as HER2, at its cell surface compared to a noncancerous cell of the same tissue type. Such overexpression may be caused by gene amplification or by enhanced transcription or translation. HER receptor overexpression can be determined in diagnostic or prognostic assays by assessing increased levels of HER protein present on the cell surface (e.g.by immunohistochemical assays; IHC). Alternatively, or in addition, the level of HER encoding nucleic acid in the cell may be measured, for example, by fluorescence in situ hybridization (FISH; see WO98/45479 published at 10 months 1998), southern blotting, or Polymerase Chain Reaction (PCR) techniques, such as timed quantitative PCR (RT-PCR). HER receptor overexpression can also be studied by measuring the exudation of antigens in biological fluids such as serum (see, e.g., U.S. Pat. No. 4,933,294 issued to 1990-06-12; WO91/05264 issued to 1991-04-18; U.S. Pat. No. 5,401,638 issued to 1995-03-28; and Sias et al J.Immunol. methods 132: 73-80 (1990)). In addition to the above assays, the skilled practitioner can also utilize various in vivo assays. For example, cells in a patient may be exposed to an antibody, which is optionally labeled with a detectable label, such as a radioisotope, and binding of the antibody to cells in the patient may be assessed, for example, by external scanning for radioactivity or by analysis of biopsy tissue taken from a patient previously exposed to the antibody.

In contrast, a cancer that "does not overexpress the HER2 receptor" is one that does not express higher than normal levels of HER2 receptor as compared to a noncancerous cell of the same tissue type.

A cancer that "overexpresses" a HER ligand is one that produces significantly higher levels of the ligand as compared to non-cancerous cells of the same tissue type. Such overexpression may be caused by gene amplification or by enhanced transcription or translation. Overexpression of a HER ligand can be determined diagnostically by assessing the level of the ligand (or nucleic acid encoding it) in the patient, for example in tumor biopsy or by various diagnostic assays such as IHC, FISH, southern blot, PCR or the in vivo assays described above.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cellular function and/or causes cellular destruction. The term is intended to includeRadionuclides (e.g. At)²¹¹，I¹³¹，I¹²⁵，Y⁹⁰，Re¹⁸⁶，Re¹⁸⁸，Sm¹⁵³，Bi²¹²，P³²And radioactive isotopes of lutetium), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and/or variants thereof.

"chemotherapeutic agents" are chemical compounds that can be used to treat cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide (cyclophosphamide) ((TM)) ) (ii) a Alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benaodopa, carboquone, meturedopa and uretonimine; ethyleneimine and melamines include altretamine (altretamine), triethylenemelamine (triethylenemelamine), triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine (trimetylomelamine); acetogenins (in particular bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol,) (ii) a beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analogue topotecan); CPT-11 (irinotecan,) (ii) a Acetyl camptothecin, scopoletin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin), and bizelesin (bizelesin) synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (especially cryptophycin1 and cryptophycin 8); dolastatin (dolastatin); duocarmycins (including synthetic analogs, KW-2189 and CBI-TMI); (ii) an elutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, cholorfamide (cholephosphamide), estramustine Nitrogen mustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), melphalan (melphalan), mechlorethamine (novembichin), cholesteryl phenylacetate, prednimustine (prednimustine), trofosfamide (trofosfamide), uracil mustard; nitrosoureas (nitrosureas) such as nitrosourea mustard (carmustine), chlorozotocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ramustine (ranimustine); antibiotics such as enediol antibiotics (e.g., calicheamicin, especially calicheamicin γ 1 and calicheamicin ω 1, see, e.g., Agnew Chem int. ed. engl.33: 183- Morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinyl-doxorubicin, doxorubicin HCl liposome injectionLiposome adriamycin TLC D-99Pegylated liposomal doxorubicinAnd doxorabicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), phleomycin (marcelomycin), mitomycins such as mitomycin C, mycophenolic acid, nogomycin (nogalamycin), olivomycin (olivomycin), pelomomycin (peplomycin), potfiromycin, puromycin, triiron doxorubicin (quelamycin), rodobicin (rodorubicin), streptonigrin; streptozotocin (streptozocin)Tubercidin, ubenimex (ubenimex), azinostatin (zinostatin), zorubicin (zorubicin); antimetabolites such as methotrexate, 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine (fludarabine), 6-mercaptopurine, thiamine, thioguanine; pyrimidine analogs, such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine, carmofur (carmofur), cytarabine, dideoxyuridine, doxifluridine (doxifluridine), enocitabine (enocitabine), floxuridine; anti-adrenal agents, such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements such as frolicic acid; aceglucomannan lactone; (ii) an aldophosphamide glycoside; aminolevulinic acid (aminolevulinic acid); amsacrine (amsacrine); bestrabuucil; bisantrene; edatrexate (edatraxate); desphosphamide (defofamine); colchicine; diazaquinone (diaziqutone); elfornitine; ammonium etitanium acetate; an epothilone; etoglut (etoglucid); gallium nitrate; a hydroxyurea; lentinan (lentinan); lonidamine (lonidamine); maytansinoids (maytansinoids), including maytansinoids and ansamitocins, mitoguazone; mitoxantrone (mitoxantrone); mopidamol (mopidamol); nifurthradine (nitracrine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); podophyllinic acid (podophyllinic acid); 2-ethyl hydrazide; procarbazine (procarbazine); Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane (rizoxane); rhizoxin; sisofilan (sizofiran); germanium spiroamines (spirogyranium); alternarionic acid; a tri-imine quinone; 2, 2', 2 "-trichlorotriethylamine (trichlororrothialylamine); trichothecene toxins (especially T-2 toxin, veracurin a, bacillocin a, serpentine); urethane (urethan); dacarbazine (dacarbazine); mannitol mustard; dibromomannitol (mitobronitol); dibromodulcitol; pipobolBromoalkane (pipobroman); a polycytidysine; arabinoside ("Ara-C"); thiotepa (thiotepa); taxanes (taxoids), such as taxol (R) ((R))Albumin-modified nanoparticle formulation of paclitaxel (ABRAXANETM), and docetaxelchlororanibucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin and carboplatin; vinblastine, which prevents tubulin polymerization to form microtubules, comprises vinblastine (A)) Vincristine (a)) Vindesine (elderine)) And Vinorelbine (NAVELBINE)) (ii) a Etoposide (VP-16); ifosfamide; mitoxantrone; leucovovin; nuantro (novantrone); edatrexae; daunorubicin; aminopterin; ibandronate; topoisomerase inhibitor RFS2000, Difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene Bisphosphonates such as chlorodiphosphates (e.g., BONEFOS)Or OSTAC) Etidronate sodium (DIDROCAL)) NE-58095, zoledronic acid/zoledronateAlendronate sodium saltPamidronic acid sodium saltTilurophosphonic acid saltOr risedronateTroxacitabine (troxacitabine) (1, 3-dioxolane nucleoside cytosine analogue); antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways involved in uncontrolled cell proliferation, such as, for example, PKC- α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such asVaccines and gene therapy vaccines, e.g. ALLOVECTINVaccine, LEUVECTINVaccine, and VAXIDA vaccine; topoisomerase 1 inhibitors (e.g., lutotecan @)) (ii) a rmRH (e.g., ABARELIX)) (ii) a BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine (perifosine), COX-2 inhibitors (e.g., celecoxib (celecoxib) or etoricoxib (etoricoxib)), proteosome inhibitors (e.g., PS 341); bortezomibCCI-779; tipifarnib (R11577); orafenaib, ABT 510; bcl-2 inhibitors such as oblimersen sodium (b:)) (ii) a pixantrone; EGFR inhibitors(see definitions below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the above agents, such as CHOP, cyclophosphamide, doxorubicin, vincristine, and hydrocortisone, and FOLFOX, with oxaliplatin (ELOXATIN) ^TM) Abbreviations for treatment regimens incorporating 5-FU and leucovin.

This definition also includes anti-hormonal agents that act to modulate or inhibit the action of hormones on tumors, such as anti-estrogen agents that have a mixed agonist/antagonist profile, including tamoxifen4-hydroxy tamoxifen, toremifeneIdoxifene, droloxifene, raloxifeneTrioxifene, keoxifene, and Selective Estrogen Receptor Modulators (SERMs) such as SERM 3; pure antiestrogens without agonist properties such as fulvestrantAnd EM800 (such agents may suppress Estrogen Receptor (ER) dimerization, inhibit DNA binding, enhance ER turnover, and/or inhibit ER levels); aromatase inhibitors, including steroidal aromatase inhibitor such as formestane and exemestane (exemestane)And non-steroidal aromatase inhibitors such as anastrozoleLetrozoleAnd aminoglutethimide, and other aromatase inhibitors including vorozole (vorozole)Megestrol acetate saltfadrozole, imidazole; luteinizing hormone releasing hormone agonists including Leuprolide (LUPRON)And ELIGARD) Goserelin, buserelin, and tripterelin; sex steroids including progestogens such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and pramlinum (premarin), and androgens/retinoids such as fluoxymesterone, all transretinic acids and fenretinide; onapristone (onapristone); an anti-progestin agent; estrogen Receptor Downregulators (ERDs); antiandrogens such as flutamide, nilutamide and bicalutamide (bicalutamide); testosterone; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the above agents.

As used herein, the term "EGFR-targeting agent" refers to a therapeutic agent that binds to EGFR and, optionally, inhibits EGFR activation. Examples of such agents include antibodies and small molecules that bind EGFR. Examples of EGFR-binding antibodies include MAb 579(ATCC CRL HB 8506), MAb 455(ATCC CRL HB 8507), MAb 225(ATCC CRL 8508), MAb 528(ATCC CRL8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn et al) and variants thereof, such as chimeric 225(C225 or Cetuximab; ERBUTIX) And reconstituted human 225(H225) (see, WO96/40210, Imclone Systems Inc.); antibodies that bind type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized and chimeric antibodies that bind EGFR as described in U.S. patent No. 5,891,996; and human antibodies that bind EGFR, such as ABX-EGF (see WO98/50433, Abgenix). anti-EGFR antibodies can be conjugated with cytotoxic agents, thereby generating immunoconjugates (see, e.g., EP659, 439a2, Merck Patent GmbH). Examples of small molecules that bind EGFR include ZD1839 or Gefitinib (IRESSA)^TM(ii) a Astra Zeneca), CP-358774 or Erlotinib HCL (TARCEVA)^TM(ii) a Genentech/OSI) and AG1478,AG1571(SU5271；Sugen)。

a "tyrosine kinase inhibitor" is a molecule that inhibits to some extent the tyrosine kinase activity of tyrosine kinases, such as HER receptors. Examples of such inhibitors include the EGFR-targeting drugs mentioned in the preceding paragraphs as well as small molecule HER2 tyrosine kinase inhibitors such as TAK165 available from Takeda, dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially bind EGFR but inhibit HER2 and EGFR-overexpressing cells, oral HER2GW572016 (available from Glaxo) and EGFR tyrosine kinase inhibitors, and PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); raf-1 inhibitors such as the antisense agent ISIS-5132 available from ISIS Pharmaceuticals, which inhibits Raf-1 signaling; non-HER targeted TK inhibitors such as Imatinib mesylate (Gleevac) ^TM) Obtainable from Glaxo; CI-1040, a MAPK extracellular regulated kinase I inhibitor (available from Pharmacia); quinazolines, such as PD153035, 4- (3-chloroaniline) quinazoline; pyridopyrimidines; pyrimidopyrimines; pyrrolopyramidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4- (phenylamino) -7H-pyrrolyl [2, 3-d]A pyrimidine; curcumin (dieruloyl methane, 4, 5-bis (4-para-fluoroaniline) phthalimide); tyrphostins comprising a nitrothiophene moiety; PD-0183805 (Warner-Lamber); antisense molecules (e.g., those that bind to HER-encoding nucleic acids); quinoxaline (U.S. patent No. 5,804,396); trypostins (U.S. patent No. 5,804,396); ZD6474(Astra Zeneca); PTK-787(Novartis/Schering AG); pan-HER inhibitors such as CI-1033 (Pfizer); affinitac (ISIS 3521; ISIS/Lilly); imatinib methanesulfonic acid (Gleevac; Novartis); PKI166 (Novartis); GW2016(Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); semaxinib (sugen); ZD6474 (AstraZeneca); PTK-787(Novartis/Schering AG); INC-1C11 (Imclone); or as described in any of the following patent publications: U.S. patent nos. 5,804,396; WO99/09016(American Cyanimid); WO98/43960(American Cyanamid); WO97/38983(Wamer Lambert); WO99/06378(Warner Lambert); WO99/06396 (WarnerLambert); WO96/30347(Pfizer, Inc); WO96/33978 (Zeneca); WO96/3397 (Zeneca); and WO96/33980 (Zeneca).

An "anti-angiogenic agent" refers to a compound that suppresses or interferes to some extent with angiogenesis. The anti-angiogenic factor can, for example, be a small molecule or antibody that binds to a growth factor or growth factor receptor involved in promoting angiogenesis. Preferred anti-angiogenic factors herein are antibodies that bind to Vascular Endothelial Growth Factor (VEGF), such as Bevacizumab

The term "cytokine" is a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Such cytokines include lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones, such as human growth hormone, N-methanedisulfonyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; a proinsulin; (ii) a relaxin; a prorelaxin; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), Luteinizing Hormone (LH); hepatocyte growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and beta; mullerian tube (mullerian) -inhibitor; mouse gonadotropin-related peptides; a statin; nandrolone phenylpropionate; vascular endothelial cell growth factor; an integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet growth factor; transforming Growth Factors (TGF) such as TGF-alpha and TGF-beta; insulin-like growth factors-I and-II; erythropoietin (EPO); osteoinductive factors (osteoinductive factors); interferons such as interferon- α, - β, - γ; colony Stimulating Factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); granulocyte-CSF (G-CSF); interleukins (IL) such as IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; tumor necrosis factors such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and Kit Ligand (KL). The term cytokine as used herein includes native proteins or proteins from recombinant cell culture as well as biologically active equivalents of native sequence cytokines.

The formulated antibody is preferably substantially pure and substantially homogeneous (i.e., free of contaminating proteins, etc.). By "substantially pure" antibody is meant a composition that includes at least about 90% by weight of the antibody, preferably at least about 95% by weight, based on the total weight of the composition. By "substantially homogeneous" antibody is meant a composition that includes at least about 99% by weight of the antibody, based on the total weight of the composition.

A "B cell surface marker" or "B cell surface antigen" herein is an antigen expressed on the surface of a B cell that can be targeted with an antibody bound thereto. Exemplary B cell surface markers include CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79B, CD80, CD81, CD82, CD83, CDw84, CD85 and CD86 Leukocyte surface markers (for a description see The Leukocyte Antigen Facts computers Book, 2^ndEdition.1997, ed.Barclay et al Academic Press, Harbourt Brace&Co., New York). Other B cell surface markers include RP105, FcRH2, B cell CR2, CCR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, Btig, NAG14, SLGC16270, FcRH1, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA, and 239287. The specific target B cell surface markers herein are preferably expressed on B cells as compared to other non-B cell tissues of mammals, and may be expressed on precursor B cells and mature B cells. Preferred B cell surface criteria herein are CD20 or BR 3.

"° c D20", antigen, or "CD 20", is an approximately 35-kDa, non-glycosylated phosphoprotein found on more than 90% of the B cell surface from peripheral blood or lymphoid organs. CD20 is present in normal B cells as well as malignant B cells, but is not expressed on stem cells. Other names for CD20 in the literature include "B-lymphocyte restricted antigens" and "Bp 35". CD20 antigen is described, for example, in Clark et al proc.natl.acad.sci. (USA) 82: 1766 (1985).

For purposes of this document only, "humanized 2H 7" refers to a humanized variant of the 2H7 antibody, the CDR sequences of which 2H7 antibody are disclosed in U.S. patent No. 5,500,362 (fig. 5 and 6), which is hereby incorporated by reference. Examples of humanized 2H7 antibodies herein include variants described in WO2004/056312, as well as other variants, including, but not limited to: 2H7v16, 2H7v31, 2H7v73, 2H7v75, 2H7v96, 2H7v114, 2H7v115, 2H7v116, 2H7v138, 2H7v477, 2H7v375, etc., WO2004/056312 is also incorporated herein by reference.

In one embodiment, the humanized 2H7 antibody comprises one, two, three, four, five or six of the following CDR sequences:

CDR L1 sequence RASSSSVSYXH wherein X is M or L (SEQ ID No.67), e.g. SEQ ID No.57 (FIG. 18A),

the CDR L2 sequence of SEQ ID No.58 (FIG. 18A),

CDR L3 sequence QQWXFNNPPT wherein X is S or A (SEQ ID No.68), such as SEQ ID No.59 (FIG. 18A),

the CDR H1 sequence of SEQ ID No.60 (FIG. 18B),

the CDR H2 sequence of AIYPGNXTSYNQKFKG, wherein X is D or A (SEQ ID NO.69), e.g., SEQ ID No.61 (FIG. 18B), and

the CDR H3 sequence of VVVYYSXXYWYFDV, wherein X at position 6 is N, A, Y, W or D, and X at position 7 is S or R (SEQ ID No.70), e.g. SEQ ID No.62 (FIG. 18B).

The above CDR sequences generally exist within human variable light and variable heavy framework sequences, such as substantially human light chain kappa subunit I (V)_L-I) and substantially human heavy chain subunit III (V)_HIII) human consensus FR residues. See also WO 2004/056312(Lowman et al).

The variable heavy region may be linked to a human IgG chain constant region, wherein the region may be, for example, IgG1 or IgG3, including native sequence and variant constant regions.

In a preferred embodiment, such antibodies comprise the variable heavy domain sequence of SEQ ID No.29 (v16, as shown in fig. 18B), optionally further comprising the variable light domain sequence of SEQ ID No.26 (v16, as shown in fig. 18A), which optionally comprises one or more amino acid substitutions at positions 56,100, and/or 100a, e.g. D56A, N100A or N100Y, and/or S100aR in the variable heavy domain, one or more amino acid substitutions at positions 32 and/or 92 in the variable light domain, e.g. M32L and/or S92A. Preferably, the antibody is a whole antibody comprising the light chain amino acid sequence of SEQ ID nos.63 or 64, and the heavy chain amino acid sequence of SEQ ID nos. 65, 66, 71 or 72.

A preferred humanized 2H7 antibody is ocrelizumab (genentech).

The antibodies herein may further comprise at least one amino acid substitution in the Fc region that increases ADCC activity, such as an amino acid substitution at positions 298,333, and 334, preferably S298A, E333A, and K334A, using Eu numbering of heavy chain residues. See also U.S. Pat. No. 6,737,056B1, Presta.

Any of these antibodies may comprise at least one substitution in the Fc region that enhances FcRn binding or serum half-life, for example a substitution at heavy chain position 434, such as N434W. See also U.S. Pat. No. 6,737,056B1, Presta.

Any of these antibodies may comprise at least one amino acid substitution in the Fc region that enhances CDC activity, e.g., comprising at least a substitution at position 326, preferably K326A or K326W. See also U.S. Pat. No. 6,528,624B1 (Idusogene et al).

Some preferred humanized 2H7 variants are those comprising the variable light domain of SEQ ID No.26 and the variable heavy domain of SEQ ID No.29, including variants with or without substitutions in the Fc region (if present), and those comprising the substitution N100A in SEQ ID No. 29; or D56A and N100A; or D56A, N100Y, and S100aR and having the substitution M32L in SEQ ID No. 26; or S92A; or variants of the variable light domains of M32L and S92A.

M34 in the variable heavy chain of 2H7v16 has been identified as a potential source of antibody stability and as a potential candidate for another substitution.

In a summary of some preferred embodiments of the invention, the variable region of the 2H7v 16-based variants comprises the amino acid sequence of v16, except for the amino acid substitution positions indicated in the table below. Unless otherwise specified, 2H7 variants will have the same light chain as v 16.

Exemplary humanized 2H7 antibody variants

2H7 version	Heavy chain (VH) changes	Light chain (VL) changes	Fc changes
				16 for reference
31			S298A，E333A，K334A
				73	N100A	M32L
75	N100A	M32L	S298A，E333A，K334A
				96	D56A，N100A	S92A
114	D56A，N100A	M32L，S92A	S298A，E333A，K334A
				15	D56A，N100A	M32L，S92A	S298A，E333A，K334A，E356D，M358L
16	D56A，N100A	M32L，S92A	S298A，K334A，K322A
				38	D56A，N100A	M32L，S92A	S298A，E333A，K334A，K326A
477	D56A，N100A	M32L，S92A	S298A，E333A，K334A，K326A，N434W
				375			K334L
588			S298A，E333A，K334A，K326A
				511	D56A，N100Y，S100aR	M32L，S92A	S298A，E333A，K334A，K326A

One preferred humanized 2H7 comprises the 2H7v16 variable light domain sequence:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQG

and 2H7v16 variable heavy domain sequence:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVV

when the humanized 2H7v16 antibody is a whole antibody, it may comprise the light chain amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQG TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC

and the heavy chain amino acid sequence of SEQ ID No.65 or:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ IDNo.71).

another preferred humanized 2H7 antibody comprises the 2H7v511 variable light domain sequence:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKR(SEQ ID No.73)

and 2H7v511 variable heavy domain sequence:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSYRYWYFDVWGQGTLVTVSS(SEQ IDNo.74).

when the humanized 2H7v511 antibody is a whole antibody, it may comprise the light chain amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID No.64)

and the heavy chain amino acid sequence of SEQ ID No.66 or:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSYRYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ IDNo.72)。

"B cell malignancy" herein includes non-Hodgkin's lymphoma (NHL) including low grade/follicular NHL, Small Lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cavitating NHL, bulk disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia; leukemias, including Acute Lymphoblastic Leukemia (ALL), Chronic Lymphoblastic Leukemia (CLL), hairy cell leukemia and chronic myeloblastic leukemia (myeloid leukemia); and other hematologic malignancies. These malignancies can be treated with antibodies against B cell surface markers, such as CD 20.

The term "non-hodgkin's lymphoma" or "NHL" as used herein, refers to cancers of lymphocytes other than hodgkin's lymphoma. Hodgkin's lymphoma can generally be distinguished from non-Hodgkin's lymphoma by the presence of Reed-Sternberg cells in the Hodgkin's lymphoma, but not by the absence of such cells in the Hodgkin's lymphoma. Examples of non-hodgkin's lymphomas encompassed by the terms as used herein include any disease that can be identified by one of skill in the art (e.g., an oncologist or pathologist) following art-known classification schemes such as the modified European-American Lymphoma (REAL) scheme, described in Color Atlas of clinical Hematology, Third Edition; victor Hoffbrand and John e.pettitt (eds.) (harbourt Publishers Limited 2000) (see in particular fig. 11.57, 11.58 and/or 11.59). More specific examples include, but are not limited to, relapsed or refractory NHL, frontline (front line) low-grade NHL, stage III/IV NHL, chemotherapy-resistant NHL, precursor B-lymphoblastic leukemia and/or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and/or prolymphocytic (prolymphocytic) leukemia and/or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and/or lymphoplasmacytoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone-MALT lymphoma, nodal marginal zone lymphoma, hairy cell leukemia, plasmacytoma and/or plasmacytoma, low-grade/follicular lymphoma, intermediate/follicular NHL, mantle cell lymphoma, follicular central lymphoma (folliculular), intermediate diffuse NHL, diffuse large B-cell lymphoma, invasive NHL (including infiltrating prodeline NHL and infiltrating relapsed NHL), relapsed or refractory NHL following autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, higher immunoblastic NHL, higher lymphoblastic NHL, higher small non-lymphocytic NHL, bulk disease NHL, Burkitt's lymphoma, precursor (exogenous) T-cell lymphoblastic leukemia and/or lymphoma, adult T-cell lymphoma and/or leukemia, T-cell chronic lymphocytic leukemia and/or prolymphocytic leukemia, large granular lymphocytic leukemia, mycosis fungoides and/or Sezary syndrome, extranodal natural/T-cell (rhino-type) lymphoma, intestinal T-cell lymphoma, hepatosplenic T-cell lymphoma, subcutaneous lipodermitis-like T cell lymphoma, cutaneous lymphoma, anaplastic (anaplastic) large cell lymphoma, angiocentric lymphoma, intestinal T cell lymphoma, peripheral T cell (not otherwise specified) lymphoma, and angioimmunoblastic T cell lymphoma.

An "autoimmune disease" herein is a disease or disorder that results from or is directed against an individual's own tissue or a co-isolate or clinical presentation thereof, or results from a condition thereof. Examples of autoimmune diseases or disorders include, but are not limited to, arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and ankylosing spondylitis), psoriasis, dermatitis including atopic dermatitis, chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis/dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as progressive systemic sclerosis, Inflammatory Bowel Disease (IBD) (e.g., crohn's disease, ulcerative colitis, autoimmune inflammatory bowel disease), pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis), respiratory distress syndrome, including Adult Respiratory Distress Syndrome (ARDS), meningitis, IgE-mediated diseases such as allergic and atopic rhinitis, encephalitis such as Rasmussen's encephalitis, uveitis or autoimmune uveitis, colitis such as microscopic colitis and collagenous colitis, Glomerulonephritis (GN) such as membranous GN (membranous nephropathy), congenital membranous nephropathy, Membranous Proliferative GN (MPGN), including types I and II, and rapidly progressive GN, allergic diseases, allergic reactions, eczema, asthma, diseases involving T cells and chronic inflammatory reactions, atherosclerosis, autoimmune myocarditis, hypoleukocyte adhesion, Systemic Lupus Erythematosus (SLE) such as cutaneous SLE, subacute cutaneous lupus erythematosus, lupus (including nephritic, cerebritic, juvenile, non-renal, discoid, alopecia), juvenile (type I) diabetes, including juvenile Insulin Dependent Diabetes (IDDM), adult diabetes (type II diabetes), Multiple Sclerosis (MS) such as spinal-ocular (spino-opthalmic) MS, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitis including macrovasculitis including polymyalgia rheumatica and giant cell (Takayasu's) vasculitis, intermediate vasculitis including Kawasaki disease and polyarteritis nodosa, CNS vasculitis, necrotizing systemic vasculitis, and ANCA-associated vasculitis such as Churg-Strauss vasculitis or syndrome (CSS), temporal arteritis, aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immunological hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia, Pure Red Cell Aplasia (PRCA), factor VIII deficiency, hemophilia, autoimmune neutropenia, pancytopenia, leukopenia, diseases associated with leukocyte extravasation, inflammatory diseases of the CNS, multiple organ injury syndrome, diseases mediated by antigen-antibody complexes, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Behcet's disease, Castleman syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, exudative erythema multiforme (Steven-Johnson syndrome), pemphigoids such as bullous pemphigoid, pemphigus including pemphigus vulgaris (vulgares), pemphigus foliatus, and pemphigus mucosae pemphigoid (mphigus multoceus-member, including Thrombotic Thrombocytopenic Purpura (TTP) and autoimmune or immune-mediated thrombocytopenia such as Idiopathic Thrombocytopenic Purpura (ITP) including chronic or acute ITP, autoimmune diseases of the testis or ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Addison's disease, Grave's disease, polyglandular syndromes such as autoimmune polyglandular syndrome (or polyglandular endocrine syndrome), paraneoplastic (paraneoplastic) syndrome including neurological neoplastic syndromes such as Lambert-Eatopovert syndrome or Lambert-Eaton syndrome, stiff person syndrome, encephalomyelitis such as allergic encephalomyelitis, myasthenia gravis, cerebellar degeneration, limbic and/or brainstem encephalitis, neuromyotonia, strabismus clonus or strabismus clonus syndrome (OMS), and sensory neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupus-like hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonia (lymphoid intestinalpneumonitis), bronchiolitis obliterans (non-transplant) versus NSIP, Guillain-Barr syndrome, Berger's disease (IgA nephropathy), primary biliary cirrhosis, stomatitis diarrhea (gluten enteropathy), intractable stomatitis diarrhea, dermatitis herpetiformis, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, Autoimmune Inner Ear Disease (AIED), or autoimmune hearing loss, Ocular Myoclonus Syndrome (OMS), polychondritis such as intractable polychondritis, pulmonary alveolar proteinosis, amyloidosis, giant cell hepatitis, scleritis, noncancerous lymphocytosis, primary lymphocytosis, including monoclonal B-cell lymphocytosis (e.g., benign monoclonal gammopathy and unidentified monoclonal gammopathy, MGUS), peripheral neuropathy, paraneoplastic syndrome, neural pathway diseases (channelopathies) such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and central nervous system neural pathway diseases, autism, inflammatory myopathy, Focal Segmental Glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveitis (uveoretinitis), autoimmune diseases, fibromyalgia, multiple endocrine disorders, schmidt's syndrome, adrenalitis, gastric atrophy, senile dementia, demyelinating disease, post myocardial infarction syndrome (drestler's syndrome), alopecia (alopecia) arcata, CREST syndrome (calcinosis, raynaud's phenomenon, esophageal dyskinesia (dysarthrit), dactylosis, and telangiectasia), male and female autoimmune infertility, ankylosing spondylitis, mixed connective tissue disease, southern American trypanosomiasis, rheumatic fever, recurrent abortion, farmer's lungs, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird fan's lungs, alder's syndrome, alveolitis such as allergic and fibrositis, interstitial lung disease, reaction, leprosy, malaria, leishmaniasis, kypanosisis, schistosomiasis, ascariasis, aspergillosis, campylosis syndrome, camplanian syndrome, dengue fever, endocarditis, endocardial fibrosis, endophthalmitis, erythema eleventum et deutinum, fetal erythroblastosis (erythroblastosis fetalis), eosinophilic fasciitis (eosinophilic fasciitis), Shulman syndrome, Felty syndrome, flariasis, ciliitis such as chronic cyclitis, isocyclocystitis, or Fuch cyclitis, Henoch-Schonlein purpura, Human Immunodeficiency Virus (HIV) infection, echovirus infection, cardiomyopathy, alzheimer's disease, parvovirus infection, rubella virus infection, post-immunization syndrome, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal dysfunction, Sydenham's disease, streptococcus post-infection nephritis, thromboangiitis obliterans, thyrotoxicosis (thyrotoxicosis), spinal cord tuberculosis, and polymyotic tuberculosis.

The "tumor necrosis factor receptor superfamily" or "TNF receptor superfamily" herein refers to receptor polypeptides bound by cytokines of the TNF family. Typically, these receptors are type I transmembrane receptors with one or more cysteine rich repeats in their extracellular domain. The TNF receptor superfamily can be further subdivided into (1) death receptors; (2) a decoy (decoy) receptor; and (3) a signal receptor lacking a death domain. The "death receptor" comprises in its cytoplasm or intracellular region a "death domain", i.e. a region or sequence that acts to transduce a signal in the cell that can lead to apoptosis or induction of certain genes. The "decoy receptor" lacks a functional death domain and is unable to transduce signals that lead to apoptosis. Examples of cytokines in the TNF gene family include tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor-beta (TNF-beta or lymphotoxin), CD30 ligand, CD27 ligand, CD40 ligand, OX-40 ligand, 4-1BB ligand, Apo-1 ligand (also known as Fas ligand or CD95 ligand), Apo-2 ligand (also known as TRAIL), Apo-3 ligand (also known as TWEAK), Osteoprotegerin (OPG), APRIL, RANK ligand (also known as TRANCE), and TALL-1 (also known as BlyS, BAFF or THANK). Examples of receptors in the TNF receptor superfamily include: tumor necrosis factor receptor type 1 (TNFR1), tumor necrosis factor receptor type 2 (TNFR2), p75 Nerve Growth Factor Receptor (NGFR), B cell surface antigen CD40, T cell antigen OX-40, Apo-1 receptor (also known as Fas or CD95), Apo-3 receptor (also known as DR3, swl-1, TRAMP and LARD), receptor known as "transmembrane activator and CAML-interactor" or "TACI"), BCMA protein, DR4, DR5 (or known as Apo-2; TRAIL-R2, TR6, Tango-63, hA 8, TRICK2 or KILLER), DR6, DcR1 (also known as TRID, LIT or TRAIL-R3), DcR2 (also known as TRAIL-R4 or TRUNDD), DCG, DcR3 (also known as TNFR 6 or M68), CAR 72, HVATAR 72 or TNFR1, TNFR 369872, TNFR1, TNFR 369872, TNFR and LARD, 371A1) In that respect

The terms "Apo-2 ligand", "Apo-2L", "Apo 2L", Apo-2 ligand/TRAIL "and" TRAIL "are used interchangeably herein and refer to a polypeptide sequence comprising amino acid residue 114 and 281 of the amino acid sequence shown in figure 24(SEQ ID No.46), including 95 to 281, including residues 92 to 281, including residues 91 to 281, including residues 41 to 281, including residues 39 to 281, including residues 15 to 281, or including residues 1 to 281, as well as biologically active fragments, deletions, insertions, and/or substitution variants of the above sequences. In one embodiment, the polypeptide sequence comprises residues 114-281 of FIG. 24(SEQ ID No. 46). Optionally, the polypeptide sequence comprises residues 92-281 or residues 91-281 of figure 24(SEQ ID No. 46). An Apo-2L polypeptide may be encoded by the natural nucleotide sequence shown in figure 24(SEQ ID No. 45). Optionally, the codon encoding residue Prol19 (FIG. 24; SEQ ID No.45) may be "CCT" or "CCG". Optionally, the fragment or variant is biologically active and has at least about 80% amino acid sequence identity, or at least about 90% sequence identity, or at least 95%, 96%, 97%, 98%, or 99% sequence identity to any of the above sequences. This definition includes substituted variants of Apo-2 ligand in which at least one of its natural amino acids is substituted with another amino acid, such as an alanine residue. This definition also includes naturally-occurring sequence Apo-2 ligands isolated from Apo-2 ligand sources or prepared by recombinant and/or synthetic methods. Apo-2 ligands of the invention include polypeptides known as Apo-2 ligands or TRAIL disclosed in WO97/01633 published as 1997-01-16, WO97/25428 published as 1997-07-17, WO 99/36535 published as July 22, 1999-07-22, WO 01/00832 published as 2001-01-04, WO 02/09755 published as 2002-02-07, WO 00/75191 published as 2000-12-14, and US patent No. 6,030,945 published as 2000-02-29. The term is generally used to refer to monomeric, dimeric, trimeric, hexamer or highly oligomeric forms comprising the polypeptide. The numbering used to refer to the amino acid residues of the Apo-2L sequence is according to FIG. 24(SEQ ID No.46), unless otherwise specifically stated.

"Apo-2 ligand receptors" include receptors referred to in the art as "DR 4" and "DR 5". Pan et al describe members of the TNF receptor family known as "DR 4" (Pan et al, Science, 276: 111-113 (1997); see also WO98/32856, 1998-07-30, WO 99/37684, 1999-07-29, WO 00/73349, 2000-12-07, US6,433,147, 2002-08-13, US6,461,823, 2002-10-08, and US6,342,383, 2002-01-29). Sheridan et al, Science, 277: 818, 821(1997) and Pan et al, Science, 277: 815-818(1997) describe another receptor for Apo2L/TRAIL (see also WO98/51793, published as 1998-11-19; WO98/41629, published as 1998-09-24). Such receptors are designated DR5 (or the receptor is also designated Apo-2; TRAIL-R, TR6, Tango-63, hAPO8, TRICK2 or KILLER; Screaton et al, curr. biol., 7: 693-containing 696 (1997); Walczak et al, EMBO J., 16: 5386-5387 (1997); Wu et al, Nature Genetics, 17: 141-143 (1997); WO98/35986, 1998-10-14, EP870, 827, 1998-10-22, WO98/46643, 1999-01-21, WO99/02653, 1999-02-25, WO99/09165, 1999-03-11, 2002-11791, 2002-08-13, US 0072091, 1999-2002-12-2002/0098550, 1998-2001-07, 1998-2-68508, 1998-11, US-2001-2-68502-68508, 1998-2001-07 01255540; US 2002/0160446 published 2002-10-31, US 2002/0048785 published 2002-04-25; US6,569,642 issued 2003-05-27, US6,072,047 issued 2000-06-06, US6,642,358 issued 2003-11-04). As noted above, other receptors for Apo-2L include DcR1, DcR2, and OPG. The term "Apo-2L receptor" as used herein includes native sequence receptors and receptor variants. These terms encompass Apo-2L receptors expressed in many mammals, including humans. The Apo-2L receptor may be expressed endogenously, as occurs naturally in the germline of many human tissues, or may be expressed by recombinant or synthetic means. A "native sequence Apo-2L receptor" comprises a polypeptide having the same amino acid sequence as that derived from a native Apo-2L receptor. Thus, a native sequence Apo-2L receptor may have the amino acid sequence of a naturally occurring Apo-2L receptor from any mammal, including a human. Such native sequence Apo-2L receptors may be isolated from nature or may be produced by recombinant or synthetic means. The term "native sequence Apo-2L receptor" specifically includes naturally occurring truncated or secreted forms of the receptor (e.g., comprising, for example, a soluble form of the extracellular domain sequence), naturally occurring variant forms (e.g., other spliced forms), and naturally occurring allelic variants. Receptor variants may include fragments or deletion mutants of the native sequence Apo-2L receptor. FIGS. 25A-C show the 411 amino acid sequence of the human DR5 receptor disclosed in WO98/51793 for 1998-11-19, and its nucleotide sequence (SEQ ID Nos.47 and 48). Transcriptional splice variants of the human DR5 receptor are known in the art. This splice variant encodes the 440 amino acid sequence of the human DR5 receptor shown in FIGS. 26A-C, and its nucleotide sequence (SEQ ID Nos.49 and 50), as disclosed in WO98/35986 of 1998-08-20.

As used herein, "death receptor antibody" refers generally to an antibody to a receptor in the tumor necrosis factor receptor superfamily and comprising a death domain capable of producing an apoptotic signal, such antibodies including the DR5 antibody and the DR4 antibody.

The use of "DR 5 receptor antibody", "DR 5 antibody", or "anti-DR 5 antibody" in a broad sense is intended to refer to an antibody that binds at least one form of the DR5 receptor or extracellular domain thereof. Optionally the DR5 antibody is fused or linked to a heterologous sequence or molecule. Preferably the heterologous sequence allows or facilitates formation of a higher order or oligomeric complex by the antibody. Optionally, the DR5 antibody binds to the DR5 receptor but does not bind or cross-react with any other Apo-2L receptor (e.g., DR4, DcR1, or DcR 2). Optionally the antibody is an agonist of DR5 signaling activity.

Optionally, the DR5 antibodies of the invention bind to the DR5 receptor at a concentration range of about 0.1nM to about 20mM, as measured in a BIAcore binding assay. Optionally, the DR5 antibodies of the invention exhibit IC50 values of about 0.6nM to about 18mM as measured in a BIAcore binding assay.

For purposes of this document only, the term "Apomab" refers to an agonist antibody that binds DR5 and comprises the variable heavy and variable light amino acid sequences of SEQ id nos.55 and 56. Preferably the Apomab comprises the heavy and light chains of SEQ ID nos.51 and 52, respectively.

Production of antibodies

The techniques for producing antibodies that can be formulated according to the invention are as follows.

(i) Antigen screening and preparation

Preferably, the antigen to which the antibody binds is a biologically important glycoprotein and administration of the antibody to a mammal suffering from a disease or disorder can confer a therapeutic benefit to the mammal. However, antibodies directed against non-polypeptide antigens (e.g., tumor-associated glycolipid antigens, see U.S. Pat. No. 5,091,178) are also contemplated.

Where the antigen is a polypeptide, it may be a transmembrane molecule (e.g. receptor) or a ligand such as a growth factor or the like. Examples of antigens include molecules such as renin; growth hormones, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; a lipoprotein; alpha-1-antitrypsin; insulin a-chain; insulin-B chain; a proinsulin; follicle stimulating hormone; a calcitonin; a progestin; glucagon; coagulation factors such as factor VIIIC, factor IX, Tissue Factor (TF) and von willebrands factor; anticoagulant factors such as protein C; atrial natriuretic factor; a pulmonary surfactant; plasminogen activators, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; a hematopoietic growth factor; tumor necrosis factor alpha and beta; enkephalinase; RANTES (regulates normal activation of T cell expression and secretion); human macrophage inflammatory protein (MIP-1-alpha); serum albumin, such as human serum albumin; mullerian tube (Muellian) inhibiting substances; a relaxin a chain; a relaxin B chain; pro-relaxin; mouse chorionic gonadotropin-related peptide; microbial proteins, such as beta lactamases; DNase; IgE; cytotoxic T lymphocyte-associated antigens (CTLA), such as CTLA-4; inhibin (inhibin); activin (activin); vascular Endothelial Growth Factor (VEGF); receptors for hormones or growth factors; protein A or D; rheumatoid factor; neurotrophic factors, such as Bone Derived Neurotrophic Factor (BDNF), neurotrophins-3, -4, -5 or 6(NT-3, NT-4, NT-5 or NT-6) or nerve growth factors, such as NGF-beta; platelet Derived Growth Factor (PDGF); fibroblast growth factors, such as aFGF and bFGF; epidermal Growth Factor (EGF); transforming Growth Factors (TGF), such as TGF-alpha and TGF-beta, including TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, or TGF-beta 5; insulin-like growth factors I and II (IGF-I and IGF-II); des (1-3) -IGF-I (brain IGF-I), insulin-like growth factor binding protein; CD proteins such as CD3, CD4, CD8, CD19, and CD 20; erythropoietin; osteoinductive factor (osteoinductive factor); an immunotoxin; bone morphogenetic (morphogenic) proteins (BMP); interferons, such as interferon- α, - β, and γ; colony Stimulating Factors (CSF), such as M-CSF, GM-CSF, and G-CSF; interleukins (IL), such as IL-1 through IL-10; superoxide dismutase; a T cell receptor; surface membrane proteins; decay (decay) acceleration factor; viral antigens, such as part of the AIDS envelope; a transporter protein; a homing receptor; an adhesin; a regulatory protein; integrins such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM; a tumor associated antigen, such as HER2, HER3, or HER4 receptor; and fragments of any of the above polypeptides.

Exemplary molecular targets of the antibodies encompassed by the invention include CD proteins such as CD3, CD4, CD8, CD19, CD20, CD22, CD34, and CD 40; a member of the ErbB receptor family such as the EGF receptor, HER2, HER3 or HER4 receptor; b cell surface antigens, such as CD20 or BR 3; members of the tumor necrosis receptor superfamily, including DR 5; prostate Stem Cell Antigen (PSCA); cell adhesion molecules such as LFA-1, Macl, p150.95, VLA-4, ICAM-1, VCAM, α 4/β 7 integrin, and α v/β 3 integrin, including their α or β subunits (e.g., anti-CD 11 a; anti-CD 18 or anti-CD 11b antibodies); growth factors such as VEGF and its receptors; tissue Factor (TF); tumor Necrosis Factor (TNF) such as TNF- α or TNF- β, interferon- α (IFN- α); interleukins, such as IL-8; IgE; a blood group antigen; flk2/flt3 receptor; obesity (OB) receptors; an mpl receptor; CTLA-4; protein C, and the like.

Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens for antibody production. For transmembrane molecules, such as receptors, fragments of these (e.g., the extracellular domain of the receptor) can be used as immunogens. Alternatively, cells expressing transmembrane molecules can be used as immunogens. Such cells may be from natural sources (e.g., cancer cell lines) or may be cells transformed by recombinant techniques to express the transmembrane molecule. Other antigens and their various forms that can be used to make antibodies will be apparent to those skilled in the art.

To produce HER2 antibody, the HER2 antigen for its production may be, for example, a soluble form of the extracellular domain of HER2 or a portion thereof, which comprises the desired epitope. Alternatively, a cell expressing HER2 on its cell surface (e.g., a transformed NIH-3T3 cell overexpressing HER 2; or a cancer cell line such as SK-BR-3 cell, see Stancovski et al PNAS (USA) 88: 8691-8695(1991)) can be used to generate antibodies.

(ii) Monoclonal antibodies

Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants that may be generated during monoclonal antibody production. Thus, the modifier "monoclonal" indicates that the antibody is not characteristic of a different antibody composition.

For example, Kohler, nature 256: 495(1975) or monoclonal antibodies can be prepared by recombinant DNA methods (U.S. patent No. 4816567).

In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to proteins used in the immunization. Alternatively, lymphocytes may be immunized in vitro and then fused with myeloma cells using an appropriate fusing agent such as polyethylene glycol to form hybridoma cells (Goding, monoclonal antibody: principles and applications, pp.59-103(Academic Press, 1986)).

The hybridoma cells so prepared are seeded and cultured in a suitable culture medium that preferably includes one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

Preferred myeloma cells are those which fuse efficiently, support stable production of high levels of antibodies by the selected antibody-producing cells, and are sensitive to HAT or the like medium. Among the preferred myeloma Cell lines are murine myeloma Cell lines, such as those derived from MOPC-21 and MPC-11 mouse tumors (from the Salk Institute Cell Distribution Center, san Diego, Calif., USA) and SP-2 or X63-Ag8-653 cells (American type culture Collection, Rockville, Md., USA). Human hybridoma cells and mouse-human heterogeneous hybridoma cell lines have also been reported for the production of human monoclonal antibodies (Kozbor, J. Immunol, 133: 3001 (1984); Brodeur et al, monoclonal antibody production techniques and applications, pp 51-63 (Marcel Dekker, Inc., New York, 1987)).

Determining the production of monoclonal antibodies to said antigen in hybridoma cell growth medium. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of a monoclonal antibody can be determined, for example, by Munson et al, anal. 220(1980) by Scatchard analysis.

After hybridoma cells producing antibodies with the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution methods and cultured by standard methods (Goding, monoclonal antibodies: principles and applications, pp.59-103, Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can grow in animals to form ascites tumors.

Monoclonal antibodies secreted by the subclones are suitably isolated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification methods, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

DNA encoding a monoclonal antibody can be readily isolated and sequenced by conventional methods (e.g., using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the monoclonal antibody). The hybridoma cells may serve as a preferred source of such DNA. Once the DNA has been isolated, it is ligated into an expression vector, which is then transformed into a host cell, such as an E.coli cell, simian COS cell, Chinese Hamster Ovary (CHO) cell, or myeloma cell that does not produce immunoglobulin protein, to synthesize monoclonal antibodies in the recombinant host cell. A review of DNA for recombinant expression of antibody-encoding DNA in bacteria includes Skerra et al, curr. 256-charge 262(1993) and Pl ü ckthun, Immunol. Revs., 130: 151-188(1992).

In another embodiment, monoclonal antibodies or antibody fragments may be isolated from antibody phage libraries used in McCafferty et al, nature 348: 552 and 554. Clackson et al, Nature 352: 624-: 581-597(1991) describes the isolation of murine and human antibodies using phage libraries, respectively. The subsequent literature describes the generation of high affinity (nM range) human antibodies as a strategy for constructing large phage libraries by chain shuffling (Marks et al, Bio/technology, 10: 779-2263 (1992)) and combinatorial infection and in vivo recombination (Waterhouse et al, nucleic acid research, 21: 2265-2266 (1993)). Thus, these techniques are alternatives to conventional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

The DNA may also be modified, for example, by replacing the murine homologous sequences with sequences encoding the human heavy and light chain constant regions (U.S. Pat. No. 4816567; Morrison et al, Proc. Natl. Acad. Sci. USA 81: 6851(1984)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the coding sequence for an immunoglobulin.

Typically, such immunoglobulin polypeptides have been produced as chimeric bivalent antibodies, which include one antigen binding site specific for one antigen, and another antigen binding site having specificity for a different antigen, in place of the constant region of an antibody, or in place of the variable region of one antigen binding site of an antibody.

(iii) Humanized antibodies

Methods for humanizing non-human antibodies have been described in the art. Preferably, the humanized antibody has one or more amino acid residues of non-human origin introduced. These non-human amino acid residues are often "import" residues, which are typically derived from "import" variable regions. Humanization can be substantially performed by replacing murine CDR or CDR sequences with those of the human antibody according to the method of Winter and coworkers (Jones et al, Nature, 321: 522-525 (1986); Riechmann et al, Nature 332: 323-327 (1988); Verhoeyen et al, science 239: 1534-1536 (1988)). Thus, such "humanized" antibodies are chimeric antibodies (U.S. patent No. 4816567) in which substantially less than the entire human variable region is replaced by a corresponding sequence derived from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and some FR residues are replaced by residues from analogous sites in the odontoblastic antibody.

The choice of human heavy and light chain variable regions for use in making humanized antibodies is important for reducing antigenicity. According to the so-called "best-fit" approach, a complete library of known human variable region sequences can be screened for the variable region sequences of rodent antibodies. The human sequence most closely related to the rodent sequence is then used as the Framework (FR) for the humanized antibody (Sims et al, J. Immunol. 151: 2296 (1993); Chothia et al, J. molecular biology. 196: 901 (1987)). Another approach employs specific frameworks derived from the common sequence of all human antibodies of a specific subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci. USA 89: 4285 (1992); Presta et al, J. Immunol., 151: 2623 (1993)).

It is even more important that the antibodies are humanized and retain high affinity for the antigen and other good biological activity. To achieve this, according to a preferred method, the parental sequences and various conceptual humanized products are analyzed using three-dimensional models of the parental and humanized sequences, by which method humanized antibodies are prepared. Three-dimensional immunoglobulin models are publicly available and well known to those skilled in the art. Computer programs have been developed which allow the specification and display of the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Observation of these indications allows analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind antigen. In this way, FR residues can be screened from the receptor and combined with important sequences to obtain desired antibody properties, such as enhanced affinity for the target antigen. In general, CDR residues are directly and predominantly involved in the effect on antigen binding.

WO 01/00245 describes the production of an exemplary humanized HER2 antibody that binds to HER2 and suppresses ligand activation of the HER receptor. The humanized antibodies of particular interest herein are substantially as effective as murine monoclonal antibody 2C4 (or a Fab fragment thereof) in blocking EGF, TGF- α and/or HRG mediated activation of MAPK, and/or as effective as murine monoclonal antibody 2C4 (or a Fab fragment thereof) in binding HER 2. The humanized antibodies herein may, for example, comprise non-human hypervariable region residues incorporated into a human variable heavy domain and may further comprise a Framework Region (FR) substitution at a position selected from 69H, 71H and 73H, using the variable domain numbering system given in Kabat et al, Sequence of Protein of immunological Interest, 5th ed. In one embodiment, the humanized antibody comprises FR substitutions at two or all of positions 69H, 71H and 73H.

The exemplary humanized antibodies of interest herein comprise the variable heavy domain complementarity determining residue GFTFTDYTMX, wherein X is preferably D or S (SEQ ID No. 7); DVNPNSGGSIYNQRFKG (SEQ ID No. 8); and/or NLGPSFYFDY (SEQ ID No.9), optionally comprising amino acid modifications of those CDR residues, e.g., wherein the modifications substantially retain or increase the affinity of the antibody. For example, the antibody variant of interest may have from about one to about seven or about five amino acid substitutions in the above variable heavy CDR sequences. Such antibody variants can be prepared, for example, by affinity maturation as described below. Most preferred humanized antibodies comprise the variable heavy domain amino acid sequence of SEQ ID No. 4.

In addition to those variable heavy domain CDR residues in the preceding paragraph, the humanized antibody may comprise, for example, variable light domain complementarity determining residue KASQDVSIGVA (SEQ ID No. 10); SASYXXX, wherein X at position 5 is preferably R or L, wherein X at position 6 is preferably Y or E and X at position 7 is preferably T or S (SEQ ID No. 11); and/or QQYYIYPYT (SEQ ID No. 12). Such humanized antibodies optionally comprise amino acid modifications of the above CDR residues, e.g., wherein the modifications substantially maintain or enhance the affinity of the antibody. For example, the antibody variant of interest may have from about one to about seven or about five amino acid substitutions in the above variable light CDR sequences. Such antibody variants can be prepared, for example, by affinity maturation as described below. Most preferred humanized antibodies comprise the variable light domain amino acid sequence of SEQ ID No. 3.

The application also relates to affinity matured antibodies that bind to HER2 and suppress ligand activation of the HER receptor. The parent antibody may be a human or humanized antibody, e.g., an antibody comprising variable light and/or heavy sequences of SEQ ID nos.3 and 4, respectively (i.e., variant 574). The affinity matured antibody preferably binds to the HER2 receptor with an affinity that is better than murine 2C4 or variant 574 (e.g., from about two-fold or about four-fold, to about 100-fold or about 1000-fold enhanced affinity, e.g., as assessed using HER 2-extracellular domain (ECD) ELISA). Exemplary substituted variable heavy CDR residues include H28, H30, H34, H35, H64, H96, H99, or a combination of two or more (e.g., two, three, four, five, six, or seven of these residues). Examples of variable light CDR residues that are altered include L28, L50, L53, L56, L91, L92, L93, L94, L96, L97, or combinations of two or more (e.g., two, three, four, five, up to about ten of these residues).

Various forms of humanized or affinity matured antibodies are claimed. For example, the humanized or affinity matured antibody may be an antibody fragment, such as a Fab, which is optionally conjugated to one or more cytotoxic agents to produce an immunoconjugate. Alternatively, the humanized antibody or affinity matured antibody may be a full length antibody, such as a full length IgG1 antibody.

(iv) Human antibodies

In addition to humanization, human antibodies can be prepared. For example, transgenic animals (e.g., mice) have now been generated that, upon immunization, produce all of the components of human antibodies without the production of endogenous immunoglobulins. For example, chimeric and germline (germ-line) mutant mice have been reported to have a heavy chain joining region (J) in the antibody_H) Homozygous deletion of the gene results in complete suppression of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays into such germline mutant mice will result in the production of human antibodies as a result of antigen challenge. See, e.g., Jakobovits et al, proceedings of the national academy of sciences USA, 90: 2551 (1993); jakobovits et al, Nature, 362: 255-258 (1993); bruggermann et alYear in immune.7: 33 (1993); and us patent nos. 5591669, 5589369 and 5545807.

Alternatively, phage display techniques (McCafferty et al, Nature 348: 552-553(1990)) can be used to generate human antibodies and antibody fragments in vitro from all components of immunoglobulin variable (V) region genes from unimmunized donors. According to this technique, antibody V region genes are cloned in the same framework as the major or minor capsid protein genes of filamentous bacteriophage (e.g., M13 or fd) and displayed as functional antibody fragments on the surface of the phage particle. Since the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional characteristics of the antibody also results in selection of the gene encoding the antibody exhibiting these properties. Thus, the phage mimics some of the characteristics of the B cell. Phage display can be performed in a variety of formats; for a review, see, e.g., Johnson, Kevin s. and Chiswell, David j., recent Opinion in Structural Biology (Current Opinion in Structural Biology) 3: 564-571(1993). Phage display can be performed using several sources of V gene fragments. Clackson et al, Nature, 352: 624-628(1991) A diverse array of anti-oxazolone antibodies was isolated from a small random combinatorial library of spleen-derived V genes from immunized mice. Basically, the molecular biology journal 222: 581-597(1991), or Griffith et al, EMBOJ.12: 725-734(1993) construct all the components of the V gene of an unimmunized human donor and isolate antibodies against an antigen diversity array (including self-antigens). See also U.S. patent nos. 5565332 and 5573905.

As described above, human antibodies can also be produced by activated B cells in vitro (see U.S. Pat. nos. 5,567,610 and 5,229,275).

Human anti-HER 2 antibodies are described in U.S. Pat. No. 5772997, published at 30/6/1998 and WO97/00271, published at 3/1/1997.

(v) Antibody fragments

Various techniques for generating antibody fragments have been developed. Traditionally, these fragments are eliminated by proteolysis of intact antibodiesChemosynthesis (see Morimoto et al, Journal of Biochemical and Biophysical Methods 24: 107-117(1992)) and Brennan et al, science 229: 81(1985)). But these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab '-SH fragments can be recovered directly from E.coli and chemically ligated to form F (ab')₂Fragments (Carter et al, Bio/technology 10: 163-167 (1992)). According to another method, F (ab') can be isolated directly from recombinant host cell culture₂And (3) fragment. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; us patent 5,571,894; and U.S. patent 5,587,458. The antibody fragment may also be a "linearized antibody," as described in U.S. Pat. No. 5,641,870. Such linearized antibody fragments may be monospecific or bispecific.

(vi) Bispecific antibodies

Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of the HER2 protein. Other such antibodies may combine the HER2 binding site with a binding site for EGFR, HER3, and/or HER 4. Alternatively, the anti-HER 2 arm may be bound to an arm that binds to a trigger molecule on a leukocyte, such as a T cell receptor molecule (e.g., CD2 or CD3), or an IgG Fc receptor (fcyr) such as fcyri (CD64), fcyrii (CD32), and fcyriii (CD16), thereby focusing on the cellular defense mechanisms of cells expressing HER 2. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing HER 2. These antibodies have a HER2 binding arm and an arm that binds a cytotoxic agent (e.g. saporin, anti-INF-alpha, vinca alkaloid, ricin a chain, methotrexate or radioisotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F (ab')₂Bispecific antibodies).

WO 96/16673 describes bispecific HER 2/FcyRIII antibodies, while in U.S. Pat. No. 5837234 the bispecific ErbB 2/FcyRI antibody IDM1(Osidem) is disclosed. Bispecific HER2/Fc α antibodies are described in WO 98/02463. Bispecific HER2/CD3 antibodies are taught in U.S. patent 5821337. MDX-210 is a bispecific HER2-Fc γ RIII Ab.

Methods of making bispecific antibodies are known in the art. The traditional approach to the preparation of complete bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al, Nature 305: 537-539 (1983)). Due to the random distribution of immunoglobulin heavy chain light chains, these hybridomas (cell hybridomas (quadroma)) may produce a mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule, which is usually performed by an affinity chromatography step, is very complicated and yields are low. Similar methods are described in WO93/08829 and Traunecker et al, EMBO J, 10: 3655-3659(1991).

According to different methods, the variable region of an antibody with the desired binding specificity (antibody-antigen binding site) is fused to an immunoglobulin constant region sequence. The fusion is preferably to an immunoglobulin heavy chain constant region comprising at least a portion of the hinge region, the CH2 and CH3 regions. Preferably, the first heavy chain constant region (CH1) containing the site required for light chain binding is present in at least one fusion. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain can be inserted into different expression vectors and co-transfected into an appropriate host organism. This allows, in embodiments constructed using unequal ratios of the three polypeptide chains, greater flexibility in adjusting the mutual ratios of the three polypeptide fragments to achieve optimal yields. However, it is also possible to insert the coding sequences for two or all three polypeptide chains into one expression vector when at least two polypeptide chains are expressed in equal proportions to achieve high yields or when said proportions are of no particular significance.

In a preferred embodiment of this method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure has been found to be advantageous for the separation of the desired bispecific compound from a mixture of non-essential immunoglobulin chains, since the presence of immunoglobulin light chains on only half of the bispecific molecule makes the separation easier. This method is disclosed in WO 94/04690. For further details of the preparation of bispecific antibodies, see Suresh et al, methods enzymology, 121: 210(1986).

According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers obtained from recombinant cell culture. Preferred interfaces include at least a portion of the antibody constant region CH3 domain. In this method, one or more small side chains of amino acids derived from the interface of the first antibody molecule are replaced by larger side chains (e.g., tyrosine or tryptophan). Complementary "grooves" of the same or similar size to the large side chains can be formed at the interface of the second antibody molecule by substituting the large side chain of an amino acid with a small side chain (e.g., alanine or threonine). This results in higher yields of heterodimers than the undesired end products like homodimers.

Bispecific antibodies include cross-linked antibodies or "heteroconjugated" antibodies. For example, one of the antibodies in the heteroconjugate can be conjugated to avidin and the other antibody conjugated to biotin. It is thought that such antibodies may be used to target immune cells to unwanted cells (U.S. Pat. No. 4676980), and may also be used to treat HIV infection (WO91/00360, WO92/200373, EP 03089). Heteroconjugate antibodies can be prepared by any suitable cross-linking method. Suitable crosslinking formulations and various crosslinking techniques are known in the art and are disclosed in U.S. Pat. No. 4676980.

Techniques for making bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical ligation. Brennan et al, science 229: 81(1985) the proteolytic preparation of F (ab')₂A method for fragmenting. These fragments are complexed with dimercapto as arsenicSodium is reduced in the presence of sodium to stabilize adjacent sulfhydryl groups and prevent intermolecular disulfide bond formation. The resulting Fab' fragments are converted to thio-nitrobenzoate (TNB) derivatives. One Fab ' -TNB derivative is reduced into Fab ' -thiol by mercaptoethylamine, and then mixed with other Fab ' -TNB derivatives with equal molecular weight to form the dual-specific antibody. The bispecific antibody thus produced can be used as a reagent for selective immobilization of enzymes.

Recent advances have facilitated the direct recovery of Fab' -SH fragments from E.coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al, journal of experimental medicine, 175: 217-225(1992) describes fully humanized bispecific antibodies F (ab')₂The generation of molecules. Each Fab' fragment was separately secreted from E.coli and directly chemically coupled in vitro to form bispecific antibodies. The bispecific antibody prepared in the way can be combined with cells which excessively express HER2 receptor and normal human T cells, and can also trigger the lytic activity of human cytotoxic lymphocytes on human breast tumor cells. Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies can be made with leucine zippers. Kostelny et al, J Immunol, 148 (5): 1547-1553(1992)). Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers are reduced to monomers in the hinge region and then re-oxidized to form antibody heterodimers. The method can also be used to prepare antibody homodimers. By Hollinger et al, proceedings of the national academy of sciences USA, 90: 6444- > 6448(1993)) provides an alternative method for the preparation of bispecific antibody fragments. The fragment contains heavy chain variable region (V) _H) Which is linked to the light chain variable region (V) via a linker_L) Connected, the linker is very short, making pairing between the two domains of the same strand impossible. Thus, V on the same fragment_HAnd V_LThe domains are forced to complement V on the other fragment_LAnd V_HThe domains pair, thereby forming two antigen binding sites. Another report has been madeStrategy for the preparation of bispecific antibodies using single chain fv (sFv) dimers. See Gruber et al, journal of immunology, 152: 5368(1994).

Antibodies of more than two valencies are contemplated. For example, a trispecific antibody can be prepared. Tutt et al, journal of immunology, 147: 60(1991).

(vii) Other amino acid sequence modifications

Amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological characteristics of an antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate nucleotide changes in the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired properties. The amino acid changes can also alter post-translational processing of the antibody, for example, changing the number or position of glycosylation sites.

One useful method of identifying certain residues or regions of an antibody at preferred positions for mutagenesis is Cunningham and Wells, science 244: 1081-1085(1989) described in "alanine scanning mutagenesis". Here, a residue or group of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine and glutamic acid) are identified and substituted with neutral or negatively charged amino acids (most preferably alanine or polyalanine) in order to affect the interaction of the amino acid with the antigen. Those amino acid positions that demonstrate functional sensitivity to substitution are improved by introducing further or additional variants at the point of substitution. Thus, while the site of introduction of an amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the effect of a mutation at a given site, alanine scanning or random mutagenesis is performed at the target codon or region and the expressed antibody variants are screened for the desired activity.

Amino acid sequence insertions include amino-and/or carboxy-terminal fusions ranging in length from one residue to polypeptides comprising 100 or more residues, as well as insertions of single or multiple amino acid residues within the sequence. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or such an antibody fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include the fusion of the N-or C-terminus of the antibody to an enzyme or polypeptide that increases the serum half-life of the antibody.

Another class of variants are amino acid substitution variants. These variants have at least one amino acid residue in the antibody molecule substituted with a different residue. Sites of greatest interest for substitution mutagenesis include hypervariable regions, and changes in FR can also be considered. Conservative substitutions are shown in table 1 under the heading "preferred substitutions". If these substitutions result in a change in biological activity, more substantial changes from the "substitution examples" column in Table 1, or further more substantial changes described in the amino acid classification below, can be introduced and the products screened.

TABLE 1

Starting residue	Exemplary substitutions	Preferred substitutions
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Asp，Lys；Arg	Gln
Asp(D)	Glu；Asn	Glu
			Cys(C)	Ser；Ala	Ser
Gln(Q)	Asn；Glu	Asn
			Glu(E)	Asp；Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu; val; met; ala; phe; norleucine	Leu
			Leu(L)	Norleucine; ile; val; met; ala; phe (Phe)	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Trp；Leu；Val；Ile；Ala；Tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Val；Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile; leu; met; phe; ala; norleucine	Leu

Substantial modification of antibody biological properties is achieved by selecting substitutions that differ significantly in their effect of maintaining (a) the polypeptide backbone structure of the substitution region, e.g., as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the side chain. Amino acids can be grouped according to the similarity of their side chain properties (in A.L. Lehninger, in Biochemistry, second ed., pp.73-75, Worth Publishers, New York (1975)):

(1) Non-polar: ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M)

(2) Uncharged polarity: gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q)

(3) Acidity: asp (D), Glu (E)

(4) Alkalinity: lys (K), Arg (R), His (H)

Alternatively, naturally occurring residues may be divided into groups based on common side chain properties:

(1) hydrophobic type: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic type: cys, ser, thr;

(3) acidity: asp, glu;

(4) alkalinity: asn, gln, his, lys, arg;

(5) chain orientation affecting residues: gly, pro; and

(6) an aromatic group: trp, tyr, phe.

Non-conservative substitutions will define the replacement of a member of one of the above classes by another class.

Any cysteine residues not involved in maintaining the correct conformation of the antibody may also be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine linkages may be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment such as an FV fragment).

A particularly preferred type of substitution variant comprises substitution of one or more residues of a hypervariable region of the parent antibody. Typically, the variant selected for further development should have improved biological activity relative to its parent antibody. One convenient way to generate such substitution variants is to use affinity maturation by phage display. Briefly, several sites (e.g., 6-7 sites) of a hypervariable region are mutated to generate all possible amino acid substitutions at each site. The antibody variants so produced are displayed in monovalent form on filamentous phage particles as fusions to the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for biological activity (e.g., binding affinity) as described herein. To identify alternative hypervariable region modification sites, residues of a hypervariable region which contribute predominantly to antigen binding can be identified by alanine scanning mutagenesis. Furthermore, it is also advantageous to analyze the crystal structure of the antigen-antibody complex to determine the contact points between the antigen and the antibody. These contact residues and their adjacent residues are candidates for substitution according to the techniques described herein. Once such variants are generated, they are all screened as described herein, and antibodies with advantageous properties in one or more relevant experiments are selected for further development.

Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. The alteration is the removal of one or more carbohydrate moieties from the antibody and/or the addition of one or more glycosylation sites not otherwise present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to linking a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of a carbohydrate moiety to an asparagine side chain. Thus, the presence of any of the above tripeptide sequences in a polypeptide may create potential glycosylation sites. O-linked glycosylation refers to the attachment of N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, primarily serine, threonine, but 5-hydroxyproline and 5-hydroxylysine may also be used.

The addition of glycosylation sites to the antibody can be achieved by altering the amino acid sequence to include one or more of the above-described tripeptide sequences (in the case of the addition of an N-linked glycosylation site). Such changes may also be achieved by adding or substituting one or more serine or threonine residues in the sequence of the original antibody (in the case of addition of an O-linkage).

When the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. For example, antibodies having a mature sugar structure lacking trehalose attached to the Fc region of the antibody are described in U.S. patent application No. US 2003/0157108a1, Presta, L. See also US2004/0093621a1(Kyowa Hakko Kogyo co., Ltd). Antibodies having bisecting N-acetylglucosamine (GlcNAc) in the sugar attached to the Fc region of the antibody are mentioned in WO03/011878, Jean-Mairet et al and U.S. Pat. No. 6,602,684, Umana et al. Antibodies having at least one galactose residue in an oligosaccharide attached to the Fc region of an antibody are reported in WO97/30087, Patel et al. See also, WO98/58964(Raju, S.) and WO99/22764(Raju, S.), which relate to antibodies having varying sugars attached to their Fc regions.

Nucleic acid molecules encoding amino acid sequence variants of antibodies are prepared by various methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of natural amino acid sequence variants), or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or non-variant form of the anti-ErbB 2 antibody.

(viii) Screening for antibodies with desired Properties

Techniques for generating antibodies have been described above. Antibodies having certain biological properties may be further selected as desired.

To identify an antibody that inhibits ligand activation of a HER receptor, the antibody can be assayed for its ability to suppress binding of the HER ligand to a cell expressing the HER receptor (e.g., to bind to another HER receptor that forms a HER hetero-oligomer with the HER receptor of interest). For example, cells that naturally express, or that after transfection express HER receptor of HER hetero-oligomers may be incubated with an antibody and subsequently exposed to a labeled HER ligand. The HER2 antibody can then be evaluated for its ability to inhibit ligand binding to HER receptors in HER heteromers.

For example, inhibition of HRG binding to MCF7 breast tumor cell line by HER2 antibody can be performed using a monolayer on ice MCF7 culture in 24-well plates essentially as described in WO 01/00245. The HER2 monoclonal antibody can be added to each well and incubated for 30 minutes. May subsequently be added¹²⁵I-labelled rHRG beta 1_177-224(25pm), the incubation may be continued for 4 to 16 hours. Dose response curves can be prepared and IC calculated for the target antibody₅₀The value is obtained. In one embodiment, an antibody that suppresses ligand activation of a HER receptor will have an IC of about 50nM or less, more preferably 10nM or less, in such an assay ₅₀To inhibit HRG binding to MCF7 cells. Wherein the antibody is an antibody fragment such as a Fab fragment, inhibiting HRG binding to IC of MCF7 cells in such an assay₅₀May be, for example, about 100nM or less, more preferably 50nM or less.

Alternatively, or in addition, the ability of the HER2 antibody to suppress HER ligand-stimulated tyrosine phosphorylation of a HER receptor present in a HER heteromultimer can be assessed. For example, cells endogenously expressing HER receptors or expressing them after transfection can be incubated with antibodies and subsequently assayed for HER ligand-dependent tyrosine phosphorylation activity using an anti-phosphotyrosine monoclonal, which is optionally conjugated with a detectable label. HER receptor activation and suppression of this activity by antibodies can also be determined using the kinase receptor activation assay described in U.S. patent No. 5,766,863.

In one embodiment, antibodies can be screened for that inhibit HRG stimulation of p180 tyrosine phosphorylation in MCF7 cells substantially as described in WO 01/00245. For example,MCF7 cells can be seeded in 24-well plates and monoclonal antibodies against HER2 can be added to each well and incubated for 30 minutes at room temperature; rHRG beta 1 may then be added _177-244Added to each tube to a final concentration of 0.2nM and incubation may be continued for 8 minutes. The medium was aspirated from each well and the reaction was stopped by adding 100. mu.l of SDS sample buffer (5% SDS, 25mM DTT, and 25mM Tris-HCl, pH 6.8). Each sample (25. mu.l) can be electrophoresed on a 4-12% gradient gel (Novex) and subsequently electrophoresed onto a polyvinylidene fluoride membrane. Anti-phosphotyrosine (at 1. mu.g/ml) immunoblots can be developed and M can be quantified by reflection densitometry_rThe predominance over 180,000 reflects the intensity of the band. The selected antibodies will preferably significantly inhibit HRG stimulation of p180 tyrosine phosphorylation to approximately 0-35% of the control in this assay. HRG-stimulated inhibitory dose response curves for p180 tyrosine phosphorylation as measured by reflection densitometry can be prepared and IC's for the target antibodies can be calculated₅₀. In one embodiment, an antibody that suppresses ligand activation of a HER receptor will have an IC of about 50nM or less, more preferably 10nM or less₅₀To inhibit HRG stimulation of p180 tyrosine phosphorylation in this assay. When the antibody is an antibody fragment, such as a Fab fragment, IC₅₀About 100nM or less, more preferably 100nM or less, in order to inhibit HRG stimulation of p180 tyrosine phosphorylation in this assay.

The growth inhibitory effect of the antibodies on MDA-MB-175 cells may also be assessed, for example, substantially as described in Schaefer et al Oncogene 15: 1385-1394 (1997). According to this assay, MDA-MB-175 cells may be treated with HER2 monoclonal antibody (10. mu.g/mL) for 4 days and stained with crystal violet. Incubation with HER2 antibody may show growth inhibitory effects on this cell line, similar to that demonstrated for monoclonal antibody 2C 4. In another embodiment, exogenous HRG will not significantly reverse this inhibition. Preferably, the antibody will be capable of inhibiting cell proliferation of MDA-MB-175 cells to a greater extent than monoclonal antibody 4D5 (optionally to a greater extent than monoclonal antibody 7F 3) in the presence and absence of exogenous HRG.

In one embodiment, the HER2 antibody of interest may suppress the heregulin-dependent association of HER2 with HER3 in MCF7 and SK-BR-3 cells as determined in a co-immunoprecipitation experiment as described in WO01/00245, which suppression is substantially more effective than monoclonal antibody 4D5, and preferably substantially more effective than monoclonal antibody 7F 3.

To identify growth inhibitory HER2 antibodies, antibodies can be screened that inhibit the growth of cancer cells that overexpress HER 2. In one embodiment, the growth inhibitory antibody is selected to be capable of inhibiting the growth of SK-BR-3 cells in cell culture by about 20-100% and preferably by about 50-100%, with an antibody concentration of about 0.5-30 μ g/ml. To identify such antibodies, an SK-BR-3 assay as described in U.S. Pat. No. 5,677,171 can be performed. According to this assay, SK-BR-3 cells were grown in a 1:1 mixture of F12 and DMEM medium supplemented with 10% fetal bovine serum, glutamate and penicillin streptomycin. SK-BR-3 cells were seeded at 20,000 cells in 35mm cell culture dishes (2mls/35mm dish). HER2 antibody was added at 0.5-30. mu.g/ml per dish. Six days later, the electronic COULTER is utilized ^TMThe cell counter compares the number of cells to the number of untreated cells. Those antibodies that inhibit the growth of SK-BR-3 cells by about 20-100% or by about 50-100% can be selected as growth inhibitory antibodies. Assays for screening growth inhibitory antibodies, such as 4D5 and 3E8, are described in U.S. patent No. 5,677,171.

To select HER2 antibodies that induce apoptosis, annexin binding assays can be performed using BT474 cells. BT474 cells were cultured and plated on petri dishes as described in the previous paragraph. The medium was then removed and replaced with fresh medium alone or medium containing 10 μ g/ml monoclonal antibody. After a three-day incubation period, the monolayers were washed with PBS and detached by trypsinization. The cells were subsequently centrifuged and resuspended in Ca²⁺Combined in buffer and aliquoted into tubes as discussed above for the cell death assay. The tubes then receive labeled annexin (e.g., annexin)White V-FTIC) (1. mu.g/ml). Can utilize FACSCAN^TMFlow cytometer and FACSCVERT^TMCellQuest software (Becton Dickinson) was used to analyze the samples. Those antibodies that induced a statistically significant level of annexin binding relative to the control were selected as apoptosis-inducing antibodies. In addition to the annexin binding assay, a DNA staining assay using BT474 cells can also be utilized. To perform this assay, BT474 cells treated with the antibody of interest as described in the previous two paragraphs were incubated with 9. mu.g/ml HOECHST33342 ^TMIncubating at 37 deg.C for 2hr, and then using MODFIT LT^TMSoftware (Verity software House) at EPICS ELITE^TMAnalysis was performed on a flow cytometer (Coulter Corporation). Using this assay, antibodies can be selected as pro-apoptotic antibodies that induce a 2-fold or greater (preferably 3-fold or greater) change in the percentage of apoptotic cells than would be the case with untreated cells (up to 100% apoptotic cells). Screening for HER2 antibodies that induce apoptosis, such as assays for 7C2 and 7F3, is described in WO 98/17797.

To screen for antibodies to an epitope on HER2 that is bound by an Antibody of interest, a conventional cross-suppression assay can be performed, as described in Antibody, a Laboratory Manual, Cold Spring harbor Laboratory, Ed harbor and David Lane (1988), to assess whether an Antibody cross-suppresses an Antibody, such as 2C4 or Pertuzumab, binding to HER 2. Alternatively, or in addition, epitope mapping may be performed by methods known in the art and/or the antibody-HER 2 structure (Franklin et al Cancer Cell 5: 317-328(2004)) may be studied to see what domain of HER2 is bound by the antibody.

(ix) Immunoconjugates

The invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic drug such as a chemotherapeutic drug, a toxin (e.g., a small molecule toxin or an enzymatically active toxin of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof), or a radioisotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in preparing such immunoconjugates are as described above. Also contemplated herein are conjugates of an antibody and one or more small molecule toxins, such as calicheamicin, maytansine (U.S. Pat. No. 5,208,020), trichothecene (trichothene), CC 1065.

In a preferred embodiment of the invention, the antibody is conjugated to one or more maytansine molecules (e.g., about 1 to about 10 maytansine molecules per antibody molecule). Maytansinoid can be converted to May-SS-Me and then reduced to May-SH3 and reacted with modified antibodies (Chari et al, cancer Res. 52: 127-131(1992)) to produce maytansinoid-antibody conjugates.

Another immunoconjugate of interest comprises an HER2 antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-pM concentration levels. Calicheamicin structural analogs that can be used include, but are not limited to gamma₁ ¹、α₂ ¹、α₃ ¹N-acetyl-gamma₁ ¹PSAG and θ¹ ₁(Hinmam et al, cancer research 53: 3336-3342(1993) and Lode et al, cancer research 58: 2925-2928 (1998)). See also U.S. Pat. nos. 5,714,586; 5,712,374; 5,264,586 and 5,773,001, which are incorporated herein by reference.

Enzymatically active toxins and fragments thereof that may be used include: diphtheria toxin a chain, non-binding active fragments of diphtheria toxin, exotoxin a chain (from pseudomonas aeruginosa), ricin a chain, abrin a chain, modeccin a chain, alpha-sarcin, aleurites fordii protein, dianthin protein, phytolacca americana protein (PAPI, PAPII, PAP-S), momordica charantia (momordia) inhibitor, curcin, crotin, saponaria officinalis (sapaonaria officinalis) inhibitor, gelonin, mitomycin (mitogellin), restrictocin, phenomycin, enomycin, and trichothecenes (tricothecenes). See, for example, WO93/21232, published on month 10 and 28, 1993.

The invention also relates to an immunoconjugate formed between an antibody and a compound having nucleolytic activity (such as a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

A variety of radioisotopes are useful in preparing radioconjugated antibodies, examples include At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²And radioactive isotopes of Lu.

Conjugates of the antibody and cytotoxic agent may be attached by a variety of bifunctional protein coupling agents such as: n-succinimidyl-3- (2-pyridyldimercapto) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, Iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl imidoadipate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as tolylene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene). For example, a ricin immunotoxin may be identified as vietta et al, science 238: 1098 (1987). C14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminopentaacetic acid ester (MX-DTPA) is an exemplary coupling agent for coupling radionucleotide to an antibody. See WO 94/11026. Such a linker may be a "breakable linker" which facilitates release of the cytotoxic drug within the cell. For example, acid labile linkers, peptidase-sensitive linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, cancer Res. 52: 127- "131 (1992)).

In another embodiment, the antibody may be conjugated to a "receptor" (e.g., streptavidin) for use in tumor pretargeting, and the antibody-receptor conjugate administered to the patient, followed by removal of unbound conjugate from circulation with a clearing agent, and administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionucleotide).

(x) Other antibody modifications

Additional modifications to the antibodies are contemplated herein. For example, the antibody may be crosslinked with a non-protein polymer, such as polyethylene glycol, polypropylene glycol, polyoxyalkylene (polyoxyalkylene), or a copolymer of polyethylene glycol and polypropylene glycol. Antibodies can also be coated in microcapsules prepared by coacervation techniques or interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly- (methylmethacylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin spheroids, microemulsions, nanoparticles, and nanocapsules), or macroemulsions (macroemulsions) as disclosed in Remingtons' Pharmaceutical Sciences, 16 th edition, Oslo, a.

It may be desirable to modify the antibodies of the invention in terms of effector function to enhance the antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively or in addition, cysteine residues may be introduced in the Fc region, thereby forming interchain disulfide bonds in this region. The homodimeric antibody so produced may have improved internalization capacity and/or increased complement-mediated cell killing capacity and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, j.exp med.176: 1191-1195(1992) and shop, B.J.Immunol.148: 2918-2922(1992). Also, for example, Wolff et al Cancer Research 53: 2560 2565(1993), heterodimeric antibodies with enhanced antitumor activity were prepared using heterodimeric functional cross-linkers. Alternatively, antibodies can be engineered to have a dual Fc region, potentially enhancing complement lysis and ADCC capabilities. See Stevenson et al, Anti-Cancer Drug Design 3: 219-230(1989).

WO00/42072(Presta, L) describes antibodies with improved ADCC function in the presence of human effector cells, wherein the antibodies comprise amino acid substitutions in their Fc region. Preferably, the antibody with increased ADCC comprises a substitution at position 298, 333, and/or 334 of the Fc region. Preferably the altered Fc region is a human IgG1Fc region comprising or consisting of a substitution at one, two or three of these positions.

Antibodies with altered C1q binding and/or Complement Dependent Cytotoxicity (CDC) are described in WO99/51642, U.S. patent No. 6,194,551B1, U.S. patent No. 6,242,195B1, U.S. patent No. 6,528,624B1, and U.S. patent No. 6,538,124(Idusogie et al). The antibody comprises an amino acid substitution at one or more of amino acid positions 270, 322, 326, 327, 329, 313, 333 and/or 334 of its Fc region.

For example, to increase the serum half-life of an antibody, a salvage receptor binding epitope can be incorporated into an antibody (particularly an antibody fragment) as described in U.S. patent No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule responsible for increasing serum half-life in vivo (e.g., IgG₁，IgG₂，IgG₃Or IgG ₄). Antibodies with substitutions in their Fc region and increased serum half-life are also described in WO00/42072(Presta, L).

Engineered antibodies having three or more (preferably four) functional antigen binding sites are also contemplated (U.S. application No. US2002/0004587a1, Miller et al).

The HER2 antibodies disclosed herein can also be formulated as immunoliposomes. The compositions are prepared by methods known in the art, such as those described in Epstein et al, proc.natl.acad.sci.usa, 82: 3688 (1985); hwang et al, proc.natl acad.sci.usa, 77: 4030 (1980); U.S. patent nos. 4,485,045 and 4,544,545; and methods described in WO97/38731 published on days 1997-1-23, to prepare antibody-containing liposomes. Liposomes with improved turnover times are disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by reverse phase evaporation methods with lipid compositions comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are pressed through a filter of defined pore size to produce liposomes of the desired diameter. As in Martin et al j.biol.chem.257: 286-288(1982), Fab' fragments of the antibodies of the invention may be coupled to liposomes by disulfide interactions. Optionally, a chemotherapeutic agent is contained in the liposomes. See Gabizon et al J.national Cancer Inst.81(19)1484 (1989).

(ix) Exemplary antibodies

Exemplary antibodies that may be formulated according to the invention include, but are not limited to, the following:

anti-ErbB antibodies, including anti-HER 2 antibodies, such as those described in more detail herein;

antibodies that bind to B cell surface markers such as CD19, CD20 (e.g., Rituximab)And humanized 2H7), CD22, CD40 or BR 3;

IgE-binding antibodies, including Omalizumab commercially available from GenentechE26 (fig. 17A-B herein), HAE1 (fig. 17A-B herein), IgE antibody with amino acid substitutions at position 265 of its Fc region (US 2004/0191244a1), Hu-901 (fig. 17A-B herein), IgE antibody in WO2004/070011, or antibodies comprising the variable domain of any of these IgE antibodies (including antibody fragments and full length antibodies). See also, Presta et al, j.immunol.151: 2623. 2632 (1993); international publication nos. WO 95/19181; U.S. Pat. No. 5,714,338, published in 1998-02-03; U.S. Pat. No. 5,091,313, issued in 1992-02-25; WO 93/04173 published in 1993-03-04; WO 99/01556 published in 1999-01-14; and U.S. patent No. 5,714,338;

antibodies that bind Vascular Endothelial Growth Factor (VEGF) or its receptor, including Bevacizumab (AVASTIN), commercially available from Genentech ^TM) And Ranibizumab (LUCENTIS)^TM)；

anti-IL-8 antibodies (St John et al, Chest, 103: 932(1993), and International publication No. WO 95/23865);

anti-PSCA antibodies (WO 01/40309);

anti-CD 40 antibodies, including S2C6 and humanized variants thereof (WO 00/75348);

anti-CD 11a antibodies, including efalizumab(U.S. Pat. No. 5,622,700, WO 98/23761, Steppe et al, Transplantation Intl.4: 3-7(1991), and Hourmant et al, Transplantation 58: 377-380 (1994)); anti-CD 18 antibody (U.S. Pat. No. 5,622,700 issued to 1997-04-22, or WO 97/26912 published to 1997-07-31);

anti-Apo-2 receptor antibodies (WO 98/51793 published by 1998-11-19);

anti-TNF-alpha antibodies include cA2CDP571 and MAK-195 (see, U.S. Pat. No. 5,672,347 issued 1997-09-30, Lorenz et al J.Immunol.156 (4): 1646-1653(1996), and Dhamaut et al crit. Care Med.23 (9): 1461-1469 (1995));

anti-Tissue Factor (TF) (granted by European patent No. 0420937B 1, 1994-11-09);

anti-human alpha 4 beta₇Integrins (WO 98/06248 published by 1998-02-19);

anti-EGFR antibodies, including the chimeric or humanized 225 antibody of WO 96/40210, published in 1996-12-19;

anti-CD 3 antibodies, such as OKT3 (U.S. patent No. 4,515,893 issued 1985-05-07);

anti-CD 25 or anti-tac antibodies such as CHI-621And(see U.S. Pat. No. 5,693,762 issued to 1997-12-02);

anti-CD 4 antibodies such as the cM-7412 antibody (Choy et al Arthritis Rheum 39 (1): 52-56 (1996));

anti-CD 52 antibodies such as CAMPATH-1H (Riechmann et al Nature 332: 323-337 (1988);

anti-Fc receptor antibodies such as Graziano et al j.immunol.155 (10): 4996 anti-Fc γ RI M22 antibody in 5002 (1995);

anti-carcinoembryonic antigen (CEA) antibodies such as hMN-14(Sharkey et al Cancer Res.55(23 Suppl): 5935s-5945s (1995);

antibodies against mammary epithelial cells include huBrE-3, hu-Mc 3 and CHL 6(Ceriani et al Cancer Res.55 (23): 5852s-5856s (1995); and Richman et al Cancer Res.55(23 Supp): 5916s-5920s (1995));

antibodies that bind colon cancer cells such as C242(Litton et al Eur J. Immunol.26 (1): 1-9 (1996));

anti-CD 38 antibodies, such as AT13/5(Ellis et al J.Immunol.155 (2): 925-937 (1995));

anti-CD 33 antibodies such as Hu M195(Jurcic et al Cancer Res 55(23 Suppl): 5908s-5910s (1995) and CMA-676 or CDP 771;

anti-CD 22 antibodies such as LL2 or LymphoCide (Juweid et al Cancer Res 55(23 Suppl): 5899s-5907s (1995);

anti-EpCAM antibodies such as 17-1A

anti-GpIIb/IIIa antibodies such as abciximab or c7E3Fab

anti-RSV antibodies such as MEDI-493

anti-CMV antibodies such as PROTOVIR

anti-HIV antibodies such as PRO 542;

anti-hepatitis antibodies such as anti-Hep B antibody OSTAVIR

anti-CA 125 antibody OvaRex;

anti-idiotypic GD3 epitope antibody BEC 2;

anti-alpha v beta 3 antibody VITAXIN

Anti-human renal cell carcinoma antibodies such as ch-G250; ING-1;

anti-human 17-1A antibody (3622W 94);

anti-human colorectal tumor antibody (a 33);

anti-human melanoma antibody R24 against GD3 ganglioside;

anti-human squamous cell carcinoma (SF-25); and

anti-Human Leukocyte Antigen (HLA) antibodies such as Smart ID10 and anti-HLA DR antibody Oncolym (Lym-1).

(xi) Antibody variant compositions

In at least one aspect, the invention relates to a formulation comprising a composition comprising a mixture of a major species of antibody and one or more variants thereof. When the main species antibody binds HER2, preferably the HER2 antibody (one or both of the main component HER2 antibody and antibody variants thereof) is an antibody that binds to domain II of HER2, inhibits HER dimerization more effectively than Trastuzumab, and/or binds to the heterodimeric binding site of HER 2. Preferred embodiments of the main species of antibody herein are antibodies comprising the variable light and variable heavy amino acid sequences of SEQ ID Nos.3 and 4, most preferably comprising a light chain amino acid sequence selected from SEQ ID Nos. 15 and 23, and a heavy chain amino acid sequence selected from SEQ ID Nos.16 and 24.

In one embodiment, the formulated HER2 antibody composition comprises a mixture of the major component HER2 antibody and amino acid sequence variants thereof comprising an amino-terminal directed extension. Preferably, the amino-terminal guide extends on a light chain of the antibody variant (e.g., on one or both light chains of the antibody variant). The main component HER2 antibody or antibody variant may be a full-length antibody or antibody fragment (e.g. Fab of a F (ab') 2 fragment), but preferably both are full-length antibodies. The antibody variants herein may comprise an amino-terminal directed extension on any one or more of their heavy or light chains. Preferably, the amino-terminal guide extends on one or both light chains of the antibody. The amino-terminal directed extension preferably comprises or consists of VHS-. The presence of an amino-terminal-directed extension in a composition can be detected by a variety of analytical techniques, including, but not limited to, N-terminal sequence analysis, charge heterogeneity determination (e.g., cation exchange chromatography or capillary zone electrophoresis), mass spectrometry, and the like. The amount of antibody variant in the composition typically ranges from an amount that constitutes the detection limit of any assay used to detect the variant (preferably N-terminal sequence analysis) to an amount that is less than the main species of antibody. Typically about 20% or less (e.g., from about 1% to about 15%, e.g., from 5% to about 15%) of the antibody molecules in the composition comprise an amino-terminal leader extension. Preferably using quantitative N-terminal sequence analysis or cation exchange analysis (preferably using a high resolution, weak cation exchange column, such as PROPAC WCX-10 ^TMCation exchange column) to determine such percentage amounts. In addition to amino-terminal directed extension variants, other major classes of antibodies and/or variants are included with amino acid sequence variations, including but not limited to antibodies comprising a C-terminal lysine residue on one or both of its heavy chains, deamidated antibody variants, and the like.

In addition, the main species antibody or variant may further comprise glycosylation variations, non-limiting examples include HER2 antibody comprising a G1 or G2 oligosaccharide structure attached to its Fc region, HER2 antibody comprising a sugar moiety attached to its light chain (e.g., one or two sugar moieties attached to one or two light chains of the antibody), HER2 antibody comprising an aglycosylated heavy chain.

Preparation of the formulations

In a first aspect, the present invention provides a stable pharmaceutical formulation comprising a monoclonal antibody, preferably a full length human or humanized IgG1 antibody, in histidine-acetate buffer at ph5.5-6.5, preferably at ph 5.8-6.2. However, the antibody in the formulation may be an antibody fragment comprising an antigen-binding region, such as a Fab or F (ab') 2 fragment.

In another embodiment, the present invention relates to a pharmaceutical formulation comprising or consisting essentially of: from about 10mg/mL to about 250mg/mL of full-length IgG1 antibody that is susceptible to deamidation or aggregation; histidine-acetate buffer, pH5.5-6.5; a sugar selected from trehalose and sucrose in an amount of about 60mM to about 250 mM; and polysorbate 20 in an amount of about 0.01% to about 0.1%.

In yet another embodiment, the invention provides a pharmaceutical formulation comprising an antibody that binds to domain II of HER2, a sugar and a surfactant in a histidine buffer at a pH of from about 5.5 to about 6.5. For example, the formulation may comprise Pertuzumab in an amount from about 20mg/mL to about 40mg/mL, a histidine-acetate buffer, sucrose, and polysorbate 20, wherein the pH of the formulation is from about 5.5 to about 6.5.

In another aspect, the present invention provides a pharmaceutical formulation comprising a DR5 antibody in histidine buffer at a pH of from about 5.5 to about 6.5, a sugar and a surfactant. Such a formulation may, for example, comprise Apomab, histidine-acetate buffer, trehalose, and polysorbate 20 in an amount from about 10mg/mL to about 30mg/mL, wherein the pH of the formulation is from about 5.5 to about 6.5.

The formulation is particularly useful for antibodies that are prone to deamidation and/or aggregation and/or fragmentation, as the buffer blocks deamidation and/or aggregation and/or fragmentation of the antibody formulated therein. Furthermore, unlike other histidine buffers prepared using HCl, the histidine-acetate buffer lacks the chloride ions found beneficial herein because such buffers have the same protective effect on antibodies as polysorbate 20 when combined with sugars, and are stable and can be stored in stainless steel jars. Thus, in addition to formulations that themselves contain antibodies that are susceptible to deamidation and/or aggregation and/or fragmentation, the invention also provides methods of attenuating deamidation, aggregation and/or fragmentation of a therapeutic monoclonal antibody (e.g., relative to a composition at a different pH or in a different buffer), comprising formulating the antibody in a histidine-acetate buffer, pH 5.5-6.5. In this embodiment, deamidation, aggregation and/or fragmentation may be determined or measured before or after formulating an antibody that exhibits acceptable deamidation, aggregation and/or fragmentation in the formulation and upon storage thereof.

The antibody in the formulation may bind to an antigen, including but not limited to: HER2, CD20, IgE, DR5, BR3 and VEGF.

When the formulated antibody binds HER2, it preferably binds to domain II of HER2, inhibits HER dimerization more effectively than Trastuzumab, and/or binds to the heterodimeric binding site of HER 2. A preferred embodiment of the HER2 antibody herein is an antibody comprising the variable light and variable heavy amino acid sequences in SEQ ID nos.3 and 4, most preferably the light and heavy chain amino acid sequences in SEQ ID nos.15 and 16 (Pertuzumab).

Examples of CD20 antibodies that may be formulated herein include: "C2B 8", which is now called "Rituximab"Commercially available from Genentech (see also U.S. patent No. 5,736,137, hereby incorporated by reference); yttrium- [90 ]]Labeled 2B8 murine antibody, named "Y2B 8" or "Ibritumomab Tiuxetan" ZEVALINCommercially available from Biogen-Idec (see also U.S. Pat. No. 5,736,137, hereby incorporated by reference); murine IgG2a "B1," also known as "Tositumomab," optionally labeled¹³¹I to generate the "131I-B1" antibody (I iodine, 131tositumomab, BEXXAR)^TM) (U.S. Pat. No. 5,595,721, herein incorporated by reference); murine monoclonal antibody "1F 5" (Press et al blood 69 (2): 584-591(1987) and variants thereof comprising the "framework patched" or humanized 1F5 (WO03/002607, Leung, S.); ATCC accession No. HB-96450); murine 2H7 and chimeric 2H7 antibodies (Clark et al PNAS 82: 1766-; humanized 2H 7; huMax-CD20(WO04/035607, Genmab, Denmark); AME-133(Applied Molecular Evolution); a20 antibody or variants thereof such as chimeric or humanized a20 antibody (cA 20, hA20, respectively) (US2003/0219433, immunology); and monoclonal antibodies L27, G28-2, 93-1B3, B-C1 or NU-B2, which are obtainable from the International Leukocyte Typing Workshop (Valentine et al, In: Leukocyte Typing III (McMichael, Ed., p.440, Oxford University Press (1987)).

In a preferred embodiment of the formulated CD20 antibody, the CD20 antibody is a humanized 2H7 antibody. Preferred humanized 2H7 antibodies herein are 2H7v16 and 2H7v 511. Humanized 2H7v16 can be an intact antibody or an antibody fragment comprising variable light and variable heavy sequences (SEQ ID nos.26 and 29). When the humanized 2H7v16 antibody is a full length antibody, preferably it comprises the light and heavy chain amino acid sequences of SEQ ID nos.63 and 65.

When the antibody binds VEGF, preferably it comprises the variable domain sequences shown in figure 19. The most preferred anti-VEGF antibody is the full length humanized IgG1 antibody, Bevacizumab (AVASTIN)^TM) Commercially available from Genentech.

When the formulated antibody binds IgE, preferably it is selected from the group consisting of: e25, Omalizumab commercially available from Genentech(alsoSee fig. 17A-B), E26 (fig. 17A-B herein), HAE1 (fig. 17A-B herein), IgE antibodies with amino acid substitutions at position 265 of their Fc region (US 2004/0191244a1), Hu-901 (fig. 17A-B herein), IgE antibodies in WO2004/070011, or antibodies (including antibody fragments and full length antibodies) that include the variable domains of any of those IgE antibodies.

When the antibody binds to a receptor in the Tumor Necrosis Factor (TNF) superfamily or a death receptor, it preferably binds to DR5, and is preferably an agonist antibody. Publications in this area include sheeridan et al, Science, 277: 818, 821(1997), Pan et al, Science, 277: 815-818(1997), WO98/51793, published in 1998-11-19; WO98/41629 disclosed in 1998-09-24; screenton et al, curr. biol., 7: 693-696 (1997); walczak et al, EMBO j., 16: 5386-5387 (1997); wu et al, Nature Genetics, 17: 141-143 (1997); WO98/35986 disclosed in 1998-08-20; EP870, 827, published by 1998-10-14; WO98/46643, published under 1998-10-22; WO99/02653 disclosed in 1999-01-21; WO99/09165 disclosed in 1999-02-25; WO99/11791 disclosed in 1998-03-11; US2002/0072091 published 2002-08-13; US 2002/0098550 published 2001-12-07; US6,313,269 issued 2001-12-06; US 2001/0010924 published 2001-08-02; US2003/01255540 published under 2003-07-03; US 2002/0160446 published 2002-10-31, US 2002/0048785 published 2002-04-25; US6,342,369 issued 2002-02; US6,569,642 published 2003-05-27, US6,072,047 published 2000-06-06, US6,642,358 published 2003-11-04; US6,743,625 issued 2004-06-01. The most preferred DR5 antibody is Apomab.

Each of the above mentioned formulations comprises a buffer, preferably a histidine buffer, most preferably a histidine-acetate buffer, ph5.5-6.5, preferably 5.8-6.2, e.g. about 6.0. The concentration of the buffer is dictated at least in part by the desired pH. Exemplary concentrations of the buffer are from about 1mM to about 200mM, preferably from about 10mM to about 40mM, and most preferably about 20 mM.

The concentration of antibody in the formulation is preferably from about 10mg/mL to about 250 mg/mL. Antibody concentrations can be determined according to the desired use and mode of administration of the formulation. For example, when the formulation is for IV administration (e.g., HER2 antibody), the concentration of antibody in the formulation is preferably from about 20mg/mL to about 40 mg/mL. In an exemplary formulation of Pertuzumab intended for Intravenous (IV) administration, the antibody concentration is from about 20mg/mL to about 40mg/mL, most preferably about 30 mg/mL.

Higher concentrations of antibody may be required when the antibody is used for SQ or IM administration (e.g., for anti-IgE antibodies). Such substantially high antibody concentrations may be from about 50mg/mL to about 250mg/mL, or from about 80mg/mL to about 250mg/mL, or from about 100mg/mL to about 200 mg/mL.

When the formulation comprises a DR5 antibody, such as Apomab, exemplary antibody concentrations are from about 10mg/mL to about 30mg/mL, e.g., about 20mg/mL DR5 antibody; such formulations may be used for intravenous administration.

The formulation for administration is preferably an aqueous formulation (not lyophilized) and has not been previously lyophilized. Although the formulation may be lyophilized, it is preferred that it not be subjected to lyophilization. However, the freezing of aqueous preparations which are not simultaneously dried during the lyophilization process is particularly claimed, which facilitates their longer term storage, for example in stainless steel cans.

The formulation preferably further comprises a sugar, most preferably a disaccharide such as trehalose or sucrose. Typically contain an amount of sugar that reduces the formation of soluble aggregates such as those that occur after freezing/thawing. Exemplary sugar concentrations are from about 10mM to about 1M, for example from about 60mM to about 250mM, most preferably about 120mM, for a HER2 antibody formulation, and about 240mM for a DR5 antibody formulation.

Although the formulation comprising histidine-acetate buffer and sugar is found herein to be stable, the formulation optionally further comprises a surfactant, such as a polysorbate, most preferably polysorbate 20. Typically comprising an amount of surfactant that reduces soluble aggregate formation (as occurs upon shaking or transport). The surfactant concentration is preferably from about 0.0001% to about 1.0%, most preferably from about 0.01% to about 0.1%, for example about 0.02%.

Optionally, the formulation does not contain substantial amounts of salts such as sodium chloride.

The formulations are generally sterile and this may be accomplished according to methods known to those skilled in the art for making pharmaceutical formulations suitable for administration to human subjects, including filtration through sterile filtration membranes, either before or after preparation of the formulation.

In addition, it is desirable that the formulation is stable upon storage. The skilled artisan can determine the stability of the formulation using a variety of stability assays. For example, the formulation may be a formulation that is stable under the following storage conditions: at about 40 ℃ for at least 4 weeks; at about 5 ℃ or about 15 ℃ for at least 3 months or at least 1 year; and/or about-20 ℃ for at least 3 months. Stability can be tested by assessing the physical stability, chemical stability, and/or biological activity of the antibody in the formulation, either at formulation or after storage at the temperature. Physical and/or stability can be assessed qualitatively and/or quantitatively in a number of different ways, including assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); (ii) assessing charge heterogeneity by using cation exchange chromatography or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometry analysis; SDS-PAGE analysis to compare reduced and intact antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; assessing the biological activity or antigen binding function of the antibody; and so on. Instability can lead to aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), cleavage/hydrolysis/fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines(s), N-terminal extension, C-terminal processing, differences in glycosylation, and the like. Various techniques available to the skilled artisan can be used to assess biological activity or antigen binding function.

As mentioned above, freezing of the formulation is particularly contemplated. Thus, the formulations can be tested for stability after freezing and thawing.

Accordingly, the invention also provides a method of preparing a pharmaceutical formulation comprising preparing a formulation as described herein, and assessing the physical stability, chemical stability, or biological activity of the monoclonal antibody in the formulation.

In a preferred embodiment, the formulation is provided in a preferably aqueous form in a vial having a cap capable of being pierced by a syringe. The vial is desirably stored at about 2-8 ℃ until it is administered to a subject in need thereof. The vial may be, for example, a 20cc vial (e.g., for a 420mg dose) or a 50cc vial (e.g., for a 1050mg dose). For DR5 antibodies, such as Apomab, the formulation can be provided in a 5cc glass vial (e.g., 5.5ml full).

In another embodiment, the formulation is provided in a stainless steel can. The formulation in the stainless steel can is optionally frozen and not lyophilized.

One or more other pharmaceutically acceptable carriers, excipients or stabilizers may be included in the formulation, such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, a.ed. (1980), provided that they do not adversely affect the desired properties of the formulation. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include: other buffer solutions; a co-solvent; antioxidants include ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers such as polyesters; a preservative; and/or salt-forming counterions such as sodium.

Treatment with antibody formulations

In one embodiment, the present invention provides a method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of a formulation described herein to treat the disease or disorder.

When the antibody in the formulation binds to HER2, it is preferably used for the treatment of cancer. The cancer will typically comprise HER 2-expressing cells, thereby enabling the HER2 antibodies herein to bind to cancer cells. Thus, in this embodiment the invention relates to a method of treating a HER2 expressing cancer in a subject comprising administering to the subject an effective amount of a HER2 antibody pharmaceutical formulation to treat the cancer. Various cancers that can be treated with the composition are listed in the definition section above.

It is also contemplated that the HER2 antibody formulation may be used in the treatment and treatment of non-malignant diseases or disorders, including autoimmune diseases (e.g. psoriasis); endometriosis; scleroderma; restenosis; polyps such as colon polyps, nasal polyps or gastrointestinal polyps; fibroadenoma; respiratory tract diseases (see definition above); cholecystitis (cholecystitis); neurofibromatosis; polycystic kidney disease; inflammatory diseases; skin diseases including psoriasis and dermatitis; vascular disease (see definition above); disorders involving abnormal proliferation of vascular epithelial cells; gastrointestinal ulcers; menetrier's disease, secretory adenoma or protein loss syndrome; renal disease; angiogenic diseases; eye diseases such as age-related macular degeneration, ocular pseudohistoplasmosis syndrome, retinal neovascularization due to proliferative diabetic retinopathy, retinal angiogenesis, diabetic retinopathy, or age-related macular degeneration; bone-related pathologies such as osteoarthritis, osteomalacia and osteoporosis; injury following a cerebral ischemic event; fibrotic or edematous (edemia) diseases such as cirrhosis of the liver, pulmonary fibrosis, carpoidosis, thyroiditis, systemic hyperviscosity syndrome, Osler Weber-Rendu disease, chronic occlusive lung disease, or edema following burns, trauma, radiation, stroke, hypoxia or ischemia; skin allergic reactions; diabetic retinopathy and diabetic nephropathy; Guillain-Barre syndrome; graft versus host disease or transplant rejection; paget's disease; bone or arthritis; photoaging (e.g., caused by UV radiation to human skin); benign prostatic hyperplasia; infection by certain microorganisms, including microbial pathogens selected from the group consisting of adenovirus, hantavirus, Borrelia burgdorferi (Borrelia burgdorferi), Yersinia spp (Yersinia spp.) and Bordetella pertussis (Bordetella pertussis); thrombosis caused by platelet aggregation; reproductive diseases such as endometriosis, ovarian hyperstimulation syndrome, preeclampsia, dysfunctional uterine bleeding, or menorrhagia; synovitis; atheroma (atherorma); acute and chronic kidney disease (including proliferative glomerulonephritis and diabetes-induced nephropathy); eczema; hypertrophic scarring; endotoxic shock and fungal infections; familial adenomatous polyposis; neurodegenerative diseases (e.g., alzheimer's disease, aids-related dementia, parkinsonism tremor, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy (spinal muscular atrophy), and cerebellar degeneration); myelodysplastic syndrome; aplastic anemia; ischemic injury; fibrosis of the lung, kidney or liver; t cell mediated allergic diseases; hypertrophic pyloric stenosis in infants; urinary disorder syndrome; psoriatic arthritis; and hashimoto's thyroiditis. Preferred non-malignant indications for treatment herein include psoriasis, endometriosis, scleroderma, vascular diseases (e.g. restenosis, atherosclerosis, coronary artery disease, or hypertension), colonic polyps, fibroadenomas or respiratory diseases (e.g. asthma, chronic bronchitis, bronchiectasis or cystic fibrosis).

Where the antibody in the formulation binds a B cell surface marker such as CD20 or BR3, the formulation may be used to treat a B cell malignancy, such as NHL or CLL, an autoimmune disease, transplant rejection, or suppress an immune response to a foreign antigen, such as an antibody, toxin, gene therapy viral vector, graft, infectious agent, or alloantigen (see WO 01/03734, Grillo-Lopez et al).

When the antibody in the formulation is an IgE antibody, it may be used to treat IgE-mediated disorders (USSN 2004/0197324a1, Liu and Shire), such as allergic asthma, allergic rhinitis, atopic (atopic) dermatitis, allergic gastrointestinal disease (allergic gastroenteropathy), allergy, eczema, urticaria, allergic bronchopulmonary aspergillosis (bronchus aspergillosis), parasitic diseases, hyper- (hyper) -IgE condensation syndrome, ataxia-telangiectasia (ataxia), weio-Aldrich syndrome, thymic lymphodysplasia (thymic lymphoplasia), myeloma IgE, and graft-versus-host reactions.

Antibodies that bind to receptors in the TNF superfamily (e.g., bind to DR5), or antibodies that bind to VEGF (or its receptor), may be used to treat cancer, various forms of which are described in the definition section above. Preferably, the cancer treated by the DR5 antibody preparation is a solid tumor or NHL.

When the indication is cancer, the patient may be treated with a combination of the antibody preparation and a chemotherapeutic agent. Combination administration includes co-administration or simultaneous administration using separate formulations or a single pharmaceutical formulation, and sequential administration in either order, wherein two (or all) active agents exert their biological activities simultaneously over a period of time. Thus, the chemotherapeutic agent may be administered before or after administration of the composition. In this embodiment, the time between at least one administration of the chemotherapeutic agent and at least one administration of the composition is preferably about 1 month or less, most preferably about 2 weeks or less. Alternatively, the chemotherapeutic agent and the composition are administered to the patient simultaneously in a single formulation or separate formulations.

Treatment with the formulation will result in an improvement in the signs or symptoms of the cancer or disease. For example, where the disease being treated is cancer, such treatment may result in an improvement in survival (overall survival and/or progression-free survival) and/or may result in a targeted clinical response (partial or complete). In addition, treatment with a combination of chemotherapeutic agents and antibody preparations may deliver synergistic or greater than additive therapeutic benefits to the patient.

Preferably, the antibody in the administered formulation is a naked antibody. However, the administered antibody may be conjugated to a cytotoxic agent. Preferably, the immunoconjugate and/or antigen to which it is bound is/are internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugate in killing the cancer cell to which it binds. In preferred embodiments, the cytotoxic agent targets or interferes with nucleic acids in cancer cells. Examples of such cytotoxic agents include maytansinoids (maytansinoids), calicheamicins (calicheamicins), ribonucleases and DNA endonucleases.

The formulations are administered to a human patient according to known methods, such as intravenous administration, e.g., as a bolus, or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Preferably, the antibody composition is administered intravenously, intramuscularly or subcutaneously, most preferably intravenously.

For subcutaneous delivery, the delivery may be by syringe; injection devices (e.g., injection-EASETM and genjectm devices); injection pens (e.g., genpen); needleless devices (e.g., MEDIJECTORTM and BIOJECTORTM); or a subcutaneous patch delivery system.

For the prevention and treatment of disease, the appropriate dosage of antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, approximately 1. mu.g/kg to 50mg/kg (e.g. 0.1-20mg/kg) of HER2 or DR5 antibody is the initial candidate dose for administration to a patient, which may be by one or more separate administrations, or by continuous perfusion. The dosage of antibody is generally from about 0.05mg/kg to about 10 mg/kg. If a chemotherapeutic agent is administered, it is generally administered at its known dose, or optionally in a reduced amount, because administration of the chemotherapeutic agent can result in combined or side effects of the drug. The preparation and dosage regimen for such chemotherapeutic agents can be used according to manufacturer's instructions or as determined empirically by a physician. The preparation and dosage regimen for such chemotherapies is also described in Chemotherapy Service ed, m.c. perry, Williams & Wilkins, Baltimore, MD (1992).

Other therapeutic regimens may be combined with the antibody, including, but not limited to: a second (third, fourth, etc.) chemotherapeutic agent (i.e., a mixture of different chemotherapeutic agents); another monoclonal antibody; a growth inhibitor; a cytotoxic agent; a chemotherapeutic agent; an EGFR-targeting drug; tyrosine kinase inhibitors; an anti-angiogenic agent; and/or a cytokine; and so on.

In addition to the above treatment regimens, the patient may also undergo surgical removal of cancer cells and/or radiation therapy.

V. manufactured goods

In another embodiment of the invention, an article of manufacture comprising a pharmaceutical formulation of the invention is provided, together with instructions for use thereof. The article of manufacture includes a container. Suitable containers include, for example, bottles, vials (e.g., dual chamber vials), syringes (e.g., dual chamber syringes), and test tubes. The container may be formed from a number of materials such as glass or plastic. The container holds the formulation and indicia on or associated with the container may indicate instructions for use. The container containing the formulation may be a multi-purpose vial which allows repeated administration of the reconstituted formulation (e.g. 2-6 administrations). The article of manufacture may further comprise materials required from other commercial and user standpoints, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use as described in the preceding section.

The present invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the invention. All documents and patents cited are incorporated herein by reference.

Examples

Stable liquid Pertuzumab formulation

These examples describe the development and stability testing of stable liquid formulations comprising Pertuzumab at a protein concentration of about 10mg/mL to 180 mg/mL. The selected formulations have low turbidity and are physically and chemically stable. Chloride ions are removed from the formulation to reduce the risk of erosion. The formulation is isotonic and suitable for subcutaneous or intramuscular delivery. Formation of insoluble aggregates following shaking (aggregation stress) was prevented using histidine-acetate and sucrose formulations, without the need to include polysorbate 20.

Analytical method

Color, appearance and clarity (CAC)

The color, appearance and clarity of the samples were determined by visual inspection of the vials against white and black backgrounds at room temperature under white fluorescence.

UV concentration measurement

The liquid product is first diluted with formulation buffer so that the Amax is 0.5-1.0 absorbance units near 278 nm. The UV absorbance of the diluted samples was measured on an HP8453 spectrophotometer in a 1cm diameter quartz cup. The absorbance at 278nm and 320nm was measured. The absorbance from 320nm was used to correct for background light scattering due to larger aggregates, bubbles and particles. The measurements were zeroed relative to the formulation buffer. Protein concentration was determined using absorbance of 1.50(mg/mL) -1 cm-1.

pH measurement

Utilizing RADIOMETER COPENHAGEN PHM82^TMThe pH meter measures pH at room temperature. The detector used was a glass/reference electrode (Sigma, Cat # E-5759) in combination with a radiometer connector. The pH meter was calibrated using standard solutions of pH4.01 and pH7.00(EM Science).

Ion exchange chromatography (IEX)

The change in charge variable was measured using cation exchange chromatography. This assay was performed in HP1100^TMDIONEX PROPAC WCX-10 on HPLC system^TMAnd (3) a column. The sample was diluted to 1mg/mL with mobile phase A containing 20mM MES pH 6.0. 50mL of the diluted sample was then loaded onto a column maintained at ambient temperature. The peak was eluted with a shallow NaCl gradient using mobile phase B containing 20mM MES, 250mM NaCl, pH 6.0. The eluate was monitored at 280 nm.Using HPCHEMStation^TMThe software (Rev a08.03) analyzed the data.

Capillary Zone Electrophoresis (CZE)

The purity of Fab and F (ab') 2 fragments was determined by CZE. The assay was performed in BIORADBIOFOCUS^TM3000^TMBIOCAP XL on capillary electrophoresis system^TMCapillary, 50 μm i.d., 44.6cm full length, and run at 40cm distance from the detector.

Size Exclusion Chromatography (SEC)

Size exclusion chromatography was used to quantify aggregates and fragments. This assay utilizes TSK G3000SWXL ^TM7.8X300mm column and in HP1100^TMRun on HPLC system. The sample was diluted to 10mg/mL with the mobile phase and the injection volume was 20. mu.L. The mobile phase is 100mMK with pH6.8₂HPO₄And the protein was eluted with an isocratic gradient at 0.5mL/min for 45 minutes. The absorbance of the eluate was monitored at 280 nm. Using HP CHEMSSTATION^TMThe software (Rev A08.03) was integrated.

Biological activity

The biological activity of Pertuzumab was determined by measuring its ability to inhibit proliferation of the human breast cancer cell line MDA-MB-175-VII.

Example 1

Pertuzumab Fab and F (ab')₂Antibody fragment:

10mM citrate, 140mM NaCl, pH 4.0;

10mM succinate, 140mM NaCl, pH 5.0;

10mM succinate, 140mM NaCl, pH 6.0;

10mM histidine, 140mM NaCl, pH 7.0; and

10mM glycylglycine (glyylglycine), 140mM NaCl, pH 8.0.

Each formulation was filtered and aliquoted to 3cc WHEATON^TMUSP type I glass bottles coated with TEFLON^TMThe gray butyl synthetic rubber stopper of (1) was sealed. The samples were stored at 40. + -. 2 ℃. Stability analysis of the drug product showed Fab and F (ab')₂Is most stable between pH5.0 and 6.0.

TABLE 2 pH vs. Fab or F (ab') stored at 40 ℃)₂Effect of degradation of

Example 2

Pertuzumab was formulated with 120mM sucrose and 0.02% polysorbate 20 into 20mM histidine-acetate buffer. The pH of the formulation was adjusted with acetic acid to a final pH between 5.0 and 7.0. The protein concentration was 30 mg/mL. Each formulation was filled into 3cc USP type I glass bottles and stored at 40 ℃ for stability analysis. The results showed that Pertuzumab was most stable around ph6.0.

TABLE 3 influence of pH on the degradation of Pertuzumab stored at 40 ℃

pH of the formulation	Temperature (. degree.C.)	Storage time (wks)	SEC% monomer	IEX% main peak
					5.0	40	2	99.4	57.4
5.5	40	2	99.4	59.2
					6.0	40	2	99.4	60.6
6.5	40	2	99.3	60.5
					7.0	40	2	99.1	54.0
5.0	40	4	97.3	48.1
					5.5	40	4	99.1	50.5
6.0	40	4	99.1	53.3

6.5	40	4	99.0	52.3
					7.0	40	4	98.6	42.3

Example 3

A formulation of Pertuzumab with a protein concentration of 100mg/mL was prepared in the following excipients:

(1)10mM histidine-HCl, 240mM sucrose, 0.02% polysorbate 20, pH 6.0;

(2)10mM histidine-acetate (histidine-acetate), 240mM sucrose, 0.02% polysorbate 20, ph 6.0;

(3)10mM histidine-phosphate (histidine-phosphate), 240mM sucrose, 0.02% polysorbate 20, ph 6.0;

(4)10mM histidine-sulfate (histidine-sulfate), 240mM sucrose, 0.02% polysorbate 20, pH6.0.

Filling each preparation into 3cc of formula VITRUM^TMUSP type I glass bottle for FLUUROTEC ^TMFace (finished) butyl rubber stopper seal. Samples were stored at 30 ℃ and 40 ℃ and evaluated for stability for quality (CAC) and purity (SEC, IEC). The stability results show that Pertuzumab in histidine-phosphate buffer degraded much faster after storage at 40 ℃ than in other histidine buffers (fig. 8 and 9).

Example 4

Pertuzumab was concentrated to various concentrations by ultrafiltration/diafiltration in the following buffer:

(1)20mM histidine-acetate, pH 6.0;

(2)10mM histidine-HClpH6.0, and

(3)10mM histidine-sulfate, pH 6.0.

Turbidity was measured for each formulation prior to filtration. As a result, as shown in fig. 10, it was revealed that Pertuzumab samples formulated in histidine-acetate and histidine-HCl had a lower amount of insoluble aggregates than samples in histidine-sulfate buffer.

Example 5

Pertuzumab was formulated at 30mg/mL in 20mM histidine-acetate, 120mM sucrose, 0.02% polysorbate 20, ph 6.0. Pertuzmab was filled in 316L and HASTELLOYTM stainless steel micro-tanks. All samples were stored at-20 ℃ and 5 ℃ and evaluated for quality (CAC), purity (SEC, IEC) and intensity (UV-Vis). Stability analysis showed that Pertuzumab was stable in this formulation after storage at-20 ℃ and 5 ℃ for at least 3 months. The chloride-free formulation was compatible with 316L and HASTELLOYTM stainless steel cans.

TABLE 4 stability of Pertuzumab in stainless Steel tanks

^a.Passage of color, appearance and transparency: clear to a micro-emulsion white, colorless to a light yellow solution.

Example 6

Pertuzumab was formulated using Tangential Flow Filtration (TFF). The final formulation contained 20mM histidine-acetate, 120mM sucrose, 0.02% polysorbate 20, ph6.0, and a protein concentration of 30 mg/mL. The sample was filled into 20M1FORMA VITRUM^TMUSP type I glass bottle for 20mm FLUUROTEC^TMThe faced butyl rubber stopper was capped and capped with an aluminum sheet. All samples were stored at-70 ℃, 5 ℃, 15 ℃ and stability was assessed by mass (CAC), purity (SEC, IEC), intensity (UV-Vis), and potential energy (Bio assay). The results show that Pertuzumab is stable in this formulation after storage at 5 ℃ and 15 ℃ for at least 3 months.

TABLE 5 stability of Pertuzumab in glass vials

Temp(℃)	Time (moon)	CAC	UV Spec.(mg/mL)	SEC (% monomer)	IEC (% Main Peak)	Bio assay (% specific activity)
								0	By passing	29.2	99.8	64.1	83
-70	1	By passing	29.7	99.8	65.2	92
								3	By passing	30.7	99.8	67.0	93
5	3	By passing	30.4	99.7	67.2	90
							15	1	By passing	29.7	99.7	64.4	78
	3	By passing	30.4	99.7	65.5	93

Example 7

100mg/mL of Pertuzumab was prepared in the following buffer conditions:

(1)10mM histidine-HCl, pH 6.0;

(2)10mM histidine-HCl, 240mM sucrose, pH 6.0;

(3)20mM succinate pH 6.0; and

(4)20mM succinate, 240mM sucrose pH6.0.

Each formulation was added with different concentrations of polysorbate 20. All samples were filled into 3ccUSP type I glass bottles and stirred horizontally at 70rpm for 7 days at room temperature. The stability of each sample was evaluated with respect to turbidity at the 7 th day time point. The results show that the use of polysorbate 20 in the final formulation is effective in preventing the formation of insoluble aggregates. See fig. 11.

Example 8

Pertuzumab was prepared in the following formulation:

(1)25mg/mL Pertuzumab, 10mM histidine-HCl, 240mM sucrose, pH 6.0;

(2)50mg/mL Pertuzumab, 10mM histidine-HCl, 240mM sucrose, pH 6.0;

(3)60mg/mL Pertuzumab, 20mM histidine-acetate, 120mM sucrose, pH6.0.

Various amounts of polysorbate 20 were added to each formulation. All samples were filled into 3ccUSP type I glass bottles and agitated horizontally at 70rpm for 7 days at room temperature. The physical stability of each sample was evaluated with respect to turbidity at the 7 day time point. The results show that the use of polysorbate 20 in histidine-HCl and sucrose formulations effectively prevented the formation of insoluble particles. The formulation containing histidine-acetate and sucrose appeared to have the same protective effect on proteins as polysorbate 20. See fig. 12.

Example 9

Pertuzumab was formulated as follows:

(1)100mg/mL protein, 10mM histidine-HCl, pH 6.0;

(2)100mg/mL protein, 20mM succinate, pH 6.0;

(3)60mg/mL protein, 20mM histidine-acetate, pH 6.0.

Each formulation was mixed with different amounts of sucrose. All samples were aseptically filled into 3cc USP type I glass vials. They were subsequently frozen at-70 ℃ and thawed three times at 5 ℃. The physical stability of each sample was determined after three cycles of freeze-thawing. The results indicate that sucrose prevents the formation of soluble aggregates during freeze-thawing. See fig. 13.

Example 10

A preferred formulation of Pertuzumab for therapeutic use consists essentially of 20mM histidine acetate, 120mM sucrose, 0.02% polysorbate 20, 30mg/mL Pertuzumab in ph 6.0.

Compound (I)	Concentration of	amount/L
			Pertuzumab	30mg/mL	30g
L-histidine MW 155.16g/mol	20mM	3.10g
			Glacial acetic acidMW＝60.05g/molDensity 1.05g/cm³	11.6mM	0.66mL
SucroseMW＝342.3g/mol	120mM	41.1g
			Polysorbate 20 density is 1.012g/cm³	0.02％(w/v)	0.2mL

MW: molecular weight

420mg dose vial configuration:

vial: 20cc Formal Vitrum Type I glass

And (3) plugging: 20mm DAIKYO GREY^TMFluorine-resin pressing

A cap: 20mm top aluminum sheet

Full volume: 14.50mL

Delivery article: 14.0mL of Pertuzumab in saline IV bag.

1050mg dose vial configuration:

vial: 50cc FormalVitrem Type I glass

And (3) plugging: 20mm DAIKYO GREY^TMFluorine-resin pressing

A cap: 20mm top aluminum sheet

Full volume: 36.0mL

Delivery article: 35.0mL Pertuzumab in saline IV bag.

Example 11

This example relates to another formulation of Pertuzumab that has been used in phase I and phase II clinical trials. The composition consists of 25mg/ml Pertuzumab, 10mM histidine-HCl buffer, 240mM sucrose, 0.02% polysorbate 20, pH 6.0.

Composition (I)	Concentration of
		Pertuzumab	25mg/ml
L-His HCI.H₂O(MW209.6)	1.12mg/ml(0.0125M)
		L-His(MW155.2)	0.72mgml(0.0099M)
Sucrose (MW342.3)	82.15mg/ml(0.240M)
		Polysorbate 20	0.2mg/ml(0.02％)

Example 12

Apoptosis is mediated by both intrinsic and extrinsic pathways. Chemotherapy can trigger cellular damage and may promote apoptosis by responding to the intrinsic pathways of cellular damage. However, cancer cells often develop resistance to chemotherapy through mutations in the p53 tumor suppressor gene (Ashkenazi A. targeting Death and decoy Receptors of the tumor-Necrosis Factor Superfamily. Nature Reviews 2: 420-430 (2002)). Death receptors located on the cell surface, such as DR4 and DR5, promote apoptosis through extrinsic pathways that do not include p 53. Agonistic molecules, such as Apo2L, bind to the DR4 and DR5 receptors and activate caspases (caspases) 8 and 10 through the Fas-associated death domain. Caspases 8 and 10 subsequently activate caspases 3, 6, and 7 to induce apoptosis. The molecular signaling of death receptors on tumor cells is therapeutically effective for eliminating cancer cells resistant to traditional therapies and molecules like Apo2L, which are currently being evaluated clinically.

"Apomab" is a full-length CHO-derived humanized IgG1 constructed with a lambda light chain. It is an agonist antibody against DR5, which has been shown to induce apoptosis in various cancer cell lines. Preclinical studies using murine tumor implantation models have shown that Apomab has a similar or enhanced tumor reduction compared to Apo 2L. Apomab is being evaluated as an anti-cancer agent for use in advanced solid tumors and non-Hodgkin's lymphoma (NHL). The heavy and light chain amino acid sequences of Apomab used in these experiments are shown in fig. 27 and 28.

Preparation of antibody formulations

Recombinantly produced Apomab has a very dilute protein concentration and a high pH. The material was concentrated to about 20mg/mL and subjected to Millipore Labscale cut flow filtration (TFF) using Millipore Labscale cut flow filtration (TFF) system to form a Millipore Pellicon^TMXL, PLCGC10, 50cm membrane exchanged into 20mM sodium acetate, pH5.0 buffer. Use of trehalose-free and TWEENSodium acetate, histidine acetate, and sodium phosphate Apomab samples were formulated into various buffer systems, pH from 4.0 to 7.0, and dialyzed against a 10,000Da molecular weight exclusion membrane (Pierce, Inc). In the final dialysis 240mM trehalose was added. After dialysis, 0.02% TWEEN20TM was added to the formulation and the sample was filtered with a 0.22 μm filter (Millipore, Inc.). A volume of 0.5mL Apomab was loaded into a sterile 3cc glass vial (form Vitrum, Inc.) and closed with a 13mm stopper (Daikyo, Inc). Protein stability was assessed by storage at-70 ℃, 5 ℃, 30 ℃, and 40 ℃ for 3 months.

Stability of Apomab formulations

For the drug product stability test, the Apomab formulation was charged to 5cc FORMAIn a glass bottle. Vials were filled with 5.5mL of the formulated antibody, using 20mmThe stopper was closed and stored in a head-up position at-70 ℃, 5 ℃, 30 ℃, and 40 ℃.

For drug substance stability testing, the Apomab formulation was sterile filtered through a 0.22 μm filter and 10mL were loaded into a autoclaved 20cc316L stainless steel canister. The small can is placed vertically at-20 deg.C and 5 deg.C. Aliquots of 1mL were aseptically removed from the canister at specified time intervals to assess protein quality. The control vial was a 1mL aliquot stored in a 3cc glass vial at-20 ℃.

Color, appearance and clarity

The clarity, appearance and color of the samples were visually assessed using a white and black background stage under room temperature white fluorescence. To analyze the drug substance, the canister sample was transferred to a 3cc glass vial for examination.

pH

For measuring the THERMO ORION SURE-FLOW ROSS of a buffer^TMSemi-micro pH electrode or THERMO ORION GLS for measuring protein pH of screening sample^TMA Beckman microelectrode for toxicologically stable samples combined with a micro-pH electrode measures pH at room temperature. METERLAB was calibrated daily with buffer standards (EM Science) at pH7 and pH4 ^TMpHM240 pH/Ionic apparatus (Radiometer Analytical).

Concentration of

The protein concentration was measured by ultraviolet absorption spectroscopy using an aglent 8453TM spectrophotometer. The samples were diluted with the appropriate blank formulation buffer to give an absorbance from 0.5 to 1.0. The instrument was zeroed with the dilution solution and the spectrum was scanned from 240 to 500 nm. The absorbance value at 320nm was subtracted from the absorbance at 279nm to correct for offset and light scattering. The protein concentration was calculated by the following equation:

concentration (mg/mL) ═ concentration(A279-A320) X dilution factor

Absorption coefficient cm^-1(mg/mL)^-1

The sequence-based absorption coefficient was initially determined to be 1.32cm^-1(mg/mL)^-1And this value was used in a pH screening study. The latter value, determined by amino acid analysis and protein hydrolysis, was 1.7cm^-1(mg/mL)^-1And this value was used in toxicology studiesStability analysis of Apomab (a).

Ion exchange chromatography

Ion exchange chromatography was performed on a 1100 series HPLC (Agilent Technologies, Inc.) equipped with a diode array detector. In PROPAC WCX-10^TMChromatography was performed on a (Dionex) column (4X250mm) at a flow rate of 0.5mL/min, column temperature 40 ℃. The mobile phase A was 25mM sodium phosphate, pH 6.5. Mobile phase B was 100mM sodium chloride in the same buffer as mobile phase a. The column was equilibrated with 100% mobile phase a. For the pH-screened samples, an amount of 20mg of Apomab was applied to the column and the absorbance was monitored at 214 nm. The protein was eluted from the column with the following gradient:

Time (min) ％A ％B

0 100 0

50 0 100

51 100 0

70 100 0

For stability analysis of the substances used in toxicological studies, Apomab in an amount of 30mg was applied to the column and the absorbance was monitored at 280 nm. The protein was eluted from the column with the following gradient:

gradient:Time (min) ％A ％B

0 100 0

40.0 40 60

41.0 0 100

45.0 0 100

45.1 100 0

60.0 100 0

size exclusion chromatography

Size exclusion chromatography was performed on a 1100 series HPLC (Agilent Technologies, Inc.) equipped with a diode array detector. Apomab in an amount of 50. mu.g was applied to a TSK gel 3000SWXLTM (7.8X300mm) column and run at a flow rate of 0.9mL/min for 20 minutes for the pH screening sample and 0.5mL/min for 30 minutes for the toxicologically stable sample with 0.20M potassium phosphate, 0.25M potassium chloride, pH6.2 as the mobile phase. The absorbance was monitored at 280 nm.

Efficiency of

The purpose of the potency bioassay was to use ALAMARBLUE^TMThe ability of Apomab to kill Colo205 cells was measured. Colo205 is a colon cancer cell line that expresses DR5 and DR4 death receptors. Such assays include fluorescent/colorimetric growth indicators based on the detection of metabolic activity. ALAMARBLUE^TMIs a redox dye that is blue and non-fluorescent in the oxidized state. Intracellular metabolic reduction converts it to a red color, which is also fluorescent. The color and fluorescence change is proportional to the metabolic activity and number of living cells. The signal decreases when the cell dies. Apomab was diluted in culture medium with anti-Fc, and Colo205 cells were subsequently added to the Apomab sample and incubated at 37 ℃ for 48 hours. Adding ALAMARBLUE for the last 2-3 hr ^TM. Plates were read at 530nm excitation and 590nm emission to obtain Relative Fluorescence Units (RFU). By KALEIDAGRAPH^TMThe data is analyzed. A dilution curve of killing was generated.

Results

Formulation pH screening study

The effect of pH on antibody stability was studied using Apomab produced by a stable, non-proliferating cell line. For this assay, Apomab was formulated at 20mg/mL antibody in 20mM sodium acetate buffer, pH4.0, 4.5, 5.0, 5.5; 20mM histidine acetate buffer, pH6.0 and 6.5; and 20mM sodium phosphate bufferpH 7.0. All formulations contained 240mM trehalose and 0.02% TWEENThe formulations were stored at-70 ℃, 5 ℃, 30 ℃, and 40 ℃ for up to 3 months and protein stability was determined by various analytical assays including CAC, pH, concentration, SEC, and IEC. No significant change in CAC, pH or protein concentration was observed during sample storage.

Analysis of the samples by SEC showed no significant change during storage at 5 ℃ and-70 ℃. However, degradation was observed during storage at 30 ℃ and 40 ℃, which was indicative of the formation of antibody fragments and soluble aggregates (fig. 20). To compare the formulations, the kinetics of the antibody monomers during storage were monitored and the first order rate constants were calculated. The pH rate curve obtained for antibody monomer loss is shown in figure 21. Optimal conditions for antibody monomer stability were obtained by formulation in histidine acetate buffer at ph 6.0.

Apomab charge heterogeneity was monitored by IEC. No significant change in IEC curve occurred during storage at 5 ℃ and-70 ℃. However, degradation was observed, which appeared to form acidic or basic variants depending on the formulation (fig. 22). In general, the base-enhancing variants form at lower formulation pH and the more acidic variants form at higher formulation pH. To compare the formulations, the kinetics of the IEC main peak were monitored during storage and the first order rate constant was calculated. The pH rate curve obtained for IEC main peak loss is shown in figure 23. The rate constants observed by IEC were approximately 10 times higher than those observed by SEC (figure 21). Therefore, the loss of the IEC main peak is a preliminary degradation of the antibody, which will ultimately limit the shelf life of the product. In addition, it was observed by SEC that the best antibody stability to stabilize the IEC main peak was obtained by formulating in histidine acetate buffer at ph 6.0.

Following analysis of the pH screening data described above, Apomab formulations were selected containing 20mg/mL antibody in 20mM histidine acetate, 240mM trehalose, 0.02% polysorbate 20, pH 6.0. For pharmaceutical productsThe medicine bottle is prepared by packing in 5cc of formula VITRUM ^TM5.5mL in a vial with 20mM DAIKYO^TMWest plug. Apomab was stored in stainless steel tanks.

Stability of Apomab drug product was evaluated in the above 5cc glass vial configuration. Vials were stored at-70 ℃ (control), 5 ℃, 30 ℃, and 40 ℃. Samples were withdrawn at specific time intervals and analyzed by the following assay: color, appearance, clarity (CAC), pH, protein concentration, SEC, IEC, and potency. The results of these measurements for the samples stored at-70 ℃ and 5 ℃ are shown in Table 6, while the results for the samples stored at 30 ℃ and 40 ℃ are shown in Table 7.

TABLE 6 stability data for Apomab stored at-70 ℃ and 5 ℃

IECTemp concentration SEC (% Main potency (. degree. C.) clarity color pH (mg/mL) (% monomer) peak) (% specific activity) at time point
	6.0 acceptance criteria: report. + -. 0.320. + -.2>95% report 60-140%
NA T0 clear colorless 5.920.299.86394-701 month clear colorless 6.020.599.86386-702 month clear colorless 6.020.499.76491-703 month clear colorless 6.020.599.763 Clear and colorless at 83-706 months 6.020.499.76485-709 months 6.020.499.86589-7012 months 6.020.899.763107
	Clear and colorless at 51 month 6.020.599.7638952 month 6.020.499.7649953 month, 6.020.699.7638456 month, 6.020.599.7649359 month, 6.020.699.76488512 month, 6.020.799.664106

TABLE 7 stability data for Apomab stored at 30 ℃ and 40 ℃

Concentration SEC IEC Effect Temp (. degree. C.) time Point clarity color pH (mg/mL) (% monomer) (% Main Peak) (% specific Activity)
	Acceptance criteria: report 6.0. + -. 0.320. + -. 2>95% report 60-140%
Clear and colorless at month 301, 6.020.698.25991302, 6.020.397.45480303, 6.020.697.24974306, 6.020.294.13751309, 6.020.493.231553012, and 6.020.691.62559

Clear and colorless at 401 month 6.020.496.64479402 month, clear and colorless at 6.020.093.73164403 month, clear and light yellow at 5.920.391.52253406 month, clear and light yellow at 6.020.283.9 NTYellow 5.920.378.8 NT 254012 clear yellow 5.920.571.4 NT 31 clear in the month 26409

NT is not quantified

No change in protein quality was observed after 12 months of storage at-70 ℃ and 5 ℃. For example, the pH was maintained at 6.0 ± 0.3, Apomab was present as a clear and colorless liquid, the protein concentration was maintained at 20.0 ± 2.0mg/mL, and% monomer was unchanged. In addition, the% IEC main peak was not significantly changed and the% specific activity determined by cell killing potency assay was within the accuracy of the determination of 60% to 140% specific activity. The results show that Apomab stored in a 5cc glass vial is stable at 5 ℃ for at least 12 months.

Table 7 shows that the change in protein mass occurs at 30 ℃ and 40 ℃. SEC showed a decrease in% monomer with an increase in predominantly fragmented material. Aggregates increased as with higher temperatures, but at a much lower rate. However, aggregates increased significantly after 6 months at 40 ℃. The IEC% main peak was reduced with a corresponding increase in acidic variants. The alkaline peak decreased slightly after 40 ℃, 2 months and after 9 months at 30 ℃. After six months of storage at 40 ℃, a decrease occurs to the point where the IEC main peak can no longer integrate. Cell killing bioassay shows that longer storage times at higher temperatures result in a loss of% specific activity. Protein concentration and pH were unchanged. The solution turned pale yellow after 9 months at 40 ℃, 3 and 30 ℃ and yellow after 9 months at 40 ℃.

Drug substance stability

The freeze-thaw stability data of the drug substance is shown in table 8.

TABLE 8 Freeze-thaw stability data for Apomab contained in small stainless steel cans

Temp (. degree. C.) Freeze-thaw concentration SEC (freeze/thaw) cycle number clarity color pH (mg/mL) (% monomer)
	Acceptance criteria: report 6.0. + -. 0.320.0. + -. 2.095%
Control (unfrozen) fruit 0 clear colorless 6.020.999.6-20/251 clear colorless 6.020.899.6-20/252 clear colorless 6.020.899.6-20/253 clear colorless 6.020.999.6

TABLE 9 stability data for Apomab in small stainless steel tanks

Temp concentration SEC IEC time Point (C.)Clarity color pH (mg/mL) (% monomer) (% Main Peak) (% specific Activity)
	6.0 acceptance criteria: report. + -. 0.320. + -.2>95% report 60-140%
NA T0 clear colorless 5.920.099.76388-201 month clear colorless 6.020.699.763107-203 month clear colorless 6.020.699.76382-206 month clear colorless 6.020.399.76492-209 month clear colorless 6.020.699.76492-2012 month clear colorless 6.021.299.76594

Clear and colorless at 51 months 6.020.599.7629553 months, clear and colorless at 6.020.799.6627156 months, clear and colorless at 6.020.499.5628459 months, clear and colorless at 6.020.899.461845 months Clear and colorless at 12 months 6.021.399.25982

No significant change in the chemical properties of the protein was observed after freezing at-20 ℃ for at least 15 hours and three thawing at ambient temperature. For example, Apomab appears as a clear and colorless liquid, with the pH maintained at 6.0 ± 0.3, while the SEC monomer peak percentage is unchanged.

Apomab stability in stainless steel containers was evaluated at-20 ℃ and 5 ℃ (Table 9).

Samples were drawn from the canister at specific intervals under sterile conditions and analyzed.

Apomab showed no change in protein mass with respect to pH, CAC, protein concentration and% main peak obtained by IEC, but lost 0.1% of monomer every 3 months as seen by SEC. A decrease in efficacy was observed during storage at 5 ℃ for 3 months. However, the efficacy of the samples increased again at the 6 month and 9 month time points. Therefore, the difference in potency at the 3 month time point was attributed to assay bias. Apomab was seen to show no change in protein mass and no significant change in potency by pH, CAC, protein concentration,% monomer by SEC,% main peak by IEC. The stability data show that Apomab is stable at-20 ℃ for at least 1 year and at 5 ℃ for three months.

Conclusion

Formulation screening studies were performed to select Apomab formulations. pH screens covering a pH range of 4.0 to 7.0 using sodium acetate, histidine acetate, and sodium phosphate, together with 240mM trehalose dihydrate and 0.02% polysorbate 20 as buffers, showed that Apomab was most stable in solution at pH 6.0. Therefore, a formulation consisting of 20mM histidine acetate, 240mM trehalose, 0.02% polysorbate 2, ph6.0 was developed and proved to be stable by experiments. With this formulation, Apomab was shown to be stable at 5 ℃ for at least 12 months. Additionally, Apomab was shown to be stable at-20 ℃ for at least 12 months and at 5 ℃ for three months when stored in 316L stainless steel containers. Apomab was also shown to be stable when subjected to up to 3 freeze-thaw cycles.

Sequence listing

<110> Jiantaike Biotechnology Co., Ltd (GENENTECH, INC.)

ANDYA，JAMES

GWEE，SHIANG C.

LIU，JUN

SHEN，YE

<120> preparation of antibodies in histidine-acetate buffer

<130>P2104R1 PCT

<140>PCT/US2005/037471

<141>2005-10-19

<150>US 60/620,413

<151>2004-10-20

<160>74

<210>1

<211>107

<212>PRT

<213> mouse (Mus musculus)

<400>1

Asp Thr Val Met Thr Gln Ser His Lys Ile Met Ser Thr Ser Val

1 5 10 15

Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser

20 25 30

Ile Gly Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Lys

35 40 45

Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp

50 55 60

Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile

65 70 75

Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

80 85 90

Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu

95 100 105

Ile Lys

<210>2

<211>119

<212>PRT

<213> mouse (Mus musculus)

<400>2

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly

1 5 10 15

Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr

20 25 30

Asp Tyr Thr Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu

35 40 45

Glu Trp Ile Gly Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr

50 55 60

Asn Gln Arg Phe Lys Gly Lys Ala Ser Leu Thr Val Asp Arg Ser

65 70 75

Ser Arg Ile Val Tyr Met Glu Leu Arg Ser Leu Thr Phe Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr

95 100 105

Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

110 115

<210>3

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>3

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser

20 25 30

Ile Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

80 85 90

Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu

95 100 105

Ile Lys

<210>4

<211>119

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>4

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr

20 25 30

Asp Tyr Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr

50 55 60

Asn Gln Arg Phe Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser

65 70 75

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr

95 100 105

Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

110 115

<210>5

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>5

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser

20 25 30

Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Leu Leu Ile Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

80 85 90

Tyr Asn Ser Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu

95 100 105

Ile Lys

<210>6

<211>119

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>6

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Ala Val Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr

50 55 60

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

65 70 75

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg Val Gly Tyr Ser Leu

95 100 105

Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

110 115

<210>7

<211>10

<212>PRT

<213> Artificial sequence

<220>

The <223> sequence was synthetic.

<220>

<221>Xaa

<222>10

<223> Xaa is preferably D or S

<400>7

Gly Phe Thr Phe Thr Asp Tyr Thr Met Xaa

5 10

<210>8

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>8

Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe

1 5 10 15

Lys Gly

<210>9

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>9

Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr

5 10

<210>10

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>10

Lys Ala Ser Gln Asp Val Ser Ile Gly Val Ala

5 10

<210>11

<211>7

<212>PRT

<213> Artificial sequence

<220>

The <223> sequence was synthetic.

<220>

<221>Xaa

<222>5

<223> Xaa is preferably R or L

<220>

<221>Xaa

<222>6

<223> Xaa is preferably Y or E

<220>

<221>Xaa

<222>7

<223> Xaa is preferably T or S

<400>11

Ser Ala Ser Tyr Xaa Xaa Xaa

5

<210>12

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>12

Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr

5

<210>13

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>13

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn

20 25 30

Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Ly sAla Pro Lys

35 40 45

Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

80 85 90

His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu

95 100 105

Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

110 115 120

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

125 130 135

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

140 145 150

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu

155 160 165

Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

170 175 180

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

185 190 195

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

200 205 210

Arg Gly Glu Cys

<210>14

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>14

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys

20 25 30

Asp Thr Tyr Ile Hi s Trp ValArg Gln Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr

50 55 60

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser

65 70 75

Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cy sSer Arg Trp Gly Gly Asp Gly Phe Tyr

95 100 105

Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

110 115 120

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

125 130 135

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

140 145 150

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

155 160 165

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

170 175 180

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

185 190 195

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

200 205 210

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

215 220 225

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

320 325 330

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

335 340 345

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

350 355 360

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

365 370 375

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

380 385 390

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

395 400 405

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

410 415 420

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

425 430 435

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

440 445

<210>15

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>15

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser

20 25 30

Ile Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

80 85 90

Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu

95 100 105

Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

110 115 120

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

125 130 135

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

140 145 150

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu

155 160 165

Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

170 175 180

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

185 190 195

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

200 205 210

Arg Gly Glu Cys

<210>16

<211>448

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>16

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr

20 25 30

Asp Tyr Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr

50 55 60

Asn Gln Arg Phe Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser

65 70 75

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr

95 100 105

Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

110 115 120

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

125 130 135

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

140 145 150

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

155 160 165

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

170 175 180

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

185 190 195

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

200 205 210

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

215 220 225

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315

Leu Asn Gly Lys Glu Tyr Lys Cys Lys yal Ser Asn Lys Ala Leu

320 325 330

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

335 340 345

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

350 355 360

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

365 370 375

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

380 385 390

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

395 400 405

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

410 415 420

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

425 430 435

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

440 445

<210>17

<211>233

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>17

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr

1 5 10 15

Gly Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Gln Asp Val Ser Ile Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly

50 55 60

Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr

65 70 75

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

80 85 90

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr

95 100 105

Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly

110 115 120

Thr Lys Val Glu Ile Lys Arg Thr Val Ala A1a Pro Ser Val Phe

125 130 135

Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

140 145 150

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

155 160 165

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser G y Asn Ser Gln Glu

170 175 180

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

185 190 195

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

200 205 210

TyrAla Cys Glu Val Thr Hi s Gln Gly Leu Ser Ser Pro Val Thr

215 220 225

Lys Ser Phe Asn Arg Gly G1u Cys

230

<210>18

<211>467

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>18

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr

1 5 10 15

Gly Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

20 25 30

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

35 40 45

Phe Thr Phe Thr Asp Tyr Thr Met Asp Trp Val Arg Gln Ala Pro

50 55 60

Gly Lys Gly Leu Glu Trp Val Ala Asp Val Asn Pro Asn Ser Gly

65 70 75

Gly Ser Ile Tyr Asn Gln Arg Phe Lys Gly Arg Phe Thr Leu Ser

80 85 90

Val Asp Arg Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

95 100 105

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu G1y

110 115 120

Pro Ser Phe Tyr Phe Asp Tyr Trp Gly G1n Gly Thr Leu Val Thr

125 130 135

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

140 145 150

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

155 160 165

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

170 175 180

Ser Gly Ala Leu Thr Ser Gly Val His Thr Ple Pro Ala Val Leu

185 190 195

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

200 205 210

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

215 220 225

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

230 235 240

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

245 250 255

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

275 280 285

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

290 295 300

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

305 310 315

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

320 325 330

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

335 340 345

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

350 355 360

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

365 370 375

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

380 385 390

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

395 400 405

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

410 415 420

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

425 430 435

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

440 445 450

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

455 460 465

Pro Gly

<210>19

<211>195

<212>PRT

<213> human (Homo sapiens)

<400>19

Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala

1 5 10 15

Ser Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly

20 25 30

Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr

35 40 45

Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly

50 55 60

Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu Gln

65 70 75

Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr

80 85 90

Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr

95 100 105

Pro Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu

110 115 120

Arg Ser Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg

125 130 135

Asn Pro Gln Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile

140 145 150

Phe His Lys Asn Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn

155 160 165

Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met Cys Lys Gly Ser

170 175 180

Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser Leu Thr Arg

185 190 195

<210>20

<211>124

<212>PRT

<213> human (Homo sapiens)

<400>20

Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro Leu Pro

1 5 10 15

Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly Pro

20 25 30

Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly

35 40 45

Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp

50 55 60

Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly

65 70 75

Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp

80 85 90

Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln Glu Val

95 100 105

Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro

110 115 120

Cys Ala Arg Val

<210>21

<211>169

<212>PRT

<213> human (Homo sapiens)

<400>21

Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val

1 5 10 15

Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe

20 25 30

Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala

35 40 45

Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu

50 55 60

Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro

65 70 75

Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile

80 85 90

Arg Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln

95 100 105

Gly Leu Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu

110 115 120

Gly Ser Gly Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe

125 130 135

Val His Thr Val Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln

140 145 150

Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp Glu Cys Val Gly

155 160 165

Glu Gly Leu Ala

<210>22

<211>142

<212>PRT

<213> human (Homo sapiens)

<400>22

Cys His Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro

1 5 10 15

Thr Gln Cys Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys

20 25 30

Val Glu Glu Cys Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val

35 40 45

Asn Ala Arg His Cys Leu Pro Cys His Pro Glu Cys Gln Pro Gln

50 55 60

Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp Gln Cys Val

65 70 75

Ala CysAla Hi s Tyr Lys Asp Pro Pro Phe Cys Val Ala Arg Cys

80 85 90

Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile Trp Lys

95 100 105

Phe Pro Asp Glu Glu GlyAla Cys Gln Pro Cys Pro Ile Asn Cys

110 115120

Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu

125 130 135

Gln Arg Ala Ser Pro Leu Thr

140

<210>23

<211>217

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>23

Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

1 5 10 15

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln

20 25 30

Asp Val Ser Ile Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly

50 55 60

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

65 70 75

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

80 85 90

Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly Thr

95 100 105

Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile

110 115 120

Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val

125 130 135

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln

140 145 150

Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

155 160 165

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

170 175 180

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

185 190 195

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

200 205 210

Ser Phe Asn Arg Gly Glu Cys

215

<210>24

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>24

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr

20 25 30

Asp Tyr Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr

50 55 60

Asn Gln Arg Phe Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser

65 70 75

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr

95 100 105

Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

110 115 120

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

125 130 135

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

140 145 150

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

155 160 165

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

170 175 180

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

185 190 195

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

200 205 210

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

215 220 225

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

320 325 330

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

335 340 345

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

350 355 360

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

365 370 375

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

380 385 390

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

395 400 405

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

410 415 420

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

425 430 435

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

440 445

<210>25

<211>107

<212>PRT

<213> mouse (Mus musculus)

<400>25

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro

1 5 10 15

Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser

20 25 30

Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro

35 40 45

Trp Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ala Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser

65 70 75

Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp

80 85 90

Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

95 100 105

Lys Arg

<210>26

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>26

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser

20 25 30

Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro

35 40 45

Leu Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp

80 85 90

Ser Phe Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

95 100 105

Lys Arg

<210>27

<211>108

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>27

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser

20 25 30

Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Leu Leu Ile Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

80 85 90

Tyr Asn Ser Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu

95 100 105

Ile Lys Arg

<210>28

<211>122

<212>PRT

<213> mouse (Mus musculus)

<400>28

Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly

1 5 10 15

Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

20 25 30

Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Arg Gln Gly Leu

35 40 45

Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr

50 55 60

Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser

65 70 75

Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp

80 85 90

Ser Ala Val Tyr Phe Cys Ala Arg Val Val Tyr Tyr Ser Asn Ser

95 100 105

Tyr Trp Tyr Phe Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val

110 115 120

Ser Ser

<210>29

<211>122

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>29

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr

20 25 30

Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr

50 55 60

Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser

65 70 75

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Asn Ser

95 100 105

Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val

110 115 120

Ser Ser

<210>30

<211>119

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>30

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Ala Val Ile Ser Gly Asp Gly G v Ser Thr Tyr Tyr

50 55 60

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

65 70 75

Ly s Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg Val Gly Tyr Ser Leu

95 100 105

Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

110 115

<210>31

<211>107

<212>PRT

<213> Artificial sequence

<220>

The <223> sequence was synthetic.

<400>31

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu

1 5 10 15

Gly Asp Arg Val Ile Ile Ser Cys Ser Ala Ser Gln Asp Ile Ser

20 25 30

Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys

35 40 45

Val Leu Ile Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

65 70 75

Ser Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln

80 85 90

Tyr Ser Thr Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu

95 100 105

Ile Lys

<210>32

<211>123

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>32

Glu Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Gln Pro Gly

1 5 10 15

Glu Thr Val Arg Ile Ser Cys Lys Ala Ser G v Tyr Thr Phe Thr

20 25 30

Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu

35 40 45

Lys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr

50 55 60

Ala Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu Glu Thr Ser

65 70 75

Ala Ser Thr Ala Tyr Leu Gln Ile Ser Asn Leu Lys Asn Asp Asp

80 85 90

Thr Ala Thr Tyr Phe Cys Ala Lys Tyr Pro His Tyr Tyr Gly Ser

95 100 105

Ser His Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr

110 115 120

Val Ser Ser

<210>33

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>33

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser

20 25 30

Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Val Leu Ile Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75

Ser Ser Leu Gln Pro 6lu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

80 85 90

Tyr Ser Thr Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu

95 100 105

Ile Lys

<210>34

<211>123

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>34

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr

20 25 30

Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr

50 55 60

Ala Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser

65 70 75

Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Lys Tyr Pro His Tyr Tyr Gly Ser

95 100 105

Ser His Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr

110 115 120

Val Ser Ser

<210>35

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>35

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser

20 25 30

Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Val Leu Ile Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

80 85 90

Tyr Ser Thr Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu

95 100 105

Ile Lys

<210>36

<211>123

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>36

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr

20 25 30

His Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr

50 55 60

Ala Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser

65 70 75

Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr

95 100 105

Ser His Trp Tyr Phe Asp Val Trp Gly Gln G v Thr Leu Val Thr

110 115 120

Val Ser Ser

<210>37

<211>218

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>37

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

1 5 10 15

140 145 150

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

155 160 165

Ser Phe Asn Arg Gly Glu Cys Asp Glu Gln Leu Lys Ser Gly Thr

170 175 180

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

185 190 195

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

200 205 210

Gln Glu Ser Val Thr Glu Gln Asp

215

<210>39

<211>218

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>39

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Pro Val Asp

20 25 30

Gly Glu Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly

35 40 45

Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser

50 55 60

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

65 70 75

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr

80 85 90

Tyr Cys Gln Gln Ser His Glu Asp Pro Tyr Thr Phe Gly Gln Gly

95 100 105

Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe

110 115 120

Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

125 130 135

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

140 145 150

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

155 160 165

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

170 175 180

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

185 190 195

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

200 205 210

Lys Ser Phe Asn Arg Gly Glu Cys

215

<210>40

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp

20 25 30

Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly

35 40 45

Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser

50 55 60

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

65 70 75

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr

80 85 90

Tyr Cys Gln Gln Ser His Glu Asp Pro Tyr Thr Phe Gly Gln Gly

95 100 105

Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe

110 115 120

Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

125 130 135

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

140 145 150

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

155 160 165

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

170 175 180

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

185 190 195

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

200 205 210

Lys Ser Phe Asn Arg Gly Glu Cys

215

<210>38

<211>218

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>38

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Pro Val Asp

20 25 30

Gly Glu Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly

35 40 45

Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser

50 55 60

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

65 70 75

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr

80 85 90

Tyr Cys Gln Gln Ser His Glu Asp Pro Tyr Thr Phe Gly Gln Gly

95 100 105

Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe

110 115 120

Ile Phe Pro Pro Ser Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

125 130 135

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

<400>40

Asp Ile Leu Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro

1 5 10 15

Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly

20 25 30

Thr Asn Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg

35 40 45

Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75

Ser Arg Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln

80 85 90

Ser Asp Ser Trp Pro Thr Thr Phe Gly Gln Gly Thr Lys Val Glu

95 100 105

Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

110 115 120

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

125 130 135

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

140 145 150

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu

155 160 165

Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

170 175 180

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

185 190 195

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

200 205 210

Arg Gly Glu Cys

<210>41

<211>451

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>41

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr

20 25 30

Ser Gly Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly

35 40 45

Leu Glu Trp Val Ala Ser Ile Thr Tyr Asp Gly Ser Thr Asn Tyr

50 55 60

Asn Pro Ser Val Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp Ser

65 70 75

Lys Asn Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ser His Tyr Phe Gly His

95 100 105

Trp His Phe Ala Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser

110 115 120

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

125 130 135

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cy s Leu Val

140 145 150

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

155 160 165

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

170 175 180

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

185 190 195

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

200 205 210

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

215 220 225

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser Hi s Glu Asp Pro Glu Val Lys PheAsn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

305 310 315

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

320 325 330

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

335 340 345

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

350 355 360

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

365 370 375

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp G u Ser Asn Gly Gln

380 385 390

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

395 400 405

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

410 415 420

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

425 430 435

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

440 445 450

Lys

<210>42

<211>451

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Val Ser G y Tyr Ser Ile Thr

20 25 30

Ser Gly Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly

35 40 45

Leu Glu Trp Val Ala Ser Ile Thr Tyr Asp Gly Ser Thr Asn Tyr

50 55 60

Asn Pro Ser Val Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp Ser

65 70 75

Lys Asn Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ser His Tyr Phe Gly His

95 100 105

Trp His Phe Ala Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser

110 115 120

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

125 130 135

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

140 145 150

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

155 160 165

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

170 175 180

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

185 190 195

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

200 205 210

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

215 220 225

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Tnr Val Leu His Gln

305 310 315

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

320 325 330

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

335 340 345

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

350 355 360

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

365 370 375

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

380 385 390

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

395 400 405

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

410 415 420

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

425 430 435

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

440 445 450

Lys

<210>43

<211>451

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>43

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr

20 25 30

Ser Gly Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly

35 40 45

Leu Glu Trp Val Ala Ser Ile Lys Tyr Ser Gly Glu Thr Lys Tyr

50 55 60

Asn Pro Ser Val Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp Ser

65 70 75

Lys Asn Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ser His Tyr Phe Gly His

95 100 105

Trp His Phe Ala Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser

110 115 120

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

125 130 135

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

140 145 150

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

155 160 165

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

170 175 180

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

185 190 195

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

200 205 210

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

215 220 225

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

305 310 315

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

320 325 330

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

335 340 345

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

350 355 360

Glu Met Thr Lys Asn Gln yal Ser Leu Thr Cys Leu Val Lys Gly

365 370 375

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

380 385 390

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

395 400 405

GlySer Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Ly s Ser Arg

410 415 420

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

425 430 435

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

440 445 450

Lys

<210>44

<211>453

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>44

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

1 5 10 15

Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser

20 25 30

Met Tyr Trp Leu Glu Trp Val Arg Gln Ala Pro Gly His Gly Leu

35 40 45

Glu Trp Val Gly Glu Ile Ser Pro Gly Thr Phe Thr Thr Asn Tyr

50 55 60

Asn Glu Lys Phe Lys AlaA rg Ala Thr Phe Thr Ala Asp Thr Ser

65 70 75

Thr Asn Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Arg Phe Ser His Phe Ser Gly Ser

95 100 105

Asn Tyr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

110 115 120

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

125 130 135

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

140 145 150

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

155 160 165

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

170 175 180

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

185 190 195

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

200 205 210

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

215 220 225

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

275 280 285

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

290 295 300

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

305 310 315

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

320 325 330

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

335 340 345

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

350 355 360

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

365 370 375

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

380 385 390

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

395 400 405

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

410 415 420

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

425 430 435

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

440 445 450

Pro Gly Lys

<210>45

<211>1042

<212>DNA

<213> human (Homo sapiens)

<220>

<221> indeterminacy

<222>447

<223> N is T or G

<400>45

tttcctcact gactataaaa gaatagagaa ggaagggctt cagtgaccgg 50

ctgcctggct gacttacagc agtcagactc tgacaggatc atggctatga 100

tggaggtcca ggggggaccc agcctgggac agacctgcgt gctgatcgtg 150

atcttcacag tgctcctgca gtctctctgt gtggctgtaa cttacgtgta 200

ctttaccaac gagctgaagc agatgcagga caagtactcc aaaagtggca 250

ttgcttgttt cttaaaagaa gatgacagtt attgggaccc caatgacgaa 300

gagagtatga acagcccctg ctggcaagtc aagtggcaac tccgtcagct 350

cgttagaaag atgattttga gaacctctga ggaaaccatt tctacagttc 400

aagaaaagca acaaaatatt tctcccctag tgagagaaag aggtccncag 450

agagtagcag ctcacataac tgggaccaga ggaagaagca acacattgtc 500

ttctccaaac tccaagaatg aaaaggctct gggccgcaaa ataaactcct 550

gggaatcatc aaggagtggg cattcattcc tgagcaactt gcacttgagg 600

aatggtgaac tggtcatcca tgaaaaaggg ttttactaca tctattccca 650

aacatacttt cgatttcagg aggaaataaa agaaaacaca aagaacgaca 700

aacaaatggt ccaatatatt tacaaataca caagttatcc tgaccctata 750

ttgttgatga aaagtgctag aaatagttgt tggtctaaag atgcagaata 800

tggactctat tccatctatc aagggggaat atttgagctt aaggaaaatg 850

acagaatttt tgtttctgta acaaatgagc acttgataga catggaccat 900

gaagccagtt ttttcggggc ctttttagtt ggctaactga cctggaaaga 950

aaaagcaata acctcaaagt gactattcag ttttcaggat gatacactat 1000

gaagatgttt caaaaaatct gaccaaaaca aacaaacaga aa 1042

<210>46

<211>281

<212>PRT

<213> human (Homo sapiens)

<400>46

Met Ala Met Met Glu Val Gln Gly Gly Pro Ser Leu Gly Gln Thr

1 5 10 15

Cys Val Leu Ile Val Ile Phe Thr Val Leu Leu Gln Ser Leu Cys

20 25 30

Val Ala Val Thr Tyr Val Tyr Phe Thr Asn Glu Leu Lys Gln Met

35 40 45

Gln Asp Lys Tyr Ser Lys Ser Gly Ile Ala Cys Phe Leu Lys Glu

50 55 60

Asp Asp Ser Tyr Trp Asp Pro Asn Asp Glu Glu Ser Met Asn Ser

65 70 75

Pro Cys Trp Gln Val Lys Trp Gln Leu Arg Gln Leu Val Arg Lys

80 85 90

Met Ile Leu Arg Thr Ser Glu Glu Thr Ile Ser Thr Val Gln Glu

95 100 105

Lys Gln Gln Asn Ile Ser Pro Leu Val Arg Glu Arg Gly Pro Gln

110 115 120

Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr

125 130 135

Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys

140 145 150

Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser

155 160 165

Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly

170 175 180

Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu

185 190 195

Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln Tyr Ile

200 205 210

Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser

215 220 225

Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr

230 235 240

Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg

245 250 255

Ile Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His

260 265 270

Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly

275 280

<210>47

<211>411

<212>PRT

<213> human (Homo sapiens)

<220>

<221>Xaa

<222>410

<223> Xaa is L or M

<400>47

Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala Arg

1 5 10 15

Lys Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro

20 25 30

Gly Leu Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala Val

35 40 45

Leu Leu Leu Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln Asp

50 55 60

Leu Ala Pro Gln Gln Arg Ala Ala Pro Gln Gln Lys Arg Ser Ser

65 70 75

Pro Ser Glu Gly Leu Cys Pro Pro Gly His His Ile Ser Glu Asp

80 85 90

Gly Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr Ser Thr

95 100 105

His Trp Asn Asp Leu Leu Phe Cys Leu Arg Cys Thr Arg Cys Asp

110 115 120

Ser Gly Glu Val Glu Leu Ser Pro Cys Thr Thr Thr Arg Asn Thr

125 130 135

Val Cys Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro

140 145 150

Glu Met Cys Arg Lys Cys Arg Thr Gly Cys Pro Arg Gly Met Val

155 160 165

Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys Val His

170 175 180

Lys Glu Ser Gly Ile Ile Ile Gly Val Thr Val Ala Ala Val Val

185 190 195

Leu Ile Val Ala Val Phe Val Cys Lys Ser Leu Leu Trp Lys Lys

200 205 210

Val Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gly Gly Gly Asp

215 220 225

Pro Glu Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Ala Glu Asp

230 235 240

Asn Val Leu Asn Glu Ile Val Ser Ile Leu Gln Pro Thr Gln Val

245 250 255

Pro Glu Gln Glu Met Glu Val Gln Glu Pro Ala Glu Pro Thr Gly

260 265 270

Val Asn Met Leu Ser Pro Gly Glu Ser Glu His Leu Leu Glu Pro

275 280 285

Ala Glu Ala Glu Arg Ser Gln Arg Arg Arg Leu Leu Val Pro Ala

290 295 300

Asn Glu Gly Asp Pro Thr Glu Thr Leu Arg Gln Cys Phe Asp Asp

305 310 315

Phe Ala Asp Leu Val Pro Phe Asp Ser Trp Glu Pro Leu Met Arg

320 325 330

Lys Leu Gly Leu Met Asp Asn Glu Ile Lys Val Ala Lys Ala Glu

335 340 345

Ala Ala Gly His Arg Asp Thr Leu Tyr Thr Met Leu Ile Lys Trp

350 355 360

Val Asn Lys Thr Gly Arg Asp Ala Ser Val His Thr Leu Leu Asp

365 370 375

Ala Leu Glu Thr Leu Gly Glu Arg Leu Ala Lys Gln Lys Ile Glu

380 385 390

Asp His Leu Leu Ser Ser Gly Lys Phe Met Tyr Leu Glu Gly Asn

395 400 405

Ala Asp Ser Ala Xaa Ser

410

<210>48

<211>1799

<212>DNA

<213> human (Homo sapiens)

<400>48

cccacgcgtc cgcataaatc agcacgcggc cggagaaccc cgcaatctct 50

gcgcccacaa aatacaccga cgatgcccga tctactttaa gggctgaaac 100

ccacgggcct gagagactat aagagcgttc cctaccgcca tggaacaacg 150

gggacagaac gccccggccg cttcgggggc ccggaaaagg cacggcccag 200

gacccaggga ggcgcgggga gccaggcctg ggctccgggt ccccaagacc 250

cttgtgctcg ttgtcgccgc ggtcctgctg ttggtctcag ctgagtctgc 300

tctgatcacc caacaagacc tagctcccca gcagagagcg gccccacaac 350

aaaagaggtc cagcccctca gagggattgt gtccacctgg acaccatatc 400

tcagaagacg gtagagattg catctcctgc aaatatggac aggactatag 450

cactcactgg aatgacctcc ttttctgctt gcgctgcacc aggtgtgatt 500

caggtgaagt ggagctaagt ccctgcacca cgaccagaaa cacagtgtgt 550

cagtgcgaag aaggcacctt ccgggaagaa gattctcctg agatgtgccg 600

gaagtgccgc acagggtgtc ccagagggat ggtcaaggtc ggtgattgta 650

caccctggag tgacatcgaa tgtgtccaca aagaatcagg catcatcata 700

ggagtcacag ttgcagccgt agtcttgatt gtggctgtgt ttgtttgcaa 750

gtctttactg tggaagaaag tccttcctta cctgaaaggc atctgctcag 800

gtggtggtgg ggaccctgag cgtgtggaca gaagctcaca acgacctggg 850

gctgaggaca atgtcctcaa tgagatcgtg agtatcttgc agcccaccca 900

ggtccctgag caggaaatgg aagtccagga gccagcagag ccaacaggtg 950

tcaacatgtt gtcccccggg gagtcagagc atctgctgga accggcagaa 1000

gctgaaaggt ctcagaggag gaggctgctg gttccagcaa atgaaggtga 1050

tcccactgag actctgagac agtgcttcga tgactttgca gacttggtgc 1100

cctttgactc ctgggagccg ctcatgagga agttgggcct catggacaat 1150

gagataaagg tggctaaagc tgaggcagcg ggccacaggg acaccttgta 1200

cacgatgctg ataaagtggg tcaacaaaac cgggcgagat gcctctgtcc 1250

acaccctgct ggatgccttg gagacgctgg gagagagact tgccaagcag 1300

aagattgagg accacttgtt gagctctgga aagttcatgt atctagaagg 1350

taatgcagac tctgccwtgt cctaagtgtg attctcttca ggaagtgaga 1400

ccttccctgg tttacctttt ttctggaaaa agcccaactg gactccagtc 1450

agtaggaaag tgccacaatt gtcacatgac cggtactgga agaaactctc 1500

ccatccaaca tcacccagtg gatggaacat cctgtaactt ttcactgcac 1550

ttggcattat ttttataagc tgaatgtgat aataaggaca ctatggaaat 1600

gtctggatca ttccgtttgt gcgtactttg agatttggtt tgggatgtca 1650

ttgttttcac agcacttttt tatcctaatg taaatgcttt atttatttat 1700

ttgggctaca ttgtaagatc catctacaaa aaaaaaaaaa aaaaaaaaag 1750

ggcggccgcg actctagagt cgacctgcag aagcttggcc gccatggcc 1799

<210>49

<211>440

<212>PRT

<213> human (Homo sapiens)

<400>49

Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala Arg

1 5 10 15

Lys Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro

20 25 30

Gly Pro Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala Val

35 40 45

Leu Leu Leu Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln Asp

50 55 60

Leu Ala Pro Gln Gln Arg Ala Ala Pro Gln Gln Lys Arg Ser Ser

65 70 75

Pro Ser Glu Gly Leu Cys Pro Pro Gly His His Ile Ser Glu Asp

80 85 90

Gly Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr Ser Thr

95 100 105

His Trp Asn Asp Leu Leu Phe Cys Leu Arg Cys Thr Arg Cys Asp

110 115 120

Ser Gly Glu Val Glu Leu Ser Pro Cys Thr Thr Thr Arg Asn Thr

125 130 135

Val Cys Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro

140 145 150

Glu Met Cys Arg Lys Cys Arg Thr Gly Cys Pro Arg Gly Met Val

155 160 165

Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys Val His

170 175 180

Lys Glu Ser Gly Thr Lys His Ser Gly Glu Ala Pro Ala Val Glu

185 190 195

Glu Thr Val Thr Ser Ser Pro Gly Thr Pro Ala Ser Pro Cys Ser

200 205 210

Leu Ser Gly Ile Ile Ile Gly Val Thr Val Ala Ala Val Val Leu

215 220 225

Ile Val Ala Val Phe Val Cys Lys Ser Leu Leu Trp Lys Lys Val

230 235 240

Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gly Gly Gly Asp Pro

245 250 255

Glu Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Ala Glu Asp Asn

260 265 270

Val Leu Asn Glu Ile Val Ser Ile Leu Gln Pro Thr Gln Val Pro

275 280 285

Glu Gln Glu Met Glu Val Gln Glu Pro Ala Glu Pro Thr Gly Val

290 295 300

Asn Met Leu Ser Pro Gly Glu Ser Glu His Leu Leu Glu Pro Ala

305 310 315

Glu Ala Glu Arg Ser Gln Arg Arg Arg Leu Leu Val Pro Ala Asn

320 325 330

Glu Gly Asp Pro Thr Glu Thr Leu Arg Gln Cys Phe Asp Asp Phe

335 340 345

Ala Asp Leu Val Pro Phe Asp Ser Trp Glu Pro Leu Met Arg Lys

350 355 360

Leu Gly Leu Met Asp Asn Glu Ile Lys Val Ala Lys Ala Glu Ala

365 370 375

Ala Gly His Arg Asp Thr Leu Tyr Thr Met Leu Ile Lys Trp Val

380 385 390

Asn Lys Thr Gly Arg Asp Ala Ser Val His Thr Leu Leu Asp Ala

395 400 405

Leu Glu Thr Leu Gly Glu Arg Leu Ala Lys Gln Lys Ile Glu Asp

410 415 420

His Leu Leu Ser Ser Gly Lys Phe Met Tyr Leu Glu Gly Asn Ala

425 430 435

Asp Ser Ala Met Ser

440

<210>50

<211>1323

<212>DNA

<213> human (Homo sapiens)

<400>50

atggaacaac ggggacagaa cgccccggcc gcttcggggg cccggaaaag 50

gcacggccca ggacccaggg aggcgcgggg agccaggcct gggccccggg 100

tccccaagac ccttgtgctc gttgtcgccg cggtcctgct gttggtctca 150

gctgagtctg ctctgatcac ccaacaagac ctagctcccc agcagagagc 200

ggccccacaa caaaagaggt ccagcccctc agagggattg tgtccacctg 250

gacaccatat ctcagaagac ggtagagatt gcatctcctg caaatatgga 300

caggactata gcactcactg gaatgacctc cttttctgct tgcgctgcac 350

caggtgtgat tcaggtgaag tggagctaag tccgtgcacc acgaccagaa 400

acacagtgtg tcagtgcgaa gaaggcacct tccgggaaga agattctcct 450

gagatgtgcc ggaagtgccg cacagggtgt cccagaggga tggtcaaggt 500

cggtgattgt acaccctgga gtgacatcga atgtgtccac aaagaatcag 550

gtacaaagca cagtggggaa gccccagctg tggaggagac ggtgacctcc 600

agcccaggga ctcctgcctc tccctgttct ctctcaggca tcatcatagg 650

agtcacagtt gcagccgtag tcttgattgt ggctgtgttt gtttgcaagt 700

ctttactgtg gaagaaagtc cttccttacc tgaaaggcat ctgctcaggt 750

ggtggtgggg accctgagcg tgtggacaga agctcacaac gacctggggc 800

tgaggacaat gtcctcaatg agatcgtgag tatcttgcag cccacccagg 850

tccctgagca ggaaatggaa gtccaggagc cagcagagcc aacaggtgtc 900

aacatgttgt cccccgggga gtcagagcat ctgctggaac cggcagaagc 950

tgaaaggtct cagaggagga ggctgctggt tccagcaaat gaaggtgatc 1000

ccactgagac tctgagacag tgcttcgatg actttgcaga cttggtgccc 1050

tttgactcct gggagccgct catgaggaag ttgggcctca tggacaatga 1100

gataaaggtg gctaaagctg aggcagcggg ccacagggac accttgtaca 1150

cgatgctgat aaagtgggtc aacaaaaccg ggcgagatgc ctctgtccac 1200

accctgctgg atgccttgga gacgctggga gagagacttg ccaagcagaa 1250

gattgaggac cacttgttga gctctggaaa gttcatgtat ctagaaggta 1300

atgcagactc tgccatgtcc taa 1323

<210>51

<211>451

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>51

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Glu Arg Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp

20 25 30

Asp Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Ser Gly Ile Asn Trp Gln Gly Gly Ser Thr Gly Tyr

50 55 60

Ala Asp Ser Val Lys Gly Arg Val Thr Ile Ser Arg Asp Asn Ala

65 70 75

Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Lys Ile Leu Gly Ala Gly Arg Gly

95 100 105

Trp Tyr Phe Asp Tyr Trp Gly Lys Gly Thr Thr Val Thr Val Ser

110 115 120

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

125 130 135

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

140 145 150

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

155 160 165

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

170 175 180

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

185 190 195

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

200 205 210

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

215 220 225

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

305 310 315

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

320 325 330

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

335 340 345

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

350 355 360

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

365 370 375

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

380 385 390

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

395 400 405

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

410 415 420

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

425 430 435

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

440 445 450

Lys

<210>52

<211>213

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>52

Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Ser Gly Asp Ser Leu Arg Ser Tyr Tyr

20 25 30

Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val

35 40 45

Ile Tyr Gly Ala Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe

50 55 60

Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly

65 70 75

Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Ala Asp

80 85 90

Ser Ser Gly Asn His Val Val Phe Gly Gly Gly Thr Lys Leu Thr

95 100 105

Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro

110 115 120

Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys

125 130 135

Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys

140 145 150

Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro

155 160 165

Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser

170 175 180

Leu Thr Pro Glu Gln Trp Lys Ser His Lys Ser Tyr Ser Cys Gln

185 190 195

Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr

200 205 210

Glu Cys Ser

<210>53

<211>451

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>53

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Glu Arg Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp

20 25 30

Asp Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr

50 55 60

Ala Asp Ser Val Lys Gly Arg Val Thr Ile Ser Arg Asp Asn Ala

65 70 75

Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Lys Ile Leu Gly Ala Gly Arg Gly

95 100 105

Trp Tyr Phe Asp Leu Trp Gly Lys Gly Thr Thr Val Thr Val Ser

110 115 120

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

125 130 135

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

140 145 150

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

155 160 165

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

170 175 180

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

185 190 195

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

200 205 210

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

215 220 225

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

305 310 315

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

320 325 330

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

335 340 345

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

350 355 360

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

365 370 375

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

380 385 390

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

395 400 405

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

410 415 420

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

425 430 435

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

440 445 450

Lys

<210>54

<211>213

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>54

Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr

20 25 30

Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val

35 40 45

Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe

50 55 60

Ser Gly Ser Se rSer Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly

65 70 75

Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp

80 85 90

Ser Ser Gly Asn His Val Val Phe Gly Gly Gly Thr Lys Leu Thr

95 100 105

Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro

110 115 120

Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys

125 130 135

Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys

140 145 150

Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro

155 160 165

Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser

170 175 180

Leu Thr Pro Glu Gln Trp Lys Ser His Lys Ser Tyr Ser Cys Gln

185 190 195

Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr

200 205 210

Glu Cys Ser

<210>55

<211>126

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>55

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Glu Arg Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp

20 25 30

Asp Tyr Ala Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Gly Ile Asn Trp Gln Gly Gly Ser Thr Gly Tyr Ala

50 55 60

Asp Ser Val Lys Gly Arg Val Thr Ile Ser Arg Asp Asn Ala Lys

65 70 75

Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

80 85 90

Ala Val Tyr Tyr Cys Ala Lys Ile Leu Gly Ala Gly Arg Gly Trp

95 100 105

Tyr Phe Asp Tyr Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser

110 115 120

Ala Ser Thr Lys Gly Pro

125

<210>56

<211>108

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>56

Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Ser Gly Asp Ser Leu Arg Ser Tyr Tyr

20 25 30

Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val

35 40 45

Ile Tyr Gly Ala Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe

50 55 60

Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly

65 70 75

Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Ala Asp

80 85 90

Ser Ser Gly Asn His Val Val Phe Gly Gly Gly Thr Lys Leu Thr

95 100 105

Val Leu Gly

<210>57

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>57

Arg Ala Ser Ser Ser Val Ser Tyr Met His

5 10

<210>58

<211>7

<212>PRT

<213> Artificial sequence

<220>

The <223> sequence was synthetic.

<400>58

Ala Pro Ser Asn Leu Ala Ser

5

<210>59

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>59

Gln Gln Trp Ser Phe Asn Pro Pro Thr

5

<210>60

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>60

Gly Tyr Thr Phe Thr Ser Tyr Asn Met His

5 10

<210>61

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>61

Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

1 5 10 15

Lys Gly

<210>62

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>62

Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val

5 10

<210>63

<211>213

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>63

Aso Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser

20 25 30

Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro

35 40 45

Leu Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp

80 85 90

Ser Phe Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

95 100 105

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser

110 115 120

Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

125 130 135

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

140 145 150

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

155 160 165

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu

170 175 180

Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

185 190 195

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

200 205 210

Gly Glu Cys

<210>64

<211>213

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>64

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro

35 40 45

Leu Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp

80 85 90

Ala Phe Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

95 100 105

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser

110 115 120

Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

125 130 135

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

140 145 150

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

155 160 165

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu

170 175 180

Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

185 190 195

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

200 205 210

Gly Glu Cys

<210>65

<211>452

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>65

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr

20 25 30

Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr

50 55 60

Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser

65 70 75

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Asn Ser

95 100 105

Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val

110 115 120

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

125 130 135

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

140 145 150

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

155 160 165

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

170 175 180

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

185 190 195

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

200 205 210

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

215 220 225

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

290 295 300

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

305 310 315

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

320 325 330

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

335 340 345

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

350 355 360

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

365 370 375

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

380 385 390

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

395 400 405

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

410 415 420

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

425 430 435

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

440 445 450

Gly Lys

<210>66

<211>452

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>66

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr

20 25 30

Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr

50 55 60

Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser

65 70 75

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Tyr Arg

95 100 105

Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val

110 115 120

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

125 130 135

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

140 145 150

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

155 160 165

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

170 175 180

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

185 190 195

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

200 205 210

Pro Ser Asn Thr Lys yal Asp Lys Lys Val Glu Pro Lys Ser Cys

215 220 225

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

290 295 300

Tyr Asn Ala Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

305 310 315

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

320 325 330

Ala Ala Leu Pro Ala Pro Ile Ala Ala Thr Ile Ser Lys Ala Lys

335 340 345

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

350 355 360

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

365 370 375

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

380 385 390

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

395 400 405

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

410 415 420

Arg Trp Gln Gln Gly Asn yal Phe Ser Cys Ser Val Met His Glu

425 430 435

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

440 445 450

Gly Lys

<210>67

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<220>

<221>Xaa

<222>9

<223> Xaa is M or L

<400>67

Arg Ala Ser Ser Ser Val Ser Tyr Xaa His

5 10

<210>68

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<220>

<221>Xaa

<222>4

<223> Xaa is S or A

<400>68

Gln Gln Trp Xaa Phe Asn Pro Pro Thr

5

<210>69

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<220>

<221>Xaa

<222>8

<223> Xaa is D or A

<400>69

Ala Ile Tyr Pro Gly Asn Gly Xaa Thr Ser Tyr Asn Gln Lys Phe

1 5 10 15

Lys Gly

<210>70

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<220>

<221>Xaa

<222>6

<223> Xaa is N, A, Y, W or D

<220>

<221>Xaa

<222>7

<223> Xaa is S or R

<400>70

Val Val Tyr Tyr Ser Xaa Xaa Tyr Trp Tyr Phe Asp Val

5 10

<210>71

<211>451

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>71

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr

20 25 30

Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr

50 55 60

Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser

65 70 75

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Asn Ser

95 100 105

Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val

110 115 120

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

125 130 135

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

140 145 150

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

155 160 165

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

170 175 180

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

185 190 195

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

200 205 210

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

215 220 225

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

290 295 300

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

305 310 315

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

320 325 330

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

335 340 345

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

350 355 360

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

365 370 375

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

380 385 390

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

395 400 405

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

410 415 420

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

425 430 435

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

440 445 450

Gly

<210>72

<211>451

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>72

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr

20 25 30

Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr

50 55 60

Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser

65 70 75

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Tyr Arg

95 100 105

Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val

110 115 120

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

125 130 135

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala A a Leu Gly Cys Leu

140 145 150

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

155 160 165

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

170 175 180

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

185 190 195

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

200 205 210

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

215 220 225

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

290 295 300

Tyr Asn Ala Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

305 310 315

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

320 325 330

Ala Ala Leu Pro Ala Pro Ile Ala Ala Thr Ile Ser Lys Ala Lys

335 340 345

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

350 355 360

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

365 370 375

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser ASn Gly

380 385 390

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

395 400 405

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

410 415 420

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

425 430 435

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

440 445 450

Gly

<210>73

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>73

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro

35 40 45

Leu Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp

80 85 90

Ala Phe Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

95 100 105

Lys Arg

<210>74

<211>122

<212>PRT

<213> Artificial sequence

<220>

<223> the sequence is synthetic

<400>74

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr

20 25 30

Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr

50 55 60

Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser

65 70 75

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Tyr Arg

95 100 105

Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val

110 115 120

Ser Ser

Claims

1. A stable pharmaceutical formulation comprising a monoclonal antibody in histidine-acetate buffer, pH 5.5-6.5, wherein the monoclonal antibody is a full length humanized IgG antibody and is susceptible to deamidation or aggregation, and the monoclonal antibody binds to HER 2.

2. The formulation of claim 1, wherein the pH is from 5.8 to 6.2.

3. The formulation of claim 1, wherein the histidine-acetate buffer concentration is from 1mM to 200 mM.

4. The formulation of claim 3, wherein the histidine-acetate buffer concentration is from 10mM to 40 mM.

5. The formulation of claim 1, wherein the antibody concentration is from 10mg/mL to 250 mg/mL.

6. The formulation of claim 5, wherein the monoclonal antibody concentration is from 20mg/mL to 40 mg/mL.

7. The formulation of claim 5, wherein the monoclonal antibody concentration is from 80mg/mL to 250 mg/mL.

8. The formulation of claim 1, further comprising a sugar, wherein the sugar is trehalose or sucrose.

9. The formulation of claim 8, wherein the sugar concentration is from 10mM to 1M.

10. The formulation of claim 9, wherein the sugar concentration is from 60mM to 250 mM.

11. The formulation of claim 1, further comprising a surfactant, wherein the surfactant is a polysorbate.

12. The formulation of claim 11, wherein the surfactant is polysorbate 20.

13. The formulation of claim 11, wherein the concentration of the surfactant is from 0.0001% to 1.0%.

14. The formulation of claim 13, wherein the concentration of the surfactant is from 0.01% to 0.1%.

15. The formulation of claim 1 which is sterile.

16. The formulation of claim 1 which is stable upon storage at 40 ℃ for at least 4 weeks.

17. The formulation of claim 1 which is stable upon storage at 5 ℃ or 15 ℃ for at least 3 months.

18. The formulation of claim 1 which is stable upon storage at-20 ℃ for at least 3 months.

19. The formulation of claim 1, which is stable under freezing and thawing conditions.

20. The formulation of claim 1 which is aqueous.

21. The formulation of claim 1 which is frozen.

22. The formulation of claim 1 which is not lyophilized and has not been previously lyophilized.

23. The formulation of claim 22, which is aqueous and administrable to a subject.

24. The formulation of claim 23, wherein the formulation is for Intravenous (IV), Subcutaneous (SQ), or Intramuscular (IM) administration.

25. The formulation of claim 24 for IV administration, the antibody concentration being from 20mg/mL to 40 mg/mL.

26. The formulation of claim 24 for SQ administration, wherein the antibody concentration is from 80mg/mL to 250 mg/mL.

27. A vial with a stopper pierceable by a syringe, the vial containing the formulation of claim 1 therein.

28. The vial of claim 27 stored at 2-8 ℃.

29. The vial of claim 27, which is a 20cc or 50cc vial.

30. A stainless steel can comprising the formulation of claim 1 in the can.

31. The can of claim 30, wherein the formulation is frozen.

32. A pharmaceutical formulation, comprising:

(a) a full length humanized IgG1 antibody that is susceptible to deamidation or aggregation and which binds to HER2 in an amount of 10mg/mL to 250 mg/mL;

(b) histidine-acetate buffer, pH 5.5-6.5;

(c) a sugar selected from the group consisting of trehalose and sucrose in an amount of 60mM to 250 mM; and

(d) polysorbate 20 in an amount of 0.01% to 0.1%.

33. A method of reducing deamidation or aggregation of a therapeutic monoclonal antibody comprising formulating the antibody in histidine-acetate buffer, pH 5.5-6.5, wherein said antibody binds HER 2.

34. The method of claim 33, comprising assessing any antibody deamidation or aggregation prior to or after formulation of the antibody.

35. A pharmaceutical formulation comprising an antibody that binds to domain II of HER2 in histidine-acetate buffer at ph5.5 to 6.5, a sugar and a surfactant, wherein the sugar is trehalose or sucrose and the surfactant is a polysorbate.

36. The formulation of claim 35, wherein the buffer is histidine-acetate.

37. The formulation of claim 35 wherein the HER2 antibody comprises the variable light chain amino acid sequence and the variable heavy chain amino acid sequence in SEQ ID nos.3 and 4, respectively.

38. The formulation of claim 37 wherein the HER2 antibody comprises a light chain amino acid sequence selected from the group consisting of SEQ ID nos. 15 and 23, and a heavy chain amino acid sequence selected from the group consisting of SEQ ID nos. 16 and 24.

39. The formulation of claim 35, wherein the formulation has a pH of 5.8 to 6.2.

40. The formulation of claim 35, wherein the antibody is a full length antibody.

41. The formulation of claim 35, wherein the antibody concentration is 20mg/mL to 40 mg/mL.

42. A pharmaceutical formulation comprising Pertuzumab in an amount of 20mg/mL to 40mg/mL, a histidine-acetate buffer, sucrose and polysorbate 20, wherein the pH of the formulation is from 5.5 to 6.5.

43. The formulation of claim 42 comprising 30mg/mL Pertuzumab, 20mM histidine-acetate, 120mM sucrose, and 0.02% polysorbate 20, wherein the formulation has a pH of 6.0.

44. A vial with a stopper pierceable by a syringe containing the formulation of claim 35.

45. A stainless steel can comprising the formulation of claim 35 in the can.

46. A method of preparing a pharmaceutical formulation comprising:

(a) preparing the formulation of claim 1; and

(b) the physical stability, chemical stability or biological activity of the monoclonal antibodies in the preparation was evaluated.