Antibody, pharmaceutical composition, conjugate, isolated nucleic acid, vector, host cell and method for antibody production

The invention relates to an antibody, a pharmaceutical composition, a conjugate, an isolated nucleic acid, a vector, a host cell, and a method for producing the antibody.

The ErbB family of receptors with tyrosine kinase activities includes important mediators of cell growth, differentiation and survival. This family of receptors includes four distinct members, including the epidermal growth factor receptor (EGFR or ErbB1), HER2 (ErbB2 or p185^neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).

EGFR, encoded by the erbB1 gene, is causally associated with human malignancies. In particular, increased expression of EGFR has been observed in breast, bladder, lung, head, neck and stomach cancer as well as in glioblastomas. Increased expression of the EGFR receptor is often accompanied by increased production by the same tumor cells of the EGFR ligand transforming growth factor alpha (TGF-α), as a result of which the receptor is activated on the autocrine stimulation pathway. Baselga and Mandelsohn, Pharmac. Ther. 64: 127-154 (1994). Monoclonal antibodies directed against EGFR or its ligands, TGF-α and EGF, have been evaluated as therapeutic agents in the treatment of such malignancies. See, e.g., Baselga and Mandelsohn, supra; Masui et al., Cancer Research 44: 1002-1007 (1984) and Wu et al., J. Clin. Invest. 95: 1897-1905 (1995).

The second member of the ErbB family, p185^neu, originally identified as the transforming neuroblastoma gene product of chemically treated rats. The activated form of the neu proto-oncogene is formed as a result of a point mutation (valine to glutamic acid) in the transmembrane region of the encoded protein. Human neu homolog amplification is observed in breast and ovarian cancers and is correlated with poor prognosis (Slamon et al., Science, 235: 177-182 (1987); Slamon et al., Science 244: 707-712 (1989) and U.S. Patent No. 4,968,603). So far, no point mutation analogous to that in the neu proto-oncogene has been reported for human tumors. ErbB2 overexpression (often but not always 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, inter alia, King et al., Science, 229: 974 (1985); Yokota et al. Lancet: 1: 765-767 (1986); Fukushigi et al., Mol. Cell. Biol., 6: 955-958 (1986); Geurin 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: 421-425 (1990); Kern et al., Cancer Res. 50: 5184 (1990); Park et al., Cancer Res., 49: 6605 (1989); Zhau et al., Mol. Carcinog., 3: 354-357 (1990); Aasland et al., Br. J. Cancer. 57: 358-363 (1988); Williams et al., Pathiobiology 59: 46-52 (1991) and McCann et al., Cancer 65: 88-92 (1990). ErbB2 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 (1993)).

Antibodies to the p185 protein products have been described^neu rat and human ErbB2. Drebin et al. Obtained antibodies directed against the rat neu gene product, p185^neu. See, for example, 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 that as a result of using mixtures of antibodies reactive with two distinct regions of p185^neu synergistic anti-tumor effects are obtained against neu-transformed NIH-3T3 cells implanted into nude mice. See also U.S. Patent No. 5,824,311, issued October 20, 1998.

Hudziak et al., Mol. Cell. Biol. 9 (3): 1165-1172 (1989) describe the generation of a panel of anti-ErbB2 antibodies that were characterized using the human breast tumor cell line SK-BR-3. The relative cell proliferation of SK-BR-3 cells after antibody exposure was determined by crystal violet staining for the monolayers after 72 hours. Using this assay, the highest inhibition was obtained with the antibody called 4D5, which inhibited cell proliferation by 56%. The other antibodies in the kit reduced cell proliferation to a lesser extent in this assay. In addition, the 4D5 antibody has been found to sensitize ErbB2 overexpressing breast cancer cell lines to the cytotoxic effects of TNF-α. See also U.S. Patent No. 5,677,171, issued October 14, 1997. Discussed in Hudziak et al. anti-ErbB2 antibodies are further characterized in Fendly et al., Cancer Research 50: 1550-1558 (1990); Kotts et al., In Vitro 26 (3): 59A (1990); Sarup et al., Growth Regulation 1: 72-82 (1991); Shepard et al., J. Clin. Immunol. 11 (3): 117-127 (1991); Kumar et al., Mol. Cell ..

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Biol. 11 (2): 979-986 (1991); Lewis et al., Cancer Immunol. Immunother. 37: 255-263 (1993); 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: 7202-7206 (1994); Lewis et al., Cancer Research 56: 1457-1465 (1996) and Schaefer et al., Oncogene 15: 1385-1394 (1997).

Recombinant humanized version of the murine anti-ErbB2 antibody 4D5 (huMab4D5-8, rhuMAb HER2 or HERCEPTIN^®, U.S. Patent No. 5,821,337) is clinically active in ErbB2 overexpressing metastatic breast cancer patients who have previously undergone extensive anti-cancer therapy (Baselga et al., J. Clin. Oncol. 14: 737-744 (1996) ). HERCEPTTN^® was approved for sale by the Food and Drug Administration on September 25, 1998 for the treatment of patients with metastatic breast cancer that overexpresses the ErbB2 protein.

Other anti-ErbB2 antibodies with different properties are described in Tagliabue et al., Int. J. Cancer 47: 933-937 (1991); McKenzie et al., Oncogene 4: 543-548 (1989); Maier et al., Cancer Res. 51: 5361-5369 (1991); Bacus et al., Molecular Carcinogenesis 3: 350-362 (1990); Stancovski et al., PNAS (USA) 88: 8691-8695 (1991); Bacus et al., Cancer Research 52: 2580-2589 (1992); Xu et al., Int. J. Cancer 53: 401-408 (1993); international patent application number 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: 1367-1373 (1994); Arteaga et al., Cancer Res. 54: 3758-3765 (1994); Harwerth et al., J. Biol. Chem. 267: 15160-15167 (1992); U.S. Patent No. 5,783,186 and Klapper et al., Oncogene 14: 2099-2109 (1997).

A homology search identified two other members of the ErbB receptor family, ErbB3 (US Patent Nos. 5,183,884 and 5,480,968, as well as Kraus et al., PNAS (USA) 86: 9193-9197 (1989) ) and ErbB4 (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)). Both of these receptors are upregulated in at least some breast cancer cell lines.

ErbB receptors generally exist in various combinations in cells and heterodimerization is believed to increase the diversity of cellular responses to a wide variety of ErbB ligands (Earp et al., Breast Cancer Research and Treatment 35: 115-132 (1995)). EGFR is bound by six different ligands: epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), amphiregulin, heparin-binding epidermal growth factor, betacellulin and epiregulin (Groenen et al., Growth Factors 11: 235-257 ( 1994)). The ligands ErbB3 and ErbB4 are the family of heregulin proteins, resulting from the alternative splicing of a single gene. The heregulin family includes the alpha, beta, and gamma heregulins (Holmes et al., Science 256: 1205-1210 (1992); U.S. Patent No. 5,641,869 and Schaefer et al., Oncogene 15: 1385-1394 (1997)) ; neu differentiation factors (NDF); glial growth factors (GGF); Acetylcholine receptor inducing activity (ARIA) and sensory and motor neuron factor (SMDF). For a review see Groenen et al., Growth Factors 11: 235-257 (1994); Lemke, G., Molec. & Cell .. Neurosci. 7: 247-262 (1996) and Lee et al., Pharm. Rev. 47: 51-85 (1995). Three additional ErbB ligands have recently been identified: neuregulin-2 (NUMERG-2), which has been reported to bind to either ErbB3 or ErbB4 (Chang et al., Nature 387: 509-512 (1997) and Carraway et al. Nature 387: 512-516 (1997)); neuregulin-3, which binds ErbB4 (Zhang et al., PNAS (USA) 94 (18): 9562-7 (1997), and neuregulin-4, which binds ErbB4 (Hararii et al., Oncogene 18: 2681-89 (1999) ; HB-EGF, betacellulin and epiregulin also bind to ErbB4.

While EGF and TGF-α do not bind to ErbB2, EGF stimulates the formation of the EGFR and ErbB2 heterodimer, which activates EGFR and results in ErbB2 transphosphorylation in the heterodimer. Dimerization and / or transphosphorylation have been shown to activate ErbB2 tyrosine kinase. See Earp et al., Supra. Similarly, when ErbB3 is co-expressed with ErbB2, an active signaling complex is formed and anti-ErbB2 antibodies are capable of disrupting this complex (Sliwkowski et al., J. Biol. Chem. 269 (20): 14661-14665 (1994)). Additionally, the affinity of ErbB3 for heregulin (HRG) increases to a higher affinity state when it is co-expressed with ErbB2. Regarding the ErbB2-ErbB3 protein complex, see also Levi et al., Journal of Neuroscience 15: 1329-1340 (1995); Morrissey et al., Proc. Natl. Acad. Sci. USA 92: 1431-1435 (1995) and Lewis et al., Cancer Res. 56: 1457-1465 (1996). ErbB4, like ErbB3, forms an active signaling complex with ErbB2 (Carraway and Cantley, Cell. 78: 5-8 (1994)).

PL 203 326 B1

The invention relates to an antibody which has an amino acid sequence in SEQ ID number: 4 of a heavy variable domain (VH) and an amino acid sequence in SEQ ID number: 3 of a light variable domain (VL).

In a preferred embodiment, the antibody is an intact IgG1 antibody, or is an antibody fragment, or may be an antibody Fab fragment.

The invention also relates to a pharmaceutical composition, characterized in that it comprises antibodies which contain an amino acid sequence in SEQ ID number: 4 of the heavy variable domain (VH) and an amino acid sequence in the sequence of SEQ ID number: 3 of a light variable domain (VL), and a pharmaceutically acceptable carrier.

In a preferred embodiment, the pharmaceutical composition according to the invention is an aqueous solution, or it may be a lyophilized composition.

The invention further provides a conjugate which comprises antibodies as defined above conjugated to a cytotoxic agent.

Further, the invention also relates to an isolated nucleic acid encoding an antibody as defined above.

The invention also relates to a vector comprising an antibody coding nucleic acid of the invention.

The invention also relates to a host cell containing a vector of the invention as defined above.

The host cell is preferably a mammalian cell, and more preferably a Chinese Hamster Ovary (CHO) cell.

The invention also relates to a method of producing an antibody, characterized in that it comprises culturing a host cell containing a nucleic acid encoding an antibody that comprises the amino acid sequence in SEQ ID number: 4 of the heavy variable domain (VH) and the amino acid sequence in SEQ ID number: 3 of the light sequence variable domain (VL, such that expression of the nucleic acid and production of the antibody take place. In a preferred embodiment of the method, the antibody is recovered from the host cell culture, or from the host cell culture medium.

In the method of the invention, the host cell is a Chinese Hamster Ovary (CHO) cell.

In a most preferred aspect of the method of the invention, the recovered antibodies are mixed with a pharmaceutically acceptable carrier, excipient, or stabilizer to prepare a pharmaceutical composition containing said antibody.

Further advantages are obtained when the antibody used for therapy blocks ligand activation of the ErbB receptor and / or has the characteristics of monoclonal antibody 2C4. For example, while EGFR-targeting drugs interfere with the functions of only EGFR, the antibodies of particular interest in the present invention (e.g. 2C4, including its humanized and / or affinity maturity variants) will interfere with the function of the EGFR / ErbB2, ErbB3 / ErbB4 and ErbB2 / ErbB3 heterodimers. In addition, the antibodies of the present invention that bind to ErbB2 and block ligand activation of the ErbB receptor will be complementary to EGFR targeting drugs, while EGFR targeting drugs are not complementary to each other.

The claimed antibody can be used to treat human cancer, which cancer is characterized or not by overexpression of the ErbB2 receptor. Cancer that can be treated is breast cancer. It could also be metastatic breast cancer. The drug can be administered to a human with a chemotherapeutic agent. Such a chemotherapeutic agent may be agents belonging to the group consisting of anthracycline antibiotics, cyclophosphomide, taxane, nevelbine, xselod, mitomycin C, oxaliplatin, gemcitabine and the platinum compound.

Also described is the use of (a) a first antibody that binds to ErbB2 and inhibits the growth of cancer cells that overexpress ErbB2; and (b) a second antibody that binds ErbB2 and blocks ligand activation of the ErbB receptor, for the manufacture of a medicament for treating cancer in a human. Preferably, the first antibody for use in the invention comprises monoclonal 4D5 or humanized 4D5 and the second antibody comprises monoclonal 2C4 or humanized 2C4.

PL 203 326 B1

Antibodies that bind to ErbB2 and block ligand activation of the ErbB receptor may also be used in the manufacture of a medicament for the treatment of human cancer, which cancer is colon, rectum, colon and rectum.

The antibody of the invention may be a component of an article of manufacture that comprises a container and a composition contained therein, wherein the composition comprises an antibody that binds to ErbB2 and blocks ligand activation of the ErbB receptor, and further includes a package insert indicating that the composition can be used to treat cancer. wherein the cancer is not characterized by overexpression of the ErbB2 receptor. It can also be an article that includes (a) a first container with a composition contained therein, wherein the composition comprises a first antibody that binds to ErbB2 and inhibits the growth of ErbB2 overexpressing cancer cells, and (b) therein the composition, wherein the composition comprises a second antibody that binds to ErbB2 and blocks ligand activation of the ErbB receptor.

The present specification considers the various benefits of using an antibody that binds ErbB2 to treat cancer as opposed to drugs targeting EGFR. In particular, EGFR is highly expressed in the liver and skin, and this is a powerful signal for an active drug if the drug binds to EGFR. In addition, skin toxicity has been observed with other EGFR-targeting drugs such as the anti-EGFR chimeric antibody C225 and the small molecule drug ZD1839 which binds EGFR. Antibodies that bind ErbB2 are expected to have a better safety profile than such drugs.

Further advantages are obtained when the antibody used for therapy blocks ligand activation of the ErbB receptor and / or has the characteristics of monoclonal antibody 2C4. For example, while EGFR-targeting drugs interfere with the functions of only EGFR, the antibodies of particular interest in the present invention (e.g. 2C4, including its humanized and / or affinity maturity variants) will interfere with the function of the EGFR / ErbB2, ErbB3 / ErbB4 and ErbB2 / ErbB3 heterodimers. In addition, the antibodies of the present invention that bind to ErbB2 and block ligand activation of the ErbB receptor will be complementary to EGFR targeting drugs, while EGFR targeting drugs are not complementary to each other.

Brief description of the drawings

Figures 1A and 1B show epitope mapping of residues 22-645 within the extracellular domain (ECD) of ErbB2 (amino acid sequence, including signal sequence, is shown in Figure 1A; SEQ ID NO: 1) as determined by analysis of truncated mutants and site-directed mutagenesis. (Nakamura et al., J. of Virology 67 (10): 6179-6191 (1993) and Renz et al., J. Cell. Biol. 125 (6): 1395-1406 (1994)). Various truncated versions or point mutations of the ErbB2 ECD were prepared from the cDNA using the polymerase chain reaction technique. ErbB2 mutants were expressed as gD fusion proteins in a mammalian expression plasmid. The expression plasmid uses the cytomegalovirus promoter / enhancer and SV40 termination and polyadenylation signals downstream of the inserted sequence. Plasmid DNA was transfected into 293 cells. One day after transfection, cells were metabolically labeled overnight in methionine and cysteine-free low glucose DMEM medium containing 1% dialyzed fetal bovine serum and 25 μCi of each. ³⁵S-methionine and ³⁵S-cysteine. Supernatants were harvested and either anti-ErbB2 monoclonal antibodies or control antibodies were added to the supernatant and incubated at 4 ° C for 2-4 hours. The complexes were precipitated, loaded onto a gel with a 10-20% gradient of acrylamide in Tricine with SDS, and electrophoresis was run at 100 V. The gel was transferred to the membrane by electroblotting and analyzed by autoradiography. As shown in Fig. 1B, the anti-ErbB2 antibodies 7C2, 7F3, 2C4, 7D3, 3E8, 4D5, 2H11, and 3H4 bind to different ErbB2 ECD epitopes.

Figures 2A and 2B show the effect of anti-ErbB2 antibodies 2C4 and 7F3 on rHRGe1 activation of MCF7 cells. Figure 2 shows dose-response curves for 2C4 or 7Fs inhibition of HRG stimulation of tyrosine phosphorylation. Figure 2B shows dose-response curves for inhibition of labeled binding by 2C4 or 7F3¹²⁵And rHRGe1 _177-244 with MCF7 cells.

Figure 3 shows inhibition of labeled binding ¹²⁵And rHRGe1 _177-244 with a panel of human tumor cell lines by the anti-ErbB2 monoclonal antibodies 2C4 or 7F3. The controls for the monoclonal antibodies are mouse monoclonal antibodies of the same isotype that do not block rHRG binding. Non-specific labeled binding

PL 203 326 B1 ¹²⁵And rHRGei 177-244 was determined in parallel incubations performed in the presence of 10 nM rHRGei. Non-specific labeled binding values¹²⁵And rHRGe1 177-244 were lower than 1% of the total for all cell lines tested.

Figures 4A and 4B show the effect of monoclonal antibodies 2C4 and 4D5 on the proliferation of MDA-MB-175 (Figure 4A) and SK-BR-3 (Figure 4B) cells. MDA-MB-175 and SK-BR-3 cells were seeded in 96-well plates and allowed to adhere for 2 hours. The experiment was carried out in medium containing 1% serum. Anti-ErbB2 antibodies or medium alone were added and the cells were incubated for hours at 37 ° C. Then rHRGe1 (1 nM) or medium alone was added and cells were incubated for 4 days. Monolayers were washed and stained / fixed with 0.5% crystal violet. Absorbance was measured at 540 nm to determine cell proliferation.

Figures 5A and 5B show the effect of monoclonal antibody 2C4, HERCEPTIN® antibody, or anti-EGFR antibody on the heregulin-dependent (HRG) ErbB2 association with ErbB3 in MCF7 cells expressing low / normal levels of ErbB2 (Figure 5A), and in cells SK-BR-3 which expressed high levels of ErbB2 (Fig. 5B); see example 2 below.

Figures 6A and 6B compare the activities of intact murine monoclonal antibody 2C4 (mu 2C4) and the chimeric Fab fragment of 2C4. Fig. 6A shows binding inhibition¹²⁵I-HGR with MCF7 cells by chimeric 2C4 Fab or intact murine monoclonal antibody 2C4. MCF7 cells were seeded in 24-well plates (1 x 10⁵ cells / well) and grown to approximately 85% compact layer within two days. Binding experiments were performed as described in Lewis et al., Cancer Research 56: 1457-1465 (1996). Figure 6B shows the inhibition of rHRGe1 activation of p180 tyrosine phosphorylation in MCF7 cells performed as described in Lewis et al., Cancer Research 56: 1457-1465 (1996).

Figures 7A and 7B show the amino acid sequence alignments of the light chain variable (VL) domains (Figure 7A) and the heavy chain variable (VH) domains (Fig. 7B) murine monoclonal antibody 2C4 (SEQ ID NOS: 1 and 2, respectively); the VL and VH domains of the humanized version of 2C4 (SEQ ID NOS: 3 and 4, respectively) and the human VL and VH framework consensus sequences (hum κ1, subgroup I kappa light chain, humIII, subgroup III heavy chain) (respectively SEQ ID NOS: 5 and 6). Asterisks indicate the differences between the humanized version 2C4, 574 and the murine monoclonal antibody 2C4 or between the humanized version 574 of 2C4 and the human framework sequence. Complementarity Determining Regions (CDRs) are in parentheses.

Figures 8A through 8C show the binding of a chimeric 2C4 Fab (Fab.v1) and several humanized 2C4 variants to the extracellular domain of ErbB2 (ECD) as determined by ELISA in Example 3.

Figure 9 is a ribbon diagram of the VL and VH domains of monoclonal antibody 2C4 with the white CDR backbone labeled (L1, L21 L3, HI, H2, H3). V H side chains evaluated by mutagenesis during humanization are also shown (see example 3, table 2).

Figure 10 shows the effect of a monoclonal antibody^® 2C4 or HERCEPTIN^® on activation of Mitogen Activated Protein Kinase (MAPK) mediated by EGF, TGF-α or HRG.

Figure 11 is a bar graph showing the effect of anti-ErbB2 antibodies (alone or in combination) on Calu3 lung adenocarcinoma xenografts (3+ ErbB2 overexpression). Note: Treatment was stopped on day 24.

Figure 12 shows the effect of recombinant humanized monoclonal antibody 2C4 (rhuMAb 2C4) or HERCEPTIN^® on MDA-175 cell growth as assessed by Almar Blue assay.

Figure 13 shows the efficacy of rhuMAb 2C4 against MCF7 xenografts.

Detailed description of beneficial solutions

1. Definitions

The ErbB receptor is a receptor with protein kinase activity that belongs to the ErbB receptor family and includes EGFR, ErbB2, ErbB3 and ErbB4 receptors and other members of this family to be identified in the future. The ErbB receptor will generally contain an extracellular domain that can bind an ErbB ligand; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxy-terminal signaling domain that carries several tyrosine residues which may be phosphorylated. The ErbB receptor may be native

ErbB receptor sequence or amino acid sequence variant thereof. Preferably, the ErbB receptor is native sequence human ErbB receptor.

The terms ErbB1, epidermal growth receptor receptor, or EGFR are used interchangeably herein to refer to the EGFR disclosed in, for example, Carpenter et al., Ann. Rev. Biochem. 56: 881-914 (1987), including its naturally occurring mutant forms (e.g., an EGFR deletion mutant as in Humphrey et al., PNAS (USA) 87: 4207-4211 (1990)). erbB1 refers to the gene encoding the EGFR protein product.

The expressions ErbB2 and HER2 are used interchangeably herein and refer to the human HER2 proteins described, for example, in Semba et al., PNAS (USA) 82: 6497-6501 (1985) and Yamamoto et al., Nature 319: 230-234 (1986) (Genbank accession number X03363). The term erbB2 refers to the gene encoding human ErbB2 and neu refers to the gene encoding p185^neurat. A preferred ErbB2 sequence is native human ErbB2 sequence.

ErbB3 and HER3 refer to a receptor polypeptide disclosed, for example, in US Patent Nos. 5,183,884 and 5,480,968, as well as in Kraus et al., PNAS (USA) 86: 9193-9197 (1989).

The terms ErbB4 and HER4 refer to a receptor polypeptide disclosed, 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 isoforms thereof, e.g., disclosed in International Patent Application No. 99/19488, published April 22, 1999.

By ErbB ligand is meant a polypeptide that binds to and / or activates an ErbB receptor. An ErbB ligand of particular interest herein is the native sequence of a human ErbB ligand, such as epidermal growth factor (EGF) (Savage et al.,

J. Biol. Chem. 247: 7612-7621 (1972), transforming growth factor alpha (TGF-α) (Marquardt et al., Science 223: 1079-1082 (1984); amphiregulin, also known as neuroblastoma or keratinocyte autocrine growth factor (Shoyab 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), betacellulin (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); 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 (NUMERG-2) (Carraway et al., Nature 387: 512-516 (1997) )), neuregulin-3 (NUMERG-3) (Zhang et al., Proc. Natl. Acad. Sci. 94: 9562-9567 (1997)), neuregulin-4 (NUMERG-4) (Harari et al., Oncogene 18: 2681-89 (1999) or cripto (CR-1) (Kannani et al., J. Biol. Chem. 272 (6): 3330-3335 (1997)) ErbB ligands that bind EGFR include EGF, TGF-α, amphiregulin, betacellulin, HB-EGF, and epiregulin ErbB ligands that bind ErbB3 include heregulins ErbB ligands ErbB capable of binding ErbB4 include betacellulin, epiregulin, HB-EGF, NUMERG-2, NUMERG-3, NUMERG-4, and heregulins.

Heregulin (HRG) when used herein refers to a polypeptide product encoded by the heregulin gene as disclosed in U.S. Patent No. 5,641,869 or Marchionni et al., Nature, 362: 312-318 (1993 ). Examples of heregulin include α heregulin, β1 heregulin, β2 heregulin, and β3 heregulin (Holmes et al., Science, 256: 1205-1210 (1992) and US Patent No. 5,641,869), neu differentiation factor (NDF) (Peles et al., Cell. 69: 205-216 (1992)); Acetylcholine receptor (ARIA) inducing activity (Falls et al., Cell. 72: 801-815 (1993)); glial growth factors (GGF) (Marchionni et al., Nature, 362: 312-318 (1993); sensory and motor neuron factor (SMDF) (Ho et al., J. Biol. Chem. 270: 14523-14532 (1995) )); heregulin γ (Schaefer et al., Oncogene 15: 1385-1394 (1997)) The term encompasses biologically active fragments and / or amino acid sequence variants of the native sequence of the HRG polypeptide, such as a fragment of the EGF-like domain (e.g., HRGe1_177-244 ).

The ErbB hetero-oligomer of the present invention is non-covalently linked to an oligomer comprising at least two different ErbB receptors. Such complexes can form when a cell expressing two or more receptors is exposed to the ErbB ligand, and can be isolated by immunoprecipitation and analyzed by SDS-PAGE as described, for example, in Sliwkowski et al., J. Biol. . Chem., 269 (20): 14661-14665 (1994). Examples of such ErbB heterooligomers include the EGFR-ErbB2, ErbB2-ErbB3 and ErbB3-ErbB4 complexes. In addition, the ErbB heterooligomer may contain two or more ErbB2 receptors linked to another ErbB receptor.

Such as ErbB3, ErbB4 or EGFR. The heterooligomer may include other proteins, such as a cytokine receptor subunit (e.g., gp130).

By ligand activation of the ErbB receptor is meant signal transduction (e.g., that caused by an intracellular domain with ErbB receptor kinase activity, phosphorylating tyrosine residues of an ErbB receptor or a polypeptide substrate) mediated by the binding of an ErbB ligand to an ErbB heterooligomer containing the ErbB receptor of interest. Generally, it will involve the binding of an ErbB ligand to an ErbB hetero-oligomer that activates a kinase domain of one or more of the ErbB receptors in the hetero-oligomer and thus phosphorylation of tyrosine residues on one or more of the ErbB receptors and / or phosphorylation of tyrosine residues in the additional (s) ) a polypeptide substrate. ErbB receptor activation can be quantified using various tyrosine phosphorylation assays.

A onative sequence polypeptide is a polypeptide that has the same amino acid sequence as a naturally derived polypeptide (e.g., an ErbB receptor or an ErbB ligand). Such native sequence polypeptides can be isolated from their natural source, or can be produced by recombinant or synthetic means. Thus, a native sequence polypeptide may have the amino acid sequence of a naturally occurring human polypeptide, a murine polypeptide, or a polypeptide of any other mammalian species.

The term amino acid sequence variant refers to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide. Typically, the amino acid sequence variants will have at least about 70% homology to at least one native ErbB receptor binding domain or the at least one native ErbB ligand binding domain, and preferably will be at least about 80%, more preferably at least about 90%. % homologous to such receptor or ligand binding domains. The amino acid sequence variants have substitutions, deletions, and / or insertions at certain positions in the amino acid sequence with respect to the native amino acid sequence.

Homology is defined as the percentage of the residues in an amino acid sequence variant that are identical after aligning the sequences and introducing gaps, if necessary, yielding the maximum percentage of homology. Assignment methods and computer programs are well known in the art. One such computer program is Align 2 by Genentech, Inc. which is filed with the user documentation at the United States Copyright Office, Washington, DC 20559 on December 10, 1991.

The term antibody as used herein is used in the broadest sense and specifically includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) made of at least two intact antibodies, and antibody fragments so far having the desired biological activity.

The term monoclonal antibody as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie the individual antibodies making up the population are identical, except for possible naturally occurring mutations which may be present in minor amounts. Monoclonal antibodies are highly specific, directed against single antigenic sites. Moreover, unlike polyclonal antibody preparations that include different antibodies to different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized not contaminated with other antibodies. The term monoclonal indicates the nature of the antibody obtained from a substantially homogeneous population of antibodies, and is not intended to require production of the antibody by any particular method. For example, monoclonal antibodies for use in the present invention can be made by the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or can be made by recombinant DNA methods (see e.g. U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage display libraries using the techniques described in, for example, Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991).

Monoclonal antibodies herein, in particular, include chimeric antibodies in which a portion of the heavy and / or light chain is identical to or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies. while the remainder of the chain (s) are identical to or homologous to the corresponding sequences in antibodies derived from a different species or belonging to a different class or subclass of antibodies, as well as fragments of such antibodies, so far they exhibit the desired biological activity (description U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). Chimeric antibodies of interest herein include primate-like antibodies containing antigen-binding variable domain sequences derived from non-human primates (e.g., Old World monkeys, great apes, etc.) and human constant region sequences.

Antibody fragments include a portion of an intact antibody, preferably include its antigen binding or variable region. Examples of antibody fragments include Fab, Fab ', F (ab') 2, and Fv fragments; diabodies; linear antibodies; single chain antibody molecules; and multispecific antibodies formed from the antibody fragment (s).

An intact antibody is an antibody that comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2, and CH3. The constant domains may be native sequence constant domains (human native sequence constant domains) or an amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

Antibody effector functions relate to those biological activities that are attributable to the Fc region (native sequence Fc region or amino acid sequence variant Fc region) of the antibody. Examples of antibody effector functions include C1q binding, complement dependent cytotoxicity, Fc receptor binding, antibody dependent cellular cytotoxicity (ADCC), phagocytosis, depletion of cell surface receptors (e.g. B cell receptor, BCR) etc.

Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of them can be further subdivided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, respectively. γ and μ. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

Antibody dependent cellular cytotoxicity and ADCC refer to a cell mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g. NK cells, neutrophils, and macrophages) recognize an antibody bound on a target cell and then lysis of the target cell. The main cells involved in ADCC, NK cells, express only FcyIII, while monocytes express FcyRI, FcyII and FcyIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991). In order to assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or 5,821,337, can be performed. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs). and NK cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g. in an animal model such as that disclosed in Clynes et al., PNAS (USA) 95: 652-656 (1998).

Human effector cells are leukocytes which express one or more FcRs and which perform effector functions. Preferably, the cells express at least FyRIII and perform an ADCC effector function. Examples of human leukocytes that are involved in ADCC include peripheral blood mononuclear cells (PBMCs), NK cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and NK cells being particularly preferred. Effector cells can be isolated from their natural source, e.g., blood or PBMCs as described herein.

The terms Fc receptor or FcR are used to describe a receptor that binds to the Fc region of an antibody. A preferred FcR is the native sequence human FcR. Moreover, a preferred FcR is one that binds an IgG antibody (a gamma receptor) and has receptors of the subclasses FcyRI, FcyRII, and FcyRIII, including allelic variants and alternate splicing forms of these receptors. FcyRII receptors include FcyRIIA (activating receptor) and FcyRIIB (inhibitory receptor), which have similar amino acid sequences and differ mainly in their domains

With cytoplasmic cells. The activating receptor FcyRIIA contains a tyrosine-based immunoreceptor activation (ITAM) motif in its cytoplasmic domain. Inhibiting the FcyRIIB receptor in its cytoplasmic domain, the tyrosine-based immunoreceptor inhibition motif (see for review in M. Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcR is reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994) and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). The term FcR as used herein includes other FcRs, including those to be identified in the future. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of 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) complexed with the appropriate antigen. To assess complement activation, a CDC assay can be performed, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996).

Native antibodies are typically heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intra-chain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The light chain constant domain is located parallel to the first heavy chain constant domain and the light chain variable domain is located parallel to the heavy chain variable domain. Certain amino acid residues are believed to form the boundary between the light chain and heavy chain variable domains.

The term variable refers to the fact that certain portions of a variable domain vary significantly in sequence between antibodies and are used in the binding and specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments, called hypervariable regions, in both the light chain and the heavy chain variable domains. The more conserved portions of the variable domains are called framework regions (FR). The native heavy and light chain variable domains each contain four FRs, most of them taking the β structure configuration, connected by three hypervariable regions that form connecting loops and in some cases forming part of the β structure. The hypervariable regions in each chain are held in close proximity by the FRs and, with the hypervariable regions of the other chain, are involved in the formation of the antigen-binding site of the antibodies (see Kabat et al., Sequences of Proteins of Immunological Interst, 5th ed., Public Health Service, pp. National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but have various effector functions, such as antibody involvement in antibody dependent cellular cytotoxicity (ADCC).

The term hypervariable region as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally includes amino acid residues from the complementarity determining region or CDR (e.g. residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the variable domain heavy chain Kabat et al., Sequences of Proteins of Immunological Interst, ed. 5, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or the rest of the hypervariable loop (e.g. residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the variable domain heavy chain; Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). Framework or FR residues are those variable domain residues which are different from the hypervariable region residues as defined herein.

Papain digestion of antibodies produces two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site, and a residual Fc fragment, whose name reflects its ability to crystallize readily. Treatment

Pepsin yields an F (ab ') 2 fragment that has two antigen binding sites and is still capable of cross-linking antigen.

Fv is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent bonding. It is configured such that the three hypervariable regions of each variable domain interact to define an antigen binding site on the surface of the V H -V L dimer. In total, the six hypervariable regions impart antigen binding specificity to the antibody. However, even a single variable domain (or a half of an Fv containing only three antigen-specific hypervariable regions) has the ability to recognize and bind an antigen, albeit with a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab 'fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab 'in which the cysteine ​​residue (s) of the constant domains have at least one free thiol group. F (ab ') 2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical coupling methods for antibody fragments are also known.

The light chains of antibodies from any vertebrate species can, based on the amino acid sequences of their constant domains, fall into one of two clearly distinct types, termed kappa (κ) and lambda (λ).

Single-chain Fv or scFv antibody fragments contain antibody VH and VL domains, which domains occur on a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding. For a review of scFv see Pliickthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994). Anti-ErbB2 scFv antibody fragments are described in WO 93/16185, US Patent No. 5,571,894 and US Patent No. 5,587,458.

The term diabody refers to small antibody fragments with two antigen binding sites, which fragments contain a heavy chain variable domain (VH) linked to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains of the same chain, pairing is forced with the complementary domains of another chain and the formation of two antigen binding sites. Diabodies are described more fully in, for example, EP 404,097, WO 93/11161, and in Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

Humanized forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human (recipient antibody) immunoglobulins in which the recipient's hypervariable region residues have been replaced with non-human (donor antibody) hypervariable region residues, such as mouse, rat, rabbit, or non-human primate antibodies. a human having the desired specificity, affinity and binding capacity. In some instances, human immunoglobulin framework region residues are replaced with corresponding non-human species residues. In addition, humanized antibodies may contain residues that are not present in either the antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will contain 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. Optionally, the humanized antibody will also include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details see Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-327 (1988) and Presta, Curr. Op. Stirs. Biol. 2: 593-596 (1992).

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Humanized anti-ErbB2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7, and huMAb4D5-8 (HERCEPTIN^®), as described in Table 3 of US Patent No. 5,821,337, the humanized antibody 520C9 (WO 93/21319) and the humanized antibodies described herein below.

An isolated antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are materials that could interfere with the diagnostic or therapeutic use of the antibody, and can include enzymes, hormones, and other protein and non-protein solutes. In preferred embodiments, the antibody will be purified (1) to a degree greater than 95% by weight of the antibody as determined by the Lowry method, and most preferably greater than 99% by weight, (2) to a degree sufficient to obtain a sequence of at least 15 N-terminal residues. and internal using a spin sequencer, or (3) for homogeneity on SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Typically, however, an isolated antibody will be prepared by at least one purification step.

An antibody that binds an antigen of interest, e.g., an ErbB2 antigen, is an antibody capable of binding the antigen with sufficient affinity such that the antibody is useful as a therapeutic targeting agent for cells expressing the antigen. If the antibody is an antibody that binds ErbB2, it will typically preferentially bind ErbB2 as opposed to other ErbB receptors, and may be one that does not significantly cross-react with other proteins such as EGFR, ErbB3, or ErbB4. In such embodiments, the degree of binding to these non-ErbB2 proteins (e.g. binding to endogenous cell surface receptors) will be less than 10% as determined by fluorescence activated cell sorting (FACS) or radioimmunoprecipitation (RIA) analysis. Occasionally, an anti-ErbB2 antibody will not significantly react with rat neu protein, e.g., as described in Schecter et al., Nature 312: 513 (1984) and Drebin et al., Nature 312: 545-548 (1984).

An antibody that blocks activation by an ErbB receptor ligand is an antibody that reduces or prevents such activation as hereinbefore defined, wherein the antibody is capable of blocking ligand activation of the ErbB receptor significantly more effectively than the monoclonal 4D5 antibody, e.g. approximately as effective as monoclonal antibody 7F3 or 2C4 or Fab fragments thereof, and preferably approximately as effective as monoclonal antibody 2C4 or Fab fragment thereof. For example, the antibody that blocks ligand activation of the ErbB receptor can be an antibody that is about 50-100% more effective than 4D5 at blocking ErbB heterooligomer formation. Blocking ligand activation of the ErbB receptor may occur in other ways, e.g. by interfering with ligand binding to ErbB receptor, ErbB complex formation, receptor tyrosine kinase activity in the ErbB complex, and / or phosphorylation of tyrosine kinase residues (s) at or through the ErbB receptor. Examples of antibodies that block ligand activation of the ErbB receptor include monoclonal antibodies 2C4 and 7F3 (which block the activation of ErbB2 / ErbB3 and ErbB2 / ErbB4 heterooligomers by HRG and activation of the EGFR / ErbB2 heterooligomer by EGF, TGF-α, HB-EGF, and / or epiregulin) and antibodies L26, L96 and L288 (Klapper et al., Oncogene 14: 2099-2109 (1977)) that block EGF and NDF binding to T47D cells expressing EGFR, ErbB2, ErbB3 and ErbB4 .

An antibody having a biological property of a particular antibody, such as a monoclonal antibody designated 2C4, is an antibody that has one or more of that antibody's biological properties that distinguish it from other antibodies that bind to the same antigen (e.g., ErbB2). For example, 2C4 antibodies can block activation of an ErbB heterooligomer including ErbB2 and ErbB3 or ErbB4 by HRG; block activation of an ErbB receptor including EGFR and ErbB2 by EGF, TGF-α, HB-EGF, epiregulin and / or amphiregulin; block TGF-α and / or HRG mediated activation of MAPK and / or bind to the same epitope in the extracellular domain of ErbB2 that binds to 2C4 (e.g. thereby blocking the binding of monoclonal antibody 2C4 to ErbB2).

Unless otherwise indicated, the phrase monoclonal antibody 2C4 refers to an antibody that has the antigen-binding residues of the murine 2C4 antibody described in the examples below.

Or derived therefrom. For example, the monoclonal antibody 2C4 can be the murine monoclonal antibody 2C4 or a variant thereof, such as humanized antibody 2C4, having antigen binding amino acid residues of the murine monoclonal antibody 2C4. Examples of humanized 2C4 antibodies are provided in Example 3 below. Unless otherwise indicated, the phrase rhuMAb 2C4 as used herein refers to an antibody having a light chain (VL) variable and a heavy chain (VH) variable sequence of SEQ ID NOS: 3 and 4, respectively, fused to human IgG1 light and heavy chain constant region sequences (allo-type non-A) possibly expressed in a Chinese hamster ovary (CHO) cell.

Unless otherwise indicated, the term monoclonal antibody 4D5 refers to an antibody that has antigen binding residues from, or derived from, the murine 4D5 antibody (ATCC CRL 10463). For example, the monoclonal antibody 4D5 can be the murine monoclonal antibody 4D5 or a variant thereof, such as the humanized antibody 4D5, having the antigen-binding residue of the murine monoclonal antibody 4D5. Exemplary humanized 4D5 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7, and huMAb4D5-8 (HERCEPTIN^®), as in U.S. Patent No. 5,821,337, wherein huMAb4D5-8 (HERCEPTIN^®) is a preferred humanized 4D5 antibody.

Growth inhibitory agent as used herein refers to a compound or composition that inhibits the growth of a cell, especially an ErbB-expressing cancer cell, either in vitro or in vivo. Thus, the growth inhibitory factor may be a factor that significantly reduces the percentage of ErbB-expressing cells in the S phase. Examples of growth inhibitory factors include factors that block cell cycle progression (non-S phase) such as factors that induce G1 arrest and M phase arrest. Classical M phase blocking agents include vinca alkaloids (vincristine and vinblastine) , taxanes and topoisomerase II inhibitors such as doxorubicin, epirubicin, etoposide and bleomycin. The action of those agents that cause G1 arrest, for example DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, chlormethine, cisplatin, methotrexate, 5-fluorouracil and ara-C, also extend to S phase retention. see The Molecular Basis of Cancer, Mendelsohn and Israel, eds. Chapter 1 titled Cell. cycle regulation, oncogenes, and antineoplastic drugs by Murakami et al. (WB Saunders, Philadelphia, 1995), especially on page 13.

Examples of growth inhibitory antibodies are those that bind to ErbB2 and inhibit the growth of cancer cells that overexpress ErbB2. Preferred growth inhibitory anti-ErbB2 antibodies inhibit the growth of SK-BR-3 breast tumor cells in cell culture by more than 20%, preferably more than 50% (e.g. about 50% to about 100%) at an antibody concentration of about 0.5 to 30 μg / ml when growth inhibition is determined after six days of exposure of SK-BR-3 cells to the antibody (see U.S. Patent No. 5 677 171, issued October 14, 1997). The SK-BR-3 cell growth inhibition assay is described in more detail in this patent and hereinbelow. A preferred growth inhibitory antibody is monoclonal 4D5, e.g. humanized 4D5.

An antibody that induces cell death is an antibody that makes a viable cell non-viable. A cell is generally a cell that expresses the ErbB2 receptor, especially if the cell overexpresses the ErbB2 receptor. Preferably, the cell is a cancer cell, e.g. a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cell. In vitro, the cell may be an SK-BR-3, BT474, Calu 3, MDA-MB-453, MDA-MB-361, or SK0V3 cell. In vitro cell death can be determined in the absence of complement and immune effector cells to differentiate cell death induced by antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). A zate , a cell death assay can be performed using a heat-inactivated serum (i.e. in the absence of complement) and in the absence of immune effector cells. To determine if an antibody is capable of inducing cell death, loss of cell membrane integrity as assessed by uptake of Propidium Iodide (PI), Triptan Blue (see Moore et al., Cytotechnology 17: 1-11 (1995)) 7AAD can be assessed against to untreated cells. Preferred cell death-inducing antibodies are those that induce PI uptake by BT474 cells in a PI uptake assay (see below).

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The antibody that induces apoptosis is one that induces programmed cell death as determined by annexin V binding, DNA fragmentation, contraction, expansion of the endoplasmic reticulum, cell fragmentation, and / or formation of membrane vesicles (called apoptotic bodies). The cell is usually one which overexpresses the ErbB2 receptor. Preferably, the cell is a cancer cell, e.g. a cell in the breast, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas, or bladder. In vitro, the cell may be an SK-BR-3, BT474, Calu 3, MDA-MB-453, MDA-MB361, or SKOV3 cell. Various methods are available for assessing cellular apoptosis events. For example, phosphatidylserine (PS) translocation can be measured by annexin binding; DNA fragmentation can be assessed by observing DNA fragments on an electrophoretic gel, and nuclear / chromatin condensation together with DNA fragmentation can be assessed by increasing the number of hypodiploid cells. Preferably, the apoptosis-inducing antibody is an antibody that induces annexin binding that is about 2- to 50-fold greater, preferably about 5- to 50-fold, and most preferably about 10 to 50-fold more potent than the cells. untreated in an annexin binding assay using BT474 cells (see below). Occasionally, the pro-apoptotic antibody will be an antibody that further blocks the activation of the ErbB receptor by an ErbB ligand (e.g. antibody 7F3); ie, the antibody has the same biological property as monoclonal antibody 2C4. In other situations, the antibody is an antibody that does not significantly block ErbB receptor activation by ErbB ligand (e.g., 7C2). The antibody may be a 7C2-like antibody which, while it induces apoptosis, does not induce a large decrease in the percentage of cells in the S phase (e.g., an antibody that induces only about 0-10% decrease in the percentage of these cells relative to control.

The 2C4 epitope is the region in the extracellular domain of ErbB2 to which the 2C4 antibody binds. To screen for antibodies that bind to the 2C4 epitope, routine cross-blocking assays can be performed, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow, and David Lane (1988). Alternatively, epitope mapping can be performed to assess whether the antibody binds to the 2C4 epitope of ErbB2 (e.g. any or more residues in the region from about residue 22 to about residue 584ErbB2, inclusive; see Figs. 1A-B).

The 4D5 epitope is the region in the extracellular domain of ErbB2 to which antibody 4D5 (ATCC CRL 10463) binds. This epitope is close to the ErbB2 transmembrane domain. To screen for antibodies that bind to the 4D5 epitope, routine cross-blocking assays can be performed, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow, and David Lane (1988). Alternatively, epitope mapping may be performed to assess whether the antibody binds to the 4D5 epitope of ErbB2 (e.g., any or more residues in the region from about residue 529 to about residue 625 of ErbB2, inclusive; see Figures 1A-B).

The 3H4 epitope is the region in the extracellular domain of ErbB2 to which the 3H4 antibody binds. This epitope includes residues from about 541 to about 599, inclusive, in the amino acid sequence of ErbB2's extracellular domain; see Figs. 1A-B.

The 72C / 7F3 epitope is the region at the N terminus of the extracellular domain of ErbB2 to which the 7C2 and / or 7F3 antibodies (each deposited with the ATCC, see below) bind. To screen for antibodies that bind to the 72C / 7F3 epitope, routine cross-blocking assays can be performed, such as that described in Anibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively, epitope mapping may be performed to assess whether the antibody binds to the ErbB2 epitope 72C / 7F3 (e.g., any or more residues in the region from about residue 22 to about residue 53 of ErbB2, inclusive; see Figures 1A-B).

Treatment refers to both therapeutic and prophylactic treatment, or measurements for prophylactic purposes. Individuals in need of treatment include those who already have the disorder as well as those who are preventing the disorder. Therefore, the mammal to be treated according to the present invention may already be diagnosed as having the disorder, or may be predisposed or prone to the disorder.

A mammal for treatment purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo animals, sports animals, or domestic pets such as dogs, horses, cats, cows, etc. Preferably. , a mammal is a human.

A disorder is any condition that would improve with treatment with the anti-ErbB2 antibody. It includes chronic or acute disorders or diseases, including those pathological conditions which predispose the mammal to the disorder of interest. Non-limiting examples of disorders that will be treated in accordance with the present invention include benign and malignant neoplasms, leukemias and malignant neoplasms of lymphoid tissue; neuronal, astrocytic, hypothalamic or other glandular, macrophage, epithelial, stromal, and blastocellic disorders, and inflammatory, angiogenic and immune disorders.

The term therapeutically effective amount refers to an amount of a drug effective to treat a disease or disorder in a mammal. In cancer, a therapeutically effective amount of the drug may reduce the number of cancer cells, reduce the size of the tumor, inhibit (i.e. slow to some extent, and preferably stop) cancer cell infiltration into peripheral organs, inhibit (i.e. to slow to some extent, and preferably to arrest) tumor metastasis, to inhibit to some extent tumor growth, and / or to alleviate to some extent one or more of the symptoms associated with the cancer. To some extent, the drug may prevent growth and / or kill existing tumor cells, and may be cytostatic and / or cytotoxic. In cancer therapy, efficacy can be measured, for example, by assessing disease development time (TTP) and / or determining rate of response (RR).

The terms cancer and cancerous refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, cancer, lymphoma, blastoma, sarcoma and leukemia, or a malignant tumor of the lymphoid tissue. More specific examples of such cancers include squamous cell carcinoma (e.g. squamous cell carcinoma), lung cancer, including small cell carcinoma, non-small cell carcinoma of the lung, adenocarcinoma of the lung and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer, including gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer , liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colon and rectal cancer, cancer of the endometrium or uterus, cancer of the salivary glands, kidney cancer, prostate cancer, vulva cancer, thyroid cancer, liver cancer, rectal cancer, penile cancer, as well as head and neck cancer.

An ErbB-expressing cancer is a cancer that contains cells that have the ErbB protein on their cell surfaces. An ErbB2-expressing cancer is a cancer that produces sufficient levels of ErbB2 on its cell surface so that an anti-ErbB2 antibody can bind to them and exert a therapeutic effect on the cancer.

A cancer characterized by over-activation of the ErbB receptor is cancer in which the degree of ErbB receptor activation in the cancer cells is significantly greater than that in non-cancer cells of the same tissue type. Such excessive activation may result from overexpression of the ErbB receptor and / or higher than normal levels of ErbB ligand available for activation of the ErbB receptor on cancer cells. Such over-activation may cause and / or be caused by the malignant state of the cancer cells. In some embodiments, the cancer will undergo a diagnostic or prognostic assay to determine whether there is an upregulation or overexpression of the ErbB receptor that results in such excessive activation of the ErbB receptor. Alternatively, or additionally, the cancer may be subjected to a diagnostic or prognostic assay to determine whether the cancer is experiencing an upregulation and / or overexpression of the ErbB ligand attributed to excessive receptor activation. In a subset of such cancers, receptor overactivation may be the result of an autocrine stimulation pathway.

The autocrine stimulation pathway involves self-stimulation of cancer cells that produce both the ErbB ligand and the corresponding ErbB receptor. For example, cancer may express or overexpress an EGFR ligand (e.g., EGF, TGF-α or HB-EGF). In another embodiment, cancer may express or overexpress heregulin (e.g., γ-HRG).

A cancer which overexpresses the ErbB receptor is cancer which has significantly higher levels of the ErbB receptor, such as ErbB2, on its cell surfaces as compared to a non-cancerous tissue cell of the same type. Such overexpression may be due to gene amplification or increased transcription or translation. ErbB receptor overexpression can be determined in a diagnostic or prognostic assay by identifying increased levels.

ErbB protein present on the surface of cells (e.g., in an immunohistochemical assay, IHC). Alternatively, or additionally, levels of the ErbB-encoding nucleic acid in the cell can be measured, e.g., by in situ fluorescence probe hybridization techniques (FISH; see International Patent Application No. 98/45479, published October 1998), Southern blotting or polymerase chain reaction ( PCR) such as quantitative real-time PCR (RT-PCR). ErbB receptor overexpression may also be examined by measuring shed antigen (e.g., the extracellular domain of ErbB) in a biological fluid such as serum (see e.g., U.S. Patent No. 4,933,294, issued June 12, 1990, International Patent Application No. 91 / 05264, published April 18, 1991, U.S. Patent No. 5,401,638, issued March 28, 1995, and Sias et al., J. Immunol. Methods 132: 73-80 (1990)). In addition to the above assays, numerous in vivo assays are available to those skilled in the art. For example, cells in the patient's body can be exposed to an antibody that is optionally labeled with a detectable label, e.g., a radioactive isotope, and the binding of the antibody to the patient's cells can be assessed, e.g., by external radioactivity testing, or by analyzing a biopsy taken from a patient previously exposed to the antibody. .

In contrast, a cancer that does not overexpress the ErbB2 receptor is one that does not express higher than normal levels of the ErbB2 receptor in a diagnostic assay as compared to non-cancerous tissue cells of the same type.

A cancer that overexpresses the ErbB ligand is one that produces significantly higher levels of this ligand as compared to a non-cancerous tissue cell of the same type. Such overexpression may be due to gene amplification or increased transcription or translation. ErbB ligand overexpression can be determined for diagnostic purposes by identifying increased levels of the ligand (or its encoding nucleic acid) in a patient, e.g. in a tumor biopsy or in various diagnostic assays such as IHC, FISH, PCR, or the in vivo assays described above.

Hormone independent cancer is cancer in which cell proliferation is not dependent on the presence of a hormone that binds to a receptor expressed by the cancer cells. In such cancers, there is no clinical regression following the use of pharmacological or surgical strategies that lower the concentration of the hormone in or near the tumor. Examples of hormone independent cancers include androgen independent prostate cancer, estrogen independent breast cancer, endometrial cancer, and ovarian cancer. Such cancers may initially develop as hormone dependent cancers and progress from a hormone sensitive stage to a hormone insensitive neoplasm following antihormone therapy.

The term cytotoxic agent as used herein refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells.

211 131 125 90 186 188

The term intent includes radioactive isotopes (e.g., At²¹¹, And¹³¹ AND¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸,

Sm¹⁵³, Bi²¹², p³² and radioactive Lu isotopes), chemotherapeutic agents and toxins such as low molecular weight or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof.

A chemotherapeutic agent is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN ™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide, and trimethylol melamine; nitrogen mustard derivatives such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycin, actinomycin, autramycin, azaserin, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carcinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diaorinzorubicin, epubicin, 6-diaorubicin, oxorubicin, 6-diaorubicin, doxorubicin, epubicin, lorubicin-5-doxorubicin idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercapto purine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, phloxouridine, 5-FU; androgens such as calusterone, propioniandromostanolone, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglaton ,; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucyl; byzantrene; edatract; defofamine; demecolcin; diazykwon; elfornithine; Eliptinium Acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenarmet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK^®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triazonone; 2,2 ', 2-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosin; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; reel; nowantron, teniposide; daunomycin; aminopterin; xselode; ibandronate; CPT-11; RFS 2000 topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoic acid; Esperamicin; capecitabine; and pharmaceutically acceptable salts, acids and derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit the action of hormones on tumors such as antiestrogens including, for example, tamoxifen, raloxifene, 4 (5) -imidazole inhibitory aromatase, 4-hydroxy tamoxifen, trioxifene, keoxifen, LY 117018, onapristone and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin, and pharmaceutically acceptable salts, acids and derivatives of any of the above.

As used herein, the term EGFR targeting drug 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 to EGFR. Examples of antibodies that bind to EGFR include monoclonal antibody 579 (ATCC CRL HB 8506), monoclonal antibody 455 (ATCC CRL HB8507), monoclonal antibody 225 (ATCC CRL 8508), monoclonal antibody 528 (ATCC CRL 8509) (see Patent Specification Of the United States of America 4,943,533, Mandelsohn et al.) And variants thereof, such as chimeric 225 (C225) and transformed human 225 (H225) (see International Application 96/40210, Imclone Systems Inc.), antibodies, which binds EGFR type II mutant (U.S. Patent 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 (U.S. Application international number 98/50433, Abgenix). An anti-EGFR antibody can be conjugated to a cytotoxic agent, thereby forming an immunoconjugate (see e.g. EP 659 439A2, Merck Patent GmbH). Examples of small molecules that bind to EGFR include ZD1839 (Astra Zeneca), CP-358774 (OSl / Pfizer), and AG1478.

An anti-angiogenic factor refers to a compound that blocks, or interferes to some extent, the development of blood vessels. The anti-angiogenic factor can be, for example, a small molecule or antibody that binds to a growth factor or growth factor receptor involved in promoting angiogenesis. A preferred anti-angiogenic agent as used herein is an antibody that binds to vascular endothelial growth factor (VEGF).

The term cytokine is a general term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. The cytokines include growth hormone such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyrotropin hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor α and β; a substance that inhibits the development of Milerian ducts; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor such as NGF-β; platelet growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor I and II; erythropoietin (EPO); bone induction factors; interferons such as interferons α, β, and γ; two18 stimulating factors

Colony Stimulating Factor (CSF), such as Macrophage Colony Stimulating Factor (M-CSF), Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and Granulocyte Colony Stimulating Factor (G-CSF); interleukins such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL- 11, IL-12; tumor necrosis factor such as TNF-α or TNF-β, and other polypeptide factors including LIF and Kit receptor ligand (KL). As used herein, the term cytokine includes proteins from natural sources or recombinant cell culture and biologically active equivalents of native sequences of cytokines.

The term prodrug as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into a more active parent form. See, e.g., Wilman, Prodrugs in Cancer Chemotherapy, Biochemical Society Transactions, 14, pp. 375-382, 615.^th Meeting, Belfast (1986) and Stella et al., Prodrugs: A Chemical Approach to Targeted Drug Delivery, Directed Drug Delivery, Borchardt et al. (eds.), pp. 247-267, Humana Press (1985). Prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, phosphorothioate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, β-lactam ring prodrugs, arbitrarily substituted prodrugs containing a phenoxyacetamide group or optionally substituted prodrugs containing a phenylacetamide group,

5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted into more active cytotoxic free drugs. Examples of cytotoxic drugs that can be derivatized into a prodrug for use in this invention include, but are not limited to, the chemotherapeutic agents described above.

A liposome is a small vesicle composed of various types of lipids, phospholipids and / or surfactants useful for delivering a drug (such as the anti-ErbB2 antibodies disclosed herein and, optionally, a chemotherapeutic agent) to a mammal. The components of the liposome are usually arranged in two layers, similar to the arrangement of lipids in biological membranes.

The term package insert is used to refer to the instructions usually included in commercial packages of therapeutic products that contain information about the indications, use, dosage, administration, contraindications and / or warnings regarding the use of such therapeutic products.

A heart protective factor is a compound or composition that prevents or reduces myocardial dysfunction (i.e., cardiomyopathy and / or congestive heart failure) associated with the administration of a drug to a patient such as an anthracycline antibiotic and / or an anti-ErbB2 antibody. For example, a heart protective agent can block or reduce the free radical cytotoxic effect and / or prevent or reduce damage from oxidative stress. Examples of heart protective agents covered by this definition include the iron chelating factor dexrazoxane (ICRF-187 (Seifert et al., The Annals of Pharmacotherapy 28: 1063-1072 (1994)); a lipid lowering agent and / or an antioxidant such as probucol (Singal et al., J. Mol. Cell. Cardiol. 27: 1055-1063 (1995)); amifostine (2 - [(3-aminopropyl) amino] ethanethiol dihydrogenphosphate acid aminothiol ester, also called WR-2721, and its dephosphorylated cellular form called WR-1065) and 5-3- (3-methylaminopropylamino) propylphosphorothioic acid (WR-2721) 151327); see Green et al., Cancer Research 54: 738-741 (1994); digoxin (Bristow, MR, in: Bristow, M. R., ed., Drug-Induced Heart Disease New York: Elsevier, 191-215 (1980)); beta blockers such as metoprolol (Hjalmarson et al., Drugs 47: Suppl. 4: 31-9 (1994) and Shaddy et al., Am. Heart J. 129: 197-9 (1995)); Vitamin E; ascorbic acid (vitamin C); free radical scavengers such as oleanic acid, ursolinic acid, and N-acetylcysteine ​​(NAC); spin trapping compounds such as alpha-phenyl-tert-butyl nitrone (PBN); (Paracchini et al., Anticancer Res. 13: 1607-1612 (1993)); organoselenium compounds such as P251 (Elbesen) and the like.

An isolated nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it is typically associated in the natural nucleic acid source of an antibody. An isolated nucleic acid molecule is in a different form or arrangement in which it occurs in nature. Therefore, isolated nucleic acid molecules are distinguished from the nucleic acid molecules that exist in natural cells. However, an isolated acid molecule

Nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody, where, for example, the nucleic acid molecule is in a chromosomal position different from that of natural cells.

The expression control sequences refers to DNA sequences necessary for the expression of operably linked coding sequences in a particular host organism. Control sequences which are suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide, the promoter or enhancer is operably linked to the coding sequence if the sequence is transcribed. , or the ribosome binding site is operably linked to the coding sequence, if it is located in a manner that facilitates translation. Generally operably linked means that the linked DNA sequences are contiguous and, in the case of the secretory leader, are contiguous and in the same reading phase. However, the enhancer sequences need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. In the absence of such sites, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

As used herein, the expressions cell, cell line and cell culture are used interchangeably and all such terms are progeny. Thus, the words transformants and transformed cells include the original cell of interest and cultures derived therefrom, regardless of the number of passages. It is also understood that all offspring may not be exactly identical in terms of DNA content due to deliberate or involuntary mutations. The term includes mutant progeny that possess the same function or biological activity as was screened for in the originally transformed cell. If other meanings are intended, they will be apparent from the context.

II. Production of anti-ErbB2 antibodies

Exemplary techniques for producing the antibodies used in the present invention are described below. The ErbB2 antigen to be used in the production of antibodies can be, for example, a soluble form of the extracellular domain of ErbB2 or a portion thereof containing the desired epitope. Alternatively, cells that express ErbB2 on their cell surfaces (e.g. NIH-3T3 cells transformed to overexpress ErbB2 or a cancer cell line such as SK-BR-3 cells, see Stancovsky et al., PNAS (USA) 88: 8691-8695 (1991)). Other forms of ErbB2 useful for generating antibodies will be apparent to those skilled in the art.

(i) Polyclonal antibodies

The production of polyclonal antibodies in animals is preferably stimulated by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the appropriate antigen and adjuvant. It may be useful to conjugate an appropriate antigen to a protein which is immunogenic in the species to be immunized, e.g. Horsetail haemocyanin, serum albumin, bovine thyroglobulin or soybean inhibitor, using a bifunctional or derivatized agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via succinyl-sulfaldehyde, SOC-2-succinimide)¹N = C = NUMBER, where R and R¹ are different alkyl groups.

Animals are immunized with the antigen, immunogenic conjugates or derivatives by combining e.g. 100 µg or 5 µg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at several sites. One month later the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at several sites. After 7 to 14 days, blood is drawn from the animals and the serum antibody titer is determined. Animals are boosted until their antibody levels are constant. Preferably, the animals are boosted with the conjugate of the same antigen, but conjugated to a different protein and / or via a different cross-linking reagent. Conjugates can be made in culture

Recombinant cells as fusion proteins. Aggregating agents such as alum can also be suitably used to enhance the immune response.

(ii) Monoclonal antibodies

Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies making up the population are identical, except for possible naturally occurring mutations which may be present in minor amounts. Thus, the term monoclonal indicates that the antibody is not a mixture of different antibodies.

For example, monoclonal antibodies can be made using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or can be made by recombinant DNA methods (US Patent No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to induce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusion inducer such as polyethylene glycol to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which preferably contains one or more substances that inhibit the growth or survival of the unfused myeloma parental cells. For example, if myeloma stem cells are deficient in the enzyme guanine-hypoxanthine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which substances will prevent growth of cells lacking HGPPRT.

Preferred myeloma cells are those that fuse efficiently, support stable, high-level antibody production by the selected antibody-producing cells, and are sensitive to a substrate such as HAT substrate. Among these, preferred myeloma cell lines are mouse myeloma cell lines, such as those derived from the murine tumors MOPC-21 and MPC-11, available from Salk Institute Cell. Distribution Center, California, USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland, USA. Human myeloma and murine-human heterosteloma cell lines for the production of human monoclonal antibodies have also been described (Kozbor, J. Immunol., 133: 3001 (1984) and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

In the culture medium in which the hybridoma cells were grown, the production of monoclonal antibodies directed against the antigen is determined. Preferably, the binding specificity of the monoclonal antibody hybridoma produced by the cells is determined by immunoprecipitation or by an in vitro binding assay, such as a radioimmunoassay (RIA) or an enzymatic immunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem. 107: 220 (1980).

Once hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity have been identified, clones can be subcloned according to a serial dilution procedure and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986) )). Suitable culture media for this purpose include, for example, D-DMEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as exudative tumors in animals.

The monoclonal antibodies secreted by the subclones are suitably isolated from the culture medium, exudate fluid, or serum according to conventional antibody purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be inserted into expression vectors which are then transfected into host cells such as E. coli, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce an antibody protein, to obtain the synthesis of monoclonal antibodies in recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Pluckthun, Immunol. Revs, 130: 151-188 (1992).

In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348: 552-554 (1990). Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Later publications describe the production of high affinity human antibodies (nM range) by chain shuffling (Marks et al., Bio / Technology 10: 779-783 (1992)) as well as combinatorial infection and in vivo recombination as a strategy for constructing very large libraries. phage (Waterhouse et al., Nuc. Acids Res. 21: 2265-2266 (1993)). Thus, these techniques provide good alternatives to traditional hybridoma monoclonal antibody techniques for isolating monoclonal antibodies.

The DNA can also be modified, for example, by substituting the heavy and light chain constant domain encoding sequences for homologous murine sequences (U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851 (1984)) or by covalently linking all or a portion of the non-immunoglobulin polypeptide coding sequence to an immunoglobulin coding sequence.

Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or are substituted for the variable domains of one antigen binding site, to form a chimeric bivalent antibody having one antigen-specific binding site and a second antigen binding site specific for another antigen.

(iii) Humanized antibodies

Methods for humanizing non-human antibodies have been described in the art. Preferably, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as import residues, which are typically obtained from an import variable domain. Humanization can be essentially carried out according to the method of Winter et al. (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting the sequences of the hypervariable regions corresponding to the sequence of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (US Patent No. 4,816,567) in which substantially less than an intact human variable domain has been substituted with the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues have been substituted with residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy chains, to be used in making the humanized antibodies is very important in reducing antigenicity. According to the so-called best fit method, the sequence of the variable domain of a rodent antibody is used to search the entire library of known human variable domain sequences. The human sequence which is most similar to that of the rodent is then accepted as the human framework (FR) region of the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993); Chothia et al., J. Mol. Biol., 196: 901 (1987)). Other methods use a specific framework region derived from the consensus sequence of all human antibodies of a particular light or heavy chain subgroup. 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)) .

In addition, it is important that the humanized antibodies retain high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by analyzing the parental sequences and various theoretical humanized products using spatial models of the parent and humanized sequences. Spatial models of immunoglobulins are widely available and known to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. The analysis of these structures enables a probable analysis

The role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of the residues which influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and linked to the recipient and import sequences to obtain the characteristics of the desired antibody, such as increased affinity for the target antigen (s). In general, the hypervariable region residues are directly and largely involved in influencing antigen binding.

Example 3 below describes the production of exemplary humanized anti-ErbB2 antibodies that bind to ErbB2 and block ligand activation of the ErbB receptor. Humanized antibodies of particular interest herein block EGF, TGF-α and / or HRG mediated MAPK activation substantially as effectively as murine monoclonal antibody 2C4 (or a Fab fragment thereof) and / or bind ErbB2 substantially as effectively as murine monoclonal antibody 2C4 (or Fab fragment thereof). The humanized antibody of the present invention may, for example, contain non-human hypervariable region residues introduced into the human heavy chain variable domain and may further have a substitution in the framework region (FR) at a position selected from the group consisting of 69H, 71H and 73H using the variable domain numbering system given in Kabat et al., Sequences of Proteins of Immunological Interest., ed. 5, Public Health Service, National Institutes of Health, Bethesda, MD (1991). In one embodiment, the humanized antibody comprises FR substitutions at two or all of positions 69H, 71H, and 73H.

An exemplary humanized antibody of interest herein contains heavy chain variable domain complementarity determining residues GFTFTDYTMX, where X is preferably D or S (SEQ ID NO: 7); DVNPNSGGSIYNQRFKG (SEQ ID NO: 8) and / or NLGPSFYFDY (SEQ ID NO: 9), optionally containing amino acid modifications to these CDR residues, e.g. if the modifications substantially maintain or improve the affinity of the antibody. For example, an antibody variant of interest may have from about one to about seven or about five amino acid substitutions in the foregoing heavy chain CDR sequences. Such antibody variants can be prepared by affinity maturation, e.g., as described below. A most preferred monoclonal antibody comprises the amino acid sequence of a heavy chain variable domain shown in SEQ ID NO: 4.

The humanized antibody may contain complementarity determining residues of the light chain variable domain KASQDVSIGVA (SEQ ID NO: 10); SASYX¹X²X³where X¹ preferably it is R or L, X² preferably Y or E is and X is preferred³ preferably it is T or S (SEQ ID NO: 11) and / or QQYYIYPYT (SEQ ID NO: 12), except for those heavy chain variable domain CDR residues given in the preceding paragraph. Such humanized antibodies optionally contain amino acid modifications to the above CDR residues, e.g., if the modifications substantially maintain or improve the affinity of the antibody. For example, an antibody variant of interest may have from about one to about seven or about five amino acid substitutions in the above light chain CDR sequences. Such antibody variants can be prepared by affinity maturation, e.g., as described below. A most preferred monoclonal antibody comprises the amino acid sequence of a light chain variable domain shown in SEQ ID NO: 3.

The present application also contemplates affinity maturation antibodies that bind to ErbB2 and block ligand activation of the ErbB receptor. The parental antibody can be a human antibody or a humanized antibody, e.g., an antibody comprising the light and / or heavy chain variable sequences set forth in SEQ ID NOS: 3 and 4 (i.e., variant 574), respectively. The obtained antibody by affinity maturation preferably binds to the ErbB2 receptor with an affinity greater than that of murine 2C4 or variant 574 (e.g., an affinity improved from about two or about fourfold to about a hundredfold or about a thousandfold, e.g. as assessed by ELISA using the extracellular domain. ErbB2 (ECD). Exemplary heavy chain variable domain CDR residues for substitution include H28, H30, H34, H35, H64, H96, H99, or combinations of two or more (e.g., two to three, four, five, or up to about ten of these residues).

Various forms of the humanized antibody or affinity maturation antibody are contemplated. For example, the humanized antibody or affinity maturation antibody can be an antibody fragment, such as a Fab, that has been optionally conjugated to one or more cytotoxic agents to form an immunoconjugate. Alternatively, the humanized antibody or affinity maturation antibody can be an intact antibody, such as an intact IgG1 antibody.

(iv) Human antibodies

As an alternative to humanization, human antibodies can be made. For example, it is now possible to create transgenic animals (e.g., mice) that are capable, upon immunization, of producing a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene system into such mutant germline mice will result in the production of human antibodies upon antigen stimulation. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993) and U.S. Patent Nos. 5,591,669, 5,589,369, and 5,545,807.

Alternatively, a phage display technique can be used to generate human antibodies or antibody fragments from a set of immunoglobulin variable (V) domain genes from unimmunized donors (McCafferty et al., Nature 348: 552-553 (1990)). According to this technique, antibody V domain genes are cloned in frame with either a major or a secondary coat protein of a filamentous bacteriophage, such as 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 properties of the antibody will also result in selection of the gene encoding the antibody exhibiting these properties. Thus, the phage mimics certain properties of the B cell. Phage display can be performed in a variety of arrangements: for these see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3: 564-571 ( 1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991) isolated various anti-oxazolone antibodies from a small random combinatorial V gene library obtained from the spleen of immunized mice. A panel of V genes from unimmunized human donors can be constructed, and antibodies to a wide variety of antigens (including self-antigens) can be isolated essentially according to the techniques described in Marks et al., J. Mol. Biol. 222: 581-597 (1991) or Griffith et al., EMBO J. 12: 725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905.

As discussed above, human antibodies can also be produced by in vitro activated B cells (see US Patent Nos. 5,676,610 and 5,229,275).

Human anti-ErbB2 antibodies are described in U.S. Patent No. 5,772,997, issued June 30, 1998, and International Patent Application No. 97/00271, published January 3, 1997.

(v) Antibody fragments

Various techniques have been developed for the generation of antibody fragments. Traditionally, these fragments have been obtained by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science, 229: 81 (1985)). However, nowadays these fragments can be directly produced by recombinant host cells. For example, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F (ab ') 2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another approach, F (ab) 2 fragments can be isolated directly from recombinant host cell culture. Other techniques for producing antibody fragments will be apparent to those working in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See International Patent Application No. 93/16185 and U.S. Patent Nos. 5,571,894 and 5,587,458. The antibody fragment may also be a linear antibody, for example as described in U.S. Patent No. 5,641,870. Such linear antibody fragments can 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 ErbB2 protein. Other such antibodies may combine the ErbB2 binding site with the EGFR, ErbB3, and / or ErbB4 binding site (s). Alternatively, the anti-ErbB2 arm may be attached to an arm that binds to an excitatory molecule on a leukocyte such as a T cell receptor molecule (e.g. CD2 or CD3) or IgG receptors (FcyR) such as FcyRI (CD64), FcyRII ( CD32) and FcyRIII (CD16) so as to concentrate the cellular defense mechanisms on cells expressing ErbB2. Bispecific antibodies can also be used to deliver cytotoxic agents to cells expressing ErbB2. These antibodies have an ErbB2 binding arm and an arm that binds a cytotoxic agent (e.g., saporin, anti-interferon-a, vinca alkaloid, ricin A chain, methotrexate, or radioactive hapten. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F (ab ') 2 bispecific antibodies).

International Patent Application No. 96/16673 describes a bispecific anti-ErbB2 / FcYRIII antibody and US Patent No. 5,837,234 discloses a bispecific anti-ErbB2 / FcYRI antibody. A bispecific anti-ErbB2 / Fca antibody is disclosed in International Patent Application No. 98/02463. U.S. Patent No. 5,821,337 describes a bispecific anti-ErbB2 / CD3 antibody.

Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies relies on the co-expression of two immunoglobulin heavy chain / light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305: 537-539 (1983)). Due to the random selection of immunoglobulin heavy and light chains, these hybridomas (quadromes) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, which is usually accomplished by affinity chromatography steps, is rather cumbersome and the product yields are low. Similar procedures are disclosed in International Patent Application No. 93/08829 and in Traunecker et al., EMBO J., 10: 3655-3659 (1991).

According to a different approach, antibody variable domains with the desired specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion is preferably with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred that the first heavy chain constant region (CHI) containing the site necessary for light chain binding is present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusions and, if desired, light chain fusions, are inserted into separate vectors and co-transfected into an appropriate host organism. This provides great flexibility to align the ratios of the three polypeptide chains in embodiments where unequal ratios of the three polypeptide chains used in construction ensure optimal performance. However, it is possible to insert sequences encoding two or three polypeptide chains into one expression vector if the expression of at least two polypeptide chains in equal ratios results in high yield or if the ratios are not of great importance.

In a preferred embodiment of this approach, the bispecific antibodies consist 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. It has been found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for an easy means of separation. This approach is disclosed in International Patent Application No. 94/04690. For further details on generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

According to another approach, described in US Patent 5,731,168, the interface of a pair of antibody molecules can be constructed to maximize the percentage of heterodimers that are recovered from recombinant cell culture. A preferred contact region comprises at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Corresponding recesses of identical or similar size are created in the interface of the second antibody molecule by replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over undesirable end products such as homodimers.

Bispecific antibodies include cross-linked or heteroconjugate antibodies. For example, one of the heteroconjugate antibodies can be conjugated to avidin and the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (US Patent No. 4,676,980) and for the treatment of HIV infection (WO 91/00360 and 92/200373 and EP 03089). Heteroconjugate antibodies can be prepared using any suitable cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in US Patent No. 4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using a chemical bond. Brennan et al., Science, 229: 81 (1985) describe a procedure by which intact antibodies are proteolytically cleaved to generate F (ab ') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent the formation of intermolecular disulfide bonds. The Fab 'fragments generated are converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then converted back to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies formed can be used as agents for the selective immobilization of enzymes.

Recent advances have facilitated the direct recovery of Fab'-SH fragments from E. coli that can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of a fully humanized F (ab ') bispecific antibody molecule. Each of the Fab 'fragments is separately secreted from E. coli and subjected to direct in vitro chemical coupling to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against targeted human breast tumors.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies were generated using leucine zippers. Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992). The 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 were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be used to generate antibody homodimers. The diabody technique described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993), has provided an alternative mechanism for making bispecific antibody fragments. The fragments contain a heavy chain variable domain (VH) linked to the light chain variable domain (VL) via a linker that is too short to allow pairing between the two domains of the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of the other fragment, thereby creating two antigen binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152: 5368 (1994).

Antibodies with more than two valencies are also contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60 (1991).

(vii) Other modifications of the amino acid sequence

Amino acid sequence modification (s) of the anti-ErbB2 antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the anti-ErbB2 antibody are prepared by introducing appropriate nucleotide changes into the nucleic acid of the anti-ErbB2 antibody, or by peptide synthesis.

PL 203 326 B1

Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the anti- ErbB2 antibody. Any combination of deletions, insertions and substitutions may be used to obtain the final product, provided that the final product has the desired properties. Amino acid changes can also alter post-translational modifications of the anti-ErbB2 antibody, such as changes in the number or position of glycosylation sites.

A useful method for identifying certain residues or regions of an anti-ErbB2 antibody that are preferred sites for mutagenesis is called alanine screening mutagenesis, as described by Cunningham and Wells, Science, 244: 1081-1085 (1989). This method identifies a residue or a group of target residues (e.g. charged residues such as arg, asp, his, lys and glu) and replaced with neutral or negatively charged amino acids (most preferably alanine or phenylalanine) to alter the interaction of the amino acids with the ErbB2 antigen. Those amino acid positions which show functional sensitivity to substitutions are then analyzed in more detail by introducing further or different variants at or behind the substitution site. Thus, while the site for introducing an amino acid sequence difference is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the role of a mutation at a given site, alanine screening or random mutagenesis of the target codon or region is performed and the expressed anti-ErbB2 antibody variants are screened for the desired activity.

Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing hundreds or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an anti-ErbB2 antibody with an N-terminal methionyl residue or an antibody fused to a cytotoxic polypeptide. Other insertional variants of the anti-ErbB2 antibody molecule include the fusion to the N or C terminus of an anti-ErbB2 antibody of an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the anti-ErbB2 antibody molecule replaced by a different residue. The sites most desirable for substitution mutagenesis include the hypervariable regions, but changes in the FR are also contemplated. Conservative substitutions are shown in Table 1 under the heading Preferred Substitutions. If such substitutions result in a change in biological activity, then more radical changes as set forth in Table 1 Exemplary Substitutions can be made and products searched.

Table 1

<td>The rest is original</td><td>Exemplary substitutions</td><td>Preferred substitutions</td>

<td> 1</td><td> 2</td><td> 3</td>

<td>Ala (A)</td><td>val, leu, ile</td><td>val</td>

<td>Arg (R)</td><td>lys, gln, asn</td><td>lys</td>

<td>Asn (N)</td><td>gln, his, asp, lys, arg</td><td>main</td>

<td>Asp (D)</td><td>glu, asn</td><td>glu</td>

<td>Cys (C)</td><td>cheese, ala</td><td>cheese</td>

<td>Gln (Q)</td><td>asn, glu</td><td>asn</td>

<td>Glu (E)</td><td>asp, gln</td><td>asp</td>

<td>Gly (G)</td><td>ala</td><td>ala</td>

<td>His (H)</td><td>asn, gln, lys, arg</td><td>arg</td>

<td>How much (I)</td><td>leu, val, met, ala, phe, norleucine</td><td></td>

<td>Leu (L)</td><td>norleucine, ile, val, met, ala, phe</td><td>how much</td>

<td>Lys (K)</td><td>arg, gn, asn</td><td>arg</td>

<td>Met (M)</td><td>leu, phe, how much</td><td>leu</td>

PL 203 326 B1 cont. table 1

<td> 1</td><td> 2</td><td> 3</td>

<td>Phe (F)</td><td>leu, val, ile, ala, tyr</td><td>tyr</td>

<td>Pro (P)</td><td>ala</td><td>ala</td>

<td>Cheese (S)</td><td>thr</td><td>thr</td>

<td>Thr (T)</td><td>cheese</td><td>cheese</td>

<td>Trp (W)</td><td>tyr, phe</td><td>tyr</td>

<td>Tyr (Y)</td><td>trp, phe, thr, ser</td><td>phe</td>

<td>Val (V)</td><td>ile, leu, met, phe, ala, norleucine</td><td>leu</td>

Substantial modifications to the biological properties of the antibody are made by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the region of substitution, for example, conformation of the β or helical structure, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the side chain. Naturally occurring residues have been divided into groups based on common side chain properties:

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

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

(3) acidic: asp, glu;

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

(5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions require a member of one of these classes to be replaced with a member of another.

Any cysteine ​​residue not involved in maintaining the proper conformation of the anti-ErbB2 antibody may also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent inappropriate crosslinking. In contrast, cysteine ​​linkage (s) can additionally be introduced into 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 involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). In general, the resulting variant (s) selected for further development will have improved biological properties relative to the parent antibody from which it was generated. A convenient way to generate such substitution variants involves affinity maturation using phage display. Briefly, several sites in the hypervariable region (e.g., 6-7 sites) are mutated to create all possible amino acid substitutions at each site. The antibody variants thus formed are displayed in a monovalent fashion on the filamentous phage particles as fusion proteins with the M13 gene III product, packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. binding affinity) as disclosed herein. In order to identify potential hypervariable region sites for modification, alanine screening mutagenesis can be performed to identify hypervariable region regions significantly contributing to antigen binding. Alternatively, or additionally, it may be advantageous to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and human ErbB2. Such contact and adjacent residues are potential substitution residues in accordance with the techniques developed herein. After such variants are generated, the set of variants are screened as described herein and antibodies with improved properties in one or more suitable assays can be selected for further development.

Another type of amino acid variant alters the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate groups present in the antibody and / or adding one or more glycosylation sites not present in the antibody.

PL 203 326 B1

Typically, the glycosylation of antibodies is N-glycosylation or O-glycosylation. N-linked glycosylation refers to the attachment of a carbohydrate group to the side chain of an asparagine residue. The recognition sequences for enzymatic attachment of a carbohydrate group to the asparagine side chain are the tri-peptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-glycosylation refers to combining one of the sugars of N-acetylgalactosamine, galactose or xylose with a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

Introducing additional glycosylation sites into an antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (N-linked glycosylation sites). Variation can also be made by adding or substituting one or more serine or threonine residues to the original antibody sequence (for O-glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of an anti-ErbB2 antibody are prepared by a variety of methods known in the art, these methods include, but are not limited to, isolation from a natural source (for naturally occurring amino acid sequence variants) and preparation by oligonucleotide-mediated (or site-directed) mutagenesis. ), mutagenesis by PCR and cassette mutagenesis of a previously prepared variant or non-variant version of the anti-ErbB2 antibody.

It may be desirable to modify the antibody of the invention for effector function, e.g., such as for enhancing antigen dependent cellular cytotoxicity (ADCC) and / or complement dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions into the Fc region of the antibody. Alternatively, or in addition, a cysteine ​​residue (s) may be introduced into the Fc region, thereby allowing the formation of an interchain disulfide bond in that region. The homodimeric antibody thus formed may exhibit improved ability to internalize and / or enhanced complement dependent cell killing and antibody dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp. Med. 176: 1191-1195 (1992) and Shopes, B., J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53: 2560-2565 (1993). Alternatively, one can construct an antibody that has dual Fc regions and therefore can have enhanced complement-dependent lysis and ADCC capabilities. See Stevanson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).

A salvage receptor binding epitope may be introduced into the antibody (specifically a fragment of the antibody) to increase the serum half-life of the antibody, as described, for example, in U.S. Patent No. 5,773,9277. As used herein, the term salvage receptor binding epitope refers to to an epitope of the Fc region of an IgG molecule (e.g. IgG1, IgG2, IgG3 or IgG4) which is responsible for increasing the serum half-life of the IgG molecule in vivo.

(viii) Screening for the antibody with the desired properties

Techniques for generating antibodies have been described above. Antibodies can be further selected for certain desirable biological properties.

To identify an antibody that blocks ligand activation of the ErbB2 receptor, the ability of the antibody to block ErbB ligand binding to ErbB receptor-expressing cells (e.g., in combination with another ErbB receptor with which the ErbB receptor of interest forms a heterooligomer) can be determined. ErbB). For example, cells naturally expressed or transfected to express the ErbB receptors of the ErbB heterooligomer may be incubated with the antibody and then exposed to labeled ErbB ligand. The ability of the anti-ErbB2 antibody to block ligand binding to the ErbB receptor in an ErbB hetero-oligomer can then be assessed.

For example, inhibition of HRG binding to the MCF7 breast tumor cell line by anti-ErbB2 antibodies can be performed by using MCF7 monolayer cultures on ice in a 24-well plate substantially as described in Example 1 below. Anti-ErbB2 monoclonal antibodies can be added to each well and incubated for a

Take minutes. The next ones can be added tagged¹²⁵And rHRGe1_177-224 (25 pm) and incubation may be continued for 4 to 16 hours. Dose-response curves can be prepared and an IC50 value calculated for the antibody of interest. In one embodiment, an antibody that blocks ligand activation of the ErbB receptor will have an IC50 in this assay for inhibiting HRG binding to MCF7 cells of about 50 nM or less, more preferably 10 nM or less. Where the antibody is an antibody fragment such as a Fab fragment, the IC50 for inhibiting HRG binding to MCF7 cells in this assay may be about 100 nM or less, more preferably 50 nM or less.

Alternatively, or in addition, the ability of the anti-ErbB2 antibody to block ErbB ligand stimulated tyrosine phosphorylation of the ErbB receptor present in an ErbB heterooligomer can be assessed. For example, cells endogenously expressing or transfecting ErbB receptors can be incubated with the antibody, and the ErbB ligand-dependent tyrosine phosphorylation activity determined using an anti-phosphotyrosine antibody (which is optionally conjugated to a detectable tag). A receptor activation assay with kinase activity as described in US Patent No. 5,766,863 is also available for determining ErbB receptor activation and blocking this activity by an antibody.

In one embodiment, screening for an antibody that inhibits HRG stimulation of p180 tyrosine phosphorylation in MCF7 cells can be performed essentially as described in Example 1 below. For example, MCF7 cells can be plated in 24-well plates and the anti-ErbB2 monoclonal antibodies added to each well and incubated at room temperature for 30 minutes; rHRGe1 can then be added to each well₁₇₇=₂₄₄ to 0.2 nM final concentration and incubation may continue for 8 minutes. The media from each well can be drained and reactions can be stopped by adding 100 µl of SDS sample buffer (5% SDS, 25 mM DTT and 25 mM Tris-HCl, pH 6.8). Each sample (25 μθ can be electrophoresed on 4-12% polyacrylamide gradient gels (Novex) and then electrophoretically transferred to a polyvinylidene fluoride membrane. Immunoblots can be induced with an anti-phosphotyrosine antibody (concentration 1 μg / ml) and the intensity of the main a reactive band with a Mr of about 180,000 can be quantified by reflected light densitometry. The selected antibody will preferably inhibit the stimulation of p180 tyrosine phosphorylation by HRG to about 0-35% of the control value in this assay. A dose-response curve can be prepared for the inhibition of HRG stimulation of p180 tyrosine phosphorylation as determined by reflectance densitometry, and an IC50 for the antibody of interest can be calculated. In one embodiment, an antibody that blocks ligand activation of the ErbB receptor will have an IC50 in this assay for inhibiting HRG p180 tyrosine stimulation of about 50 nM or less, more preferably 10 nM or less. Where the antibody is an antibody fragment such as a Fab fragment, the IC50 for inhibiting HRG stimulation of p180 tyrosine phosphorylation in this assay may be, for example, about 100 nM or less, more preferably 50 nM or less.

The growth inhibitory effect of the antibody on MDA-MB175 cells may also be assessed, e.g., essentially as described in Schaefer et al., Oncogene 15: 1385-1394 (1997). According to this assay, MDA-MB-175 cells can be treated with anti-ErbB2 monoclonal antibody (10 µg / ml) for 4 days and stained with crystal violet. Incubation with the anti-ErbB2 antibody may have a growth inhibitory effect on this cell line similar to that displayed by the monoclonal antibody 2C4. In a further embodiment, exogenous HRG will not significantly reverse this inhibition. Preferably, the antibody will be able to inhibit cellular proliferation of MDA-MB-175 cells to a greater extent than monoclonal antibody 4D5 (and possibly to a greater extent than monoclonal antibody 7F3), both in the presence and absence of exogenous HRG.

In one embodiment, the anti-ErbB2 antibody of interest can block the heregulin-dependent association of ErbB2 with ErbB3 in both MCF7 and SK-BR-3 cells, determined in a co-immunoprecipitation experiment such as that described in Example 2, much more effectively than monoclonal the antibody 4D5, and preferably significantly more effective than monoclonal antibody 7F3.

To identify growth inhibitory antibodies to ErbB2, antibodies can be screened for those that inhibit the growth of ErbB2 overexpressing tumor cells. In one embodiment, the selected growth inhibitory antibody is capable

To inhibit the growth of SK-BR-3 cells in cell culture by about 20-100%, and preferably about 50-100%, at an antibody concentration of about 0.5 to 30 µg / ml. To identify such antibodies, the SK-BR-3 cell assay described in US Patent No. 5,677,171 can be performed. According to this assay, SK-BR-3 cells are grown in a 1: 1 mixture of F12 and DMEM supplemented with 10% fetal bovine serum, glutamine and penicillin / streptomycin. SK-BR-3 cells are seeded at 20,000 cells in 35 mm cell culture dishes (2 ml / 35 mm dish). 0.5 to 30 µg / ml of the anti-ErbB2 antibody is added to the dish. After six days, the number of cells is counted, as compared to untreated cells, using a COULTER ™ electronic cell counter. 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.

To select antibodies that induce cell death, loss of membrane integrity as indicated by PI, tryptane blue, or 7AAD uptake can be assessed against controls. A preferred assay is a PI uptake assay using BT474 cells. According to this assay, BT474 cells (which can be obtained from the American Type Culture Collection (Rockville, MD)) are grown in Dulbecco's Modified Eagle Medium (D-MEM): Ham's F-12 (50:50), supplemented with 10% inactivated heat treatment with FBS (Hyclone) and 2mM L-glutamine. (Thus, the assay is performed in the absence of complement and immune effector cells). BT474 cells are seeded at a density of 3 x 10⁶ cells per dish in 100 x 20 mm dishes and allow attachment overnight. The medium is then removed and replaced with fresh medium alone or medium containing 10 µg / ml of the appropriate monoclonal antibody. Cells are incubated for a period of 3 days. After each treatment, the monolayers are washed with PBS and detached using trypsin. The cells are then centrifuged at 1200 rpm for 5 minutes at 4 ° C, the pellet resuspended in 3 ml ice-cold Ca-binding buffer.²⁺(10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) and distribute to 12 x 75 tubes sealed with a 35 mm filter (1 ml per tube, 3 tubes per treatment group) to remove cell aggregates. Tubes then receive PI (10 µg / ml). Samples can be analyzed using a FACSCAN ™ flow cytometer and FACSCONVERT ™ Cell software. Quest (Becton Dickinson). Those antibodies that induce statistically significant levels of cell death as determined by PI uptake can be selected as cell death inducing antibodies.

An annexin binding assay using BT474 cells is available for selection for antibodies that induce apoptosis. BT474 cells are grown and plated in plates as discussed in the previous paragraph. The medium is then removed and replaced with fresh medium alone or medium containing 10 µg / ml monoclonal antibody. Cells are incubated for a period of 3 days, monolayers are washed with PBS and detached using trypsin. Cells are then centrifuged, resuspended in Ca binding buffer²⁺ and arranges into test tubes as discussed above for the determination of cell death. Tubes then receive labeled annexin (e.g. annexin V-FITC) (1 µg / ml). Samples can be analyzed using a FACSCAN ™ flow cytometer and FACSCONVERT ™ Cell software. Cjuest (Becton Dickinson). Those antibodies that induce statistically significant levels of annexin binding relative to controls are selected as apoptosis-inducing antibodies.

In addition to the annexin binding assay, a DNA staining assay using BT474 cells is available. To perform this assay, BT474 cells that were treated with the antibody of interest as described in the previous two paragraphs are incubated with 9 μg / ml HOECHST 33342 ™ for two hours at 37 ° C and then analyzed on an EPICS flow cytometer ELITE ™ (Coulter Corporation) using MODFIT LT ™ software (Verity Software House). Using the assay, antibodies that induce a percentage change in apoptotic cells that is 2 times or greater (and preferably 3 times or greater) than that of untreated cells (up to 100% apoptotic cells) can be selected as pro-apoptotic antibodies.

To screen for antibodies that bind to an epitope bound by the antibody of interest, routine cross-blocking assays can be performed, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow, and David Lane (1988). Alternatively, or additionally, epitope mapping can be performed by methods known in the art (see, e.g., Figs. 1A and 1B herein).

(Ix) Immunoconjugates

The invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, 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. radioconjugate).

Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Conjugates of the antibody with one or more small molecule toxins such as calicheamicin, maytansine (US Patent No. 5,208,020), trichotene, and CC1065 are also contemplated herein.

In one preferred embodiment of the invention, the antibody is conjugated to one or more maytansine molecules (e.g., from about 1 to about 10 maytansine molecules per antibody molecule). Maytansine can, for example, be converted to May-SS-Me, which can be reduced to May-SH3 and reacted with a modified antibody (Chari et al., Cancer Research 52: 127-131 (1992)) to form a maytansinoid-antibody immunoconjugate .

Another immunoconjugate of interest comprises an anti-ErbB2 antibody conjugated to one or more calichamicin molecules. The calicheamicin family of antibiotics is characterized by the ability to disrupt double-stranded DNA at sub-picomolar concentrations. Structural calicheamicin analogs that can be used include, but are not limited to, γ₁¹, α2¹, as¹, N-acetyl-γ-ι¹, PSAG and θ1¹ (Hinman et al., Cancer Research 53: 3336-3342 (1993) and Lode et al., Cancer Research 58: 2928 (1998)). See also U.S. Patent Nos. 5,714,586; 5,712.74; 5,264,586; and 5,773,001; formally incorporated herein by reference.

Enzymatically active toxins and fragments thereof that can be used include diphtheria toxin A chain, non-binding diphtheria toxin active fragments, endotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, moddecin A chain, alpha-sacrin, Aleurites proteins Fordii, diantin proteins, Phytolaca americana proteins (PAPI, PAPU and PAP-S), Momordica charantia inhibitor, curcine, crotin, Saponaria officinalis inhibitor, gelonin, mitogelin, restricotocin, phenomycin, enomycin and tricothecenes. See, for example, International Application Number 93/21232, published October 28, 1993.

The present invention further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as deoxyribonuclease, DNAse).

A number of isotopes are available for the production of radioactive-conjugated anti-ErbB2 antibodies. Examples include At²¹¹, And¹³¹, And¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive Lu isotopes.

Antibody and cytotoxic agent conjugates can be prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridylthio) propionate, succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolate (IT) , bifunctional imide ester derivatives (such as dimethyl adipimidate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniobenzoyl) ethylenediamine), diisocyanates (such as 2,6-toliene diisocyanate) and di-active fluorine compounds ( such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Labeled C.¹⁴ 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DPTA) is an exemplary chelating agent for conjugating a ribonucleotide to an antibody. See International Patent Application No. 94/11026. The linker may be a cleavable linker that facilitates release of the cytotoxic drug in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52: 127-131 (1992)) may be used.

Alternatively, a fusion protein may be prepared comprising the anti-ErbB2 antibody and a cytotoxic agent, e.g. by recombinant techniques or peptide synthesis.

In yet another embodiment, the antibody can be conjugated to a receptor (such as streptavidin) for use in tumor pre-targeting in which the patient is administered an antibody-receptor conjugate, and then the unbound conjugate is removed from the circulation.

After using a scavenger, a ligand (e.g., avidin) that has been conjugated to a cytotoxic agent (e.g., a radionuclide) is administered.

(x) Antibody-dependent enzyme-mediated prodrug therapy (ADEPT)

The antibodies of the present invention can also be used in ADEPT by conjugating the antibody to a prodrug activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see International Patent Application No. 81/01145) into an active anti-cancer drug. See, for example, International Patent Application No. 88/07378 and U.S. Patent No. 4,975,278.

The enzymatic component of an immunoconjugate useful in ADEPT includes any enzyme capable of acting on a prodrug in such a way that it converts it into a more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs to free drugs, arylsulfatase useful for converting sulfate-containing drugs to free drugs, cytosine deamianase useful for converting non-toxic 5 into free drugs. fluorocytosine in the anti-cancer drug, 5-fluorouracil, proteases such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L) which are useful in converting peptide-containing prodrugs into free drugs, D-alanyl carboxypeptidases useful in converting prodrugs that contain D-amino acid substituents, carbohydrate-splitting enzymes such as β-galactosidase and neuraminidase useful in converting glycosylated prodrugs into free drugs, β-lactamase useful for converting β-lactam modified drugs to free drugs and penicillin amidases such as penicillin V amidase or penicillin G amidase useful for converting drugs modified at amino nitrogen atoms with phenoxyacetyl or phenylacetyl groups, respectively, to free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as abzymes, can be used to convert the prodrugs of the invention into free active drugs (see, e.g., Massey, Nature, 328: 457-458 (1987)). Antibody-abzyme conjugates can be prepared as described herein for the purpose of delivering the abzyme to a tumor cell population.

The enzymes of this invention can be covalently linked to anti-ErbB2 antibodies using techniques well known in the art, such as the use of the heterobifunctional cross-linking reagents discussed above. Alternatively, using recombinant DNA techniques well known in the art, fusion proteins can be constructed comprising at least the antigen-binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention (see, e.g., Neuberger et al., Nature, 312: 604-). 608 (1984).

(xi) Other antibody modifications

Other modifications to the antibodies are contemplated herein. For example, the antibody can be combined with one of a variety of non-protein polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The antibody may also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, in hydroxymethylcellulose or gelatin microcapsules and polymethylmetacylate microcapsules, and in colloidal drug delivery systems (for example, liposomes, microcellulemes, microcellulosomes, nanoparticles and nanocapsules) or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A. Ed. (1980).

The anti-ErbB2 antibodies disclosed herein can also be formulated as immunoliposomes. Antibody-containing liposomes are prepared by methods known in the art, such as 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 International Patent Application No. 97/38731, published October 23, 1997. Liposomes with extended circulating lifetime are disclosed in U.S. Patent No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phase evaporation method from a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-modified phosphatidylethanolamine (PEG-PE). Liposomes were passed through filters with defined pore sizes to obtain liposomes of the desired diameter. Fab 'fragments of the antibodies of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem. 257: 286-288 (1982)

Due to the disulfide exchange reaction. The liposomes optionally contain a chemotherapeutic agent. See Gabizon et al., J. National Cancer Inst. 81 (19), 1484 (1989).

III. Vectors, host cells and methods of recombination

The invention also provides isolated nucleic acid encoding a humanized anti-ErbB2 antibody, vectors and host cells containing the nucleic acid, and recombinant techniques for producing the antibody.

To recombinantly produce the antibody, the nucleic acid encoding it is isolated and inserted into a replication competent vector for further cloning (DNA amplification) or expression. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of binding specifically to the genes encoding the heavy and light chains of the antibody). Many vectors are available. Components of a vector generally include, but are not limited to, one or more of the following elements: a signal sequence, an origin of replication, one or more marker genes, an enhancer sequence, a promoter, and a transcription termination sequence.

(i) Signal sequence

The anti-ErbB2 antibody of this invention can be recombinantly produced not only directly but also as a polypeptide fused with a heterologous polypeptide which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected is that which is recognized and modified (i.e. cleaved by signal peptidase) in the host cell. For prokaryotic host cells that do not recognize and modify the native signal sequence of the anti-ErbB2 antibody, the signal sequence is substituted with a prokaryotic signal sequence selected, for example, from the group of alkaline phosphatase, penicillinase, lpp, or heat stable enterotoxin II leaders. For yeast secretion, a native signal sequence can be substituted e.g. the leader of yeast invertase, the leader of the α factor (including the leaders of the α factors Saccharomyces and Kluyveromyces) or the leader of acid phosphatase, the leader of C. albicans glucoamylase, or the signal described in International Patent Application No. 90/13646. For expression in mammalian cells, mammalian signal sequences as well as viral secretory leaders, for example the herpes virus gD signal, are available.

The DNA for this precursor region is ligated in reading frame to DNA encoding the anti-ErbB2 antibody.

(ii) Replication origin

Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is a sequence that allows the virus to replicate independently of the host chromosomal DNA, and includes origin of replication or autonomously replicating sequences. Such sequences are well known for a wide variety of bacteria, yeasts, and viruses. The origin of replication from the pBR322 plasmid is suitable for most Gram-negative bacteria, the origin of replication from the 2μ plasmid is suitable for yeast, and various viral replication origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. In general, an origin of replication is not needed in mammalian expression vectors (typically the SV40 origin of replication may be used, but only because it contains an early promoter).

(iii) The selection gene

Expression and cloning vectors may contain a selection gene, also called a selectable marker. Common selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate or tetracycline), (b) supplement auxotrophic deficiencies, or (c) provide essential nutrients not available in complex media , e.g., the gene encoding the D-alanine racemase in the case of Bacilli.

In one example of selection schemes, a drug is used to arrest growth of the host cell. Those cells that have been successfully transformed with the heterologous gene produce the drug resistance protein and thus survive the selection regime. Examples of such frequently used selections include drugs such as neomycin, mycophenolic acid, and hygromycin.

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Other examples of suitable selectable markers for mammalian cells are those which allow the identification of cells competent for the uptake of the nucleic acid encoding the anti-ErbB2 antibody, such as the genes DHFR, thymidine kinase, metallothioneins I and II, preferably primate metallothioneins, adenosine deaminase, ornithine decarboxylase, etc. .

For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a culture medium that contains methotrexate (Mtx), a competing antagonist of DHFR. When wild-type DHFR is used, a suitable host cell is a Chinese hamster ovary (CHO) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding the anti-ErbB2 antibody, wild-type DHFR protein, or other selectable markers such as aminoglycoside 3'-phosphotransferase (APH) can be selected by cell growth in a medium containing a selection agent for a selectable marker such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin or G418. See U.S. Patent No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene, present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 (1979). The trp1 gene provides a selection marker for a mutant strain of yeast deficient in the ability to grow in the presence of tryptophan, for example ATCC No. 44076 or PEP4-1 Jones, Genetics, 85: 12 (1977). The presence of a trp1 damage in the genome of a yeast host cell thus provides an efficient genetic environment for detecting transformation by growth in the absence of tryptophan. Likewise, mutant yeast strains lacking Leu2 (ATCC 20 622 or 38 626) are complemented by known plasmids carrying the Leu2 gene.

In addition, vectors derived from the 1.6 µm circular plasmid, pKD1, can be used to transform Kluyveromyces yeast. Alternatively, an expression system for large-scale production of recombinant calf rennet has been reported for K. lactis. Van der Berg, Bio / Technology, 8: 135 (1990). Stable, high-copy expression vectors for the secretion of mature, recombinant human serum albumin by industrial Kluyveromyces strains have also been disclosed. Fleer et al., Bio / Technology, 9: 968-975 (1991).

(iv) Promoter

Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid encoding the anti-ErbB2 antibody.

Promoters suitable for use with prokaryotic hosts include the phoA promoter, the β-lactamase and lactose promoter systems, the alkaline phosphatase promoter, the tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are suitable. Promoters for use in bacterial systems will also contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the anti-ErbB2 antibody.

Promoter sequences for eukaryotes are known. Virtually all eukaryotic genes have an AT-rich region approximately 25 to 30 bases upstream of where transcription begins. Another sequence, 70 to 80 bases upstream of the start of transcription of many genes, is a CNCAAT region where N can be any nucleotide. At the 3 'end of most eukaryotic genes there is an AATAAA sequence, which may be a signal to add the polyA segment to the 3' end of the coding sequence. All of these sequences are suitable for insertion into eukaryotic expression vectors.

Examples of suitable promoter sequences for use in yeast hosts include promoters of 3-phosphoglycerate kinase and other glycolytic enzymes such as enolase, glyceraldehyde 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphoofructokinase, glucose-6-phosphate mutantase, phosphate-6-phosphate mutantase. pyruvate, triosephosphate isomerase, phosphorglucose isomerase and glucokinase.

Other yeast promoters that are inducible promoters with the added advantage of controlling transcription by growth conditions are the alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, catabolic enzymes related to nitrogen metabolism, metallothionein, aldehyde-3-phosphate dehydrogenase, and glycerol-3 enzyme promoter regions. maltose and galactose. Vectors and promoters suitable for

Use in yeast expression is further described in EP 73,657. It is also advantageous to use yeast enhancer sequences with yeast promoters.

The transcription of anti-ErbB2 antibody from vectors in yeast cells is controlled by, for example, promoters obtained from viral genomes such as polyoma virus, avipox virus, adenovirus (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and most preferably simian virus 40 (SV40), heterologous mammalian promoters, e.g. an actin promoter or an immunoglobulin promoter, heat shock protein promoters, provided that such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using bovine papillomavirus as a vector is described in US Patent No. 4,419,446. A modification of this system is described in US Patent No. 4,601,987. See also Reyes et al., Nature, 297: 598-601 (1982) for expression of human interferon β cDNA in mouse cells under the control of the herpes virus thymidine kinase promoter. Alternatively, Rous sarcoma virus long terminal repeats can be used as a promoter.

(v) Reinforcing element

Transcription of the DNA encoding the anti-ErbB2 antibody of this invention by higher eukaryotic organisms is often enhanced by insertion of an enhancer sequence into the vector. Many enhancer sequences are known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, an enhancer from a eukaryotic cell virus will be used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature, 297: 17-18 (1982) for enhancer elements for activating eukaryotic promoters. The enhancer sequence may be incorporated into the vector 5 'or 3' of the anti-ErbB2 antibody coding sequence, but is preferably 5 'to the promoter.

(vi) Transcription termination signal

Expression vectors used in eukaryotic host cells (yeast, fungal, insect, animal, human or nuclear cells from other multicellular organisms) will also contain sequences necessary for transcription termination and for mRNA stabilization. Such sequences are commonly available from the 5 'and sometimes 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide stretches transcribed as a polyadenylated fragment in the untranslated portion of the mRNA encoding the anti-ErbB2 antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See International Patent Application No. 94/11026 and the expression vector disclosed therein.

(vii) Selection and transformation of host cells

Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryotic, yeast, or higher eukaryotic cells described above. Suitable prokaryotic organisms for this purpose include bacteria such as gram-negative or gram-positive organisms, e.g. Enterobacteriaceae, such as Escherichia, e.g. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g. Salmonella typhimurium, Serratia, e.g. Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g. B. licheniformis 41P, disclosed in DD 266 710, published April 12, 1989), Pseudomonas , such as P. aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31 446), although other strains such as E. coli B, E. coli X1776 (ATCC 31 537) and E. coli W3110 are suitable. These examples are illustrative rather than limiting.

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In addition to prokaryotes, eukaryotic organisms such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-ErbB2 antibody vectors. Of the lower prokaryotic host microorganisms, Saccharomyces cerevisiae, or common baker's yeast, is most commonly used. However, numerous other genera, species, and strains, such as Saccharomyces pombe; Kluyveromyces hosts, such as K. lactis,

K. fragilis (ATCC 12 424), K. bulgaricus, (ATCC 16 045), K. wickermania (ATCC 24 178), K. waltii (ATCC 56 500), K. drosophilarum (ATCC 36 906), K. thermotolerans and K. marxianus; yarrowia (EP 402 226); Pichia pastoris (EP Patent No. 183,070); Candida; Trichoderma reesia (EP 244 234), Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis and filamentous fungi such as Neurospora, Penicillium, Tolypocladium and Aspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of a glycosylated anti-ErbB2 antibody are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants and corresponding recommended insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypki (mosquito), Aedes albopictus (mosquito), Drosphila melanogaster (supple dwarf) and Bombyx mori have been identified. Various viral strains for transfection are widely available, e.g. Autographa californica NPV variant Ll and Bombyx mori strain Bm-5 NPV, and such variants can be used as a virus of the present invention, particularly for transfecting Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

However, vertebrate cells are of greater interest and the expansion of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CVI line transformed with SV40 (COS-7, ATCC CRL 1651), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol . 36: 59 (1977); baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1989)), mouse Sertoli cells (TM4, Mather , Biol. Reprod. 23: 243-251 (1989)); monkey kidney cells (CVI, ATCC CCL 70); green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Bufallo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse breast cancer (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transformed with the expression or cloning vectors described above to produce the anti-ErbB2 antibody and grown in conventional nutrient media suitably modified to induce promoters, select transformants, or amplify genes encoding desired sequences.

(viii) Culturing the host cells

The host cells used to produce the anti-ErbB2 antibody of this invention can be grown in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essentail Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM) (Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. May be used as culture media for host cells. Enz. 58: 44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, International Patent Application Nos. 90/03430 and 87/00195 or reissue United States of America number 30 985. Each of these media can be supplemented as necessary with hormones and growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN ™), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range) and glucose or an equivalent energy source. Any other necessary supplements may also be included in appropriate concentrations which should be known

To those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used for the host cells selected for expression, and will be known to the person skilled in the art.

(ix) Purification of the anti-ErbB2 antibody

When using recombinant techniques, the antibody may be produced intracellularly, in the periplasmic space, or may be directly secreted into the medium. If the antibody is produced intracellularly, in a first step, partial residues, either host cells or lysed fragments, are removed, for example by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describe a procedure for isolating antibodies that are secreted into the periplasmic space of E. coli.

Briefly, cell mass is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) over approximately 30 minutes. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example an Amicon or Millipore Pellicon ultrafiltration unit. In each of the above steps, a protease inhibitor such as PMSF can be included to inhibit proteolysis and antibiotics to prevent the growth of accidental contaminants.

The antibody composition prepared from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred technique. The suitability of protein A as an affinity ligand depends on the species and isotype of the immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γ1, γ2, or γ4 chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). The G protein is recommended for all mouse and human γ3 isotypes (Guss et al., EMBO J. 5: 1567-1576 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for higher flow rates and shorter processing times than can be achieved with agarose. When the antibody contains a CH3 domain, Bakerbond ABX ™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other protein purification techniques are also available, such as fractionation on an ion exchange column, ethanol precipitation, reverse phase HPL.C, silica chromatography, heparin-SEPHAROSE ™ chromatography, anion or cation exchange resin chromatography (such as a polyaspartic acid column) ), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation depending on the antibody recovered.

Following any purification step (s), the mixture containing the antibody of interest and impurities may be subjected to hydrophobic interaction chromatography at low pH using an elution buffer having a pH between about 2.5-4.5, preferably performed at low salt concentration (e.g. from about 0 to 0.25 M salt).

IV. Pharmaceutical forms

The therapeutic forms of the antibodies used in the present invention are prepared for storage by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizing agents (Remington's Pharmaceutical Sciences, 16th edition, Osol, A., eds. (1980)) into lyophilized forms. or aqueous solutions. Acceptable carriers, excipients, or stabilizing agents are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzothonium chloride, phenol, butyl or benzyl alcohols, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-c-cresolhexanol); low molecular weight (less than 10 residues in length) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, and dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium; metal complexes (e.g. Zn-protein complexes) and / or nonionic surfactants such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG).

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Preferred freeze-dried forms of the anti-ErbB2 antibody are described in the international patent application.

The embodiment of the present invention may also contain more than one active compound as necessary for the particular indication being treated, preferably compounds with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide antibodies that bind to EGFR, ErbB2 (e.g., antibodies that bind to a different epitope on ErbB2), ErbB3, ErbB4, or vascular endothelial factor (VEGF) in a single formulation. Alternatively, or additionally, the composition may further comprise a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitory agent, an antihormonal agent, an EGFR targeting drug, an anti-angiogenic agent, and / or a cardioprotective agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules, prepared, for example, by coacervation techniques or by interfacial polymerization, for example hydroxymethylcellulose or gelatin microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, albumin liposomes, microspheres, microspheres and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include antibody-containing semipermeable arrays of solid hydrophobic polymers in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethylmethacrylate) or polyvinyl alcohol, polylactides (U.S. Patent No. 3,773,919), L-glutamic acid and γ-ethyl-L-glutamine copolymers, non-degradable acetate ethylene-vinyl, degradable copolymers lactic acid-glycolic acid, such as LUPRON DEPOT ™ (injectable microspheres composed of a lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D - (-) - 3-hydroxybutyric acid.

Forms intended for in vivo administration must be sterile. This is simply achieved by filtering through sterile filtration membranes.

V. Treatment with anti-ErbB2 antibodies

It is believed that in accordance with the present invention, the anti-ErbB2 antibodies can be used to treat a variety of diseases or disorders. Exemplary conditions or disorders include benign and malignant neoplasms, leukemias and malignant neoplasms of lymphoid tissue, other disorders such as neuronal, neuroglial, astrocytic, hypothalamic, glandular, macrophage, epithelial, stromal, blastocellic, inflammatory, angiogenic, and immunological disorders.

Generally, the disease or disorder that will be treated is cancer. Examples of cancer that will be treated in accordance with the present invention include, but are not limited to, cancer, lymphoma, blastoma, sarcoma and leukemia, or a malignant tumor of lymphoid tissue. More particularly examples of such cancers include squamous cell carcinoma (e.g. squamous cell carcinoma), lung cancer, including small cell lung cancer, non-small cell carcinoma of the lung, adenocarcinoma of the lung and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer, including gastrointestinal cancer, pancreatic cancer, glioma, cancer cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colon and rectal cancer, cancer of the endometrium or uterus, cancer of the salivary glands, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, as well as head and neck cancer.

The cancer will generally include cells that express ErbB2 such that the anti-ErbB2 antibody of the present invention is capable of binding to cancer.

While the cancer may be characterized by the ErbB2 receptor overexpression, the present application further provides a method of treating cancer that is not considered to be ErbB2 overexpressing cancer. Various diagnostic / prognostic assays are available for determining ErbB2 expression in cancer. In one embodiment, ErbB2 overexpression can be analyzed by IHC, e.g. using HERCEPTEST^® (Dako). Paraffin-embedded tissue sections from a tumor biopsy can be tested for IHC and the following criteria for the intensity of ErbB2 protein staining were adopted:

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Rating 0

No staining is observed, or there is membrane staining in less than 10% of the tumor cells.

A rating of 1+ detects faint / barely discernible membrane staining in more than 10% of tumor cells. Only part of the cell membrane is stained.

A rating of 2+, there is weak to moderate staining of entire membranes in more than 10% of neoplastic cells.

A rating of 3+, moderate to strong staining of entire membranes is observed in more than 10% of tumor cells.

Tumors with a rating of 0 to 1+ for ErbB2 overexpression can be characterized as non-ErbB2 overexpression, while tumors with a rating of 2+ to 3+ can be characterized as ErbB2 overexpression.

Alternatively, or in addition, FISH assays such as INFORM ™ (marketed by Ventana, Arizona) or PATHVISION ™ (Vysis, Illinois) may be performed on formalin-fixed, paraffin-embedded tumor tissue to determine the degree (if any) of ErbB2 overexpression. in the tumor.

In one embodiment, the cancer will be EGFR expressing (and may be overexpressing) cancer. Examples of cancers that may express / overexpress EGFR include squamous cell carcinoma (e.g. squamous cell carcinoma), lung cancer, including small cell carcinoma, non-small cell carcinoma of the lung, adenocarcinoma of the lung and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer, including gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer , ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colon and rectal cancer, cancer of the endometrium or uterus, cancer of the salivary glands, kidney cancer, prostate cancer, vulva cancer, thyroid cancer, liver cancer, rectal cancer, penile cancer, as well as head and neck cancer.

The cancer treated according to the present invention may be a cancer characterized by over-activation of the ErbB receptor, e.g. EGFR. Such over-activation may be due to overexpression or increased production of the ErbB receptor or ErbB ligand. In one embodiment of the invention, a diagnostic or prognostic assay can be performed to determine whether a patient's cancer is characterized by over-activation of the ErbB receptor. For example, ErbB gene amplification and / or ErbB receptor overexpression may be determined. Various assays are available in the art to determine such amplification / overexpression, and include the IHC, FISH, and antigen shedding assays described above. Alternatively, or additionally, the levels of an ErbB ligand, such as TGF-α, in or associated with a tumor may be determined in accordance with known procedures. Such assays can detect the protein or the nucleic acid encoding it in the sample to be tested. In one embodiment, tumor levels of ErbB ligand can be determined using immunohistochemistry (IHC); see, for example, Scher et al., Clin. Cancer Research 1: 545-550 (1995). Alternatively, or in addition, the levels of the ErbB ligand encoding nucleic acid in the sample to be tested may be assessed, e.g., by FISH, Southern blotting, or PCR techniques.

Moreover, furthermore, overexpression or amplification of an ErbB receptor or ErbB ligand can be assessed using an in vivo diagnostic assay, e.g. by administering a molecule (such as an antibody) that binds to the molecule to be detected and is labeled with a detectable label (e.g. a radioisotope) and by externally examining the patient for the location of the marker.

If the cancer to be treated is hormone independent cancer, expression of a hormone (e.g. androgen) and / or its corresponding receptor in the tumor can be assessed using any of the various assays available, e.g. as described above. Alternatively, or additionally, a patient may be diagnosed as having hormone independent cancer due to the fact that he no longer responds to antiandrogen therapy.

In some embodiments, the patient is administered an immunoconjugate comprising an anti-ErbB2 antibody conjugated to a cytotoxic agent. Preferably, the immunoconjugate and / or the ErbB2 protein to which it binds is / are internalized into the cell, resulting in increased therapeutic efficacy of the immunoconjugate in killing cancer cells.

With which it is associated. In a preferred embodiment, the cytotoxic agent targets or interferes with the nucleic acid in the cancer cell. Examples of such cytotoxic agents include maytansinoids, calicheamicins, ribonuclease, and DNA endonuclease.

The anti-ErbB2 antibodies or immunoconjugates are administered to a human patient according to known methods such as intravenous administration, e.g. intraocular, oral, topical or inhalation. Intravenous or subcutaneous administration is preferred.

Other therapeutic regimens can be combined with the administration of the anti-ErbB2 antibody. Combined administration includes simultaneous administration using separate forms or a single pharmaceutical form, and sequential administration in any order, preferably there is a period during which both (or all) active agents simultaneously exert their biological activities.

In a preferred embodiment, the patient is treated with two different anti-ErbB2 antibodies. For example, a patient can be treated with a first anti-ErbB2 antibody that blocks ligand activation of the ErbB receptor or an antibody having the biological properties of monoclonal antibody 2C4, as well as a second anti-ErbB2 antibody that is a growth inhibitory antibody (e.g., HERCEPTIN).^®), or an anti-ErbB2 antibody that induces apoptosis in cells which overexpress ErbB2 (e.g. 7C2, 7F3 or humanized variants thereof). Preferably, such combination therapy results in a synergistic therapeutic effect. For example, a HERCEPTIN patient can be treated^®then treat with rhuMAb 2C4, e.g., if a patient is unresponsive to HERCEPTIN therapy^®. In another embodiment, the patient may first be treated with rhuMAb 2C4 followed by HERCEPTIN therapy.^®. In yet a further embodiment, the patient may be treated with rhuMAb 2C4 and HERCEPTIN simultaneously^®.

It may also be desirable to combine administration of the antibody or antibodies directed against ErbB2, with administration of the antibody directed against another tumor associated antigen. The other antibody in this case may bind, for example, to EGFR, ErbB3, ErbB4, or vascular endothelial growth factor (VEGF).

In one embodiment, the treatment of the present invention comprises the combined administration of the anti-ErbB2 antibody (or antibodies) and one or more chemotherapeutic agents or growth inhibitory agents, including the simultaneous administration of cocktails of different chemotherapeutic agents. Preferred chemotherapeutic agents include taxanes (such as paclitaxel and docetaxel) and / or anthracycline antibiotics. The formulations and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by a practitioner in the art. Formulations and dosing schedules for such chemotherapy are also described in the Chemotherapy Service, edited by MC Perry, Williams &amp; amp; Wilkins, Baltimore, MD (1992).

The antibody can be combined with an anti-hormonal compound, e.g., an anti-estrogenic compound such as tamoxifen, an anti-progesterone such as onapristone (see European Patent Specification No. 616,812), or an anti-androgen such as flutamide, at dosages known for such molecules. If the cancer to be treated is hormone independent cancer, the patient may undergo antihormone therapy beforehand, and after the cancer becomes hormone independent, the patient may be administered an anti-ErbB2 antibody (and optionally other agents as described herein). description).

At times, it may also be beneficial to co-administer a cardiac protective agent to the patient (to prevent or reduce therapy-related myocardial dysfunction, or one or more cytokines. A drug targeting EGFR or an anti-angiogenic agent may also be administered concurrently. surgical removal of cancer cells and / or radiation therapy.

The anti-ErbB2 antibodies of the present invention can also be combined with an EGFR-targeting drug such as those discussed above in the Definitions section to provide a complementary, and potentially synergistic, therapeutic effect.

The appropriate dosage for each of the above co-administered agents is as currently used, and may be reduced due to the combined effect (synergy) of the agent and the anti-ErbB2 antibody.

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The appropriate dosage of the antibody to prevent or treat a disease will depend on the type of disease being treated, as defined above, the severity and course of the disease, whether the antibody is being administered for preventive or therapeutic purposes, prior treatment, the patient's clinical history and response to the antibody, and discretion. attending physician. The antibody is suitably administered to the patient once or in series during the course of treatment. Depending on the nature and severity of the disease, the initial proposed dose for administration to a patient is approximately from 1 μg / kg to 15 mg / kg (e.g. 0.1 mg / kg 20 mg / kg) of the antibody, depending on, for example, whether there is it is one or more separate administrations or administration by continuous infusion. A typical daily dosage might range from about 1 µg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, treatment is continued until a desired suppression of disease symptoms occurs. A preferred dosage of the antibody will range from about 0.05 mg / kg to about 10 mg / kg. Thus, a patient may be administered one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof). Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, e.g. about six, doses of the anti-ErbB2 antibody). A higher loading dose may be administered initially, followed by one or more lower doses. An exemplary dosing regimen involves administering an initial loading dose of about 4 mg / kg, followed by a weekly maintenance dose of about 2 mg / kg of the anti-ErbB2 antibody. However, other dosing regimens may be useful. The progress of this therapy is readily monitored using conventional techniques and assays.

In addition to administering an antibody protein to a patient, the present application contemplates administering the antibody by gene therapy. Such administration of the nucleic acid encoding the antibody is encompassed by the expression administering a therapeutically effective amount of the antibody. See, for example, International Patent Application No. 96/07321, published March 14, 1996 relating to the use of gene therapy to generate intracellular antibodies.

There are two main approaches to delivering a nucleic acid (possibly contained in a vector) into a patient's cells: in vivo and ex vivo. For in vivo delivery, the nucleic acid is injected directly into the patient, usually at the site where the antibody is required. For ex vivo treatment, the patient's cells are removed, nucleic acid is introduced into these isolated cells, and the modified cells are administered to the patient either directly or, for example, encapsulated in porous membranes that are implanted into the patient (see, e.g., U.S. patents). with numbers 4 892 538 and 5 283 187). There are various techniques available for introducing nucleic acids into viable cells. Techniques differ depending on whether the nucleic acid is transferred into cultured cells in vitro or in vivo in the cells of the intended host. Suitable techniques for transferring nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation methods, etc. A vector commonly used for ex vivo gene delivery is retrovirus.

Currently preferred in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, herpes simplex virus I, or adeno-associated virus) and lipid-based systems (useful lipids for lipid mediated gene transfer are, for example, DOTMA, DOPE, and DC-Chol) . In some situations, it is desirable to provide a nucleic acid source with an agent that targets target cells, such as an antibody specific for a target cell surface membrane protein, a receptor ligand on target cells, etc. When liposomes are used, it may be used to direct and / or facilitate uptake. proteins that bind to the cell surface membrane protein associated with endocytosis, e.g. capsid proteins or fragments thereof which are tropic towards a particular cell type, antibodies directed against proteins that are internalized in the cycle of action, and proteins that target the intracellular position and increase the intracellular half-life. The technique of receptor dependent endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262: 4429-4432 (1987) and Wagner et al., Proc. Natl. Acad. Sci. USA 87: 3410-3414 (1990). For a review of currently known gene marking and gene therapy protocols see Anderson et al., Science 256: 808-813 (1992). See also International Patent Application No. 93/25673 and the references cited therein.

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VI. Products

The invention allows for the creation of an article containing materials useful for the treatment of the described disorders. The article of manufacture comprises a container and a label or package insert on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The containers can be formed from a variety of materials, such as glass or plastic. The container holds a composition that is effective in treating the condition and may have a sterile access site (for example, the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-ErbB2 antibody. The label or package insert indicates that the composition is used to treat the selected condition, such as cancer. In one embodiment, the label or package insert indicates that the composition comprising an ErbB2 binding antibody can be used to treat a cancer that expresses an ErbB receptor selected from the group consisting of epidermal growth factor receptor (EGFR), ErbB3, and ErbB4, preferably EGFR. . Furthermore, the label or package insert may indicate that the patient that can be treated is a patient having a cancer characterized by over-activation of an ErbB receptor selected from EGFR, ErbB3, or ErbB4. For example, the cancer may be a cancer which overexpresses these receptors and / or which overexpresses an ErbB ligand (such as TGF-α). The label or package insert may also indicate that the composition can be used to treat cancer that is not characterized by overexpression of the ErbB2 receptor. For example, while the information sheet for HERCEPTIN^® indicates that the antibody is used to treat patients with metastatic breast cancer that overexpress ErbB2 protein, the package insert of the present invention may indicate that the antibody or composition is used to treat cancer regardless of the degree of ErbB2 overexpression. In other embodiments, the package insert may indicate that the antibody or composition can be used to treat breast cancer (e.g. metastatic breast cancer), hormone independent cancer, prostate cancer (e.g. androgen independent prostate cancer), lung cancer (e.g. non-small cell lung cancer), colon cancer, rectal cancer or colorectal cancer, or any of the other diseases or disorders disclosed herein. In addition, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises a first antibody that binds to ErbB2 and inhibits the growth of ErbB2-overexpressing cancer cells, and (b) a second container with the composition contained therein. wherein the composition comprises a second antibody that binds to ErbB2 and blocks ligand activation of the ErbB receptor. The article of manufacture in this embodiment of the invention may further include a package insert indicating that the first and second antibody compositions can be used to treat cancer. In addition, the package insert may contain instructions for the user of the composition (comprising an antibody that binds ErbB2 and blocks ligand activation of the ErbB receptor) for combination therapy with the antibody and any of the adjunctive therapies described in the preceding sections (e.g. a chemotherapeutic agent, an EGFR targeting drug, an anti-angiogenic agent, an antihormonal compound, a cardioprotective agent and / or a cytokine). Alternatively, or additionally, the article may further comprise a second (or third) container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial or user standpoint, including other buffers, diluents, filters, needles, and syringes.

VII. Non-therapeutic uses of the anti-ErbB2 antibody

The antibodies (e.g., humanized anti-ErbB2 antibodies) also have non-therapeutic applications.

For example, the antibodies can be used as affinity purification agents. In this method, the antibodies are immobilized on a solid phase such as Sephadex resin or filter paper using methods known in the art. The immobilized antibody is contacted with the sample containing the anti-ErbB2 protein (or fragment thereof) to be purified, and the support is then washed with a suitable solvent that will remove substantially all material from the sample, except for the ErbB2 protein which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0, that will release the ErbB2 protein from the antibody.

Anti-ErbB2 antibodies may also be useful in diagnostic assays of the ErbB2 protein, e.g., detecting its expression in specific cells, tissues or serum.

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For diagnostic applications, the antibody will typically be labeled with a detectable group. There are a number of tags available, which can be broadly grouped into the following categories:

(a) Radioactive isotopes, such as ³⁵S, ¹⁴C, ¹²⁵AND, ³H i ¹³¹I. Antibody can be radiolabeled using the techniques described in, for example, Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Eds., Wiley-Interscience, New York, New York (1991), and radioactivity can be measured using a counter. scintillation.

(b) Fluorescent markers such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin and Texas Red. Fluorescent labels can be conjugated to the antibody using the techniques disclosed in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate tags are available and a review of some of them is provided in United States Patent No. 4,275,149. The enzyme generally catalyzes a chemical alteration of a chromogenic substrate that can be measured using various techniques. For example, an enzyme can catalyze a color change of a substrate that can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying the change in fluorescence are described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and can then emit light that can be measured (using a chemiluminometer, for example) or provides energy to a fluorescent acceptor. Examples of enzyme labels include luciferases (e.g. firefly luciferase or bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, oxidases (e.g. glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase and the like. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al. , Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in: Methods in Enzym. (Eds. J. Langone and H. Van Vunakis), Academic Press, New York, 73: 147-166 (1981).

Examples of enzyme-substrate combinations include, for example:

(i) horseradish peroxidase (HRPO) with hydrogen peroxide as substrate, wherein the hydrogen peroxide oxidizes a dye precursor (e.g., orthophenylene diamine (OPD) or 3,3 ', 5,5'-tetramethylbenzidine hydrochloride (TMB);

(ii) alkaline phosphatase (AP) with paranitrophenylphosphate as chromogenic substrate and (iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g. p-nitrophenyl-eD-galactoside or the fluorogenic substrate 4-methylumbeliferyl-eD -galactoside.

Numerous other enzyme-substrate combinations are available to those skilled in the art. See U.S. Patent Nos. 4,275,149 and 4,318,980 for a general overview.

Sometimes the label is indirectly conjugated to the antibody. One skilled in the art will be aware of the various techniques by which this may be achieved. For example, the antibody can be conjugated with biotin and avidin can be conjugated with any of the three broad categories listed above, or vice versa. Biotin binds selectively to avidin and thus the label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the tag with the antibody, the antibody is conjugated with a small hapten (e.g., digoxin) and one of the various types of labels listed above is conjugated with an anti-hapten antibody (e.g., an anti-digoxin antibody). Thus, indirect conjugation of the label with the antibody can be achieved.

In another embodiment of the invention, the anti-ErbB2 antibody need not be labeled, and its presence can be detected using a labeled antibody that binds to the anti-ErbB2 antibody.

The antibodies of the present invention can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

The tumor sample for immunocytochemistry may be fresh or frozen, or may be paraffin-embedded and fixed with a preservative such as, for example, formalin.

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The antibodies can also be used in in vivo diagnostic assays. Generally, the antibody is labeled with a radionuclide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹AND, ¹²⁵AND, ³H, ³²P or ³⁵S) so that the tumor can be located using immunoscintigraphy. For convenience, the antibodies of the present invention may be provided in a kit, i.e. a packaged kit of reagents in predetermined amounts with instructions for performing a diagnostic assay. If the antibody has been labeled with an enzyme, the kit will contain the substrates and cofactors required by the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives such as stabilizing agents, buffers (e.g., blocking buffer or lysis buffer), and the like may be included. The relative amount of the various reagents can be varied widely to provide concentrations of the reagents in the solution that substantially optimize the sensitivity of the assay. In particular, the reagents may be provided as dry powders, usually lyophilized, including excipients which, when reconstituted, will provide the correct solution of the reagents.

VIII. Material depository The following hybridoma cell lines have been deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, United States of America (ATCC):

<td>Mark</td><td>ATCC number</td><td>Deposit date</td>

<td>Antibodies</td><td></td><td></td>

<td>7C2</td><td>ATCC HB-12215</td><td>October 17, 1996</td>

<td>7F3</td><td>ATCC HB-12216</td><td>October 17, 1996</td>

<td>4D5</td><td>ATCC CRL 10464</td><td>May 24, 1990</td>

<td>2C4</td><td>ATCC HB-12697</td><td>April 8, 1999</td>

Further details of the invention are illustrated by the following non-limiting Examples. The disclosures of all citations in the specification are formally incorporated herein by reference.

Example 1

Production and characterization of the monoclonal antibody 2C4

Mouse monoclonal antibodies 2C4, 7F3 and 4D5 that specifically bind to the extracellular domain of ErbB2 were generated as described in Fendly et al., Cancer Research, 50: 1550-1558 (1990). Briefly, NIH 3T3 / HER2-3400 cells (which express approximately 1 x 10⁵ ErbB2 molecules / cell) obtained as described by Hudziak et al., Proc. Natl. Acad. Sci. (USA) 84: 7158-7163 (1987) was harvested using phosphate buffered saline (PBS) containing 25 mM EDTA and used to immunize BALB / c mice. Mice received intraperitoneal injections 10⁷ cells in 0.5 ml PBS at weeks 0, 2, 5 and 7. Mice with antisera that immunoprecipitated labeled ³²P ErbB2, received intraperitoneal injections of ErbB2 membrane extract purified on Sepharose with Immobilized Wheat Germ Agglutinin (WBA) at weeks 9 and 13. Subsequently, 0.1 ml of ErbB2 was injected intravenously and spleen lymphocytes were fused with the mouse myeloma X63-Ag8.633 line. .

Hybridoma supernatants were screened for ErbB2 binding by ELISA and radioimmunoprecipitation.

ErbB2 epitopes bound by monoclonal antibodies 4D5, 7F3 and 2C4 were determined by competitive binding analysis (Fendly et al., Cancer Research 50: 1550-1558 (1990)). Cross-blocking studies were performed on antibodies by direct fluorescence of intact cells using PANDEX ™ Screen Malachite to quantify the fluorescence. Each of the monoclonal antibodies was conjugated to fluorescein isothiocyanate (FITC) using established procedures (Wofsy et al., Selected Methods in Cellular Immunology, page 287, Mishel and Schiigi (ed.) San Francisco: WJ Freeman Co. (1980)). Coherent monolayers of NIH 3T3 / HER2-3400 cells were trypsinized, washed once and resuspended at a density of 1.75 x 10⁶ cells / ml in cold PBS containing 0.5% Bovine Serum Albumin (BSA) and 0.1% NaN3. Latex particles (IDC, Portland, OR) were added to 1% final concentration to reduce clogging of the PANDEX ™ platelet membranes. Cells in suspension, 20 µ], and 20 µl of purified monoclonal antibodies (100 µg / ml to 0.1 µg / ml) were added to the wells of a PANDEX ™ plate and incubated on ice for 30 minutes. A predetermined dilution of 20 µL of FITC-labeled monoclonal antibodies was added to each well, incubated for 30 minutes, washed and fluorescence quantified using PANDEX ™. Monoclonal antibodies were considered to share a common epitope if they blocked their mutual binding by 50% or more compared to the control monoclonal antibody. In this experiment, the monoclonal antibodies 4D5, 7F3 and 2C4 were assigned to epitopes I, G / F and F, respectively.

The growth inhibitory properties of monoclonal antibodies 2C4, 7F3 and 4D5 were assessed using the breast tumor cell line SK-BR-3 (see Hudziak et al., Molec. Cell. Biol. 9 (3): 1165-1172 (1989)). Briefly, SK-BR-3 cells were detached from the medium using 0.25% (v / v) trypsin and resuspended in complete medium at a density of 4 x 10⁵ cells per ml. Volumes of 100 μl (4 x 10⁴ cells) were plated in 96-well microdilution plates, the cells were allowed to adhere, and then 100 μl of medium alone or medium containing the monoclonal antibody was added (final concentration 5 μg / ml). After 72 hours, the plates were washed twice with PBS (pH 7.5), stained with crystal violet (0.5% in methanol) and analyzed for relative cell proliferation as described in Sugarman et al., Science, 230: 943-945 (1985 ). Monoclonal antibodies 2C4 and 7F3 inhibited the relative proliferation of SK-BR-3 cells by about 20% and about 30%, respectively, compared to about 56% inhibition achieved with monoclonal antibody 4D5.

Monoclonal antibodies 2C4, 4D5, and 7F3 were evaluated for their ability to inhibit HRG-stimulated tyrosine phosphorylation of 180,000 proteins from MCF7 whole cell lysates (Lewis et al., Cancer Research 56: 1457-1465 (1996)). MCF7 cells have been reported to express all known ErbB receptors, but at relatively low levels, since ErbB2, ErbB3 and ErbB4 have nearly identical molecular sizes, it is not possible to distinguish which tyrosine proteins were phosphorylated when whole cell lysates were assessed by blotting analysis Western type.

However, these cells are ideal for assays of HRG-stimulated tyrosine phosphorylation because under the assay conditions used, in the absence of exogenously added HRG, they show only low or undetectable levels of protein tyrosine phosphorylation with a Mr in the range of 180,000.

MCF7 cells were seeded in 24-well plates, anti-ErbB2 monoclonal antibodies were added to each well and incubated for 30 minutes at room temperature. Then rHRGe1 was added to each well_177-244 to 0.2 nM final concentration and incubation continued for 8 minutes. Media was carefully aspirated from each well and the reaction was stopped by adding 100 µl of SDS sample buffer (5% SDS, 25 mM DTT and 25 mM Tris-HCl, pH 6.8). Each sample (25 μl was subjected to 4-12% gradient gel electrophoresis (Novex) and then electrophoretically transferred to a polyvinylidene fluoride membrane. Immunoblots were developed using an anti-phosphotyrosine antibody (4G10, with UBI, used at a concentration of 1 (µg / ml) and the intensity of the major reactive band of about 180,000 Mr was quantified by reflected light densitometry as previously described (Holmes et al., Science , 256: 1205-1210 (1992); Sliwkowski et al., J. Biol. Chem., 269: 14661-14665 (1994)).

Monoclonal antibodies 2C4, 7F3, and 4D5 significantly inhibited signal generation of HRG-induced tyrosine phosphorylation at Mr 180,000. In the absence of HRG, neither of these antibodies were able to stimulate protein tyrosine phosphorylation with Mr 180,000. These antibodies also did not cross-react with EGFR. (Fendly et al., Cancer Research 50: 1550-1558 (1990)), ErbB3 or ErbB4. The 2C4 and 7F3 antibodies significantly inhibited the HRG stimulation of p180 tyrosine phosphorylation to &lt;25% of control. The 4D5 monoclonal antibody was able to block the stimulation of tyrosine phosphorylation by HRG by approximately 50%. Figure 2A shows dose-response curves for 2C4 and 7F3 inhibition of HRG-stimulated p180 tyrosine phosphorylation as determined by reflectance densitometry.

Analysis of these inhibition curves using 4-parameter fit yielded IC⁵⁰ of 2.8 0.7 nM and 29.0 4.1 nM for 2C4 and 7F3, respectively.

Studies of the inhibition of HRG binding by anti-ErbB2 antibodies to MCF7 breast tumor cell lines were performed with monolayer cultures on ice in a 24-well plate (Lewis et al., Cancer Research, 56: 1457-1465 (1996)). Anti-ErbB2 monoclonal antibodies were added to each well and incubated for 30 minutes. Tagged was added¹²⁵I rHRGe1177-244 (25 pm) and incubation was continued for 4 to 16 hours. Figure 2B shows dose-response curves for 2C4 or 7F3 inhibition of HRG binding to MCF7 cells. Varying concentrations of 2c4 or 7F3 were incubated with MCF7 cells in the presence of labeled¹²⁵I rHRGe1 and the inhibition curves are shown in Fig. 2B. Analysis of these data gave IC50s of 2.4 0.3 nM and 19.0 7.3 nM for 2C4 and 7F3, respectively. Maximum inhibition of about 74% for 2C4 and 7F3 was consistent with the data for inhibition of tyrosine phosphorylation.

In order to determine whether the effect of anti-ErbB2 antibodies on MCF7 cells was a general phenomenon, human tumor cell lines were incubated with 2C4 and 7F3

The degree of specific labeled binding was monitored ¹²⁵I rHRGe1 (Lewis et al., Cancer Research 56: 1457-1465 (1996)). The results of these studies are shown in Figure 3. Labeled Binding¹²⁵And rHRGe1 can be significantly inhibited by either 2C4 or 7F3 in all cell lines except the MDA-MB-468 breast cancer cell line, which has been reported to express little or no ErbB2. ErbB2 expression has been reported for the remaining cell lines, with a wide degree of variation in the expression level. Indeed, the extent of ErbB2 expression in the cell lines tested varies by more than two orders of magnitude. For example, about 10 are expressed on BT-20, MCF7 and Caov lines⁴ ErbB2 receptors / cell, while in BT-474 and SK-BR-3 around 10⁶ ErbB2 receptors / cell. Given the wide expression range of ErbB2 in these cells and the above data, it was concluded that the interaction between ErbB2 and ErbB3 or ErbB4 was itself a high affinity interaction that occurs on the surface of the plasma membrane.

The growth inhibitory effect of monoclonal antibodies 2C4 and 4D5 on MDAMB-175 and SK-BR-3 cells in the presence or absence of exogenous rHRGe1 was assessed (Schaefer et al., Oncogene 15: 1385-1394 (19997)). ErbB2 levels in MDA-MB-175 cells are 4-6 times higher than those found in normal breast epithelial cells, and ErbB2-ErbB4 receptor tyrosine is constitutively phosphorylated in MDA-MB-175 cells. MDA-MB-175 cells were treated with the anti-ErbB2 monoclonal antibodies 2C4 and 4D5 (10 µg / ml) for 4 days. In the crystal violet staining assay, incubation with 2C4 showed a strong growth inhibitory effect on this cell line (Fig. 4A). Exogenous HRG did not significantly reverse this inhibition. On the other hand, 2C4 showed no inhibitory effect on the SK-BR-3 cell line which overexpresses ErbB2 (Fig. 4B). Monoclonal antibody 2C4 was able to inhibit MDA-MB-175 cell proliferation to a greater extent than monoclonal antibody 4D5, both in the presence and absence of exogenous HRG. Inhibition of cell proliferation by 4D5 is dependent on the expression level of ErbB2 (Lewis et al., Cancer Immunol. Immunother. 37: 255-263 (1993)). A maximum 66% inhibition could be detected in SK-BR-3 cells (Fig. 4B). However, this effect could be overcome by endogenous HRG.

Example 2

2C4 monoclonal antibody blocks HRG-dependent binding of ErbB2 to ErbB3

The ability of ErbB3 to associate with ErbB2 was tested in a co-immunoprecipitation experiment. 1.0 x 10⁶ MCF7 or SK-BR-3 cells were plated in six-well cell culture plates in DMEM / Ham's F12 medium at a 50:50 ratio containing 10% Fetal Bovine Serum (FBS) and 10 mM HEPES, pH 7.2 (growth medium) and allowed to attachment to the ground overnight. Before starting the experiment, cells were starved for two hours in serum-free growth medium.

Cells were washed briefly with phosphate buffered saline (PBS) and then incubated with either 100 nM of the indicated antibody diluted in 0.2% w / v bovine serum albumin (BSA) in RPMI medium with 10 mM HEPES, pH 7.2 (buffer for binding), or with binding buffer alone (control). After one hour at room temperature, HRG was added to half of the (+) wells to a final concentration of 5 nM. A similar volume of binding buffer was added to the other (-) wells. Incubation was continued for 10 minutes.

Supernatants were removed by aspiration and cells were lysed in RPMI, 10 mM HEPES, pH 7.2, 1.0% v / v TRITON-X100 ™, 1.0% w / v CHAPS (lysis buffer) containing 0.2 mM PMSF , 10 μg / ml leupeptin and 10 TU / ml aprotinin. Lysates were cleared of insoluble material by centrifugation.

ErbB2 was immunoprecipitated using a monoclonal antibody covalently coupled to an affinity gel (Affi-Prep 10, Bio-Rad). This antibody (Ab-3, Oncogene Sciences) recognizes an epitope of the cytoplasmic domain. Immunoprecipitation was carried out by adding 10 µl of the gel suspension containing approximately 8.5 µg of immobilized antibody to each lysate, and allowing the samples to mix at room temperature for two hours. The gels were then collected by centrifugation. The gels were periodically washed three times with lysis buffer to remove unbound material. SDS sample buffer was then added and the samples were briefly heated in a boiling water bath.

The supernatants were electrophoresed on 4-12% polyacrylamide gels and electroblotted onto nitrocellulose membranes. The presence of ErbB3 was assessed as a blot probe using a polyclonal antibody directed against epitopes of its cytoplasmic domain (C-17, Santa Cruz Botech). The blots were visualized using a chemiluminescent substrate (ECL, Amersham).

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As shown in the control lanes in Figures 5A and 5B for MCF-7 and SK-BR-3 cells, respectively, ErbB3 was present in ErbB2 immunoprecipitates only when cells were stimulated with HRG. If cells were first incubated with monoclonal antibody 2C4, the ErbB3 signal was suppressed in MCF7 cells (Fig. 5A, lane 2C4 +) or significantly decreased in SK-BR-3 cells (Fig. 5B, lane 2C4 +). As shown in Fig. 5A-B, monoclonal antibody 2C4 blocks heregulin-dependent binding of ErbB3 to ErbB2 in both MCF7 and SK-BR-3 cells much more effectively than HERCEPTIN^®. Pre-incubation with HERCEPTIN^® decreased the ErbB3 signal in MCF7 lysates, but had little or no effect on the amount of ErbB3 co-precipitating from SK-BR-3 lysates. Pre-incubation with an anti-EGF receptor antibody (Ab-1, Oncogene Sciences) had no effect on the ability of ErbB3 to co-immunoprecipitate with ErbB2 for any of the cell lines.

Example 3

Humanized 2C4 antibodies

The variable domains of the murine monoclonal antibody 2C4 were first cloned into a vector that allows the production of a chimeric mouse / human Fab fragment. Total RNA was isolated from the hybridoma cells using the Stratagene extraction kit according to the manufacturer's protocols. The variable domains were amplified by RT-PCR, gel purified and inserted into a derivative of a pUC119 based plasmid containing a human kappa constant domain and a human CH1 domain as previously described (Carter et al., PNAS (USA) 89: 4285 (1992) and description U.S. Patent No. 5,821,337). The resulting plasmid was transformed into the E. coli 16C9 strain to express the Fab fragment. The cultivation, induction of protein expression and purification of the Fab fragment were performed as previously described (Werther et al., I. Immunol. 157: 4986-4995 (1996), Presta et al., Cancer Research 57: 4593-4599 (1997). ).

Purified chimeric 2C4 Fab fragments were compared to the murine parental antibody 2C4 for its ability to inhibit binding ¹²⁵I-HRG with MCF7 cells and inhibition of HRG activation of p180 tyrosine phosphorylation in MCF7 cells. As shown in Figure 6A, the chimeric 2C4 Fab fragment is very effective in disrupting the formation of the high affinity ErbB2-ErbB3 binding site on the human breast cancer cell line, MCF7. The relative IC50 value calculated for intact murine 2C4 is 4.0 ± 0.4 nM, while the value for the Fab fragment is 1.7 ± 1.1 nM. As shown in Fig. 6B, the monovalent chimeric 2C4 Fab fragment is very effective in disrupting the HRG dependent activation of ErbB2-ErbB3. The IC50 value calculated for intact murine monoclonal antibody 2C4 is 6.0 ± 2 nM, while the value for the Fab fragment is 15.0 ± 2 nM.

DNA sequencing of a chimeric clone made it possible to identify CDR residues (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)) (Figures 7A and B). Using oligonucleotide directed mutagenesis, all six of these CDRs were introduced into the complete human framework (kappa VL subgroup I and VH subgroup III) contained in plasmid VX4 as previously described (Presta et al., Cancer Research, 57: 4593-4599 (1997) ). The protein was expressed from the resulting sequences with the replaced CDRs and purified as above. Binding studies were performed to compare the two versions. Briefly, a NUNC MAXISORP ™ plate was coated with 1 microgram per ml of ErbB2 extracellular domain (ECD; manufactured as described in International Patent Application No. 90/14357) in 50 mM carbonate buffer, pH 9.6, overnight at 4 ° C. and then blocked with ELISA diluent (0.5% BSA, 0.05% polysorbate 20, PBS) at room temperature for 1 hour. Serial dilutions of the samples in ELISA Diluent were incubated in the plates for 2 hours. After washing, the bound Fab fragment was detected using a biotinylated mouse anti-human kappa chain antibody (ICN 634771) followed by streptavidin-conjugated horseradish peroxidase (Sigma) and 3,3 ', 5,5'-tetramethylbenzidine (Kirkegaard &amp; Perry Laboratories, Gaithersburg , MD) as a substrate. Absorbance was read at 450 nm. As shown in Fig. 8A, all binding was lost upon construction of a human Fab fragment with the swapped CDRs.

To restore humanized Fab binding, mutants were constructed using CDR-replaced DNA as template. Using a computer generated model (Figure 9), these mutations were designed to change human framework region residues to their murine counterparts at positions where the change would affect CDR conformation or the antibody-antigen interface. The mutants are shown in Table 2.

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Table 2

Design of humanized 2C4 FR mutations

<td>Mutant number</td><td>Framework Region Substitutions (FR)</td>

<td> 560</td><td>ArgH71Val</td>

<td> 561</td><td>As pH 73 Arg</td>

<td> 562</td><td>ArgH71Val, AspH73Arg</td>

<td> 568</td><td>ArgH71Val, AspH73Arg, AlaH49Gly</td>

<td> 569</td><td>ArgH71Val, AspH73Arg, PheH67Ala</td>

<td> 570</td><td>ArgH71Val, AspH73Arg, AsnH76Arg</td>

<td> 571</td><td>ArgH71Val, AspH7 3Arg, LeuH7 8Val</td>

<td> 574</td><td>ArgH71Val, AspH73Arg, IleH69Leu</td>

<td> 56869</td><td>ArgH71Val, AspH73Arg, AlaH49Gly, PheH67Ala</td>

Binding curves for various mutants are shown in Figures 8A-C. Version 574 of the humanized Fab with changes to Ar-gH71Val, AspH73Arg and IleH69Leu was found to have restored binding to this original chimeric 2C4 Fab fragment. Additional FR and / or CDR residues such as L2, L54, L55, L56, H35 and / or H48 (e.g. substitute as follows - IleL2Thr, ArgL54Leu, TyrL55-Glu, ThrL56Ser, AspH35Ser and ValH48Ile). Alternatively, or additionally, the humanized antibody may be affinity matured (see above) to further improve its affinity and / or other biological activities.

Humanized 2C4 version 574 was affinity matured using the phage display method. Briefly, Humanized Fab 2C4.574 was cloned into a phage display vector fused to gene III. When the phage particles are induced by infection with the helper phage M13K07, this fusion allows the display of the Fab at the N-terminus of the tail fiber protein product of gene III (Baca et al., J. Biol. Chem., 272: 10678-10678 (1997)).

Individual libraries were constructed for each of the 6 CDRs identified above. In these libraries, the amino acids in the CDRs that were identified using a computer model (Figure 9) to be potentially important in binding to ErbB2 were randomly mutated using oligonucleotides containing NNS as codons. Libraries were then selected against ECD ErbB2 coated NUNC MAXIS0RP ™ plates with 3% milk powder in PBS with 0.2% TWEEN 20^® (MPBST) used in place of all blocking solutions. In order to select phages with affinities higher than that of 2C4.574, soluble ECD ErbB2 or soluble Fab 2C4.574 were added as a competitor during the 3, 4 and 5 selection rounds during the washing steps. The washing time was extended to 1 hour at room temperature.

After 5 rounds of selection, individual clones were reanalyzed by phage ELISA. Single clones were grown in U-bottom 96-well tissue culture plates, Costar, and phage were induced by addition of helper phage. After overnight growth, E. coli cells were pelleted and the phage-containing supernatants were transferred to 96-well plates where phage were blocked with MPBST for one hour at room temperature. NUNC MAXISORP ™ plates coated with EDC ErbB2 were also blocked with MPBST as above. Blocked phage were incubated on the plates for 2 hours. After washing, bound phages were detected using horseradish peroxidase-conjugated anti-M13 monoclonal antibody (Amersham Pharmacia Biotech, Inc., 27-9421-01) diluted 1: 5000 in MPBST followed by 3,3 ', 5,5'-tetramethylbenzidine as a substrate. Absorbance was read at 450 nm.

The 48 clones from each library giving the highest signals were sequenced. The clones with the most frequent sequences were subcloned into the vector described above, which enables the expression of soluble Fabs. These Fabs were induced, proteins were purified and the purified Fabs were analyzed for binding in the ELISA assay described above and the binding was compared to that of the starting humanized version of 2C4.574.

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After the mutations of interest in individual CDRs had been identified, additional mutants were constructed and tested as described above which had different combinations of these mutations. Mutants that showed improved affinity over 574 are described in Table 3.

Table 3

Design of mutants obtained by affinity maturation of 2C4.574

<td>The name of the mutant</td><td>Change from 574</td><td>Mutant / 574 *</td>

<td>H3.A1</td><td>serH99trp, metH34leu</td><td> 0,380</td>

<td>L2.F5</td><td>serL50trp, tyrL53gly, metH34leu</td><td> 0,087</td>

<td>HI.3.B3</td><td>thrH28gln, thrH30ser, metH34leu</td><td> 0,572</td>

<td>L3.G6</td><td>tyrL92pro, ileL931ys, metH34leu</td><td> 0,569</td>

<td>L3.G11</td><td>tyrL92ser, ileL93arg, tyrL94gly, metH34leu</td><td> 0,561</td>

<td>L3.29</td><td>tyrL92phe, tyr96asn, metH34leu</td><td> 0,552</td>

<td>L3.36</td><td>tyrL92phe, tyrL94leu, tyrL96pro, metH34leu</td><td> 0,215</td>

<td> 654</td><td>serL50trp, metH34leu</td><td> 0,176</td>

<td> 655</td><td>metH34ser</td><td> 0,542</td>

<td> 659</td><td>serL50trp, metH34ser</td><td> 0,076</td>

<td>L2.F5.H3.A1</td><td>serL50trp, tyrL53gly, metH34leu, serH99trp</td><td> 0,175</td>

<td>L3G6.H3.A1</td><td>tyrL92pro, ile931ys, metH34leu, serH99trp</td><td> 0,218</td>

<td>H1.3.B3.H3.A1</td><td>thrH28gln, thrH30ser, metH34leu, serH99trp</td><td> 0,306</td>

<td>L3.G11.H3.A1</td><td>tyrL92ser, ile93arg, tyrL94gly, metH34leu, serH99trp</td><td> 0,248</td>

<td>654.H3.A1</td><td>serL50trp, metH34leu, serH99trp</td><td> 0,133</td>

<td>654.L3.G6</td><td>serL50trp, metH34leu, tyrL92pro, ileL93lys</td><td> 0,213</td>

<td>654.L3.29</td><td>serL50trp, metH34leu, tyrL92phe, tyrL96asn</td><td> 0,236</td>

<td>654.L3.36</td><td>serL50trp, metH351eu, tyrL92phe, tyrL94leu, tyrL96pro</td><td> 0,141</td>

Ratio of the amount of mutant needed to get the mean OD of the standard curve to the amount of 574 needed to get the mean OD of the standard curve in the Erb2 ECD ELISA. A number less than 1.0 indicates that the mutant binds Erb2 better than 574.

The following mutants have also been constructed and are currently being assessed:

659.L3.G6

659.L3.11

659.L3.29

659.L3.36

L2F5.L3G6

L2F5.L3G11

L2F5.L29

L2F5.L36

L2F5.L3G6.655

L2F5.L3G11.655

L2F5.L29.655 serL50trp, metH34ser, tyrL92pro, ileL93lys serL50trp, metH34ser, tyrL92ser, ileL93arg, tyrL94gly serL50trp, metH34ser, tyrL92phe, tyrL96asn serL50trp, metH34ser, tyrL92phe, tyrL94leu, tyrL96pro serL50trp, tyrL53gly, metH34leu, tyrL92pro, ile93lys serL50trp, tyrL53gly, metH34leu, tyrL92ser, ile93arg, tyrL94gly serL50trp, tyrL53gly, metH34leu, tyrL92phe, tyrL96asn serL50trp, tyrL53gly, metH34leu, tyrL92phe, tyrL94leu, tyrL96pro, tyrL96pro serL53trply ile93lys serL50trp, tyrL53gly, metH34ser, tyrL92ser, ileL93arg, tyrL94gly serL50trp, tyrL53gly, metH34ser, tyrL92phe, tyrL96asn L2F5.

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The following mutants are currently being constructed and a homology search is suggested:

678

679

680

681

682

683

684

685

686

687

688

689 thrH30ala thrH30ser lysH64arg leuH96val thrL97ala thrL97ser tyrL96phe tyrL91ala tyrL91phe thrL56ala glnL28ala glnL28glu

A preferred amino acid at position H34 could be methionine. A change to leucine can be made if oxidation is to be performed at this position.

AsnH52 and asnH53 were found to be very favorable for binding. Changing these residues to alanine or aspartic acid dramatically reduced binding.

An intact antibody was prepared containing the light and heavy chain variable domains of humanized version 574 with the heavy chain constant region of human IgG1 (see US Patent No. 5,821,337). The intact antibody is produced by Chinese hamster ovary (CHO) cells. This molecule has been designated rhuMAb 2C4 herein.

Example 4

The monoclonal antibody 2C4 blocks the activation of MAPK by EGF, TGF-α or HRG

Many growth factor receptors signal through the mitogen-activated protein kinase (MAPK) pathway. These dual specificity kinases are one of the key targets of the signal transduction pathways that ultimately trigger cancer cell division. The ability of the monoclonal antibody 2C4 or HERCEPTIN® to inhibit MAPK activation by EGFJGF-α or HRG was assessed as follows.

MCF7 cells (10⁵ cells / well) was plated in serum-containing media in 12-well cell culture plates. The next day, the media was removed and fresh media containing 0.1% serum was added to each well. This procedure was then repeated the next day and the medium was replaced with serum-free binding buffer (Jonesin., J. Biol. Chem. 273: 11667-74 (1998) and Schaefer et al., J. Biol. Chem. 273: 859 before the determination of the medium. -66 (1999)). Cells were allowed to equilibrate to room temperature and then incubated for 30 minutes with 0.5 ml of 200 nM HERCEPTIN * or monoclonal antibody 2C4. The cells were then treated with 1 nM EGF, 1 nM TGF-α, or 0.2 nM HRG for 15 minutes. The reaction was stopped by aspirating the medium, then 0.2 ml of SDS-PAGE sample buffer containing 1% DTT was added. MAPK activation was assessed by Western blotting using an anti-active MAPK antibody (Promega) as previously described (Jones et al., J. Biol. Chem., 273: 11667-74 (1998)).

As shown in Figure 10, monoclonal antibody 2C4 significantly blocked EGF, TGF-α and HRG activation of MAPK to a greater extent than HERCEPTIN®. These data suggest that monoclonal antibody 2C4 binds to the ErbB2 surface that is used to bind it to either EGFR or ErbB3, and thus prevents formation of a signaling receptor complex.

The monoclonal antibody 2C4 has also been shown to inhibit HRG-dependent activation of Akt. Activation of the PI3 kinase signaling pathway is important for cell survival (Carraway

PL 203 326 B1 et al., J. Biol. Chem., 270: 7111-6 (1995)). In cancer cells, activation of PI3 kinase may play a role in the invasive phenotype (Tan et al., Cancer Research, 59: 1620-1625 (1999)). The survival pathway is mediated primarily by the serine / threonine kinase AKT (Bos et al., Trends Blochem. Sci., 20: 441-442 (1995)). Complexes formed between ErbB2 and either ErbB3 or EGFR can initiate these pathways in response to heregulin or EGF, respectively (Olayioye et al., Mol. &amp; Cell .. Biol., 18: 5042-51 (1998); Karunagaran et al., EMBO Journal, 15: 254-264 (1996) and Krymskaya et al., Am. J. Physiol., 276: L246-55 (1999)). Incubation of MCF7 breast cancer cells with 2C4 inhibits heregulin mediated ATK activation. Moreover, the basal ATK activation level present in the absence of heregulin addition is further lowered by the addition of 2C4. These data suggest that 2C4 can inhibit the activation of PI3 kinase by ErbB ligand and that this inhibition can lead to apoptosis. The increased sensitivity to apoptosis may be manifested by the increased sensitivity of neoplastic cells to the toxic effects of chemotherapy.

Thus, monoclonal antibody 2C4 inhibits ligand-initiated ErbB signaling through two major pathways - MAP kinases (major proliferation pathway) and PI3 kinases (major survival / anti-apoptotic pathways).

Example 5

In vivo combination of monoclonal antibody 2C4 and HERCEPTIN

A xenograft model using the lung adenocarcinoma cell line, Calu-3, was used to evaluate the efficacy of anti-HER2 monoclonal antibodies, either alone or in combination, in inhibiting tumor growth. Female NCR nude mice were inoculated subcutaneously with 20 x 10⁶ cells in 0.1 ml. Tumor measurements were performed twice a week and after the tumor nodules had reached a volume of 100 mm³ animals were randomized into 7 treatment groups. The treatment groups were as follows:

(a) a control monoclonal antibody, MAb 1766;

(b) HERCEPTIN^®, 10 mg / kg;

(c) monoclonal antibody 7C2, 10 mg / kg;

(d) monoclonal antibody 2C4, 10 mg / kg;

(e) HERCEPTIN^® and 7C2 each 10 mg / kg (f) HERCEPTIN^® and 2C4 each 10 mg / kg, and (g) monoclonal antibodies 2C4 and 7C2 each 10 mg / kg.

Animals were treated twice a week to day 24. Tumor volumes were measured twice a week to day 38.

As shown in the bar graph in Figure 11, treatment with 2C4 or HERCEPTIN^® Calu-3 tumor bearing mice significantly inhibited tumor growth. HERCEPTIN combination^®and 2C4 or HERCEPTIN^® and 7C2 was superior to either monoclonal antibody administered separately.

Example 6

Treatment of colon and rectal cancer with the monoclonal antibody 2C4

Human colon rectal cancer cell lines such as HCA-7, LS174T, or CaCo-2 are subcutaneously implanted in athymic nude mice as described in Sheng et al., J. Clin. Invest., 99: 2254-2259 (1997). After the tumors have reached a volume of approximately 100 mm³, groups of animals are treated with 10-50 mg / kg of monoclonal antibody 2C4 administered twice weekly by injection into the peritoneal cavity. The monoclonal antibody 2C4 inhibits the growth of colon and rectal cancer xenografts in vivo.

Example 7

Treatment of breast cancer with humanized 2C4

The effect of rhuMAb 2C4 or HERCEPTIN® on ErbB2 non-overexpressing human breast cancer cells was assessed in a 3-day assay using Alamar Blue (Ahmed, SA, Immunol Methods, 170: 211-224 (1994) and Page et al. , Int. J. Oncol. 3: 473-476 (1994)). The cells used in this assay were MDA-175 human breast cancer cells expressing ErbB2 at the 1+ level. As shown in Fig. 12, the growth of the MDA-175 breast cancer cell line is significantly inhibited in a dose-dependent manner by the addition of rhuMAb 2C4 compared to treatment with HERCEPTIN^®.

The efficacy of rhuMAb 2C4 was assessed against MCF7 xenografts which are estrogen receptor (ER +) positive and which express low levels of ErbB2. Female mice supplemented with estrogens were used. rhuMAb 2C4 was administered at dose

Mg / kg every week. As shown in Figure 13, rhuMAb 2C4 was effective in inhibiting the growth of breast cancer in vivo, where the breast cancer was not characterized by ErbB2 overexpression.

Example 8

The pharmacokinetics, metabolism and toxicology of 2C4 rhuMAb 2C4 was stable in human serum. There was no evidence of aggregation or complex formation in the biological arrays. In mice, rhuMAb 2C4 was cleared from the body faster than HERCEPTIN^®. Pharmacokinetic studies indicate that weekly dosing of about 2-6 mg / kg rhuMAb 2C4 should achieve serum concentrations similar to HERCEPTIN^® at the doses currently used. The obtained concentration of 2C4 in serum should significantly exceed the IC50 determined in vitro.

The toxicological studies were carried out on Javanese macaques (2 males and 2 females per group). rhuMAb 2C4 was administered intravenously at doses of 0, 10, 50 or 100 mg / kg twice a week for 4 weeks. The toxicology studies included body weight measurements (weeks -2, -1 and weekly thereafter); food consumption studies (qualitative, daily); medical examinations with blood pressure, electrocardiogram (ECG) and body temperature assessment (weeks -2, -1 and weeks 2 and 4, 4 hours after the second dose in a given week); ultrasound examinations of the heart (after first dose at week 1 and at end of study, week 4); laboratory diagnostics (baseline and end of weeks 2 and 4); urinalysis (baseline and end of weeks 2 and 4); sampling for antibody analysis (baseline and end of weeks 2 and 4) as well as dissection and histopathological analysis.

All animals in all groups survived to the end of the study. There were no significant clinical observations or differences between groups. The autopsy results showed no significant major organ abnormalities in any of the animals. No significant microscopic tissue abnormalities were observed in any of the animals. There were no significant changes in the ECG from baseline to completion of the study. Moreover, there were no apparent differences between the groups.

Example 9

Increasing the dose

Cancer patients are administered a first dose of rhuMAb 2C4 at one of five dose levels (0.05, 0.5, 2.0, 4.0 or 10 mg / kg; 6 subjects per dose level) followed by 4 weeks without administration. In week 5, patients receive the same dose four times, followed by another 4-week period without treatment. Patients with complete response, partial response, or stable disease are selected for extended studies.

Example 10

Treatment of relapsed or refractory prostate cancer

RhuMAb 2C4 is a full-length humanized monoclonal antibody (produced by CHO cells) against ErbB2. RhuMAb 2C4 blocks ErbB2 from connecting to other members of the ErbB family, thereby starting intracellular signaling along the ErbB pathway. Unlike HERCEPTIN^®, rhuMAb 2C4 not only inhibits the growth of ErbB2 overexpressing tumors, but also blocks the growth of tumors that require signaling dependent on ErbB ligands.

RhuMAb 2C4 is indicated as a single agent for the treatment of patients with hormone (androgen independent) refractory prostate cancer. Primary endpoints for efficacy include overall survival versus best available treatment (Mitoxantrone / Prednisone) when used as a single agent, and safety. Secondary targets for efficacy include time to disease progression, speed of response, quality of life, pain, and / or duration of response. RhuMAb 2C4 is administered intravenously (IV) every week or every three weeks at doses of 2 or 4 mg / kg, respectively, while the disease progresses. The antibody is supplied as a multi-dose liquid formulation (20 ml fill at a concentration of 20 mg / ml or greater).

RhuMAb 2C4 is also indicated in combination with chemotherapy for the treatment of hormone refractory (androgen independent) prostate cancer. Primary endpoints for efficacy include overall survival compared to chemotherapy, and safety. Secondary targets for efficacy include time to disease progression, speed of response, quality of life, pain, and / or duration of response. RhuMAb 2C4 is administered intravenously (IV) every week or every three weeks at doses of 2 or 4 mg / kg, respectively, while the disease progresses.

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The antibody is supplied as a multi-dose liquid formulation (20 ml fill at a concentration of 20 mg / ml or greater).

Examples of drugs that can be combined with an anti-ErbB2 antibody (which blocks ligand activation of the ErbB2 receptor) in the treatment of prostate cancer (e.g., androgen independent prostate cancer) include a farnesyl transferase inhibitor, an anti-angiogenic factor (e.g. an anti-VEGF antibody) , a drug targeting EGFR (e.g. C225 or ZD1839), another anti-ErbB2 antibody (e.g. a growth inhibitory anti-ErbB2 antibody such as HERCEPTIN^®, or an anti-ErbB2 antibody that induces apoptosis, such as 7C2 or 7F3, including humanized and / or affinity maturation variants thereof), a cytokine (e.g. IL-2, IL-12, G-CSF, or GM-CSF), an anti-androgen (such as flutamide or cyproterone acetate), leuprolide, suramin, a chemotherapeutic agent such as vinblastine, estramustine, mitoxantrone, liarozole (a factor that blocks the metabolism of retinolic acid) , cyclophosphamide, anthracycline antibiotics such as doxorubicin, a taxane (e.g. paclitaxel or docetaxel) or methotrexate, or any combination thereof such as vinblastine / estramustine or cyclophosphamide / doxorubicin / methotrexate, prednisone, hydrocortisone or combinations thereof. Standard doses of these various drugs can be administered, e.g. 40 mg / m2²/ wk docetaxel (TAZOTERE^®); 6 (AUC) of carboplatin and 200 mg / m² paclitaxel (TAXOL^®).

Example 11

Therapy of metastatic breast cancer

RhuMAb 2C4 is indicated as a single agent for the treatment of patients with metastatic breast cancer. The primary objectives for effectiveness are response speed and safety. Secondary targets for efficacy include overall survival, time to disease progression, quality of life, and / or duration of response. RhuMAb 2C4 is administered intravenously (IV) every week or every three weeks at doses of 2 or 4 mg / kg, respectively, while the disease progresses. The antibody is supplied as a multi-dose liquid formulation (20 ml fill at a concentration of 20 mg / ml or greater).

RhuMAb 2C4 is also indicated in combination with chemotherapy for the treatment of patients with metastatic breast cancer that does not overexpress ErbB2. Primary endpoints for efficacy include overall survival compared to chemotherapy alone, and safety. Secondary targets for efficacy include time to disease progression, speed of response, quality of life, and / or duration of response. RhuMAb 2C4 is administered intravenously (IV) every week or every three weeks at doses of 2 or 4 mg / kg, respectively, while the disease progresses. The antibody is supplied as a multi-dose liquid formulation (20 ml fill at a concentration of 20 mg / ml or greater).

Examples of drugs that can be combined with an anti-ErbB2 antibody (which blocks ligand activation of the ErbB2 receptor) in the treatment of breast cancer (e.g., metastatic breast cancer that does not overexpress ErbB2) include chemotherapeutic agents such as anthracycline antibiotics (e.g., doxorubicin) , cyclophosphamide, taxane (e.g. paclitaxel or docetaxel), carrelbin, xselod, mitomycin C, platinum compounds, oxaliplatin, gemcitabine, or combinations of two or more of these, such as doxorubicin / cyclophosphamide, another anti-ErbB2 antibody (e.g. a growth inhibitory anti-ErbB2 antibody such as HERCEPTIN, or an anti-ErbB2 antibody that induces apoptosis such as 7C2 or 7F3, including humanized and / or affinity maturation variants thereof), antiestrogen (e.g. tamoxifen), transferase inhibitor farnesyl, anti-angiogenic agent (e.g. anti-VEGF antibody), EGFR targeting drug (C225 or ZD1839), cytokine (e.g. IL-2, IL-12, G-CSF or GM-CSF) or combinations of the above. Standard doses of these additional drugs can be administered.

RhuMAb 2C4 is additionally indicated in combination with HERCEPTIN^® for the treatment of patients with ErbB2 overexpressing metastatic breast cancer. The primary objectives for effectiveness are response speed and safety. Secondary targets for efficacy include: time to disease progression, overall survival compared to HERCEPTIN alone '^®, quality of life and / or duration of response. RhuMAb 2C4 is administered intravenously (IV) every week or every three weeks at doses of 2 or 4 mg / kg, respectively, while the disease progresses. The antibody is supplied as a multi-dose liquid formulation (20 ml fill at a concentration of 20 mg / ml or greater). HERCEPTIN^® IV is given as an initial loading dose of 4 mg / kg,

The weekly dose is then maintained at 2 mg / kg. HERCEPTIN^® supplied as a lyophilized powder. Each vial of HERCEPTIN^® contains 440 mg HERCEPTIN^®, 9.9 mg L-histidine hydrochloride, 6.4 mg L-histidine, 400 mg α-α-trehalose dihydrate and 1.8 mg polysorbate 20. By dissolving 20 ml bacteriostatic water for injection (BWFI) containing 1.1% benyl alcohol as a preservative is prepared in 21 ml of a multi-dose solution containing 21 mg / ml HERCEPTIN^® with a pH of about 6.0.

Example 12

Lung cancer therapy

RhuMAb 2C4 is indicated as a single agent for the treatment of stage IIIb or stage IV of non-small cell lung cancer (NSCLC). The primary objectives for effectiveness are response speed and safety. Secondary targets for efficacy include overall survival, time to disease progression, quality of life, and / or duration of response. RhuMAb 2C4 is administered intravenously (IV) every week or every three weeks at doses of 2 or 4 mg / kg, respectively, while the disease progresses. The antibody is supplied as a multi-dose liquid formulation (20 ml fill at a concentration of 20 mg / ml or greater).

RhuMAb 2C4 is also indicated in combination with chemotherapy for the treatment of patients with metastatic non-small cell lung cancer. Primary endpoints for efficacy include overall survival compared to standard therapy and safety. Secondary targets for efficacy include time to disease progression, speed of response, quality of life, and / or duration of response. RhuMAb 2C4 is administered intravenously (IV) every week or every three weeks at doses of 2 or 4 mg / kg, respectively, while the disease progresses. The antibody is supplied as a multi-dose liquid formulation (20 ml fill at a concentration of 20 mg / ml or greater).

Examples of additional drugs that can be combined with an anti-ErbB2 antibody (which binds to ErbB2 and blocks ligand activation of the ErbB2 receptor) in the treatment of lung cancer include chemotherapeutic agents such as carboplatin, a taxane (e.g. paclitaxel or docetaxel), gemcitabine, relbine , cisplatin, oxaliplatin, or combinations of two or more of these, such as carboplatin / docetaxel, another anti-ErbB2 antibody (e.g. a growth inhibitory anti-ErbB2 antibody such as HERCEPTIN^®, or an anti-ErbB2 antibody that induces apoptosis such as 7C2 or 7F3, including humanized and / or affinity maturation variants thereof), farnesyl transferase inhibitor, anti-angiogenic agent (e.g. anti-VEGF antibody), EGFR targeting drug (C225 or ZD1839), a cytokine (e.g., IL-2, IL-12, G-CSF or GM-CSF), or combinations of the above.

Example 13

Colon and rectal cancer therapy

RhuMAb 2C4 is indicated as a single agent in the treatment of metastatic cancer of the colon and rectum. The primary objectives for effectiveness are response speed and safety. Secondary targets for efficacy include overall survival, time to disease progression, quality of life, and / or duration of response. RhuMAb 2C4 is administered intravenously (IV) every week or every three weeks at doses of 2 or 4 mg / kg, respectively, while the disease progresses. The antibody is supplied as a multi-dose liquid formulation (20 ml fill at a concentration of 20 mg / ml or greater).

RhuMAb 2C4 is also indicated in combination with chemotherapy for the treatment of patients with metastatic cancer of the colon and rectum. Primary endpoints for efficacy include overall survival compared to standard therapy and safety. Secondary targets for efficacy include time to disease progression, speed of response, quality of life, and / or duration of response. RhuMAb 2C4 is administered intravenously (IV) every week or every three weeks at doses of 2 or 4 mg / kg, respectively, while the disease progresses. The antibody is supplied as a multi-dose liquid formulation (20 ml fill at a concentration of 20 mg / ml or greater).

Examples of chemotherapeutic agents used to treat colon and rectal cancer that can be combined with an antibody that binds to ErbB2 and blocks ligand activation of the ErbB2 receptor include 5-fluorouracil (5-FU), leucovorin (LV), CPT-11, levamisole, or a combination of any two or more of them, e.g. F-FU / LV / CPT-11. Standard doses of such chemotherapeutic agents may be administered. Other drugs that can be combined with the antibody

Anti-ErbB2 in the treatment of colon and rectal cancer include a farnesyl transferase inhibitor, an anti-angiogenic factor (e.g. an anti-VEGF antibody), an EGFR-targeting drug (e.g. C225 or ZD1839), a cytokine (e.g. IL-2, IL-12, G-CSF, or GM-CSF), another anti-ErbB2 antibody (e.g., a growth inhibitory anti-ErbB2 antibody such as HERCEPTIN^®, or an anti-ErbB2 antibody that induces apoptosis such as 7C2 or 7F3, including humanized and / or affinity maturation variants thereof) or combinations of the above.

SEQUENCE LIST

<td> &lt;110&gt;</td><td>Genencech, Inc.</td><td></td><td></td>

<td> &lt;120&gt;</td><td>Humanized directed antibodies</td><td>against</td><td>ErbB2 and</td>

<td></td><td>treatment with directed antibodies</td><td>against</td><td>ErbB2</td>

<td> &lt;130&gt;</td><td>P1467R2PCT</td><td></td><td></td>

<td> &lt;141&gt;</td><td> 2000-06-23</td><td></td><td></td>

<td> &lt;150&gt;</td><td>US 60 / 141,316</td><td></td><td></td>

<td> &lt;151&gt;</td><td> 1999-06-25</td><td></td><td></td>

<td> &lt;160&gt;</td><td> 13</td><td></td><td></td>

<td> &lt;210&gt;</td><td></td><td></td><td></td>

<td> &lt;211&gt;</td><td> 107</td><td></td><td></td>

<td> &lt;212&gt;</td><td>PRT</td><td></td><td></td>

&lt;2i3&gt; House mouse &lt;400&gt; 1

<td>Asp 1</td><td>Thr</td><td>Val</td><td>Underworld</td><td>Thr 5</td><td>Main</td><td>Cheese</td><td>His</td><td>Lys</td><td>How much 10</td><td>Underworld</td><td>Cheese</td><td>Thr</td><td>Cheese</td><td>Val 15</td>

<td>Gly</td><td>Asp</td><td>Arg</td><td>Val</td><td>Cheese twenty</td><td>How much</td><td>Thr</td><td>Cys</td><td>Lys</td><td>Ala 25</td><td>Cheese</td><td>Main</td><td>Asp</td><td>Val</td><td>Cheese thirty</td>

<td>How much</td><td>Gly</td><td>Val</td><td>Ala</td><td>Trp 35</td><td>Tyr</td><td>Main</td><td>Main</td><td>Arg</td><td>Pro 40</td><td>Gly</td><td>Main</td><td>Cheese</td><td>Pro</td><td>Lys 45</td>

<td>Leu</td><td>Leu</td><td>How much</td><td>Tyr</td><td>Cheese 50</td><td>Ala</td><td>Cheese</td><td>Tyr</td><td>Arg</td><td>Tyr 55</td><td>Thr</td><td>Gly</td><td>Val</td><td>Pro</td><td>Asp 60</td>

<td>Arg</td><td>Phe</td><td>Thr</td><td>Gly</td><td>Cheese 65</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp 70</td><td>Phe</td><td>Thr</td><td>Phe</td><td>Thr</td><td>How much 75</td>

<td>Cheese</td><td>Cheese</td><td>Val</td><td>Main</td><td>Ala 80</td><td>Glu</td><td>Asp</td><td>Leu</td><td>Ala</td><td>Val 85</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Main</td><td>Main 90</td>

<td>Tyr</td><td>Tyr</td><td>How much</td><td>Tyr</td><td>Pro</td><td>Tyr</td><td>Thr</td><td>Phe</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Thr</td><td>Lys</td><td>Leu</td><td>Glu</td>

100 105

How many Lys &lt;210&gt; 2 &lt;211&gt; 119 &lt;212&gt; PRT &lt;2i3&gt; House mouse

<td>&lt;400&gt; 2 Glu Val 1</td><td>Main</td><td>Leu</td><td>Main 5</td><td>Main</td><td>Cheese</td><td>Gly</td><td>Pro</td><td>Glu 10</td><td>Leu</td><td>Val</td><td>Lys</td><td>Pro</td><td>Gly 15</td>

<td>Thr Ser</td><td>Val</td><td>Lys</td><td>How much twenty</td><td>Cheese</td><td>Cys</td><td>Lys</td><td>Ala</td><td>Cheese 25</td><td>Gly</td><td>Phe</td><td>Thr</td><td>Phe</td><td>Thr thirty</td>

<td>Asp Tyr</td><td>Thr</td><td>Underworld</td><td>Asp 35</td><td>Trp</td><td>Val</td><td>Lys</td><td>Main</td><td>Cheese 40</td><td>His</td><td>Gly</td><td>Lys</td><td>Cheese</td><td>Leu 45</td>

<td>Glu Trp</td><td>How much</td><td>Gly</td><td>Asp</td><td>Val</td><td>Asn</td><td>Pro</td><td>Asn</td><td>Cheese</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>How much</td><td>Tyr</td>

55 60

PL 203 326 B1

<td>Asn</td><td>Gin</td><td>Arg</td><td>Phe</td><td>Lys 65</td><td>Gly</td><td>Lys</td><td>Ala</td><td>Cheese</td><td>Leu 70</td><td>Thr</td><td>Val</td><td>Asp</td><td>Arg</td><td>Cheese 75</td>

<td>Cheese</td><td>Arg</td><td>How much</td><td>Val</td><td>Tyr 80</td><td>Underworld</td><td>Glu</td><td>Leu</td><td>Arg</td><td>Cheese 85</td><td>Leu</td><td>Thr</td><td>Phe</td><td>Glu</td><td>Asp 90</td>

<td>Thr</td><td>Ala</td><td>Val</td><td>Tyr</td><td>Tyr 95</td><td>Cys</td><td>Ala</td><td>Arg</td><td>Asn</td><td>Leu 100</td><td>Gly</td><td>Pro</td><td>Cheese</td><td>Phe</td><td>Tyr 105</td>

<td>Phe</td><td>Asp</td><td>Tyr</td><td>Trp</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Val</td><td>Cheese</td><td>Cheese</td><td></td>

110 115 &lt;210&gt; 3 &lt;211&gt; 107 &lt;212&gt; PRT &lt;2ΐ3&gt; artificial

<td colspan="2"> &lt;400&gt; 3</td><td rowspan="2">Gin</td><td rowspan="2">Underworld</td><td rowspan="2">Thr 5</td><td rowspan="2">Gin</td><td rowspan="2">Cheese</td><td rowspan="2">Pro</td><td rowspan="2">Cheese</td><td rowspan="2">Leu cheese 10</td><td rowspan="2">Ala cheese</td><td rowspan="2">Cheese</td><td rowspan="2">Val 15</td>

<td>Asp 1</td><td>How much</td>

<td>Gly</td><td>Asp</td><td>Arg</td><td>Val</td><td>Thr twenty</td><td>How much</td><td>Thr</td><td>Cys</td><td>Lys</td><td>Ala Ser 25</td><td>Gin Asp</td><td>Val</td><td>Cheese thirty</td>

<td>How much</td><td>Gly</td><td>Val</td><td>Ala</td><td>Trp 35</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>Lys</td><td>Pro Gly 40</td><td>Lys Ala</td><td>Pro</td><td>Lys 45</td>

<td>Leu</td><td>Leu</td><td>How much</td><td>Tyr</td><td>Cheese 50</td><td>Ala</td><td>Cheese</td><td>Tyr</td><td>Arg</td><td>Tyr Thr 55</td><td>Gly Val</td><td>Pro</td><td>Cheese 60</td>

<td>Arg</td><td>Phe</td><td>Cheese</td><td>Gly</td><td>Cheese 65</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp Phe 70</td><td>Thr Leu</td><td>Thr</td><td>How much 75</td>

<td>Cheese</td><td>Cheese</td><td>Leu</td><td>Gin</td><td>Pro</td><td>Glu</td><td>Δ Cr ·,</td><td>Phe</td><td>Ala</td><td>Πν, ν-</td><td></td><td>Gin</td><td>Gin</td>

<td></td><td></td><td></td><td></td><td> 80</td><td></td><td></td><td></td><td></td><td>Λ j- j. 85</td><td>and J- i- -r&gt;</td><td></td><td> 90</td>

<td>Tyr</td><td>Tyr</td><td>How much</td><td>Tyr</td><td>Pro</td><td>Tyr</td><td>Thr</td><td>Phe</td><td>Gly</td><td>Gin Gly</td><td>Thr Lys</td><td>Val</td><td>Glu</td>

100 105

How many Lys &lt;210&gt; 4 ο i ι τ τ η '-ώΙΙΧ &lt;212&gt; PRT &lt;2ΐ3&gt; artificial &lt;220&gt;

&lt;22ΐ&gt; artificial &lt;222&gt; 1-119 &lt;223&gt; Fab 574 VH &lt;400&gt; 4

Glu Val.Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Giy 15 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 Gin Ala Pro Gly Lys Gly Leu 35 40 45

Glu Trp Val Ala Asp Val Asn Pro Asn Ser Glv Gly Ser Ile Tyr 50 55 '' 60

PL 203 326 B1

<td>Asn</td><td>Gin</td><td>Arg</td><td>Phe</td><td>Lys 65</td><td>Gly</td><td>Arg</td><td>Phe</td><td>Thr</td><td>Leu 70</td><td>Cheese</td><td>Val</td><td>Asp</td><td>Arg</td><td>Cheese 75</td>

<td>Lys</td><td>Asn</td><td>Thr</td><td>Leu</td><td>Tyr 80</td><td>Leu</td><td>Gin</td><td>Underworld</td><td>Asn</td><td>Cheese 85</td><td>Leu</td><td>Arg</td><td>Ala</td><td>Glu</td><td>Asp 90</td>

<td>Thr</td><td>Ala</td><td>Val</td><td>Tyr</td><td>Tyr 95</td><td>Cys</td><td>Ala</td><td>Arg</td><td>Asn</td><td>Leu 100</td><td>Gly</td><td>Pro</td><td>Cheese</td><td>Phe</td><td>Tyr 105</td>

<td>Phe</td><td>Asp</td><td>Tyr</td><td>Trp</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>Leu</td><td>Val</td><td>Thr</td><td>Val</td><td>Cheese</td><td>Cheese</td><td></td>

110 115 &lt;210&gt; 5 &lt;211&gt; 107 &lt;212&gt; PRT &lt;2ΐ3&gt; artificial

<td colspan="12"> &lt;400&gt; 5</td><td rowspan="2">Ala</td><td rowspan="2">Cheese</td><td rowspan="2">Val 15</td>

<td>Asp 1</td><td>How much</td><td>Gin</td><td>Underworld</td><td>Thr 5</td><td>Gin</td><td>Cheese</td><td>Pro</td><td>Cheese</td><td>Cheese 10</td><td>Leu</td><td>Cheese</td>

<td>Gly</td><td>Asp</td><td>Arg</td><td>Val</td><td>Thr twenty</td><td>How much</td><td>Thr</td><td>Cys</td><td>Arg</td><td>Ala 25</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>How much</td><td>Cheese thirty</td>

<td>Asn</td><td>Tyr</td><td>Leu</td><td>Ala</td><td>Trp 35</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>Lys</td><td>Pro 40</td><td>Gly</td><td>Lys</td><td>Ala</td><td>Pro</td><td>Lys 45</td>

<td>Leu</td><td>Leu</td><td>How much</td><td>Tyr</td><td>Ala 50</td><td>Ala</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Glu 55</td><td>Cheese</td><td>Gly</td><td>Val</td><td>Pro</td><td>Cheese 60</td>

<td>Arg</td><td>Phe</td><td>Cheese</td><td>Gly</td><td>Cheese 65</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp 70</td><td>Phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>How much 75</td>

<td>Cheese</td><td>Cheese</td><td>Leu</td><td>Gin</td><td>Pro 80</td><td>Glu</td><td>Asp</td><td>Phe</td><td>Ala</td><td>Thr 85</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gin</td><td>Gin 90</td>

<td>Tyr</td><td>Aśn</td><td>Cheese'</td><td>Leu</td><td>Pro</td><td>Trp</td><td>Thr</td><td>Phe</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>Lys</td><td>Val</td><td>Glu</td>

100 105

How many Lys &lt;210&gt; 6 &lt;211&gt; 119 &lt;212&gt; PRT &lt;2ΐ3&gt; artificial

<td colspan="4"> &lt;400&gt; 6</td><td rowspan="2">Val 5</td><td rowspan="2">Glu</td><td rowspan="2">Cheese</td><td rowspan="2">Gly</td><td colspan="2" rowspan="2">Gly Gly 10</td><td rowspan="2">Leu</td><td colspan="4" rowspan="2">Val Gin Pro Gly 15</td>

<td>Glu 1</td><td>Val</td><td>Gin</td><td>Leu</td>

<td>Gly</td><td>Cheese</td><td>Leu</td><td>Arg</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>Ala</td><td>Ala</td><td>Cheese</td><td>Gly</td><td>Phe</td><td>Thr</td><td>Phe</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td>

<td>Cheese</td><td>Tyr</td><td>Ala</td><td>Underworld</td><td>Cheese</td><td>Trp</td><td>Val</td><td>Arg</td><td>Gin</td><td>Ala</td><td>Pro</td><td>Gly</td><td>Lys</td><td>Gly</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td>

<td>Glu</td><td>Teepee</td><td>Val</td><td>Ala</td><td>Val</td><td>How much</td><td>Cheese</td><td>Gly</td><td>Asp</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>Thr</td><td>Tyr</td><td>Tyr</td>

<td></td><td></td><td></td><td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td>

<td>Ala</td><td>Asp</td><td>Cheese</td><td>Val</td><td>Lys</td><td>Gly</td><td>Arg</td><td>Phe</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Asp</td><td>Asn</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td>

<td>Lys</td><td>Asn</td><td>Thr</td><td>Leu</td><td>Tyr</td><td>Leu</td><td>Gin</td><td>Underworld</td><td>Asn</td><td>Cheese</td><td>Leu</td><td>Arg</td><td>Ala</td><td>Glu</td><td>Asp</td>

PL 203 326 B1

<td>Thr</td><td>Ala</td><td>Val</td><td>Tyr</td><td>Tyr 95</td><td>Cys</td><td>Ala</td><td>Arg</td><td>Gly</td><td>Arg 100</td><td>Val</td><td>Gly</td><td>Tyr</td><td>Cheese</td><td>Leu 105</td>

<td>Tyr</td><td>Asp</td><td>Tyr</td><td>Trp</td><td>Gly 110</td><td>Gin</td><td>Gly</td><td>Thr</td><td>Leu</td><td>Val 115</td><td>Thr</td><td>Val</td><td>Cheese</td><td>Cheese</td><td></td>

&lt;210&gt; 7 &lt;211&gt; 10 &lt;212&gt; PRT &lt;2ΐ3&gt; House mouse &lt;220&gt;

^&lt;22ΐ^&gt; uncertain &lt;222&gt; ίο &lt;223&gt; unknown amino acid &lt;400&gt; -7

Gly Phe Thr Phe Thr Asp Tyr Thr Met Xaa 15 10

<td> &lt;210&gt;</td><td> 8</td>

<td> &lt;211&gt;</td><td> 17</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>House mouse</td>

<td> &lt;400&gt;</td><td> 3</td>

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

10 15

Lys Gly &lt;210&gt; 9 &lt;211&gt; 10 &lt;212&gt; ERT.

&lt;2i3&gt; House mouse &lt;400&gt; 9

Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr 15 10 &lt;210&gt; 10 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; House mouse &lt;400&gt; 10

Lys Ala Ser Gin Asp Val Ser Ile Gly Val Ala 15 10 &lt;210&gt; 11 &lt;211&gt; 7 &lt;212&gt; PRT '&lt;2i3&gt; House mouse &lt;220&gt;

&lt;221&gt; uncertain &lt;222&gt; 5-7 &lt;223&gt; unknown amino acid &lt;400&gt; 11

Ser Ala Ser Tyr Xaa Xaa Xaa

PL 203 326 B1

<td> &lt;210&gt;</td><td> 12</td>

<td> &lt;211&gt;</td><td> 9</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>House mouse</td>

<td> &lt;400&gt;</td><td> 12</td>

<td colspan="3">Gin Gin Tyr Tyr</td><td>' How much 5</td><td>Tyr</td><td>Pro</td><td>Tyr</td><td colspan="7">Thr</td>

<td> &lt;210&gt; 13</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2">&lt;211&gt; 645 &lt;212&gt; PRT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="3">&lt;2i3&gt; human</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;400&gt; 13</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>MeC Glu</td><td>Leu</td><td>Ala</td><td>Ala</td><td>Leu</td><td>Cys</td><td>Arg</td><td>Trp</td><td>Gly</td><td>Leu</td><td>Leu</td><td>Leu</td><td>Ala</td><td>Leu</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td>

<td>Leu Pro</td><td>Pro</td><td>Gly</td><td>Ala</td><td>Ala</td><td>Cheese</td><td>Thr</td><td>Gin</td><td>Val</td><td>Cys</td><td>Thr</td><td>Gly</td><td>Thr</td><td>Asp</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td>

<td>MeC Lys</td><td>Leu</td><td>Aręt</td><td>Leu</td><td>Pro</td><td>Ala</td><td>Cheese</td><td>Pro</td><td>Glu</td><td>Thr</td><td>His</td><td>Leu</td><td>Asp</td><td>MeC</td>

<td></td><td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td>

<td>Leu Arg</td><td>His</td><td>Leu</td><td>Tyr</td><td>Gin</td><td>Gly</td><td>Cys</td><td>Gin</td><td>Val</td><td>Val</td><td>Gin</td><td>Gly</td><td>Asn</td><td>Leu</td>

<td></td><td></td><td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td>

<td>Glu Leu</td><td>Thr</td><td>Tyr</td><td>Leu</td><td>Pro</td><td>Thr</td><td>Asn</td><td>Ala</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Phe</td><td>Leu</td><td>Gin</td>

<td></td><td></td><td></td><td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td>

<td>Asp Ile</td><td>Gin</td><td>Glu</td><td>Val</td><td>Gin</td><td>Gly</td><td>Tyr</td><td>Val</td><td>Leu</td><td>How much</td><td>Ala</td><td>His</td><td>Asn</td><td>Gin</td>

<td></td><td></td><td></td><td> 80</td><td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td>

<td>Val Arg</td><td>Gin</td><td>Val</td><td>Pro</td><td>Leu</td><td>Gin</td><td>Arg</td><td>Leu</td><td>Arg</td><td>How much</td><td>Val</td><td>Arg</td><td>Gly</td><td>Thr</td>

<td></td><td></td><td></td><td> 95</td><td></td><td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td>

<td>Gin Leu</td><td>Phe</td><td>Glu</td><td>Δ c? T ~&gt;</td><td>Asn</td><td>yy * J '*</td><td>.Ala</td><td>Leu</td><td>Ala</td><td>Val</td><td>L.^andat</td><td>Asp</td><td>Asn</td><td>r-1. . centipede</td>

<td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td>

<td>Asp Pro</td><td>Leu</td><td>Asn</td><td>Asn</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Val</td><td>Thr</td><td>Gly</td><td>Ala</td><td>Cheese</td><td>Pro</td><td>Gly</td>

<td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td><td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td>

<td>Gly Leu</td><td>Arg</td><td>Glu</td><td>Leu</td><td>Gin</td><td>Leu</td><td>Arg</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Glu</td><td>How much</td><td>Leu</td><td>Lys</td>

<td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td><td> 145</td><td></td><td></td><td></td><td></td><td> 150</td>

<td>Gly Gly</td><td>Val</td><td>Leu</td><td>How much</td><td>Gin</td><td>Arg</td><td>Asn</td><td>Pro</td><td>Gin</td><td>Leu</td><td>Cys</td><td>Tyr</td><td>Gin</td><td>Asp</td>

<td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td><td></td><td></td><td></td><td></td><td> 165</td>

<td>Thr Ile</td><td>Leu</td><td>Trp</td><td>Lys</td><td>Asp</td><td>How much</td><td>Phe</td><td>His</td><td>Lys</td><td>Asn</td><td>Asn</td><td>Gin</td><td>Leu</td><td>Ala</td>

<td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td><td></td><td></td><td></td><td> 180</td>

<td>Leu Thr</td><td>Leu</td><td>How much</td><td>Asp</td><td>Thr</td><td>Asn</td><td>Arg</td><td>Cheese</td><td>Arg</td><td>Ala</td><td>Cys</td><td>His</td><td>Pro</td><td>Cys</td>

<td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td><td></td><td></td><td> 195</td>

<td>Pro cheese</td><td>Underworld</td><td>Cys</td><td>Lys</td><td>Gly</td><td>Cheese</td><td>Arg</td><td>Cys</td><td>Trp</td><td>Gly</td><td>Glu</td><td>Cheese</td><td>Cheese</td><td>Glu</td>

<td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td><td></td><td> 210</td>

<td>Asp Cys</td><td>Gin</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Arg</td><td>Thr</td><td>Val</td><td>Cys</td><td>Ala</td><td>Gly</td><td>Gly</td><td>Cys</td><td>Ala</td>

<td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td><td> 225</td>

<td>Arg Cys</td><td>Lys</td><td>fil \ r</td><td>Pro</td><td>Leu</td><td>Pro</td><td>Thr</td><td>A &lt;? Rł</td><td></td><td rowspan="2"> —</td><td>His</td><td>Glu</td><td>Gin</td><td></td>

<td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td> 240</td>

PL 203 326 B1

Ala Ala Gly Cys Thr 245

Leu His Phe Asn His 260

Leu Val Thr Tyr Asn 275

Glu Gly Arg Tyr Thr 290

Tyr Asn Tyr Leu Ser 305

Pro Leu His Asn Gin 320

Cys Glu Lys Cys Ser 335

Gly Met Glu His Leu 350

How Much Gin Glu Phe Ala 365

Phe Leu Pro Glu Ser 380

Pro Leu Gin Pro-Glu 395

Ile Thr Gly Tyr Leu 410

Asp Leu Ser Val Phe '425

Leu His Asn Gly Ala 440

Trp Leu Gly Leu Cheese '455

Ala Leu Ile His His 470

Pro Trp Asp Gin Leu 485

Thr Ala Ash Arg Pro 500

Cys His..Gin Leu Cys 515

Thr Gin Cys Val Asn 530

Val Glu Glu Cys Arg 545

Asn Ala Arg His Cys

<td>Gly</td><td>Pro</td><td>Lys</td><td>His</td><td>Cheese 250</td>

<td>Cheese</td><td>Gly</td><td>How much</td><td>Cys</td><td>Glu 265</td>

<td>Thr</td><td>Asp</td><td>Thr</td><td>Phe</td><td>Glu 280</td>

<td>Phe</td><td>Gly</td><td>Ala</td><td>Cheese</td><td>Cys 295</td>

<td>Thr</td><td>Asp</td><td>Val</td><td>Gly</td><td>Cheese 310</td>

<td>Glu</td><td>Val</td><td>Thr</td><td>Ala</td><td>Glu 325</td>

<td>Lys</td><td>Pro</td><td>Cys</td><td>Ala</td><td>Arg 340</td>

<td>Arg</td><td>Glu</td><td>Val</td><td>Arg</td><td>Ala 355</td>

<td>Gly</td><td>Cys</td><td>Lys</td><td>Lys</td><td>How much 370</td>

<td>Phe</td><td>Asp</td><td>Gly</td><td>Asp</td><td>Pro 385</td>

<td>Gin</td><td>Leu</td><td>Gin</td><td>Val</td><td>Phe 400</td>

<td>Tyr</td><td>How much</td><td>Cheese</td><td>Ala</td><td>Trp 415</td>

<td>Gin</td><td>Asn</td><td>Leu</td><td>Gin</td><td>Val 430</td>

<td>Tyr</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Leu 445</td>

<td>Arg</td><td>Cheese</td><td>Leu</td><td>Arg</td><td>Glu 460</td>

<td>Asn</td><td>Thr</td><td>His</td><td>Leu</td><td>Cys 475</td>

<td>Phe</td><td>Arg</td><td>Asn</td><td>Pro</td><td>His 490</td>

<td>Glu</td><td>Asp</td><td>Glu</td><td>Cys</td><td>Val 505</td>

<td>Ala</td><td>Arg</td><td>Gly</td><td>His</td><td>Cys 520</td>

<td>Cys</td><td>Cheese</td><td>Gin</td><td>Phe</td><td>Leu 535</td>

<td>Val</td><td>Leu</td><td>Gin</td><td>Gly</td><td>Leu 550</td>

<td>Leu</td><td>Pro</td><td>Cys</td><td>His</td><td>Pro</td>

Asp Cys Leu Ala Cys 255

Leu His Cys Pro Ala 270

Ser Met Pro Asn Pro 285

Val Thr Ala Cys Pro 300

Cys Thr Leu Val Cys 315

Asp Gly Thr Gin Arg 330

Val Cys Tyr Gly Leu 345

Val Thr Ser Ala Asn 360

Phe Gly Ser Leu Ala 375

Ala Ser Asn Thr Ala 390

Glu Thr Leu Glu Glu 405

Pro Asp Ser Leu Pro 420

How Much Arg Gly Arg Ile 435

Gin Gly Leu Gly Ile 450

Leu Gly Ser Gly Leu 465

Phe Val His Thr Val 480

Gin Ala Leu Leu His 495

Gly Glu Gly Leu Ala 510

Trp Gly Pro Gly Pro 525

Arg Gly Gin Glu Cys 540

Pro Arg Glu Tyr Val 555

Glu Cys Gin Pro Gin

PL 203 326 B1

560 565 570

<td>Asn</td><td>Gly</td><td>Cheese</td><td>Val</td><td>Thr 575</td><td>Cys</td><td>Phe</td><td>Gly</td><td>Pro</td><td>Glu 580</td><td>Ala</td><td>Asp</td><td>Gin</td><td>Cys</td><td>Val 585</td>

<td>Ala</td><td>Cys</td><td>Ala</td><td>His</td><td>Tyr 590</td><td>Lys</td><td>Asp</td><td>Pro</td><td>Pro</td><td>Phe 595</td><td>Cys</td><td>Val</td><td>Ala</td><td>Arg</td><td>Cys 600</td>

<td>Pro</td><td>Cheese</td><td>Gly</td><td>Val</td><td>Lys 605</td><td>Pro</td><td>Asp</td><td>Leu</td><td>Cheese</td><td>Tyr 610</td><td>Underworld</td><td>Pro</td><td>How much</td><td>Trp</td><td>Lys 615</td>

<td>Phe</td><td>Pro</td><td>Asp</td><td>Glu</td><td>Glu 620</td><td>Gly</td><td>Ala</td><td>Cys</td><td>Gin</td><td>Pro 625</td><td>Cys</td><td>Pro</td><td>How much</td><td>Asn</td><td>Cys 630</td>

<td>Thr</td><td>His</td><td>Cheese</td><td>Cys</td><td>Val</td><td>Asp</td><td>Leu</td><td>Asp</td><td>Asp</td><td>Lys</td><td>Gly</td><td>Cys</td><td>Pro</td><td>Ala</td><td>Glu</td>

635 640 645