ANTI-DLL3 ANTIBODY

Abstract

It is intended to disclose an antibody which binds to DLL3 protein. Preferably, the antibody of the present invention recognizes a region from amino acids 216 to 492 in human DLL3 having the amino acid sequence as set forth in SEQ ID NO: 1. The present invention also provides a pharmaceutical composition, for example, an anticancer agent, comprising the antibody of the present invention as an active ingredient. The present invention further discloses a method for diagnosing cancer using the antibody of the present invention and a diagnostic drug for cancer comprising the antibody of the present invention.

Description

[0001] The present application is a Continuation of U.S. application Ser. No. 13/575,861, filed Jul. 27, 2012, which is a National Stage application filed under .sctn.371 of PCT Application No. PCT/JP2011/000485, filed Jan. 28, 2011, which claims the priority based on Japanese Patent Application No. 2010-019391 (filed on Jan. 29, 2010). The contents thereof are incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates to an anticancer agent and a method for diagnosing cancer.

BACKGROUND ART

[0003] Small-cell lung cancer accounts for approximately 20% of all lung cancer incidence. The small-cell lung cancer rapidly progresses and is difficult to be surgically removed because lymph node metastasis or distant metastasis has already occurred at the time of diagnosis in many cases. This cancer exhibits high response rates to an anticancer agent in its early stage. Thus, chemotherapy is considered as the first choice for treating the cancer. The cancer, however, immediately becomes resistant to chemotherapy and recurs, resulting in a 3-year survival rate of 5% or lower. Hence, new therapy has been demanded.

[0004] Delta-like 3 (DLL3) is a type I membrane protein belonging to Notch ligand family members. DLL3 is necessary for normal somite formation and patterning. Mutations in DLL3 cause rib defects or spondylolysis in autosomal recessive spondylocostal dysostosis patients [Non Patent Literatures 1 and 2]. DLL1 localizes on cell surface and binds to Notch, whereas DLL3 predominantly localizes in the Golgi apparatus and does not bind to Notch [Non Patent Literatures 3 and 4].

[0005] There exist previous studies reporting the amplification of the DLL3 gene on chromosome and increased expression of this gene in pancreatic cancer cell lines [Non Patent Literature 5] and increased DLL3 expression in some glioma cases [Non Patent Literature 6]. However, the number of the DLL3 protein on cell surface has not yet been reported. The expression of 10.sup.5 or more antigen molecules for unmodified antibodies or antigen molecules of 10.sup.4 order even for defucosylated antibodies having the enhanced ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) is required for targeting the antigen molecules on cell surface using such antibodies or for killing cancer cells under the anti-tumor mechanism of the ADCC activity [Non Patent Literature 7]. Thus, it is uncertain whether DLL3 is suitable as a therapeutic target based on antibodies.

[0006] Mouse anti-DLL3 monoclonal antibodies (MAB4315, R&D Systems, Inc.) are already commercially available as a research reagent.

CITATION LIST

Non Patent Literature

[0007] Non Patent Literature 1: Bulman, M. P. et al. (2000) Nat Genet 24, 438-441.

[0008] Non Patent Literature 2: Turnpenny, P. D. et al. (2003) J Med Genet 40, 333-339.

[0009] Non Patent Literature 3: Geffers, I. et al. (2007) J Cell Biol 178, 465-476.

[0010] Non Patent Literature 4: Ladi, E. et al. (2005) J Cell Biol 170, 983-992.

[0011] Non Patent Literature 5: Phillips, H. S. (2006) Cancer Cell 9, 157-173.

[0012] Non Patent Literature 6: Mulledndore, M. E. (2009) Clin Cancer Res 15, 2291-2301.

[0013] Non Patent Literature 7: Kenya Shitara (2009) YAKUGAKU ZASSHI 129, 3-9.

SUMMARY OF INVENTION

Technical Problem

[0014] An object of the present invention is to provide a novel antibody, an anticancer agent comprising the same, and a method for diagnosing cancer using the same.

Solution to Problem

[0015] The present inventors found that DLL3 mRNA expression was increased in small-cell lung cancer. Its expression was low in all normal tissues except for the fetal brain. The present inventors prepared monoclonal antibodies against the DLL3 protein. The antigen level on cell surface measured based on the capability of binding to the antibody was only less than 10.sup.4 per expressing cell. Unexpectedly, the present inventors found that an antibody that bound to DLL3 via a characteristic epitope in the vicinity of the C terminus of the extracellular domain stably resided on the cell membrane and had an ADCC-inducing activity. Specifically, the present inventors successively screened for an antibody having an anti-tumor activity. Moreover, the present inventors found that an antibody conjugated with toxin had a cytotoxic activity against DLL3-expressing cells. From these findings, the present inventors found that the anti-DLL3 antibody was useful in the diagnosis, prevention, and treatment of primary or metastatic cancer expressing DLL3. Based on these findings, the present invention has been completed.

[0016] The present invention provides an antibody which binds to DLL3 protein. Preferably, the antibody of the present invention has a cytotoxic activity. Particularly preferably, the cytotoxic activity is an antibody-dependent cell-mediated cytotoxic activity (ADCC activity). Also preferably, the antibody of the present invention has an internalization activity. Also preferably, the antibody of the present invention is conjugated with a cytotoxic substance. Also preferably, the antibody of the present invention recognizes a region from amino acids 216 to 492 in human DLL3 having the amino acid sequence as set forth in SEQ ID NO: 1.

[0017] In another aspect, the present invention provides an antibody which binds to DLL3 protein described in any of the following:

(1) an antibody comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 12, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 13, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 14; (2) an antibody comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 24, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 25, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 26; (3) an antibody comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 36, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 37, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 38; (4) an antibody comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 48, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 49, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 50; (5) an antibody comprising a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 18, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 19, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 20; (6) an antibody comprising a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 30, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 31, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 32; (7) an antibody comprising a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 42, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 43, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 44; (8) an antibody comprising a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 54, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 55, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 56; (9) an antibody comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 12, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 13, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 14, and a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 18, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 19, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 20; (10) an antibody comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 24, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 25, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 26, and a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 30, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 31, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 32; (11) an antibody comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 36, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 37, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 38, and a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 42, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 43, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 44; (12) an antibody comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 48, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 49, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 50, and a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 54, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 55, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 56; (13) an antibody comprising one or more amino acid substitutions, deletions, additions, and/or insertions in the antibody of any one of (1) to (12) and has an activity equivalent to that of any of the antibodies (1) to (12); and (14) an antibody which binds to the same epitope as that in DLL3 protein to which any of the antibodies (1) to (12) bind.

[0018] In a further alternative aspect, the present invention provides a pharmaceutical composition comprising any of the antibodies described above as an active ingredient. Preferably, the pharmaceutical composition of the present invention is an anticancer agent. Particularly preferably, the anticancer agent targets lung cancer.

[0019] In a further alternative aspect, the present invention provides a method for diagnosing cancer, comprising the following steps:

(a) providing a sample isolated from a test subject; and (b) detecting the expression level of DLL3 protein or DLL3 gene in the sample. Preferably, the diagnosis method of the present invention is intended for the diagnosis of lung cancer. The present invention also provides a diagnostic agent for cancer comprising any of the antibodies described above.

BRIEF DESCRIPTION OF DRAWINGS

[0020] FIG. 1 shows increased DLL3 expression in small-cell lung cancer and the fetal brain.

[0021] FIG. 2 shows the binding of an anti-DLL3 monoclonal antibody to a soluble DLL3 protein.

[0022] FIG. 3 shows the binding of an antibody to the DLL3 protein on a cell membrane and the turnover of the cell-bound antibody.

[0023] FIG. 4 shows the induction of ADCC by an anti-DLL3 antibody (concentration: 2.5 .mu.g/ml).

[0024] FIG. 5 shows the antibody concentration dependence of an ADCC activity against target cells DLL3/BaF3 (a) and NCI-H1184 (b).

[0025] FIG. 6 shows the inhibition of cell growth by the uptake of an anti-DLL3 antibody and an anti-mouse secondary antibody Mab-ZAP labeled with toxin.

[0026] FIG. 7 shows the binding of a recombinant anti-DLL3 antibody to the DLL3 protein on cell surface.

[0027] FIG. 8 shows the binding of a recombinant anti-DLL3 human chimeric antibody to a soluble DLL3 protein and the binding competition of an anti-DLL3 chimeric antibody with a mouse antibody against a soluble DLL3 protein.

[0028] FIG. 9 shows the schematic structure of full-length and soluble DLL3 proteins and a site recognized by an anti-DLL3 antibody.

[0029] FIG. 10 shows the binding of a recombinant anti-DLL3 human chimeric antibody DL306 to the DLL3 protein on cell surface.

[0030] FIG. 11 shows the antibody concentration dependence of the ADCC activity of an anti-DLL3 human chimeric antibody against target cells DLL3/BaF3 and NCI-H1184.

[0031] FIG. 12 shows the antibody concentration dependence of the ADCC activity of an anti-DLL3 mouse low-fucose antibody against target cells DLL3/BaF3 and NCI-H1184.

DESCRIPTION OF EMBODIMENTS

DLL3

[0032] The amino acid sequence of DLL3 (Delta-like 3) is known in the art. For example, the amino acid sequence of human DLL3 is as set forth in SEQ ID NO: 1 (NM_016941), and the amino acid sequence of mouse DLL3 is as set forth in SEQ ID NO: 2 (NM_007866).

[0033] The DLL3 protein used in the present invention may be a DLL3 protein having the sequence described above or may be a modified protein having a sequence derived from the sequence described above by the modification of one or more amino acids. Examples of the modified protein having a sequence derived from the sequence described above by the modification of one or more amino acids can include polypeptides having 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more homology to the amino acid sequence. Alternatively, partial peptides of these DLL3 proteins may be used.

[0034] The DLL3 protein used in the present invention is not limited by its origin and is preferably a human DLL3 protein.

Anti-DLL3 Antibody

[0035] The anti-DLL3 antibody used in the present invention needs only to bind to the DLL3 protein and is not particularly limited by its origin, type, shape, etc. Specifically, an antibody can be used, such as a non-human animal-derived antibody (e.g., a mouse, rat, or camel antibody), a human-derived antibody, a chimeric antibody, or a humanized antibody. The anti-DLL3 antibody used in the present invention may be a polyclonal or monoclonal antibody and is preferably a monoclonal antibody.

[0036] The anti-DLL3 antibody used in the present invention is preferably an anti-human DLL3 antibody. The anti-human DLL3 antibody may be an antibody which specifically binds to human DLL3 or may be an antibody which binds to human DLL3 as well as non-human animal-derived DLL3 (e.g., mouse DLL3).

[0037] The anti-DLL3 antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using means known in the art. The anti-DLL3 antibody used in the present invention is particularly preferably a mammal-derived monoclonal antibody. The mammal-derived monoclonal antibody encompasses, for example, those produced by hybridomas and those produced by hosts transformed with expression vectors containing an antibody gene by a genetic engineering approach.

[0038] The anti-DLL3 antibody of the present invention may be modified with various molecules such as polyethylene glycol (PEG). As described later, the anti-DLL3 antibody of the present invention may also be modified with a chemotherapeutic agent, a radioactive chemical, or the like, having a cytotoxic activity.

[0039] Examples of the antibody used in the present invention, which recognizes DLL3 and binds thereto, can include the following antibodies:

(1) an antibody (DL301) comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 12, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 13, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 14; (2) an antibody (DL306) comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 24, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 25, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 26; (3) an antibody (DL309) comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 36, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 37, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 38; (4) an antibody (DL312) comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 48, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 49, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 50; (5) an antibody (DL301) comprising a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 18, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 19, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 20; (6) an antibody (DL306) comprising a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 30, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 31, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 32; (7) an antibody (DL309) comprising a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 42, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 43, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 44; (8) an antibody (DL312) comprising a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 54, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 55, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 56; (9) an antibody (DL301) comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 12, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 13, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 14, and a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 18, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 19, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 20; (10) an antibody (DL306) comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 24, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 25, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 26, and a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 30, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 31, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 32; (11) an antibody (DL309) comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 36, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 37, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 38, and a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 42, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 43, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 44; (12) an antibody (DL312) comprising a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 48, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 49, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 50, and a light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 54, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 55, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 56; (13) an antibody that is derived from any of the antibodies (1) to (12) by the substitution, deletion, addition, and/or insertion of one or more amino acids and has an activity equivalent to that of any of the antibodies (1) to (12); and (14) an antibody which binds to the same epitope as that in DLL3 protein to which any of the antibodies (1) to (12) bind.

[0040] The antibodies (1) to (12) may contain constant regions. The constant regions used are not particularly limited, and any constant region may be used. Preferable examples of the constant regions used in the present invention can include human-derived constant regions. For example, a human IgG1-derived, human IgG2-derived, human IgG3-derived, or human IgG4-derived constant region can be used as a heavy chain constant region. Also, for example, human .kappa. chain-derived or human .lamda. chain-derived constant region can be used as a light chain constant region. The constant regions used in the present invention may be constant regions having a native sequence or may be modified constant regions having a sequence derived from the native sequence by the modification of one or more amino acids.

[0041] The antibodies (1) to (12) may contain FRs. The FRs used are not particularly limited, and any FR may be used as long as the resulting antibody maintains its binding activity against human DLL3. Preferable examples of the FRs used in the present invention can include human antibody-derived FRs. Since the technique of FR replacement with the antigen binding activity of an antibody maintained is known in the art, those skilled in the art can appropriately select FRs. The FRs used in the present invention may be FRs having a native sequence or may be FRs having a sequence derived from the native sequence by the modification of one or more amino acids.

[0042] Examples of the heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 12, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 13, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 14 can include a heavy chain variable region having the amino acid sequence as set forth in SEQ ID NO: 9. Also, examples of the heavy chain comprising the heavy chain variable region can include a heavy chain having the amino acid sequence of SEQ ID NO: 10 or 11.

[0043] Examples of the heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 24, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 25, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 26 can include a heavy chain variable region having the amino acid sequence as set forth in SEQ ID NO: 21. Also, examples of the heavy chain comprising the heavy chain variable region can include a heavy chain having the amino acid sequence of SEQ ID NO: 22 or 23.

[0044] Examples of the heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 36, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 37, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 38 can include a heavy chain variable region having the amino acid sequence as set forth in SEQ ID NO: 33. Also, examples of the heavy chain comprising the heavy chain variable region can include a heavy chain having the amino acid sequence of SEQ ID NO: 34 or 35.

[0045] Examples of the heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 48, heavy chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 49, and heavy chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 50 can include a heavy chain variable region having the amino acid sequence as set forth in SEQ ID NO: 45. Also, examples of the heavy chain comprising the heavy chain variable region can include a heavy chain having the amino acid sequence of SEQ ID NO: 46 or 47.

[0046] Examples of the light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 18, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 19, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 20 can include a light chain variable region having the amino acid sequence as set forth in SEQ ID NO: 15. Also, examples of the light chain comprising the light chain variable region can include a light chain having the amino acid sequence of SEQ ID NO: 16 or 17.

[0047] Examples of the light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 30, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 31, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 32 can include a light chain variable region having the amino acid sequence as set forth in SEQ ID NO: 27. Also, examples of the light chain comprising the light chain variable region can include a light chain having the amino acid sequence of SEQ ID NO: 28 or 29.

[0048] Examples of the light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 42, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 43, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 44 can include a light chain variable region having the amino acid sequence as set forth in SEQ ID NO: 39. Also, examples of the light chain comprising the light chain variable region can include a light chain having the amino acid sequence of SEQ ID NO: 40 or 41.

[0049] Examples of the light chain variable region comprising light chain CDR1 having the amino acid sequence as set forth in SEQ ID NO: 54, light chain CDR2 having the amino acid sequence as set forth in SEQ ID NO: 55, and light chain CDR3 having the amino acid sequence as set forth in SEQ ID NO: 56 can include a light chain variable region having the amino acid sequence as set forth in SEQ ID NO: 51. Also, examples of the light chain comprising the light chain variable region can include a light chain having the amino acid sequence of SEQ ID NO: 52 or 53.

[0050] In the present invention, the phrase "having an activity equivalent to that of the antibody of the present invention" refers to having a DLL3 binding activity, an internalization activity, and/or a cytotoxic activity (ADCC activity, etc.) against DLL3-expressing cells equivalent thereto. In the present invention, the equivalent activity is not necessarily required to be an identical activity and may be, for example, 50% or more, preferably 70% or more, more preferably 90% or more activity compared with the activity of any of the antibodies (1) to (12). Examples of the upper limit of the activity can include, but not particularly limited to, 1000% or less, 500% or less, 300% or less, 150% or less, and 100% or less.

[0051] An antibody derived from the antibody of the present invention by the substitution, deletion, addition, and/or insertion of one or more amino acids is also incorporated in the scope of the present invention and may be prepared artificially or occur naturally. Examples of a method for introducing a mutation in the polypeptide include site-directed mutagenesis (Hashimoto-Gotoh, T. et al. (1995) Gene 152, 271-275, Zoller, M J, and Smith, M. (1983) Methods Enzymol. 100, 468-500, Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441-9456, Kramer W, and Fritz H J (1987) Methods. Enzymol. 154, 350-367, Kunkel, T A (1985) Proc Natl Acad Sci USA. 82, 488-492, Kunkel (1988) Methods Enzymol. 85, 2763-2766). This is one of methods well known by those skilled in the art for preparing a polypeptide functionally equivalent to a certain polypeptide. Those skilled in the art can appropriately introduce a mutation in the antibody of the present invention using such a method and thereby prepare an antibody functionally equivalent to the antibody. Moreover, amino acid mutations may occur in the natural world. Such an antibody that has an amino acid sequence derived from the amino acid sequence of the antibody of the present invention by the mutation of one or more amino acids and is functionally equivalent to the antibody is also encompassed by the antibody of the present invention.

[0052] The number of amino acids mutated in such a variant is usually within 50 amino acids, preferably within 30 amino acids, more preferably within 10 amino acids (e.g., within 5 amino acids).

[0053] For amino acid residues to be mutated, it is preferred that this mutation should be performed conservatively between amino acids having the same side chain property. For example, the following classification based on the properties of amino acid side chains has been established:

hydrophobic amino acids (A, I, L, M, F, P, W, Y, and V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, and T), amino acids having an aliphatic side chain (G, A, V, L, I, and P), amino acids having a hydroxy group-containing side chain (S, T, and Y), amino acids having a sulfur atom-containing side chain (C and M), amino acids having a side chain containing carboxylic acid and amide (D, N, E, and Q), amino acids having a base-containing side chain (R, K, and H), and amino acids having an aromatic group-containing side chain (H, F, Y, and W) (all symbols within the parentheses represent single letter codes of amino acids).

[0054] A polypeptide having an amino acid sequence modified from a certain amino acid sequence by the deletion and/or addition of one or more amino acid residue(s) and/or the substitution thereof by other amino acids is already known to maintain the biological activity of the original polypeptide (Mark, D. F. et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666, Zoller, M. J. and Smith, M., Nucleic Acids Research (1982) 10, 6487-6500, Wang, A. et al., Science 224, 1431-1433, Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413). Specifically, when amino acids in an amino acid sequence constituting a certain polypeptide are substituted by amino acids classified in the same group thereas, it is generally said that the polypeptide is likely to maintain its activity. In the present invention, the substitution between amino acids within the same amino acid group described above is referred to as conservative substitution.

[0055] The present invention also provides an antibody which binds to the same epitope as that to which any of the antibodies (1) to (12) bind. Specific examples of the antibodies (1) to (12) can include antibodies DL301, DL306, DL309, and DL312 described below in Examples. Specifically, the present invention also provides an antibody recognizing the same epitope as that recognized by any of these antibodies.

[0056] Whether or not an analyte antibody shares an epitope with a certain antibody can be confirmed based on their competition for the same epitope. The competition between the antibodies is detected by cross-blocking assay or the like. The cross-blocking assay is preferably, for example, competitive ELISA assay.

[0057] Specifically, the cross-blocking assay involves preincubating DLL3 proteins coated on the wells of a microtiter plate in the presence or absence of a candidate competing antibody and then adding thereto the anti-DLL3 antibody of the present invention. The amount of the anti-DLL3 antibody of the present invention bound to the DLL3 protein in each well indirectly correlates with the binding capability of the candidate competing antibody (analyte antibody) that competes therewith for binding to the same epitope. Specifically, the higher affinity of the analyte antibody for the same epitope results in the smaller amount of the anti-DLL3 antibody of the present invention bound to the DLL3 protein-coated well and instead, the larger amount of the analyte antibody bound to the DLL3 protein-coated well.

[0058] The amount of each antibody bound to the well can be determined easily by labeling the antibody in advance. For example, a biotinylated antibody can be assayed using an avidin-peroxidase conjugate and an appropriate substrate. The cross-blocking assay using enzyme (e.g., peroxidase) labeling is particularly called competitive ELISA assay. The antibody may be labeled with any of other detectable or measurable labeling materials. Specifically, for example, radiolabeling or fluorescent labeling is known in the art.

[0059] Furthermore, when the analyte antibody has constant regions derived from a species different from that of the anti-DLL3 antibody of the present invention, the amount of any antibody bound to the well can also be measured using a labeled antibody that recognizes any constant region. Alternatively, even antibodies differing in class, albeit derived from the same species, can be measured for their respective amounts bound to the well using antibodies that discriminate each class.

[0060] Provided that the candidate antibody can block the binding of the anti-DLL3 antibody by at least 20%, preferably at least 30%, more preferably at least 50%, even more preferably at least 80%, compared to the binding activity obtained in the control test performed in the absence of the competing antibody, this candidate antibody is determined as an antibody that binds to substantially the same epitope as that to which the anti-DLL3 antibody of the present invention binds or as an antibody that competes therewith for the binding to the same epitope.

[0061] Further examples of the antibody of the present invention can include an antibody recognizing a region from amino acids 27 to 175 in human DLL3 (SEQ ID NO: 1) and an antibody recognizing a region from amino acids 216 to 492 in human DLL3. Examples of another aspect of the antibody of the present invention can include an antibody that binds to human DLL3 but does not bind to a polypeptide (DLL3delta1-Fc) consisting of the amino acid sequence of SEQ ID NO: 6 or a polypeptide (DLL3delta2-Fc) consisting of the amino acid sequence of SEQ ID NO: 7, and an antibody that binds to human DLL3 and also binds to the polypeptide (DLL3delta1-Fc) consisting of the amino acid sequence of SEQ ID NO: 6 and the polypeptide (DLL3delta2-Fc) consisting of the amino acid sequence of SEQ ID NO: 7.

[0062] The antibody of the present invention has activities such as an ADCC activity and an internalization activity and as such, is useful as a pharmaceutical drug, particularly, an anticancer agent.

Genetically Recombinant Anti-DLL3 Antibody

[0063] The antibody to be administered to humans can be converted to a genetically recombinant antibody that has been engineered artificially, for example, for the purpose of reducing heteroantigenicity in humans. The genetically recombinant antibody encompasses, for example, chimeric antibodies and humanized antibodies. These engineered antibodies can be produced using a method known in the art.

(1) Chimeric Antibody

[0064] The chimeric antibodies refer to antibodies comprising variable and constant regions of different origins ligated with each other. For example, mouse-human heterogeneous chimeric antibodies are antibodies comprising the heavy and light chain variable regions of a mouse antibody and the heavy and light chain constant regions of a human antibody. Mouse antibody variable region-encoding DNAs are ligated with human antibody constant region-encoding DNAs, and the ligation products can be incorporated into expression vectors to prepare chimeric antibody-expressing recombinant vectors. Cells transformed with these vectors (recombinant cells) can be cultured for the expression of the DNA insert to obtain the chimeric antibodies produced during the culture.

[0065] Human antibody constant regions are used as the constant regions of the chimeric antibodies. For example, C.gamma.1, C.gamma.2, C.gamma.3, C.gamma.4, C.mu., C.delta., C.alpha.1, C.alpha.2, and C.epsilon. can be used as heavy chain constant regions. Moreover, C.kappa. and C.lamda. can be used as light chain constant regions. The amino acid sequences of these constant regions and nucleotide sequences encoding them are known in the art. Moreover, one or more amino acids in the human antibody constant regions can be substituted, deleted, added, and/or inserted for improving the stability of the antibody itself or of its production.

(2) Humanized Antibody

[0066] In general, the chimeric antibodies comprise non-human animal-derived antibody variable regions and human antibody-derived constant regions. By contrast, the humanized antibodies comprise non-human animal-derived antibody complementarity-determining regions (CDRs), human antibody-derived framework regions (FRs), and human antibody-derived constant regions. The humanized antibodies are also called reshaped human antibodies. Specifically, for example, humanized antibodies comprising non-human animal (e.g., mouse) antibody CDRs grafted in human antibodies are known in the art. The humanized antibodies are useful as active ingredients for a therapeutic agent of the present invention, owing to their reduced antigenicity in the human body.

[0067] Each antibody variable region usually comprises 3 CDRs flanked by 4 FRs. The CDR regions substantially determine the binding specificity of the antibody. The CDRs have diverse amino acid sequences. On the other hand, amino acid sequences constituting the FRs often exhibit high homology among antibodies having different binding specificities. Therefore, in general, the binding specificity of a certain antibody can allegedly be transplanted to other antibodies through CDR grafting.

[0068] General gene recombination approaches are also known for obtaining the humanized antibodies. Specifically, for example, Overlap Extension PCR is known in the art as a method for grafting mouse antibody CDRs into human FRs. The Overlap Extension PCR employs primers for human antibody FR synthesis comprising an additional nucleotide sequence encoding each mouse antibody CDR to be grafted. The primers are prepared for each of the 4 FRs. In the mouse CDR grafting into the human FRs, in general, it is allegedly advantageous to select human FRs highly homologous to mouse FRs for maintaining the CDR functions. Specifically, it is generally preferred to use human FRs comprising amino acid sequences highly homologous to those of the FRs adjacent to the mouse CDRs to be grafted.

[0069] Moreover, the nucleotide sequences to be ligated are designed such that they are connected in frame. The human FR-encoding nucleotide sequences are individually synthesized using their respective primers. As a result, products are obtained, which comprise the mouse CDR-encoding DNA added to each FR-encoding sequence. The mouse CDR-encoding nucleotide sequence in each product is designed such that the nucleotide sequence overlaps with another. Subsequently, the overlapping CDR portions are annealed to each other for complementary strand synthesis reaction. Through this reaction, the human FR sequences are ligated via the mouse CDR sequences.

[0070] Finally, the full-length gene of the variable region comprising 3 CDRs and 4 FRs ligated is amplified with primers that respectively anneal to the 5' and 3' ends thereof and comprise an additional recognition sequence for an appropriate restriction enzyme. The DNA thus obtained and human antibody constant region-encoding DNA can be inserted into expression vectors such that they are fused in frame to prepare vectors for humanized antibody expression. Hosts are transformed with these vectors to establish recombinant cells, which are then cultured for the expression of the humanized antibody-encoding DNA to produce the humanized antibodies into the cultures of the cultured cells (see European Patent Publication No. EP 239400 and International Publication No. WO 96/02576).

[0071] The humanized antibodies thus prepared can be evaluated for their binding activities for the antigen by qualitative or quantitative assay. As a result, human antibody FRs can be selected preferably such that they allow CDRs to form a favorable antigen-binding site when ligated via the CDRs. If necessary, FR amino acid residue(s) may be substituted such that the CDRs of the humanized antibody form an appropriate antigen-binding site. For example, a mutation can be introduced in the amino acid sequence of FR by applying the PCR method used in the mouse CDR grafting into the human FRs. Specifically, a mutation of a partial nucleotide sequence can be introduced in the primers annealing to the FR nucleotide sequence. The FR nucleotide sequence synthesized using such primers contains the mutation thus introduced. The variant antibodies having the substituted amino acid(s) can be evaluated for their binding activities for the antigen by the same assay as above to select variant FR sequences having the desired property (Sato, K. et al., Cancer Res, 1993, 53, 851-856).

(3) Polyvalent Antibody

[0072] The antibody of the present invention encompasses not only bivalent antibodies typified by IgG (IgG1, IgG2, IgG4, etc.) but also monovalent antibodies or polyvalent antibodies typified by IgM as long as these antibodies bind to the DLL3 protein. The polyvalent antibody of the present invention encompasses polyvalent antibodies having antigen-binding sites, all of which are the same as each other or some or all of which are different from each other.

(4) Low-Molecular Antibody

[0073] The antibody of the present invention is not limited to whole antibody molecules and may be a low-molecular antibody or a modified form thereof as long as the antibody binds to the DLL3 protein.

[0074] The low-molecular antibody encompasses an antibody fragment deficient in a portion of the whole antibody (e.g., whole IgG). Such partial deficiency of the antibody molecule is accepted as long as the resultant antibody fragment is capable of binding to the DLL3 antigen. It is preferred that the antibody fragment according to the present invention should contain one or both of heavy chain variable (VH) and light chain variable (VL) regions. It is also preferred that the antibody fragment according to the present invention should contain CDRs. The number of CDRs contained in the antibody fragment of the present invention is not particularly limited and is preferably at least 6 CDRs: heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3.

[0075] The amino acid sequence of VH or VL can contain substitution, deletion, addition, and/or insertion. Furthermore, the antibody fragment of the present invention may be deficient in a portion of one or both of VH and VL as long as the resultant antibody fragment is capable of binding to the DLL3 antigen. Moreover, its variable region may be chimerized or humanized. Specific examples of the antibody fragment can include Fab, Fab', F(ab')2, and Fv. Moreover, specific examples of the low-molecular antibody can include Fab, Fab', F(ab')2, Fv, scFv (single chain Fv), Diabody, sc(Fv)2 (single chain (Fv)2), and scFv-Fc. In the present invention, the low-molecular antibody is preferably Diabody or sc(Fv)2. These antibody multimers (e.g., dimmers, trimers, tetramers, and polymers) are also encompassed by the low-molecular antibody of the present invention.

[0076] Such fragments of the antibody can be obtained by enzymatically treating the antibody to form antibody fragments. The digestive enzymes cleave the antibody fragment at a particular position to give antibody fragments having a particular structure. For example, papain, pepsin, or plasmin is known in the art as the enzyme for forming the antibody fragments. The papain digestion gives F(ab)2 or Fab, while the pepsin digestion gives F(ab')2 or Fab'. Alternatively, genes encoding these antibody fragments are constructed, and these genes can be introduced into expression vectors and then expressed in appropriate host cells (see e.g., Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976, Better, M. & Horwitz, A. H. Methods in Enzymology (1989) 178, 476-496, Plueckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 497-515, Lamoyi, E., Methods in Enzymology (1986) 121, 652-663, Rousseaux, J. et al., Methods in Enzymology (1986) 121, 663-669, Bird, R. E. et al., TIBTECH (1991) 9, 132-137).

[0077] The use of a genetic engineering approach for the enzymatically obtained antibody fragments can delete an arbitrary portion of the antibody. The low-molecular antibody according to the present invention may lack an arbitrary region as long as the resulting antibody fragment has binding affinity for DLL3.

i) Diabody

[0078] The Diabody refers to a bivalent antibody fragment constructed by gene fusion (e.g., Holliger P et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP404,097, and WO 93/11161). The Diabody is a dimer comprising two polypeptide chains. Usually, each of the polypeptide chains constituting the dimer comprises heavy and light chain variable regions linked via a linker on the same chain. The linker in the Diabody is generally too short to allow paring between heavy and light chain variable regions on the same chain. Specifically, the number of amino acid residues constituting the linker is, for example, approximately 5 residues. Therefore, heavy and light chain variable regions encoded on the same polypeptide chain cannot together form a single chain variable region fragment. Instead, they form a dimer by pairing with another single chain variable region fragment. As a result, the Diabody has two antigen-binding sites.

ii) scFv

[0079] The scFv is obtained by linking heavy and light chain variable regions of the antibody. In the scFv, the heavy and light chain variable regions are linked via a linker, preferably, a peptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A, 1988, 85, 5879-5883). The heavy and light chain variable regions in the scFv can be derived from any of the antibodies described in the present specification. The peptide linker that links the variable regions is not particularly limited. For example, an arbitrary single chain peptide of approximately 3 to 25 residues can be used as the linker. Specifically, for example, a peptide linker described later can be used.

[0080] The variable regions of both the chains can be linked, for example, by PCR. First, of DNA sequences encoding the heavy chain or heavy chain variable region of the antibody and DNA sequences encoding the light chain or light chain variable region of the antibody, DNAs encoding the whole or desired partial amino acid sequence are used as templates for linking the variable regions by PCR.

[0081] The heavy chain variable region-encoding DNA and the light chain variable region-encoding DNA are separately amplified by PCR using a pair of primers having sequences corresponding to both terminal sequences of each DNA to be amplified. Subsequently, DNA encoding the peptide linker portion is prepared. The DNA encoding the peptide linker can also be synthesized using PCR. Nucleotide sequences that can be linked to the amplification product of each variable region separately synthesized are respectively added to the 5' sequences of primers used in this PCR. Subsequently, PCR reaction is performed using each DNA of [heavy chain variable region DNA]-[peptide linker DNA]-[light chain variable region DNA] and primers for assembly PCR.

[0082] The primers for assembly PCR comprise the combination of a primer annealing to the 5' sequence of the [heavy chain variable region DNA] and a primer annealing to the 3' sequence of the [light chain variable region DNA]. Specifically, the primers for assembly PCR are a primer set that is capable of amplifying DNA encoding the full-length sequence of the scFv to be synthesized. By contrast, the [peptide linker DNA] contains an additional nucleotide sequence that can be linked to each variable region DNA. As a result, these DNAs are linked and, further, finally prepared into a full-length scFv amplification product using the primers for assembly PCR. Once the scFv-encoding DNA is prepared, expression vectors containing this DNA and cells transformed with the expression vectors (recombinant cells) can be obtained according to a routine method. Moreover, the resultant recombinant cells can be cultured for the expression of the scFv-encoding DNA to obtain the scFv.

iii) scFv-Fc

[0083] The scFv-Fc is a low-molecular antibody comprising an Fc region fused to scFv (Cellular & Molecular Immunology 2006; 3: 439-443). The origin of the scFv used in the scFv-Fc is not particularly limited, and, for example, scFv derived from IgM can be used. Moreover, the origin of the Fc is not particularly limited, and, for example, Fc derived from human IgG (human IgG1, etc.) can be used. Thus, examples of a preferable aspect of the scFv-Fc can include scFv-Fc comprising an IgM antibody scFv fragment linked to human IgG1 CH2 (e.g., C.gamma.2) and CH3 (e.g., C.gamma.3) via the hinge region (H.gamma.) of human IgG1.

iv) sc(Fv)2

[0084] The sc(Fv)2 is a low-molecular antibody having a single chain comprising two heavy chain variable regions (VHs) and two light chain variable regions (VLs) linked via linkers or the like (Hudson et al., J Immunol. Methods 1999; 231: 177-189). The sc(Fv)2 can be prepared, for example, by linking scFvs via a linker. Three linkers are usually necessary for linking four antibody variable regions.

[0085] Moreover, the sc(Fv)2 is preferably an antibody wherein two VHs and two VLs are aligned as VH, VL, VH, and VL (i.e., [VH]-linker-[VL]-linker-[VH]-linker-[VL]) in this order starting at the N-terminus of the single chain polypeptide.

[0086] The order of two VHs and two VLs is not particularly limited to the arrangement described above and may be any order of arrangement. Examples thereof can also include the following arrangements:

[VL]-linker-[VH]-linker-[VH]-linker-[VL] [VH]-linker-[VL]-linker-[VL]-linker-[VH] [VH]-linker-[VH]-linker-[VL]-linker-[VL] [VL]-linker-[VL]-linker-[VH]-linker-[VH] [VL]-linker-[VH]-linker-[VL]-linker-[VH]

[0087] For example, an arbitrary peptide linker or synthetic compound linker (e.g., linkers disclosed in the reference Protein Engineering, 9 (3), 299-305, 1996) that can be introduced by genetic engineering can be used as the linker that links the antibody variable regions. A plurality of the same or different linkers may be used. In the present invention, the peptide linker is preferable. The length of the peptide linker is not particularly limited and can be selected appropriately by those skilled in the art according to the purpose. The number of amino acid residues constituting the peptide linker is usually 1 to 100 amino acids, preferably 3 to 50 amino acids, more preferably 5 to 30 amino acids, particularly preferably 12 to 18 amino acids (e.g., 15 amino acids).

[0088] The amino acid sequence constituting the peptide linker can be an arbitrary sequence as long as this sequence does not inhibit the binding effect of the scFv. For example, the following amino acid sequences can be used for the peptide linker:

TABLE-US-00001 Ser Gly .cndot. Ser Gly .cndot. Gly .cndot. Ser Ser .cndot. Gly .cndot. Gly (SEQ ID NO: 61) Gly .cndot. Gly .cndot. Gly .cndot. Ser (SEQ ID NO: 62) Ser .cndot. Gly .cndot. Gly .cndot. Gly (SEQ ID NO: 63) Gly .cndot. Gly .cndot. Gly .cndot. Gly .cndot. Ser (SEQ ID NO: 64) Ser .cndot. Gly .cndot. Gly .cndot. Gly .cndot. Gly (SEQ ID NO: 65) Gly .cndot. Gly .cndot. Gly .cndot. Gly .cndot. Gly .cndot. Ser (SEQ ID NO: 66) Ser .cndot. Gly .cndot. Gly .cndot. Gly .cndot. Gly .cndot. Gly (SEQ ID NO: 67) Gly .cndot. Gly .cndot. Gly .cndot. Gly .cndot. Gly .cndot. Gly .cndot. Ser (SEQ ID NO: 68) Ser .cndot. Gly .cndot. Gly .cndot. Gly .cndot. Gly .cndot. Gly .cndot. Gly (Gly .cndot. Gly .cndot. Gly .cndot. Gly .cndot. Ser)n (Ser .cndot. Gly .cndot. Gly .cndot. Gly .cndot. Gly)n [n represents an integer of 1 or more].

[0089] The amino acid sequence of the peptide linker can be selected appropriately by those skilled in the art according to the purpose. For example, the integer n that determines the length of the peptide linker is usually 1 to 5, preferably 1 to 3, more preferably 1 or 2.

[0090] Accordingly, examples of a particularly preferable aspect of the sc(Fv)2 according to the present invention can include the following sc(Fv)2: [VH]-peptide linker (15 amino acids)-[VL]-peptide linker (15 amino acids)-[VH]-peptide linker (15 amino acids)-[VL].

[0091] Alternatively, the variable regions can also be linked using the chemically synthesized linker (chemical cross-linking agent). Cross-linking agents usually used in the cross-link of peptide compounds or the like can be used in the present invention. For example, chemical cross-linking agents as shown below are known in the art. These cross-linking agents are commercially available:

N-hydroxysuccinimide (NHS),

[0092] disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES), etc.

Activity of Anti-DLL3 Antibody

(1) Cytotoxic Activity

[0093] For the treatment of cell-proliferative disease such as cancer, it is preferred that the antibody should maintain its effector activity. Specifically, the preferable antibody according to the present invention has both of a binding affinity for DLL3 and effector functions. The effector functions of the antibody encompass an antibody-dependent cell-mediated cytotoxic (ADCC) activity and a complement-dependent cytotoxic (CDC) activity. The therapeutic antibody according to the present invention particularly preferably possesses an ADCC activity as effector functions.

[0094] The antibody of the present invention used for the therapeutic purpose is preferably an antibody having a cytotoxic activity.

[0095] Examples of the cytotoxic activity according to the present invention can include ADCC and CDC activities. In the present invention, the ADCC activity means the activity of damaging target cells through the binding of Fc.gamma. receptor-bearing cells (immunocytes, etc.) via the Fc.gamma. receptors to the Fc domains of antibodies specifically attached to the cell surface antigens of the target cells. On the other hand, the CDC activity means a cytotoxic activity mediated by the complement system.

[0096] Whether or not the anti-DLL3 antibody has an ADCC activity or has a CDC activity can be determined by a method known in the art (e.g., Current protocols in Immunology, Chapter 7. Immunologic studies in humans, Editor, John E, Coligan et al., John Wiley & Sons, Inc., (1993)). Specifically, effector cells, a complement solution, and target cells are first prepared.

i) Preparation of Effector Cells

[0097] The spleens are excised from CBA/N mice or the like, and spleen cells are separated therefrom in an RPMI1640 medium (manufactured by Invitrogen Corp.). The cells can be washed with this medium containing 10% fetal bovine serum (FBS, manufactured by HyClone Laboratories, Inc.) and then adjusted to a cell concentration of 5.times.10.sup.6 cells/ml to prepare effector cells.

ii) Preparation of Complement Solution

[0098] Baby Rabbit Complement (manufactured by CEDARLANE Laboratories Ltd.) can be diluted 10-fold with a medium (manufactured by Invitrogen Corp.) containing 10% FBS to prepare a complement solution.

iii) Preparation of Target Cell

[0099] Cells expressing DLL3 proteins can be cultured at 37.degree. C. for 1 hour, together with 0.2 mCi .sup.51Cr-sodium chromate (manufactured by GE Healthcare Bio-Sciences Corp.), in a DMEM medium containing 10% FBS to radiolabel the target cells. Cells transformed with DLL3 protein-encoding genes, small-cell lung cancer cell lines, or the like can be used as the cells expressing DLL3 proteins. The cells thus radiolabeled can be washed three times with an RPMI1640 medium containing 10% FBS and adjusted to a cell concentration of 2.times.10.sup.5 cells/ml to prepare the target cells.

[0100] The ADCC or CDC activity can be assayed by a method described below. For the ADCC activity assay, the target cells and the anti-DLL3 antibody (50 .mu.l each) are added to a U-bottom 96-well plate (manufactured by Becton, Dickinson and Company) and reacted for 15 minutes on ice. Then, 100 .mu.l of the effector cells is added to the plate, and the cells are cultured for 4 hours in a CO.sub.2 incubator. The final concentration of the antibody is set to 0 or 10 .mu.g/ml. After the culture, 100 .mu.l of the supernatant is collected, and the radioactivity is measured using a gamma counter (COBRA II AUTO-GAMMA, MODEL D5005, manufactured by Packard Instrument Company). The cytotoxic activity (%) can be calculated based on the calculation formula (A-C)/(B-C).times.100 using the obtained value. In the formula, A represents radioactivity (cpm) from each sample; B represents radioactivity (cpm) from a sample supplemented with 1% NP-40 (manufactured by Nacalai Tesque, Inc.); and C represents radioactivity (cpm) from a sample containing only the target cells.

[0101] On the other hand, for the CDC activity assay, the target cells and the anti-DLL3 antibody (50 .mu.l each) are added to a flat-bottomed 96-well plate (manufactured by Becton, Dickinson and Company) and reacted for 15 minutes on ice. Then, 100 .mu.l of the complement solution is added to the plate, and the cells are cultured for 4 hours in a CO.sub.2 incubator. The final concentration of the antibody is set to 0 or 3 .mu.g/ml. After the culture, 100 .mu.l of the supernatant is collected, and the radioactivity is measured using a gamma counter. The cytotoxic activity can be calculated in the same way as in the ADCC activity assay.

[0102] By contrast, in the cytotoxic activity assay using antibody conjugates, the target cells and the anti-DLL3 antibody conjugates (50 .mu.l each) are added to a flat-bottomed 96-well plate (manufactured by Becton, Dickinson and Company) and reacted for 15 minutes on ice. The cells are cultured for 1 to 4 hours in a CO.sub.2 incubator. The final concentration of the antibody is set to 0 or 3 .mu.g/ml. After the culture, 100 .mu.l of the supernatant is collected, and the radioactivity is measured using a gamma counter. The cytotoxic activity can be calculated in the same way as in the ADCC activity assay.

(2) Conjugated Antibody

[0103] The antibody may be conjugated with a cytotoxic substance such as a chemotherapeutic agent, a toxic peptide, or a radioactive chemical. Such a modified antibody (hereinafter, referred to as an antibody conjugate) can be obtained by chemically modifying the obtained antibody. A method for the antibody modification has already been established in the art.

[0104] Examples of the chemotherapeutic agent whose cytotoxic activity functions through the conjugation to the anti-DLL3 antibody can include the following chemotherapeutic agents: azaribine, anastrozole, azacytidine, bleomycin, bortezomib, bryostatin-1, busulfan, camptothecin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin, irinotecan, carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunomycin glucuronide, daunorubicin, dexamethasone, diethylstilbestrol, doxorubicin, doxorubicin glucuronide, epirubicin, ethinyl estradiol, estramustine, etoposide, etoposide glucuronide, floxuridine, fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide, leucovorin, lomustine, mechlorethamine, medroxyprogesterone acetate, megestrol acetate, melphalan, mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, phenylbutyrate, prednisone, procarbazine, paclitaxel, pentostatin, semustine, streptozocin, tamoxifen, taxanes, taxol, testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vinblastine, vinorelbine, vincristine.

[0105] The chemotherapeutic agent is preferably a low-molecular chemotherapeutic agent. The low-molecular chemotherapeutic agent is unlikely to interfere with the antibody functions even after its conjugation to the antibody. In the present invention, the low-molecular chemotherapeutic agent usually has a molecular weight of 100 to 2000, preferably 200 to 1000. All of the chemotherapeutic agents exemplified above are low-molecular chemotherapeutic agents. These chemotherapeutic agents according to the present invention encompass prodrugs that are converted in vivo to active chemotherapeutic agents. The prodrug activation may be enzymatic conversion or nonenzymatic conversion.

[0106] Moreover, the antibody may be modified with the toxic peptide. Examples of the toxic peptide can include the following: Diphtheria toxin A Chain (Langone J. J., et al., Methods in Enzymology, 93, 307-308, 1983), Pseudomonas Exotoxin (Nature Medicine, 2, 350-353, 1996), Ricin A Chain (Fulton R. J., et al., J. Biol. Chem., 261, 5314-5319, 1986; Sivam G., et al., Cancer Res., 47, 3169-3173, 1987; Cumber A. J. et al., J. Immunol. Methods, 135, 15-24, 1990; Wawrzynczak E. J., et al., Cancer Res., 50, 7519-7562, 1990; Gheeite V., et al., J. Immunol. Methods, 142, 223-230, 1991); Deglicosylated Ricin A Chain (Thorpe P. E., et al., Cancer Res., 47, 5924-5931, 1987); Abrin A Chain (Wawrzynczak E. J., et al., Br. J. Cancer, 66, 361-366, 1992; Wawrzynczak E. J., et al., Cancer Res., 50, 7519-7562, 1990; Sivam G., et al., Cancer Res., 47, 3169-3173, 1987; Thorpe P. E., et al., Cancer Res., 47, 5924-5931, 1987); Gelonin (Sivam G., et al., Cancer Res., 47, 3169-3173, 1987; Cumber A. J. et al., J. Immunol. Methods, 135, 15-24, 1990; WawrzynczakE. J., et al., Cancer Res., 50, 7519-7562, 1990; Bolognesi A., et al., Clin. exp. Immunol., 89, 341-346, 1992); PAP-s; Pokeweed anti-viral protein from seeds (Bolognesi A., et al., Clin. exp. Immunol., 89, 341-346, 1992); Briodin (Bolognesi A., et al., Clin. exp. Immunol., 89, 341-346, 1992); Saporin (Bolognesi A., et al., Clin. exp. Immunol., 89, 341-346, 1992); Momordin (Cumber A. J., et al., J. Immunol. Methods, 135, 15-24, 1990; Wawrzynczak E. J., et al., Cancer Res., 50, 7519-7562, 1990; Bolognesi A., et al., Clin. exp. Immunol., 89, 341-346, 1992); Momorcochin (Bolognesi A., et al., Clin. exp. Immunol., 89, 341-346, 1992); Dianthin 32 (Bolognesi A., et al., Clin. exp. Immunol., 89, 341-346, 1992); Dianthin 30 (Stirpe F., Barbieri L., FEBS letter 195, 1-8, 1986); Modeccin (Stirpe F., Barbieri L., FEBS letter 195, 1-8, 1986); Viscumin (Stirpe F., Barbieri L., FEBS letter 195, 1-8, 1986); Volkesin (Stirpe F., Barbieri L., FEBS letter 195, 1-8, 1986); Dodecandrin (Stirpe F., Barbieri L., FEBS letter 195, 1-8, 1986); Tritin (Stirpe F., Barbieri L., FEBS letter 195, 1-8, 1986); Luffin (Stirpe F., Barbieri L., FEBS letter 195, 1-8, 1986); Trichokirin (Casellas P., et al., Eur. J. Biochem. 176, 581-588, 1988; Bolognesi A., et al., Clin. exp. Immunol., 89, 341-346, 1992).

[0107] In the present invention, the radioactive chemical refers to a chemical containing a radioisotope. The radioisotope is not particularly limited, and any radioisotope may be used. For example, .sup.32c, .sup.14C, .sup.125I, .sup.3H, .sup.131I, .sup.186Re, or .sup.188Re can be used.

[0108] In another aspect, one or two or more low-molecular chemotherapeutic agents and one or two or more toxic peptides can be used in combination in the antibody modification. The anti-DLL3 antibody can be conjugated to the low-molecular chemotherapeutic agent via a covalent or noncovalent bond. A method for preparing such a chemotherapeutic agent-conjugated antibody is known in the art.

[0109] A proteinous agent or toxin can be conjugated to the antibody by a genetic engineering approach. Specifically, for example, DNA encoding the toxic peptide and DNA encoding the anti-DLL3 antibody are fused in frame with each other, and this fused DNA can be incorporated into expression vectors to construct recombinant vectors. The vectors are introduced into appropriate host cells, and the resultant transformed cells are cultured. The DNA insert can be expressed by the cells to obtain toxic peptide-conjugated anti-DLL3 antibodies as fusion proteins. For obtaining antibody-fusion proteins, the proteinous agent or toxin is generally located on the C-terminal side of the antibody. A peptide linker may be allowed to intervene between the antibody and the proteinous agent or toxin.

(3) Bispecific Antibody

[0110] Furthermore, the antibody of the present invention may be a bispecific antibody. The bispecific antibody refers to an antibody having, in the same antibody molecule, variable regions that recognize different epitopes. In the present invention, the bispecific antibody can have antigen-binding sites that recognize different epitopes on the DLL3 molecule. Thus, two such bispecific antibody molecules can bind to one DLL3 molecule. As a result, stronger cytotoxic effect can be expected.

[0111] Alternatively, the bispecific antibody of the present invention may have antigen-binding sites, one of which recognizes DLL3 and the other of which recognizes a cytotoxic substance. The cytotoxic substance specifically encompasses, for example, a chemotherapeutic agent, a toxic peptide, and a radioactive chemical. Such a bispecific antibody binds to cells expressing DLL3, while it captures the cytotoxic substance. As a result, the cytotoxic substance can be allowed to directly act on the cells expressing DLL3. Specifically, the bispecific antibody that recognizes the cytotoxic substance can specifically damage tumor cells and inhibit the growth of the tumor cells.

[0112] Moreover, in the present invention, a bispecific antibody comprising a DLL3-binding site combined with an antigen-binding site that recognizes an antigen other than DLL3 may be used. The antigen-binding site that can be combined therewith in such a bispecific antibody recognizes, for example, an antigen that is specifically expressed on the surface of target cancer cells, as with DLL3, but is different from DLL3.

[0113] A method for producing the bispecific antibody is known in the art. For example, two antibodies differing in antigen recognized thereby can be bound to prepare the bispecific antibody. Each of the antibodies bound may be a 1/2 molecule having heavy and light chains or may be a 1/4 molecule consisting of heavy chains. Alternatively, different monoclonal antibody-producing hybridomas may be fused to prepare fusion cells producing bispecific antibodies. Furthermore, the bispecific antibody can be prepared by a genetic engineering approach.

[0114] The antigen binding activity of the antibody can be determined using means known in the art (Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988). For example, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay), or fluoroimmunoassay can be used.

(4) Modification of Sugar Chain

[0115] The antibody of the present invention may be an antibody having a modified sugar chain. It is known that the cytotoxic activities of antibodies can be enhanced by modifying their sugar chains. For example, glycosylated antibodies (WO 99/54342, etc.), antibodies deficient in fucose added to their sugar chains (WO 00/61739, WO 02/31140, etc.), and antibodies having a sugar chain having bisecting GlcNAc (WO 02/79255, etc.) are known in the art as the antibody having a modified sugar chain.

(5) Internalization Activity

[0116] Moreover, the antibody of the present invention may have an internalization activity. In the present invention, the "antibody having an internalization activity" means an antibody that is transported into cells (cytoplasms, vesicles, other organelles, etc.) through its binding to DLL3.

[0117] Whether or not the antibody has an internalization activity can be confirmed by a method generally known by those skilled in the art and can be confirmed by, for example, a method involving contacting labeling material-bound anti-DLL3 antibodies with DLL3-expressing cells and confirming whether or not the labeling material is incorporated into the cells, or a method involving contacting cytotoxic substance-conjugated anti-DLL3 antibodies with DLL3-expressing cells and confirming whether or not the death of the DLL3-expressing cells is induced.

[0118] More specifically, the internalization activity of the anti-DLL3 antibody can be assayed by, for example, a method described in Examples.

[0119] The antibody having an internalization activity can be conjugated with, for example, the cytotoxic substance and used as a pharmaceutical composition such as an anticancer agent described later.

Preparation of Anti-DLL3 Antibody

1. Preparation of Anti-DLL3 Antibody Using Monoclonal Antibody-Producing Hybridoma

[0120] Monoclonal antibody-producing hybridomas can be prepared according to a technique known in the art as follows: first, animals are immunized with DLL3 proteins or partial peptides thereof (which will be described later) used as sensitizing antigens according to a usual immunization method. The obtained immunocytes are fused with parental cells known in the art by a usual cell fusion method to obtain hybridomas. These hybridomas are further screened for cells producing the antibody of interest by a usual screening method to select hybridomas producing the anti-DLL3 antibody. The desired anti-DLL3 monoclonal antibody is obtained from the selected hybridomas. Specifically, the anti-DLL3 monoclonal antibody is prepared as follows:

(1) Preparation of DLL3 Protein

[0121] First, DLL3 genes can be expressed to obtain DLL3 proteins used as sensitizing antigens for antibody obtainment. Specifically, the DLL3-encoding gene sequence is inserted into expression vectors known in the art, with which appropriate host cells are then transformed. Then, the human DLL3 proteins of interest are purified from the host cells or a culture supernatant thereof by a method known in the art. Purified natural DLL3 proteins or fusion proteins comprising the desired partial polypeptide of the DLL3 protein fused with a different polypeptide may be used as immunogens. For example, antibody Fc fragments, peptide tags, and so on can be used for producing the fusion proteins used as immunogens. Expression vectors for the fusion proteins can be prepared by fusing, in frame, two or more genes respectively encoding the desired polypeptide fragments and inserting this fusion gene into expression vectors. The method for preparing the fusion proteins is described in Molecular Cloning 2nd ed. (Sambrook, J. et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. Press, 1989).

[0122] The DLL3 proteins thus purified can be used as sensitizing antigens for the immunization of mammals. Partial peptides of DLL3 can also be used as sensitizing antigens. For example, the following peptides can be used as sensitizing antigens:

[0123] The region and size of the partial peptide of DLL3 used are not limited. The number of amino acids constituting the peptide serving as a sensitizing antigen is preferably at least 3 or more, for example, 5 or more or 6 or more. More specifically, peptides of 8 to 50 residues, preferably 10 to 30 residues can be used as sensitizing antigens.

(2) Immunization with DLL3 Protein

[0124] Mammals are immunized with the DLL3 proteins or partial peptides thereof as sensitizing antigens. The immunized mammals are not particularly limited. For obtaining the monoclonal antibody by the cell fusion method, it is preferred that the immunized animals should be selected in consideration of compatibility with the parental cells used in cell fusion. In general, rodents are preferable as the immunized animals. Specifically, mice, rats, hamsters, or rabbits can be used as the immunized animals. In addition, monkeys or the like may be used as the immunized animals.

[0125] These animals can be immunized with the sensitizing antigens according to a method known in the art. For example, a general method can involve immunizing the mammals with the sensitizing antigens by intraperitoneal or subcutaneous injection. Specifically, the sensitizing antigens are administered to the mammals several times at 4- to 21-day intervals. The sensitizing antigens are diluted with PBS (phosphate-buffered saline), saline, or the like at an appropriate dilution ratio and used in the immunization. Furthermore, the sensitizing antigens may be administered together with an adjuvant. For example, the antigens can be mixed with a Freund's complete adjuvant for emulsification to prepare sensitizing antigens. Moreover, an appropriate carrier can be used in the immunization with the sensitizing antigens. Particularly, when partial peptides having a small molecular weight are used as the sensitizing antigens, it is preferred that the sensitizing antigen peptides should be bound to carrier proteins such as albumin or keyhole limpet hemocyanin and used in the immunization.

(3) DNA Immunization

[0126] The monoclonal antibody can also be obtained by DNA immunization. The DNA immunization is an immunostimulation method involving: immunizing animals by the administration of vector DNA that has been constructed in a form capable of expressing antigenic protein-encoding genes in the immunized animals; and allowing the immunized animals to express the immunizing antigens in vivo. The DNA immunization can be expected to be superior to general immunization methods using the administration of protein antigens as follows:

[0127] it can provide immunostimulation with membrane protein (e.g., DLL3) structures maintained; and

[0128] it eliminates the need of purifying immunizing antigens.

[0129] For obtaining the monoclonal antibody of the present invention by the DNA immunization, first, animals are immunized by the administration of DLL3 protein expression vector DNA. DLL3-encoding DNA can be synthesized by a method known in the art such as PCR. The obtained DNA is inserted into appropriate expression vectors, with which animals are immunized by administration. For example, commercially available expression vectors such as pcDNA3.1 can be used as the expression vectors. Likewise, a method generally used can be used for administering the vectors to the animals. For example, gold particles with the expression vectors adsorbed onto can be inserted into cells using a gene gun to perform DNA immunization.

(4) Preparation of Hybridoma

[0130] A rise in the amount of the desired antibody is confirmed in the serum of the mammals thus immunized. Then, immunocytes are collected from the mammals and subjected to cell fusion. Particularly, spleen cells can be used as preferable immunocytes.

[0131] Mammalian myeloma cells are used in the cell fusion with the immunocytes. It is preferred that the myeloma cells should have an appropriate selection marker for screening. The selection marker refers to a character that can survive (or cannot survive) under particular culture conditions. For example, hypoxanthine-guanine phosphoribosyltransferase deficiency (hereinafter, abbreviated to HGPRT deficiency) or thymidine kinase deficiency (hereinafter, abbreviated to TK deficiency) is known in the art as the selection marker. Cells having the HGPRT or TK deficiency are sensitive to hypoxanthine-aminopterin-thymidine (hereinafter, abbreviated to HAT-sensitive). The HAT-sensitive cells are killed in a HAT selective medium because they cannot synthesize DNA. By contrast, these cells, when fused with normal cells, can grow even in the HAT selective medium because they can continue DNA synthesis by use of the salvage pathway of the normal cells.

[0132] The cells having the HGPRT or TK deficiency can be selected in a medium containing 6-thioguanine or 8-azaguanine (hereinafter, abbreviated to 8AG) for the HGPRT deficiency or 5'-bromodeoxyuridine for the TK deficiency. The normal cells are killed in such a medium because they incorporate these pyrimidine analogs into their DNAs. By contrast, the cells deficient in these enzymes can survive in the selective medium because they cannot incorporate the pyrimidine analogs therein. In addition, a selection marker called G418 resistance imparts, to cells, 2-deoxystreptamine antibiotic (gentamicin analog) resistance via a neomycin resistance gene. Various myeloma cells suitable for the cell fusion are known in the art. For example, the following myeloma cells can be used in the production of the monoclonal antibody according to the present invention:

[0133] P3 (P3x63Ag8. 653) (J. Immunol. (1979) 123, 1548-1550),

[0134] P3x63Ag8U. 1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7),

[0135] NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519),

[0136] MPC-11 (Margulies. D. H. et al., Cell (1976) 8, 405-415), SP2/0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (de St. Groth, S. F. et al., J. Immunol. Methods (1980) 35, 1-21),

[0137] S194 (Trowbridge, I. S. J. Exp. Med. (1978) 148, 313-323), R210 (Galfre, G. et al., Nature (1979) 277, 131-133) or the like.

[0138] Basically, the cell fusion of the immunocytes with the myeloma cells is performed according to a method known in the art, for example, the method of Kohler and Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46).

[0139] More specifically, the cell fusion can be performed, for example, in a usual nutrient culture medium in the presence of a cell fusion promoter. For example, polyethylene glycol (PEG) or hemagglutinating virus of Japan (HVJ) can be used as the fusion promoter. Furthermore, an auxiliary such as dimethyl sulfoxide can also be added thereto, if desired, for enhancing fusion efficiency.

[0140] The ratio between the immunocytes and the myeloma cells used can be set arbitrarily. For example, it is preferred that the amount of the immunocytes should be set to 1 to 10 times that of the myeloma cells. For example, an RPMI1640 or MEM culture medium suitable for the growth of the myeloma cell line as well as a usual culture medium used in this kind of cell culture can be used as the culture medium in the cell fusion. Furthermore, a solution supplemented with serum (e.g., fetal calf serum (FCS)) can be added to the culture medium.

[0141] For the cell fusion, the immunocytes and the myeloma cells are well mixed in the predetermined amounts in the culture medium and then mixed with a PEG solution preheated to approximately 37.degree. C. to form the fusion cells (hybridomas) of interest. In the cell fusion method, for example, PEG with an average molecular weight on the order of 1000 to 6000 can usually be added at a concentration of 30 to 60% (w/v). Subsequently, the appropriate culture medium exemplified above is sequentially added to the hybridomas, and the mixture is centrifuged, followed by removal of the supernatant. This procedure is repeated to remove the cell fusion agents or the like unfavorable for hybridoma growth.

[0142] The hybridomas thus obtained can be selected by use of a selective culture medium appropriate for the selection marker of the myeloma cells used in the cell fusion. For example, the cells having the HGPRT or TK deficiency can be selected by culturing the hybridomas in a HAT culture medium (culture medium containing hypoxanthine, aminopterin, and thymidine). Specifically, when HAT-sensitive myeloma cells are used in the cell fusion, only cells successfully fused with normal cells can be grown selectively in the HAT culture medium. The culture using the HAT culture medium is continued for a time long enough to kill cells (non-fused cells) other than the hybridomas of interest. Specifically, the culture can generally be performed for a few days to a few weeks to select the hybridomas of interest. Subsequently, hybridomas producing the antibody of interest can be screened for and cloned as single clones by a usual limiting dilution method.

[0143] The screening of the antibody of interest and cloning as single clones thereof can be performed preferably by a screening method based on antigen-antibody reaction known in the art. For example, the antigens are bound to a carrier such as beads made of polystyrene or the like or a commercially available 96-well microtiter plate and reacted with the culture supernatant of the hybridomas. Subsequently, the carrier is washed and then reacted with enzyme-labeled secondary antibodies or the like. When the culture supernatant contains the antibody of interest reactive with the sensitizing antigens, the secondary antibodies bind to the carrier via this antibody. Finally, the secondary antibodies bound with the carrier can be detected to determine the presence of the antibody of interest in the culture supernatant. The hybridomas producing the desired antibody capable of binding to the antigen can be cloned by a limiting dilution method or the like. In this screening, the DLL3 proteins used in the immunization or DLL3 proteins substantially identical thereto can be used preferably as the antigens. For example, cell lines expressing DLL3, soluble DLL3, or the like can be used as the antigens.

[0144] A method described in International Publication No. WO 03/104453 may be used in the production of the antibody against human DLL3.

[0145] Moreover, in addition to the method for obtaining the hybridomas by immunizing non-human animals with the antigens, human lymphocytes may be sensitized with the antigens to obtain the antibody of interest. Specifically, the human lymphocytes are first sensitized with the DLL3 proteins in vitro. Subsequently, the sensitized lymphocytes are fused with appropriate fusion partners. For example, human-derived myeloma cells capable of dividing throughout their lives can be used as the fusion partners (see Japanese Patent Publication No. 1-59878).

[0146] Furthermore, the anti-DLL3 human antibody can also be obtained by administering the DLL3 proteins as antigens to transgenic animals having all repertoires of human antibody genes or by immunizing the animals with DNA that has been constructed to express DLL3 in the animals. Antibody-producing cells from the immunized animals can be immortalized by treatment such as cell fusion with appropriate fusion partners or infection with Epstein-Barr virus. From the immortalized cells thus obtained, human antibodies against the DLL3 protein can be isolated (see International Publication Nos. WO 94/25585, WO 93/12227, WO 92/03918, and WO 94/02602). Furthermore, the immortalized cells can also be cloned as cells producing antibodies having the reaction specificity of interest. When transgenic animals are used as the immunized animals, the immune systems of the animals recognize human DLL3 as foreigners. Thus, the human antibodies against human DLL3 can be obtained easily.

(5) Obtainment of Monoclonal Antibody from Hybridoma

[0147] The monoclonal antibody-producing hybridomas thus prepared can be subcultured in a usual culture medium. Moreover, the hybridomas can also be stored over a long period in liquid nitrogen.

[0148] The hybridomas are cultured according to a usual method, and the monoclonal antibody of interest can be obtained from the culture supernatant thereof. Alternatively, the hybridomas are administered to mammals compatible therewith and grown, and the monoclonal antibody can also be obtained in the form of ascitic fluids. The former method is suitable for obtaining highly pure antibodies.

2. Preparation of Anti-DLL3 Antibody by Genetic Engineering Approach

(1) Cloning of Antibody Gene

[0149] The antibody may be prepared by a genetic engineering approach using antibody genes cloned from antibody-producing cells. The cloned antibody genes can be incorporated into appropriate vectors and expressed as antibodies by the transformation of hosts. Methods for the antibody gene isolation, the introduction into vectors, and the transformation of host cells have already been established (see e.g., Vandamme, A. M. et al., Eur. J. Biochem. (1990) 192, 767-775).

[0150] For example, cDNAs encoding the variable regions of the anti-DLL3 antibody can be obtained from the anti-DLL3 antibody-producing hybridoma cells. For this purpose, usually, total RNAs are first extracted from the hybridomas. For example, the following methods can be used for mRNA extraction from the cells:

[0151] guanidine ultracentrifugation method (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299), and

[0152] AGPC method (Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-159).

[0153] The extracted mRNAs can be purified using mRNA Purification Kit (manufactured by GE Healthcare Bio-Sciences Corp.) or the like. Alternatively, a kit for directly extracting total mRNAs from cells is also commercially available, such as QuickPrep mRNA Purification Kit (manufactured by GE Healthcare Bio-Sciences Corp.). The total mRNAs may be obtained from the hybridomas using such a kit. From the obtained mRNAs, antibody variable region-encoding cDNAs can be synthesized using reverse transcriptase. In this procedure, arbitrary 15- to 30-base sequences selected from sequences common to the antibody genes can be used as primers. The cDNAs can be synthesized using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Seikagaku Corp.) or the like. Moreover, 5'-Ampli FINDER RACE Kit (manufactured by Clontech Laboratories, Inc.) and 5'-RACE PCR (Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; and Belyaysky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) can be used for the cDNA synthesis and amplification. Furthermore, appropriate restriction enzyme sites described later can be introduced into both ends of the cDNAs in the course of such cDNA synthesis.

[0154] From the obtained PCR products, the cDNA fragments of interest are purified and subsequently ligated with vector DNAs. The recombinant vectors thus prepared are introduced into E. coli or the like. After colony selection, the desired recombinant vectors can be prepared from E. coli that has formed the colony. Then, the cDNA can be sequenced by a method known in the art, for example, a dideoxynucleotide chain termination method.

[0155] Moreover, cDNA libraries may be used for obtaining the antibody variable region-encoding genes. First, cDNAs are synthesized with mRNAs extracted from the antibody-producing cells as templates to obtain cDNA libraries. A commercially available kit is conveniently used in the cDNA library synthesis. In actuality, mRNAs from only a small number of cells are obtained in very small amounts. Therefore, direct purification thereof results in low yields. Thus, carrier RNAs shown to be free from the antibody genes are usually added thereto, followed by mRNA purification. Alternatively, when RNAs can be extracted in given amounts from the antibody-producing cells, efficient extraction can be achieved without using carrier RNAs. The addition of the carrier RNAs may be unnecessary for RNA extraction from, for example, 10 or more or 30 or more, preferably 50 or more antibody-producing cells.

[0156] The antibody genes are amplified by PCR with the obtained cDNA libraries as templates. Primers for the PCR amplification of the antibody genes are known in the art. For example, primers for human antibody gene amplification can be designed based on the disclosure of the paper (J. Mol. Biol. (1991) 222, 581-597) or the like. These primers have a nucleotide sequence differing on an immunoglobulin subclass basis. Thus, when cDNA libraries whose subclass is unknown are used as templates, PCR is performed by selecting primers in consideration of every possibility.

[0157] Specifically, for example, for the purpose of obtaining human IgG-encoding genes, primers can be used, which are capable of amplifying each of genes encoding .gamma.1 to .gamma.4 heavy chains and .kappa. and .lamda. light chains. Primers annealing to a portion corresponding to the hinge region are generally used as 3' primers for amplifying IgG variable region genes. On the other hand, primers appropriate for each subclass can be used as 5' primers.

[0158] The PCR products obtained from the primers for gene amplification for these heavy and light chain subclasses are prepared as their respective independent libraries. The libraries thus synthesized can be used to reshape immunoglobulins comprising the heavy and light chains in combination. The antibody of interest can be screened for with the binding activities of the reshaped immunoglobulins for DLL3 as an index.

(2) Introduction of Antibody Gene into Host Cell

[0159] For producing the anti-DLL3 antibody, the cloned antibody genes can be incorporated into expression vectors such that these genes are expressed under the control of expression control regions. The expression control regions for antibody expression encompass, for example, enhancers and promoters. Subsequently, appropriate host cells can be transformed with these expression vectors to obtain recombinant cells expressing the anti-DLL3 antibody-encoding DNA.

[0160] For the antibody gene expression, the antibody heavy chain- and light chain-encoding DNAs can be incorporated separately in different expression vectors. The same host cell can be co-transfected with the heavy chain- and light chain-incorporated vectors and thereby allowed to express antibody molecules comprising the heavy and light chains. Alternatively, the heavy chain- and light chain-encoding DNAs may be incorporated in single expression vectors, with which host cells are transformed (see International Publication No. WO 94/11523).

[0161] Many combinations of hosts and expression vectors are known in the art for introducing the isolated antibody genes into appropriate hosts for antibody preparation. All of these expression systems can be applied to the present invention. When eukaryotic cells are used as the hosts, animal, plant, or fungus cells can be used. Specifically, examples of the animal cells that can be used in the present invention can include the following cells:

i) mammalian cells such as CHO, COS, myeloma, BHK (baby hamster kidney), Hela, Vero, HEK293, Ba/F3, HL-60, Jurkat, and SK-HEP1 cells; ii) amphibian cells such as Xenopus oocytes; and iii) insect cells such as: sf9, sf21, and Tn5 cells.

[0162] For the plant cells, antibody gene expression systems are known in the art, which involve cells derived from the genus Nicotiana (e.g., Nicotiana tabacum). Cultured callus cells can be used in the plant cell transformation.

[0163] Furthermore, the following cells can be used as the fungus cells:

cells derived from yeasts such as the genus Saccharomyces (e.g., Saccharomyces cerevisiae) and filamentous fungi of the genus Pichia (e.g., Pichia pastoris), and cell derived from the genus Aspergillus (e.g., Aspergillus niger).

[0164] Alternatively, antibody gene expression systems using prokaryotic cells are also known in the art. For example, when bacterial cells are used, bacterial cells derived from E. coli, Bacillus subtilis, or the like can be used in the present invention.

[0165] For the gene expression using mammalian cells, a useful promoter routinely used, the antibody gene to be expressed, and a poly A signal located 3'-downstream thereof can be ligated functionally. Examples of the promoter/enhancer can include a human cytomegalovirus immediate early promoter/enhancer.

[0166] In addition, for example, virus promoters/enhancers or mammalian cell-derived promoters/enhancers (e.g., human elongation factor 1.alpha. (HEF1.alpha.)) can be used in the antibody expression. Examples of the viruses whose promoter/enhancer can be used can specifically include retrovirus, polyomavirus, adenovirus, and simian virus 40 (SV40).

[0167] The SV40 promoter/enhancer can be used according to the method of Mulligan et al. (Nature (1979) 277, 108). Moreover, the HEF1.alpha. promoter/enhancer can be used easily in the gene expression of interest by the method of Mizushima et al. (Nucleic Acids Res. (1990) 18, 5322).

[0168] When antibodies are produced using animal cells, the signal sequence of the heavy or light chain gene of the antibody is preferably used as a signal sequence required for extracellular secretion. Moreover, the signal sequence of a secretory protein such as IL-3 or IL-6 may be used.

[0169] For the gene expression using E. coli, a useful promoter routinely used, a signal sequence for antibody secretion, and the antibody gene to be expressed can be ligated functionally. Examples of the promoter can include lacZ and araB promoters. The lacZ promoter can be used according to the method of Ward et al. (Nature (1989) 341, 544-546; and FASEBJ. (1992) 6, 2422-2427). Alternatively, the araB promoter can be used in the gene expression of interest by the method of Better et al. (Science (1988) 240, 1041-1043).

[0170] When antibodies are produced in E. coli periplasm, a pe1B signal sequence (Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379) may be used for antibody secretion. Then, the antibodies produced in the periplasm are separated and then refolded by use of protein denaturants such as urea and guanidine hydrochloride such that the resultant antibodies have the desired binding activity.

[0171] A replication origin derived from SV40, polyomavirus, adenovirus, bovine papillomavirus (BPV), or the like can be inserted in the expression vectors. Furthermore, a selection marker can be inserted in the expression vectors for increasing a gene copy number in the host cell systems. Specifically, selection markers can be used, such as aminoglycoside phosphotransferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, and dihydrofolate reductase (dhfr) gene.

(3) Obtainment of Antibody from Host Cell

[0172] The host cells are transformed with these expression vectors, and the transformed host cells are then cultured in vitro or in vivo to produce the antibody of interest. The culture of the host cells is performed according to a method known in the art. For example, a DMEM, MEM, RPMI1640, or IMDM culture medium can be used and may be used in combination with a solution supplemented with serum such as fetal calf serum (FCS).

[0173] The antibodies thus expressed and produced can be purified by using, alone or in appropriate combination, usual protein purification methods known in the art. For example, affinity or chromatography columns (e.g., protein A columns), filters, ultrafiltration, salting-out, and dialysis can be selected and combined appropriately to separate and purify the antibodies (Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).

[0174] Thus, the present invention provides a gene encoding the antibody of the present invention. The present invention also provides a vector comprising the gene. The present invention further provides a host cell carrying the vector. The present invention further provides a method for producing an antibody encoded by the gene, comprising the step of culturing the host cell.

3. Antibody Production by Transgenic Animal

[0175] In addition to the host cells, transgenic animals can also be used in the recombinant antibody production. Specifically, the antibody of interest can be obtained from animals transfected with the genes encoding this antibody of interest. For example, the antibody genes can be inserted in frame into genes encoding proteins specifically produced in milk to construct fusion genes. For example, goat .beta. casein can be used as the proteins secreted into milk. DNA fragments containing the fusion genes having the antibody gene insert are injected into goat embryos, which are in turn introduced into female goats. From milk produced by transgenic goats (or progeny thereof) brought forth by the goats that have received the embryos, the desired antibody can be obtained as a fusion protein with the milk protein. Moreover, in the transgenic goats, hormone can be used appropriately for increasing the amount of milk containing the desired antibody produced from the transgenic goats (Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).

Pharmaceutical Composition

[0176] Since DLL3 is highly expressed in small-cell lung cancer tissues, the anti-DLL3 antibody has a cancer cell-specific cytotoxic activity. Thus, the anti-DLL3 antibody is useful in the treatment of cancer expressing DLL3.

[0177] Specifically, the present invention provides a pharmaceutical composition comprising an antibody which binds to DLL3 protein as an active ingredient. In an embodiment, the pharmaceutical composition is a cell growth inhibitor, particularly, an anticancer agent. Preferably, the cell growth inhibitor and the anticancer agent of the present invention are administered to a subject having cancer or possibly having cancer.

[0178] The anti-DLL3 antibody used in the pharmaceutical composition (e.g., anticancer agent) of the present invention is not particularly limited, and, for example, any of the anti-DLL3 antibodies described above can be used.

[0179] In the present invention, the phrase "comprising the antibody which binds to DLL3 as an active ingredient" means comprising the anti-DLL3 antibody as a main active ingredient and does not limit the content of the anti-DLL3 antibody.

[0180] The pharmaceutical composition of the present invention may comprise a cytotoxic substance-conjugated anti-DLL3 antibody as an active ingredient. This pharmaceutical composition can be used as, for example, a cell growth inhibitor, particularly, an anticancer agent. Preferably, the cell growth inhibitor and the anticancer agent of the present invention are administered to a subject having cancer or possibly having cancer.

[0181] In the present invention, the phrase "comprising the cytotoxic substance-conjugated anti-DLL3 antibody as an active ingredient" means comprising the cytotoxic substance-conjugated anti-DLL3 antibody as a main active ingredient and does not limit the content of the cytotoxic substance-conjugated anti-DLL3 antibody.

[0182] When the disease targeted by the pharmaceutical composition of the present invention is cancer, the targeted cancer is not particularly limited and is preferably lung cancer, particularly, small-cell lung cancer. The cancer may be any of primary foci and metastatic foci.

[0183] The pharmaceutical composition of the present invention can be administered either orally or parenterally to a patient. Parenteral administration is preferable. Specific examples of such an administration method include injection, transnasal, pulmonary, and transdermal administrations. Examples of the injection administration include intravenous, intramuscular, intraperitoneal, and subcutaneous injections, through which the pharmaceutical composition of the present invention can be administered systemically or locally. Moreover, the administration method can be selected appropriately according to the age or symptoms of the patient. The dose of the pharmaceutical composition of the present invention can be selected from among a dose range of, for example, 0.0001 mg to 1000 mg per kg body weight per dosing. Alternatively, the dose can be selected from among a range of, for example, 0.001 to 100000 mg per body. However, the pharmaceutical composition of the present invention is not limited to these doses.

[0184] The pharmaceutical composition of the present invention can be formulated according to a standard method (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A) and may additionally contain pharmaceutically acceptable carriers or additives. Examples thereof include, but not limited thereto, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffers, suspending agents, tonicity agents, binders, disintegrants, lubricants, flow promoters, and corrigents. Other carriers routinely used can be used appropriately. Specific examples of the carriers can include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl acetal diethylaminoacetate, polyvinyl pyrrolidone, gelatin, middle chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, white sugar, carboxymethylcellulose, corn starch, and inorganic salts.

[0185] Upon contact with DLL3-expressing cells, the anti-DLL3 antibody of the present invention can damage the DLL3-expressing cells or inhibit their growth. Such a method using the anti-DLL3 antibody is also incorporated in the scope of the present invention. The antibody used is not particularly limited, and, for example, any of the antibodies described above can be used. The cells to which the anti-DLL3 antibody binds are not particularly limited as long as the cells express DLL3. In the present invention, the DLL3-expressing cells are preferably cancer cells, more preferably lung cancer cells, particularly preferably small-cell lung cancer cells.

[0186] In the present invention, the "contact" is performed, for example, by adding the antibody to a culture medium of DLL3-expressing cells cultured in vitro. In the present invention, the "contact" is also performed by administering the anti-DLL3 antibody to non-human animals implanted with DLL3-expressing cells in their bodies or to animals endogenously having cancer cells expressing DLL3.

[0187] Methods shown below are preferably used for evaluating or determining cytotoxicity caused in the DLL3-expressing cells by the contact of the anti-DLL3 antibody. Examples of the methods for evaluating or determining the cytotoxic activity in vitro can include the ADCC or CDC activity assay described above. Whether or not the anti-DLL3 antibody has an ADCC activity or has a CDC activity can be determined by a method known in the art (e.g., Current protocols in Immunology, Chapter 7. Immunologic studies in humans, Editor, John E, Coligan et al., John Wiley & Sons, Inc., (1993)). In the activity assay, antibodies that have an isotype identical to that of the anti-DLL3 antibody and do not bind to the cells are used as control antibodies in the same way as in the anti-DLL3 antibody. When the anti-DLL3 antibody exhibits a stronger cytotoxic activity than that of the control antibodies, the anti-DLL3 antibody can be determined to have the activity.

[0188] The isotype of an antibody is defined based on the sequence of the heavy chain constant region in the amino acid sequence of this antibody. The antibody isotype is finally determined depending on class switching caused by genetic recombination on the chromosome during the maturation of antibody-producing B cells in vivo. Difference in isotype reflects difference between the physiological/pathological functions of antibodies. Specifically, it is known that, for example, the strength of the cytotoxic activity is influenced not only by antigen expression levels but by antibody isotypes. Thus, for the cytotoxic activity assay described above, it is preferred that the antibodies used as controls should have an isotype identical to that of the analyte antibody.

[0189] Moreover, for evaluating or determining the cytotoxic activity in vivo, for example, DLL3-expressing cancer cells are intradermally or subcutaneously transplanted to non-human test animals. Then, the analyte antibody is intravenously or intraperitoneally administered thereto on a daily basis or at a few day-intervals from the administration day or the next day. The cytotoxic activity can be determined by measuring tumor sizes over time. Control antibodies having an isotype identical thereto are administered in the same way in the in vitro evaluation. When the anti-DLL3 antibody-administered group has a significantly smaller tumor size than that of the control antibody-administered group, the anti-DLL3 antibody can be determined to have the cytotoxic activity. When mice are used as the non-human test animals, nude (nu/nu) mice can be used preferably, which are genetically deficient in thymus gland and thus lack the functions of T lymphocytes. The use of these mice can exclude the involvement of the endogenous T lymphocytes of the test animals in the evaluation/determination of the cytotoxic activity of administered antibodies.

Diagnostic Drug (Diagnosis Method)

[0190] The present invention also provides a method for diagnosing cancer, comprising detecting the DLL3 protein or a gene encoding the DLL3 protein. DLL3 expression has been confirmed to be remarkably increased in cancer tissues or cancer cell lines. Thus, DLL3 is useful as a marker for specific detection of cancer.

[0191] One specific example of the diagnosis method of the present invention can include a method for diagnosing cancer, comprising the following steps:

(a) providing a sample isolated from a test subject; and (b) detecting the expression level of DLL3 protein or DLL3 gene in the sample.

[0192] The method of the present invention may further comprise the step of

(c) evaluating the possibility that the test subject has cancer, based on the expression level of the DLL3 protein or the DLL3 gene.

Detection of DLL3 Protein or Gene Encoding DLL3 Protein

[0193] In one aspect of the method of the present invention, cancer is diagnosed by detecting the DLL3 protein in a sample. It is preferred that the DLL3 protein detection should be performed using an antibody that recognizes the DLL3 protein.

[0194] In the present invention, the detection encompasses quantitative or qualitative detection. Examples of the qualitative detection can include the following assays:

[0195] assay to simply determine the presence or absence of the DLL3 protein,

[0196] assay to determine the presence or absence of more than a predetermined amount of the DLL3 protein, and

[0197] assay to compare the amount of the DLL3 protein with that contained in another sample (e.g., a control sample).

[0198] On the other hand, examples of the quantitative detection can include measurement of a DLL3 protein concentration and measurement of the amount of the DLL3 protein.

[0199] The test sample according to the present invention is not particularly limited as long as the sample is likely to contain the DLL3 protein. Specifically, samples collected from living bodies such as mammals are preferable. Samples collected from humans are more preferable. Specific examples of the test sample can include blood, interstitial fluid, plasma, extravascular fluid, cerebrospinal fluid, synovial fluid, pleural fluid, serum, lymph, saliva, urine, and tissues. The sample is preferably a sample obtained from the test sample, such as a preparation in which tissues or cells collected from a living body are fixed, or a cell culture medium.

[0200] The cancer diagnosed by the present invention may be any cancer without particular limitations. Specific examples thereof can include lung cancer, particularly, small-cell lung cancer. In the present invention, any of primary foci and metastatic foci of these cancers can be diagnosed.

[0201] In the present invention, when the protein is detected in the test sample, cancer is diagnosed with its level as an index. Specifically, when the amount of the DLL3 protein detected in the test sample is larger than that of a negative control or a healthy individual, the test subject is shown to have cancer or highly possibly have cancer in the future. Specifically, the present invention relates to a method for diagnosing cancer, comprising the following steps:

(1) detecting the expression level of DLL3 in a biological sample collected from a test subject, and (2) comparing the expression level of DLL3 detected in step (1) with that of a control, wherein when the expression level of DLL3 is higher than that of the control, the test subject is determined to have cancer.

[0202] In the present invention, the control refers to a reference sample for comparison and encompasses negative controls and biological samples of healthy individuals. The negative controls can be obtained by collecting biological samples of healthy individuals and mixing them, if necessary. The expression level of DLL3 in the control can be detected in parallel with the detection of the expression level of DLL3 in the biological sample of the test subject. Alternatively, the expression level of DLL3 in a large number of biological samples of healthy individuals can be detected in advance to statistically determine the standard expression level in the healthy individuals. Specifically, for example, mean.+-.2.times.standard deviation (S.D.) or mean.+-.3.times.standard deviation (S.D.) can also be used as the standard value. Statistically, the mean.+-.2.times.standard deviation (S.D.) and the mean.+-.3.times.standard deviation (S.D.) include values of 80% and 90% of the healthy individuals, respectively.

[0203] Alternatively, the expression level of DLL3 in the control can be set using an ROC curve. The ROC curve, or receiver operating characteristic curve, is a graph showing detection sensitivity in the ordinate and false positive rates (i.e., "1-specificity") in the abscissa. In the present invention, the ROC curve can be obtained by plotting changes in sensitivity and false positive rate at a series of varying reference values for determining the expression level of DLL3 in biological samples.

[0204] The "reference value" for obtaining the ROC curve is a numeric value temporarily used for statistical analysis. In general, the "reference value" for obtaining the ROC curve is serially varied within a range which can cover all selectable reference values. For example, the reference value can be varied between the minimal and maximal measured values of DLL3 in a population to be analyzed.

[0205] A standard value that can be expected to offer the desired detection sensitivity and precision can be selected based on the obtained ROC curve. The standard value statistically set based on the ROC curve or the like is also called a cut-off value. In a method for detecting cancer based on the cut-off value, step (2) described above comprises comparing the expression level of DLL3 detected in step (1), with the cut-off value. Then, when the expression level of DLL3 detected in step (1) is higher than the cut-off value, cancer is detected in the test subject.

[0206] In the present invention, the expression level of DLL3 can be determined by an arbitrary method. Specifically, the expression level of DLL3 can be determined by evaluating the amount of DLL3 mRNA, the amount of the DLL3 protein, or the biological activity of the DLL3 protein. The amount of the DLL3 mRNA or protein can be determined by a method as described in the present specification.

[0207] In the present invention, the test subject is particularly preferably a human. When a non-human animal is used as the test subject, the DLL3 protein to be detected is derived from this animal species.

[0208] A method for detecting the DLL3 protein contained in the test sample is not particularly limited and is preferably an immunological detection method using the anti-DLL3 antibody as exemplified below:

enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluoroimmunoassay (FIA), luminescent immunoassay (LIA), immunoprecipitation (IP), turbidimetric immunoassay (TIA), Western blotting (WB), immunohistochemical (IHC) method, single radial immunodiffusion (SRID), dot blot, and slot blot.

[0209] Among these approaches, the immunohistochemical (IHC) method is a preferable immunological assay method for diagnosing cancer, comprising the step of detecting DLL3 proteins in sections in which tissues or cells obtained from a patient having cancer are fixed. The immunological methods described above, such as the immunohistochemical (IHC) method, are generally known by those skilled in the art.

[0210] Since DLL3 is a membrane protein whose expression is enhanced in a cancer cell-specific manner, cancer cells or cancer tissues can be detected using the anti-DLL3 antibody. Cancer cells contained in cells or tissues collected from living bodies are detected by the immunohistological analysis.

[0211] In another preferable aspect, cancer tissues can also be detected in vivo using the anti-DLL3 antibody. This method specifically comprises the steps of: (1) administering, to a test subject, a labeling material (e.g., radioisotope)-labeled antibody which binds to DLL3 protein; and (2) detecting the accumulation of the labeling material. The antibody can be labeled detectably for tracing the antibody administered into the living body. For example, the antibody can be labeled with a fluorescent or luminescent material or a radioisotope, and its in vivo behavior can be traced. The antibody labeled with the fluorescent or luminescent material can be observed using an endoscope or peritoneoscope. The localization of the antibody can be imaged by tracing the radioactivity of the radioisotope. In the present invention, the in vivo localization of the anti-DLL3 antibody represents the presence of cancer cells.

[0212] A positron-emitting nuclide can be used as the radioisotope for labeling the antibody for in vivo cancer detection. For example, the antibody can be labeled with a positron-emitting nuclide such as 18F, 55Co, 64Cu, 66Ga, 68Ga, 76Br, 89Zr, and 1241. A method known in the art (Acta Oncol. 32, 825-830, 1993) can be used in the labeling of the anti-DLL3 antibody with these positron-emitting nuclides.

[0213] The anti-DLL3 antibody labeled with the positron-emitting nuclide is administered to humans or animals. Then, radiation emitted by the radionuclide is measured non-invasively using PET (positron emission tomograph) and converted to images by a computed tomographic approach. The PET apparatus is intended to noninvasively obtain data about in vivo drug behavior or the like. The PET can quantitatively image radiation intensity as signal intensity. By such use of the PET, antigen molecules highly expressed in particular cancer can be detected without collecting samples from patients. The anti-DLL3 antibody may be radiolabeled with a short-life nuclide using a positron-emitting nuclide such as 11C, 13N, 15O, 18F, and 45Ti, in addition to the nuclides described above.

[0214] Research and development have been pursued as to, for example, techniques of producing short-life nuclides using a medical cyclotron and the nuclides described above or producing short-life radiolabeling compounds. The anti-DLL3 antibody can be labeled with various radioisotopes by these techniques. The anti-DLL3 antibody administered to patients accumulates in primary foci and metastatic foci according to the specificity of the anti-DLL3 antibody for pathological tissues at each site. When the anti-DLL3 antibody is labeled with the positron-emitting nuclide, its radioactivity can be detected to detect the presence of the primary foci and the metastatic foci based on the localization of the radioactivity. An active value of gamma radiation or positron emission of 25 to 4000 keV can be used appropriately for the diagnostic use. Moreover, therapeutic effect can also be expected by selecting an appropriate nuclide and administering the selected nuclide in larger amounts. A nuclide that provides a value of gamma radiation or positron emission of 70 to 700 keV can be used for obtaining anticancer effect attributed to radiation.

Detection of Polynucleotide Encoding DLL3 Protein

[0215] In an alternative aspect of the method of the present invention, the expression of the DLL3 polynucleotide is detected. In the present invention, the detected polynucleotide is not particularly limited and is preferably mRNA. In the present invention, the detection encompasses quantitative or qualitative detection. Examples of the qualitative detection can include the following assay procedures:

[0216] assay to simply determine the presence or absence of the DLL3 mRNA,

[0217] assay to determine the presence or absence of more than a predetermined amount of the DLL3 mRNA, and

[0218] assay to compare the amount of the DLL3 mRNA with that contained in another sample (e.g., a control sample).

[0219] On the other hand, examples of the quantitative detection can include measurement of a DLL3 mRNA concentration and measurement of the amount of the DLL3 mRNA.

[0220] In the present invention, an arbitrary sample likely to contain the DLL3 mRNA can be used as the test sample. Samples collected from living bodies such as mammals are preferable. Samples collected from humans are more preferable. Specific examples of the test sample can include blood, interstitial fluid, plasma, extravascular fluid, cerebrospinal fluid, synovial fluid, pleural fluid, serum, lymph, saliva, urine, and tissues. The sample is preferably a sample obtained from the test sample, such as a preparation in which tissues or cells collected from a living body are fixed, or a cell culture medium. These samples are encompassed by the test sample of the present invention.

[0221] In situ hybridization is preferably used for the sample obtained from the test sample, such as a preparation in which tissues or cells collected from a living body are fixed, or a cell culture medium. The in situ hybridization has been evolved as an approach for confirming the presence or absence or distribution of particular DNA or RNA in cells or tissues, and the strength of its expression. This method employs the principles on which a probe nucleic acid having a nucleotide sequence complementary to an intracellular particular nucleic acid sequence has the property of specifically forming a complex. The probe is labeled in advance with a radioisotope (RI), an antigenic substance (hapten), or the like. As a result, the hybridization site can be distinguished through the detection of the label. Thus, the in situ hybridization is used in, for example, the detection of intracellular DNA or RNA, or the like. Preferably, RI can be used for the probe labeling. For example, fluorescence labeling with a nonradioactive substance such as biotin or hapten (e.g., digoxigenin) can be used more preferably. For example, a detection method by fluorescence in situ hybridization called FISH is particularly preferably used.

[0222] The cancer diagnosed in the present invention is not particularly limited. Specific examples thereof can include lung cancer, particularly, small-cell lung cancer. In the present invention, any of primary foci and metastatic foci of these cancers can be diagnosed.

[0223] In the present invention, an arbitrary animal species expressing the DLL3 gene can be used as the test subject. The test subject is particularly preferably a human. When a non-human animal species is used as the test subject, the DLL3 gene to be detected is derived from this animal species.

[0224] Hereinafter, a specific aspect of the detection method will be described. First, a sample is prepared from a test subject. Subsequently, DLL3 mRNA contained in the sample is detected. In the present invention, cDNA synthesized from the mRNA can also be detected. In the present invention, when DLL3 mRNA or DLL3-encoding cDNA is detected in the test sample, the test subject is diagnosed as possibly having cancer. For example, when the amount of the DLL3 mRNA or DLL3-encoding cDNA detected in the test sample is larger than that in negative controls or healthy individuals, the test subject is shown to have cancer or highly possible have cancer in the future.

[0225] A method for detecting the mRNA is known in the art. Specific examples of the method that can be used in the present invention include: nucleic acid hybridization using samples immobilized on a solid phase selected from gene chips, cDNA arrays, and membrane filters; RT-PCR; real-time PCR; subtraction method; differential display method; differential hybridization; and cross hybridization.

[0226] The detection method of the present invention may be automated using various automatic detectors. Such automation achieves detection of a large number of samples in a short time.

Kit for Cancer Diagnosis

[0227] The present invention also provides a diagnostic drug or a kit for cancer diagnosis, comprising a reagent for detecting DLL3 protein in a test sample. The diagnostic drug of the present invention comprises at least the anti-DLL3 antibody.

[0228] The reagent for cancer diagnosis of the present invention can be combined with other factors used in DLL3 detection to prepare a kit for cancer diagnosis. Specifically, the present invention relates to a kit for cancer diagnosis, which comprises: an antibody which binds to DLL3; and a reagent for detecting the binding of the antibody to DLL3 and may further comprise a control sample comprising a biological sample containing DLL3. A manual for instruction of assay procedures may further be included in the kit of the present invention.

EXAMPLES

[0229] Hereinafter, the present invention will be described more specifically with reference to Examples. However, the present invention is not intended to be limited to these Examples.

Example 1

Increased Transcription of DLL3 (Delta-Like 3) in Small-Cell Lung Cancer

[0230] The gene expression distribution of human DLL3 mRNA in clinical cancers, cancer cell lines, and various normal organs was analyzed using Human Exon 1.0 ST Array (Affymetrix, Inc.). In the expression analysis, the total RNAs used were derived from tumor sites in 13 cases of isolated small-cell lung cancer tissues, 3 types of small-cell lung cancer cell lines, and 49 types of normal tissues (purchased from Clontech Laboratories, Inc., Ambion, Inc., Stratagene Corp., Cell Applications, Inc., Panomics, Inc., CHEMICON, and BioChain Institute, Inc.). All of tumor sites in isolated clinical cancer tissues and cancer cell lines (purchased from ATCC) were subjected to total RNA extraction using Trizol (Invitrogen Corp.) according to the protocol included in the product. The experiment of gene expression analysis was conducted using 1 .mu.g of each total RNA thus obtained according to GeneChip Whole Transcript (WT) Sense Target Labeling Assay Manual (Affymetrix, Inc.). Human Exon 1.0 ST Array Data was digitized using ExACT (Exon Array Computational Tool) software provided by Affymetrix, Inc.

[0231] 13 core probe sets for DLL3 were present on Human Exon 1.0 ST Array. The mean of expression values was determined from these probe sets, and the gene expression level was compared among tissues. The expression data obtained from the normal tissues, the tumor sites in isolated small-cell lung cancer tissues, and the small-cell lung cancer cell lines is shown in FIG. 1.

[0232] It was found that human DLL3 gene expression was remarkably increased in the tumor sites in isolated small-cell lung cancer tissues and the small-cell lung cancer cell lines, compared with the normal tissues except for the fetal brain. These results promise the effectiveness of therapy using an antitumor agent molecularly targeting human DLL3, i.e., the possibility of reducing the size of tumor without damaging normal tissues.

Example 2

cDNA Cloning and Preparation of Recombinant Cell

[0233] The human DLL3 cDNA (NM_016941) as set forth in SEQ ID NOs: 1 and 57 was amplified by PCR from a human fetal brain cDNA library and cloned into expression vectors pMCN for mammals. The pMCN vector achieves the expression of a foreign gene under the control of a mouse CMV promoter (GenBank: U68299). The pMCN vector has a Geneticin resistance gene. A CHO cell line DG44 (Invitrogen Corp.) was transformed with the human DLL3 expression vector. Drug-resistant cells were selected in the presence of Geneticin. Cloned cells stably expressing the human DLL3 protein were selected using commercially available anti-DLL3 antibodies (R&D Systems, Inc., MAB4315) to establish human DLL3/DG cells. Likewise, a mouse IL-3-dependent pro B cell line Ba/F3 was transformed the human DLL3 expression vector to establish human DLL3/BaF3 cells.

[0234] The mouse DLL3 cDNA (NM_007866) as set forth in SEQ ID NOs: 2 and 58 was amplified by PCR from a mouse fetal cDNA library and cloned into expression vectors pMCN for mammals. A cell line Ba/F3 was transformed with the mouse DLL3 expression vector. Drug-resistant cells were selected in the presence of Geneticin. Cells expressing the mouse DLL3 protein were selected using commercially available antibodies (R&D Systems, Inc., MAB4315) to establish mouse DLL3/BaF3 cells.

[0235] The human DLL1 cDNA (NM_005618) as set forth in SEQ ID NOs: 3 and 59 was amplified by PCR from a human spleen cDNA library and cloned into expression vectors pMCN for mammals. A cell line Ba/F3 was transformed with the human DLL1 expression vector. Drug-resistant cells were selected in the presence of Geneticin. Cells expressing the human DLL1 protein were selected using commercially available antibodies (R&D Systems, Inc., MAB1818) to establish human DLL1/BaF3 cells.

[0236] The human Notch1 cDNA (NM_017617) as set forth in SEQ ID NOs: 4 and 60 was amplified by PCR from a breast cancer cell line DU4475 cDNA library and cloned into expression vectors pMCN for mammals. A cell line DG44 was transformed with the human Notch1 expression vector. Drug-resistant cells were selected in the presence of Geneticin. Cells expressing the human Notch1 protein were selected using commercially available antibodies (GeneTex Inc., GTX23294) to establish human Notch1/DG44 cells.

[0237] Cell lines producing a soluble DLL3 protein or its N-terminal deletion variant protein were prepared for the purpose of obtaining immunogens for anti-DLL3 antibody obtainment and determining epitopes for the obtained antibodies. The human DLL3 extracellular sequence is composed of a Notch receptor-binding motif DSL domain (Nos. 176-215 in the amino acid sequence) and six EGF-like domains (1: 216-249, 2: 274-310, 3: 312-351, 4: 353-389, 5: 391-427, and 6: 429-465).

[0238] cDNA encoding a chimeric molecule human DLL3-Fc (SEQ ID NO: 5) consisting of a human DLL3 extracellular region (27-492 in the amino acid sequence of SEQ ID NO: 1) and mouse IgG2a antibody constant region sequences was prepared and cloned into expression vectors pMCN for mammals. A cell line DG44 was transformed with the human DLL3-Fc expression vector. Drug-resistant cells were selected in the presence of Geneticin. Clones highly expressing the human DLL3-Fc protein were selected by ELISA using anti-mouse antibodies to establish human DLL3-Fc-producing DG44 cells.

[0239] cDNA encoding a chimeric molecule human DLL3delta1-Fc (SEQ ID NO: 6) consisting of a partial sequence of the human DLL3 extracellular region (176-492 in the amino acid sequence of SEQ ID NO: 1) and mouse IgG2a antibody constant region sequences was prepared and cloned into expression vectors pMCN for mammals. A cell line DG44 was transformed with the human DLL3delta1-Fc expression vector. Drug-resistant cells were selected in the presence of Geneticin. Clones highly expressing the human DLL3delta1delta1-Fc protein were selected by ELISA using anti-mouse antibodies to establish human DLL3delta1-Fc-producing DG44 cells.

[0240] cDNA encoding a chimeric molecule human DLL3delta2-Fc (SEQ ID NO: 7) consisting of a partial sequence of the human DLL3 extracellular region (216-492 in the amino acid sequence of SEQ ID NO: 1) and mouse IgG2a antibody constant region sequences was prepared and cloned into expression vectors pMCN for mammals. A cell line DG44 was transformed with the human DLL3delta2-Fc expression vector. Drug-resistant cells were selected in the presence of Geneticin. Clones highly expressing the human DLL3delta1delta2-Fc protein were selected by ELISA using anti-mouse antibodies to establish human DLL3delta2-Fc-producing DG44 cells.

[0241] cDNA encoding a chimeric molecule mouse DLL3-Fc (SEQ ID NO: 8) consisting of a mouse DLL3 extracellular region (33-490 in the amino acid sequence of SEQ ID NO: 2) and mouse IgG2a antibody constant region sequences was prepared and cloned into expression vectors pMCN for mammals. A cell line DG44 was transformed with the mouse DLL3-Fc expression vector. Drug-resistant cells were selected in the presence of Geneticin. Clones highly expressing the mouse DLL3-Fc protein were selected by ELISA using an anti-mouse antibody to establish mouse DLL3-Fc-producing DG44 cells.

[0242] Each protein of interest was purified from the culture supernatant of the Fc fusion protein-producing cell line by Protein G affinity column chromatography and gel filtration chromatography. The concentration of the purified protein was determined by DC protein assay (Bio-Rad Laboratories, Inc.) with the IgG known for its concentration as a standard.

Example 3

Obtainment of Anti-DLL3 Antibody and Analysis of Epitope and Antibody Internalization

[0243] Six- to seven-week-old Balb/c (Charles River Laboratories Japan, Inc.) and MRL/MpJJmsSlc-1pr/1pr (Japan SLC, Inc.) mice were immunized. For the initial challenge, by prepared into an emulsion using a Freund's complete adjuvant (Becton, Dickinson and Company), the antigenic proteins were subcutaneously administered thereto at a dose of 0.1 mg human DLL3-Fc/head. Two weeks later, an antigen emulsion prepared using a Freund's incomplete adjuvant was subcutaneously administered thereto at a dose of 0.05 mg/head a total of 3 to 6 times on a once-a-week basis. 0.05 mg of the antigenic proteins was intravenously administered to each mouse individual confirmed to have a rise in antibody titer in its serum. Three days later, their spleen cells were extracted and mixed with mouse myeloma cells P3-X63Ag8U1 (ATCC) at a cell count ratio of approximately 3:1. These cells were fused by the polyethylene glycol (PEG) method. After removal of PEG by centrifugation, the cells were suspended in an RPMI1640 medium containing 1.times.HAT media supplement (Sigma-Aldrich Corp.), 0.5.times.BM-Condimed H1 Hybridoma cloning supplement (Roche Diagnostics GmbH), and 10% fetal bovine serum to adjust the cell concentration. Then, the cells were seeded to each well of a 96-well plate. Hybridoma colony formation was confirmed, and the presence or absence of anti-DLL3 antibodies contained in the culture supernatant was then analyzed by ELISA using a plate coated with human DLL3-Fc. Hybridoma cells contained in positive wells were cloned by the limiting dilution method to establish hybridoma lines producing the anti-DLL3 antibodies. The monoclonal antibodies were isotyped using IsoStrip (Roche Diagnostics GmbH).

[0244] Each IgG monoclonal antibody was purified from the culture supernatants of the established hybridomas by Protein G affinity column chromatography and desalting treatment. The concentration of the purified antibody was determined by DC protein assay.

[0245] Human DLL3-Fc, mouse DLL3-Fc, human DLL3delta1-Fc, and human DLL3delta2-Fc were separately immobilized on a Nunc immunoplate (439454). Subsequently, the unreacted surface of the plate was blocked with a solution containing bovine serum albumin. After washing, each diluted antibody solution adjusted to an appropriate concentration was added thereto and incubated at room temperature for 1 hour. The reaction solution was removed from the plate. After washing with TBS containing Tween 20, alkaline phosphatase-labeled anti-mouse IgK antibodies were added to the plate and incubated for 1 hour. After washing of the plate, an alkaline phosphatase substrate Sigma 104 was added thereto and incubated at room temperature. After the incubation, the absorbance was measured at a wavelength of 405 nm and a reference wavelength of 655 nm. The results are shown in FIG. 2. All the purified antibodies bound to human DLL3-Fc in a dose-dependent manner. The monoclonal antibodies DL301, DL302, DL306, and DL312 and the commercially available antibody MAB4315 bound to mouse DLL3-Fc. The commercially available antibody MAB4315 bound to human DLL3delta1-Fc, but did not bind to human DLL3delta2-Fc, demonstrating that its epitope was located in the DSL domain. The DL303, DL304, DL307, and DL311 antibodies bound to neither human DLL3delta1-Fc nor human DLL3delta2-Fc. Thus, predicted epitopes for these antibodies are located between residues 27 and 175 in the amino acid sequence of SEQ ID NO: 1. DL301, DL302, DL305, DL306, DL308, DL309, and DL312 bound to both human DLL3delta1-Fc and human DLL3delta2-Fc, demonstrating that epitopes for these antibodies were located in residues 216-492 in the amino acid sequence of SEQ ID NO: 1. The schematic structure of the full-length DLL3 protein and the soluble DLL3-Fc fusion protein and a site recognized by each anti-DLL3 antibody are shown in FIG. 9.

[0246] The binding of each monoclonal antibody to human DLL3 expressed on a cell membrane and the behavior of a human DLL3-monoclonal antibody complex on the cell membrane were analyzed by flow cytometry. Human DLL3/BaF3 cells were suspended in a FACS buffer (PBS containing 1% fetal bovine serum and 0.05% sodium azide). The cell suspension of 1.times.10.sup.6 cells/ml was reacted with the monoclonal antibody (final concentration: 5 .mu.g/ml) at 4.degree. C. for 30 minutes. After centrifugation and supernatant removal, the cells were washed once with a FACS buffer. FITC-labeled anti-mouse IgG (H+L) antibodies (Beckman Coulter, Inc.) were added thereto and incubated at 4.degree. C. for 30 minutes. Unreacted FITC antibodies were removed by centrifugation. Then, the cells were resuspended and analyzed using a flow cytometer FACSCalibur (Becton, Dickinson and Company). For the purpose of analyzing the movement or disappearance of the DLL3-antibody complex from the cell membrane, human DLL3/BaF3 cells were suspended in an RPMI1640 culture medium containing 10% fetal bovine serum (FBS) and mouse IL3. The cell suspension of 1.times.10.sup.6 cells/ml was mixed with the monoclonal antibody (final concentration: 5 .mu.g/ml) and reacted at 37.degree. C. for 1 hour or 4 hours in a CO2 incubator. After the reaction, the amount of the antibody bound onto the cell membrane was analyzed by flow cytometry in the same way as above. The geometric mean of fluorescence intensity values (X Geo Mean) in a cell histogram plot was determined using the analytical software CELLQuest Pro included in FACSCalibur. All the isolated anti-DLL3 monoclonal antibodies bound to DLL3 on the cells (FIG. 3(a)). The reaction of the antibody with the cells at 4.degree. C. inhibits membrane fluidity such as the cellular uptake of the DLL3-antibody complex from the cell membrane. The reaction of the antibody with the cells at 37.degree. C. may cause the cellular uptake of the DLL3-antibody complex and shading (release from the cell membrane) resulting from protease digestion or the like. The amount of each monoclonal antibody bound onto the cell membrane as a result of incubation at 37.degree. C. for 1 hour or 4 hours is shown in FIG. 3(b) as a relative value compared with that at 4.degree. C. As a result of incubation at 37.degree. C., the amount of the antibody DL303, DL304, DL305, DL308, DL309, or MAB4315 on the surface of the cell membrane was decreased. These results suggest the internalization or shading of the DLL3-antibody complex. By contrast, as a result of incubation at 37.degree. C., the amount of the antibody DL301, DL306, DL307, DL311, or DL312 on the surface of the cell membrane was hardly changed or was increased, on the contrary. The latter result demonstrated that these antibodies were able to stably reside in the form of a DLL3 complex on the cell membrane.

[0247] The number of the DLL3 protein on cell surface was determined using a QIFIKIT (DAKO, F0479) for the quantitative determination of cell surface antigen by flow cytometry. The analysis was conducted with the anti-DLL3 antibody DL303 (final concentration: 5 .mu.g/ml) as a primary antibody according to the protocol included therein. The numbers of the antigen on the surfaces of the human DLL3/BaF3, NCI-H1184 (ATCC), NCI-H1436 (ATCC), and Y79 (Riken Cell Bank) cells were approximately 9000, 7000, 6000, and 3000, respectively.

Example 4

Induction of ADCC Activity by Anti-DLL3 Antibody and Growth Inhibition Mediated by Antibody-Toxin Complex

[0248] Each anti-DLL3 antibody was examined for its antibody-dependent cell-mediated cytotoxicity (ADCC)-inducing activity against DLL3-expressing cells labeled with calcein. DLL3-expressing human DLL3/BaF3 and small-cell lung cancer NCI-H1184 (ATCC) cell lines were separately cultured for 90 minutes in the presence of 20 .mu.g/ml Calcein-AM (Dojindo, 349-07201), then centrifuged, and washed to prepare calcein-labeled target cells. The target cells were seeded at 1.times.10.sup.4 cells/well to a 96-well plate (Coster 3799). Subsequently, the antibody adjusted to an appropriate final concentration was added thereto and incubated at room temperature for 15 minutes. Effector cells were added thereto at 5.times.10.sup.4 cells/well. The reaction plate was incubated at 37.degree. C. in a CO2 incubator. The effector cells used were NK92 cells expressing a mouse Fc.gamma.R3-human Fc.gamma.R3 chimeric molecule (WO 2008/093688). After 4-hour incubation, the plate was centrifuged, and 100 .mu.l of the culture supernatant was collected from each well. The fluorescence intensity was measured using ARVO SX (Wallac).

[0249] The ADCC-inducing activity was calculated according to the following formula:

ADCC [%]=(A-C)/(B-C).times.100, wherein

A represents fluorescence intensity in each well; B represents the mean of fluorescence intensity in the supernatant of cells lysed with Nonidet P-40 with the final concentration of 1%; and C represents the mean of fluorescence intensity in a well supplemented with only a medium. The mean and standard deviation were calculated from three measurements under each experimental condition. FIG. 4 shows the ADCC-inducing activity of the antibody added at the final concentration of 2.5 .mu.g/ml against the DLL3/BaF3 cells. No ADCC activity was confirmed control antibodies IgG1 and IgG2b, whereas the ADCC-inducing activity was confirmed in DL301, DL306, and DL312. No distinct ADCC-inducing activity was confirmed in the commercially available monoclonal antibody MAB4315. FIG. 5 shows that the DL301, DL306, and D312 antibodies induce ADCC against DLL3/BaF3 and NCI-H1184 in a dose-dependent manner.

[0250] Each anti-DLL3 monoclonal antibody was evaluated for its cellular uptake using a toxin-labeled anti-mouse secondary antibody Mab-ZAP (Advanced Targeting Systems). DLL3/BaF3 cells were seeded at 5.times.10.sup.3 cells/well to a 96-well plate. Subsequently, the anti-DLL3 mouse monoclonal antibody and Mab-ZAP (final concentration: 1 .mu.g/ml) were added thereto and incubated at 37.degree. C. in a CO2 incubator. Four days later, Cell Count Reagent SF (Nacalai Tesque, Inc.) was added thereto. The absorbance was measured at 450 nm and at a control wavelength of 620 nm using a microplate reader to determine cell growth (FIG. 6). The addition of the anti-DLL3 monoclonal antibody and Mab-ZAP to the cells inhibited cell growth. The DL301, DL306, and DL312 antibodies confirmed to have an ADCC-inducing activity remained in large amounts on DLL3-expressing cells after being mixed with the cells and cultured at 37.degree. C., and exhibited low growth inhibitory effect brought about by the cellular uptake of the Mab-ZAP complex.

Example 5

Cloning of Antibody Variable Region and Preparation of Recombinant Antibody

[0251] A Smart 5'-RACE cDNA library (Clontech Laboratories, Inc.) was prepared from the total RNAs of hybridomas producing each anti-DLL3 antibody. The total RNA preparation was performed using RNeasy Mini column (Qiagen). The cDNA library preparation followed the instruction of the manufacturer. Sequences encoding antibody variable regions (VH and VL) were amplified by PCR using primers complementary to sequences encoding antibody constant regions. The fragments thus amplified by PCR were cloned into pCR2.1TOPO and sequenced. Chimeric antibody expression vectors containing the obtained VH- and VL-encoding sequences and human IgG1 constant region-encoding sequences were constructed. The light chain- and heavy chain-encoding sequences were both incorporated in one expression vector for mammalian cells and transcribed under the control of a mouse CMV promoter. Table 1 shows SEQ ID NOs of the identified antibody variable region sequences and their CDR sequences, full-length mouse antibody sequences, and chimeric antibody sequences.

TABLE-US-00002 TABLE 1 CDR1 CDR2 CDR3 DL301 Heavy chain NYLIE VMNPGSGGTHYSEKFRG SDYDYVTYAMDY (SEQ ID NO: 12) (SEQ ID NO: 13) (SEQ ID NO: 14) Light chain KASQDINSYLI RTNRLVD LQYDEFPFT (SEQ ID NO: 18) (SEQ ID NO: 19) (SEQ ID NO: 20) DL306 Heavy chain DYYMK DINPNNGDTFYNQKFKG DGNYAYFDY (SEQ ID NO: 24) (SEQ ID NO: 25) (SEQ ID NO: 26) Light chain RASKSVSTSGYSYMH LASNLES QHSRHLPWT (SEQ ID NO: 30) (SEQ ID NO: 31) (SEQ ID NO: 32) DL309 Heavy chain NYYIE EILPGSGSTTYNEKFKG WGAREPGFPY (SEQ ID NO: 36) (SEQ ID NO: 37) (SEQ ID NO: 38) Light chain KASQNVGTNVA SASYRYS QQYNNYPLT (SEQ ID NO: 42) (SEQ ID NO: 43) (SEQ ID NO: 44) DL312 Heavy chain DYYMN LIRNKANGYTTEYNASVKG DSDGYYEYYFDY (SEQ IDNO: 48) (SEQ ID NO: 49) (SEQ ID NO: 50) Light chain RASQEISDYLS AASTLDS LQYASYPYT (SEQ ID NO: 54) (SEQ ID NO: 55) (SEQ ID NO: 56) Variable region Mouse antibody Chimeric antibody sequence sequence sequence DL301 Heavy chain (SEQ ID NO: 9) (SEQ ID NO: 10) (SEQ ID NO: 11) Light chain (SEQ ID NO: 15) (SEQ ID NO: 16) (SEQ ID NO: 17) DL306 Heavy chain (SEQ ID NO: 21) (SEQ ID NO: 22) (SEQ ID NO: 23) Light chain (SEQ ID NO: 27) (SEQ ID NO: 28) (SEQ ID NO: 29) DL309 Heavy chain (SEQ ID NO: 33) (SEQ ID NO: 34) (SEQ ID NO: 35) Light chain (SEQ ID NO: 39) (SEQ ID NO: 40) (SEQ ID NO: 41) DL312 Heavy chain (SEQ ID NO: 45) (SEQ ID NO: 46) (SEQ ID NO: 47) Light chain (SEQ ID NO: 51) (SEQ ID NO: 52) (SEQ ID NO: 53)

[0252] COS-7 cells were transfected with each chimeric antibody expression vector and allowed to transiently express the chimeric antibody. The chimeric antibody in the culture supernatant of COS-7 was confirmed to bind to the human DLL3 protein by flow cytometry and ELISA. The chimeric antibody concentration in the culture supernatant of COS-7 was calculated by sandwich ELISA. In this concentration calculation, a human chimeric antibody known for its concentration was used as a standard. The chimeric antibody (final concentration: 1 .mu.g/ml) was added to human DLL3/BaF3 cells suspended in a FACS buffer (PBS containing 1% fetal bovine serum). After incubation at 4.degree. C. for 30 minutes and washing, the cells were reacted with FITC-labeled anti-human antibodies (Beckman Coulter, Inc.), and the binding of the chimeric antibody was analyzed using a flow cytometer FACSCalibur. As shown in FIG. 7, the chimeric antibodies DL301, DL309, and DL312 bound to human DLL3/BaF3. None of the chimeric antibodies bound to Ba/F3 cells, which were a parental line of the forced expressing cells. Human DLL3-Fc was immobilized on a Nunc immunoplate, and the binding of each chimeric antibody to the immobilized protein was analyzed. For detecting antigen-bound chimeric antibodies, the chimeric antibody was added thereto, and alkaline phosphatase-labeled anti-human antibodies (Biosource, AHI0305) and an alkaline phosphatase substrate Sigma 104 were then added in this order. The absorbance was determined. The DL301, DL309, and DL312 chimeric antibodies bound to human DLL3-Fc in a dose-dependent manner (FIG. 8(a)).

Example 6

Grouping of Epitope by Competitive ELISA

[0253] The competition of a chimeric antibody and a mouse antibody for binding to the antigen molecule was analyzed by ELISA (FIG. 8(b)). Human DLL3-Fc was added at 10 ng/well to a Nunc immunoplate and immobilized thereon. A mixture (final concentration: 50 ng/ml) of the chimeric antibody and the appropriately diluted mouse antibody was added to the DLL3-Fc protein-immobilized plate. The plate was incubated at room temperature for 1 hour and then washed. Alkaline phosphatase-labeled anti-human antibodies were added thereto and incubated. After the incubation, Sigma 104 was added thereto, and the absorbance was measured. The binding of the chimeric antibody to the antigen molecule competed with that of the original mouse monoclonal antibody thereto. The antibodies other than the DL301 mouse antibody did not inhibit the binding of the DL301 chimeric antibody to the antigen. The antibodies other than the DL312 mouse antibody did not inhibit the binding of the DL312 chimeric antibody to the antigen. These results demonstrated that DL301 and DL312 bound to their respective unique epitopes. The DL305, DL308, and DL309 mouse antibodies inhibited the binding of the DL309 chimeric antibody to the antigen at almost the same level, suggesting that an epitope was the same among these 3 antibodies. The DL306 mouse antibody confirmed to have an ADCC-inducing activity did not inhibit the binding of the DL301, DL309, or DL312 chimeric antibody to the antigen. These results demonstrated that DL306 recognized an epitope independent of that for DL301, DL309, or DL312.

Example 7

Establishment of Cell Line Expressing Recombinant Anti-DLL3 Antibody and Purification of Recombinant Antibody

[0254] COS-7 cells were transfected with the DL306 chimeric antibody expression vector and allowed to transiently express the chimeric antibody. The chimeric antibody in the culture supernatant of COS-7 was confirmed to bind to the human DLL3 protein by flow cytometry. The chimeric antibody concentration in the culture supernatant of COS-7 was calculated by sandwich ELISA. In this concentration calculation, a human chimeric antibody known for its concentration was used as a standard. The chimeric antibody (final concentration: 1 .mu.g/ml) was added to human DLL3/BaF3 cells suspended in a FACS buffer (PBS containing 1% fetal bovine serum). After incubation at 4.degree. C. for 30 minutes and washing, the cells were reacted with FITC-labeled anti-human antibodies (Beckman Coulter, Inc.), and the binding of the chimeric antibody was analyzed using a flow cytometer FACSCalibur. As shown in FIG. 10, the chimeric antibody DL306 bound to human DLL3/BaF3 and did not bind to Ba/F3 cells, which were a parental line.

[0255] A cell line DG44 was transformed with the DL301, DL306, DL309, or DL312 human chimeric antibody expression vector. Drug-resistant cells were selected in the presence of Geneticin. Clones highly expressing the human chimeric antibody were selected by ELISA using anti-human antibodies to establish human chimeric antibody-producing DG44 cells.

[0256] Each protein of interest was purified from the culture supernatant of the human chimeric antibody-producing cell line by rProtein A affinity column chromatography and gel filtration chromatography. The concentration of the purified protein was determined by DC protein assay (Bio-Rad Laboratories, Inc.) with the IgG known for its concentration as a standard.

[0257] Mouse IgG2a antibody expression vector containing the VH- and VL-encoding sequences of DL301, DL306, or DL312 and mouse IgG2a constant region-encoding sequences were constructed. The light chain- and heavy chain-encoding sequences were both incorporated in one expression vector for mammalian cells and transcribed under the control of a mouse CMV promoter. A CHO cell line deficient in fucose transporter (WO2005/017155) was transformed with the mouse IgG2a antibody expression vector. Drug-resistant cells were selected in the presence of Geneticin. Clones highly expressing the mouse IgG2a antibody were selected by ELISA using anti-mouse IgG2a antibodies to establish mouse low-fucose IgG2a antibody-producing CHO cells. Each protein of interest was purified from the culture supernatant of the mouse low-fucose IgG2a antibody-producing cell line by Protein G affinity column chromatography and gel filtration chromatography. The concentration of the purified protein was determined by DC protein assay with the IgG known for its concentration as a standard.

TABLE-US-00003 TABLE 2 DL301 Heavy chain (SEQ ID NO: 69) Light chain (SEQ ID NO: 16) DL306 Heavy chain (SEQ ID NO: 70) Light chain (SEQ ID NO: 28) DL312 Heavy chain (SEQ ID NO: 71) Light chain (SEQ ID NO: 52)

Example 8

Induction of ADCC Activity by Recombinant Anti-DLL3 Antibody

[0258] Human chimeric and mouse IgG2a recombinant anti-DLL3 antibodies were examined for their antibody-dependent cell-mediated cytotoxicity (ADCC)-inducing activities against DLL3-expressing cells labeled with calcein. DLL3-expressing human DLL3/BaF3 and small-cell lung cancer NCI-H1184 (ATCC) cell lines were separately cultured for 90 minutes in the presence of 20 .mu.g/ml Calcein-AM (Dojindo, 349-07201), then centrifuged, and washed to prepare calcein-labeled target cells. The target cells were seeded at 1.times.10.sup.4 cells/well to a 96-well plate (Coster 3799). Subsequently, the antibody adjusted to an appropriate final concentration was added thereto and incubated at room temperature for 15 minutes. Effector cells were added thereto at 5.times.10.sup.4 cells/well. The reaction plate was incubated at 37.degree. C. in a CO2 incubator. The effector cells used for the recombinant mouse low-fucose IgG2a antibody were NK92 cells expressing a mouse Fc.gamma.R4-human Fc.gamma.R3 chimeric molecule (WO 2008/093688). The effector cells used for the human chimeric recombinant antibody were NK92 cells expressing a human Fc.gamma.R3 molecule. After 4-hour incubation, the plate was centrifuged, and 100 .mu.l of the culture supernatant was collected from each well. The fluorescence intensity was measured using ARVO SX. The ADCC-inducing activity was calculated by the method described in Example 4.

[0259] FIG. 11 shows that the recombinant human chimeric antibodies DL301, DL306, DL309, and D312 induce ADCC against DLL3/BaF3 and NCI-H1184 in a dose-dependent manner.

[0260] FIG. 12 shows that the recombinant mouse low-fucose IgG2a antibodies DL301, DL306, and D312 induce ADCC against DLL3/BaF3 and NCI-H1184 in a dose-dependent manner.

[0261] All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Sequence CWU 1

1

711618PRThomo sapiens 1Met Val Ser Pro Arg Met Ser Gly Leu Leu Ser Gln Thr Val Ile Leu 1 5 10 15 Ala Leu Ile Phe Leu Pro Gln Thr Arg Pro Ala Gly Val Phe Glu Leu 20 25 30 Gln Ile His Ser Phe Gly Pro Gly Pro Gly Pro Gly Ala Pro Arg Ser 35 40 45 Pro Cys Ser Ala Arg Leu Pro Cys Arg Leu Phe Phe Arg Val Cys Leu 50 55 60 Lys Pro Gly Leu Ser Glu Glu Ala Ala Glu Ser Pro Cys Ala Leu Gly 65 70 75 80 Ala Ala Leu Ser Ala Arg Gly Pro Val Tyr Thr Glu Gln Pro Gly Ala 85 90 95 Pro Ala Pro Asp Leu Pro Leu Pro Asp Gly Leu Leu Gln Val Pro Phe 100 105 110 Arg Asp Ala Trp Pro Gly Thr Phe Ser Phe Ile Ile Glu Thr Trp Arg 115 120 125 Glu Glu Leu Gly Asp Gln Ile Gly Gly Pro Ala Trp Ser Leu Leu Ala 130 135 140 Arg Val Ala Gly Arg Arg Arg Leu Ala Ala Gly Gly Pro Trp Ala Arg 145 150 155 160 Asp Ile Gln Arg Ala Gly Ala Trp Glu Leu Arg Phe Ser Tyr Arg Ala 165 170 175 Arg Cys Glu Pro Pro Ala Val Gly Thr Ala Cys Thr Arg Leu Cys Arg 180 185 190 Pro Arg Ser Ala Pro Ser Arg Cys Gly Pro Gly Leu Arg Pro Cys Ala 195 200 205 Pro Leu Glu Asp Glu Cys Glu Ala Pro Leu Val Cys Arg Ala Gly Cys 210 215 220 Ser Pro Glu His Gly Phe Cys Glu Gln Pro Gly Glu Cys Arg Cys Leu 225 230 235 240 Glu Gly Trp Thr Gly Pro Leu Cys Thr Val Pro Val Ser Thr Ser Ser 245 250 255 Cys Leu Ser Pro Arg Gly Pro Ser Ser Ala Thr Thr Gly Cys Leu Val 260 265 270 Pro Gly Pro Gly Pro Cys Asp Gly Asn Pro Cys Ala Asn Gly Gly Ser 275 280 285 Cys Ser Glu Thr Pro Arg Ser Phe Glu Cys Thr Cys Pro Arg Gly Phe 290 295 300 Tyr Gly Leu Arg Cys Glu Val Ser Gly Val Thr Cys Ala Asp Gly Pro 305 310 315 320 Cys Phe Asn Gly Gly Leu Cys Val Gly Gly Ala Asp Pro Asp Ser Ala 325 330 335 Tyr Ile Cys His Cys Pro Pro Gly Phe Gln Gly Ser Asn Cys Glu Lys 340 345 350 Arg Val Asp Arg Cys Ser Leu Gln Pro Cys Arg Asn Gly Gly Leu Cys 355 360 365 Leu Asp Leu Gly His Ala Leu Arg Cys Arg Cys Arg Ala Gly Phe Ala 370 375 380 Gly Pro Arg Cys Glu His Asp Leu Asp Asp Cys Ala Gly Arg Ala Cys 385 390 395 400 Ala Asn Gly Gly Thr Cys Val Glu Gly Gly Gly Ala His Arg Cys Ser 405 410 415 Cys Ala Leu Gly Phe Gly Gly Arg Asp Cys Arg Glu Arg Ala Asp Pro 420 425 430 Cys Ala Ala Arg Pro Cys Ala His Gly Gly Arg Cys Tyr Ala His Phe 435 440 445 Ser Gly Leu Val Cys Ala Cys Ala Pro Gly Tyr Met Gly Ala Arg Cys 450 455 460 Glu Phe Pro Val His Pro Asp Gly Ala Ser Ala Leu Pro Ala Ala Pro 465 470 475 480 Pro Gly Leu Arg Pro Gly Asp Pro Gln Arg Tyr Leu Leu Pro Pro Ala 485 490 495 Leu Gly Leu Leu Val Ala Ala Gly Val Ala Gly Ala Ala Leu Leu Leu 500 505 510 Val His Val Arg Arg Arg Gly His Ser Gln Asp Ala Gly Ser Arg Leu 515 520 525 Leu Ala Gly Thr Pro Glu Pro Ser Val His Ala Leu Pro Asp Ala Leu 530 535 540 Asn Asn Leu Arg Thr Gln Glu Gly Ser Gly Asp Gly Pro Ser Ser Ser 545 550 555 560 Val Asp Trp Asn Arg Pro Glu Asp Val Asp Pro Gln Gly Ile Tyr Val 565 570 575 Ile Ser Ala Pro Ser Ile Tyr Ala Arg Glu Val Ala Thr Pro Leu Phe 580 585 590 Pro Pro Leu His Thr Gly Arg Ala Gly Gln Arg Gln His Leu Leu Phe 595 600 605 Pro Tyr Pro Ser Ser Ile Leu Ser Val Lys 610 615 2585PRTMus musculus 2Met Val Ser Leu Gln Val Ser Pro Leu Ser Gln Thr Leu Ile Leu Ala 1 5 10 15 Phe Leu Leu Pro Gln Ala Leu Pro Ala Gly Val Phe Glu Leu Gln Ile 20 25 30 His Ser Phe Gly Pro Gly Pro Gly Leu Gly Thr Pro Arg Ser Pro Cys 35 40 45 Asn Ala Arg Gly Pro Cys Arg Leu Phe Phe Arg Val Cys Leu Lys Pro 50 55 60 Gly Val Ser Gln Glu Ala Thr Glu Ser Leu Cys Ala Leu Gly Ala Ala 65 70 75 80 Leu Ser Thr Ser Val Pro Val Tyr Thr Glu His Pro Gly Glu Ser Ala 85 90 95 Ala Ala Leu Pro Leu Pro Asp Gly Leu Val Arg Val Pro Phe Arg Asp 100 105 110 Ala Trp Pro Gly Thr Phe Ser Leu Val Ile Glu Thr Trp Arg Glu Gln 115 120 125 Leu Gly Glu His Ala Gly Gly Pro Ala Trp Asn Leu Leu Ala Arg Val 130 135 140 Val Gly Arg Arg Arg Leu Ala Ala Gly Gly Pro Trp Ala Arg Asp Val 145 150 155 160 Gln Arg Thr Gly Thr Trp Glu Leu His Phe Ser Tyr Arg Ala Arg Cys 165 170 175 Glu Pro Pro Ala Val Gly Ala Ala Cys Ala Arg Leu Cys Arg Ser Arg 180 185 190 Ser Ala Pro Ser Arg Cys Gly Pro Gly Leu Arg Pro Cys Thr Pro Phe 195 200 205 Pro Asp Glu Cys Glu Ala Pro Ser Val Cys Arg Pro Gly Cys Ser Pro 210 215 220 Glu His Gly Tyr Cys Glu Glu Pro Asp Glu Cys Arg Cys Leu Glu Gly 225 230 235 240 Trp Thr Gly Pro Leu Cys Thr Val Pro Val Ser Thr Ser Ser Cys Leu 245 250 255 Asn Ser Arg Val Pro Gly Pro Ala Ser Thr Gly Cys Leu Leu Pro Gly 260 265 270 Pro Gly Pro Cys Asp Gly Asn Pro Cys Ala Asn Gly Gly Ser Cys Ser 275 280 285 Glu Thr Ser Gly Ser Phe Glu Cys Ala Cys Pro Arg Gly Phe Tyr Gly 290 295 300 Leu Arg Cys Glu Val Ser Gly Val Thr Cys Ala Asp Gly Pro Cys Phe 305 310 315 320 Asn Gly Gly Leu Cys Val Gly Gly Glu Asp Pro Asp Ser Ala Tyr Val 325 330 335 Cys His Cys Pro Pro Gly Phe Gln Gly Ser Asn Cys Glu Lys Arg Val 340 345 350 Asp Arg Cys Ser Leu Gln Pro Cys Gln Asn Gly Gly Leu Cys Leu Asp 355 360 365 Leu Gly His Ala Leu Arg Cys Arg Cys Arg Ala Gly Phe Ala Gly Pro 370 375 380 Arg Cys Glu His Asp Leu Asp Asp Cys Ala Gly Arg Ala Cys Ala Asn 385 390 395 400 Gly Gly Thr Cys Val Glu Gly Gly Gly Ser Arg Arg Cys Ser Cys Ala 405 410 415 Leu Gly Phe Gly Gly Arg Asp Cys Arg Glu Arg Ala Asp Pro Cys Ala 420 425 430 Ser Arg Pro Cys Ala His Gly Gly Arg Cys Tyr Ala His Phe Ser Gly 435 440 445 Leu Val Cys Ala Cys Ala Pro Gly Tyr Met Gly Val Arg Cys Glu Phe 450 455 460 Ala Val Arg Pro Asp Gly Ala Asp Ala Val Pro Ala Ala Pro Arg Gly 465 470 475 480 Leu Arg Gln Ala Asp Pro Gln Arg Phe Leu Leu Pro Pro Ala Leu Gly 485 490 495 Leu Leu Val Ala Ala Gly Leu Ala Gly Ala Ala Leu Leu Val Ile His 500 505 510 Val Arg Arg Arg Gly Pro Gly Gln Asp Thr Gly Thr Arg Leu Leu Ser 515 520 525 Gly Thr Arg Glu Pro Ser Val His Thr Leu Pro Asp Ala Leu Asn Asn 530 535 540 Leu Arg Leu Gln Asp Gly Ala Gly Asp Gly Pro Ser Ser Ser Ala Asp 545 550 555 560 Trp Asn His Pro Glu Asp Gly Asp Ser Arg Ser Ile Tyr Val Ile Pro 565 570 575 Ala Pro Ser Ile Tyr Ala Arg Glu Ala 580 585 3723PRThomo sapiens 3Met Gly Ser Arg Cys Ala Leu Ala Leu Ala Val Leu Ser Ala Leu Leu 1 5 10 15 Cys Gln Val Trp Ser Ser Gly Val Phe Glu Leu Lys Leu Gln Glu Phe 20 25 30 Val Asn Lys Lys Gly Leu Leu Gly Asn Arg Asn Cys Cys Arg Gly Gly 35 40 45 Ala Gly Pro Pro Pro Cys Ala Cys Arg Thr Phe Phe Arg Val Cys Leu 50 55 60 Lys His Tyr Gln Ala Ser Val Ser Pro Glu Pro Pro Cys Thr Tyr Gly 65 70 75 80 Ser Ala Val Thr Pro Val Leu Gly Val Asp Ser Phe Ser Leu Pro Asp 85 90 95 Gly Gly Gly Ala Asp Ser Ala Phe Ser Asn Pro Ile Arg Phe Pro Phe 100 105 110 Gly Phe Thr Trp Pro Gly Thr Phe Ser Leu Ile Ile Glu Ala Leu His 115 120 125 Thr Asp Ser Pro Asp Asp Leu Ala Thr Glu Asn Pro Glu Arg Leu Ile 130 135 140 Ser Arg Leu Ala Thr Gln Arg His Leu Thr Val Gly Glu Glu Trp Ser 145 150 155 160 Gln Asp Leu His Ser Ser Gly Arg Thr Asp Leu Lys Tyr Ser Tyr Arg 165 170 175 Phe Val Cys Asp Glu His Tyr Tyr Gly Glu Gly Cys Ser Val Phe Cys 180 185 190 Arg Pro Arg Asp Asp Ala Phe Gly His Phe Thr Cys Gly Glu Arg Gly 195 200 205 Glu Lys Val Cys Asn Pro Gly Trp Lys Gly Pro Tyr Cys Thr Glu Pro 210 215 220 Ile Cys Leu Pro Gly Cys Asp Glu Gln His Gly Phe Cys Asp Lys Pro 225 230 235 240 Gly Glu Cys Lys Cys Arg Val Gly Trp Gln Gly Arg Tyr Cys Asp Glu 245 250 255 Cys Ile Arg Tyr Pro Gly Cys Leu His Gly Thr Cys Gln Gln Pro Trp 260 265 270 Gln Cys Asn Cys Gln Glu Gly Trp Gly Gly Leu Phe Cys Asn Gln Asp 275 280 285 Leu Asn Tyr Cys Thr His His Lys Pro Cys Lys Asn Gly Ala Thr Cys 290 295 300 Thr Asn Thr Gly Gln Gly Ser Tyr Thr Cys Ser Cys Arg Pro Gly Tyr 305 310 315 320 Thr Gly Ala Thr Cys Glu Leu Gly Ile Asp Glu Cys Asp Pro Ser Pro 325 330 335 Cys Lys Asn Gly Gly Ser Cys Thr Asp Leu Glu Asn Ser Tyr Ser Cys 340 345 350 Thr Cys Pro Pro Gly Phe Tyr Gly Lys Ile Cys Glu Leu Ser Ala Met 355 360 365 Thr Cys Ala Asp Gly Pro Cys Phe Asn Gly Gly Arg Cys Ser Asp Ser 370 375 380 Pro Asp Gly Gly Tyr Ser Cys Arg Cys Pro Val Gly Tyr Ser Gly Phe 385 390 395 400 Asn Cys Glu Lys Lys Ile Asp Tyr Cys Ser Ser Ser Pro Cys Ser Asn 405 410 415 Gly Ala Lys Cys Val Asp Leu Gly Asp Ala Tyr Leu Cys Arg Cys Gln 420 425 430 Ala Gly Phe Ser Gly Arg His Cys Asp Asp Asn Val Asp Asp Cys Ala 435 440 445 Ser Ser Pro Cys Ala Asn Gly Gly Thr Cys Arg Asp Gly Val Asn Asp 450 455 460 Phe Ser Cys Thr Cys Pro Pro Gly Tyr Thr Gly Arg Asn Cys Ser Ala 465 470 475 480 Pro Val Ser Arg Cys Glu His Ala Pro Cys His Asn Gly Ala Thr Cys 485 490 495 His Glu Arg Gly His Arg Tyr Val Cys Glu Cys Ala Arg Gly Tyr Gly 500 505 510 Gly Pro Asn Cys Gln Phe Leu Leu Pro Glu Leu Pro Pro Gly Pro Ala 515 520 525 Val Val Asp Leu Thr Glu Lys Leu Glu Gly Gln Gly Gly Pro Phe Pro 530 535 540 Trp Val Ala Val Cys Ala Gly Val Ile Leu Val Leu Met Leu Leu Leu 545 550 555 560 Gly Cys Ala Ala Val Val Val Cys Val Arg Leu Arg Leu Gln Lys His 565 570 575 Arg Pro Pro Ala Asp Pro Cys Arg Gly Glu Thr Glu Thr Met Asn Asn 580 585 590 Leu Ala Asn Cys Gln Arg Glu Lys Asp Ile Ser Val Ser Ile Ile Gly 595 600 605 Ala Thr Gln Ile Lys Asn Thr Asn Lys Lys Ala Asp Phe His Gly Asp 610 615 620 His Ser Ala Asp Lys Asn Gly Phe Lys Ala Arg Tyr Pro Ala Val Asp 625 630 635 640 Tyr Asn Leu Val Gln Asp Leu Lys Gly Asp Asp Thr Ala Val Arg Asp 645 650 655 Ala His Ser Lys Arg Asp Thr Lys Cys Gln Pro Gln Gly Ser Ser Gly 660 665 670 Glu Glu Lys Gly Thr Pro Thr Thr Leu Arg Gly Gly Glu Ala Ser Glu 675 680 685 Arg Lys Arg Pro Asp Ser Gly Cys Ser Thr Ser Lys Asp Thr Lys Tyr 690 695 700 Gln Ser Val Tyr Val Ile Ser Glu Glu Lys Asp Glu Cys Val Ile Ala 705 710 715 720 Thr Glu Val 42555PRThomo sapiens 4Met Pro Pro Leu Leu Ala Pro Leu Leu Cys Leu Ala Leu Leu Pro Ala 1 5 10 15 Leu Ala Ala Arg Gly Pro Arg Cys Ser Gln Pro Gly Glu Thr Cys Leu 20 25 30 Asn Gly Gly Lys Cys Glu Ala Ala Asn Gly Thr Glu Ala Cys Val Cys 35 40 45 Gly Gly Ala Phe Val Gly Pro Arg Cys Gln Asp Pro Asn Pro Cys Leu 50 55 60 Ser Thr Pro Cys Lys Asn Ala Gly Thr Cys His Val Val Asp Arg Arg 65 70 75 80 Gly Val Ala Asp Tyr Ala Cys Ser Cys Ala Leu Gly Phe Ser Gly Pro 85 90 95 Leu Cys Leu Thr Pro Leu Asp Asn Ala Cys Leu Thr Asn Pro Cys Arg 100 105 110 Asn Gly Gly Thr Cys Asp Leu Leu Thr Leu Thr Glu Tyr Lys Cys Arg 115 120 125 Cys Pro Pro Gly Trp Ser Gly Lys Ser Cys Gln Gln Ala Asp Pro Cys 130 135 140 Ala Ser Asn Pro Cys Ala Asn Gly Gly Gln Cys Leu Pro Phe Glu Ala 145 150 155 160 Ser Tyr Ile Cys His Cys Pro Pro Ser Phe His Gly Pro Thr Cys Arg 165 170 175 Gln Asp Val Asn Glu Cys Gly Gln Lys Pro Gly Leu Cys Arg His Gly 180 185 190 Gly Thr Cys His Asn Glu Val Gly Ser Tyr Arg Cys Val Cys Arg Ala 195 200 205 Thr His Thr Gly Pro Asn Cys Glu Arg Pro Tyr Val Pro Cys Ser Pro 210 215 220 Ser Pro Cys Gln Asn Gly Gly Thr Cys Arg Pro Thr Gly Asp Val Thr 225 230 235 240 His Glu Cys Ala Cys Leu Pro Gly Phe Thr Gly Gln Asn Cys Glu Glu 245 250 255 Asn Ile Asp Asp Cys Pro Gly Asn Asn Cys Lys Asn Gly Gly Ala Cys 260 265 270 Val Asp Gly Val Asn Thr Tyr Asn Cys Arg Cys Pro Pro Glu Trp Thr 275 280 285 Gly Gln Tyr Cys Thr Glu Asp Val Asp Glu Cys Gln Leu Met Pro Asn 290 295 300 Ala Cys Gln Asn Gly Gly Thr Cys His Asn Thr His Gly Gly Tyr Asn 305 310 315 320 Cys Val Cys Val Asn Gly Trp Thr Gly Glu Asp Cys Ser Glu Asn Ile 325 330 335 Asp Asp Cys Ala Ser Ala Ala Cys Phe His Gly Ala Thr Cys His Asp 340 345 350 Arg Val Ala Ser Phe Tyr Cys Glu Cys Pro His Gly Arg Thr Gly Leu 355 360 365 Leu Cys His Leu Asn Asp Ala Cys Ile Ser Asn Pro Cys

Asn Glu Gly 370 375 380 Ser Asn Cys Asp Thr Asn Pro Val Asn Gly Lys Ala Ile Cys Thr Cys 385 390 395 400 Pro Ser Gly Tyr Thr Gly Pro Ala Cys Ser Gln Asp Val Asp Glu Cys 405 410 415 Ser Leu Gly Ala Asn Pro Cys Glu His Ala Gly Lys Cys Ile Asn Thr 420 425 430 Leu Gly Ser Phe Glu Cys Gln Cys Leu Gln Gly Tyr Thr Gly Pro Arg 435 440 445 Cys Glu Ile Asp Val Asn Glu Cys Val Ser Asn Pro Cys Gln Asn Asp 450 455 460 Ala Thr Cys Leu Asp Gln Ile Gly Glu Phe Gln Cys Ile Cys Met Pro 465 470 475 480 Gly Tyr Glu Gly Val His Cys Glu Val Asn Thr Asp Glu Cys Ala Ser 485 490 495 Ser Pro Cys Leu His Asn Gly Arg Cys Leu Asp Lys Ile Asn Glu Phe 500 505 510 Gln Cys Glu Cys Pro Thr Gly Phe Thr Gly His Leu Cys Gln Tyr Asp 515 520 525 Val Asp Glu Cys Ala Ser Thr Pro Cys Lys Asn Gly Ala Lys Cys Leu 530 535 540 Asp Gly Pro Asn Thr Tyr Thr Cys Val Cys Thr Glu Gly Tyr Thr Gly 545 550 555 560 Thr His Cys Glu Val Asp Ile Asp Glu Cys Asp Pro Asp Pro Cys His 565 570 575 Tyr Gly Ser Cys Lys Asp Gly Val Ala Thr Phe Thr Cys Leu Cys Arg 580 585 590 Pro Gly Tyr Thr Gly His His Cys Glu Thr Asn Ile Asn Glu Cys Ser 595 600 605 Ser Gln Pro Cys Arg His Gly Gly Thr Cys Gln Asp Arg Asp Asn Ala 610 615 620 Tyr Leu Cys Phe Cys Leu Lys Gly Thr Thr Gly Pro Asn Cys Glu Ile 625 630 635 640 Asn Leu Asp Asp Cys Ala Ser Ser Pro Cys Asp Ser Gly Thr Cys Leu 645 650 655 Asp Lys Ile Asp Gly Tyr Glu Cys Ala Cys Glu Pro Gly Tyr Thr Gly 660 665 670 Ser Met Cys Asn Ile Asn Ile Asp Glu Cys Ala Gly Asn Pro Cys His 675 680 685 Asn Gly Gly Thr Cys Glu Asp Gly Ile Asn Gly Phe Thr Cys Arg Cys 690 695 700 Pro Glu Gly Tyr His Asp Pro Thr Cys Leu Ser Glu Val Asn Glu Cys 705 710 715 720 Asn Ser Asn Pro Cys Val His Gly Ala Cys Arg Asp Ser Leu Asn Gly 725 730 735 Tyr Lys Cys Asp Cys Asp Pro Gly Trp Ser Gly Thr Asn Cys Asp Ile 740 745 750 Asn Asn Asn Glu Cys Glu Ser Asn Pro Cys Val Asn Gly Gly Thr Cys 755 760 765 Lys Asp Met Thr Ser Gly Tyr Val Cys Thr Cys Arg Glu Gly Phe Ser 770 775 780 Gly Pro Asn Cys Gln Thr Asn Ile Asn Glu Cys Ala Ser Asn Pro Cys 785 790 795 800 Leu Asn Gln Gly Thr Cys Ile Asp Asp Val Ala Gly Tyr Lys Cys Asn 805 810 815 Cys Leu Leu Pro Tyr Thr Gly Ala Thr Cys Glu Val Val Leu Ala Pro 820 825 830 Cys Ala Pro Ser Pro Cys Arg Asn Gly Gly Glu Cys Arg Gln Ser Glu 835 840 845 Asp Tyr Glu Ser Phe Ser Cys Val Cys Pro Thr Gly Trp Gln Gly Gln 850 855 860 Thr Cys Glu Val Asp Ile Asn Glu Cys Val Leu Ser Pro Cys Arg His 865 870 875 880 Gly Ala Ser Cys Gln Asn Thr His Gly Gly Tyr Arg Cys His Cys Gln 885 890 895 Ala Gly Tyr Ser Gly Arg Asn Cys Glu Thr Asp Ile Asp Asp Cys Arg 900 905 910 Pro Asn Pro Cys His Asn Gly Gly Ser Cys Thr Asp Gly Ile Asn Thr 915 920 925 Ala Phe Cys Asp Cys Leu Pro Gly Phe Arg Gly Thr Phe Cys Glu Glu 930 935 940 Asp Ile Asn Glu Cys Ala Ser Asp Pro Cys Arg Asn Gly Ala Asn Cys 945 950 955 960 Thr Asp Cys Val Asp Ser Tyr Thr Cys Thr Cys Pro Ala Gly Phe Ser 965 970 975 Gly Ile His Cys Glu Asn Asn Thr Pro Asp Cys Thr Glu Ser Ser Cys 980 985 990 Phe Asn Gly Gly Thr Cys Val Asp Gly Ile Asn Ser Phe Thr Cys Leu 995 1000 1005 Cys Pro Pro Gly Phe Thr Gly Ser Tyr Cys Gln His Asp Val Asn 1010 1015 1020 Glu Cys Asp Ser Gln Pro Cys Leu His Gly Gly Thr Cys Gln Asp 1025 1030 1035 Gly Cys Gly Ser Tyr Arg Cys Thr Cys Pro Gln Gly Tyr Thr Gly 1040 1045 1050 Pro Asn Cys Gln Asn Leu Val His Trp Cys Asp Ser Ser Pro Cys 1055 1060 1065 Lys Asn Gly Gly Lys Cys Trp Gln Thr His Thr Gln Tyr Arg Cys 1070 1075 1080 Glu Cys Pro Ser Gly Trp Thr Gly Leu Tyr Cys Asp Val Pro Ser 1085 1090 1095 Val Ser Cys Glu Val Ala Ala Gln Arg Gln Gly Val Asp Val Ala 1100 1105 1110 Arg Leu Cys Gln His Gly Gly Leu Cys Val Asp Ala Gly Asn Thr 1115 1120 1125 His His Cys Arg Cys Gln Ala Gly Tyr Thr Gly Ser Tyr Cys Glu 1130 1135 1140 Asp Leu Val Asp Glu Cys Ser Pro Ser Pro Cys Gln Asn Gly Ala 1145 1150 1155 Thr Cys Thr Asp Tyr Leu Gly Gly Tyr Ser Cys Lys Cys Val Ala 1160 1165 1170 Gly Tyr His Gly Val Asn Cys Ser Glu Glu Ile Asp Glu Cys Leu 1175 1180 1185 Ser His Pro Cys Gln Asn Gly Gly Thr Cys Leu Asp Leu Pro Asn 1190 1195 1200 Thr Tyr Lys Cys Ser Cys Pro Arg Gly Thr Gln Gly Val His Cys 1205 1210 1215 Glu Ile Asn Val Asp Asp Cys Asn Pro Pro Val Asp Pro Val Ser 1220 1225 1230 Arg Ser Pro Lys Cys Phe Asn Asn Gly Thr Cys Val Asp Gln Val 1235 1240 1245 Gly Gly Tyr Ser Cys Thr Cys Pro Pro Gly Phe Val Gly Glu Arg 1250 1255 1260 Cys Glu Gly Asp Val Asn Glu Cys Leu Ser Asn Pro Cys Asp Ala 1265 1270 1275 Arg Gly Thr Gln Asn Cys Val Gln Arg Val Asn Asp Phe His Cys 1280 1285 1290 Glu Cys Arg Ala Gly His Thr Gly Arg Arg Cys Glu Ser Val Ile 1295 1300 1305 Asn Gly Cys Lys Gly Lys Pro Cys Lys Asn Gly Gly Thr Cys Ala 1310 1315 1320 Val Ala Ser Asn Thr Ala Arg Gly Phe Ile Cys Lys Cys Pro Ala 1325 1330 1335 Gly Phe Glu Gly Ala Thr Cys Glu Asn Asp Ala Arg Thr Cys Gly 1340 1345 1350 Ser Leu Arg Cys Leu Asn Gly Gly Thr Cys Ile Ser Gly Pro Arg 1355 1360 1365 Ser Pro Thr Cys Leu Cys Leu Gly Pro Phe Thr Gly Pro Glu Cys 1370 1375 1380 Gln Phe Pro Ala Ser Ser Pro Cys Leu Gly Gly Asn Pro Cys Tyr 1385 1390 1395 Asn Gln Gly Thr Cys Glu Pro Thr Ser Glu Ser Pro Phe Tyr Arg 1400 1405 1410 Cys Leu Cys Pro Ala Lys Phe Asn Gly Leu Leu Cys His Ile Leu 1415 1420 1425 Asp Tyr Ser Phe Gly Gly Gly Ala Gly Arg Asp Ile Pro Pro Pro 1430 1435 1440 Leu Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Glu Asp Ala 1445 1450 1455 Gly Asn Lys Val Cys Ser Leu Gln Cys Asn Asn His Ala Cys Gly 1460 1465 1470 Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys 1475 1480 1485 Asn Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly 1490 1495 1500 His Cys Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly 1505 1510 1515 Phe Asp Cys Gln Arg Ala Glu Gly Gln Cys Asn Pro Leu Tyr Asp 1520 1525 1530 Gln Tyr Cys Lys Asp His Phe Ser Asp Gly His Cys Asp Gln Gly 1535 1540 1545 Cys Asn Ser Ala Glu Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu 1550 1555 1560 His Val Pro Glu Arg Leu Ala Ala Gly Thr Leu Val Val Val Val 1565 1570 1575 Leu Met Pro Pro Glu Gln Leu Arg Asn Ser Ser Phe His Phe Leu 1580 1585 1590 Arg Glu Leu Ser Arg Val Leu His Thr Asn Val Val Phe Lys Arg 1595 1600 1605 Asp Ala His Gly Gln Gln Met Ile Phe Pro Tyr Tyr Gly Arg Glu 1610 1615 1620 Glu Glu Leu Arg Lys His Pro Ile Lys Arg Ala Ala Glu Gly Trp 1625 1630 1635 Ala Ala Pro Asp Ala Leu Leu Gly Gln Val Lys Ala Ser Leu Leu 1640 1645 1650 Pro Gly Gly Ser Glu Gly Gly Arg Arg Arg Arg Glu Leu Asp Pro 1655 1660 1665 Met Asp Val Arg Gly Ser Ile Val Tyr Leu Glu Ile Asp Asn Arg 1670 1675 1680 Gln Cys Val Gln Ala Ser Ser Gln Cys Phe Gln Ser Ala Thr Asp 1685 1690 1695 Val Ala Ala Phe Leu Gly Ala Leu Ala Ser Leu Gly Ser Leu Asn 1700 1705 1710 Ile Pro Tyr Lys Ile Glu Ala Val Gln Ser Glu Thr Val Glu Pro 1715 1720 1725 Pro Pro Pro Ala Gln Leu His Phe Met Tyr Val Ala Ala Ala Ala 1730 1735 1740 Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu Leu Ser Arg 1745 1750 1755 Lys Arg Arg Arg Gln His Gly Gln Leu Trp Phe Pro Glu Gly Phe 1760 1765 1770 Lys Val Ser Glu Ala Ser Lys Lys Lys Arg Arg Glu Pro Leu Gly 1775 1780 1785 Glu Asp Ser Val Gly Leu Lys Pro Leu Lys Asn Ala Ser Asp Gly 1790 1795 1800 Ala Leu Met Asp Asp Asn Gln Asn Glu Trp Gly Asp Glu Asp Leu 1805 1810 1815 Glu Thr Lys Lys Phe Arg Phe Glu Glu Pro Val Val Leu Pro Asp 1820 1825 1830 Leu Asp Asp Gln Thr Asp His Arg Gln Trp Thr Gln Gln His Leu 1835 1840 1845 Asp Ala Ala Asp Leu Arg Met Ser Ala Met Ala Pro Thr Pro Pro 1850 1855 1860 Gln Gly Glu Val Asp Ala Asp Cys Met Asp Val Asn Val Arg Gly 1865 1870 1875 Pro Asp Gly Phe Thr Pro Leu Met Ile Ala Ser Cys Ser Gly Gly 1880 1885 1890 Gly Leu Glu Thr Gly Asn Ser Glu Glu Glu Glu Asp Ala Pro Ala 1895 1900 1905 Val Ile Ser Asp Phe Ile Tyr Gln Gly Ala Ser Leu His Asn Gln 1910 1915 1920 Thr Asp Arg Thr Gly Glu Thr Ala Leu His Leu Ala Ala Arg Tyr 1925 1930 1935 Ser Arg Ser Asp Ala Ala Lys Arg Leu Leu Glu Ala Ser Ala Asp 1940 1945 1950 Ala Asn Ile Gln Asp Asn Met Gly Arg Thr Pro Leu His Ala Ala 1955 1960 1965 Val Ser Ala Asp Ala Gln Gly Val Phe Gln Ile Leu Ile Arg Asn 1970 1975 1980 Arg Ala Thr Asp Leu Asp Ala Arg Met His Asp Gly Thr Thr Pro 1985 1990 1995 Leu Ile Leu Ala Ala Arg Leu Ala Val Glu Gly Met Leu Glu Asp 2000 2005 2010 Leu Ile Asn Ser His Ala Asp Val Asn Ala Val Asp Asp Leu Gly 2015 2020 2025 Lys Ser Ala Leu His Trp Ala Ala Ala Val Asn Asn Val Asp Ala 2030 2035 2040 Ala Val Val Leu Leu Lys Asn Gly Ala Asn Lys Asp Met Gln Asn 2045 2050 2055 Asn Arg Glu Glu Thr Pro Leu Phe Leu Ala Ala Arg Glu Gly Ser 2060 2065 2070 Tyr Glu Thr Ala Lys Val Leu Leu Asp His Phe Ala Asn Arg Asp 2075 2080 2085 Ile Thr Asp His Met Asp Arg Leu Pro Arg Asp Ile Ala Gln Glu 2090 2095 2100 Arg Met His His Asp Ile Val Arg Leu Leu Asp Glu Tyr Asn Leu 2105 2110 2115 Val Arg Ser Pro Gln Leu His Gly Ala Pro Leu Gly Gly Thr Pro 2120 2125 2130 Thr Leu Ser Pro Pro Leu Cys Ser Pro Asn Gly Tyr Leu Gly Ser 2135 2140 2145 Leu Lys Pro Gly Val Gln Gly Lys Lys Val Arg Lys Pro Ser Ser 2150 2155 2160 Lys Gly Leu Ala Cys Gly Ser Lys Glu Ala Lys Asp Leu Lys Ala 2165 2170 2175 Arg Arg Lys Lys Ser Gln Asp Gly Lys Gly Cys Leu Leu Asp Ser 2180 2185 2190 Ser Gly Met Leu Ser Pro Val Asp Ser Leu Glu Ser Pro His Gly 2195 2200 2205 Tyr Leu Ser Asp Val Ala Ser Pro Pro Leu Leu Pro Ser Pro Phe 2210 2215 2220 Gln Gln Ser Pro Ser Val Pro Leu Asn His Leu Pro Gly Met Pro 2225 2230 2235 Asp Thr His Leu Gly Ile Gly His Leu Asn Val Ala Ala Lys Pro 2240 2245 2250 Glu Met Ala Ala Leu Gly Gly Gly Gly Arg Leu Ala Phe Glu Thr 2255 2260 2265 Gly Pro Pro Arg Leu Ser His Leu Pro Val Ala Ser Gly Thr Ser 2270 2275 2280 Thr Val Leu Gly Ser Ser Ser Gly Gly Ala Leu Asn Phe Thr Val 2285 2290 2295 Gly Gly Ser Thr Ser Leu Asn Gly Gln Cys Glu Trp Leu Ser Arg 2300 2305 2310 Leu Gln Ser Gly Met Val Pro Asn Gln Tyr Asn Pro Leu Arg Gly 2315 2320 2325 Ser Val Ala Pro Gly Pro Leu Ser Thr Gln Ala Pro Ser Leu Gln 2330 2335 2340 His Gly Met Val Gly Pro Leu His Ser Ser Leu Ala Ala Ser Ala 2345 2350 2355 Leu Ser Gln Met Met Ser Tyr Gln Gly Leu Pro Ser Thr Arg Leu 2360 2365 2370 Ala Thr Gln Pro His Leu Val Gln Thr Gln Gln Val Gln Pro Gln 2375 2380 2385 Asn Leu Gln Met Gln Gln Gln Asn Leu Gln Pro Ala Asn Ile Gln 2390 2395 2400 Gln Gln Gln Ser Leu Gln Pro Pro Pro Pro Pro Pro Gln Pro His 2405 2410 2415 Leu Gly Val Ser Ser Ala Ala Ser Gly His Leu Gly Arg Ser Phe 2420 2425 2430 Leu Ser Gly Glu Pro Ser Gln Ala Asp Val Gln Pro Leu Gly Pro 2435 2440 2445 Ser Ser Leu Ala Val His Thr Ile Leu Pro Gln Glu Ser Pro Ala 2450 2455 2460 Leu Pro Thr Ser Leu Pro Ser Ser Leu Val Pro Pro Val Thr Ala 2465 2470 2475 Ala Gln Phe Leu Thr Pro Pro Ser Gln His Ser Tyr Ser Ser Pro 2480 2485 2490 Val Asp Asn Thr Pro Ser His Gln Leu Gln Val Pro Glu His Pro 2495 2500 2505 Phe Leu Thr Pro Ser Pro Glu Ser Pro Asp Gln Trp Ser Ser Ser 2510 2515 2520 Ser Pro His Ser Asn Val Ser Asp Trp Ser Glu Gly Val Ser Ser 2525 2530 2535 Pro Pro Thr Ser Met Gln Ser Gln Ile Ala Arg Ile Pro Glu Ala 2540 2545 2550 Phe Lys 2555 5724PRTArtificial Sequencehuman-mouse chimeric sequence 5Met Val Ser Pro Arg Met Ser Gly Leu Leu Ser Gln Thr Val Ile Leu 1 5 10 15 Ala Leu Ile Phe Leu Pro Gln Thr Arg Pro Ala Gly Val Phe Glu Leu 20 25 30 Gln Ile His Ser Phe Gly Pro Gly Pro Gly Pro Gly Ala Pro

Arg Ser 35 40 45 Pro Cys Ser Ala Arg Leu Pro Cys Arg Leu Phe Phe Arg Val Cys Leu 50 55 60 Lys Pro Gly Leu Ser Glu Glu Ala Ala Glu Ser Pro Cys Ala Leu Gly 65 70 75 80 Ala Ala Leu Ser Ala Arg Gly Pro Val Tyr Thr Glu Gln Pro Gly Ala 85 90 95 Pro Ala Pro Asp Leu Pro Leu Pro Asp Gly Leu Leu Gln Val Pro Phe 100 105 110 Arg Asp Ala Trp Pro Gly Thr Phe Ser Phe Ile Ile Glu Thr Trp Arg 115 120 125 Glu Glu Leu Gly Asp Gln Ile Gly Gly Pro Ala Trp Ser Leu Leu Ala 130 135 140 Arg Val Ala Gly Arg Arg Arg Leu Ala Ala Gly Gly Pro Trp Ala Arg 145 150 155 160 Asp Ile Gln Arg Ala Gly Ala Trp Glu Leu Arg Phe Ser Tyr Arg Ala 165 170 175 Arg Cys Glu Pro Pro Ala Val Gly Thr Ala Cys Thr Arg Leu Cys Arg 180 185 190 Pro Arg Ser Ala Pro Ser Arg Cys Gly Pro Gly Leu Arg Pro Cys Ala 195 200 205 Pro Leu Glu Asp Glu Cys Glu Ala Pro Leu Val Cys Arg Ala Gly Cys 210 215 220 Ser Pro Glu His Gly Phe Cys Glu Gln Pro Gly Glu Cys Arg Cys Leu 225 230 235 240 Glu Gly Trp Thr Gly Pro Leu Cys Thr Val Pro Val Ser Thr Ser Ser 245 250 255 Cys Leu Ser Pro Arg Gly Pro Ser Ser Ala Thr Thr Gly Cys Leu Val 260 265 270 Pro Gly Pro Gly Pro Cys Asp Gly Asn Pro Cys Ala Asn Gly Gly Ser 275 280 285 Cys Ser Glu Thr Pro Arg Ser Phe Glu Cys Thr Cys Pro Arg Gly Phe 290 295 300 Tyr Gly Leu Arg Cys Glu Val Ser Gly Val Thr Cys Ala Asp Gly Pro 305 310 315 320 Cys Phe Asn Gly Gly Leu Cys Val Gly Gly Ala Asp Pro Asp Ser Ala 325 330 335 Tyr Ile Cys His Cys Pro Pro Gly Phe Gln Gly Ser Asn Cys Glu Lys 340 345 350 Arg Val Asp Arg Cys Ser Leu Gln Pro Cys Arg Asn Gly Gly Leu Cys 355 360 365 Leu Asp Leu Gly His Ala Leu Arg Cys Arg Cys Arg Ala Gly Phe Ala 370 375 380 Gly Pro Arg Cys Glu His Asp Leu Asp Asp Cys Ala Gly Arg Ala Cys 385 390 395 400 Ala Asn Gly Gly Thr Cys Val Glu Gly Gly Gly Ala His Arg Cys Ser 405 410 415 Cys Ala Leu Gly Phe Gly Gly Arg Asp Cys Arg Glu Arg Ala Asp Pro 420 425 430 Cys Ala Ala Arg Pro Cys Ala His Gly Gly Arg Cys Tyr Ala His Phe 435 440 445 Ser Gly Leu Val Cys Ala Cys Ala Pro Gly Tyr Met Gly Ala Arg Cys 450 455 460 Glu Phe Pro Val His Pro Asp Gly Ala Ser Ala Leu Pro Ala Ala Pro 465 470 475 480 Pro Gly Leu Arg Pro Gly Asp Pro Gln Arg Tyr Leu Ala Arg Gly Pro 485 490 495 Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu 500 505 510 Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu 515 520 525 Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser 530 535 540 Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu 545 550 555 560 Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr 565 570 575 Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser 580 585 590 Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro 595 600 605 Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln 610 615 620 Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val 625 630 635 640 Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val 645 650 655 Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu 660 665 670 Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg 675 680 685 Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val 690 695 700 Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg 705 710 715 720 Thr Pro Gly Lys 6575PRTArtificial Sequencehuman-mouse chimeric sequence 6Met Val Leu Ala Ser Ser Thr Thr Ser Ile His Thr Met Leu Leu Leu 1 5 10 15 Leu Leu Met Leu Ala Gln Pro Ala Met Ala Ala Arg Cys Glu Pro Pro 20 25 30 Ala Val Gly Thr Ala Cys Thr Arg Leu Cys Arg Pro Arg Ser Ala Pro 35 40 45 Ser Arg Cys Gly Pro Gly Leu Arg Pro Cys Ala Pro Leu Glu Asp Glu 50 55 60 Cys Glu Ala Pro Leu Val Cys Arg Ala Gly Cys Ser Pro Glu His Gly 65 70 75 80 Phe Cys Glu Gln Pro Gly Glu Cys Arg Cys Leu Glu Gly Trp Thr Gly 85 90 95 Pro Leu Cys Thr Val Pro Val Ser Thr Ser Ser Cys Leu Ser Pro Arg 100 105 110 Gly Pro Ser Ser Ala Thr Thr Gly Cys Leu Val Pro Gly Pro Gly Pro 115 120 125 Cys Asp Gly Asn Pro Cys Ala Asn Gly Gly Ser Cys Ser Glu Thr Pro 130 135 140 Arg Ser Phe Glu Cys Thr Cys Pro Arg Gly Phe Tyr Gly Leu Arg Cys 145 150 155 160 Glu Val Ser Gly Val Thr Cys Ala Asp Gly Pro Cys Phe Asn Gly Gly 165 170 175 Leu Cys Val Gly Gly Ala Asp Pro Asp Ser Ala Tyr Ile Cys His Cys 180 185 190 Pro Pro Gly Phe Gln Gly Ser Asn Cys Glu Lys Arg Val Asp Arg Cys 195 200 205 Ser Leu Gln Pro Cys Arg Asn Gly Gly Leu Cys Leu Asp Leu Gly His 210 215 220 Ala Leu Arg Cys Arg Cys Arg Ala Gly Phe Ala Gly Pro Arg Cys Glu 225 230 235 240 His Asp Leu Asp Asp Cys Ala Gly Arg Ala Cys Ala Asn Gly Gly Thr 245 250 255 Cys Val Glu Gly Gly Gly Ala His Arg Cys Ser Cys Ala Leu Gly Phe 260 265 270 Gly Gly Arg Asp Cys Arg Glu Arg Ala Asp Pro Cys Ala Ala Arg Pro 275 280 285 Cys Ala His Gly Gly Arg Cys Tyr Ala His Phe Ser Gly Leu Val Cys 290 295 300 Ala Cys Ala Pro Gly Tyr Met Gly Ala Arg Cys Glu Phe Pro Val His 305 310 315 320 Pro Asp Gly Ala Ser Ala Leu Pro Ala Ala Pro Pro Gly Leu Arg Pro 325 330 335 Gly Asp Pro Gln Arg Tyr Leu Ala Arg Gly Pro Thr Ile Lys Pro Cys 340 345 350 Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val 355 360 365 Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser 370 375 380 Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp 385 390 395 400 Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln 405 410 415 Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser 420 425 430 Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys 435 440 445 Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile 450 455 460 Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro 465 470 475 480 Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met 485 490 495 Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn 500 505 510 Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser 515 520 525 Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn 530 535 540 Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu 545 550 555 560 His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 565 570 575 7535PRTArtificial Sequencehuman-mouse chimeric sequence 7Met Val Leu Ala Ser Ser Thr Thr Ser Ile His Thr Met Leu Leu Leu 1 5 10 15 Leu Leu Met Leu Ala Gln Pro Ala Met Ala Ala Pro Leu Val Cys Arg 20 25 30 Ala Gly Cys Ser Pro Glu His Gly Phe Cys Glu Gln Pro Gly Glu Cys 35 40 45 Arg Cys Leu Glu Gly Trp Thr Gly Pro Leu Cys Thr Val Pro Val Ser 50 55 60 Thr Ser Ser Cys Leu Ser Pro Arg Gly Pro Ser Ser Ala Thr Thr Gly 65 70 75 80 Cys Leu Val Pro Gly Pro Gly Pro Cys Asp Gly Asn Pro Cys Ala Asn 85 90 95 Gly Gly Ser Cys Ser Glu Thr Pro Arg Ser Phe Glu Cys Thr Cys Pro 100 105 110 Arg Gly Phe Tyr Gly Leu Arg Cys Glu Val Ser Gly Val Thr Cys Ala 115 120 125 Asp Gly Pro Cys Phe Asn Gly Gly Leu Cys Val Gly Gly Ala Asp Pro 130 135 140 Asp Ser Ala Tyr Ile Cys His Cys Pro Pro Gly Phe Gln Gly Ser Asn 145 150 155 160 Cys Glu Lys Arg Val Asp Arg Cys Ser Leu Gln Pro Cys Arg Asn Gly 165 170 175 Gly Leu Cys Leu Asp Leu Gly His Ala Leu Arg Cys Arg Cys Arg Ala 180 185 190 Gly Phe Ala Gly Pro Arg Cys Glu His Asp Leu Asp Asp Cys Ala Gly 195 200 205 Arg Ala Cys Ala Asn Gly Gly Thr Cys Val Glu Gly Gly Gly Ala His 210 215 220 Arg Cys Ser Cys Ala Leu Gly Phe Gly Gly Arg Asp Cys Arg Glu Arg 225 230 235 240 Ala Asp Pro Cys Ala Ala Arg Pro Cys Ala His Gly Gly Arg Cys Tyr 245 250 255 Ala His Phe Ser Gly Leu Val Cys Ala Cys Ala Pro Gly Tyr Met Gly 260 265 270 Ala Arg Cys Glu Phe Pro Val His Pro Asp Gly Ala Ser Ala Leu Pro 275 280 285 Ala Ala Pro Pro Gly Leu Arg Pro Gly Asp Pro Gln Arg Tyr Leu Ala 290 295 300 Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro 305 310 315 320 Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys 325 330 335 Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val 340 345 350 Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn 355 360 365 Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr 370 375 380 Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp 385 390 395 400 Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu 405 410 415 Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg 420 425 430 Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys 435 440 445 Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp 450 455 460 Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys 465 470 475 480 Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser 485 490 495 Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser 500 505 510 Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser 515 520 525 Phe Ser Arg Thr Pro Gly Lys 530 535 8718PRTArtificial Sequencehuman-mouse chimeric sequence 8Met Val Ser Leu Gln Val Ser Pro Leu Ser Gln Thr Leu Ile Leu Ala 1 5 10 15 Phe Leu Leu Pro Gln Ala Leu Pro Ala Gly Val Phe Glu Leu Gln Ile 20 25 30 His Ser Phe Gly Pro Gly Pro Gly Leu Gly Thr Pro Arg Ser Pro Cys 35 40 45 Asn Ala Arg Gly Pro Cys Arg Leu Phe Phe Arg Val Cys Leu Lys Pro 50 55 60 Gly Val Ser Gln Glu Ala Thr Glu Ser Leu Cys Ala Leu Gly Ala Ala 65 70 75 80 Leu Ser Thr Ser Val Pro Val Tyr Thr Glu His Pro Gly Glu Ser Ala 85 90 95 Ala Ala Leu Pro Leu Pro Asp Gly Leu Val Arg Val Pro Phe Arg Asp 100 105 110 Ala Trp Pro Gly Thr Phe Ser Leu Val Ile Glu Thr Trp Arg Glu Gln 115 120 125 Leu Gly Glu His Ala Gly Gly Pro Ala Trp Asn Leu Leu Ala Arg Val 130 135 140 Val Gly Arg Arg Arg Leu Ala Ala Gly Gly Pro Trp Ala Arg Asp Val 145 150 155 160 Gln Arg Thr Gly Thr Trp Glu Leu His Phe Ser Tyr Arg Ala Arg Cys 165 170 175 Glu Pro Pro Ala Val Gly Ala Ala Cys Ala Arg Leu Cys Arg Ser Arg 180 185 190 Ser Ala Pro Ser Arg Cys Gly Pro Gly Leu Arg Pro Cys Thr Pro Phe 195 200 205 Pro Asp Glu Cys Glu Ala Pro Ser Val Cys Arg Pro Gly Cys Ser Pro 210 215 220 Glu His Gly Tyr Cys Glu Glu Pro Asp Glu Cys Arg Cys Leu Glu Gly 225 230 235 240 Trp Thr Gly Pro Leu Cys Thr Val Pro Val Ser Thr Ser Ser Cys Leu 245 250 255 Asn Ser Arg Val Pro Gly Pro Ala Ser Thr Gly Cys Leu Leu Pro Gly 260 265 270 Pro Gly Pro Cys Asp Gly Asn Pro Cys Ala Asn Gly Gly Ser Cys Ser 275 280 285 Glu Thr Ser Gly Ser Phe Glu Cys Ala Cys Pro Arg Gly Phe Tyr Gly 290 295 300 Leu Arg Cys Glu Val Ser Gly Val Thr Cys Ala Asp Gly Pro Cys Phe 305 310 315 320 Asn Gly Gly Leu Cys Val Gly Gly Glu Asp Pro Asp Ser Ala Tyr Val 325 330 335 Cys His Cys Pro Pro Gly Phe Gln Gly Ser Asn Cys Glu Lys Arg Val 340 345 350 Asp Arg Cys Ser Leu Gln Pro Cys Gln Asn Gly Gly Leu Cys Leu Asp 355 360 365 Leu Gly His Ala Leu Arg Cys Arg Cys Arg Ala Gly Phe Ala Gly Pro 370 375 380 Arg Cys Glu His Asp Leu Asp Asp Cys Ala Gly Arg Ala Cys Ala Asn 385 390 395 400 Gly Gly Thr Cys Val Glu Gly Gly Gly Ser Arg Arg Cys Ser Cys Ala 405 410 415 Leu Gly Phe Gly Gly Arg Asp Cys Arg Glu Arg Ala Asp Pro Cys Ala 420 425 430 Ser Arg Pro Cys Ala His Gly Gly Arg Cys Tyr Ala His Phe Ser Gly 435 440 445 Leu Val Cys Ala Cys Ala Pro Gly Tyr Met Gly Val Arg Cys Glu Phe 450 455 460 Ala Val Arg Pro Asp Gly Ala Asp Ala Val Pro Ala Ala Pro Arg Gly 465 470 475 480 Leu Arg Gln Ala Asp Pro Gln Arg Gly Pro Thr Ile Lys Pro Cys Pro 485

490 495 Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe 500 505 510 Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro 515 520 525 Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val 530 535 540 Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr 545 550 555 560 Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala 565 570 575 Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys 580 585 590 Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser 595 600 605 Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro 610 615 620 Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val 625 630 635 640 Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly 645 650 655 Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp 660 665 670 Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp 675 680 685 Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His 690 695 700 Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 705 710 715 9121PRTMus musculus 9Gln Val Gln Leu Gln Gln Ser Gly Ala Asp Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30 Leu Ile Glu Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Asn Pro Gly Ser Gly Gly Thr His Tyr Ser Glu Lys Phe 50 55 60 Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ile Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ser Asp Tyr Asp Tyr Val Thr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 10464PRTMus musculus 10Met Glu Trp Ser Gly Val Phe Ile Phe Leu Leu Ser Val Thr Ala Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Asp Leu Val Arg 20 25 30 Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe 35 40 45 Thr Asn Tyr Leu Ile Glu Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Met Asn Pro Gly Ser Gly Gly Thr His Tyr Ser 65 70 75 80 Glu Lys Phe Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ile Ser Leu Thr Ser Asp Asp Ser Ala Val 100 105 110 Tyr Phe Cys Ala Arg Ser Asp Tyr Asp Tyr Val Thr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr 130 135 140 Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn 145 150 155 160 Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val 195 200 205 Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val 210 215 220 Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg 225 230 235 240 Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser 245 250 255 Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu 260 265 270 Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro 275 280 285 Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala 290 295 300 Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val 305 310 315 320 Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe 325 330 335 Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr 340 345 350 Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile 355 360 365 Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys 370 375 380 Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp 385 390 395 400 Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asn 405 410 415 Thr Asn Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser 420 425 430 Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly 435 440 445 Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 450 455 460 11470PRTArtificial Sequencechimeric antibody heavy chain 11Met Glu Trp Ser Gly Val Phe Ile Phe Leu Leu Ser Val Thr Ala Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Asp Leu Val Arg 20 25 30 Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe 35 40 45 Thr Asn Tyr Leu Ile Glu Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Met Asn Pro Gly Ser Gly Gly Thr His Tyr Ser 65 70 75 80 Glu Lys Phe Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ile Ser Leu Thr Ser Asp Asp Ser Ala Val 100 105 110 Tyr Phe Cys Ala Arg Ser Asp Tyr Asp Tyr Val Thr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 125PRTMus musculus 12Asn Tyr Leu Ile Glu 1 5 1317PRTMus musculus 13Val Met Asn Pro Gly Ser Gly Gly Thr His Tyr Ser Glu Lys Phe Arg 1 5 10 15 Gly 1412PRTMus musculus 14Ser Asp Tyr Asp Tyr Val Thr Tyr Ala Met Asp Tyr 1 5 10 15107PRTMus musculus 15Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Ile Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Thr Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr 65 70 75 80 Gly Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 16236PRTMus musculus 16Met Asp Met Arg Thr Pro Ala Gln Phe Leu Gly Ile Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ile Lys Cys Asp Ile Lys Met Thr Gln Ser Pro Ser Ser 20 25 30 Met Tyr Ala Ser Leu Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser 35 40 45 Gln Asp Ile Asn Ser Tyr Leu Ile Trp Phe Gln Gln Lys Pro Gly Lys 50 55 60 Ser Pro Lys Thr Leu Ile Tyr Arg Thr Asn Arg Leu Val Asp Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr 85 90 95 Ile Ser Ser Leu Glu Tyr Gly Asp Met Gly Ile Tyr Tyr Cys Leu Gln 100 105 110 Tyr Asp Glu Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 115 120 125 Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser 130 135 140 Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn 145 150 155 160 Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu 165 170 175 Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr 195 200 205 Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr 210 215 220 Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 235 17236PRTArtificial Sequencechimeric antibody light chain 17Met Asp Met Arg Thr Pro Ala Gln Phe Leu Gly Ile Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ile Lys Cys Asp Ile Lys Met Thr Gln Ser Pro Ser Ser 20 25 30 Met Tyr Ala Ser Leu Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser 35 40 45 Gln Asp Ile Asn Ser Tyr Leu Ile Trp Phe Gln Gln Lys Pro Gly Lys 50 55 60 Ser Pro Lys Thr Leu Ile Tyr Arg Thr Asn Arg Leu Val Asp Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr 85 90 95 Ile Ser Ser Leu Glu Tyr Gly Asp Met Gly Ile Tyr Tyr Cys Leu Gln 100 105 110 Tyr Asp Glu Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 1811PRTMus musculus 18Lys Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ile 1 5 10 197PRTMus musculus 19Arg Thr Asn Arg Leu Val Asp 1 5 209PRTMus musculus 20Leu Gln Tyr Asp Glu Phe Pro Phe Thr 1 5 21118PRTMus musculus 21Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Arg Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Lys Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro Asn Asn Gly Asp Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ile Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Asn Tyr Ala Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 22461PRTMus musculus 22Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Gly Gly 1 5 10 15 Val Leu Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Arg Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Tyr Met Lys Trp Val Lys Gln Ser His Gly Lys Ser Leu 50 55 60 Glu Trp Ile Gly Asp Ile Asn Pro Asn Asn Gly Asp Thr Phe Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ile Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Asn Ser Leu Thr Ser Asp Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Asp Gly Asn Tyr Ala Tyr Phe Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser 130 135 140 Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val 145 150 155 160 Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro 195 200 205 Ser Ser Thr Trp Pro Ser Glu

Thr Val Thr Cys Asn Val Ala His Pro 210 215 220 Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly 225 230 235 240 Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile 245 250 255 Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys 260 265 270 Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln 275 280 285 Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln 290 295 300 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu 305 310 315 320 Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg 325 330 335 Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 340 345 350 Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro 355 360 365 Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr 370 375 380 Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln 385 390 395 400 Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asn Thr Asn Gly 405 410 415 Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu 420 425 430 Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn 435 440 445 His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 450 455 460 23467PRTArtificial Sequencechimeric antibody heavy chain 23Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Gly Gly 1 5 10 15 Val Leu Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Arg Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Tyr Met Lys Trp Val Lys Gln Ser His Gly Lys Ser Leu 50 55 60 Glu Trp Ile Gly Asp Ile Asn Pro Asn Asn Gly Asp Thr Phe Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ile Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Asn Ser Leu Thr Ser Asp Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Asp Gly Asn Tyr Ala Tyr Phe Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 245PRTMus musculus 24Asp Tyr Tyr Met Lys 1 5 2517PRTMus musculus 25Asp Ile Asn Pro Asn Asn Gly Asp Thr Phe Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly 269PRTMus musculus 26Asp Gly Asn Tyr Ala Tyr Phe Asp Tyr 1 5 27111PRTMus musculus 27Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Phe Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 His Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 28238PRTMus musculus 28Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala 20 25 30 Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser 35 40 45 Val Ser Thr Ser Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Phe Leu Ala Ser Asn Leu Glu Ser 65 70 75 80 Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Gln His Ser Arg His Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125 Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro 130 135 140 Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu 145 150 155 160 Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly 165 170 175 Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp 195 200 205 Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr 210 215 220 Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 235 29238PRTArtificial Sequencechimeric antibody light chain 29Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala 20 25 30 Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser 35 40 45 Val Ser Thr Ser Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Phe Leu Ala Ser Asn Leu Glu Ser 65 70 75 80 Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Gln His Ser Arg His Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125 Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 3015PRTMus musculus 30Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His 1 5 10 15 317PRTMus musculus 31Leu Ala Ser Asn Leu Glu Ser 1 5 329PRTMus musculus 32Gln His Ser Arg His Leu Pro Trp Thr 1 5 33119PRTMus musculus 33Gln Val Gln Leu Gln Gln Ser Gly Gly Asp Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ala Gly Tyr Thr Phe Ser Asn Tyr 20 25 30 Tyr Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Thr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Ser Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Ala Arg Glu Pro Gly Phe Pro Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 34462PRTMus musculus 34Met Glu Trp Thr Trp Val Phe Leu Phe Leu Leu Ser Val Thr Ala Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Gly Asp Leu Met Lys 20 25 30 Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ala Gly Tyr Thr Phe 35 40 45 Ser Asn Tyr Tyr Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu 50 55 60 Glu Trp Ile Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Thr Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Lys Ala Ser Phe Thr Ala Asp Thr Ser Ser Asn 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Trp Gly Ala Arg Glu Pro Gly Phe Pro Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro 130 135 140 Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met 145 150 155 160 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val 195 200 205 Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His 210 215 220 Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys 225 230 235 240 Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe 245 250 255 Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro 260 265 270 Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val 275 280 285 Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr 290 295 300 Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu 305 310 315 320 Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys 325 330 335 Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser 340 345 350 Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro 355 360 365 Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile 370 375 380 Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly 385 390 395 400 Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asn Thr Asn 405 410 415 Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp 420 425 430 Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His 435 440 445 Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 450 455 460 35468PRTArtificial Sequencechimeric antibody heavy chain 35Met Glu Trp Thr Trp Val Phe Leu Phe Leu Leu Ser Val Thr Ala Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Gly Asp Leu Met Lys 20 25 30 Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ala Gly Tyr Thr Phe 35 40 45 Ser Asn Tyr Tyr Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu 50 55 60 Glu Trp Ile Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Thr Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Lys Ala Ser Phe Thr Ala Asp Thr Ser Ser Asn 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Trp Gly Ala Arg Glu Pro Gly Phe Pro Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 225 230 235 240 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Gly Lys 465 365PRTMus musculus 36Asn Tyr Tyr Ile Glu 1 5 3717PRTMus musculus 37Glu Ile Leu Pro Gly Ser Gly Ser Thr Thr Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly 3810PRTMus musculus 38Trp Gly Ala Arg Glu Pro Gly Phe Pro Tyr 1 5 10 39107PRTMus musculus 39Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 40238PRTMus musculus 40Met Gly Ile Lys Met Glu Ser Gln Thr Gln Val Phe Val Tyr Met Leu 1 5 10 15 Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser Gln 20 25 30 Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys 35 40 45 Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Ser Pro Lys Ala Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Glu Tyr Phe Cys 100 105 110 Gln Gln Tyr Asn Asn Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu 115 120 125 Glu Leu Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro 130 135 140 Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu 145 150 155 160 Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly 165 170 175 Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp 195 200 205 Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr 210 215 220 Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 235 41238PRTArtificial Sequencechimeric antibody light chain 41Met Gly Ile Lys Met Glu Ser Gln Thr Gln Val Phe Val Tyr Met Leu 1 5 10 15 Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser Gln 20 25 30 Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys 35 40 45 Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Ser Pro Lys Ala Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Glu Tyr Phe Cys 100 105 110 Gln Gln Tyr Asn Asn Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu 115 120 125 Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 4211PRTMus musculus 42Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala 1 5 10 437PRTMus musculus 43Ser Ala Ser Tyr Arg Tyr Ser 1 5 449PRTMus musculus 44Gln Gln Tyr Asn Asn Tyr Pro Leu Thr 1 5 45123PRTMus musculus 45Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Ala Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Asn Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Asn Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Ser Ala Thr Tyr 85 90 95 Tyr Cys Ala Arg Asp Ser Asp Gly Tyr Tyr Glu Tyr Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 46466PRTMus musculus 46Met Lys Leu Trp Leu Asn Trp Ile Phe Leu Val Thr Leu Leu Asn Gly 1 5 10 15 Ile Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Thr Asp Tyr Tyr Met Asn Trp Val Arg Gln Pro Pro Gly Lys Ala Leu 50 55 60 Glu Trp Leu Ala Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu 65 70 75 80 Tyr Asn Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 85 90 95 Gln Asn Ile Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Ser 100 105 110 Ala Thr Tyr Tyr Cys Ala Arg Asp Ser Asp Gly Tyr Tyr Glu Tyr Tyr 115 120 125 Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys 130 135 140 Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln 145 150 155 160 Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro 165 170 175 Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val 180 185 190 His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser 195 200 205 Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys 210 215 220 Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val 225 230 235 240 Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val 245 250 255 Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile 260 265 270 Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp 275 280 285 Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His 290 295 300 Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg 305 310 315 320 Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr 355 360 365 Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu 370 375 380 Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp 385 390 395 400 Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile 405 410 415 Met Asn Thr Asn Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln 420 425 430 Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His 435 440 445 Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro 450 455 460 Gly Lys 465 47472PRTArtificial Sequencechimeric antibody heavy chain 47Met Lys Leu Trp Leu Asn Trp Ile Phe Leu Val Thr Leu Leu Asn Gly 1 5 10 15 Ile Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Thr Asp Tyr Tyr Met Asn Trp Val Arg Gln Pro Pro Gly Lys Ala Leu 50 55 60 Glu Trp Leu Ala Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu 65 70 75 80 Tyr Asn Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 85 90 95 Gln Asn Ile Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Ser 100 105 110 Ala Thr Tyr Tyr Cys Ala Arg Asp Ser Asp Gly Tyr Tyr Glu Tyr Tyr 115 120 125 Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser 130 135 140 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 145 150 155 160 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 165 170 175 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 180 185 190 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 195 200 205 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 210 215 220 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 225 230 235 240 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265 270 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340 345 350 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360 365 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 370 375 380 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 420 425 430 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460 Ser Leu Ser Leu Ser Pro Gly Lys 465 470 485PRTMus musculus 48Asp Tyr Tyr Met Asn 1 5 4919PRTMus musculus 49Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Asn Ala Ser 1 5 10 15 Val Lys Gly 5012PRTMus musculus 50Asp Ser Asp Gly Tyr Tyr Glu Tyr Tyr Phe Asp Tyr 1 5 10 51107PRTMus musculus 51Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Asp Tyr 20 25 30 Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile 35 40 45 Phe Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Phe Ser Leu Ser Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 52236PRTMus musculus 52Met Asp Met Arg Val Pro Ala His Val Phe Gly Phe Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Thr Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Leu Gly Glu Arg Val Ser Leu Thr Cys Arg Ala Ser 35 40 45 Gln Glu Ile Ser Asp Tyr Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly 50 55 60 Thr Ile Lys Arg Leu Ile Phe Ala Ala Ser Thr Leu Asp Ser Gly Val 65 70 75 80 Pro Lys Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Phe Ser Leu Ser 85 90 95 Ile Ser Ser Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln 100 105 110 Tyr Ala Ser Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 115 120 125 Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser 130 135 140 Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn 145 150 155 160 Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu 165 170 175 Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr 195 200 205 Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr 210 215 220 Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 235

53236PRTArtificial Sequencechimeric antibody light chain 53Met Asp Met Arg Val Pro Ala His Val Phe Gly Phe Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Thr Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Leu Gly Glu Arg Val Ser Leu Thr Cys Arg Ala Ser 35 40 45 Gln Glu Ile Ser Asp Tyr Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly 50 55 60 Thr Ile Lys Arg Leu Ile Phe Ala Ala Ser Thr Leu Asp Ser Gly Val 65 70 75 80 Pro Lys Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Phe Ser Leu Ser 85 90 95 Ile Ser Ser Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln 100 105 110 Tyr Ala Ser Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 5411PRTMus musculus 54Arg Ala Ser Gln Glu Ile Ser Asp Tyr Leu Ser 1 5 10 557PRTMus musculus 55Ala Ala Ser Thr Leu Asp Ser 1 5 569PRTMus musculus 56Leu Gln Tyr Ala Ser Tyr Pro Tyr Thr 1 5 571857DNAhomo sapiens 57atggtctccc cacggatgtc cgggctcctc tcccagactg tgatcctagc gctcattttc 60ctcccccaga cacggcccgc tggcgtcttc gagctgcaga tccactcttt cgggccgggt 120ccaggccctg gggccccgcg gtccccctgc agcgcccggc tcccctgccg cctcttcttc 180agagtctgcc tgaagcctgg gctctcagag gaggccgccg agtccccgtg cgccctgggc 240gcggcgctga gtgcgcgcgg accggtctac accgagcagc ccggagcgcc cgcgcctgat 300ctcccactgc ccgacggcct cttgcaggtg cccttccggg acgcctggcc tggcaccttc 360tctttcatca tcgaaacctg gagagaggag ttaggagacc agattggagg gcccgcctgg 420agcctgctgg cgcgcgtggc tggcaggcgg cgcttggcag ccggaggccc gtgggcccgg 480gacattcagc gcgcaggcgc ctgggagctg cgcttctcgt accgcgcgcg ctgcgagccg 540cctgccgtcg ggaccgcgtg cacgcgcctc tgccgtccgc gcagcgcccc ctcgcggtgc 600ggtccgggac tgcgcccctg cgcaccgctc gaggacgaat gtgaggcgcc gctggtgtgc 660cgagcaggct gcagccctga gcatggcttc tgtgaacagc ccggtgaatg ccgatgccta 720gagggctgga ctggacccct ctgcacggtc cctgtctcca ccagcagctg cctcagcccc 780aggggcccgt cctctgctac caccggatgc cttgtccctg ggcctgggcc ctgtgacggg 840aacccgtgtg ccaatggagg cagctgtagt gagacaccca ggtcctttga atgcacctgc 900ccgcgtgggt tctacgggct gcggtgtgag gtgagcgggg tgacatgtgc agatggaccc 960tgcttcaacg gcggcttgtg tgtcgggggt gcagaccctg actctgccta catctgccac 1020tgcccacccg gtttccaagg ctccaactgt gagaagaggg tggaccggtg cagcctgcag 1080ccatgccgca atggcggact ctgcctggac ctgggccacg ccctgcgctg ccgctgccgc 1140gccggcttcg cgggtcctcg ctgcgagcac gacctggacg actgcgcggg ccgcgcctgc 1200gctaacggcg gcacgtgtgt ggagggcggc ggcgcgcacc gctgctcctg cgcgctgggc 1260ttcggcggcc gcgactgccg cgagcgcgcg gacccgtgcg ccgcgcgccc ctgtgctcac 1320ggcggccgct gctacgccca cttctccggc ctcgtctgcg cttgcgctcc cggctacatg 1380ggagcgcggt gtgagttccc agtgcacccc gacggcgcaa gcgccttgcc cgcggccccg 1440ccgggcctca ggcccgggga ccctcagcgc taccttttgc ctccggctct gggactgctc 1500gtggccgcgg gcgtggccgg cgctgcgctc ttgctggtcc acgtgcgccg ccgtggccac 1560tcccaggatg ctgggtctcg cttgctggct gggaccccgg agccgtcagt ccacgcactc 1620ccggatgcac tcaacaacct aaggacgcag gagggttccg gggatggtcc gagctcgtcc 1680gtagattgga atcgccctga agatgtagac cctcaaggga tttatgtcat atctgctcct 1740tccatctacg ctcgggaggt agcgacgccc cttttccccc cgctacacac tgggcgcgct 1800gggcagaggc agcacctgct ttttccctac ccttcctcga ttctgtccgt gaaatga 1857581758DNAMus musculus 58atggtctctc tgcaggtgtc tccgctttcc cagacgctga tcctggcttt tcttcttcct 60caggcactgc cagctggtgt cttcgagcta caaattcatt ctttcgggcc aggcccaggc 120ctcgggaccc cacgctcccc ctgcaacgcc cgaggccctt gccgcctctt cttcagggtc 180tgcctgaagc ccggagtctc ccaggaggcc accgagtccc tgtgcgccct gggcgcagca 240ctgagcacga gcgtcccggt ctatacggag caccccggag agtcagcggc tgccctgccg 300ctgcctgatg gcctcgtacg tgtgcccttc cgcgatgctt ggccgggcac cttctccctc 360gtcattgaaa cctggagaga gcagctggga gagcatgctg gagggcccgc ctggaacctg 420ctagcacgtg tggtcggccg tagacgcctg gcggctgggg gcccgtgggc ccgcgatgtg 480cagcgcacag gcacatggga gttgcacttc tcctaccgcg cgcggtgcga gccgcccgcc 540gtcggggccg cctgcgcgcg cctgtgccgc tcacgcagtg ccccctcgcg gtgtggcccg 600ggactgcgac cctgcacgcc attcccagac gagtgcgaag ccccgtctgt gtgtcgacca 660ggctgcagcc ccgagcacgg ctactgtgaa gagcctgatg aatgccgttg cctggagggc 720tggactggac ccctctgcac ggtccctgtc tccaccagta gctgcctgaa ctccagggtt 780cctggtcctg ccagcactgg atgcctttta cctgggcctg gaccttgtga tgggaaccca 840tgtgccaatg ggggcagctg tagtgaaacc tctggctcct ttgaatgtgc ctgtccccgg 900ggattctacg ggcttcgatg tgaggtgagc ggggtcacgt gcgcagatgg accctgcttc 960aatggcggct tgtgtgttgg cggtgaagat cctgactctg cctatgtctg tcattgccca 1020cctggtttcc aaggctctaa ctgtgagaag agggtggacc gctgtagcct gcagccatgt 1080cagaatggcg gcctctgcct ggacctgggc cacgcgttgc gctgccgctg tcgcgcggga 1140ttcgccgggc cgcgctgcga gcacgacctg gacgactgcg ccggccgcgc ctgtgccaac 1200ggcggcacgt gcgtggaggg cggcggctcg cgccgctgct cctgtgcgct gggcttcggc 1260gggcgcgact gccgagaacg cgccgacccc tgcgcctccc gcccctgcgc gcatggaggc 1320cgttgctacg cccacttctc tggcctggtc tgcgcctgcg cgcccggcta catgggcgtg 1380agatgcgagt tcgctgtgcg cccggacggc gcggacgcgg tgcccgccgc cccgcggggc 1440ctgaggcagg cggatccaca gcgctttctt ctgcctcccg ccttggggct gctggtggcc 1500gccggtttgg ctggcgccgc actcttggtc atccacgttc gccgccgagg tcctggccag 1560gataccggga ctcgcctgct ttctgggacc cgggagcctt cggtccacac gctcccggat 1620gcactcaaca acctgaggtt acaagacggt gctggggatg gccccagttc gtcggctgac 1680tggaatcatc ctgaagatgg agactctaga tccatttatg tcataccagc cccttccatt 1740tatgcacgag aggcctga 1758592172DNAhomo sapiens 59atgggcagtc ggtgcgcgct ggccctggcg gtgctctcgg ccttgctgtg tcaggtctgg 60agctctgggg tgttcgaact gaagctgcag gagttcgtca acaagaaggg gctgctgggg 120aaccgcaact gctgccgcgg gggcgcgggg ccaccgccgt gcgcctgccg gaccttcttc 180cgcgtgtgcc tcaagcacta ccaggccagc gtgtcccccg agccgccctg cacctacggc 240agcgccgtca cccccgtgct gggcgtcgac tccttcagtc tgcccgacgg cgggggcgcc 300gactccgcgt tcagcaaccc catccgcttc cccttcggct tcacctggcc gggcaccttc 360tctctgatta ttgaagctct ccacacagat tctcctgatg acctcgcaac agaaaaccca 420gaaagactca tcagccgcct ggccacccag aggcacctga cggtgggcga ggagtggtcc 480caggacctgc acagcagcgg ccgcacggac ctcaagtact cctaccgctt cgtgtgtgac 540gaacactact acggagaggg ctgctccgtt ttctgccgtc cccgggacga tgccttcggc 600cacttcacct gtggggagcg tggggagaaa gtgtgcaacc ctggctggaa agggccctac 660tgcacagagc cgatctgcct gcctggatgt gatgagcagc atggattttg tgacaaacca 720ggggaatgca agtgcagagt gggctggcag ggccggtact gtgacgagtg tatccgctat 780ccaggctgtc tccatggcac ctgccagcag ccctggcagt gcaactgcca ggaaggctgg 840gggggccttt tctgcaacca ggacctgaac tactgcacac accataagcc ctgcaagaat 900ggagccacct gcaccaacac gggccagggg agctacactt gctcttgccg gcctgggtac 960acaggtgcca cctgcgagct ggggattgac gagtgtgacc ccagcccttg taagaacgga 1020gggagctgca cggatctcga gaacagctac tcctgtacct gcccacccgg cttctacggc 1080aaaatctgtg aattgagtgc catgacctgt gcggacggcc cttgctttaa cgggggtcgg 1140tgctcagaca gccccgatgg agggtacagc tgccgctgcc ccgtgggcta ctccggcttc 1200aactgtgaga agaaaattga ctactgcagc tcttcaccct gttctaatgg tgccaagtgt 1260gtggacctcg gtgatgccta cctgtgccgc tgccaggccg gcttctcggg gaggcactgt 1320gacgacaacg tggacgactg cgcctcctcc ccgtgcgcca acgggggcac ctgccgggat 1380ggcgtgaacg acttctcctg cacctgcccg cctggctaca cgggcaggaa ctgcagtgcc 1440cccgtcagca ggtgcgagca cgcaccctgc cacaatgggg ccacctgcca cgagaggggc 1500caccgctatg tgtgcgagtg tgcccgaggc tacgggggtc ccaactgcca gttcctgctc 1560cccgagctgc ccccgggccc agcggtggtg gacctcactg agaagctaga gggccagggc 1620gggccattcc cctgggtggc cgtgtgcgcc ggggtcatcc ttgtcctcat gctgctgctg 1680ggctgtgccg ctgtggtggt ctgcgtccgg ctgaggctgc agaagcaccg gcccccagcc 1740gacccctgcc ggggggagac ggagaccatg aacaacctgg ccaactgcca gcgtgagaag 1800gacatctcag tcagcatcat cggggccacg cagatcaaga acaccaacaa gaaggcggac 1860ttccacgggg accacagcgc cgacaagaat ggcttcaagg cccgctaccc agcggtggac 1920tataacctcg tgcaggacct caagggtgac gacaccgccg tcagggacgc gcacagcaag 1980cgtgacacca agtgccagcc ccagggctcc tcaggggagg agaaggggac cccgaccaca 2040ctcaggggtg gagaagcatc tgaaagaaaa aggccggact cgggctgttc aacttcaaaa 2100gacaccaagt accagtcggt gtacgtcata tccgaggaga aggatgagtg cgtcatagca 2160actgaggtgt aa 2172607668DNAhomo sapiens 60atgccgccgc tcctggcgcc cctgctctgc ctggcgctgc tgcccgcgct cgccgcacga 60ggcccgcgat gctcccagcc cggtgagacc tgcctgaatg gcgggaagtg tgaagcggcc 120aatggcacgg aggcctgcgt ctgtggcggg gccttcgtgg gcccgcgatg ccaggacccc 180aacccgtgcc tcagcacccc ctgcaagaac gccgggacat gccacgtggt ggaccgcaga 240ggcgtggcag actatgcctg cagctgtgcc ctgggcttct ctgggcccct ctgcctgaca 300cccctggaca atgcctgcct caccaacccc tgccgcaacg ggggcacctg cgacctgctc 360acgctgacgg agtacaagtg ccgctgcccg cccggctggt cagggaaatc gtgccagcag 420gctgacccgt gcgcctccaa cccctgcgcc aacggtggcc agtgcctgcc cttcgaggcc 480tcctacatct gccactgccc acccagcttc catggcccca cctgccggca ggatgtcaac 540gagtgtggcc agaagcccgg gctttgccgc cacggaggca cctgccacaa cgaggtcggc 600tcctaccgct gcgtctgccg cgccacccac actggcccca actgcgagcg gccctacgtg 660ccctgcagcc cctcgccctg ccagaacggg ggcacctgcc gccccacggg cgacgtcacc 720cacgagtgtg cctgcctgcc aggcttcacc ggccagaact gtgaggaaaa tatcgacgat 780tgtccaggaa acaactgcaa gaacgggggt gcctgtgtgg acggcgtgaa cacctacaac 840tgccgctgcc cgccagagtg gacaggtcag tactgtaccg aggatgtgga cgagtgccag 900ctgatgccaa atgcctgcca gaacggcggg acctgccaca acacccacgg tggctacaac 960tgcgtgtgtg tcaacggctg gactggtgag gactgcagcg agaacattga tgactgtgcc 1020agcgccgcct gcttccacgg cgccacctgc catgaccgtg tggcctcctt ctactgcgag 1080tgtccccatg gccgcacagg tctgctgtgc cacctcaacg acgcatgcat cagcaacccc 1140tgtaacgagg gctccaactg cgacaccaac cctgtcaatg gcaaggccat ctgcacctgc 1200ccctcggggt acacgggccc ggcctgcagc caggacgtgg atgagtgctc gctgggtgcc 1260aacccctgcg agcatgcggg caagtgcatc aacacgctgg gctccttcga gtgccagtgt 1320ctgcagggct acacgggccc ccgatgcgag atcgacgtca acgagtgcgt ctcgaacccg 1380tgccagaacg acgccacctg cctggaccag attggggagt tccagtgcat ctgcatgccc 1440ggctacgagg gtgtgcactg cgaggtcaac acagacgagt gtgccagcag cccctgcctg 1500cacaatggcc gctgcctgga caagatcaat gagttccagt gcgagtgccc cacgggcttc 1560actgggcatc tgtgccagta cgatgtggac gagtgtgcca gcaccccctg caagaatggt 1620gccaagtgcc tggacggacc caacacttac acctgtgtgt gcacggaagg gtacacgggg 1680acgcactgcg aggtggacat cgatgagtgc gaccccgacc cctgccacta cggctcctgc 1740aaggacggcg tcgccacctt cacctgcctc tgccgcccag gctacacggg ccaccactgc 1800gagaccaaca tcaacgagtg ctccagccag ccctgccgcc acgggggcac ctgccaggac 1860cgcgacaacg cctacctctg cttctgcctg aaggggacca caggacccaa ctgcgagatc 1920aacctggatg actgtgccag cagcccctgc gactcgggca cctgtctgga caagatcgat 1980ggctacgagt gtgcctgtga gccgggctac acagggagca tgtgtaacat caacatcgat 2040gagtgtgcgg gcaacccctg ccacaacggg ggcacctgcg aggacggcat caatggcttc 2100acctgccgct gccccgaggg ctaccacgac cccacctgcc tgtctgaggt caatgagtgc 2160aacagcaacc cctgcgtcca cggggcctgc cgggacagcc tcaacgggta caagtgcgac 2220tgtgaccctg ggtggagtgg gaccaactgt gacatcaaca acaatgagtg tgaatccaac 2280ccttgtgtca acggcggcac ctgcaaagac atgaccagtg gctacgtgtg cacctgccgg 2340gagggcttca gcggtcccaa ctgccagacc aacatcaacg agtgtgcgtc caacccatgt 2400ctgaaccagg gcacgtgtat tgacgacgtt gccgggtaca agtgcaactg cctgctgccc 2460tacacaggtg ccacgtgtga ggtggtgctg gccccgtgtg cccccagccc ctgcagaaac 2520ggcggggagt gcaggcaatc cgaggactat gagagcttct cctgtgtctg ccccacgggc 2580tggcaagggc agacctgtga ggtcgacatc aacgagtgcg ttctgagccc gtgccggcac 2640ggcgcatcct gccagaacac ccacggcggc taccgctgcc actgccaggc cggctacagt 2700gggcgcaact gcgagaccga catcgacgac tgccggccca acccgtgtca caacgggggc 2760tcctgcacag acggcatcaa cacggccttc tgcgactgcc tgcccggctt ccggggcact 2820ttctgtgagg aggacatcaa cgagtgtgcc agtgacccct gccgcaacgg ggccaactgc 2880acggactgcg tggacagcta cacgtgcacc tgccccgcag gcttcagcgg gatccactgt 2940gagaacaaca cgcctgactg cacagagagc tcctgcttca acggtggcac ctgcgtggac 3000ggcatcaact cgttcacctg cctgtgtcca cccggcttca cgggcagcta ctgccagcac 3060gatgtcaatg agtgcgactc acagccctgc ctgcatggcg gcacctgtca ggacggctgc 3120ggctcctaca ggtgcacctg cccccagggc tacactggcc ccaactgcca gaaccttgtg 3180cactggtgtg actcctcgcc ctgcaagaac ggcggcaaat gctggcagac ccacacccag 3240taccgctgcg agtgccccag cggctggacc ggcctttact gcgacgtgcc cagcgtgtcc 3300tgtgaggtgg ctgcgcagcg acaaggtgtt gacgttgccc gcctgtgcca gcatggaggg 3360ctctgtgtgg acgcgggcaa cacgcaccac tgccgctgcc aggcgggcta cacaggcagc 3420tactgtgagg acctggtgga cgagtgctca cccagcccct gccagaacgg ggccacctgc 3480acggactacc tgggcggcta ctcctgcaag tgcgtggccg gctaccacgg ggtgaactgc 3540tctgaggaga tcgacgagtg cctctcccac ccctgccaga acgggggcac ctgcctcgac 3600ctccccaaca cctacaagtg ctcctgccca cggggcactc agggtgtgca ctgtgagatc 3660aacgtggacg actgcaatcc ccccgttgac cccgtgtccc ggagccccaa gtgctttaac 3720aacggcacct gcgtggacca ggtgggcggc tacagctgca cctgcccgcc gggcttcgtg 3780ggtgagcgct gtgaggggga tgtcaacgag tgcctgtcca atccctgcga cgcccgtggc 3840acccagaact gcgtgcagcg cgtcaatgac ttccactgcg agtgccgtgc tggtcacacc 3900gggcgccgct gcgagtccgt catcaatggc tgcaaaggca agccctgcaa gaatgggggc 3960acctgcgccg tggcctccaa caccgcccgc gggttcatct gcaagtgccc tgcgggcttc 4020gagggcgcca cgtgtgagaa tgacgctcgt acctgcggca gcctgcgctg cctcaacggc 4080ggcacatgca tctccggccc gcgcagcccc acctgcctgt gcctgggccc cttcacgggc 4140cccgaatgcc agttcccggc cagcagcccc tgcctgggcg gcaacccctg ctacaaccag 4200gggacctgtg agcccacatc cgagagcccc ttctaccgtt gcctgtgccc cgccaaattc 4260aacgggctct tgtgccacat cctggactac agcttcgggg gtggggccgg gcgcgacatc 4320cccccgccgc tgatcgagga ggcgtgcgag ctgcccgagt gccaggagga cgcgggcaac 4380aaggtctgca gcctgcagtg caacaaccac gcgtgcggct gggacggcgg tgactgctcc 4440ctcaacttca atgacccctg gaagaactgc acgcagtctc tgcagtgctg gaagtacttc 4500agtgacggcc actgtgacag ccagtgcaac tcagccggct gcctcttcga cggctttgac 4560tgccagcgtg cggaaggcca gtgcaacccc ctgtacgacc agtactgcaa ggaccacttc 4620agcgacgggc actgcgacca gggctgcaac agcgcggagt gcgagtggga cgggctggac 4680tgtgcggagc atgtacccga gaggctggcg gccggcacgc tggtggtggt ggtgctgatg 4740ccgccggagc agctgcgcaa cagctccttc cacttcctgc gggagctcag ccgcgtgctg 4800cacaccaacg tggtcttcaa gcgtgacgca cacggccagc agatgatctt cccctactac 4860ggccgcgagg aggagctgcg caagcacccc atcaagcgtg ccgccgaggg ctgggccgca 4920cctgacgccc tgctgggcca ggtgaaggcc tcgctgctcc ctggtggcag cgagggtggg 4980cggcggcgga gggagctgga ccccatggac gtccgcggct ccatcgtcta cctggagatt 5040gacaaccggc agtgtgtgca ggcctcctcg cagtgcttcc agagtgccac cgacgtggcc 5100gcattcctgg gagcgctcgc ctcgctgggc agcctcaaca tcccctacaa gatcgaggcc 5160gtgcagagtg agaccgtgga gccgcccccg ccggcgcagc tgcacttcat gtacgtggcg 5220gcggccgcct ttgtgcttct gttcttcgtg ggctgcgggg tgctgctgtc ccgcaagcgc 5280cggcggcagc atggccagct ctggttccct gagggcttca aagtgtctga ggccagcaag 5340aagaagcggc gggagcccct cggcgaggac tccgtgggcc tcaagcccct gaagaacgct 5400tcagacggtg ccctcatgga cgacaaccag aatgagtggg gggacgagga cctggagacc 5460aagaagttcc ggttcgagga gcccgtggtt ctgcctgacc tggacgacca gacagaccac 5520cggcagtgga ctcagcagca cctggatgcc gctgacctgc gcatgtctgc catggccccc 5580acaccgcccc agggtgaggt tgacgccgac tgcatggacg tcaatgtccg cgggcctgat 5640ggcttcaccc cgctcatgat cgcctcctgc agcgggggcg gcctggagac gggcaacagc 5700gaggaagagg aggacgcgcc ggccgtcatc tccgacttca tctaccaggg cgccagcctg 5760cacaaccaga cagaccgcac gggcgagacc gccttgcacc tggccgcccg ctactcacgc 5820tctgatgccg ccaagcgcct gctggaggcc agcgcagatg ccaacatcca ggacaacatg 5880ggccgcaccc cgctgcatgc ggctgtgtct gccgacgcac aaggtgtctt ccagatcctg 5940atccggaacc gagccacaga cctggatgcc cgcatgcatg atggcacgac gccactgatc 6000ctggctgccc gcctggccgt ggagggcatg ctggaggacc tcatcaactc acacgccgac 6060gtcaacgccg tagatgacct gggcaagtcc gccctgcact gggccgccgc cgtgaacaat 6120gtggatgccg cagttgtgct cctgaagaac ggggctaaca aagatatgca gaacaacagg 6180gaggagacac ccctgtttct ggccgcccgg gagggcagct acgagaccgc caaggtgctg 6240ctggaccact ttgccaaccg ggacatcacg gatcatatgg accgcctgcc gcgcgacatc 6300gcacaggagc gcatgcatca cgacatcgtg aggctgctgg acgagtacaa cctggtgcgc 6360agcccgcagc tgcacggagc cccgctgggg ggcacgccca ccctgtcgcc cccgctctgc 6420tcgcccaacg gctacctggg cagcctcaag cccggcgtgc agggcaagaa ggtccgcaag 6480cccagcagca aaggcctggc ctgtggaagc aaggaggcca aggacctcaa ggcacggagg 6540aagaagtccc aggacggcaa gggctgcctg ctggacagct ccggcatgct ctcgcccgtg 6600gactccctgg agtcacccca tggctacctg tcagacgtgg cctcgccgcc actgctgccc 6660tccccgttcc agcagtctcc gtccgtgccc ctcaaccacc tgcctgggat gcccgacacc 6720cacctgggca tcgggcacct gaacgtggcg gccaagcccg agatggcggc gctgggtggg 6780ggcggccggc tggcctttga gactggccca cctcgtctct cccacctgcc tgtggcctct 6840ggcaccagca ccgtcctggg ctccagcagc ggaggggccc tgaatttcac tgtgggcggg 6900tccaccagtt tgaatggtca atgcgagtgg ctgtcccggc tgcagagcgg catggtgccg 6960aaccaataca accctctgcg ggggagtgtg gcaccaggcc ccctgagcac acaggccccc 7020tccctgcagc atggcatggt aggcccgctg cacagtagcc ttgctgccag cgccctgtcc 7080cagatgatga gctaccaggg cctgcccagc acccggctgg ccacccagcc tcacctggtg 7140cagacccagc aggtgcagcc acaaaactta cagatgcagc agcagaacct gcagccagca 7200aacatccagc agcagcaaag cctgcagccg ccaccaccac caccacagcc gcaccttggc 7260gtgagctcag

cagccagcgg ccacctgggc cggagcttcc tgagtggaga gccgagccag 7320gcagacgtgc agccactggg ccccagcagc ctggcggtgc acactattct gccccaggag 7380agccccgccc tgcccacgtc gctgccatcc tcgctggtcc cacccgtgac cgcagcccag 7440ttcctgacgc ccccctcgca gcacagctac tcctcgcctg tggacaacac ccccagccac 7500cagctacagg tgcctgagca ccccttcctc accccgtccc ctgagtcccc tgaccagtgg 7560tccagctcgt ccccgcattc caacgtctcc gactggtccg agggcgtctc cagccctccc 7620accagcatgc agtcccagat cgcccgcatt ccggaggcct tcaagtaa 7668614PRTartificialartificial linker 61Gly Gly Gly Ser 1 624PRTartificialartificial linker 62Ser Gly Gly Gly 1 635PRTartificialartificial linker 63Gly Gly Gly Gly Ser 1 5 645PRTartificialartificial linker 64Ser Gly Gly Gly Gly 1 5 656PRTartificialartificial linker 65Gly Gly Gly Gly Gly Ser 1 5 666PRTartificialartificial linker 66Ser Gly Gly Gly Gly Gly 1 5 677PRTartificialartificial linker 67Gly Gly Gly Gly Gly Gly Ser 1 5 687PRTartificialartificial linker 68Ser Gly Gly Gly Gly Gly Gly 1 5 69475PRTMus musculus 69Met Glu Trp Ser Gly Val Phe Ile Phe Leu Leu Ser Val Thr Ala Gly1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Asp Leu Val Arg 20 25 30 Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe 35 40 45 Thr Asn Tyr Leu Ile Glu Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Met Asn Pro Gly Ser Gly Gly Thr His Tyr Ser65 70 75 80Glu Lys Phe Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ile Ser Leu Thr Ser Asp Asp Ser Ala Val 100 105 110 Tyr Phe Cys Ala Arg Ser Asp Tyr Asp Tyr Val Thr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr 130 135 140 Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Gly Thr Thr Gly145 150 155 160Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro 165 170 175 Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr 180 185 190 Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Leu Ser Ser Ser Val 195 200 205 Thr Val Thr Ser Asn Thr Trp Pro Ser Gln Thr Ile Thr Cys Asn Val 210 215 220 Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg225 230 235 240Val Pro Ile Thr Gln Asn Pro Cys Pro Pro Leu Lys Glu Cys Pro Pro 245 250 255 Cys Ala Ala Pro Asp Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro 260 265 270 Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Met Val Thr 275 280 285 Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser 290 295 300 Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His305 310 315 320Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile 325 330 335 Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn 340 345 350 Asn Arg Ala Leu Pro Ser Pro Ile Glu Lys Thr Ile Ser Lys Pro Arg 355 360 365 Gly Pro Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Ala Glu 370 375 380 Glu Met Thr Lys Lys Glu Phe Ser Leu Thr Cys Met Ile Thr Gly Phe385 390 395 400Leu Pro Ala Glu Ile Ala Val Asp Trp Thr Ser Asn Gly Arg Thr Glu 405 410 415 Gln Asn Tyr Lys Asn Thr Ala Thr Val Leu Asp Ser Asp Gly Ser Tyr 420 425 430 Phe Met Tyr Ser Lys Leu Arg Val Gln Lys Ser Thr Trp Glu Arg Gly 435 440 445 Ser Leu Phe Ala Cys Ser Val Val His Glu Gly Leu His Asn His Leu 450 455 460 Thr Thr Lys Thr Ile Ser Arg Ser Leu Gly Lys465 470 47570472PRTMus musculus 70Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Gly Gly1 5 10 15 Val Leu Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Arg Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Tyr Met Lys Trp Val Lys Gln Ser His Gly Lys Ser Leu 50 55 60 Glu Trp Ile Gly Asp Ile Asn Pro Asn Asn Gly Asp Thr Phe Tyr Asn65 70 75 80Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ile Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Asn Ser Leu Thr Ser Asp Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Asp Gly Asn Tyr Ala Tyr Phe Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser 130 135 140 Val Tyr Pro Leu Ala Pro Val Cys Gly Gly Thr Thr Gly Ser Ser Val145 150 155 160Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu 165 170 175 Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala 180 185 190 Leu Leu Gln Ser Gly Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr 195 200 205 Ser Asn Thr Trp Pro Ser Gln Thr Ile Thr Cys Asn Val Ala His Pro 210 215 220 Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Val Pro Ile225 230 235 240Thr Gln Asn Pro Cys Pro Pro Leu Lys Glu Cys Pro Pro Cys Ala Ala 245 250 255 Pro Asp Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile 260 265 270 Lys Asp Val Leu Met Ile Ser Leu Ser Pro Met Val Thr Cys Val Val 275 280 285 Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val 290 295 300 Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp305 310 315 320Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln 325 330 335 Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Arg Ala 340 345 350 Leu Pro Ser Pro Ile Glu Lys Thr Ile Ser Lys Pro Arg Gly Pro Val 355 360 365 Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Ala Glu Glu Met Thr 370 375 380 Lys Lys Glu Phe Ser Leu Thr Cys Met Ile Thr Gly Phe Leu Pro Ala385 390 395 400Glu Ile Ala Val Asp Trp Thr Ser Asn Gly Arg Thr Glu Gln Asn Tyr 405 410 415 Lys Asn Thr Ala Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr 420 425 430 Ser Lys Leu Arg Val Gln Lys Ser Thr Trp Glu Arg Gly Ser Leu Phe 435 440 445 Ala Cys Ser Val Val His Glu Gly Leu His Asn His Leu Thr Thr Lys 450 455 460 Thr Ile Ser Arg Ser Leu Gly Lys465 470 71477PRTMus musculus 71Met Lys Leu Trp Leu Asn Trp Ile Phe Leu Val Thr Leu Leu Asn Gly1 5 10 15 Ile Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Thr Asp Tyr Tyr Met Asn Trp Val Arg Gln Pro Pro Gly Lys Ala Leu 50 55 60 Glu Trp Leu Ala Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu65 70 75 80Tyr Asn Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 85 90 95 Gln Asn Ile Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Ser 100 105 110 Ala Thr Tyr Tyr Cys Ala Arg Asp Ser Asp Gly Tyr Tyr Glu Tyr Tyr 115 120 125 Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys 130 135 140 Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Gly Thr145 150 155 160Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro 165 170 175 Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val 180 185 190 His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Leu Ser Ser 195 200 205 Ser Val Thr Val Thr Ser Asn Thr Trp Pro Ser Gln Thr Ile Thr Cys 210 215 220 Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu225 230 235 240Pro Arg Val Pro Ile Thr Gln Asn Pro Cys Pro Pro Leu Lys Glu Cys 245 250 255 Pro Pro Cys Ala Ala Pro Asp Leu Leu Gly Gly Pro Ser Val Phe Ile 260 265 270 Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Met 275 280 285 Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln 290 295 300 Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln305 310 315 320Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu 325 330 335 Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys 340 345 350 Val Asn Asn Arg Ala Leu Pro Ser Pro Ile Glu Lys Thr Ile Ser Lys 355 360 365 Pro Arg Gly Pro Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro 370 375 380 Ala Glu Glu Met Thr Lys Lys Glu Phe Ser Leu Thr Cys Met Ile Thr385 390 395 400Gly Phe Leu Pro Ala Glu Ile Ala Val Asp Trp Thr Ser Asn Gly Arg 405 410 415 Thr Glu Gln Asn Tyr Lys Asn Thr Ala Thr Val Leu Asp Ser Asp Gly 420 425 430 Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Gln Lys Ser Thr Trp Glu 435 440 445 Arg Gly Ser Leu Phe Ala Cys Ser Val Val His Glu Gly Leu His Asn 450 455 460 His Leu Thr Thr Lys Thr Ile Ser Arg Ser Leu Gly Lys465 470 475

Claims

1. An anti-DLL3 antibody which can bind to DLL3 having the amino acid sequence set forth in SEQ ID NO: 1, wherein the antibody has an internalizing activity and conjugates one or more cytotoxic substances.

2. The anti-DLL3 antibody according to claim 1, wherein the antibody can bind to an epitope located between residues 27 and 175 of SEQ ID NO: 1.

3. The anti-DLL3 antibody according to claim 1, wherein the antibody can bind to an epitope located between residues 27 and 215 of SEQ ID NO: 1.

4. The anti-DLL3 antibody according to claim 1, wherein the antibody is a chimera or humanized antibody.

5. A method of inducing death of a cell expressing DLL3 having the amino acid sequence set forth in SEQ ID NO: 1 comprising contacting the cell with an anti-DLL3 antibody conjugated to one or more cytotoxic substances, wherein the antibody has an internalizing activity.

6. The method according to claim 5, wherein the antibody can bind to an epitope located between residues 27 and 175 of SEQ ID NO: 1.

7. The method according to claim 5, wherein the antibody can bind to an epitope located between residues 27 and 215 of SEQ ID NO: 1.

8. The method according to claim 5, wherein the antibody is a chimera or humanized antibody.

9. A method of suppressing growth of a cell expressing DLL3 as set forth in SEQ ID NO: 1 comprising contacting the cell with an anti-DLL3 antibody conjugated to one or more cytotoxic substances.

10. The method according to claim 9, wherein the antibody can bind to an epitope located between residues 27 and 175 of SEQ ID NO: 1.

11. The method according to claim 9, wherein the antibody can bind to an epitope located between residues 27 and 215 of SEQ ID NO: 1.

12. The method according to claim 9, wherein the antibody is a chimera or humanized antibody.

13. A method of treating cancer comprising administering an anti-DLL3 antibody which can bind to DLL3 having the amino acid sequence set forth in SEQ ID NO: 1 to a subject having cancer or suspected of having cancer, wherein the antibody has an internalizing activity and is conjugated to one or more cytotoxic substances.

14. The method according to claim 13, wherein the antibody can bind to an epitope located between residues 27 and 175 of SEQ ID NO: 1.

15. The method according to claim 13, wherein the antibody can bind to an epitope located between residues 27 and 215 of SEQ ID NO: 1.

16. The method according to claim 13, wherein the antibody is a chimera or humanized antibody.

17. The method according to claim 13, wherein the cancer is a lung cancer.

18. The method according to claim 17, wherein the lung cancer is a small-cell lung cancer.