Therapeutic antibodies and uses thereof

Abstract

The present invention provides antibodies or antigen-binding fragments thereof specific for variant exon v9 (CD44v9) of the CD44 gene, antibody-drug conjugates (ADCs) and other derivatives comprising the antibodies or antigen-binding fragments, nucleic acid molecules encoding the antibodies or antigen-binding fragments, and methods for making the antibodies or antigen-binding fragments, pharmaceutical compositions comprising the antibodies and antigen-binding fragments, and uses of the antibodies or antigen-binding fragments in the treatment of disease or in the manufacture of a medicament for the treatment of disease, such as cancer, are also provided.

Description

[Background technology]

[0001] CD44 is a family of transmembrane glycoproteins involved in homotypic cell, cell-matrix, and cell-cytoskeleton interactions. The extracellular domain of CD44 binds to many matrix substrates: hyaluronan, ezrin, radixin, moesin, and merlin, heparin-affinity growth factor, vascular endothelial growth factor, p185HER2, epidermal growth factor, and hepatocyte growth factor. Its intracellular domain binds to the cytoskeletal substrate ankyrin, thus determining cell and tissue architectural types (Bourguignon et al., 1998, Front. Biosci. 3:D637-649; Welch et al., 1995, J. Cell. Physiol. 164:605-612).

[0002] The CD44 gene is mapped to chromosome 11, contains 20 exons spanning 60 kb, and can be subdivided into five structural domains. Ten of the 20 exons, exons 1–5 and 16–20, constitute the standard form of CD44 (CD44s or CD44std). This minimal CD44 isoform (CD44s) is ubiquitously expressed in different tissues, including epithelial cells, whereas specific CD44 splice variants (CD44v, CD44var) are expressed only on a subset of epithelial cells.

[0003] CD44 variants are generated by alternative splicing at the messenger RNA (mRNA) level, in which sequences from 10 exons (v1–v10) in the extracellular portion of the protein are completely excised in CD44s but can appear in different combinations in larger variants (Screaton et al., 1992; Toelg et al., 1993; Hofmann et al., 1991). The variants differ by the insertion of different amino acid sequences at specific sites in the extracellular portion of the protein. Theoretically, there are over 1,000 potential peptide domain combinations for CD44 variant (CD44v) isoforms. Inclusion of all variant exons results in a protein with a molecular weight of 230 kD, although most variant isoforms are less than 120 kD. Longer variant isoforms (CD44v1–10) contain one or more of exons 6–15 by splicing, but exon 6 (v1) is not expressed in humans.

[0004] Some splice variants are expressed by normal epithelial cells in a tissue-specific manner, for example, CD44v10 is expressed by normal lymphocytes (Okamoto et al., 1998, J. Natl. Cancer Inst. 90:307-315).

[0005] However, it has recently been shown that expression of specific CD44 variants is necessary and sufficient to induce the so-called spontaneous metastatic behavior of nonmetastatic rat pancreatic adenocarcinoma and fibrosarcoma cell lines (Guenthert et al., 1991). These variants can be detected in a variety of human tumor cells and tissues.

[0006] For example, Okamoto et al. (Okamoto et al., 2002, Am. J. Pathol. 160:441-447; Okamoto et al., 2001, J. Cell. Biol. 155:755-762; Murakami et al., 2003, Oncogene 22:1511-1516) have shown that in several human tumors (excluding prostate cancer), a 25-30 kD cleavage product can be detected by Western blot using an antibody against the cytoplasmic portion of CD44. The soluble portion of CD44 has been detected in serum as a 100-160 kD fragment using an anti-CD44v monoclonal antibody against the extracellular portion of the molecule (Gansauge et al., 1997, Cancer 80:1733-1739). Western blot detection of shed CD44 isoforms in the circulation can serve as a diagnostic or prognostic test for malignancies (Taylor et al., 1996, J. Soc. Gynecol. Invest. 3:289-294). Enzyme-linked serum immunoassays (ELISAs) may then be developed for sensitive and easier detection of the protein. Amplification of CD44v6 has been associated with the metastatic phenotype of pancreatic cancer (Rall and Rustgi, 1995, Cancer Res. 55:1831-1835) and with increasing grade of breast cancer (Woodman et al., 1996, Am. J. Pathol. 149:1519-1530; Bourguignon et al., 1999, Cell Motil. Cytoskeleton 43:269-287).Inclusion of single or consecutive mutant exons has been described by RT-PCR and sequencing in many benign and cancerous tissues (Okamoto et al., 1998, J. Natl. Cancer Inst. 90:307-315; Okamoto et al., 2002, Am. J. Pathol. 160:441-447; Rall and Rustgi, 1995, Cancer Res. 55:1831-1835; Woodman et al., 1996, Am. J. Pathol. 149:1519-1530; Bourguignon et al., 1999, Cell Motil. Cytoskeleton 43:269-287; Roca et al., 1998, Am. J. Pathol. 153:183-190; Franzmann et al., 2001, Otolaryngol. Head Neck Surg. 124:426-432;Terpe et al., 1996, Am. J. Pathol. 148:453-463;Christ et al., 2001, J. Leukoc. Biol. 69:343-352;Mortegani et al., 1999, Am. J. Pathol. 154:291-300;Miyake et al. 1998, Int. J. Cancer. 18:560-564; Yamaguchi et al., 1996, J. Clin. Oncol. 14:1122-1127).

[0007] During metastasis, tumor cells leave the primary site, migrate into the extracellular matrix, and invade blood and lymphatic vessels. Tumor outgrowth at metastatic sites requires attachment to new extracellular matrix through adhesion proteins such as CD44. This is consistent with the fact that many cancers have deregulated CD44 mRNA splicing, leading to the expression of novel CD44 variant isoforms that may play a role in metastasis.

[0008] Indeed, the expression of CD44 variants during colorectal carcinogenesis has recently been studied (Heider et al., 1993a). Expression of CD44 variants is absent in normal human colonic epithelium, and only weak expression is detectable in proliferating cells of the crypts. At later stages of tumor progression, e.g., in adenocarcinomas, all malignant tumors express variants of CD44. High levels of tissue expression of variant CD44 have also been shown in aggressive non-Hodgkin's lymphomas (Koopman et al., 1993).

[0009] Exon v6 appears to play a particularly special role in the process of metastatic spread (Rudy et al., 1993). In animal models, antibodies against a v6-specific epitope can prevent the establishment of metastatic cells and the growth of metastases (Seiter et al., 1993). In colon cancer, v6 expression correlates with tumor progression (Wielenga et al., 1993). In gastric cancer, v6 expression is an important diagnostic marker that distinguishes intestinal-type tumors from diffuse-type tumors (Heider et al., 1993b). In the latter two publications, v6 expression has been determined using antibodies against a v6-specific epitope.

[0010] CD44v6 has been shown to be a tumor-associated antigen with a preferred expression pattern in human tumors and normal tissues (Heider et al., 1995; Heider et al., 1996), making it the target of antibody-based diagnostic and therapeutic approaches (Heider et al., 1996; WO 95 / 33771; WO 97 / 21104).

[0011] Meanwhile, aberrant expression of CD44v9 has been associated with gastric cancer, colon cancer, breast cancer, lung cancer, and head and neck squamous cell carcinoma (US20170137810A1). Both CD44v6 and CD44v9 have previously been shown to be overexpressed in colon cancer (Wielenga et al., Am. J. Pathol., 1999, 154: 515-523). CD44v9 has also been found to be overexpressed in gastric cancer (Ue et al., Co-expression of osteopontin and CD44v9 in gastric cancer. Int J Cancer 1998; 79:127-132).

[0012] CD44v9-positive cells exhibit an enhanced ability to suppress ROS production, which subsequently leads to therapy resistance, recurrence, and tumor metastasis (Ishimoto et al., 2011; Tsugawaet et al., 2012; Yae et al., 2012). CD44v9 has also been reported to be a cancer stem cell marker in various tumor types (Aso et al., 2015).

[0013] One serious problem that arises when using non-human antibodies for human applications is that these antibodies rapidly generate human-anti-non-human reactions, which reduce the efficacy of the antibody in patients and impair continuous administration. To overcome this problem, the concept of "humanized" non-human antibodies has been developed in the art. In the first approach, attempts have been made to achieve humanization of non-human antibodies by constructing non-human / human chimeric antibodies in which non-human variable regions are linked to human constant regions (Boulianne GL, Hozumi N. and Shulman, MJ (1984) Production of functional chimeric mouse / human antibody Nature 312: 643). The chimeric antibodies thus generated retain the binding specificity and affinity of the original non-human antibody.

[0014] Although chimeric antibodies are significantly superior to murine antibodies, they can still induce anti-chimeric responses in humans (LoBuglio AF, Wheeler RH, Trang J., Haynes A., Rogers K., Harvey EB, Sun L., Ghrayeb J., and Khazaeli MB (1989) Mouse / human chimeric monoclonal antibody in man: Kinetics and immune response. Proc. Natl. Acad. Sci. 86: 4220). This approach was later improved by further reducing the amount of nonhuman sequence by grafting complementarity-determining regions (CDRs) from nonhuman variable regions onto human variable regions and then linking these "reshaped human" variable regions to human constant regions (Riechmann L., Clark M., Waldmann H., and Winter G. (1988) Reshaping human antibodies for therapy. Nature 332: 323). CDR-grafted or reshaped human antibodies contain little or no protein sequences that can be identified as being derived from mouse antibodies. Although antibodies humanized by CDR-grafting may still be able to induce some immune responses, such as anti-allotypic or anti-idiotypic responses, as even naturally occurring human antibodies can, CDR-grafted antibodies are significantly less immunogenic than mouse antibodies, thus allowing for longer-term treatment of patients.

[0015] However, it soon became apparent that CDR-grafting alone does not always produce antibodies with sufficient binding affinity. CDR-grafted antibodies sometimes have relatively poor binding properties compared to their parent nonhuman antibodies, for example, because more amino acids than those within the CDRs may be involved in antigen binding. As a result, CDR-grafted antibodies with poor binding affinity are not considered useful in therapy. Therefore, attempts have been made to generate antibodies that combine the low immunogenicity of CDR-grafted antibodies with the excellent binding properties of the nonhuman parent antibody. In addition to CDR-grafting, the concept has evolved that one or several amino acids in the humanized framework region must be retained as residues of rodent donor origin to maintain binding affinity (Queen et al. (1989) Proc. Natl. Acad. Sci. 86: 10029-10033).

[0016] Because of the great potential utility that such antibodies can have in diagnostics and therapy, there is a need for antibodies with improved properties suitable for the treatment of human diseases, such as various cancers.

[0017] One problem underlying the present invention was to provide alternative CD44v9-specific antibodies, preferably with superior properties compared to known CD44v9-specific antibodies. [Prior art documents] [Patent documents]

[0018] [Patent Document 1] WO 95 / 33771 [Patent Document 2] WO 97 / 21104 [Patent Document 3] US20170137810A1 [Non-patent literature]

[0019] [Non-Patent Document 1] Bourguignon, 1998, Front. Biosci. 3:D637-649 [Non-licensed document 2] Welch, 1995, J. Cell. Physiol. 164:605-612 [Non-licensed document 3] Screatonら、1992

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[0020] One aspect of the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof specific for CD44v9, wherein the monoclonal antibody comprises: (1) a heavy chain variable region (HCVR) comprising (a) the HCVR CDR1 sequence of SEQ ID NO: 1, wherein the HCVR comprises up to two amino acid mutations, (b) the HCVR CDR2 sequence of SEQ ID NO: 2, wherein the HCVR comprises up to two amino acid mutations, and / or (c) the HCVR CDR3 sequence of SEQ ID NO: 3, wherein the HCVR comprises up to five amino acid mutations; and / or (2) a light chain variable region (LCVR) comprising (d) the LCVR CDR1 sequence of SEQ ID NO: 4, wherein the HCVR comprises up to two amino acid mutations, (e) the LCVR CDR2 sequence of SEQ ID NO: 5, wherein the LCVR comprises up to two amino acid mutations, and / or (f) the LCVR CDR3 sequence of SEQ ID NO: 6, wherein the LCVR comprises up to two amino acid mutations; optionally, the monoclonal antibody is not mAb116.

[0021] In certain embodiments, the heavy chain variable region (HCVR) of the monoclonal antibody or antigen-binding fragment comprises (a) the HCVR CDR1 sequence of SEQ ID NO: 1; (b) the HCVR CDR2 sequence of SEQ ID NO: 2, which comprises up to one amino acid mutation at the alanine residue at position 6; and / or (c) the HCVR CDR3 sequence of SEQ ID NO: 3, which comprises up to five amino acid mutations at the arginine residue at position 2, the alanine residue at position 4, the aspartic acid residue at position 5, the asparagine residue at position 7, and the proline residue at position 8, and / or the light chain variable region (LCVR) comprises (d) the LCVR CDR1 sequence of SEQ ID NO: 4; (e) the LCVR CDR2 sequence of SEQ ID NO: 5; and / or (f) the LCVR CDR3 sequence of SEQ ID NO: 6, which comprises up to two amino acid mutations at the leucine residues at positions 1 and 8.

[0022] In certain embodiments, the heavy chain variable region (HCVR) of the monoclonal antibody or antigen-binding fragment comprises (a) the HCVR CDR1 sequence of SEQ ID NO: 1; (b) the HCVR CDR2 sequence of SEQ ID NO: 2, in which the alanine residue at position 6 is optionally substituted with glycine; and / or (c) the HCVR CDR3 sequence of SEQ ID NO: 3, in which the arginine residue at position 2 is optionally substituted with serine, the alanine residue at position 4 is optionally substituted with glycine, the aspartic acid residue at position 5 is optionally substituted with glutamic acid, the asparagine residue at position 7 is optionally substituted with threonine, and the proline residue at position 8 is optionally substituted with glycine; and / or the light chain variable region (LCVR) comprises (d) the LCVR CDR1 sequence of SEQ ID NO: 4; (e) the LCVR CDR2 sequence of SEQ ID NO: 5; and / or (f) the LCVR CDR3 sequence of SEQ ID NO: 6, in which the leucine residue at position 1 is optionally substituted with methionine and the leucine residue at position 8 is optionally substituted with phenylalanine.

[0023] In certain embodiments, the monoclonal antibody or antigen-binding fragment comprises: (1) a heavy chain variable region (HCVR) comprising (a) the HCVR CDR1 sequence of SEQ ID NO: 1, (b) the HCVR CDR2 sequence of SEQ ID NO: 2, and / or (c) the HCVR CDR3 sequence of SEQ ID NO: 3; and / or (2) a light chain variable region (LCVR) comprising (d) the LCVR CDR1 sequence of SEQ ID NO: 4, (e) the LCVR CDR2 sequence of SEQ ID NO: 5, and / or (f) the LCVR CDR3 sequence of SEQ ID NO: 6.

[0024] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof binds to said CD44v9 or a cell bearing said CD44v9 with a K of about 40 nM, 20 nM, 10 nM, about 5 nM, about 2 nM, about 1 nM or less. D Combine with.

[0025] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is a murine monoclonal antibody.

[0026] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment further comprises: (1) a heavy chain framework region sequence of FR1 of SEQ ID NO: 7, FR2 of SEQ ID NO: 8, FR3 of SEQ ID NO: 9, and / or FR4 of SEQ ID NO: 10, respectively, comprising up to two amino acid mutations in each framework region sequence; and / or (2) a light chain framework region sequence of FR1 of SEQ ID NO: 11, FR2 of SEQ ID NO: 12, FR3 of SEQ ID NO: 13, and / or FR4 of SEQ ID NO: 14, respectively, comprising up to two amino acid mutations in each framework region sequence; or (i) a heavy chain framework region sequence of FR1 of SEQ ID NO: 37, FR2 of SEQ ID NO: 38, FR3 of SEQ ID NO: 39, and FR4 of SEQ ID NO: 40, respectively, comprising up to two amino acid mutations in each framework region sequence; and (ii) a light chain framework region sequence of FR1 of SEQ ID NO: 41, FR2 of SEQ ID NO: 42, FR3 of SEQ ID NO: 43, and FR4 of SEQ ID NO: 44, respectively, comprising up to two amino acid mutations in each framework region sequence.

[0027] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises (1) a heavy chain framework region sequence of FR1 of SEQ ID NO:7, FR2 of SEQ ID NO:8, FR3 of SEQ ID NO:9, and / or FR4 of SEQ ID NO:10, respectively, and / or (2) a light chain framework region sequence of FR1 of SEQ ID NO:11, FR2 of SEQ ID NO:12, FR3 of SEQ ID NO:13, and / or FR4 of SEQ ID NO:14, respectively.

[0028] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment comprises (1) a heavy chain framework region sequence of FR1 of SEQ ID NO: 37, FR2 of SEQ ID NO: 38, FR3 of SEQ ID NO: 39, and / or FR4 of SEQ ID NO: 40, respectively, and / or (2) a light chain framework region sequence of FR1 of SEQ ID NO: 41, FR2 of SEQ ID NO: 42, FR3 of SEQ ID NO: 43, and / or FR4 of SEQ ID NO: 44, respectively.

[0029] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is a human-mouse chimeric antibody.

[0030] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is a humanized murine monoclonal antibody.

[0031] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof further comprises (1) a heavy chain framework region sequence of FR1 of SEQ ID NO: 15, FR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and / or FR4 of SEQ ID NO: 18, respectively, and / or (2) a light chain framework region sequence of FR1 of SEQ ID NO: 19, FR2 of SEQ ID NO: 20, FR3 of SEQ ID NO: 21, and / or FR4 of SEQ ID NO: 22, respectively.

[0032] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof further comprises (1) a heavy chain framework region sequence of FR1 of SEQ ID NO: 23, FR2 of SEQ ID NO: 24, FR3 of SEQ ID NO: 25, and / or FR4 of SEQ ID NO: 26, respectively, and / or (2) a light chain framework region sequence of FR1 of SEQ ID NO: 27, FR2 of SEQ ID NO: 28, FR3 of SEQ ID NO: 29, and / or FR4 of SEQ ID NO: 30, respectively.

[0033] In certain embodiments, the antigen-binding fragment is a Fab, Fab', F(ab'), F d , single chain Fv or scFv, disulfide-linked F v , V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0034] Another aspect of the invention provides polypeptides comprising the HCVR and / or LCVR of any one of the subject anti-CD44v9 antibodies or antigen-binding fragments thereof.

[0035] In certain embodiments, the polypeptide is a fusion protein (eg, a chimeric antigen T cell receptor).

[0036] Another aspect of the invention provides polynucleotides encoding any of the subject polypeptides.

[0037] Another aspect of the invention provides a vector comprising any of the subject polynucleotides.

[0038] In certain embodiments, the vector is an expression vector (eg, a mammalian expression vector, a yeast expression vector, an insect expression vector, or a bacterial expression vector).

[0039] Another aspect of the invention provides a cell comprising any of the subject anti-CD44v9 antibodies or antigen-binding fragments thereof, any of the subject polypeptides, any of the subject polynucleotides, or any of the subject vectors.

[0040] In certain embodiments, the cells express any of the subject antibodies or antigen-binding fragments thereof, or any of the subject polypeptides.

[0041] In certain embodiments, the cells are BHK cells, CHO cells, or COS cells.

[0042] In certain embodiments, the cells comprise on their cell surface either a subject anti-CD44v9 antibody or antigen-binding fragment thereof, or either a subject polypeptide.

[0043] In certain embodiments, the cell is a T cell bearing a chimeric antigen receptor (CAR-T cell) comprising any of the subject antibodies or antigen-binding fragments thereof, or any of the subject polypeptides.

[0044] Another aspect of the invention provides methods for producing a subject anti-CD44v9 antibody, or antigen-binding fragment thereof, comprising culturing a subject cell, and isolating the subject antibody, antigen-binding fragment thereof, or polypeptide from the cultured cell.

[0045] Another aspect of the present invention is a compound of the formula: Ab-[-LD] n wherein Ab is either a subject anti-CD44v9 antibody or antigen-binding fragment thereof, or a subject polypeptide thereof, covalently linked to one or more units of a linker-drug moiety -[-LD], L is a linker, and D is a cytotoxic agent; and n is an integer between 1 and 20 (e.g., 1 and 12); and each linker-drug moiety may have the same or different linker L or cytotoxic agent D.

[0046] In certain embodiments, each linker-drug moiety -[-LD] is covalently attached to the Ab through the side chain amino group of Lys.

[0047] In certain embodiments, each linker-drug moiety -[-LD] is covalently attached to an Ab through the side chain thiol group of a Cys.

[0048] In certain embodiments, each linker-drug moiety -[-LD] is covalently attached to the Ab through a site-specifically incorporated unnatural amino acid.

[0049] In certain embodiments, each linker L comprises a peptide unit.

[0050] In certain embodiments, a peptide unit comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-10, or 2-5 amino acid residues.

[0051] In certain embodiments, the linker L is not cleavable by a protease (eg, a cathepsin).

[0052] In certain embodiments, the linker L is a cleavable linker that is cleavable by a protease (eg, a cathepsin), an acidic environment, or a change in redox state.

[0053] In certain embodiments, the cytotoxic agent is a DNA intercalator, a microtubule binding agent, a topoisomerase I inhibitor, or a DNA minor groove binding agent.

[0054] In certain embodiments, the cytotoxic agent is a member of the auristatin class, e.g., monomethylauristatin E (MMAE) and MMAF, a maytansine class, e.g., DM-1, DM-3, DM-4, ​​a calicheamicin, e.g., ozogamicin, SN-38, or a PBD (pyrrolobenzodiazepine).

[0055] Another aspect of the present invention provides pharmaceutical compositions comprising a subject anti-CD44v9 antibody or antigen-binding fragment thereof, or a polypeptide thereof, or an immunoconjugate thereof, and a pharmaceutically acceptable carrier or excipient.

[0056] Another aspect of the invention provides a method for inhibiting the proliferation of a cell expressing CD44v9, the method comprising the step of contacting the cell with either a subject anti-CD44v9 antibody or antigen-binding fragment thereof, or a subject polypeptide thereof, or a subject immunoconjugate thereof, or a subject pharmaceutical composition thereof.

[0057] In certain embodiments, the cell is a tumor cell.

[0058] In certain embodiments, the tumor cells are from a lung cancer (eg, NSCLC).

[0059] In particular embodiments, the tumor cells are from colorectal cancer, breast cancer, head and neck cancer, ovarian cancer, bladder cancer, pancreatic cancer, or metastatic cancer of the brain.

[0060] Another aspect of the invention provides a method for inhibiting the proliferation of a cell expressing CD44v9, the method comprising contacting the cell with any of a subject anti-CD44v9 antibody or antigen-binding fragment thereof, or a polypeptide thereof, or an immunoconjugate thereof, or a pharmaceutical composition thereof.

[0061] In certain embodiments, the cell is a tumor cell.

[0062] In certain embodiments, the tumor cells are from a lung cancer (eg, NSCLC).

[0063] In particular embodiments, the tumor cells are from colorectal cancer, breast cancer, liver cancer, head and neck cancer, ovarian cancer, bladder cancer, pancreatic cancer, or metastatic cancer of the brain.

[0064] Another aspect of the present invention provides a method for treating a subject having cancer, wherein the cancer cells express CD44v9, the method comprising administering to the subject a therapeutically effective amount of a CD44v9 antagonist comprising a CD44v9 antibody or antigen-binding fragment thereof.

[0065] Another aspect of the invention provides a method for treating a cell proliferative disorder in a subject, wherein cells of the cell proliferative disorder express CD44v9, the method comprising administering to the subject a therapeutically effective amount of a CD44v9 antagonist comprising a CD44v9 antibody or antigen-binding fragment thereof.

[0066] In certain embodiments, the CD44v9 antagonist comprises either a subject anti-CD44v9 antibody or antigen-binding fragment thereof, or a subject polypeptide thereof, or a subject immunoconjugate thereof, or a subject pharmaceutical composition thereof.

[0067] In particular embodiments, the cancer is an epithelial carcinoma, including those of breast, lung, liver, colorectal, head and neck, esophageal, pancreatic, ovarian, bladder, stomach, skin, endometrial, ovarian, testicular, esophageal, prostatic, or renal origin; a bone and soft tissue sarcoma, e.g., osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma (MFH), leiomyoma; a hematopoietic malignancy, e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, or leukemia; a neuroectodermal tumor, e.g., peripheral nerve tumor, astrocytoma, or melanoma; or a mesothelioma.

[0068] Another aspect of the invention provides a method for determining the presence and / or abundance of CD44v9 in a sample from a subject, the method comprising contacting the sample with any of a subject anti-CD44v9 antibody or antigen-binding fragment thereof.

[0069] Another aspect of the present invention provides methods for diagnosing and treating a subject having cancer, wherein the cancer cells express CD44v9, the method comprising: (1) determining the presence and / or abundance of CD44v9 in a cancer sample from the subject using the method to identify a subject expressing CD44v9 in the cancer sample; and (2) administering to the subject a therapeutically effective amount of a subject anti-CD44v9 antibody or antigen-binding fragment thereof, or a polypeptide thereof, or an immunoconjugate thereof, or a pharmaceutical composition thereof; thereby diagnosing and treating the subject having cancer.

[0070] In certain embodiments, the antibody CDR and framework region sequences are all based on the IMGT numbering scheme.

[0071] It should be understood that any embodiment described herein, including those described only as examples and those described only under one aspect of the invention, may be combined with one or more other embodiments unless clearly prohibited or inappropriate. [Brief explanation of the drawings]

[0072] [Figure 1] Figure 1 shows the binding of monoclonal mouse antibody HTS033 to CD44 recombinant protein in the presence of different CD44 peptides. P14, a CD44v9-specific peptide, blocked the binding of HTS033 to recombinant CD44. [Figure 2] Figure 2 shows the results of FACS analysis of the monoclonal mouse antibody HTS033 on HEK293 cells overexpressing CD44-FLAG. Overexpressing cells that stained positive for FLAG also stained positive with HTS033. [Figure 3] Figure 3 shows FACS analysis of HTS033 on cells expressing and not expressing CD44v9. SKME-1 tumor cells did not bind to the HTS033 antibody, while HTS033 bound to BxPC3, NCl-H226, and NCl-H526. [Figure 4] Figures 4A-4D show that HTS033 binding to tumor cells was reduced after siRNA-mediated knockdown of CD44v9. Control or cells transfected with siRNA targeting CD44v9 or CD44v6 were incubated with HTS033 and subsequently with FITC-conjugated anti-mouse IgG. Figure 4A shows HTS033 binding to BXPC-3 cells, where the signal was reduced in knockdown cells. Quantification of the FITC signal in BXPC-3 and the % knockdown of CD44v9 are listed in Figure 4B. Similarly, Figure 4C shows HTS033 binding to HUH7 cells, where the signal was reduced in knockdown cells. Quantification of the FITC-A signal in HUH7 and the % knockdown of CD44v9 are listed in Figure 4D. [Figure 5] Figure 5 shows the antibody affinity of HTS033 measured by FACS analysis on MDA-MB-468, NCl-H226, 5637, RT-4, and NCl-H520 cells. FACS titration of HTS033 was performed by incubating the cells with serial dilutions of HTS033 (17-1,000,000 pM) and subsequently staining the cells with FITC-conjugated anti-mouse IgG. EC50 values ​​were determined to be in the range of 2-8 nM. [Figure 6] Figures 6A-6B show positive immunohistochemical staining of CD44v9 by HTS033 in several tumor samples. In Figure 6A, HTS033 staining was positive in a triple-negative breast cancer sample, with staining intensities ranging from 1+ (1), 2+ (2), and 3+ (3). Similarly, Figure 6B shows negative staining of HTS033 in a normal sample (right), while HTS033 showed positive staining in a non-small cell lung cancer sample (left). LUSCC: squamous cell carcinoma of the lung. [Figure 7] Figure 7 shows the binding of chimeric HTS033 to CD44 recombinant protein in the presence of different CD44 peptides. The CD44v9-specific peptide, P14, blocked the binding of HTS033 to recombinant CD44, while the other peptides did not. [Figure 8] Figures 8A-8B show the antibody affinity of chimeric and humanized forms of HTS033, as measured by antibody binding to PC-9 cells using FACS analysis. FACS titration of each antibody was performed by incubating cells with serial dilutions of antibody (13-750,000 pM) and subsequently staining the cells with the appropriate secondary antibody (Figure 8A). The EC50 values ​​of the antibodies are listed in Figure 8B. [Figure 9] Figure 9 shows the cytotoxicity of HTS033 and a secondary MMAE antibody-drug conjugate in pancreatic (BXPC-3), lung (NCl-H520 and NCl-H226), liver (HUH7 and SK-HEP-1), and bladder (5637) cancer cell lines. Cells were treated with serial dilutions (1.5 pM to 30 nM) of HTS033 or an IgG control along with an MMAE-conjugated anti-mouse IgG antibody. The graph shows the % cell viability compared to the untreated control. The IC50 values ​​for HTS033 ranged from 14 to 315 pM for the cell lines tested. [Figure 10] Figure 10 shows the cytotoxicity of HTS033 and a secondary MMAE antibody-drug conjugate in breast (MDA-MB-231 and MDA-MB-468) and bladder (RT-4) cancer cell lines. IC50 values ​​for HTS033 ranged from 22 to 224 pM for the cell lines tested. [Figure 11]Figure 11 shows the cytotoxicity of humanized HTS033-25-1 conjugated with MMAE in lung (PC-9), head and neck (A253), and pancreatic (BxPC-3) cancer cell lines. Cells were treated with serially diluted humanized HTS033-25-1-MMAE or MMAE-conjugated negative control antibody (1.5 pM to 30 nM). The IC50 values ​​of humanized HTS033-25-1-MMAE were 269 pM (PC-9), 203 pM (A253), and 309 pM (BxPC-3), respectively. [Figure 12] Figure 12 shows the cytotoxicity of HTS033 CAR-T against PC-9 lung cancer cells. CAR-T cells generated with the HTS033 variable domain sequence and control T cells were incubated with PC-9 cells at different E:T ratios (E:T cells, T:tumor cells) for 24 hours. The viability of PC-9 cells after T cell removal was determined by CCK-8 assay. Efficient killing of target PC-9 cells was observed at all E:T ratios. DETAILED DESCRIPTION OF THE INVENTION

[0073] 1. Overview The invention described herein is based in part on the discovery that certain anti-CD44v9 antibodies, such as those described herein, are effective for treating diseases such as cancer.

[0074] Accordingly, one aspect of the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof specific for CD44v9, comprising: (1) a heavy chain variable region (HCVR) comprising (a) the HCVR CDR1 sequence of SEQ ID NO: 1, (b) the HCVR CDR2 sequence of SEQ ID NO: 2, and / or (c) the HCVR CDR3 sequence of SEQ ID NO: 3; and / or (2) a light chain variable region (LCVR) comprising (d) the LCVR CDR1 sequence of SEQ ID NO: 4, (e) the LCVR CDR2 sequence of SEQ ID NO: 5, and / or (f) the LCVR CDR3 sequence of SEQ ID NO: 6, optionally with the proviso that the monoclonal antibody is not mAb116.

[0075] It is generally recognized that the amino acid mutation in protein can occur without affecting the binding properties and other functions of protein.This is possible when mutation occurs in the amino acid that is not essential to the structure and function of protein, or when substitution is conservative, that is, occurs between amino acids with similar properties, and therefore is non-disruptive.The prediction of conservative substitution is listed by Pechmann and Frydman (2014) PLoS Comput Biol 10(6): e1003674, the contents of which are incorporated herein by reference.

[0076] Accordingly, one aspect of the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof specific for CD44v9, wherein the monoclonal antibody comprises: (1) a heavy chain variable region (HCVR) comprising: (a) the HCVR CDR1 sequence of SEQ ID NO: 1, wherein the HCVR comprises up to two amino acid mutations; (b) the HCVR CDR2 sequence of SEQ ID NO: 2, wherein the HCVR comprises up to two amino acid mutations; and / or (c) the HCVR CDR3 sequence of SEQ ID NO: 3, wherein the HCVR comprises up to five amino acid mutations; and / or (2) a light chain variable region (LCVR) comprising: (d) the LCVR CDR1 sequence of SEQ ID NO: 4, wherein the HCVR comprises up to two amino acid mutations; (e) the LCVR CDR2 sequence of SEQ ID NO: 5, wherein the LCVR comprises up to two amino acid mutations; and / or (f) the LCVR CDR3 sequence of SEQ ID NO: 6, wherein the LCVR comprises up to two amino acid mutations.

[0077] In certain embodiments, the heavy chain variable region (HCVR) of the monoclonal antibody or antigen-binding fragment comprises (a) the HCVR CDR1 sequence of SEQ ID NO: 1; (b) the HCVR CDR2 sequence of SEQ ID NO: 2, in which the alanine residue at position 6 is optionally substituted with glycine; and / or (c) the HCVR CDR3 sequence of SEQ ID NO: 3, in which the arginine residue at position 2 is optionally substituted with serine, the alanine residue at position 4 is optionally substituted with glycine, the aspartic acid residue at position 5 is optionally substituted with glutamic acid, the asparagine residue at position 7 is optionally substituted with threonine, and the proline residue at position 8 is optionally substituted with glycine; and / or the light chain variable region (LCVR) comprises (d) the LCVR CDR1 sequence of SEQ ID NO: 4; (e) the LCVR CDR2 sequence of SEQ ID NO: 5; and / or (f) the LCVR CDR3 sequence of SEQ ID NO: 6, in which the leucine residue at position 1 is optionally substituted with methionine and the leucine residue at position 8 is optionally substituted with phenylalanine.

[0078] A monoclonal murine anti-CD44v9 antibody designated mAb116 has been generated and is described in application PCT / CN2018 / 076958, the contents of which are incorporated herein by reference. In one embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof specific for CD44v9 comprises: (1) a heavy chain variable region (HCVR) comprising (a) the HCVR CDR1 sequence of SEQ ID NO: 31, (b) the HCVR CDR2 sequence of SEQ ID NO: 32, and / or (c) the HCVR CDR2 sequence of SEQ ID NO: 33; and / or (2) a light chain variable region (LCVR) comprising (d) the LCVR CDR1 sequence of SEQ ID NO: 34, (e) the LCVR CDR2 sequence of SEQ ID NO: 35, and / or (f) the LCVR CDR3 sequence of SEQ ID NO: 36.

[0079] In certain embodiments, the isolated anti-CD44v9 monoclonal antibody or antigen-binding fragment thereof has a K of about 40 nM, 20 nM, 10 nM, about 5 nM, or about 2 nM or less to CD44v9 or a cell bearing said CD44v9. D Combine with.

[0080] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is a murine antibody.

[0081] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment comprises: (1) a heavy chain framework region sequence of FR1 of SEQ ID NO:7, FR2 of SEQ ID NO:8, FR3 of SEQ ID NO:9, and / or FR4 of SEQ ID NO:10, respectively, containing up to two amino acid mutations in each framework region sequence; and / or (2) a light chain framework region sequence of FR1 of SEQ ID NO:11, FR2 of SEQ ID NO:12, FR3 of SEQ ID NO:13, and / or FR4 of SEQ ID NO:14, respectively, containing up to two amino acid mutations in each framework region sequence.

[0082] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is a humanized antibody, for example, a humanized murine monoclonal antibody.

[0083] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof further comprises (1) a heavy chain framework region sequence of FR1 of SEQ ID NO: 15, FR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and / or FR4 of SEQ ID NO: 18, respectively; and / or (2) a light chain framework region sequence of FR1 of SEQ ID NO: 19, FR2 of SEQ ID NO: 20, FR3 of SEQ ID NO: 21, and / or FR4 of SEQ ID NO: 22, respectively.

[0084] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof further comprises (1) a heavy chain framework region sequence of FR1 of SEQ ID NO: 23, FR2 of SEQ ID NO: 24, FR3 of SEQ ID NO: 25, and / or FR4 of SEQ ID NO: 26, respectively, and / or (2) a light chain framework region sequence of FR1 of SEQ ID NO: 27, FR2 of SEQ ID NO: 28, FR3 of SEQ ID NO: 29, and / or FR4 of SEQ ID NO: 30, respectively.

[0085] In certain embodiments, the antigen-binding fragment is a Fab, Fab', F(ab'), Fd , single chain Fv or scFv, disulfide-linked F v , V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0086] In a related aspect, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, which binds to the same epitope on CD44v9 bound by a reference monoclonal antibody or competes with the reference monoclonal antibody for binding to the same epitope on CD44v9, wherein the reference monoclonal antibody comprises: (1) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 1, the HCVR CDR2 sequence of SEQ ID NO: 2, and / or the HCVR CDR3 sequence of SEQ ID NO: 3; and / or (2) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 4, the LCVR CDR2 sequence of SEQ ID NO: 5, and / or the LCVR CDR3 sequence of SEQ ID NO: 6, optionally with the proviso that the monoclonal antibody is not mAb116.

[0087] Another aspect of the invention provides polypeptides comprising the HCVR and / or LCVR of any one of the subject anti-CD44v9 antibodies or antigen-binding fragments thereof.

[0088] In certain embodiments, the polypeptide is a fusion protein (eg, a chimeric antigen T cell receptor).

[0089] Chimeric antigen T cell receptors (CAR-T) are also known as chimeric antigen receptors (CARs), chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors. They are engineered receptors that transfer arbitrary specificities onto immune effector T cells. Typically, these receptors are used to transfer the specificity of monoclonal antibodies onto T cells, and the transfer of their coding sequences is facilitated by retroviral vectors. The receptors are called chimeric because they are composed of parts from different sources. CAR-T can be used to treat cancer using adoptive cell transfer, in which T cells are removed from a patient and modified to express a receptor specific to the patient's particular cancer, such as CD44v9, which is expressed on cancer cells. The T cells, now capable of recognizing and killing cancer cells, are then reintroduced into the patient. Modification of T cells provided by a donor other than the patient can also be used similarly.

[0090] In certain embodiments, the CAR-T of the present invention is a fusion of a subject single-chain variable fragment (scFv) derived from any of the subject monoclonal anti-CD44v9 antibodies fused to a transmembrane domain (e.g., a CD3-zeta transmembrane domain) and an endodomain (e.g., a CD3-zeta endodomain).

[0091] In certain embodiments, the scFv is preceded by a signal peptide that directs the nascent protein to the endoplasmic reticulum and subsequent surface expression. Any eukaryotic signal peptide sequence may be used. In certain embodiments, the signal peptide naturally attached to the amino terminus is used (e.g., in an scFv with a light chain-linker-heavy chain orientation, the natural signal of the light chain is used).

[0092] In certain embodiments, a flexible spacer is added to allow the scFv to orient in different directions to enable optimal antigen binding. The spacer is preferably flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition. In certain embodiments, the hinge region from IgG1 is used as the spacer. In certain embodiments, the CH2CH3 region and CD3 portion of an immunoglobulin are used as the spacer. For most scFv-based constructs, an IgG1 hinge is usually sufficient.

[0093] In certain embodiments, the construct comprises a transmembrane domain, a typical hydrophobic alpha helix, derived from the original molecule of the signaling endodomain, which protrudes into the cell and transmits the desired signal. In certain embodiments, a transmembrane domain derived from the membrane-most component of the endodomain, such as the CD3-zeta transmembrane domain, is used.

[0094] In a particular embodiment, the endodomain is a CD3-zeta endodomain containing three ITAMs, which transmits an activation signal to T cells after the antigen-binding fragment of the invention binds to an antigen.

[0095] In certain embodiments, the endodomain further comprises an intracellular signaling domain from a costimulatory protein receptor (e.g., that of CD28, 41BB, ICOS) fused to the cytoplasmic tail of the construct (N- or C-terminal to the CD3-zeta domain) to provide an additional signal to the T cell.

[0096] In certain embodiments, the endodomain combines multiple signaling domains, e.g., CD3z-CD28-41BB or CD3z-CD28-OX40, to increase the strength or transmit growth / survival signals.

[0097] In certain embodiments, the chimeric antigen receptor of the present invention further comprises a Strep tag II sequence (an eight-residue minimal peptide sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits unique affinity for streptavidin) to provide the engineered T cells with an identification marker for rapid purification.

[0098] Another aspect of the invention provides polynucleotides encoding any of the subject polypeptides.

[0099] Another aspect of the invention provides a vector comprising any of the subject polynucleotides.

[0100] In certain embodiments, the vector is an expression vector (eg, a mammalian expression vector, a yeast expression vector, an insect expression vector, or a bacterial expression vector).

[0101] Another aspect of the invention provides a cell comprising any of the subject anti-CD44v9 antibodies or antigen-binding fragments thereof, any of the subject polypeptides, any of the subject polynucleotides, or any of the subject vectors.

[0102] In certain embodiments, the cells express any of the subject antibodies or antigen-binding fragments thereof, or any of the subject polypeptides.

[0103] In certain embodiments, the cells are BHK cells, CHO cells, or COS cells.

[0104] In certain embodiments, the cells comprise on their cell surface either a subject anti-CD44v9 antibody or antigen-binding fragment thereof, or either a subject polypeptide.

[0105] In certain embodiments, the cell is a T cell bearing a chimeric antigen receptor (CAR-T cell) comprising any of the subject antibodies or antigen-binding fragments thereof, or any of the subject polypeptides.

[0106] In certain embodiments, a viral vector, e.g., a retrovirus, lentivirus, or transposon, may be used to integrate a transgene carrying a subject CAR-T construct into the host cell genome.

[0107] In certain embodiments, non-integrative vectors or episomal DNA / RNA constructs, such as plasmids or mRNA, may instead be used.

[0108] In certain embodiments, vectors that are stably maintained in T cells without integrating into the genome are used, allowing long-term transgene expression without the risk of insertional mutagenesis or genotoxicity.

[0109] Another aspect of the invention provides methods for producing any of the subject anti-CD44v9 antibodies or antigen-binding fragments thereof, or any of the subject polypeptides, comprising the steps of: (a) culturing any of the subject cells; and (b) isolating the antibody, antigen-binding fragment thereof, or polypeptide from the cultured cells.

[0110] In certain embodiments, the cell is a eukaryotic cell.

[0111] Another aspect of the present invention is a compound of the formula: Ab-[-LD] n wherein Ab is either a subject anti-CD44v9 antibody or antigen-binding fragment thereof, or a subject polypeptide thereof, covalently linked to one or more units of a linker-drug moiety -[-LD], L is a linker, and D is a cytotoxic agent; and n is an integer between 1 and 20 (e.g., 1 and 12); and each linker-drug moiety may have the same or different linker L or cytotoxic agent D.

[0112] In certain embodiments, each linker-drug moiety -[-LD] is covalently attached to the Ab through the side chain amino group of Lys.

[0113] In certain embodiments, each linker-drug moiety -[-LD] is covalently attached to an Ab through the side chain thiol group of a Cys.

[0114] In certain embodiments, each linker-drug moiety -[-LD] is covalently attached to the Ab through a site-specifically incorporated unnatural amino acid.

[0115] In certain embodiments, each linker L comprises a peptide unit.

[0116] In certain embodiments, a peptide unit comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-10, or 2-5 amino acid residues.

[0117] In certain embodiments, the linker L is not cleavable by a protease (eg, a cathepsin).

[0118] In certain embodiments, the linker L is a cleavable linker that is cleavable by a protease (eg, a cathepsin), an acidic environment, or a change in redox state.

[0119] In certain embodiments, the cytotoxic agent is a DNA intercalator, a microtubule binding agent, a topoisomerase I inhibitor, or a DNA minor groove binding agent.

[0120] In certain embodiments, the cytotoxic agent is a member of the auristatin class, e.g., monomethylauristatin E (MMAE) and MMAF, a maytansine class, e.g., DM-1, DM-3, DM-4, ​​a calicheamicin, e.g., ozogamicin, SN-38, or a PBD (pyrrolobenzodiazepine).

[0121] In a related aspect, the D moiety is not itself a drug molecule, but rather an adapter molecule (e.g., FITC) to which a universal CAR-T specific for the adapter molecule can tightly bind. According to this aspect of the invention, a single universal CAR-T cell that binds with very high affinity to an adapter molecule, e.g., FITC, is co-administered with a bispecific SMDC (small molecule drug conjugate) adapter molecule to treat a variety of cancer types. These unique bispecific adapters are constructed with an adapter, e.g., an FITC molecule, and a tumor-homing molecule, e.g., an antigen-binding fragment of a target anti-CD44v9 antibody, to precisely bridge the universal CAR-T cell to the cancer cell, thereby triggering local T cell activation. Antitumor activity is induced only in the presence of both the universal CAR-T cell and the correct antigen-specific adapter molecule. Antitumor activity and toxicity can be further controlled by adjusting the administered adapter molecule dosage. Treatment of tumors with heterogeneous antigens can be achieved by administering a mixture of the desired antigen-specific adapters.

[0122] Another aspect of the present invention provides pharmaceutical compositions comprising a subject anti-CD44v9 antibody or antigen-binding fragment thereof, or a polypeptide thereof, or an immunoconjugate thereof, and a pharmaceutically acceptable carrier or excipient.

[0123] Another aspect of the invention provides a method for inhibiting the proliferation of a cell expressing CD44v9, the method comprising the step of contacting the cell with either a subject anti-CD44v9 antibody or antigen-binding fragment thereof, or a subject polypeptide thereof, or a subject immunoconjugate thereof, or a subject pharmaceutical composition thereof.

[0124] In certain embodiments, the cell is a tumor cell.

[0125] In certain embodiments, the tumor cells are from a lung cancer (eg, NSCLC).

[0126] In particular embodiments, the tumor cells are from colorectal cancer, breast cancer, head and neck cancer, ovarian cancer, bladder cancer, pancreatic cancer, or metastatic cancer of the brain.

[0127] Another aspect of the present invention provides a method for treating a subject having cancer, wherein the cancer cells express CD44v9, the method comprising administering to the subject a therapeutically effective amount of a CD44v9 antagonist comprising a CD44v9 antibody or antigen-binding fragment thereof.

[0128] Another aspect of the invention provides a method for treating a cell proliferative disorder in a subject, wherein cells of the cell proliferative disorder express CD44v9, the method comprising administering to the subject a therapeutically effective amount of a CD44v9 antagonist comprising a CD44v9 antibody or antigen-binding fragment thereof.

[0129] In certain embodiments, the CD44v9 antagonist comprises either a subject anti-CD44v9 antibody or antigen-binding fragment thereof, or a subject polypeptide thereof, or a subject immunoconjugate thereof, or a subject pharmaceutical composition thereof.

[0130] In particular embodiments, the cancer is an epithelial carcinoma, including those of breast, lung, liver, colorectal, head and neck, esophageal, pancreatic, ovarian, bladder, stomach, skin, endometrial, ovarian, testicular, esophageal, prostatic, or renal origin; a bone and soft tissue sarcoma, e.g., osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma (MFH), leiomyoma; a hematopoietic malignancy, e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, or leukemia; a neuroectodermal tumor, e.g., peripheral nerve tumor, astrocytoma, or melanoma; or a mesothelioma.

[0131] Another aspect of the invention provides a method for determining the presence and / or abundance of CD44v9 in a sample from a subject, the method comprising contacting the sample with any of a subject anti-CD44v9 antibody or antigen-binding fragment thereof.

[0132] Another aspect of the present invention provides methods for diagnosing and treating a subject having cancer, wherein the cancer cells express CD44v9, the method comprising: (1) determining the presence and / or abundance of CD44v9 in a cancer sample from the subject using a subject method to identify a subject expressing CD44v9 in the cancer sample; and (2) administering to the subject a therapeutically effective amount of a subject anti-CD44v9 antibody or antigen-binding fragment thereof, or a polypeptide thereof, or an immunoconjugate thereof, or a pharmaceutical composition thereof; thereby diagnosing and treating the subject having cancer.

[0133] While the invention is generally described above, certain specific aspects or embodiments of the invention are further described in the following sections.

[0134] 2. Definition The terms "antibody," "antibody molecule," and "antibody protein" are used interchangeably herein and should be considered equivalent. These include immunoglobulin molecules that recognize and specifically bind to a target molecule, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing, through at least one antigen recognition site within the light and / or heavy chain variable region of the immunoglobulin molecule. As used herein, the term "antibody" includes intact polyclonal antibodies, intact monoclonal antibodies, and truncated forms such as antibody fragments (e.g., Fab, Fab', F(ab'), and Fv fragments), single-chain Fv (scFv) mutants, multispecific antibodies, e.g., bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antigenic determinant of an antibody, and any other modified immunoglobulin containing an antigen recognition site, so long as the antibody exhibits the desired biological activity. Antibodies may be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, based on the identity of the heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies may be naked or conjugated to other molecules, such as toxins, radioisotopes, etc.

[0135] In some embodiments, the antibody is a non-naturally occurring, recombinantly produced antibody. In some embodiments, the antibody is purified from natural components. In some embodiments, the antibody is recombinantly produced. In some embodiments, the antibody is produced by a hybridoma or generated in an antibody library.

[0136] "Complementarity determining regions (CDRs) of a monoclonal antibody" are understood to be the amino acid sequences involved in specific antigen binding according to Kabat (Kabat EA, Wu TT, Perry HM, Gottesman KS and Foeller C. (1991) Sequences of Proteins of Immunological Interest (5th ed.). NIH Publication No. 91-3242. US Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, Md., incorporated herein by reference) in relation to Chothia and Lesk (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917, incorporated herein by reference).

[0137] As used herein, the term "framework modification" refers to the replacement, deletion, or addition of a single or multiple amino acids in the variable region surrounding each complementarity-determining region. Framework modifications may affect the immunogenicity, productivity, or binding specificity of an antibody protein.

[0138] An "antigen-binding fragment," "antigen-binding portion," or, for short, "fragment," as used herein, is encoded by a nucleic acid molecule that is shorter than the full-length sequence, but still retains its antibody binding activity (e.g., substantially the same binding specificity, but with a shorter K d "antibody" refers to a shorter version of an antibody molecule, i.e., any polypeptide subset, characterized by a slightly poorer binding affinity as measured by .gtoreq. ...

[0139] These terms refer to portions of an intact antibody and refer to the antigen-determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), and F(ab'). vAntibody fragments include linear antibodies, single-chain antibodies, and multispecific antibodies formed from antibody fragments. The term "antigen-binding fragment" of an antibody includes one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by certain fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (without limitation): (i) Fab fragments, V L , V H , C L , and C H1 (ii) a monovalent fragment consisting of a V domain (e.g., digestion of an antibody with papain produces three fragments: two antigen-binding Fab fragments, and one Fc fragment that does not bind to antigen); (iii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region (e.g., digestion of an antibody with pepsin produces two fragments: a bivalent antigen-binding F(ab')2 fragment, and a pFc' fragment that does not bind to antigen) and its associated F(ab')2 monovalent unit; (iv) a V H and C H1 F consisting of domains d (iv) a V fragment of a single arm of an antibody (i.e., a portion of the heavy chain contained in Fab); L and V H F consisting of domains v Fragments, and associated disulfide bridges F v ;(v)V H domains, dAb (domain antibody) or sdAb (single domain antibody) fragments (Ward et al., Nature 341:544-546, 1989); and (vi) isolated complementarity determining regions (CDRs).

[0140] Various techniques for producing antibody fragments are known. Traditionally, these fragments are obtained by proteolytic digestion of intact antibodies (e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117, 1993; Brennan et al., Science 229:81, 1985). In certain embodiments, antibody fragments are produced recombinantly. Fab, Fv, and scFv antibody fragments may all be expressed in and secreted from E. coli or other host cells, thus allowing for the production of large amounts of these fragments. Such antibody fragments may also be isolated from antibody phage libraries. Antibody fragments may also be linear antibodies, as described, for example, in U.S. Pat. No. 5,641,870, and may be monospecific or bispecific. Other techniques for the production of antibody fragments will be apparent to those skilled in the art.

[0141] A "monoclonal antibody" refers to a homogeneous antibody population that is responsible for highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" includes intact and full-length monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab'), F v ), single chain (scFv) mutants, fusion proteins containing an antibody portion, and any other modified immunoglobulin molecule containing an antigen recognition site. Furthermore, "monoclonal antibody" refers to antibodies made in any of a number of ways, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[0142] Monoclonal antibodies may be prepared using hybridoma techniques, such as those described in Kohler and Milstein (1975) Nature 256:495. Using the hybridoma technique, a mouse, hamster, or other suitable host animal is immunized to induce the production by lymphocytes of antibodies that specifically bind to the immunizing antigen. Lymphocytes may also be immunized in vitro. After immunization, lymphocytes are isolated and fused with a suitable myeloma cell line, for example, using polyethylene glycol, to form hybridoma cells, which may then be selected from unfused lymphocytes and myeloma cells. Hybridomas producing monoclonal antibodies specifically directed against a selected antigen, as determined by immunoprecipitation, immunoblotting, or by in vitro binding assays (e.g., radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA)), may then be grown either in in vitro culture using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986), or in vivo as ascites tumors in animals. Monoclonal antibodies may then be purified from the culture medium or ascites fluid as described for polyclonal antibodies.

[0143] Alternatively, monoclonal antibodies may be produced using recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. Polynucleotides encoding the monoclonal antibodies are isolated from mature B cells or hybridoma cells, for example, by RT-PCR using oligonucleotide primers that specifically amplify genes encoding the antibody heavy and light chains, and their sequences are determined using conventional methods. The isolated polynucleotides encoding the heavy and light chains are then cloned into appropriate expression vectors, which, when transfected into host cells that do not otherwise produce immunoglobulin proteins, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, result in the production of the monoclonal antibodies by the host cells. Recombinant monoclonal antibodies or fragments thereof of the desired species may also be isolated from phage display libraries expressing the CDRs of the desired species as described (McCafferty et al., Nature 348:552-554, 1990; Clackson et al., Nature, 352:624-628, 1991; and Marks et al., J. Mol. Biol. 222:581-597, 1991).

[0144] The polynucleotide(s) encoding a monoclonal antibody may be further modified in several different ways using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light and heavy chains of, for example, a murine monoclonal antibody may be substituted 1) for regions of a human antibody, e.g., to generate a chimeric antibody, or 2) for a non-immunoglobulin polypeptide, to generate a fusion protein. In some embodiments, the constant regions are truncated or removed to generate desired antibody fragments of the monoclonal antibody. Site-directed or high-density mutagenesis of the variable regions may be used to optimize the specificity, affinity, etc. of the monoclonal antibody.

[0145] The term "humanized antibody" refers to a form of non-human (e.g., murine) antibody that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof, containing minimal non-human (e.g., murine) sequence. Typically, humanized antibodies are human immunoglobulins in which residues from the complementarity-determining regions (CDRs) are replaced by residues from the CDRs of non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and capacity (Jones et al., Nature 321:522-525, 1986; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988).

[0146] Methods for engineering, humanizing, or resurfacing non-human or human antibodies may also be used and are well known in the art. Humanized, resurfaced, or similarly engineered antibodies may have one or more amino acid residues derived from a non-human source, such as, but not limited to, mouse, rat, rabbit, non-human primate, or other mammal. These non-human amino acid residues are typically replaced by residues taken from an "import" variable, constant, or other domain of a known human sequence, often referred to as "import" residues.

[0147] Such imported sequences may be used to reduce immunogenicity, or to reduce, enhance, or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable property known in the art. Generally, CDR residues are directly and most substantially involved in affecting CD44v9 binding. Thus, some or all of the non-human or human CDR sequences may be maintained, while the non-human sequences of the variable and constant regions may be replaced with human or other amino acids.

[0148] Antibodies may also optionally be humanized, surface-modified, engineered, or human antibodies engineered to retain high affinity for the antigen CD44v9 and to incorporate other favorable biological properties. To this end, humanized (or human) or engineered anti-CD44v9 antibodies and surface-modified antibodies may optionally be prepared by a process of analysis of the parental sequences and various conceptual humanized and engineered products using three-dimensional models of the parental, engineered, and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen, such as CD44v9. In this manner, framework (FR) residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.

[0149] Humanization, surface modification, or engineering of the antibodies of the present invention may be accomplished using methods such as, but not limited to, Winter (Jones et al., Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Sims et al., J. Immunol. 151:2296, 1993; Chothia and Lesk, J. Mol. Biol. 196:901, 1987; Carter et al., Proc. Natl. Acad. Sci. USA 89:4285, 1992; Presta et al., J. Immunol. 151:2623, 1993; Raguska et al., Proc. Natl. Acad. Sci. USA 89:4285, 1992; Presta et al., J. Immunol. 151:2623, 1993; Raguska et al., Proc. Natl. Acad. Sci. USA 89:4285, 1992), each of which is incorporated herein by reference in its entirety, including the references cited therein. 91(3):969-973, 1994; U.S. Patent Nos. 5,639,641, 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; 4,816,56 This may be carried out using any known method, such as those described in PCT / :US98 / 16280; US96 / 18978; US91 / 09630; US91 / 05939; US94 / 01234; GB89 / 01334; GB91 / 01134; GB92 / 01755; WO90 / 14443; WO90 / 14424; WO90 / 14430; EP229246; 7,557,189; 7,538,195; and 7,342,110.

[0150] In certain other embodiments, the antibody against CD44v9 is a human antibody. Human antibodies can be prepared directly using a variety of techniques known in the art. Immortalized human B lymphocytes can be generated by in vitro immunization or isolated from immunized individuals that produce antibodies directed against target antigens (see, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., 1991, J. Immunol, 147 (1):86-95; and U.S. Patent No. 5,750,373). Human antibodies may also be selected from phage libraries expressing human antibodies, as described, for example, in Vaughan et al., Nat. Biotech. 14:309-314, 1996; Sheets et al., Proc. Nat'l. Acad. Sci. 95:6157-6162, 1998; Hoogenboom and Winter, J. Mol. Biol. 227:381, 1991; and Marks et al., J. Mol. Biol. 222:581, 1991. Techniques for the production and use of antibody phage libraries are also described in U.S. Pat. Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe et al., J. Mol. Bio. doi: 10.1016 / j.jmb.2007.12.018, 2007 (each of which is incorporated herein by reference in its entirety). Affinity maturation and chain shuffling strategies (Marks et al., Bio / Technology 10:779-783, 1992, incorporated herein by reference in its entirety) are known in the art and may be used to generate high affinity human antibodies.

[0151] Humanized antibodies may also be produced in transgenic mice containing human immunoglobulin loci that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. This approach is described in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

[0152] In some instances, human immunoglobulin F v Framework region (FR) residues are replaced with corresponding residues in an antibody from a non-human species having the desired specificity, affinity, and capacity. v Humanized antibodies may be further modified by substitution of additional residues either in the framework regions and / or within the substituted non-human residues to improve and optimize antibody specificity, affinity, and / or capacity. Generally, a humanized antibody comprises substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are of human immunoglobulin consensus sequences. Humanized antibodies also comprise immunoglobulin constant regions or domains (F), typically those of a human immunoglobulin. c ) Examples of methods used to generate humanized antibodies are described in U.S. Patent Nos. 5,225,539 and 5,639,641, Roguska et al., Proc. Natl. Acad. Sci. USA 91(3):969-973, 1994; and Roguska et al., Protein Eng. 9(10):895-904, 1996 (all incorporated herein by reference). In some embodiments, a "humanized antibody" is a surface-modified antibody. In some embodiments, a "humanized antibody" is a CDR-grafted antibody.

[0153] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The heavy and light chain variable regions each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are tightly held together by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antibody's antigen-binding site. There are at least two techniques for determining CDRs: (1) an approach based on interspecies sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., 1991, National Institutes of Health, Bethesda, Md.); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., J. Molec. Biol. 273:927-948, 1997). Furthermore, a combination of these two approaches is sometimes used in the art to determine CDRs.

[0154] The Kabat numbering system is commonly used when referring to residues in the variable domain (residues 1-107 of the light chain and 1-113 of the heavy chain). (See, e.g., Kabat et al., Sequences of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0155] Amino acid position numbering as in Kabat refers to the numbering system used for the heavy or light chain variable domains of the antibody compilation in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991) (incorporated herein). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody sequence with the "standard" Kabat numbered sequence at the regions of homology. Chothia instead refers to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987). When numbered using the Kabat numbering convention, the Chothia CDR-H1 loop ends vary between H32 and H34, depending on the length of the loop. This is because the Kabat numbering scheme places an insertion between H35A and H35B: if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34. The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

[0156] [Table 1] In certain embodiments, the antibody CDR and framework region sequences are all based on the IMGT numbering scheme.

[0157] The term "human antibody" refers to an antibody produced by a human, or an antibody having an amino acid sequence corresponding to an antibody produced by a human, made using any technique known in the art. In certain embodiments, a human antibody is free of non-human sequences. This definition of a human antibody includes intact or full-length antibodies, or antigen-binding fragments thereof.

[0158] The term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecule are derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in antibodies derived from another species (usually human) to avoid or reduce the possibility of eliciting an immune response in that species (e.g., human). In certain embodiments, a chimeric antibody may include an antibody or antigen-binding fragment thereof comprising at least one human heavy and / or light chain polypeptide, e.g., an antibody comprising a murine light chain and a human heavy chain polypeptide.

[0159] For purposes of the present invention, it should be recognized that modified antibodies may comprise any type of variable region that provides for the association of the antibody with the human CD44v9 polypeptide. In this regard, the variable region may comprise or be derived from any type of mammal that can be induced to generate a humoral response and generate immunoglobulins against a desired tumor-associated antigen. As such, the variable region of the modified antibody may be of, for example, human, murine, non-human primate (e.g., cynomolgus monkey, macaque, etc.), or lupine origin. In some embodiments, both the variable and constant regions of the modified immunoglobulin are human. In other embodiments, the variable region of a suitable antibody (usually from a non-human source) may be engineered or specifically tailored to improve the binding characteristics or reduce the immunogenicity of the molecule. In this regard, variable regions useful in the present invention may be humanized or otherwise modified through the inclusion of imported amino acid sequences.

[0160] In certain embodiments, both the heavy and light chain variable domains are modified by at least partial replacement of one or more CDRs, and, if necessary, partial framework region replacement and sequence variation. The CDRs may be derived from antibodies of the same class or even the same subclass as the antibody from which the framework regions are derived, although it is contemplated that the CDRs may be derived from antibodies of different classes, and in certain embodiments, from antibodies of different species. It may not be necessary to replace all of the CDRs with the complete CDRs from the donor variable region to transfer the antigen-binding capacity of one variable domain to another. Rather, it may be necessary to transfer only those residues necessary to maintain the activity of the antigen-binding site. Given the explanations set forth in U.S. Patent Nos. 5,585,089, 5,693,761, and 5,693,762, obtaining functional antibodies with reduced immunogenicity will be well within the capabilities of one skilled in the art, either by performing routine experimentation or by trial and error testing.

[0161] Notwithstanding modifications to the variable region, those skilled in the art will recognize that the modified antibodies of the present invention include antibodies (e.g., full-length antibodies or immunoreactive fragments thereof) in which at least a portion of one or more of the constant region domains has been deleted or otherwise modified to provide desirable biochemical properties, such as increased tumor localization or decreased serum half-life, when compared to antibodies of substantially the same immunogenicity containing native or unmodified constant regions. In some embodiments, the constant region of the modified antibody comprises a human constant region. Modifications to the constant region compatible with the present invention include the addition, deletion, or substitution of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein may include modifications or alterations to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and / or to the light chain constant domain (CL). In some embodiments, modified constant regions in which one or more domains have been partially or completely deleted are contemplated. In some embodiments, the modified antibodies include domain-deleted constructs or variants in which the entire CH2 domain has been removed (ACH2 constructs). In some embodiments, the deleted constant region domain is replaced by a short amino acid spacer (e.g., 10 residues) that provides a degree of molecular flexibility typically imparted by the absent constant region.

[0162] It is noted that in certain embodiments, modified antibodies may be engineered to fuse the CH3 domain directly to the hinge region of each modified antibody. In other constructs, it may be desirable to provide a peptide spacer between the hinge region and the modified CH2 and / or CH3 domain. For example, compatible constructs may be expressed in which the CH2 domain is deleted and the remaining CH3 domain (modified or unmodified) is linked to the hinge region with a 5-20 amino acid spacer. Such a spacer may be added, for example, to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible. However, it should be noted that in some cases, amino acid spacers may be shown to be immunogenic and elicit an undesirable immune response against the construct. Therefore, in certain embodiments, any spacer added to the construct is relatively non-immunogenic or even completely removed in order to maintain the desired biochemical qualities of the modified antibody.

[0163] It will be appreciated that in addition to the deletion of entire constant region domains, antibodies of the present invention can be provided by the partial deletion or substitution of several or even single amino acids. For example, a single amino acid mutation in a selected region of the CH2 domain may be sufficient to substantially reduce Fc binding and thereby increase tumor localization. Similarly, it may be desirable to simply delete portions of one or more constant region domains that control the modulation of effector functions (e.g., complement C1Q binding). Such partial deletion of the constant region may improve selected characteristics of the antibody (e.g., serum half-life) while leaving other desired functions associated with the constant region domain of interest intact. Furthermore, as suggested above, the constant regions of the disclosed antibodies may be modified, for example, through the mutation or substitution of one or more amino acids, which may enhance the profile of the resulting construct. In this regard, it may be possible to disrupt the activity provided by a conserved binding site (e.g., Fc binding) while substantially maintaining the configuration and immunogenic profile of the modified antibody. Certain embodiments may involve the addition of one or more amino acids to the constant region to enhance a desired property, such as reduced or increased effector function, or to provide more cytotoxicity or carbohydrate attachment. In such embodiments, it may be desirable to insert or duplicate a particular sequence from a selected constant region domain.

[0164] The present invention further includes variants and equivalents that are substantially homologous to the chimeric, humanized, and human antibodies, or antibody fragments thereof, set forth herein. These may contain, for example, conservative substitution mutations, i.e., the substitution of one or more amino acids with similar amino acids. For example, a conservative substitution refers to the substitution of one amino acid for another within the same general class, such as the substitution of one acidic amino acid for another acidic amino acid, one basic amino acid for another basic amino acid, or one neutral amino acid for another neutral amino acid. What is intended by conservative amino acid substitution is well known in the art, such as those defined above.

[0165] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to the portion of an antigen capable of being recognized and specifically bound by a particular antibody. When an antigen is a polypeptide, epitopes can be formed from both contiguous amino acids and non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturation, while epitopes formed by tertiary folding are typically lost upon protein denaturation. An epitope typically includes at least 3, and more usually at least 5 or 8-10, amino acids in a unique spatial conformation.

[0166] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" herein refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K d ) or half-maximal effective concentration (EC 50 ) Affinity may be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high affinity antibodies generally bind antigens quickly and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which may be used for purposes of the present invention. Certain exemplary embodiments are described herein.

[0167] The phrases "substantially similar" or "substantially the same" are used herein to mean a comparison between two values ​​(e.g., K d"A" indicates a sufficiently high degree of similarity between two numerical values ​​(typically one associated with an antibody of the invention and the other associated with a reference / comparator antibody) that one would consider there to be little or no biological and / or statistical significance between the two values ​​within the context of the biological property measured by the antibody (value). The difference between said two values ​​is less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% as a function of the value for the reference / comparator antibody.

[0168] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some embodiments, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0169] Methods known in the art for purifying antibodies and other proteins also include those described in U.S. Patent Publication Nos. 2008 / 0312425, 2008 / 0177048, and 2009 / 0187005, each of which is incorporated herein by reference in its entirety.

[0170] As used herein, "substantially pure" refers to a material that is at least 50% pure (ie, free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0171] A "functional variant" of an antibody according to the present invention is an antibody molecule that possesses a biological activity (either functional or structural) that is substantially similar to that of an antibody molecule according to the present invention, i.e., substantially similar substrate specificity or substrate cleavage.

[0172] The term "functional variant" also includes "fragments," "allelic variants," "functional variants," "variants based on a degenerate nucleic acid code," or "chemical derivatives." Such "functional variants" may possess one or several point mutations, one or several nucleic acid exchanges, deletions, or insertions in the coding sequence, or one or several amino acid exchanges, deletions, or insertions. Said functional variants still at least partially retain a biological activity, such as antibody binding activity, or even have an improvement in said biological activity.

[0173] "Functional variants" of the antibody molecules according to the present invention may also include antibody molecules that possess biological activity (either functional or structural) that is substantially similar to that of the antibody molecules according to the present invention, i.e., substantially similar target molecule binding activity.

[0174] An "allelic variant" is a variant due to allelic variation, e.g., the difference between two alleles in humans, which still at least partially retains a biological activity, such as antibody target binding activity, or even has an improved biological activity.

[0175] "Variants based on the degeneracy of the genetic code" are variants due to the fact that a particular amino acid can be coded for by several different nucleotide triplets, which still at least partially retain biological activity, such as antibody binding activity, or even have an improved biological activity.

[0176] A "fusion molecule" may be, for example, an antibody molecule according to the present invention fused to a reporter, such as a radiolabel, a chemical molecule, such as a toxin or a fluorescent label, or any other molecule known in the art.

[0177] As used herein, a "chemical derivative" according to the present invention is an antibody molecule according to the present invention that is chemically modified or contains additional chemical moieties not normally part of the molecule. Such moieties may improve the activity of the molecule, e.g., target destruction (e.g., tumor cell killing), or may improve its solubility, absorption, biological half-life, etc.

[0178] A molecule is "substantially similar" to another molecule if both molecules have a substantially similar structure or biological activity. Thus, two molecules are considered variants, as the term variant is used herein, even if one of the molecules has a structure not found in the other molecule or the amino acid residue sequence is not identical, so long as the two molecules possess similar activity.

[0179] In certain embodiments, a "sample" or "biological sample" of the present invention is of biological origin, such as from a eukaryotic organism. In some embodiments, the sample is a human sample, although animal samples may also be used. Non-limiting sources of samples for use in the present invention include, for example, solid tissue, biopsy aspirate, ascites, liquid extract, blood, plasma, serum, spinal fluid, lymphatic fluid, external portions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, tumors, organs, cell cultures, and / or cell culture components. A "cancerous / tumor sample" is a sample containing cancerous cells. The method may be used to examine aspects of CD44v9 expression or sample status, including, but not limited to, comparing different types of cells or samples, comparing different stages of development, and detecting or determining the presence and / or type of disease or abnormality.

[0180] For many uses of the antibodies according to the invention, it is desirable to have the smallest possible antigen-binding, i.e., CD44v9-binding, unit. Therefore, in another preferred embodiment, the antibody protein according to the invention is a Fab fragment (Fragment antigen-binding = Fab). These CD44v9-specific antibody proteins according to the invention consist of the variable regions of both chains held together by adjacent constant regions. They may be formed from conventional antibodies by protease digestion, for example with papain, although similar Fab fragments may also be produced by genetic engineering. In another preferred embodiment, the antibody protein according to the invention is a F(ab')2 fragment, which may be prepared by proteolytic cleavage with pepsin.

[0181] Using genetic engineering techniques, it is possible to produce truncated antibody fragments consisting only of the heavy chain variable region (VH) and the light chain variable region (VL). These are called Fv fragments (Fragment variable = fragment of the variable part). In another preferred embodiment, the CD44v9-specific antibody molecule according to the present invention is such an Fv fragment. These Fv fragments lack the covalent bond between the two chains by the cysteines of the constant chains, and therefore are often stabilized. It is preferable to link the heavy and light chain variable regions by a short peptide fragment, for example, a fragment of 10 to 30 amino acids, preferably 15 amino acids. In this manner, a single peptide chain consisting of VH and VL linked by a peptide linker is obtained. This type of antibody protein is known as a single-chain Fv (scFv). An example of this type of scFv antibody protein known from the prior art is described in Huston et al. (1988, PNAS 16: 5879-5883). Therefore, in another preferred embodiment, the CD44v9-specific antibody protein according to the present invention is a single chain Fv protein (scFv).

[0182] In recent years, various strategies have been developed to prepare multimeric scFv derivatives. This is intended to lead to recombinant antibodies with improved pharmacokinetic and biodistribution properties, as well as increased binding avidity. To achieve multimerization of scFvs, scFvs have been prepared as fusion proteins with multimerization domains. The multimerization domain may be, for example, the CH3 region of IgG or a coiled-coil structure (helical structure), such as a leucine zipper domain. However, there are also strategies in which interactions between the VH / VL regions of scFvs are used for multimerization (e.g., dibodies, tribodies, and pentabodies). Thus, in another embodiment, the antibody protein according to the present invention is a CD44v9-specific diabody antibody fragment. By diabody, those skilled in the art mean a bivalent homodimeric scFv derivative (Hu et al., 1996, PNAS 16: 5879-5883). Shortening the linker in scFv molecules to 5-10 amino acids leads to homodimer formation, where interchain VH / NL superimposition occurs. Diabodies can be further stabilized by the incorporation of disulfide bridges. Examples of diabody antibody proteins from the prior art can be found in Perisic et al. (1994, Structure 2: 1217-1226).

[0183] By minibody, those skilled in the art mean a bivalent homodimeric scFv derivative. Minibodies consist of a fusion protein containing the CH3 region of an immunoglobulin, preferably IgG, most preferably IgG1, as a dimerization domain, linked to an scFv via a hinge region (e.g., also from IgG1) and a linker region. Disulfide bridges in the hinge region are often formed in higher order cells but not in prokaryotes. In another preferred embodiment, the antibody protein according to the present invention is a CD44v9-specific minibody antibody fragment. Examples of minibody-antibody proteins from the prior art can be found in Hu et al. (1996, Cancer Res. 56: 3055-61).

[0184] By triabody, the skilled person means: a trivalent homotrimeric scFv derivative (Kortt et al. 1997 Protein Engineering 10: 423-433). ScFv derivatives in which VH-VL are directly fused without a linker sequence lead to the formation of trimers.

[0185] Those skilled in the art are also familiar with so-called miniantibodies, which have bivalent, trivalent, or tetravalent structures and are derived from scFvs. Multimerization is achieved by dimeric, trimeric, or tetrameric coiled-coil structures (Pack et al., 1993 Biotechnology II:, 1271-1277; Lovejoy et al., 1993 Science 259: 1288-1293; Pack et al., 1995 J. Mol. Biol. 246: 28-34).

[0186] Thus, in one embodiment, the antibody according to the invention is a CD44v9-specific multimerizing molecule based on the aforementioned antibody fragments and may be, for example, a triabody, a tetravalent minibody or a pentabody.

[0187] Humanized CD44v9-specific antibody proteins may be produced by molecular biology methods known in the art.

[0188] The variable regions of the antibody proteins of the present invention are typically linked to at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Human constant region DNA sequences may be isolated from a variety of human cells, but preferably from immortalized B cells, according to well-known methods (see Kabat et al., supra, and WO 87 / 02671). Thus, the antibody proteins of the present invention may contain all of the constant region, or only a portion, so long as it exhibits specific binding to the CD44v9 antigen. The type and extent of the constant region selection depends on whether effector functions, such as complement fixation or antibody-dependent cellular cytotoxicity, are desired and on the desired pharmacological properties of the antibody protein. The antibody proteins of the present invention are typically tetramers consisting of two light / heavy chain pairs, but may also be dimers, i.e., light / heavy chain pairs, such as Fab or Fv fragments.

[0189] Thus, in a further embodiment, the present invention relates to an antibody protein according to the present invention, characterized in that it has a variable light chain region and a variable heavy chain region, each linked to a human constant region. In particular, the light chain variable region is linked to a human kappa constant region, and the heavy chain variable region is linked to a human gamma-1 constant region. Other human constant regions can also be used to chimerize the light and heavy chains.

[0190] Humanization of the variable regions of murine antibodies may be achieved using methods known in the art. EP 0239400 discloses a process for grafting the CDRs of a murine variable region into a human variable region framework. WO 90 / 07861 discloses a method for recreating a CDR-grafted variable region by introducing additional framework modifications. WO 92 / 11018 discloses a method for producing a humanized Ig by combining donor CDRs with an acceptor framework highly homologous to the donor framework. WO 92 / 05274 discloses the preparation of framework-mutated antibodies starting from a murine antibody. Further prior art references related to the humanization of murine monoclonal antibodies are EP 0368684; EP 0438310; WO 92 / 07075, or WO 92 / 22653, all of which are incorporated herein by reference.

[0191] In another embodiment, the invention relates to an antibody molecule according to the invention, characterized in that each of said light chain variable region and said heavy chain variable region is linked separately to a human constant region.

[0192] In another embodiment, the invention relates to an antibody molecule according to the invention, wherein said light chain human constant region is a human kappa constant region.

[0193] In another embodiment, the invention relates to an antibody protein according to the invention, wherein said heavy chain human constant region is a human IgG1 constant region.

[0194] The antibody protein of the present invention provides a highly specific tool for targeting therapeutic agents to the CD44v9 antigen. Therefore, in a further aspect, the present invention relates to an antibody protein according to the present invention, wherein the antibody protein is conjugated to a therapeutic agent, optionally via a linker, in an antibody-drug conjugate (ADC). Among the many therapeutic agents known in the art, preferred are therapeutic agents selected from the group consisting of radioisotopes, toxins, toxoids, inflammatory agents, enzymes, antisense molecules, peptides, cytokines, and chemotherapeutic agents. Among radioisotopes, gamma-, beta-, and alpha-emitting radioisotopes can be used as therapeutic agents. Beta-emitting radioisotopes are preferred as therapeutic radioisotopes. 186 rhenium, 188 rhenium, 131 Iodine and 90 Yttrium has proven to be a particularly useful beta-emitting isotope for achieving localized irradiation and destruction of malignant tumor cells. 186 rhenium, 188 rhenium, 131 Iodine and 90 Radioisotopes selected from the group consisting of yttrium are particularly preferred as therapeutic agents conjugated to the antibody proteins of the present invention. For example, methods such as those disclosed in WO 93 / 05804 may be used to radioiodinate the antibodies of the present invention.

[0195] The terms "immunoconjugate," "conjugate," or "ADC," as used herein, refer to a compound or derivative thereof linked to a cell-binding agent (i.e., an anti-CD44v9 antibody or fragment thereof) and defined by the general formula: ALC, where C = cytotoxin, L = linker, and A = cell-binding agent (CBA), e.g., an anti-CD44v9 antibody or antibody fragment. An immunoconjugate may also be defined by the general formula in reverse order: CLA.

[0196] A "linker" is any chemical moiety capable of linking a compound, usually a drug, e.g., a cytotoxic agent described herein, to a cell-binding agent, such as an anti-CD44v9 antibody or fragment thereof, in a stable, covalent manner. The linker may be sensitive or substantially resistant to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage under conditions under which the compound or antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid-labile groups, photolabile groups, peptidase-labile groups, and esterase-labile groups. Linkers also include charged linkers, and hydrophilic versions thereof, as described herein and known in the art.

[0197] The terms "cancer cells," "tumor cells," and grammatical equivalents refer to the total population of cells derived from a tumor or precancerous lesion, including both non-tumorigenic cells, including the bulk tumor cell population, and tumorigenic stem cells (cancer stem cells). As used herein, the term "tumor cells" is modified by the term "non-tumorigenic" when referring solely to tumor cells that lack the ability to reproduce and differentiate to differentiate tumor cells from cancer stem cells.

[0198] The term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., who is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein with reference to a human subject.

[0199] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0200] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered. Such formulations may be sterile.

[0201] An "effective amount" of an antibody or immunoconjugate as disclosed herein is an amount sufficient to carry out a particular stated purpose. An "effective amount" may be determined empirically and routinely for the stated purpose.

[0202] The term "therapeutically effective amount" refers to an amount of an antibody or other agent effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, a therapeutically effective amount of an agent may reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow to a certain extent, and in certain embodiments, stop) cancer cell invasion into peripheral organs; inhibit (i.e., slow to a certain extent, and in certain embodiments, stop) tumor metastasis; inhibit tumor growth to a certain extent; alleviate to a certain extent one or more symptoms associated with cancer; and / or produce a favorable response, such as progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), or in some cases, stable disease (SD), reduced progressive disease (PD), reduced time to progression (TTP), or any combination thereof. See the definition herein of "treatment." To the extent the drug prevents growth and / or kills existing cancer cells, it may be cytostatic and / or cytotoxic.

[0203] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, because a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be lower than the therapeutically effective amount, although this is not necessarily the case.

[0204] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Terms such as "treating" or "treatment" or "treat" or "palliative" or "palliating" refer to therapeutic measures that cure, slow, reduce the symptoms, and / or halt the progression of a diagnosed pathological condition or disorder. Thus, those in need of treatment include those for whom a disorder has already been diagnosed, and may also include those with minimal residual disease, or resistant disease, or recurrent disease. In certain embodiments, a subject has been successfully "treated" for cancer according to the methods of the present invention if the patient exhibits one or more of the following: a reduction or complete absence of cancer cell count; a reduction in tumor size; inhibition or absence of cancer cell invasion into peripheral organs, including, for example, spread of cancer into soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; alleviation of one or more symptoms associated with a particular cancer; a reduction in morbidity and mortality; an improvement in quality of life; a reduction in tumorigenicity, tumorigenic frequency, or tumorigenic potential of the tumor; a reduction in the number or frequency of cancer stem cells in the tumor; differentiation of tumorigenic cells into a non-tumorigenic state; an increase in progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), a complete response (CR), a partial response (PR), stable disease (SD), a reduction in progressive disease (PD), a reduction in time to progression (TTP), or any combination thereof.

[0205] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to a polymer of nucleotides of any length, and include DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide may also include modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps," substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as those containing uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, cabamates, etc.) and those containing charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those containing intercalating agents (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylating agents, those containing modified linkages (e.g., alpha-anomeric nucleic acids, etc.), and unmodified forms of polynucleotide(s). Additionally, any of the hydroxyl groups normally present on the sugar may be substituted, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare for further linkage to additional nucleotides, or conjugated to a solid support. The 5' and 3' terminal OH may be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups.Polynucleotides may also contain analogs of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, phosphate P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, or P(O)OR. * , CO or CH2 ("formacetyl"), where each R or R * are independently substituted by H, or substituted or unsubstituted alkyl (1-20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl, optionally containing an ether (—O—) linkage. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0206] The term "vector" refers to a construct capable of delivering one or more genes or sequences of interest to a host cell and expressing them in the cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0207] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The terms also include amino acid polymers that are naturally modified or modified by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within this definition are polypeptides containing, for example, one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. Because the polypeptides of the invention are based on antibodies, it is understood that in certain embodiments, the polypeptide can occur as single chains or associated chains. In some embodiments, the polypeptide, peptide, or protein is non-naturally occurring. In some embodiments, the polypeptide, peptide, or protein is purified from other naturally occurring components. In some embodiments, the polypeptide, peptide, or protein is recombinantly produced.

[0208] The term "identical" or percent "identity" refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotide or amino acid residues that are the same when compared and aligned for maximum correspondence (introducing gaps, if necessary) in the context of two or more nucleic acids or polypeptides, without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity may be measured using sequence comparison software or algorithms or by visual inspection. A variety of algorithms and software are known in the art that can be used to align amino acid or nucleotide sequences. One such non-limiting example of a sequence alignment algorithm is the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. 87:2264-2268, 1990, as modified by Karlin et al., Proc. Natl. Acad. Sci. 90:5873-5877, 1993, and incorporated into the NBLAST and XBLAST programs (Altschul et al., Nucleic Acids Res. 25:3389-3402, 1991). In certain embodiments, gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; BLAST-2, WU-BLAST-2 (Altschul et al., Methods in Enzymology 266:460-480, 1996), ALIGN, ALIGN-2 (Genentech, South San Francisco, CA) or Megalign (DNASTAR) are other publicly available software programs that can be used to align sequences. In certain embodiments, the GAP program in GCG software is used to determine the percent identity between two nucleotide sequences (for example, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90, and a length weight of 1, 2, 3, 4, 5, or 6).In certain other embodiments, the percent identity between two amino acid sequences may be determined using the GAP program in the GCG software package, which incorporates the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453, 1970) (e.g., using a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, or 5). Alternatively, in certain embodiments, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4:11-17, 1989). For example, percent identity may be determined using the ALIGN program (version 2.0) and using PAM120 with a residue table, a gap length penalty of 12, and a gap penalty of 4. Appropriate parameters for maximal alignment with particular alignment software can be determined by those skilled in the art. In certain embodiments, the default parameters of the alignment software are used. In certain embodiments, the percent identity "X" of a first amino acid sequence to a second sequence amino acid is calculated as 100x(Y / Z), where Y is the number of amino acid residues that score as identical matches in an alignment of the first and second sequences (when aligned by visual inspection or by a particular sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, then the percent identity of the first sequence to the second sequence will be longer than the percent identity of the second sequence to the first sequence.

[0209] As a non-limiting example, whether any particular polynucleotide has a particular percent sequence identity (e.g., at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identical) to a reference sequence may, in certain embodiments, be determined using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489, 1981, to find the highest segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence described herein, the percent identity is calculated over the entire length of the reference nucleotide sequence and parameters are set to allow gaps in homology of up to 5% of the total number of nucleotides in the reference sequence.

[0210] In some embodiments, two nucleic acids or polypeptides of the invention are substantially identical, i.e., they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence as determined using a sequence comparison algorithm or by visual inspection. In particular embodiments, identity exists over a region of the sequences that is at least about 10, about 20, about 40-60 residues in length, or any integer value therebetween, or over a region longer than 60-80 residues, such as at least 90-100 residues, or the sequences are substantially identical over the entire length of the sequences being compared, e.g., the coding regions of the nucleotide sequences.

[0211] "Conservative amino acid substitution" refers to the substitution of one amino acid residue with another amino acid residue having a similar side chain.Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).For example, the substitution of phenylalanine for tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the present invention do not inhibit the binding of the polypeptides or antibodies containing the amino acid sequences to the antigen(s), i.e., CD123 / IL-3Rα to which the polypeptides or antibodies bind. Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187, 1993; Kobayashi et al., Protein Eng. 12(10):879-884, 1999; and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417, 1997).

[0212] Another aspect of the present invention provides an antibody protein according to the present invention linked to a therapeutic agent, wherein the therapeutic agent is selected from the group consisting of a radioisotope, a toxin, a toxoid, a prodrug, and a chemotherapeutic agent.

[0213] In certain embodiments, the therapeutic agent is MAG-3 (U.S. Pat. No. 5,082,930 A, EP 0247866 B1 (page 2, lines 55-56 to page 3, lines 1-23)); MAG-2 GABA (U.S. Pat. No. 5,681,927 A, EP 0284071 B1 (page 6, lines 9-29)); and N2S2 ((=phenthioate) U.S. Pat. Nos. 4,897,255 A, 5,242,679 A, ​​EP 0188256 B1). B1 (page 2, line 38 to page 3, line 18)), (Ac)Phe-Lys(aroc)-PABC-PNP, 6-maleimidohexanoic acid N-hydroxysuccinimide ester, 6-quinoxalinecarboxylic acid, 2,3-bis(bromomethyl)-Fmoc-Val-Cit-PAB, Fmoc-Val-Cit-PAB-PNP, Mc-Val-Cit-PABC-PNP, Val-cit-PAB-OH, all of which are incorporated herein by reference.

[0214] In certain embodiments, the radioisotope is 186 rhenium, 188 rhenium, 131 Iodine and 90 yttrium.

[0215] In a particular embodiment, the antibody protein according to the present invention is labeled. Such a CD44v9-specific labeled antibody allows for the localization and / or detection of the CD44v9 antigen in vitro and / or in vivo.

[0216] A label is defined as a marker that can be directly or indirectly detectable. An indirect marker is defined as a marker that is not detectable by itself but requires an additional directly detectable marker specific to the indirect marker. A preferred label for carrying out the present invention is a detectable marker. From the wide variety of detectable markers, the detectable marker may be selected from the group consisting of enzymes, dyes, radioisotopes, digoxigenin, and biotin.

[0217] In certain embodiments, the label is a detectable marker, such as one selected from the group consisting of an enzyme, a dye, a radioisotope, digoxigenin, and biotin.

[0218] In certain embodiments, the antibody protein according to the present invention is conjugated to an imageable agent. A wide variety of imageable agents, particularly radioisotopes, are available in the art. In certain embodiments, the imageable agent is a gamma-emitting isotope, e.g. 125 In certain embodiments, the antibody protein has a specific activity of about 0.5 to about 15 mCi / mg, or about 0.5 to about 14 mCi / mg, or about 1 to about 10 mCi / mg, or about 1 to about 5 mCi / mg, and about 2 to 6 mCi / mg, or 1 to 3 mCi / mg.

[0219] 3. Compositions and Pharmaceutical Compositions The present invention includes compositions (e.g., pharmaceutical compositions) comprising a subject antibody or antigen-binding fragment thereof, or immunoconjugate thereof, as described herein, and a carrier (e.g., a pharmaceutically acceptable carrier). The present invention also includes compositions (e.g., pharmaceutical compositions) comprising a subject antibody or antigen-binding fragment thereof, or a conjugate thereof, and a carrier (e.g., a pharmaceutically acceptable carrier), and further comprising a second therapeutic agent. The compositions are useful for inhibiting abnormal cell growth or treating proliferative disorders, including hematological cancers, leukemias, or lymphomas, in mammals (e.g., humans).

[0220] In particular, the present invention provides pharmaceutical compositions comprising one or more of the CD44v9-binding agents or immunoconjugates thereof described herein. In certain embodiments, the pharmaceutical compositions further comprise a pharmaceutically acceptable vehicle. These pharmaceutical compositions find use in inhibiting tumor growth and treating cancer in human patients, including hematological cancers, leukemia, or lymphoma.

[0221] In certain embodiments, a formulation for storage and use is prepared by combining a purified antibody of the invention, or an immunoconjugate thereof, with a pharmaceutically acceptable vehicle (e.g., carrier, excipient) (Remington, The Science and Practice of Pharmacy, 20th ed., Mack Publishing, 2000). Suitable pharmaceutically acceptable vehicles include, but are not limited to, non-toxic buffers, such as phosphate, citric acid, and other organic acids; salts such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (e.g., polypeptides containing approximately 10 amino acid residues) less than 500 mg; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and non-ionic surfactants such as TWEEN® or polyethylene glycol (PEG).

[0222] Pharmaceutically acceptable carriers may contain physiologically acceptable compounds, such as those that stabilize or increase the absorption of AMPA glutamate receptor agonists, antagonists or modulators.Such physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low-molecular-weight proteins or other stabilizers or excipients (see also, for example, Remington's Pharmaceutical Sciences (1990), 18th Edition, Mack Publ., Easton).Those skilled in the art will know that the selection of pharmaceutically acceptable carriers, including physiologically acceptable compounds, depends, for example, on the route of administration of the composition.

[0223] Suitable pharmaceutically acceptable carriers, diluents, and excipients are generally well known and may be determined by those of ordinary skill in the art as clinical circumstances warrant. Examples of suitable carriers, diluents, and / or excipients include: (1) Dulbecco's phosphate-buffered saline, pH about 7.4, with or without about 1 mg / mL to 25 mg / mL human serum albumin, (2) 0.9% saline (0.9% w / v NaCl), and (3) 5% (w / v) dextrose; and may also contain antioxidants, such as tryptamine, and stabilizers, such as Tween® 20.

[0224] The pharmaceutical compositions described herein can be administered by any method for either local or systemic treatment.Administration can be topical (e.g., to mucous membranes, including vaginal and rectal delivery), for example, transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders; pulmonary (e.g., powder or aerosol inhalation or insufflation, including by nebulizer; including intrathecal, intranasal, epithelial, and transdermal); oral; or parenteral, including intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial (e.g., intrathecal or intraventricular) administration.In some specific embodiments, administration is intravenous.The pharmaceutical compositions described herein can also be used in vitro or ex vivo.

[0225] In animals or humans, it may be suitable to apply the pharmaceutical composition as described above to the tissue or organ of interest intravenously or by other routes, for example, systemically, locally or locally, depending on the type and origin of the disease or problem to be treated, such as tumor.For example, when different organs or organ systems need to be treated, such as in the case of systemic autoimmune disease, allergy, or transplantation of foreign organs or tissues, or in the case of tumors that are diffuse or difficult to locate, a systemic mode of action is desirable.When only local signs of neoplasia or immune action are expected, such as in the case of local tumors, a local mode of action is considered.

[0226] Pharmaceutical compositions containing the antibody protein of the present invention may be administered via different routes of administration known to experts, in particular by intravenous injection or direct injection into the target tissue. For systemic administration, intravenous, intravascular, intramuscular, intraarterial, intraperitoneal, oral, or intrathecal routes are preferred. More localized administration may be achieved by subcutaneous, intradermal, intracardiac, intralobar, intramedullary, intrapulmonary, or by direct administration into or near the tissue to be treated (connective tissue, bone tissue, muscle tissue, nervous tissue, epithelial tissue). Depending on the desired duration and effectiveness of the treatment, the pharmaceutical antibody composition may be administered once or several times, as well as intermittently, for example daily for several days, weeks, or months, and in different dosages.

[0227] To prepare suitable pharmaceutical compositions containing antibody preparations for the above applications, known injectable, physiologically acceptable sterile solutions may be used. To prepare ready-to-use solutions for parenteral injection or infusion, aqueous isotonic solutions, such as physiological saline or corresponding plasma protein solutions, are readily available. Pharmaceutical compositions may also exist as lyophilized or dried preparations, which may be reconstituted with known injectable solutions under sterile conditions immediately prior to use, e.g., as a kit of parts. The final preparation of the antibody composition of the present invention is prepared for injection, infusion, or perfusion by mixing the purified antibody according to the present invention with a sterile, physiologically acceptable solution, optionally supplemented with known carrier substances and / or additives (e.g., serum albumin, dextrose, sodium bisulfite, EDTA).

[0228] The amount of antibody applied depends on the nature of the disease. In cancer patients, the applied dose of the "naked" antibody contained in the pharmaceutical composition of the present invention is 0.1 to 100 mg / m 2 Between 5 and 50 mg / m per application 2 Between 10 mg / m 2 ~about 40mg / m 2 , 10 mg / m 2 ~about 30mg / m 2 , and 20 mg / m 2 ~about 30mg / m 2 , and approximately 25 mg / m 2 Body surface area may be approximately 50 mg / m 2 Body surface area antibody protein doses may also be used.

[0229] The dose of radioactivity delivered to the patient per administration must be high enough to be effective, but below dose-limiting toxicity (DLT). For example, 186For pharmaceutical compositions containing rhenium-radiolabeled antibodies, a maximum tolerated dose (MTD) must be determined, which should not be exceeded in a therapeutic setting. The radiolabeled antibody may then be administered to cancer patients by repeated (monthly or weekly) intravenous infusions of doses below the MTD (see, e.g., Welt et al. (1994) J. Clin. Oncol. 12: 1193-1203). Multiple administrations, typically at weekly intervals, are preferred; however, radiolabeled substances should be administered at longer intervals, i.e., 4 to 24 weeks apart, preferably 12 to 20 weeks apart. However, those skilled in the art may divide the administration into two or more administrations, which may be administered slightly later than each other or at some other predetermined interval, e.g., one day to one week apart.

[0230] Furthermore, the radioactivity dose applied follows the guidelines outlined below. Generally, the radioactivity dose per administration is between 30 and 75 mCi / m 2 Body surface area (BSA) is the area that will be applied to the patient. 186 rhenium, 188 rhenium, 99m technetium, 131 Iodine or 90 Labeled with yttrium, preferably 186 The amount of radiolabeled antibody in the pharmaceutical composition according to the invention that is labeled with rhenium is 10, 20, 30, 40, 50 or 60 mCi / m 2 , preferably 50 mCi / m 2 In one embodiment, the present invention provides a method for administering a radiolabeled antibody of the present invention at a dose of 50 mCi / m 2 The present invention relates to a pharmaceutical composition comprising:

[0231] In certain embodiments, the pharmaceutical compositions according to the present invention further comprise one or more radioprotectants selected from the group consisting of ascorbic acid, gentisic acid, reducing acid, erythorbic acid, p-aninobenzoic acid, 4-hydroxybenzoic acid, nicotinic acid, nicotinamide, 2,5-dihydroxy-1,4-benzenedisulfonic acid, povidone, inositol, and / or citric acid. In certain embodiments, the radioprotectant is ascorbic acid.

[0232] The antibody or immunoconjugate of the present invention may be combined with a second compound, such as one known to be effective in treating the disease or disorder of interest, in a pharmaceutical combination formulation or in a combined therapy regimen. In some embodiments, the second compound is an anti-cancer agent. In some embodiments, the method includes administering a second compound and an immunoconjugate of the present invention, resulting in greater efficacy compared to administering the immunoconjugate alone. The second compound may be administered via any of several methods, including, for example, topical, pulmonary, oral, parenteral, or intracranial administration. In some cases, administration is oral. In some embodiments, administration is intravenous. In some embodiments, administration is both oral and intravenous.

[0233] The antibody or immunoconjugate may also be combined with analgesics, or other medications, in a pharmaceutical combination formulation or dosage regimen as a combination therapy.

[0234] The antibody or immunoconjugate may be combined with a second compound having anti-cancer properties in a pharmaceutical combination or in a dosage regimen as a combination therapy. The second compound of the pharmaceutical combination or dosage regimen may have complementary activities to the ADC of the combination such that they do not adversely affect each other. Pharmaceutical compositions comprising a CD44v9-binding agent and a second anti-cancer agent are also provided.

[0235] In certain embodiments, a therapeutically effective amount of a subject antibody or antigen-binding fragment thereof, or immunoconjugate, or composition thereof described herein, alone or in combination with a second therapeutic agent, preferentially inhibits proliferation of leukemic stem cells (LSC), leukemic progenitor cells (LP), and / or leukemic blasts, over normal hematopoietic stem cells (HSC). In certain embodiments, the subject agent inhibits proliferation of leukemic stem cells (LSC), leukemic progenitor cells (LP), and / or leukemic blasts, or over normal hematopoietic stem cells (HSC). 50 The value or half-maximal concentration is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 300, 500 times lower than that for normal hematopoietic stem cells (HSCs).

[0236] 4.Treatment The present invention includes methods for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (e.g., a human), comprising administering to the mammal a subject antibody or antigen-binding fragment thereof, or immunoconjugate, or composition thereof, described herein, alone or in combination with a second therapeutic agent.

[0237] Another aspect of the present invention is the use of an antibody protein according to the present invention in the manufacture of a medicament for the treatment of cancer. Another aspect of the present invention relates to the use of an antibody protein according to the present invention conjugated with a therapeutic agent as described above for the treatment of cancer. Cancer includes any disease associated with malignant growth, such as solid tumors, sarcomas, and leukemias. A necessary prerequisite for such diseases is CD44v9 expression.

[0238] The present invention also provides a method for inducing cell death in a selected cell population, comprising contacting target cells or tissue containing target cells with an effective amount of a subject antibody or antigen-binding fragment thereof, or immunoconjugate of the invention, the target cells being cells to which the cell-binding agent of the conjugate is capable of binding.

[0239] The methods of the invention for inducing cell death, inhibiting cell proliferation, and / or treating cancer in selected cell populations may be carried out in vitro, in vivo, or ex vivo. For clinical in vivo use, the cytotoxic compounds or conjugates of the invention are supplied as solutions or lyophilized powders and tested for sterility and endotoxin levels.

[0240] In certain embodiments, the abnormal cell growth or proliferative disorder in a mammal is a disease or condition associated with or characterized by expression of CD44v9, such as cancer.

[0241] For example, the cancer may be selected from the group consisting of: epithelial carcinomas, including those of breast, lung, liver, colorectal, head and neck, esophageal, pancreatic, ovarian, bladder, stomach, skin, endometrial, ovarian, testicular, esophageal, prostatic or renal origin; bone and soft tissue sarcomas, including osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma (MFH), and leiomyoma; hematopoietic malignancies, including lymphoma and leukemia; neuroectodermal tumors, including peripheral nerve tumor, astrocytoma, and melanoma, and mesothelioma.

[0242] Cancers according to the present invention may also include, but are not limited to: 1) epithelial carcinomas, including those of breast, lung, liver, colorectal, head and neck, esophageal, pancreatic, ovarian, bladder, stomach, skin, endometrial, ovarian, testicular, esophageal, prostatic or renal origin; 2) bone and soft tissue sarcomas: osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma (MFH), leiomyoma; 3) hematopoietic malignancies: Hodgkin's lymphoma and non-Hodgkin's lymphoma, leukemia; 4) neuroectodermal tumors; peripheral nerve tumor, astrocytoma, melanoma; 5) mesothelioma.

[0243] Examples of cancerous disease states associated with solid tumors include, but are not limited to: colorectal cancer, non-small cell lung cancer, breast cancer, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, pancreatic cancer, and metastatic cancer of the brain.

[0244] In certain embodiments, the cancer has at least one negative prognostic factor.

[0245] Another aspect of the present invention relates to the use of an antibody protein according to the present invention as defined above in the manufacture of a medicament for the treatment of cancer, wherein the amount of antibody protein per application is between 0.1 and 100 mg / m 2 Between 5 and 50 mg / m 2 Between 10 mg / m 2 ~about 40mg / m 2 , 10 mg / m 2 ~about 30mg / m 2 , or 20 mg / m 2 ~about 30mg / m 2 , or 25 mg / m 2 body surface area, or approximately 50 mg / m 2 Body surface area.

[0246] In a particular embodiment, the antibody protein conjugated to a radioisotope according to the present invention as defined above is used in the manufacture of a medicament for the treatment of cancer, wherein the radioactive dose per administration is between 30 and 75 mCi / m 2 In a particular embodiment, the antibody protein according to the present invention is 186 rhenium, 188 rhenium, 99m technetium, 131 Iodine or 90 Yttrium, for example 186 In yet another aspect, the present invention relates to the use of an antibody protein conjugated with a radioisotope according to the present invention, as defined above, in the manufacture of a medicament for the treatment of cancer, wherein the antibody dose is 10, 20, 30, 40, 50 or 60 mCi / m 2 , or 50 mCi / m 2 is.

[0247] In a specific embodiment, an antibody protein conjugated to a radioisotope according to the present invention as defined above is used in the manufacture of a medicament for the treatment of cancer, wherein the antibody protein has a specific activity of about 0.5 to about 15 mCi / mg, or about 0.5 to about 14 mCi / mg, preferably about 1 to about 10 mCi / mg, preferably about 1 to about 5 mCi / mg, and most preferably 2 to 6 mCi / mg or 1 to 3 mCi / mg.

[0248] Also preferred is the use of an antibody protein conjugated to a radioisotope according to the invention as defined above in the manufacture of a medicament for the treatment of cancer, wherein said antibody or antibody derivative is in an aqueous solution at a pH of about 7 to about 8 and at a concentration of about 0.5 to about 2.0 mg / ml.

[0249] The present invention further relates to a method for treating cancer, comprising administering an antibody according to the present invention to an individual in need thereof one to several times, wherein the antibody protein selectively binds to CD44v9 and destroys tumor cells through a therapeutic agent linked to the antibody protein, and monitoring the success of the therapy. The antibody protein may be present as a naked / unmodified antibody protein, a modified antibody protein, e.g., a fusion protein, or an antibody protein conjugated to a therapeutic agent, and the method comprises contacting a tumor with an effective amount of the antibody. The method for treating tumors as described above may be effective in vitro or in vivo. The cancer may be any of the cancers described above.

[0250] Cancer therapies, including their dosages, routes of administration, and recommended methods of use, are known in the art and are described in such publications as the Physician's Desk Reference (PDR). The PDR discloses the dosages of agents used in the treatment of various cancers. The therapeutically effective dosage regimens and doses of these aforementioned chemotherapeutic agents depend on the particular cancer being treated, the severity of the disease, and other factors well known to physicians in the art and can be determined by a physician. The contents of the PDR are incorporated herein by reference in their entirety. Those skilled in the art may use one or more of the following parameters to review the PDR and determine the dosage regimens and doses of chemotherapeutic agents and conjugates that may be used in accordance with the teachings of this invention. These parameters include: General Index; Manufacturer; Product (company-registered drug name); Category Index; Generic / Chemical Index (non-trademark generic drug name); Drug Color Image; Product Information Consistent with FDA Labeling; Chemical Information; Function / Action; Indications and Contraindications; Experimental Studies, Side Effects, and Warnings.

[0251] The amount of antibody applied depends on the nature of the disease. In cancer patients, the applied dose of "naked" antibody ranges from 0.1 to 100 mg / m 2 , 5-50 mg / m per application 2 , 10 mg / m 2 ~about 40mg / m 2 , 10 mg / m 2 ~about 30mg / m 2 , and 20 mg / m 2 ~about 30mg / m 2 , and approximately 25 mg / m 2 body surface area, or approximately 50 mg / m 2 Body surface area.

[0252] Per administration, the dose of radioactivity delivered to the patient must be high enough to be effective, but below dose-limiting toxicity (DLT). For example, 186For rhenium-radiolabeled antibodies, a maximum tolerated dose (MTD) must be determined, which should not be exceeded in a therapeutic setting. Radiolabeled antibodies can then be administered to cancer patients by repeated (monthly or weekly) intravenous infusions of doses below the MTD (see, e.g., Welt et al. (1994) J. Clin. Oncol. 12: 1193-1203). Multiple administrations, typically weekly, are preferred; however, radiolabeled materials should be administered at longer intervals, i.e., 4 to 24 weeks apart, or 12 to 20 weeks apart. However, those skilled in the art may divide the administration into two or more administrations, administered slightly later than each other, or at some other predetermined interval, e.g., one day to one week apart.

[0253] Also provided is a method of cancer therapy according to the invention (see above), wherein the antibody protein conjugated to a radioisotope according to the invention, as defined above, has a specific activity of about 0.5 to about 15 mCi / mg, or about 0.5 to about 14 mCi / mg, preferably about 1 to about 10 mCi / mg, preferably about 1 to about 5 mCi / mg, and most preferably 2 to 6 mCi / mg or 1 to 3 mCi / mg.

[0254] Also provided is a method of cancer treatment according to the invention (see above), wherein the antibody protein conjugated to a radioisotope according to the invention, as defined above, is in an aqueous solution at a pH of about 7 to about 8, and at a concentration of about 0.5 to about 2.0 mg / ml.

[0255] In particular embodiments, the cancer is colorectal cancer, non-small cell lung cancer, breast cancer, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, pancreatic cancer or metastatic cancer of the brain.

[0256] The methods of the present invention also provide in vitro methods for killing cells, such as cancer cells. Examples of in vitro uses include treating autologous bone marrow before transplantation into the same patient to kill diseased or malignant cells; treating bone marrow before transplantation to kill qualified T cells and prevent graft-versus-host disease (GVHD); treating cell cultures to kill all cells except for desired variants that do not express the target antigen; or to kill variants that express undesirable antigens.

[0257] Conditions for non-clinical in vitro use are readily determined by one of ordinary skill in the art.

[0258] Examples of clinical ex vivo uses include the removal of tumor cells or lymphocytes from bone marrow prior to autologous transplantation in cancer or autoimmune disease treatment, or the removal of T cells and other lymphocytes from autologous or allogeneic bone marrow or tissue prior to transplantation to prevent graft-versus-host disease (GVHD). Treatment may be performed as follows: Bone marrow is harvested from a patient or other individual and then incubated at about 37°C for about 30 minutes to about 48 hours in serum-containing medium supplemented with a cytotoxic agent of the present invention at a concentration ranging from about 10 μM to 1 pM. The exact conditions of concentration and time of incubation, i.e., dose, are readily determined by one of ordinary skill in the art. After incubation, the bone marrow cells are washed with serum-containing medium and returned intravenously to the patient according to known methods. If the patient undergoes other treatments, such as ablative chemotherapy or a course of total-body irradiation, between bone marrow harvest and reinfusion of the treated cells, the treated bone marrow cells are cryopreserved in liquid nitrogen using standard medical equipment.

[0259] 5. Nucleic acid A further aspect of the present invention is a nucleic acid characterized in that it encodes an antibody or protein according to the present invention. The nucleic acid may be RNA or, preferably, DNA. The DNA molecule may be chemically synthesized. Appropriate oligonucleotides may be first used to generate a synthetic gene, or the gene may be synthesized by methods known in the art (e.g., Gait, MJ, 1984, Oligonucleotide Synthesis. A Practical Approach. IRL Press, Oxford, UK). Methods for generating synthetic genes are known in the art (e.g., Stemmer et al. 1995, Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides, Gene 164(1): 49-53; Ye et al. 1992, Gene synthesis and expression in E. coli for pump, a human matrix metalloproteinase, Biochem Biophys Res Commun 186(1): 143-9; Hayden and Mandecki 1988, Gene synthesis by serial cloning of oligonucleotides, DNA 7(8): 571-7). These methods may be used to synthesize any of the DNA molecules disclosed in this application.

[0260] The nucleic acids of the present invention may contain 5' or 3' or 5' and 3' untranslated regions. The nucleic acids of the present invention may contain other untranslated regions upstream and / or downstream. The untranslated regions may contain regulatory elements, such as transcription initiation units (promoters) or enhancers. The promoters may be, for example, constitutive, inducible, or developmentally regulated promoters. In particular embodiments, and without excluding other known promoters, constitutive promoters of human cytomegalovirus (CMV) and Rous sarcoma virus (RSV), as well as simian virus 40 (SV40) and herpes simplex promoters. Inducible promoters of the present invention include antibiotic resistance promoters, heat shock promoters, hormone-inducible "mammary tumor virus promoters," and metallothionein promoters. The nucleic acids of the present invention may encode fragments of antibody proteins of the present invention. This refers to portions of polypeptides of the present invention.

[0261] 6. Vector Another important aspect of the present invention is a recombinant DNA vector characterized by containing the nucleic acid according to the present invention. Examples include viral vectors such as vaccinia, Semliki Forest virus, and adenovirus. Vectors for use in COS cells contain the SV40 origin of replication, making it possible to achieve high copy numbers of the plasmid. Vectors for use in insect cells are, for example, E. coli transfer vectors and contain, for example, DNA encoding polyhedrin as a promoter.

[0262] Another aspect of the present invention is a recombinant DNA vector according to the invention, characterized in that it is an expression vector.

[0263] Another aspect of the invention is a recombinant DNA vector according to the invention, characterized in that it is the vector pAD-CMV or a functional derivative thereof, such as pAD-CMV1, pAD-CMV19 or pAD-CMV25.

[0264] The vector may be one disclosed in U.S. Patent No. 5,648,267A or 5,733,779A, which comprises the nucleotide sequence according to the invention. Another aspect of the invention is a recombinant DNA vector according to the invention, characterized in that it is the vector N5KG1 Val or a derivative thereof.

[0265] 7. Cells or host cells Another aspect is a host characterized in that it contains a vector according to the invention.

[0266] Another aspect is a host according to the invention, characterized in that it is a eukaryotic host cell, including fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma, insect cells (e.g., from Spodoptera frugiperda Sf-9 with a baculovirus expression system), plant cells such as those from Nicotiana tabacum, and mammalian cells such as COS, BHK, CHO, or myeloma cells.

[0267] The antibody proteins according to the present invention are particularly well folded and glycosylated in the progeny of immune system cells that also form antibody proteins in the human body. Mammalian host cells are preferred, preferably CHO or COS cells, such as CHO DG44 (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77(7):4216-20(1980)), or CHO-K1 (ATCC CCL-61) cells. Thus, another aspect is a host according to the present invention characterized in that it is a BHK, CHO, or COS cell, most preferably a CHO DG44 or CHO-K1 (ATCC CCL-61) cell.

[0268] In a particular embodiment, the host is a bacteriophage.

[0269] In particular embodiments, the host is a prokaryotic host cell. Examples of prokaryotic host cells are Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis.

[0270] The present invention further relates to a process for preparing an antibody protein according to the present invention, characterized in that it comprises the following steps: culturing a host according to the present invention under conditions in which the antibody protein is expressed by the host cells, and isolating the antibody protein. The antibody according to the present invention may be produced as follows: Nucleic acid molecules encoding the light and heavy chains may be chemically and enzymatically synthesized by standard methods. Appropriate oligonucleotides may first be synthesized by methods known in the art (detailed above). Methods for generating synthetic genes from oligonucleotides are known in the art (detailed above). These nucleic acid molecules encoding the antibody heavy and light chains may be cloned into expression vectors (either both chains in one vector molecule or each chain in a separate vector molecule), which are then introduced into host cells. The host cells may be mammalian host cells (detailed above), such as COS, CHO (Chinese hamster ovary), or BHK cells. The host cells are then cultured in an appropriate medium under conditions in which the antibody is produced, and the antibody is then isolated from the culture according to standard methods. Methods for producing antibodies from recombinant DNA and respective expression vectors in host cells are well known in the art (see, for example, WO 94 / 11523, WO 97 / 9351, EP 0481790).

[0271] The present invention also relates to a process in which the host is a mammalian cell, preferably a CHO or COS cell.

[0272] In a particular embodiment, the host cell is co-transfected with two plasmids carrying the expression units for the light chain or the heavy chain. [Example]

[0273] The following examples serve to further illustrate the present invention; however, should not be construed as limiting the scope of the invention disclosed herein.

[0274] Example 1 Mouse monoclonal antibody HTS033 targets human CD44v9 exons Figure 1 shows the binding of the monoclonal mouse antibody HTS033 to human CD44 recombinant protein in the presence of different CD44 peptides by standard ELISA. Of the peptides tested, the CD44v-specific peptide P14 blocked HTS033 binding to recombinant CD44, suggesting that HTS033 is specific for the CD44 v9 exon.

[0275] Example 2 Mouse monoclonal antibody HTS033 targets cell surface CD44v9 on cancer cells HTS033 was tested against control and CD44-FLAG-overexpressing HEK293 cells containing the v9 exon. Control and CD44-FLAG-overexpressing HEK293 cells were incubated with anti-FLAG and HTS033 for 30 minutes, followed by a secondary antibody for 30 minutes. FACS analysis was performed using a BD Accuri C6. As shown in Figure 2, the original HEK293 cells were not stained with either the anti-FLAG or HTS033 antibodies. However, the FLAG-positive overexpressing cells also stained positively with HTS033. This result indicates that HTS033 can bind to CD44v9 on the cell surface.

[0276] Furthermore, HTS033 selectively binds to multiple cancer cell lines. In FACS analysis, several cancer cell lines were incubated with HTS033 for 30 minutes, followed by FITC-conjugated anti-mouse IgG antibody for 30 minutes. SKMES-1 tumor cells did not bind to HTS033, while BxPC3, NCl-H266, and NCl-H256 were stained by HTS033 (see Figure 3).

[0277] HTS033 binding to cancer cells was reduced after siRNA-mediated knockdown of CD44v9. Specifically, BxPC-3 and HUH7 cells were transfected with control siRNA or siRNA targeting CD44v9 or CD44v6 for 48 hours and then subjected to FACS analysis. Both BxPC-3 and HUH7 cells treated with control siRNA (CON-HTS033) stained positively with HTS033 (Figures 4A and 4C). siRNA targeting CD44 exon 9 reduced CD44v9 expression by ~43% in BxPC3 and ~61% in HUH7 (Figures 4B and 4D), resulting in decreased surface staining intensity (Figures 4A and 4C) and a downward shift in median signal intensity (Figures 4B and 4D) in both cell lines. Some CD44 molecules appear to contain both exons 6 and 9, and therefore targeting exon 6 of CD44 also resulted in a reduction in overall CD44 levels, knocked down some expression of exon 9-containing CD44, and reduced staining with HTS033.

[0278] The binding affinity of HTS033 to MDA-MB-468, NCl-H226, 5637, RT-4, and NCl-H520 cells was measured by FACS analysis. FACS titration of HTS033 was performed by incubating the cells with serial dilutions of HTS033 (17–1,000,000 pM) and subsequently staining the cells with FITC-conjugated anti-mouse IgG. EC50 values ​​were determined to be in the range of 2–8 nM (see Figure 5).

[0279] Table A below lists the binding of HTS033 to a panel of cell lines representing multiple tumor types by FACS analysis.

[0280] Table A: HTS033 antibody FACS binding to multiple cancer cell lines

[0281] [Table 2] Example 3 CD44v9 Expression in Human Non-Small Cell Lung and Breast Cancer HTS033 staining for CD44v9 was positive in several tumor samples. In particular, immunohistochemical staining with HTS033 was positive in 13 of 25 triple-negative breast cancer patient samples (representative images shown in Figure 6A). HTS033 staining was also positive in 34 of 54 non-small cell lung cancer patient samples (representative images shown in Figure 6B). This suggests that CD44v9 expression is upregulated in breast and lung cancer patients and that CD44v9 may be useful for targeted therapy.

[0282] Example 4. Chimeric and humanized antibodies of HTS033 retain binding and specificity for the CD44 v9 exon To obtain an antibody more suitable for application in humans, chimeric and humanized versions of HTS033, designated chHTS033 and hHTS033, respectively, were generated. The two resulting hHTS033 antibodies are designated 25-1 and 25-3.

[0283] Figure 7 shows the binding of chimeric HTS033 to human CD44 recombinant protein in the presence of different CD44 peptides. The presence of most peptides did not interfere with the binding of chimeric HTS033 to recombinant CD44, with the exception of the CD44v9-specific peptide, P14, which completely blocked binding. This indicates that chimeric HTS033 binds to recombinant CD44 at the v9 exon and that the antibody is specific for the v9 exon.

[0284] The antibody affinity of chimeric and humanized HTS033 was measured by FACS analysis of antibody binding to PC-9 cells. FACS titration of each antibody was performed by incubating cells with serial dilutions of antibody (17–1,000,000 pM) and subsequently staining the cells with an Alexa-488-conjugated goat anti-mouse secondary antibody (Figure 8A). The EC50 value of chimeric HTS033 was approximately 34.7 nM. The EC50 values ​​of the two humanized HTS033 antibodies, 25-1 and 25-3, were 21.8 and 17.8 nM, respectively (Figure 8B).

[0285] Example 5 Indirect cytotoxicity of HTS033 correlates with CD44v9 expression level in cancer cells Figures 9 and 10 show the indirect cytotoxicity of HTS033 and control IgG. Cells were cultured and then treated with serial dilutions of HTS033 or IgG control along with MMAE-conjugated anti-mouse IgG antibody for 72 hours. Cell numbers were determined by CCK-8 cell viability assay. The graph shows the % viable cells compared to the untreated control. The IC of HTS033 for each cell line was 50 Overall, the cocktail mix of MMAE-conjugated secondary antibodies and HTS033 was toxic to some degree to all cell lines, while the IgG control cocktail had no effect.

[0286] Furthermore, there is a correlation between the efficacy of HTS033 and the drug cocktail and the expression levels of CD44v9 on different cell lines. Among the cell lines tested, most with higher levels of CD44v9 expression, including BxPC-3, NCl-H520, 5637, MDA-MB-468, and RT-4 (see Table A), had lower estimated IC50 values ​​in the 14-42 pM range. Cell lines with lower CD44v9 expression levels, such as HUH7, SK-HEP-1, and MDA-MB-231, had IC50 values ​​in the 126-316 pM range, almost an order of magnitude higher. This data suggests that the HTS033 antibody and antibodies derived from it may be highly effective in targeting cancers with high CD44v9 expression.

[0287] Example 6 Indirect cytotoxicity of humanized HTS033 against cancer cells Figure 11 shows a direct cytotoxicity assay using humanized HTS033 conjugated with MMAE. Cells were treated with serially diluted humanized HTS033-25-1-MMAE or negative control antibody-conjugated MMAE (1.5 pM to 30 nM) for 72 hours. Cell viability was determined by CCK-8 cell viability assay. IC of humanized HTS033-25-1 MMAE 50 The values ​​were 269 pM (PC-9), 203 pM (A253), and 309 pM (BxPC3), respectively.

[0288] Example 7 Cytotoxicity of CD44v9-targeting CAR-T cells through expression of the antigen-binding domain of HTS033 Figure 12 shows the cytotoxicity of HTS033 CAR-T against PC-9 lung cancer cells. CAR-T cells generated with the HTS033 variable domain sequence and control T cells were incubated with PC-9 cells at E:T ratios of 1:1, 3:1, and 6:1 for 24 hours. After T cell removal, PC-9 cell viability was determined by CCK-8 assay. % cytotoxicity was calculated as follows: (1 - treatment viability / control viability) x 100%. Control viability is the value obtained from target cells alone without the addition of T cells.

[0289] At all E:T ratios tested, HTS033 CAR-T was effective in killing PC-9 cells.

[0290] Example 8 Antibody Sequences The sequences of the variable regions / domains of HTS033, hHTS033 25-1 and hHTS033 25-3 are listed below.

[0291] Table 1: Variable region sequences of HTS033

[0292] [Table 3] Table 2: Variable region sequences of hHTS033, 25-1

[0293] [Table 4] Table 3: Variable region sequences of hHTS033, 25-3

[0294] [Table 5] Table 4: Variable region sequences of mAb116

[0295] [Table 6]

Claims

1. 1. An isolated monoclonal antibody or antigen-binding fragment thereof specific for CD44v9, the isolated monoclonal antibody or antigen-binding fragment thereof comprising: (1) A heavy chain variable region (HCVR) comprising: (a) the HCVR CDR1 sequence of SEQ ID NO: 1; (b) the HCVR CDR2 sequence of SEQ ID NO: 2; and (c) the HCVR CDR3 sequence of SEQ ID NO: 3 a heavy chain variable region comprising: (2) a light chain variable region (LCVR) comprising: (d) the LCVR CDR1 sequence of SEQ ID NO: 4; (e) the LCVR CDR2 sequence of SEQ ID NO: 5; and (f) the LCVR CDR3 sequence of SEQ ID NO: 6 a light chain variable region comprising 2. The isolated monoclonal antibody or antigen-binding fragment thereof, comprising:

2. 2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, comprising: (1) heavy chain framework region sequences of FR1 of SEQ ID NO: 7, FR2 of SEQ ID NO: 8, FR3 of SEQ ID NO: 9, and FR4 of SEQ ID NO: 10, respectively, containing up to two amino acid mutations in each framework region sequence; and (2) light chain framework region sequences of FR1 of SEQ ID NO: 11, FR2 of SEQ ID NO: 12, FR3 of SEQ ID NO: 13, and FR4 of SEQ ID NO: 14, respectively, containing up to two amino acid mutations in each framework region sequence; or (i) heavy chain framework region sequences of FR1 of SEQ ID NO: 37, FR2 of SEQ ID NO: 38, FR3 of SEQ ID NO: 39, and FR4 of SEQ ID NO: 40, respectively, containing up to two amino acid mutations in each framework region sequence; and (ii) light chain framework region sequences of FR1 of SEQ ID NO: 41, FR2 of SEQ ID NO: 42, FR3 of SEQ ID NO: 43, and FR4 of SEQ ID NO: 44, respectively, containing up to two amino acid mutations in each framework region sequence; The isolated monoclonal antibody or antigen-binding fragment thereof further comprises:

3. 3. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 2, comprising: (1) Heavy chain framework region sequences of FR1 of SEQ ID NO: 7, FR2 of SEQ ID NO: 8, FR3 of SEQ ID NO: 9, and FR4 of SEQ ID NO: 10, respectively, and light chain framework region sequences of FR1 of SEQ ID NO: 11, FR2 of SEQ ID NO: 12, FR3 of SEQ ID NO: 13, and FR4 of SEQ ID NO: 14, respectively. or (2) Heavy chain framework region sequences of FR1 of SEQ ID NO: 37, FR2 of SEQ ID NO: 38, FR3 of SEQ ID NO: 39, and FR4 of SEQ ID NO: 40, respectively, and light chain framework region sequences of FR1 of SEQ ID NO: 41, FR2 of SEQ ID NO: 42, FR3 of SEQ ID NO: 43, and FR4 of SEQ ID NO: 44, respectively. The isolated monoclonal antibody or antigen-binding fragment thereof, comprising:

4. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, which is a humanized murine monoclonal antibody.

5. 5. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 4, comprising: (3) Heavy chain framework region sequences of FR1 of SEQ ID NO: 15, FR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and FR4 of SEQ ID NO: 18, respectively, and light chain framework region sequences of FR1 of SEQ ID NO: 19, FR2 of SEQ ID NO: 20, FR3 of SEQ ID NO: 21, and FR4 of SEQ ID NO: 22, respectively. or (4) Heavy chain framework region sequences of FR1 of SEQ ID NO: 23, FR2 of SEQ ID NO: 24, FR3 of SEQ ID NO: 25, and FR4 of SEQ ID NO: 26, respectively, and light chain framework region sequences of FR1 of SEQ ID NO: 27, FR2 of SEQ ID NO: 28, FR3 of SEQ ID NO: 29, and FR4 of SEQ ID NO: 30, respectively. The isolated monoclonal antibody or antigen-binding fragment thereof further comprises:

6. K of 40 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM or less for CD44v9 Ｄ The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which binds to

7. The antigen-binding fragment may be Fab, Fab', F(ab') ２ , single chain Fv or scFv, disulfide-linked F ｖ , IgGΔCH ２ , minibody, F(ab') ３ , tetrabody, triabody, diabody, DVD-Ig, Fcab, mAb ２ , (scFv) ２ 7. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which is an scFv-Fc or scFv-Fc.

8. A polypeptide comprising the HCVR and LCVR of any one of claims 1 to 7, wherein the polypeptide is a fusion protein.

9. A polynucleotide or a set of polynucleotides encoding the heavy and light chains of the isolated monoclonal antibody of any one of claims 1 to 7, or the HCVR and LCVR of the antigen-binding fragment of any one of claims 1 to 7, or the polypeptide of claim 8.

10. A vector comprising the polynucleotide or set of polynucleotides of claim 9.

11. A cell comprising the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 7, the polypeptide of claim 8, the polynucleotide or set of polynucleotides of claim 9, or the vector of claim 10.

12. The cell of claim 11, comprising the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 7, or the polypeptide of claim 8, on the cell surface.

13. 13. The cell of claim 12, which is a T cell bearing a chimeric antigen receptor (CAR), wherein the CAR comprises the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 7, or the polypeptide of claim 8.

14. A method for producing the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 7, or the polypeptide of claim 8, comprising: (a) culturing the cells of claim 11; and (b) isolating the monoclonal antibody, antigen-binding fragment thereof, or polypeptide from the cultured cells. The method comprises the steps of:

15. The following formula: A-[-L-D] ｎ an immunoconjugate having the formula: During the ceremony: Ab is the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-7, or the polypeptide of claim 8, covalently linked to one or more units of a linker-drug moiety [-L-D], where L is a linker and D is a cytotoxic drug; and n is an integer from 1 to 20; and Each linker-drug moiety has the same or different linker, L, or cytotoxic drug, D; Immunoconjugates.

16. each linker-drug moiety [-LD] is (1) the side chain amino group of Lys; (2) a side chain thiol group of Cys, or (3) Site-specific incorporation of unnatural amino acids 16. The immunoconjugate of claim 15, which is covalently attached to the Ab via

17. 17. The immunoconjugate of claim 15 or 16, wherein each linker L comprises a peptide unit, wherein the peptide unit comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.

18. The immunoconjugate of any one of claims 15 to 17, wherein the linker L is not cleavable by a protease, or the linker L is a cleavable linker that can be cleaved by a protease, an acidic environment, or a change in redox state.

19. 19. The immunoconjugate of any one of claims 15 to 18, wherein the cytotoxic agent is a DNA intercalator, a microtubule binding agent, a topoisomerase I inhibitor, a DNA minor groove binder, an auristatin class agent, a maytansine class agent, a calicheamicin, SN-38, or a PBD (pyrrolobenzodiazepine).

20. 20. The immunoconjugate of claim 19, wherein the auristatin class drug is monomethylauristatin E (MMAE) or MMAF, the maytansine class drug is DM-1, DM-3, or DM-4, ​​and the calicheamicin is ozogamicin.

21. A pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 7, or the polypeptide of claim 8, or the immunoconjugate of any one of claims 15 to 20, and a pharmaceutically acceptable carrier or excipient.

22. 22. The pharmaceutical composition of claim 21 for use in inhibiting the proliferation of cells expressing CD44v9.

23. 23. The pharmaceutical composition of claim 22, wherein the cell is a tumor cell.

24. 24. The pharmaceutical composition of claim 23, wherein the tumor cells are from lung cancer, colorectal cancer, breast cancer, head and neck cancer, ovarian cancer, bladder cancer, pancreatic cancer, or metastatic cancer of the brain.

25. A CD44v9 antagonist comprising a CD44v9 antibody or antigen-binding fragment thereof for use in treating a subject having cancer or a cell proliferative disorder, wherein cells of the cancer or cell proliferative disorder express CD44v9, and the CD44v9 antagonist comprises an isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 7, or a polypeptide of claim 8, or an immunoconjugate of any one of claims 15 to 20, or a pharmaceutical composition of claim 22.

26. 26. The CD44v9 antagonist of claim 25, wherein said cancer is an epithelial carcinoma, bone and soft tissue sarcoma, hematopoietic malignancy, neuroectodermal tumor, melanoma, or mesothelioma.

27. the epithelial cancer is of breast, lung, liver, colorectal, head and neck, esophageal, pancreatic, ovarian, bladder, gastric, skin, endometrial, testicular, prostate, or renal origin; the bone and soft tissue sarcoma is osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma (MFH), or leiomyoma; the hematopoietic malignancy is Hodgkin's lymphoma, non-Hodgkin's lymphoma, or leukemia; The neuroectodermal tumor is a peripheral nerve tumor or an astrocytoma.

27. The CD44v9 antagonist of claim 26.

28. 8. A method for determining the presence and / or abundance of CD44v9 in a sample from a subject, the method comprising contacting the sample with the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 7.

29. A method for providing information for diagnosing a subject having cancer, the method comprising determining the presence and / or abundance of CD44v9 in a cancer sample previously obtained from the subject using the method of claim 28.

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