Antibodies to m(h)DM2 / 4 and uses thereof in cancer diagnosis and treatment

Abstract

To provide antibodies against M(H)DM2 / 4 and uses thereof in cancer diagnosis and treatment.SOLUTION: The invention relates to certain anti-M(H)DM2 / 4 antibodies or antigen-binding fragments thereof, pharmaceutical compositions comprising anti-M(H)DM2 / 4 antibodies or antigen-binding fragments thereof, antibody-drug conjugates comprising anti-M(H)DM2 / 4 antibodies or antigen-binding fragments thereof bound to a cytotoxic drug, and the use of such antibodies, fragments, compositions and conjugates for treating cancer and / or for preventing metastasis.SELECTED DRAWING: None

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 537,914, filed July 27, 2017, which is incorporated herein by reference in its entirety.

[0002] 2. Reference to Electronically Submitted Sequence Listings This application incorporates by reference the Sequence Listing submitted herewith as an ASCII text file entitled "14160-006-228_ST25", created on July 25, 2018, and having a size of 88,287 bytes.

[0003] 3. Field The present invention relates to certain anti-M(H)DM2 / 4 antibodies or antigen-binding fragments thereof, pharmaceutical compositions comprising the anti-M(H)DM2 / 4 antibodies or antigen-binding fragments thereof, antibody-drug conjugates comprising the anti-M(H)DM2 / 4 antibodies or antigen-binding fragments thereof linked to cytotoxic drugs, and the use of such antibodies, fragments, compositions, and conjugates for treating cancer and / or preventing metastasis. [Background technology]

[0004] 4.Background The MDM2 (MDM2 is the mouse homolog of HDM2) protein consists of 489 amino acids and contains a p53-binding domain, two nuclear localization signals (amino acids 176-182 and 464-471), and zinc finger motifs (amino acids 297-326 and 436-477) (see UniProt accession number P23804 on the UniProt website). Its human homolog, HDM2, consists of 491 amino acids and contains a p53-binding domain, two nuclear localization signals (amino acids 179-185 and 466-473), and zinc finger motifs (amino acids 299-328 and 438-479) (see UniProt accession number Q00987 on the UniProt website). The mouse protein MDM4 (also identified as MDMX) is a homolog of the MDM2 protein (see UniProt accession number O35618 on the UniProt website), and both MDM2 and MDM4 are major negative regulators of p53 (Wade et al., 2013, Nat Rev. Cancer 13:83-96; Marine et al., 2004, Cell Cycle 3:900-904; Momand et al., 2011, Gene 486:23-30). HDM4 (also identified as HDMX) is a MD M(H)DM2 is the human homolog of M4 (see UniProt accession number O15151 on the UniProt website). The most conserved domain within all M(H)DM2 and M(H)DM4 proteins is the RING domain, which is responsible for ubiquitination of their target proteins, including p53, and for heterodimerization between M(H)DM2 and M(H)DM4. M(H)DM4 is required for M(H)DM2-mediated polyubiquitination of p53. A distinct feature of M(H)DM2 and M(H)DM4 is their highly complex expression patterns. Multiple sizes of M(H)DM2 transcripts and protein products have been identified in cancer cells by several groups (Olson et al., 1993, Oncogene 8:2353-2360; Bartel et al., 2002, Cancer Cell 2:9-15 ("Bartel 2002"); Sigalas et al., 1996, Nat. Med. 2:912-917 ("Sigalas 1996"); Iwakuma & Lozano, 2003, Mol. Cancer Res. 1:993-1000 ("Iwakuma & Lozano 2003"). Many types of human cancer overexpress the MDM2 protein, and a common feature among these cancers is the increase in the associated mdm2 splice variants. These M(H)DM2 variants have been shown to be expressed in a variety of tumors, including human ovarian, breast, and astrocytic neoplasms, glioblastoma, leukemia, and pediatric rhabdomyosarcoma (Iwakuma & Lozano 2003; Rosso et al., 2014, Subcell Biochem. 85:247-61 (“Rosso 2014”)). Most interestingly, they are found to be more frequent in advanced tumors (Bartel 2002). Multiple-sized M(H)DM2 transcripts, which have been shown to be splice variant forms of M(H)DM2 mRNA, have been reported to be expressed at higher frequencies in tumor cells than in normal cells (Bartel 2002). et al., 2004, Mol. Cancer Res. 2:29 ("Bartel 2004"). It has been suggested that an mRNA surveillance system exists in cells that degrades spliced ​​transcripts and protects cells from transcriptional errors, mRNA processing, or mRNA transport, whereas in transformed cells, this system may not function properly (Bartel 2004). Furthermore, some of these variants in cancer cells encode protein products that have been shown to transform NIH3T3 cells in vitro and some of which promoted tumor formation in mouse models (Sigalas 1996; Volk et al., 2009, Mol Cancer Res. 7(6): 863-869).

[0005] To date, more than 70 different M(H)DM2 splice variants have been identified (Bartel 2002, Bartel 2004, Rosso 2014). Some fragments, such as MDM2-A and MDM2-B, are common to multiple tumor types (Sigalas 1996). Others, such as MDM2-FB25 and MDM2-FB26 in pediatric rhabdomyosarcoma, are common to multiple tumor types (Sigalas 1996). Short forms of these alternatively spliced ​​MDM2 transcripts have been found only in certain tumors, such as MDM2-FB26 and MDM2-FB26. Several of these short forms of the alternatively spliced ​​MDM2 transcripts have been correlated with high-grade malignancies in human ovarian, bladder, and astrocytic tumors (Sigalas 1996; Matsumoto et al., 1998, Cancer Res. 58:609-613; Tamborini et al., 2001, Int. J. Cancer 92:790-796; Steinman et al., 2004, JBC 279:4877-4886). It has also been shown that aberrant mdm2 (281, 254, and 219 bp) and alternative mdm2 (653 bp) splice products are strongly associated with shorter overall patient survival in breast cancer (Lukas et al., 2001, Cancer Res. 61:3212).

[0006] Several studies have evaluated the subcellular localization of various human and mouse M(H)DM2 protein variants to predict potential activity. In 25% of non-small cell lung cancers, M(H)DM2 variants were aberrantly localized to the cytoplasm (Evans et al., 2001, Oncogene 20:4041-4049). This finding led to the discovery that sequestration of full-length M(H)DM2 in the cytoplasmic compartment was due to binding and sequestration by an alternatively spliced ​​M(H)DM2 product (HDM2ALT1). In another study, MDM2-D150-230 was localized to the cytoplasm of U20S cells (Schuster et al., 2007, Mol. Cancer Res. 5:403-412 ("Schuster 2007")). Both of these M(H)DM2 protein variants lacked the portion of the NH2-terminal region that contains the nuclear localization signal (NLS), suggesting that the absence of this signal prevented these two proteins from entering the nucleus (Schuster 2007). Together, these data indicate that the subcellular localization of M(H)DM2 protein variants is highly complex.

[0007] Various M(H)DM4 protein variants have also been characterized, including the splicing variant MDMX-S (Lenos and Jochemsen, 2011, J. Biomed Biotechnol., doi:10.1155 / 2011 / 876173).

[0008] HDM2 has been found to be expressed on the plasma membrane of cancer cells (Sarafraz-Yazdi et al., 2010, PNAS 107:1918-1923 (“Sarafraz-Yazdi 2010”)). Furthermore, anti-cancer peptides PNC-27 and PNC-28 have been developed that bind to HDM2 expressed on the cancer cell membrane and kill cancer cells by inducing necrosis (Sarafraz-Yazdi 2010, Davitt et al., 2014, Annals Clin. Lab. Sci. 44:241-248) (PNC-2 The amino acid sequences of PNC-7 and PNC-28 are provided in Table I of U.S. Patent Application Publication No. 2012 / 0177566. PNC-27 has been reported to bind to amino acids 25-109 of HDM2 (Do et al., 2003, Oncogene 22(10):1431-1444 ("Do 2003"); Chene, 2003, Nat. Rev. Cancer 3(2):102-109). U.S. Patent Application Publication No. 2012 / 0177566 also discloses a method for selectively necrosing cells by administering to the cells a compound comprising a HDM-2 targeting moiety and a cytotoxic moiety conjugated to the HDM-2 targeting moiety (e.g., PNC-27 and PNC-28), the compound comprising a membrane-active form. The membrane-active function of the PNC-27 and PNC-28 peptides (containing membrane-resident peptide ("MRP") and p53 sequences) is achieved only when a cargo (i.e., a p53 sequence component) is attached to the MRP component, such that a cytotoxic structure can be formed (Kanovsky et al., 2001, PNAS 98:12438-12443 ("Kanovsky 2001"); Bowne et al., 2008, Ann Surg Oncol. 15:3588-3600 ("Bowne 2008"). When either the MRP component or the HDM-2 targeting component (i.e., the p53 component) was used separately, they were found to be non-cytotoxic to cancer cells (Kanovsky 2001; Do 2003), demonstrating the lack of activity of the MRP component and HDM-2 targeting component alone. When recombinantly expressed in cells, HDM-2 targeting components were observed to induce apoptosis (Bowne 2008). U.S. Patent No. 9,765,117 describes HDM2 No. 9,765,117 discloses targeting peptides and fusion peptides comprising a HDM2 targeting peptide and a transmembrane sequence, such as MRP, and that MRP is required for the induction of cell necrosis (see column 4, lines 27-28). U.S. Patent No. 9,765,117 shows that expression of a p53 HDM2 targeting sequence in cancer cells in the absence of MRP causes p53-dependent apoptosis but not tumor necrosis (see column 4, lines 29-32). Cell-penetrating peptides ("CPPs") (e.g., MRP, membrane transduction domain of Antennapedia, trans-activating transcriptional activator ("TAT"), and penetratin peptides) enable cell membrane delivery of peptides and peptide-linked molecules to the plasma membrane lipid bilayer, including that of normal, healthy cells. These peptides have been shown to efficiently transport a variety of biologically active molecules into the interior of living cells (Bechara et al., 2013, FEBS Lett. 587:1693-1702; Dupont et al., 2015, Methods Mol. Biol. 1324:29-37). The use of these CPPs (e.g., MRP, the membrane transduction domain of Antennapedia, TAT, and penetratin peptides) linked to various cargoes (e.g., other peptides, DNA, RNA, small molecules, antibodies, or fragments thereof) has been demonstrated to improve the pharmacokinetics, biodistribution, retention time, uptake, and delivery of these cargoes to various tumors in vitro and in vivo (Torchilin et al., 2003, PNAS 100:1972-1977; Shin et al., 2014, J. Biomed. Mater Res. A. 102:575-587; Kleemann et al., 2005, J. Control Release 109:299-316; Olson et al., 2009, Integr. Biol (Camb) 1:382-393; Jain et al., 2005, Cancer Res. 65:7840-7846; William et al., 2002, PNAS 99:10423-10428; Bolhassani, 2011, Biochim Biophys. Acta 1816:232-246). Most of these CPPs are Although penetratin and MRP peptides have been used as drug delivery entities, they have been reported to form unique cytotoxic membrane-active structures when linked to cargoes (Rosal et al., 2005, Adv Drug Deliv Rev 57:653-60 (“Rosal 2005”)). , Bowne 2008, Kanovsky 2001). PNC-27 and PNC-28 peptides are examples of penetratin- / MRP-cargo conjugates that exhibit cytotoxic function dependent on the binding and ligation of penetratin to MRP, which is required for the formation of their membrane-active structure and therefore their cytotoxic function (Kanovsky 2001, Rosal 2005, Bowne 2008). There is a long-felt unmet need in the art to effectively treat cancer, eg, metastatic cancer, and prevent metastasis in patients. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] US Patent Application Publication No. 2012 / 0177566 [Patent Document 2] U.S. Patent No. 9,765,117 [Non-patent literature]

[0010] [Non-Patent Document 1] Wade et al., 2013, Nat Rev. Cancer 13:83-96 [Non-licensed document 2] Marine et al., 2004, Cell Cycle 3:900-904 [Non-licensed document 3] Momand et al., 2011, Gene 486:23-30

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[0011] 5. Summary of the Invention In one aspect, described herein is an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4. Specifically, described herein is an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 and inhibits tumor growth in vivo (or inhibits tumor cell proliferation in vivo). In certain embodiments, the antibodies or fragments described herein are not conjugated to a cell-penetrating peptide (e.g., a membrane-resident peptide). In certain embodiments, the antibodies or fragments described herein are not conjugated to a cytotoxic moiety (i.e., are not conjugated to a cytotoxic agent).

[0012] In one aspect, described herein is an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment thereof specifically binds to a peptide, the peptide having the sequence MCNTNMSVPTDGAVT (SEQ ID NO: 1), TTSQIPASEQE (SEQ ID NO: 2), or CPVCRQPIQMIVLTYFP (SEQ ID NO: 3). Specifically, described herein is an antibody or fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., HDM2), wherein the antibody or fragment thereof specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment thereof specifically binds to a peptide, the peptide having the sequence MCNTNMSVPTDGAVT (SEQ ID NO: 1), TTSQIPASEQE (SEQ ID NO: 2), or CPVCRQPIQMIVLTYFP (SEQ ID NO: 3). In one embodiment, an antibody or fragment binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment specifically binds to a peptide having the sequence MCNTNMSVPTDGAVT (SEQ ID NO: 1). In one embodiment, an antibody or fragment binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment specifically binds to a peptide having the sequence TTSQIPASEQE (SEQ ID NO: 2). In one embodiment, an antibody or fragment binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment specifically binds to a peptide having the sequence CPVCRQPIQMIVLTYFP (SEQ ID NO: 3). In certain embodiments, described herein are antibodies or fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2 / 4 contained within the peptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In one embodiment, described herein is an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 contained within the peptide of SEQ ID NO: 1. In one embodiment, described herein is an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 contained within the peptide of SEQ ID NO: 2.In one embodiment, described herein is an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 contained within the peptide of SEQ ID NO:3.

[0013] In one embodiment, there is provided a humanized antibody or fragment thereof that specifically binds to HDM2, comprising: (i) a heavy chain variable region (VH) comprising VH complementarity determining region ("CDR") 1, VH CDR 2, and VH CDR3, wherein said VH CDR 1, VH CDR 2, and VH CDR3 Described herein is a humanized antibody or fragment thereof comprising (i) a heavy chain variable region (VH) wherein CDR 3 is a CDR of a VH having an amino acid sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 38, and SEQ ID NO: 40, or (ii) a light chain variable region (VL) comprising VL CDR 1, VL CDR 2, and VL CDR3, which are CDRs of a VL having an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 41. In one embodiment, the humanized antibody or fragment that specifically binds to HDM2 comprises a VH, wherein VH CDR 1, VH CDR 2, and VH CDR 3 are of the VH having the amino acid sequence of SEQ ID NO: 36. In one embodiment, the humanized antibody or fragment that specifically binds to HDM2 comprises a VH, wherein VH CDR 1, VH CDR 2, and VH CDR 3 are of the VH having the amino acid sequence of SEQ ID NO: 38. In one embodiment, a humanized antibody or fragment that specifically binds to HDM2 comprises a VH, wherein VH CDR1, VH CDR2, and VH CDR3 are of the VH having the amino acid sequence of SEQ ID NO: 40. In one embodiment, a humanized antibody or fragment that specifically binds to HDM2 comprises a VL, wherein VL CDR1, VL CDR2, and VL CDR3 are of the VL having the amino acid sequence of SEQ ID NO: 37 (and, optionally, a VH wherein VH CDR1, VH CDR2, and VH CDR3 are of the VH having the amino acid sequence of SEQ ID NO: 36). In one embodiment, a humanized antibody or fragment that specifically binds to HDM2 comprises a VL, wherein VL CDR1, VL CDR2, and VL CDR3 are of a VL having the amino acid sequence of SEQ ID NO: 39 (and, optionally, a VH, wherein VH CDR1, VH CDR2, and VH CDR3 are of a VH having the amino acid sequence of SEQ ID NO: 38). In one embodiment, a humanized antibody or fragment that specifically binds to HDM2 comprises a VL, wherein VL CDR1, VL CDR2, and VL CDR3 are of a VL having the amino acid sequence of SEQ ID NO: 41 (and, optionally, a VH, wherein VH CDR1, VH CDR2, and VH CDR3 are of a VH having the amino acid sequence of SEQ ID NO: 40).

[0014] In one embodiment, there is provided an antibody or fragment thereof that specifically binds to M(H)DM2 / 4, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) comprising VH complementarity determining region ("CDR") 1, VH CDR 2, and VH CDR 3; (i) the VH CDR 1 has the amino acid sequence GFTFTHY (SEQ ID NO: 18), the VH CDR 2 has the amino acid sequence RNKAKGYT (SEQ ID NO: 19), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO: 20); (ii) the VH CDR 1 has the amino acid sequence GFTFTHYYMS (SEQ ID NO: 42), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAE (SEQ ID NO: 45), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO: 20); (iii) the VH CDR 1 has the amino acid sequence HYYMS (SEQ ID NO: 43), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAEYSASVKG (SEQ ID NO: 46), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO: 20); (iv) the VH CDR 1 has the amino acid sequence THYYMS (SEQ ID NO: 44), the VH CDR 2 has the amino acid sequence WLGFIRNKAKGYTAE (SEQ ID NO: 47), and the VH CDR 3 has the amino acid sequence ARDIGD (SEQ ID NO: 48), or (v) The antibody or fragment thereof described herein, wherein the VH CDR 1 has the amino acid sequence FTFTHYY (SEQ ID NO: 144), the VH CDR 2 has the amino acid sequence IRNKAKGYTA (SEQ ID NO: 145), and the VH CDR 3 has the amino acid sequence ARDIGDN (SEQ ID NO: 146).

[0015] In one embodiment, an antibody or fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., HDM2), wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) comprising VH complementarity determining region ("CDR") 1, VH CDR 2, and VH CDR 3; (i) the VH CDR 1 has the amino acid sequence GDTLSGS (SEQ ID NO: 24), the VH CDR 2 has the amino acid sequence HLNRGT (SEQ ID NO: 25), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO: 26), (ii) the VH CDR 1 has the amino acid sequence GDTLSGSWMH (SEQ ID NO: 52), the VH CDR 2 has the amino acid sequence EIHLNRGTTN (SEQ ID NO: 55), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO: 26); (iii) the VH CDR 1 has the amino acid sequence GSWMH (SEQ ID NO: 53), the VH CDR 2 has the amino acid sequence EIHLNRGTTNYNEKFKG (SEQ ID NO: 56), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO: 26); (iv) the VH CDR 1 has the amino acid sequence SGSWMH (SEQ ID NO: 54), the VH CDR 2 has the amino acid sequence WIGEIHLNRGTTN (SEQ ID NO: 57), and the VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO: 58), or (v) The antibody or fragment thereof described herein, wherein the VH CDR 1 has the amino acid sequence GDTLSGSW (SEQ ID NO: 148), the VH CDR 2 has the amino acid sequence IHLNRGTT (SEQ ID NO: 143), and the VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO: 58).

[0016] In one embodiment, an antibody or fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., HDM2), wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) comprising VH complementarity determining region ("CDR") 1, VH CDR 2, and VH CDR 3; (i) the VH CDR 1 has the amino acid sequence GYTFTSY (SEQ ID NO: 30), the VH CDR 2 has the amino acid sequence NPRNGG (SEQ ID NO: 31), and the VH CDR 3 has SGYYAMDY (SEQ ID NO: 32), (ii) the VH CDR 1 has the amino acid sequence GYTFTSYYMY (SEQ ID NO: 62), the VH CDR 2 has the amino acid sequence GINPRNGGTN (SEQ ID NO: 65), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO: 32); (iii) the VH CDR 1 has the amino acid sequence SYYMY (SEQ ID NO: 63), the VH CDR 2 has the amino acid sequence GINPRNGGTNFNEKFKN (SEQ ID NO: 66), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO: 32), or (iv) Provided herein is an antibody or fragment thereof, wherein the VH CDR 1 has the amino acid sequence TSYYMY (SEQ ID NO: 64), the VH CDR 2 has the amino acid sequence WIGGINPRNGGTN (SEQ ID NO: 67), and the VH CDR 3 has the amino acid sequence TRSGYYAMD (SEQ ID NO: 68).

[0017] In one embodiment, there is provided a humanized antibody or fragment thereof that specifically binds to HDM2, wherein said antibody or fragment thereof comprises a heavy chain variable region (VH) comprising VH complementarity determining region ("CDR") 1, VH CDR 2, and VH CDR 3; (i) the VH CDR 1 has the amino acid sequence GFTFTHY (SEQ ID NO: 18), the VH CDR 2 has the amino acid sequence RNKAKGYT (SEQ ID NO: 19), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO: 20); (ii) the VH CDR 1 has the amino acid sequence GFTFTHYYMS (SEQ ID NO: 42), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAE (SEQ ID NO: 45), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO: 20); (iii) the VH CDR 1 has the amino acid sequence HYYMS (SEQ ID NO: 43), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAEYSASVKG (SEQ ID NO: 46), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO: 20); (iv) the VH CDR 1 has the amino acid sequence THYYMS (SEQ ID NO: 44), the VH CDR 2 has the amino acid sequence WLGFIRNKAKGYTAE (SEQ ID NO: 47), and the VH CDR 3 has the amino acid sequence ARDIGD (SEQ ID NO: 48), or (v) A humanized antibody or fragment thereof described herein, wherein the VH CDR 1 has the amino acid sequence FTFTHYY (SEQ ID NO: 144), the VH CDR 2 has the amino acid sequence IRNKAKGYTA (SEQ ID NO: 145), and the VH CDR 3 has the amino acid sequence ARDIGDN (SEQ ID NO: 146).

[0018] In one embodiment, a humanized antibody or fragment thereof that specifically binds to HDM2, comprising a VH, wherein the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3; (i) VH CDR3 has the amino acid sequence DIGDN (SEQ ID NO: 20); (ii) VH CDR3 has the amino acid sequence ARDIGD (SEQ ID NO: 48), or (iii) Described herein is a humanized antibody or fragment thereof, wherein VH CDR 3 has the amino acid sequence ARDIGDN (SEQ ID NO: 146).

[0019]

[0023] In one embodiment, there is provided a humanized antibody or fragment thereof that specifically binds to HDM2, wherein said antibody or fragment comprises VL complementarity determining region ("CDR") 1, VL CDR 2, and VL a light chain variable region (VL) comprising CDR 3, (i) the VL CDR 1 has the amino acid sequence RSSKNLLHSNGITYLY (SEQ ID NO: 21), the VL CDR 2 has the amino acid sequence RVSNLAS (SEQ ID NO: 22), and the VL CDR 3 has the amino acid sequence AQLLELPYT (SEQ ID NO: 23); (ii) the VL CDR 1 has the amino acid sequence LHSNGITYLYWY (SEQ ID NO: 49), the VL CDR 2 has the amino acid sequence LLISRVSNLA (SEQ ID NO: 50), and the VL CDR 3 has the amino acid sequence AQLLELPY (SEQ ID NO: 51), or (iii) the VL CDR 1 has the amino acid sequence KNLLHSNGITY (SEQ ID NO: 147), the VL CDR 2 has the amino acid sequence RVS, and the VL CDR Described herein is an antibody or fragment, wherein SEQ ID NO:3 has the amino acid sequence AQLLELPYT (SEQ ID NO:23).

[0020] In one embodiment, a humanized antibody or fragment thereof that specifically binds to HDM2, wherein said antibody or fragment comprises: (a) a heavy chain variable region (VH) comprising VH complementarity-determining region ("CDR") 1, VH CDR 2, and VH CDR 3; (i) the VH CDR 1 has the amino acid sequence GFTFTHY (SEQ ID NO: 18), the VH CDR 2 has the amino acid sequence RNKAKGYT (SEQ ID NO: 19), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO: 20); (ii) the VH CDR 1 has the amino acid sequence GFTFTHYYMS (SEQ ID NO: 42), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAE (SEQ ID NO: 45), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO: 20); (iii) the VH CDR 1 has the amino acid sequence HYYMS (SEQ ID NO: 43), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAEYSASVKG (SEQ ID NO: 46), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO: 20); (iv) the VH CDR 1 has the amino acid sequence THYYMS (SEQ ID NO: 44), the VH CDR 2 has the amino acid sequence WLGFIRNKAKGYTAE (SEQ ID NO: 47), and the VH CDR 3 has the amino acid sequence ARDIGD (SEQ ID NO: 48), or (v) the VH CDR 1 has the amino acid sequence FTFTHYY (SEQ ID NO: 144), the VH CDR 2 has the amino acid sequence IRNKAKGYTA (SEQ ID NO: 145), and the VH CDR 3 has the amino acid sequence ARDIGDN (SEQ ID NO: 146); A heavy chain variable region (VH); and (b) a light chain variable region (VL) comprising VL complementarity determining region ("CDR") 1, VL CDR 2, and VL CDR 3; (i) the VL CDR 1 has the amino acid sequence RSSKNLLHSNGITYLY (SEQ ID NO: 21), the VL CDR 2 has the amino acid sequence RVSNLAS (SEQ ID NO: 22), and the VL CDR 3 has the amino acid sequence AQLLELPYT (SEQ ID NO: 23); (ii) the VL CDR 1 has the amino acid sequence LHSNGITYLYWY (SEQ ID NO: 49), the VL CDR 2 has the amino acid sequence LLISRVSNLA (SEQ ID NO: 50), and the VL CDR 3 has the amino acid sequence AQLLELPY (SEQ ID NO: 51), or (iii) the VL CDR 1 has the amino acid sequence KNLLHSNGITY (SEQ ID NO: 147), the VL CDR 2 has the amino acid sequence RVS, and the VL CDR 3 has the amino acid sequence AQLLELPYT (SEQ ID NO: 23), Light chain variable region (VL) Described herein are antibodies or fragments comprising:

[0021] In one embodiment, there is provided an antibody or fragment thereof that specifically binds to HDM2, wherein said antibody or fragment thereof comprises a heavy chain variable region (VH) comprising VH complementarity determining region ("CDR") 1, VH CDR 2, and VH CDR 3; (i) the VH CDR 1 has the amino acid sequence GDTLSGS (SEQ ID NO: 24), the VH CDR 2 has the amino acid sequence HLNRGT (SEQ ID NO: 25), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO: 26), (ii) the VH CDR 1 has the amino acid sequence GDTLSGSWMH (SEQ ID NO: 52), the VH CDR 2 has the amino acid sequence EIHLNRGTTN (SEQ ID NO: 55), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO: 26); (iii) the VH CDR 1 has the amino acid sequence GSWMH (SEQ ID NO: 53), the VH CDR 2 has the amino acid sequence EIHLNRGTTNYNEKFKG (SEQ ID NO: 56), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO: 26); (iv) the VH CDR 1 has the amino acid sequence SGSWMH (SEQ ID NO: 54), the VH CDR 2 has the amino acid sequence WIGEIHLNRGTTN (SEQ ID NO: 57), and the VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO: 58), or (v) The antibody or fragment thereof described herein, wherein the VH CDR 1 has the amino acid sequence GDTLSGSW (SEQ ID NO: 148), the VH CDR 2 has the amino acid sequence IHLNRGTT (SEQ ID NO: 143), and the VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO: 58).

[0022] In one embodiment, an antibody or fragment thereof that specifically binds to HDM2, comprising a VH, wherein the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3; (i) VH CDR3 has the amino acid sequence SPGFAY (SEQ ID NO: 26), or (ii) Described herein is an antibody or fragment thereof, wherein VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO: 58).

[0023] In one embodiment, there is provided an antibody or fragment thereof that specifically binds to HDM2, wherein said antibody or fragment thereof comprises a light chain variable region (VL) comprising VL complementarity determining region ("CDR") 1, VL CDR 2, and VL CDR 3; (i) the VL CDR 1 has the amino acid sequence RSSKSLLHSNGNSYLY (SEQ ID NO: 27), the VL CDR 2 has the amino acid sequence RMSNLAS (SEQ ID NO: 28), and the VL CDR 3 has the amino acid sequence MQHLEYPFT (SEQ ID NO: 29); (ii) the VL CDR 1 has the amino acid sequence LHSNGNSYLYWF (SEQ ID NO: 59), the VL CDR 2 has the amino acid sequence LLIYRMSNLA (SEQ ID NO: 60), and the VL CDR 3 has the amino acid sequence MQHLEYPF (SEQ ID NO: 61), or (iii) the VL CDR 1 has the amino acid sequence KSLLHSNGNSY (SEQ ID NO: 141), the VL CDR 2 has the amino acid sequence RMS, and the VL CDR Described herein is an antibody or fragment thereof, wherein SEQ ID NO:3 has the amino acid sequence MQHLEYPFT (SEQ ID NO:29).

[0024] In one embodiment, an antibody or fragment thereof that specifically binds to HDM2, wherein the antibody or fragment thereof is (a) a heavy chain variable region (VH) comprising VH complementarity-determining region ("CDR") 1, VH CDR 2, and VH CDR 3; (i) the VH CDR 1 has the amino acid sequence GDTLSGS (SEQ ID NO: 24), the VH CDR 2 has the amino acid sequence HLNRGT (SEQ ID NO: 25), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO: 26), (ii) the VH CDR 1 has the amino acid sequence GDTLSGSWMH (SEQ ID NO: 52), the VH CDR 2 has the amino acid sequence EIHLNRGTTN (SEQ ID NO: 55), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO: 26); (iii) the VH CDR 1 has the amino acid sequence GSWMH (SEQ ID NO: 53), the VH CDR 2 has the amino acid sequence EIHLNRGTTNYNEKFKG (SEQ ID NO: 56), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO: 26); (iv) the VH CDR 1 has the amino acid sequence SGSWMH (SEQ ID NO: 54), the VH CDR 2 has the amino acid sequence WIGEIHLNRGTTN (SEQ ID NO: 57), and the VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO: 58), or (v) a heavy chain variable region (VH) wherein the VH CDR 1 has the amino acid sequence GDTLSGSW (SEQ ID NO: 148), the VH CDR 2 has the amino acid sequence IHLNRGTT (SEQ ID NO: 143), and the VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO: 58); and (b) a light chain variable region (VL) comprising VL complementarity determining region ("CDR") 1, VL CDR 2, and VL CDR 3; (i) the VL CDR 1 has the amino acid sequence RSSKSLLHSNGNSYLY (SEQ ID NO: 27), the VL CDR 2 has the amino acid sequence RMSNLAS (SEQ ID NO: 28), and the VL CDR 3 has the amino acid sequence MQHLEYPFT (SEQ ID NO: 29); (ii) the VL CDR 1 has the amino acid sequence LHSNGNSYLYWF (SEQ ID NO: 59), the VL CDR 2 has the amino acid sequence LLIYRMSNLA (SEQ ID NO: 60), and the VL CDR 3 has the amino acid sequence MQHLEYPF (SEQ ID NO: 61), or (iii) the VL CDR 1 has the amino acid sequence KSLLHSNGNSY (SEQ ID NO: 141), the VL CDR 2 has the amino acid sequence RMS, and the VL CDR 3 has the amino acid sequence MQHLEYPFT (SEQ ID NO: 29); Described herein are antibodies or fragments thereof comprising a light chain variable region (VL).

[0025] In one embodiment, there is provided an antibody or fragment thereof that specifically binds to HDM2, wherein said antibody or fragment thereof comprises a heavy chain variable region (VH) comprising VH complementarity determining region ("CDR") 1, VH CDR 2, and VH CDR 3; (i) the VH CDR 1 has the amino acid sequence GYTFTSY (SEQ ID NO: 30), the VH CDR 2 has the amino acid sequence NPRNGG (SEQ ID NO: 31), and the VH CDR 3 has SGYYAMDY (SEQ ID NO: 32), (ii) the VH CDR 1 has the amino acid sequence GYTFTSYYMY (SEQ ID NO: 62), the VH CDR 2 has the amino acid sequence GINPRNGGTN (SEQ ID NO: 65), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO: 32); (iii) the VH CDR 1 has the amino acid sequence SYYMY (SEQ ID NO: 63), the VH CDR 2 has the amino acid sequence GINPRNGGTNFNEKFKN (SEQ ID NO: 66), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO: 32), or (iv) The antibody or fragment thereof described herein, wherein the VH CDR 1 has the amino acid sequence TSYYMY (SEQ ID NO: 64), the VH CDR 2 has the amino acid sequence WIGGINPRNGGTN (SEQ ID NO: 67), and the VH CDR 3 has the amino acid sequence TRSGYYAMD (SEQ ID NO: 68).

[0026] In one embodiment, an antibody or fragment thereof that specifically binds to HDM2, comprising a VH, wherein the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3; (i) VH CDR3 has the amino acid sequence SGYYAMDY (SEQ ID NO: 32), or (ii) Described herein is an antibody or fragment thereof, wherein VH CDR 3 has the amino acid sequence TRSGYYAMD (SEQ ID NO: 68).

[0027] In one embodiment, a light chain variable region (VL) comprising VL complementarity determining region ("CDR") 1, VL CDR 2, and VL CDR 3, (i) the VL CDR 1 has the amino acid sequence RASQDISNFLN (SEQ ID NO: 33), the VL CDR 2 has the amino acid sequence YTSRLHS (SEQ ID NO: 34), and the VL CDR 3 has the amino acid sequence QQGNTLPRT (SEQ ID NO: 35), or (ii) The antibody or fragment described herein further comprises a light chain variable region (VL) wherein the VL CDR 1 has the amino acid sequence SNFLNWY (SEQ ID NO: 69), the VL CDR 2 has the amino acid sequence LLIYYTSRLH (SEQ ID NO: 70), and the VL CDR 3 has the amino acid sequence QQGNTLPR (SEQ ID NO: 71).

[0028] In one embodiment, an antibody or fragment thereof that specifically binds to HDM2, wherein the antibody or fragment thereof is (a) a heavy chain variable region (VH) comprising VH complementarity-determining region ("CDR") 1, VH CDR 2, and VH CDR 3; (i) the VH CDR 1 has the amino acid sequence GYTFTSY (SEQ ID NO: 30), the VH CDR 2 has the amino acid sequence NPRNGG (SEQ ID NO: 31), and the VH CDR 3 has SGYYAMDY (SEQ ID NO: 32), (ii) the VH CDR 1 has the amino acid sequence GYTFTSYYMY (SEQ ID NO: 62), the VH CDR 2 has the amino acid sequence GINPRNGGTN (SEQ ID NO: 65), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO: 32); (iii) the VH CDR 1 has the amino acid sequence SYYMY (SEQ ID NO: 63), the VH CDR 2 has the amino acid sequence GINPRNGGTNFNEKFKN (SEQ ID NO: 66), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO: 32), or (iv) the VH CDR 1 has the amino acid sequence TSYYMY (SEQ ID NO: 64), the VH CDR 2 has the amino acid sequence WIGGINPRNGGTN (SEQ ID NO: 67), and the VH CDR 3 has the amino acid sequence TRSGYYAMD (SEQ ID NO: 68); A heavy chain variable region (VH); and (b) a light chain variable region (VL) comprising VL complementarity determining region ("CDR") 1, VL CDR 2, and VL CDR 3; (i) the VL CDR 1 has the amino acid sequence RASQDISNFLN (SEQ ID NO: 33), the VL CDR 2 has the amino acid sequence YTSRLHS (SEQ ID NO: 34), and the VL CDR 3 has the amino acid sequence QQGNTLPRT (SEQ ID NO: 35), or (ii) a light chain variable region (VL), wherein the VL CDR 1 has the amino acid sequence SNFLNWY (SEQ ID NO: 69), the VL CDR 2 has the amino acid sequence LLIYYTSRLH (SEQ ID NO: 70), and the VL CDR 3 has the amino acid sequence QQGNTLPR (SEQ ID NO: 71). Described herein is an antibody or fragment thereof comprising:

[0029] In one aspect, described herein is an antibody or fragment thereof that specifically binds to HDM2, wherein the antibody or fragment thereof comprises a VH having the amino acid sequence of SEQ ID NO: 36, or a VH having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 36.

[0030] In one aspect, described herein is an antibody or fragment thereof that specifically binds to HDM2, wherein the antibody or fragment thereof comprises a VL having the amino acid sequence of SEQ ID NO: 37, or a VL having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 37 (and, optionally, a VH having the amino acid sequence of SEQ ID NO: 36, or a VH having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 36).

[0031] In one aspect, described herein is an antibody or fragment thereof that specifically binds to HDM2, wherein the antibody or fragment thereof comprises a VH having the amino acid sequence of SEQ ID NO: 38, or a VH having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 38.

[0032] In one aspect, described herein is an antibody or fragment thereof that specifically binds to HDM2, wherein the antibody or fragment thereof comprises a VL having the amino acid sequence of SEQ ID NO: 39, or a VL having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 39 (and, optionally, a VH having the amino acid sequence of SEQ ID NO: 38, or a VH having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 38).

[0033] In one aspect, described herein is an antibody or fragment thereof that specifically binds to HDM2, wherein the antibody or fragment thereof comprises a VH having the amino acid sequence of SEQ ID NO: 40, or a VH having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 40.

[0034] In one aspect, described herein is an antibody or fragment thereof that specifically binds to HDM2, wherein the antibody or fragment thereof comprises a VL having the amino acid sequence of SEQ ID NO: 41, or a VL having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 41 (and, optionally, a VH having the amino acid sequence of SEQ ID NO: 40, or a VH having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 40).

[0035] In preferred embodiments, the anti-M(H)DM2 / 4 antibodies described herein are monoclonal antibodies. In certain embodiments, the anti-M(H)DM2 / 4 antibodies described herein are human, humanized, or chimeric antibodies (e.g., human monoclonal, humanized, or chimeric monoclonal antibodies). In one embodiment, the anti-M(H)DM2 / 4 antibodies described herein are human antibodies. In one embodiment, the anti-M(H)DM2 / 4 antibodies described herein are humanized antibodies. In one embodiment, the anti-M(H)DM2 / 4 antibodies described herein are chimeric antibodies.

[0036] In certain embodiments, the anti-M(H)DM2 / 4 antibodies described herein are purified antibodies.

[0037] In certain embodiments, the anti-M(H)DM2 / 4 antibodies described herein are immunoglobulins (e.g., IgG or IgM). In one embodiment, the immunoglobulin is IgG. In one embodiment, the immunoglobulin is IgM. In certain embodiments, the immunoglobulin is of the IgG1 isotype. In other embodiments, the immunoglobulin is of the IgG3 isotype. In other embodiments, the immunoglobulin is of the IgG2 isotype. In certain embodiments, the anti-M(H)DM2 / 4 antibodies described herein comprise an Fc region, wherein the Fc region is a human IgG Fc region or a human IgM Fc region. In certain embodiments, the anti-M(H)DM2 / 4 antibodies described herein comprise an Fc region that is a human IgG1 Fc region, a human IgG2 Fc region, or a human IgG3 Fc region. In one embodiment, the anti-M(H)DM2 / 4 antibodies described herein comprise a human IgG1 Fc region. In one embodiment, the anti-M(H)DM2 / 4 antibodies described herein comprise a human IgG3 Fc region. In one embodiment, the anti-M(H)DM2 / 4 antibodies described herein comprise a human IgG2 Fc region. In one embodiment, the anti-M(H)DM2 / 4 antibodies described herein comprise a human IgM Fc region. In one embodiment, the anti-M(H)DM2 / 4 antibodies described herein comprise a human IgE Fc region.

[0038] In certain embodiments, the anti-M(H)DM2 / 4 antibodies or fragments described herein are antigen-binding fragments of anti-M(H)DM2 / 4 antibodies. In certain embodiments, the antibodies or fragments described herein are Fv fragments, Fab fragments, Fab' fragments, F(ab')2 fragments, single-chain antibody molecules, or single-chain Fvs (scFvs). In one embodiment, the antibodies or fragments described herein are Fv fragments. In one embodiment, the antibodies or fragments described herein are Fab fragments. In one embodiment, the antibodies or fragments described herein are Fab' fragments. In one embodiment, the antibodies or fragments described herein are F(ab')2 fragments. In one embodiment, the antibodies or fragments described herein are single-chain antibody molecules. In one embodiment, the antibodies or fragments described herein are single-chain Fvs (scFvs).

[0039] In certain embodiments, the anti-M(H)DM2 / 4 antibodies or antigen-binding fragments described herein mediate complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). In one embodiment, the anti-M(H)DM2 / 4 antibodies or antigen-binding fragments mediate complement-dependent cytotoxicity (CDC).

[0040] In certain embodiments, the anti-M(H)DM2 / 4 antibodies described herein are bispecific antibodies that also specifically bind to a cell surface antigen of an effector cell (e.g., a T cell, a B lymphocyte, a neutrophil, a macrophage, a natural killer cell, or a dendritic cell).

[0041] In one embodiment, an antibody or fragment described herein specifically binds to an extracellularly accessible epitope of M(H)DM2 (e.g., HDM2) but does not bind to M(H)DM4 (e.g., HDM4). In another embodiment, an antibody or fragment described herein specifically binds to an extracellularly accessible epitope of both M(H)DM2 (e.g., HDM2) and M(H)DM4 (e.g., HDM4). In one embodiment, an antibody or fragment described herein specifically binds to an extracellularly accessible epitope of HDM2, and, optionally, can also bind to an extracellularly accessible epitope of MDM2.

[0042] In certain embodiments, the anti-HDM2 antibodies or fragments described herein specifically bind to HDM2 within the amino acids of SEQ ID NO: 1 (which are amino acids 1-15 of HDM2 (SEQ ID NO: 4)). In another specific embodiment, the anti-HDM2 antibodies described herein specifically bind to HDM2 within the amino acids of SEQ ID NO: 2 (which are amino acids 15-25 of HDM2 (SEQ ID NO: 4)). In another specific embodiment, the anti-HDM2 antibodies described herein specifically bind to HDM2 within the amino acids of SEQ ID NO: 3 (which are amino acids 475-491 of HDM2 (SEQ ID NO: 4)). In another specific embodiment, the anti-HDM2 antibodies described herein specifically bind to HDM2 within amino acids 19-50 of SEQ ID NO: 4. In another specific embodiment, the anti-HDM2 antibodies described herein specifically bind to amino acids 154-167 of SEQ ID NO: 4. In yet another specific embodiment, the anti-HDM2 antibodies described herein specifically bind to amino acids 1-60 of SEQ ID NO: 4. In yet another specific embodiment, the anti-HDM2 antibodies described herein specifically bind to amino acids 1-100 of SEQ ID NO: 4. In another specific embodiment, the anti-HDM2 antibodies described herein specifically bind within amino acids 100-110 of SEQ ID NO:4. In another specific embodiment, the anti-HDM2 antibodies described herein specifically bind within amino acids 50-60 of SEQ ID NO:4. In yet another specific embodiment, the anti-HDM2 antibodies described herein specifically bind within amino acids 1-109 of SEQ ID NO:4. In another specific embodiment, the anti-HDM2 antibodies described herein specifically bind within amino acids 26-60 of SEQ ID NO:4. In one specific embodiment, the anti-HDM2 antibodies described herein specifically bind within the terminal 60 amino acids at the C-terminus of HDM2 on the plasma membrane of cancer cells. In another specific embodiment, the anti-HDM2 antibodies described herein specifically bind within the terminal 100 amino acids at the C-terminus of HDM2 on the plasma membrane of cancer cells.

[0043] In one embodiment, the anti-M(H)DM2 / 4 antibodies described herein do not bind within amino acids 101-200 of SEQ ID NO:4. In one embodiment, the anti-M(H)DM2 / 4 antibodies described herein do not bind to HDM2 or the epitope of MDM2 bound by "MDM2 Monoclonal Antibody (M01), Clone 1A7" (Abnova Catalog No. H00004193-M01). In one embodiment, the anti-M(H)DM2 / 4 antibodies described herein do not compete with "MDM2 Monoclonal Antibody (M01), Clone 1A7" (Abnova Catalog No. H00004193-M01) for binding to HDM2.

[0044] In another embodiment, the anti-M(H)DM2 / 4 antibodies described herein bind within amino acids 101-200 of SEQ ID NO:4. In one embodiment, the anti-M(H)DM2 / 4 antibodies described herein bind to HDM2 or the epitope of MDM2 bound by "MDM2 Monoclonal Antibody (M01), Clone 1A7" (Abnova Catalog No. H00004193-M01). In one embodiment, the anti-M(H)DM2 / 4 antibodies described herein compete with "MDM2 Monoclonal Antibody (M01), Clone 1A7" (Abnova Catalog No. H00004193-M01) for binding to HDM2.

[0045] In one embodiment, the anti-M(H)DM2 / 4 antibodies described herein do not bind within amino acids 153-222 of SEQ ID NO: 4. In one embodiment, the anti-M(H)DM2 / 4 antibodies described herein do not bind within amino acids 26-169 of SEQ ID NO: 4. In certain embodiments, the anti-M(H)DM2 / 4 antibodies described herein do not bind within amino acids 26-222 of SEQ ID NO: 4.

[0046] In certain embodiments, M(H)DM2 / 4 exposed on the surface of cancer cells targeted by antibodies or fragments described herein is an M(H)DM2 / 4 variant lacking one or more nuclear localization signal domains. In certain embodiments, HDM2 exposed on the surface of cancer cells targeted by antibodies or fragments described herein is an HDM2 variant lacking the sequence of amino acids 179-185 of SEQ ID NO:4 and / or the sequence of amino acids 464-471 of SEQ ID NO:4. In one embodiment, HDM2 exposed on the surface of cancer cells targeted by antibodies or fragments described herein is an HDM2 variant lacking the sequence of amino acids 181-185 of SEQ ID NO:4.

[0047] In one aspect, provided herein is an antibody or fragment thereof that competes with an anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibody or fragment thereof described herein for binding to M(H)DM2 / 4. Preferably, such an antibody or fragment that competes for binding is a monoclonal antibody or fragment thereof.

[0048] In one aspect, provided herein is an antibody or fragment thereof that competes for binding to M(H)DM2 / 4 with a mouse anti-HDM2 immunoglobulin (preferably, IgG) antibody selected from the group consisting of: (i) an antibody comprising a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 36 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 37; (ii) an antibody comprising a VH having the amino acid sequence of SEQ ID NO: 38 and a VL having the amino acid sequence of SEQ ID NO: 39; and (iii) an antibody comprising a VH having the amino acid sequence of SEQ ID NO: 40 and a VL having the amino acid sequence of SEQ ID NO: 41.

[0049] In one aspect, provided herein is an antibody or fragment thereof that (i) competes with a murine anti-HDM2 IgG1 antibody comprising a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 36 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 37 for binding to a peptide of sequence SEQ ID NO: 1, or (ii) competes with a murine anti-HDM2 IgG3 antibody comprising a VH having the amino acid sequence of SEQ ID NO: 38 and a VL having the amino acid sequence of SEQ ID NO: 39 for binding to a peptide of SEQ ID NO: 2, or (iii) competes with a murine IgM antibody comprising a VH having the amino acid sequence of SEQ ID NO: 40 and a VL having the amino acid sequence of SEQ ID NO: 41 for binding to a peptide of SEQ ID NO: 3.

[0050] In one aspect, provided herein is an antibody-drug conjugate comprising any of the antibodies or fragments described herein (e.g., an antibody-drug conjugate in which an anti-M(H)DM2 / 4 antibody or fragment described herein is covalently linked to a cytotoxic drug).

[0051] In one aspect, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of any of the antibodies or fragments described herein.

[0052] In one aspect, provided herein is a method for treating cancer in a subject in need thereof, comprising administering to the subject (i) any anti-M(H)DM2 / 4 antibody described herein, (ii) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 and that is not conjugated to a cell-penetrating peptide (e.g., a membrane-resident peptide), or an antibody-drug conjugate comprising the antibody or fragment thereof conjugated to a cytotoxic drug (i.e., the antibody or fragment not conjugated to a cell-penetrating peptide), (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one aspect, provided herein is a method for treating cancer in a subject in need thereof, comprising administering to the subject an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2) and that is not conjugated to a cytotoxic moiety. In one aspect, provided herein is a method for treating cancer in a subject in need thereof, comprising administering to the subject (i) any anti-HDM2 antibody or fragment described herein, (ii) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, the antibody or fragment thereof not being conjugated to a cell-penetrating peptide (e.g., a membrane-resident peptide), or an antibody-drug conjugate comprising the antibody or fragment thereof conjugated to a cytotoxic drug (i.e., the antibody or fragment not conjugated to a cell-penetrating peptide), (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one embodiment, the method comprises administering to the subject an antibody-drug conjugate comprising an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, the antibody or fragment thereof not being conjugated to a cell-penetrating peptide, conjugated to a cytotoxic drug.In one embodiment, provided herein is a method for treating cancer in a subject in need thereof, comprising administering to the subject an anti-M(H)DM2 / 4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment specifically binds to a peptide having the sequence MCNTNMSVPTDGAVT (SEQ ID NO: 1), TTSQIPASEQE (SEQ ID NO: 2), or CPVCRQPIQMIVLTYFP (SEQ ID NO: 3). In one embodiment, provided herein is a method for treating cancer in a subject in need thereof, comprising administering to the subject an anti-M(H)DM2 / 4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 contained within the peptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0053] In one aspect, provided herein is a method for inhibiting tumor growth in a subject in need thereof, comprising administering to the subject (i) any anti-M(H)DM2 / 4 antibody or fragment described herein, (ii) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 and that is not conjugated to a cell-penetrating peptide (e.g., a membrane-resident peptide), or an antibody-drug conjugate comprising the antibody or fragment thereof conjugated to a cytotoxic drug (i.e., the antibody or fragment not conjugated to a cell-penetrating peptide), (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one aspect, provided herein is a method for inhibiting tumor growth in a subject in need thereof, comprising administering to the subject an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2) and that is not conjugated to a cytotoxic moiety. In one aspect, provided herein is a method for inhibiting tumor growth in a subject in need thereof, comprising administering to the subject (i) any anti-HDM2 antibody or fragment described herein, (ii) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, the antibody or fragment thereof not conjugated to a cell-penetrating peptide (e.g., a membrane-resident peptide), or an antibody-drug conjugate comprising the antibody or fragment thereof conjugated to a cytotoxic drug (i.e., the antibody or fragment not conjugated to a cell-penetrating peptide), (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one embodiment, the method comprises administering to the subject an antibody-drug conjugate comprising an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, the antibody or fragment thereof not conjugated to a cell-penetrating peptide, conjugated to a cytotoxic drug.In one embodiment, provided herein is a method for inhibiting tumor growth in a subject in need thereof, comprising administering to the subject an anti-M(H)DM2 / 4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment specifically binds to a peptide having the sequence MCNTNMSVPTDGAVT (SEQ ID NO: 1), TTSQIPASEQE (SEQ ID NO: 2), or CPVCRQPIQMIVLTYFP (SEQ ID NO: 3). In one embodiment, provided herein is a method for inhibiting tumor growth in a subject in need thereof, comprising administering to the subject an anti-M(H)DM2 / 4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 contained within the peptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0054] In one aspect, provided herein is a method for inhibiting tumor progression in a subject in need thereof, comprising administering to the subject (i) any anti-M(H)DM2 / 4 antibody or fragment described herein, (ii) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 and that is not conjugated to a cell-penetrating peptide (e.g., a membrane-resident peptide), or an antibody-drug conjugate comprising the antibody or fragment thereof conjugated to a cytotoxic drug (i.e., the antibody or fragment not conjugated to a cell-penetrating peptide), (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one aspect, provided herein is a method for inhibiting tumor progression in a subject in need thereof, comprising administering to the subject an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2) and that is not conjugated to a cytotoxic moiety. In one aspect, provided herein is a method for inhibiting tumor progression in a subject in need thereof, comprising administering to the subject (i) any anti-HDM2 antibody or fragment described herein, (ii) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, the antibody or fragment thereof not being conjugated to a cell-penetrating peptide (e.g., a membrane-resident peptide), or an antibody-drug conjugate comprising the antibody or fragment thereof conjugated to a cytotoxic drug (i.e., the antibody or fragment not conjugated to a cell-penetrating peptide), (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one embodiment, the method comprises administering to the subject an antibody-drug conjugate comprising an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, the antibody or fragment thereof not being conjugated to a cell-penetrating peptide, conjugated to a cytotoxic drug.In one embodiment, provided herein is a method for inhibiting tumor progression in a subject in need thereof, comprising administering to the subject an anti-M(H)DM2 / 4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the fragment specifically binds to a peptide having the sequence MCNTNMSVPTDGAVT (SEQ ID NO: 1), TTSQIPASEQE (SEQ ID NO: 2), or CPVCRQPIQMIVLTYFP (SEQ ID NO: 3), or an antibody thereof. In one embodiment, provided herein is a method for inhibiting tumor progression in a subject in need thereof, comprising administering to the subject an anti-M(H)DM2 / 4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 contained within the peptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0055] In one aspect, provided herein is a method for preventing the recurrence or recurrence of cancer in a subject in need thereof (e.g., a subject in remission from cancer), the method comprising the step of administering to the subject: (i) any anti-M(H)DM2 / 4 antibody or fragment described herein; (ii) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment is not conjugated to a cell-penetrating peptide (e.g., a membrane-resident peptide), or an antibody-drug conjugate comprising the antibody or fragment thereof conjugated to a cytotoxic drug (i.e., the antibody or fragment not conjugated to a cell-penetrating peptide); (iii) any pharmaceutical composition described herein; or (iv) any antibody-drug conjugate described herein. In one aspect, provided herein is a method for preventing the recurrence or recurrence of cancer in a subject in need thereof (e.g., a subject in remission from cancer), the method comprising administering to the subject an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2), wherein the antibody or fragment is not conjugated to a cytotoxic moiety. In one aspect, provided herein is a method for preventing the recurrence or recurrence of cancer in a subject in need thereof (e.g., a subject in remission from cancer), the method comprising the step of administering to the subject: (i) any anti-HDM2 antibody or fragment described herein; (ii) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, wherein the antibody or fragment is not conjugated to a cell-penetrating peptide (e.g., a membrane-resident peptide), or an antibody-drug conjugate comprising the antibody or fragment thereof conjugated to a cytotoxic drug (i.e., the antibody or fragment not conjugated to a cell-penetrating peptide); (iii) any pharmaceutical composition described herein; or (iv) any antibody-drug conjugate described herein.In one embodiment, the method comprises administering to a subject an antibody-drug conjugate comprising an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment is not conjugated to a cell-penetrating peptide, conjugated to a cytotoxic drug. In one embodiment, provided herein is a method for preventing the recurrence or recurrence of cancer in a subject in need thereof (e.g., a subject in remission from cancer), comprising administering to the subject an anti-M(H)DM2 / 4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment thereof specifically binds to a peptide, the peptide having the sequence MCNTNMSVPTDGAVT (SEQ ID NO: 1), TTSQIPASEQE (SEQ ID NO: 2), or CPVCRQPIQMIVLTYFP (SEQ ID NO: 3). In one embodiment, provided herein is a method for preventing the recurrence or recurrence of cancer in a subject in need thereof (e.g., a subject in remission from cancer), the method comprising administering to the subject an anti-M(H)DM2 / 4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 contained within the peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0056] In one aspect, provided herein is a method for increasing survival in a subject having cancer (e.g., compared to a subject not treated with an anti-M(H)DM2 / 4 antibody or fragment), the method comprising administering to the subject: (i) any anti-M(H)DM2 / 4 antibody or fragment described herein; (ii) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment is not linked to a cell-penetrating peptide (e.g., a membrane-resident peptide), or an antibody-drug conjugate comprising the antibody or fragment thereof linked to a cytotoxic drug (i.e., the antibody or fragment not linked to a cell-penetrating peptide); (iii) any pharmaceutical composition described herein; or (iv) any antibody-drug conjugate described herein. In one aspect, provided herein is a method for increasing survival in a subject having cancer (e.g., compared to a subject not treated with an anti-M(H)DM2 / 4 antibody or fragment), the method comprising administering to the subject an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2), wherein the antibody or fragment is not conjugated to a cytotoxic moiety. In one aspect, provided herein is a method for increasing survival in a subject with cancer (e.g., compared to a subject not treated with an anti-M(H)DM2 / 4 antibody or fragment), the method comprising administering to the subject: (i) any anti-HDM2 antibody or fragment described herein; (ii) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, wherein the antibody or fragment is not conjugated to a cell-penetrating peptide (e.g., a membrane-resident peptide), or an antibody-drug conjugate comprising the antibody or fragment thereof conjugated to a cytotoxic drug (i.e., the antibody or fragment not conjugated to a cell-penetrating peptide); (iii) any pharmaceutical composition described herein; or (iv) any antibody-drug conjugate described herein.In one embodiment, the method comprises administering to a subject an antibody-drug conjugate comprising an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment is not conjugated to a cell-penetrating peptide, conjugated to a cytotoxic drug. In one embodiment, provided herein is a method for increasing survival in a subject with cancer (e.g., compared to a subject not treated with the anti-M(H)DM2 / 4 antibody or fragment thereof), the method comprising administering to the subject an anti-M(H)DM2 / 4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment thereof specifically binds to a peptide, the peptide having the sequence MCNTNMSVPTDGAVT (SEQ ID NO: 1), TTSQIPASEQE (SEQ ID NO: 2), or CPVCRQPIQMIVLTYFP (SEQ ID NO: 3). In one embodiment, provided herein is a method for increasing survival in a subject having cancer (e.g., compared to a subject not treated with an anti-M(H)DM2 / 4 antibody or fragment), comprising administering to the subject an anti-M(H)DM2 / 4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 contained within the peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0057] In one aspect, provided herein is a method for preventing metastasis in a subject having cancer, the method comprising administering to the subject (i) any anti-M(H)DM2 / 4 antibody or fragment described herein, (ii) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 and that is not conjugated to a cell-penetrating peptide (e.g., a membrane-resident peptide), or an antibody-drug conjugate comprising the antibody or fragment thereof conjugated to a cytotoxic drug (i.e., the antibody or fragment not conjugated to a cell-penetrating peptide), (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one aspect, provided herein is a method for preventing metastasis in a subject in need thereof, the method comprising administering to the subject an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2), the antibody or fragment thereof not conjugated to a cytotoxic moiety. In one aspect, provided herein is a method for preventing metastasis in a subject with cancer, the method comprising administering to the subject: (i) any anti-HDM2 antibody or fragment described herein, (ii) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, the antibody or fragment thereof not being conjugated to a cell-penetrating peptide (e.g., a membrane-resident peptide), or an antibody-drug conjugate comprising the antibody or fragment thereof conjugated to a cytotoxic drug (i.e., the antibody or fragment not conjugated to a cell-penetrating peptide), (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one embodiment, the method comprises administering to the subject an antibody-drug conjugate comprising an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, the antibody or fragment thereof not being conjugated to a cell-penetrating peptide, conjugated to a cytotoxic drug.In one embodiment, provided herein is a method for preventing metastasis in a subject with cancer, comprising administering to the subject an anti-M(H)DM2 / 4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment thereof specifically binds to a peptide, the peptide consisting of the sequence MCNTNMSVPTDGAVT (SEQ ID NO: 1), TTSQIPASEQE (SEQ ID NO: 2), or CPVCRQPIQMIVLTYFP (SEQ ID NO: 3). In one embodiment, provided herein is a method for preventing metastasis in a subject with cancer, comprising administering to the subject an anti-M(H)DM2 / 4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 contained within the peptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0058] In one aspect, provided herein is a method for inhibiting metastasis (e.g., reducing the number, size, or invasiveness of metastases) in a subject with metastatic cancer, comprising administering to the subject: (i) any anti-M(H)DM2 / 4 antibody or fragment described herein, (ii) an antibody or fragment thereof that specifically binds to an exposed, extracellularly accessible epitope of M(H)DM2 / 4 and that is not conjugated to a cell-penetrating peptide (e.g., a membrane-resident peptide), or an antibody-drug conjugate comprising the antibody or fragment thereof conjugated to a cytotoxic drug (i.e., the antibody or fragment not conjugated to a cell-penetrating peptide), (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one aspect, provided herein is a method for inhibiting metastasis in a subject in need thereof, comprising administering to the subject an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2), that is not conjugated to a cytotoxic moiety. In one aspect, provided herein is a method for inhibiting metastasis (e.g., reducing the number, size, or invasiveness of metastases) in a subject with metastatic cancer, the method comprising administering to the subject: (i) any anti-HDM2 antibody or fragment described herein; (ii) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, wherein the antibody or fragment is not conjugated to a cell-penetrating peptide (e.g., a membrane-resident peptide), or an antibody-drug conjugate comprising the antibody or fragment thereof conjugated to a cytotoxic drug (i.e., the antibody or fragment not conjugated to a cell-penetrating peptide); (iii) any pharmaceutical composition described herein; or (iv) any antibody-drug conjugate described herein.In one embodiment, the method comprises administering to a subject an antibody-drug conjugate comprising an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment is not conjugated to a cell-penetrating peptide, conjugated to a cytotoxic drug. In one embodiment, provided herein is a method for inhibiting metastasis (e.g., reducing the number, size, or invasiveness of metastases) in a subject with metastatic cancer, comprising administering to the subject an anti-M(H)DM2 / 4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment thereof specifically binds to a peptide, the peptide having the sequence MCNTNMSVPTDGAVT (SEQ ID NO: 1), TTSQIPASEQE (SEQ ID NO: 2), or CPVCRQPIQMIVLTYFP (SEQ ID NO: 3). In one embodiment, provided herein is a method for inhibiting metastasis (e.g., reducing the number, size, or invasiveness of metastases) in a subject with metastatic cancer, the method comprising administering to the subject an anti-M(H)DM2 / 4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 contained within the peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0059] In one aspect, a method of selecting and treating a subject (e.g., a human) with cancer includes the steps of: (a) identifying a subject with cancer, wherein an antibody or fragment thereof (e.g., a labeled antibody or fragment) that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2) binds to the surface of intact cells of the cancer; and (b) administering to the subject: (i) any M(H)DM2 / 4 (e.g., anti-HDM2) antibody or fragment described herein, or an antibody-drug antibody comprising said antibody or fragment (e.g., an anti-M(H)DM2 / 4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment specifically binds to a peptide, wherein the sequence of the peptide consists of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3). Provided herein are methods comprising administering to a subject a subject a conjugate, (ii) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2), the antibody or fragment being not conjugated to a cell-penetrating peptide (e.g., a membrane-resident peptide), or an antibody-drug conjugate comprising the antibody or fragment conjugated to a cytotoxic drug (i.e., the antibody or fragment not conjugated to a cell-penetrating peptide), (iii) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2), the antibody or fragment being not conjugated to a cytotoxic moiety, (iv) any pharmaceutical composition described herein, or (v) any antibody-drug conjugate described herein. The antibody or fragment thereof in step (b) may be the same as or different from the antibody or fragment thereof in step (a). In certain embodiments, methods are provided herein that further comprise, prior to step (b), determining whether the antibody or fragment binds to the surface of intact cells of the cancer (e.g., using FACS or cell-based ELISA analysis) (using any anti-M(H)DM2 / 4 antibody described herein).In one embodiment, provided herein is a method further comprising obtaining intact cells of the cancer (e.g., by biopsy of a cancerous tumor in the subject or by obtaining a blood sample having circulating cancer cells from the subject) prior to the determining step. In one embodiment, the method comprises administering to the subject an antibody-drug conjugate comprising an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, conjugated to a cytotoxic drug, wherein the antibody or fragment thereof is not conjugated to a cell-penetrating peptide.

[0060] In one aspect, provided herein are methods for selecting a subject (e.g., a human) for treatment and treating cancer in a subject, the methods comprising: (a) selecting a subject with cancer for treatment by (i) obtaining intact cancer cells from the subject (e.g., by biopsy of a cancerous tumor in the subject or by obtaining a blood sample having circulating cancer cells from the subject); and (ii) determining (e.g., using FACS or cell-based ELISA analysis) whether an antibody or fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., an antibody or fragment that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, e.g., any anti-M(H)DM2 / 4 antibody or fragment described herein) binds to the surface of intact cancer cells obtained from the subject; and (b) if binding is detected in step (a), administering to the subject the antibody or fragment that is not conjugated to a cell-penetrating peptide. In one aspect, provided herein is a method for selecting a subject for treatment and treating cancer in a subject, the method comprising: (a) selecting a subject with cancer for treatment by (i) obtaining intact cancer cells from the subject (e.g., by biopsy of a cancerous tumor in the subject or by obtaining a blood sample having circulating cancer cells from the subject); and (ii) determining (e.g., using FACS or cell-based ELISA analysis) whether an antibody or fragment thereof that specifically binds HDM2 (e.g., an antibody or fragment that specifically binds to an extracellularly accessible epitope of HDM2, e.g., any anti-M(H)DM2 / 4 antibody or fragment described herein) binds to the surface of intact cancer cells obtained from the subject; and (b) if binding is detected in step (a), administering to the subject the antibody or fragment that is not conjugated to a cell-penetrating peptide.

[0061] In certain embodiments, the cancer treated according to the methods described herein is a type of cancer known to metastasize. In some embodiments, the cancer treated according to the methods described herein is an advanced cancer. In other embodiments, the cancer treated according to the methods described herein is an early stage cancer. In certain embodiments, the cancer treated according to the methods described herein is a metastatic cancer. The cancer treated may be a solid cancer or a non-solid cancer (e.g., leukemia).

[0062] In certain embodiments, the cancer treated according to the methods described herein is cervical cancer, endometrial cancer, ovarian cancer, pancreatic cancer, melanoma (e.g., uveal melanoma), breast cancer, colorectal cancer (e.g., colon cancer), bladder cancer, astrocytic neoplasm, glioblastoma, pediatric rhabdomyosarcoma, or lung cancer (e.g., non-small cell lung cancer). In certain embodiments, the cancer treated according to the methods described herein is melanoma, pancreatic cancer, breast cancer, or ovarian cancer. In one embodiment, the cancer treated according to the methods described herein is lung cancer. In one embodiment, the cancer treated according to the methods described herein is colorectal cancer. In one embodiment, the cancer treated according to the methods described herein is colon cancer. In one embodiment, the cancer treated according to the methods described herein is melanoma. In one embodiment, the cancer treated according to the methods described herein is pancreatic cancer. In one embodiment, the cancer treated according to the methods described herein is breast cancer. In one embodiment, the cancer treated according to the methods described herein is ovarian cancer.

[0063] The subject treated according to the methods described herein can be a human or a non-human animal (e.g., a mammal). In a preferred embodiment, the subject is a human.

[0064] In certain embodiments, the anti-M(H)DM2 / 4 antibodies or fragments described herein are administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or intratumorally. In other embodiments, the anti-M(H)DM2 / 4 antibodies or fragments described herein are administered orally.

[0065] In certain embodiments, a subject treated according to the methods described herein is further administered an additional anti-cancer therapy (e.g., a vaccine, targeted therapy, chemotherapy, radiation therapy, surgery, or immunotherapy) different from the antibody or fragment or antibody-drug conjugate. In one embodiment, the additional therapy is a vaccine. In one embodiment, the additional therapy is targeted therapy. In one embodiment, the additional therapy is chemotherapy (e.g., gemcitabine, paclitaxel, nab-paclitaxel, or a combination of gemcitabine and nab-paclitaxel). In one embodiment, the additional therapy is immunotherapy. In one embodiment, the additional therapy is radiation therapy. In one embodiment, the additional therapy is surgery (e.g., to remove some or all of the cancerous tumor being treated). In certain embodiments, the additional therapy is an inhibitor of the function of one or more checkpoint inhibitory molecules (e.g., an inhibitor against one or more of CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, OX40, and LAG-3, e.g., an inhibitory antibody). In certain embodiments, the additional therapy is not a cell cycle inhibitor. In certain embodiments, a subject treated using the methods described herein is not administered a cell cycle inhibitor during the course of treatment with the antibody or fragment. In certain embodiments, an anti-M(H)DM2 / 4 antibody or fragment described herein is administered alone, without any additional anti-cancer therapy (e.g., a subject treated using the methods described herein is not administered an additional anti-cancer therapy during the course of treatment with the antibody or fragment).

[0066] In certain embodiments, a subject treated according to the methods described herein is further administered chemotherapy, wherein the chemotherapy is cisplatin. In certain embodiments, a subject treated according to the methods described herein is further administered chemotherapy, wherein the chemotherapy is 5-FU. In certain embodiments, a subject treated according to the methods described herein is further administered chemotherapy, wherein the chemotherapy is paclitaxel. In certain embodiments, a subject treated according to the methods described herein is further administered chemotherapy, wherein the chemotherapy is paclitaxel formulated as albumin-bound particles (e.g., ABRAXANE®). In certain embodiments, a subject treated according to the methods described herein is further administered chemotherapy, wherein the chemotherapy is gemcitabine (e.g., if the cancer being treated is pancreatic cancer). In certain embodiments, a subject treated according to the methods described herein is further administered chemotherapy, wherein the chemotherapy is nab-paclitaxel (e.g., if the cancer being treated is pancreatic cancer). In certain embodiments, the cancer is pancreatic cancer, and the subject treated according to the methods described herein is further administered chemotherapy, wherein the chemotherapy is a combination of gemcitabine and nab-paclitaxel. In certain embodiments, the gemcitabine and / or nab-paclitaxel are administered at a dose lower than that used when the gemcitabine and / or nab-paclitaxel is administered without combination with an anti-cancer antibody (e.g., an anti-M(H)DM2 / 4 antibody or fragment described herein). In certain embodiments, where the subject is a human, gemcitabine is administered at a dose of 1,500 mg / m 2 and / or nab-paclitaxel is administered at a dose lower than 300 mg / m 2 In one embodiment, in which the subject is a human, gemcitabine is administered at a dose lower than 1,000 mg / m 2 and / or nab-paclitaxel is administered at a dose equal to or lower than 125 mg / m 2In one embodiment, where the subject is a human, gemcitabine is administered at a dose equal to or less than 500 mg / m 2 and / or nab-paclitaxel is administered at a dose equal to or lower than 62.5 mg / m 2 In certain embodiments, the combination of gemcitabine and nab-paclitaxel is administered once every two weeks or less frequently.

[0067] In certain embodiments, a subject treated according to the methods described herein is resistant to other cancer therapies (e.g., vaccines, targeted therapies, chemotherapy, radiation therapy, surgery, or immunotherapy). In certain embodiments, a subject treated according to the methods described herein is resistant to chemotherapy. In one embodiment, a subject treated according to the methods described herein has chemotherapy-resistant ovarian cancer. In other embodiments, a subject treated according to the methods described herein is resistant to one or more inhibitors of inhibitory immune checkpoint molecules. In other embodiments, a subject treated according to the methods described herein is resistant to radiation therapy.

[0068] In certain embodiments, an anti-M(H)DM2 / 4 antibody or fragment used in the methods described herein specifically binds within amino acids 19-50 of SEQ ID NO:4. In another specific embodiment, an anti-M(H)DM2 / 4 antibody used in the methods described herein specifically binds within amino acids 154-167 of SEQ ID NO:4. In yet another specific embodiment, an anti-M(H)DM2 / 4 antibody used in the methods described herein specifically binds within amino acids 1-60 of SEQ ID NO:4. In yet another specific embodiment, an anti-M(H)DM2 / 4 antibody used in the methods described herein specifically binds within amino acids 1-100 of SEQ ID NO:4. In yet another specific embodiment, an anti-M(H)DM2 / 4 antibody used in the methods described herein specifically binds within amino acids 1-109 of SEQ ID NO:4. In another specific embodiment, an anti-M(H)DM2 / 4 antibody used in the methods described herein specifically binds within amino acids 26-60 of SEQ ID NO:4. In one specific embodiment, the anti-M(H)DM2 / 4 antibody used in the methods described herein specifically binds within the terminal 60 amino acids at the C-terminus of HDM2 on the plasma membrane of cancer cells. In another specific embodiment, the anti-M(H)DM2 / 4 antibody used in the methods described herein specifically binds within the terminal 100 amino acids at the C-terminus of HDM2 on the plasma membrane of cancer cells. In one specific embodiment, the anti-M(H)DM2 / 4 antibody used in the methods described herein specifically binds within amino acids 101-200 of SEQ ID NO:4.

[0069] In specific embodiments, the anti-M(H)DM2 / 4 antibodies or fragments used in the methods described herein compete with the mouse anti-HDM2 antibody OP145 for binding to M(H)DM2 / 4 (described herein, see e.g., Table 10). In other specific embodiments, the anti-M(H)DM2 / 4 antibodies or fragments used in the methods described herein compete with the mouse anti-HDM2 antibody 965 (SMP14) for binding to M(H)DM2 / 4 (described herein, see e.g., Tables 3 and 10). In yet other specific embodiments, the anti-M(H)DM2 / 4 antibodies or fragments used in the methods described herein compete with the rabbit anti-HDM2 antibody sc-813 (N-20) for binding to M(H)DM2 / 4 (described herein, see e.g., Table 10). In another embodiment, an anti-M(H)DM2 / 4 antibody or fragment used in the methods described herein competes with rabbit anti-HDM2 antibody sc-812 (C-18) for binding to M(H)DM2 / 4 (described herein, see e.g., Table 10). In another embodiment, an anti-M(H)DM2 / 4 antibody or fragment used in the methods described herein competes with mouse anti-HDM2 antibody M01, clone 1A7 for binding to M(H)DM2 / 4 (described herein, see e.g., Table 3).

[0070] In certain aspects, provided herein are methods for treating cancer or preventing metastasis in a subject in need thereof, comprising administering to the subject any of the anti-M(H)DM2 / 4 antibodies described herein (e.g., an antibody that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 on the surface of cells of the cancer), wherein the antibody comprises a human IgG Fc region that mediates complement-dependent cytotoxicity (CDC) and / or antibody-dependent cell-mediated cytotoxicity (ADCC). In some of these embodiments, the extracellular region of HDM2 targeted by the anti-HDM2 antibodies or fragments used herein is within one of the following amino acid regions of HDM2: amino acids 1-15 of SEQ ID NO:4, amino acids 15-25 of SEQ ID NO:4, amino acids 375-491 of SEQ ID NO:4, amino acids 19-50 of SEQ ID NO:4, amino acids 50-60 of SEQ ID NO:4, amino acids 100-110 of SEQ ID NO:4, amino acids 154-167 of SEQ ID NO:4, amino acids 1-60 of SEQ ID NO:4, or the terminal 60 amino acids at the C-terminus of HDM2 on the plasma membrane of cancer cells. In certain embodiments of the methods described in this paragraph, the cancer is leukemia, lung cancer, colon cancer, melanoma, pancreatic cancer, breast cancer, or ovarian cancer.

[0071] In one aspect, provided herein is a method for diagnosing cancer in a subject (e.g., a human), the method comprising: (a) detecting whether an antibody or fragment thereof (e.g., a labeled antibody or fragment) that specifically binds to M(H)DM2 / 4 (e.g., HDM2) binds to the surface of intact cells of the subject, wherein the antibody or fragment is any anti-M(H)DM2 / 4 antibody or fragment described herein (in a preferred example, the antibody or fragment is any anti-M(H)DM2 / 4 antibody or fragment that specifically binds to the peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3); and (b) diagnosing the subject with cancer if binding is detected in step (a). In one embodiment, the method of diagnosis is an ex vivo method. In one embodiment, the method of diagnosis further comprises obtaining intact cells from the subject prior to step (a). In one embodiment, the method of diagnosis comprises administering the antibody or fragment to the subject prior to the detection in step (a), wherein the detection is performed by in vivo imaging of the subject. In certain embodiments, for example, the following are provided: (Item 1) An antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment thereof specifically binds to a peptide, the sequence of which consists of MCNTNMSVPTDGAVT (SEQ ID NO: 1), TTSQIPASEQE (SEQ ID NO: 2), or CPVCRQPIQMIVLTYFP (SEQ ID NO: 3). (Item 2) 2. The antibody or fragment according to item 1, wherein the sequence of the peptide is MCNTNMSVPTDGAVT (SEQ ID NO: 1). (Item 3) 2. The antibody or fragment according to item 1, wherein the sequence of the peptide is TTSQIPASEQE (SEQ ID NO: 2). (Item 4) 2. The antibody or fragment of item 1, wherein the sequence of the peptide is CPVCRQPIQMIVLTYFP (SEQ ID NO: 3). (Item 5) A humanized antibody or fragment thereof that specifically binds to HDM2, comprising: (i) a heavy chain variable region (VH) comprising a VH complementarity-determining region ("CDR") 1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 are CDRs of the VH having an amino acid sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 38, and SEQ ID NO: 40; or (ii) a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 are CDRs of the VL having an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 41. (Item 6) 6. The humanized antibody or fragment of item 5, comprising the VH, wherein VH CDR1, VH CDR2, and VH CDR3 are those of the VH having the amino acid sequence of SEQ ID NO: 36. (Item 7) 7. The humanized antibody or fragment of item 5 or 6, comprising the VL, wherein VL CDR1, VL CDR2, and VL CDR3 are those of a VL having the amino acid sequence of SEQ ID NO: 37. (Item 8) 6. The humanized antibody or fragment of item 5, comprising the VH, wherein VH CDR1, VH CDR2, and VH CDR3 are those of the VH having the amino acid sequence of SEQ ID NO: 38. (Item 9) 9. The humanized antibody or fragment of item 5 or 8, comprising the VL, wherein VL CDR1, VL CDR2, and VL CDR3 are those of a VL having the amino acid sequence of SEQ ID NO: 39. (Item 10) 6. The humanized antibody or fragment according to item 5, comprising the VH, wherein VH CDR1, VH CDR2, and VH CDR3 are those of the VH having the amino acid sequence of SEQ ID NO: 40. (Item 11) 11. The humanized antibody or fragment of item 5 or 10, comprising the VL, wherein VL CDR1, VL CDR2, and VL CDR3 are those of a VL having the amino acid sequence of SEQ ID NO: 41. (Item 12) 1. An antibody or fragment thereof that specifically binds to HDM2, comprising: a heavy chain variable region (VH) comprising VH complementarity determining region ("CDR") 1, VH CDR 2, and VH CDR 3; (i) the VH CDR 1 has the amino acid sequence GFTFTHY (SEQ ID NO: 18), the VH CDR 2 has the amino acid sequence RNKAKGYT (SEQ ID NO: 19), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO: 20); (ii) the VH CDR 1 has the amino acid sequence GFTFTHYYMS (SEQ ID NO: 42), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAE (SEQ ID NO: 45), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO: 20); (iii) the VH CDR 1 has the amino acid sequence HYYMS (SEQ ID NO: 43), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAEYSASVKG (SEQ ID NO: 46), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO: 20); (iv) the VH CDR 1 has the amino acid sequence THYYMS (SEQ ID NO: 44), the VH CDR 2 has the amino acid sequence WLGFIRNKAKGYTAE (SEQ ID NO: 47), and the VH CDR 3 has the amino acid sequence ARDIGD (SEQ ID NO: 48), or (v) the VH CDR 1 has the amino acid sequence FTFTHYY (SEQ ID NO: 144), the VH CDR 2 has the amino acid sequence IRNKAKGYTA (SEQ ID NO: 145), and the VH CDR 3 has the amino acid sequence ARDIGDN (SEQ ID NO: 146); An antibody or fragment thereof comprising a heavy chain variable region (VH). (Item 13) 1. An antibody or fragment thereof that specifically binds to HDM2, comprising: a heavy chain variable region (VH) comprising VH complementarity determining region ("CDR") 1, VH CDR 2, and VH CDR 3; (i) the VH CDR 1 has the amino acid sequence GDTLSGS (SEQ ID NO: 24), the VH CDR 2 has the amino acid sequence HLNRGT (SEQ ID NO: 25), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO: 26), (ii) the VH CDR 1 has the amino acid sequence GDTLSGSWMH (SEQ ID NO: 52), the VH CDR 2 has the amino acid sequence EIHLNRGTTN (SEQ ID NO: 55), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO: 26); (iii) the VH CDR 1 has the amino acid sequence GSWMH (SEQ ID NO: 53), the VH CDR 2 has the amino acid sequence EIHLNRGTTNYNEKFKG (SEQ ID NO: 56), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO: 26); (iv) the VH CDR 1 has the amino acid sequence SGSWMH (SEQ ID NO: 54), the VH CDR 2 has the amino acid sequence WIGEIHLNRGTTN (SEQ ID NO: 57), and the VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO: 58), or (v) the VH CDR 1 has the amino acid sequence GDTLSGSW (SEQ ID NO: 148), the VH CDR 2 has the amino acid sequence IHLNRGTT (SEQ ID NO: 143), and the VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO: 58); An antibody or fragment thereof comprising a heavy chain variable region (VH). (Item 14) 1. An antibody or fragment thereof that specifically binds to HDM2, comprising: a heavy chain variable region (VH) comprising VH complementarity determining region ("CDR") 1, VH CDR 2, and VH CDR 3; (i) the VH CDR 1 has the amino acid sequence GYTFTSY (SEQ ID NO: 30), the VH CDR 2 has the amino acid sequence NPRNGG (SEQ ID NO: 31), and the VH CDR 3 has SGYYAMDY (SEQ ID NO: 32), (ii) the VH CDR 1 has the amino acid sequence GYTFTSYYMY (SEQ ID NO: 62), the VH CDR 2 has the amino acid sequence GINPRNGGTN (SEQ ID NO: 65), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO: 32); (iii) the VH CDR 1 has the amino acid sequence SYYMY (SEQ ID NO: 63), the VH CDR 2 has the amino acid sequence GINPRNGGTNFNEKFKN (SEQ ID NO: 66), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO: 32), or (iv) the VH CDR 1 has the amino acid sequence TSYYMY (SEQ ID NO: 64), the VH CDR 2 has the amino acid sequence WIGGINPRNGGTN (SEQ ID NO: 67), and the VH CDR 3 has the amino acid sequence TRSGYYAMD (SEQ ID NO: 68); An antibody or fragment thereof comprising a heavy chain variable region (VH). (Item 15) the antibody or fragment comprises a light chain variable region (VL) comprising VL complementarity determining region ("CDR") 1, VL CDR 2, and VL CDR 3; (i) the VL CDR 1 has the amino acid sequence RSSKNLLHSNGITYLY (SEQ ID NO: 21), the VL CDR 2 has the amino acid sequence RVSNLAS (SEQ ID NO: 22), and the VL CDR 3 has the amino acid sequence AQLLELPYT (SEQ ID NO: 23); (ii) the VL CDR 1 has the amino acid sequence LHSNGITYLYWY (SEQ ID NO: 49), the VL CDR 2 has the amino acid sequence LLISRVSNLA (SEQ ID NO: 50), and the VL CDR 3 has the amino acid sequence AQLLELPY (SEQ ID NO: 51), or (iii) the VL CDR 1 has the amino acid sequence KNLLHSNGITY (SEQ ID NO: 147), the VL CDR 2 has the amino acid sequence RVS, and the VL CDR 3 has the amino acid sequence AQLLELPYT (SEQ ID NO: 23), 13. The antibody or fragment of item 12, further comprising a light chain variable region (VL). (Item 16) the antibody or fragment comprises a light chain variable region (VL) comprising VL complementarity determining region ("CDR") 1, VL CDR 2, and VL CDR 3; (i) the VL CDR 1 has the amino acid sequence RSSKSLLHSNGNSYLY (SEQ ID NO: 27), the VL CDR 2 has the amino acid sequence RMSNLAS (SEQ ID NO: 28), and the VL CDR 3 has the amino acid sequence MQHLEYPFT (SEQ ID NO: 29); (ii) the VL CDR 1 has the amino acid sequence LHSNGNSYLYWF (SEQ ID NO: 59), the VL CDR 2 has the amino acid sequence LLIYRMSNLA (SEQ ID NO: 60), and the VL CDR 3 has the amino acid sequence MQHLEYPF (SEQ ID NO: 61), or (iii) the VL CDR 1 has the amino acid sequence KSLLHSNGNSY (SEQ ID NO: 141), the VL CDR 2 has the amino acid sequence RMS, and the VL CDR 3 has the amino acid sequence MQHLEYPFT (SEQ ID NO: 29); 14. The antibody or fragment of item 13, further comprising a light chain variable region (VL). (Item 17) the antibody or fragment comprises a light chain variable region (VL) comprising VL complementarity determining region ("CDR") 1, VL CDR 2, and VL CDR 3; (i) the VL CDR 1 has the amino acid sequence RASQDISNFLN (SEQ ID NO: 33), the VL CDR 2 has the amino acid sequence YTSRLHS (SEQ ID NO: 34), and the VL CDR 3 has the amino acid sequence QQGNTLPRT (SEQ ID NO: 35), or (ii) The antibody or fragment of item 14, further comprising a light chain variable region (VL) in which the VL CDR 1 has the amino acid sequence SNFLNWY (SEQ ID NO: 69), the VL CDR 2 has the amino acid sequence LLIYYTSRLH (SEQ ID NO: 70), and the VL CDR 3 has the amino acid sequence QQGNTLPR (SEQ ID NO: 71). (Item 18) 13. The antibody or fragment of item 12, comprising a VH having the amino acid sequence of SEQ ID NO: 36 or a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 36. (Item 19) 19. The antibody or fragment of item 1, 12, or 18, comprising a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 37, or a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 37. (Item 20) 14. The antibody or fragment of item 13, comprising a VH having the amino acid sequence of SEQ ID NO: 38 or a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 38. (Item 21) 21. The antibody or fragment of item 1, 13, or 20, comprising a VL having the amino acid sequence of SEQ ID NO: 39, or a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 39. (Item 22) 15. The antibody or fragment of item 14, comprising a VH having the amino acid sequence of SEQ ID NO: 40 or a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 40. (Item 23) 23. The antibody or fragment of item 1, 14, or 22, comprising a VL having the amino acid sequence of SEQ ID NO: 41, or a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 41. (Item 24) 24. The antibody or fragment of any one of items 1 to 4 and 12 to 23, wherein the antibody is a humanized antibody. (Item 25) 24. The antibody or fragment of any one of items 1 to 4 and 12 to 23, wherein the antibody is a human antibody. (Item 26) 24. The antibody or fragment of any one of items 1 to 4 and 12 to 23, wherein the antibody is a chimeric antibody. (Item 27) 27. The antibody or fragment of any one of items 1 to 26, wherein the antibody is a monoclonal antibody. (Item 28) 28. The antibody or fragment of any one of items 1 to 27, which is an immunoglobulin. (Item 29) 29. The antibody or fragment of item 28, wherein the immunoglobulin is an IgG. (Item 30) 29. The antibody or fragment of item 28, wherein the immunoglobulin is of the IgG1 isotype. (Item 31) 29. The antibody or fragment of item 28, wherein the immunoglobulin is of the IgG3 isotype. (Item 32) 29. The antibody or fragment of any one of items 1 to 28, comprising an Fc region, wherein the Fc region is a human IgG1, human IgG2, human IgG3, human IgG4, or human IgM Fc region. (Item 33) 33. The antibody or fragment of item 32, wherein the antibody or fragment mediates complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). (Item 34) 34. The antibody or fragment of item 33, wherein the antibody or fragment mediates complement-dependent cytotoxicity (CDC). (Item 35) 28. The antibody or fragment of any one of items 1 to 27, which is an Fv fragment, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a single-chain antibody molecule, or a single-chain Fv (scFv). (Item 36) 24. The antibody or fragment of any one of items 5 to 23, wherein the HDM2 is an HDM2 variant lacking a nuclear localization signal domain, an HDM2 variant lacking the sequence of amino acids 179 to 185 of SEQ ID NO: 4, and / or an HDM2 variant lacking the sequence of amino acids 464 to 471 of SEQ ID NO: 4. (Item 37) 37. The antibody or fragment of any one of items 1 to 36, which is purified. (Item 38) 38. The antibody or fragment of any one of items 1 to 37, which inhibits tumor cell proliferation in vivo. (Item 39) 39. An antibody or fragment thereof that competes with the antibody or fragment of any one of items 1 to 38 for binding to M(H)DM2 / 4. (Item 40) an antibody or fragment thereof that (i) competes with a murine anti-HDM2 IgG1 antibody comprising a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 36 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 37 for binding to the peptide of SEQ ID NO: 1; or (ii) competes with a murine anti-HDM2 IgG3 antibody comprising a VH having the amino acid sequence of SEQ ID NO: 38 and a VL having the amino acid sequence of SEQ ID NO: 39 for binding to the peptide of SEQ ID NO: 2; or (iii) competes with a murine IgM antibody comprising a VH having the amino acid sequence of SEQ ID NO: 40 and a VL having the amino acid sequence of SEQ ID NO: 41 for binding to the peptide of SEQ ID NO: 3. (Item 41) 41. An antibody-drug conjugate comprising the antibody or fragment of any one of items 1 to 40 conjugated to a cytotoxic drug. (Item 42) A pharmaceutical composition comprising a therapeutically effective amount of the antibody or fragment of any one of items 1 to 40 or the antibody-drug conjugate of item 41. (Item 43) 41. A method for treating cancer in a subject in need thereof, comprising administering to the subject (i) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment is not conjugated to a cell-penetrating peptide, or an antibody-drug conjugate comprising the antibody or fragment conjugated to a cytotoxic drug, (ii) the antibody or fragment of any one of items 1 to 40, (iii) the antibody-drug conjugate of item 41, or (iv) the pharmaceutical composition of item 42. (Item 44) A method of treating cancer in a subject in need thereof, comprising administering to the subject an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment thereof is not conjugated to a cytotoxic moiety. (Item 45) 41. A method for inhibiting or preventing metastasis in a subject having cancer, the method comprising the step of administering to the subject (i) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment is not conjugated to a cell-penetrating peptide, or an antibody-drug conjugate comprising the antibody or fragment conjugated to a cytotoxic drug, (ii) the antibody or fragment of any one of items 1 to 40, (iii) the antibody-drug conjugate of item 41, or (iv) the pharmaceutical composition of item 42. (Item 46) A method for inhibiting or preventing metastasis in a subject with cancer, comprising administering to the subject an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment is not conjugated to a cytotoxic moiety. (Item 47) 41. A method for preventing cancer recurrence or preventing cancer recurrence in a subject in need thereof, comprising administering to the subject (i) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment is not conjugated to a cell-penetrating peptide, or an antibody-drug conjugate comprising the antibody or fragment conjugated to a cytotoxic drug, (ii) the antibody or fragment of any one of items 1 to 40, (iii) the antibody-drug conjugate of item 41, or (iv) the pharmaceutical composition of item 42. (Item 48) A method for preventing cancer recurrence or recurrence in a subject with cancer, comprising administering to the subject an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment is not conjugated to a cytotoxic moiety. (Item 49) 50. A method for increasing survival in a subject with cancer compared to a subject not treated with an anti-M(H)DM2 / 4 antibody or fragment, comprising administering to the subject (i) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment is not conjugated to a cell-penetrating peptide, or an antibody-drug conjugate comprising the antibody or fragment conjugated to a cytotoxic drug, (ii) the antibody or fragment of any one of items 1 to 40, (iii) the antibody-drug conjugate of item 41, or (iv) the pharmaceutical composition of item 42. A method for increasing survival in a subject having cancer compared to a subject not treated with an anti-M(H)DM2 / 4 antibody or fragment, comprising administering to the subject an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment is not conjugated to a cytotoxic moiety. (Item 51) 51. The method of any one of items 43 to 50, comprising administering to the subject the antibody or fragment of any one of items 1 to 40. (Item 52) 50. The method of any one of items 43, 45, 47, and 49, comprising administering to the subject the antibody or fragment of any one of items 1 to 40, the antibody-drug conjugate of item 41, or the pharmaceutical composition of item 42. (Item 53) 53. The method of claim 52, wherein the antibody or fragment is not conjugated to a cell-penetrating peptide. (Item 54) 50. The method of any one of items 43, 45, 47, and 49, comprising administering to the subject an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment thereof is not conjugated to a cell-penetrating peptide. (Item 55) 57. The method of any one of items 43 to 52 and 56, wherein the antibody or fragment is a humanized antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4. (Item 56) 55. The method of any one of items 43 to 50 and 54, wherein the antibody or fragment is a human antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4. (Item 57) 55. The method of any one of items 43 to 50 and 54, wherein the antibody or fragment is a chimeric antibody. (Item 58) 58. The method of any one of items 43 to 50 and 54 to 57, wherein the antibody or fragment is a monoclonal antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4. (Item 59) 59. The method of any one of items 43 to 50 and 54 to 58, wherein the antibody or fragment is an immunoglobulin. (Item 60) 60. The method of claim 59, wherein the immunoglobulin is IgG. (Item 61) Item 62. The method of Item 59, wherein the immunoglobulin is of the IgG1 isotype. Item 63. The method of Item 59, wherein the immunoglobulin is of the IgG3 isotype. 60. The method of any one of items 43 to 50 and 54 to 59, wherein the antibody or fragment comprises an Fc region, and the Fc region is a human IgG1, human IgG2, human IgG3, human IgG4, or human IgM Fc region. (Item 64) 64. The method of claim 63, wherein the antibody or fragment mediates complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). (Item 65) 65. The method of claim 64, wherein the antibody or fragment mediates complement-dependent cytotoxicity (CDC). (Item 66) 59. The method of any one of items 43 to 50 and 54 to 58, wherein the antibody or fragment is an Fv fragment, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a single-chain antibody molecule, or a single-chain Fv (scFv). (Item 67) 67. The method of any one of items 43 to 50 and 54 to 66, wherein the extracellularly accessible epitope of M(H)DM2 / 4 is within amino acids 19 to 50 of SEQ ID NO: 4. (Item 68) 67. The method of any one of items 43 to 50 and 54 to 66, wherein the extracellularly accessible epitope of M(H)DM2 / 4 is within amino acids 154 to 167 of SEQ ID NO: 4. (Item 69) 69. The method of any one of items 43 to 52 and 56 to 68, wherein the extracellularly accessible epitope of M(H)DM2 / 4 is within amino acids 1 to 60 of SEQ ID NO: 4. (Item 70) 67. The method of any one of items 43 to 50 and 54 to 66, wherein the extracellularly accessible epitope of M(H)DM2 / 4 is within amino acids 26 to 60 of SEQ ID NO: 4. (Item 71) 67. The method of any one of items 43 to 50 and 54 to 66, wherein the extracellularly accessible epitope of M(H)DM2 / 4 is within amino acids 101 to 200 of SEQ ID NO: 4. (Item 72) 67. The method of any one of items 43 to 50 and 54 to 66, wherein the extracellularly accessible epitope of M(H)DM2 / 4 is within amino acids 50 to 60 of SEQ ID NO: 4. (Item 73) 67. The method of any one of items 43 to 50 and 54 to 66, wherein the extracellularly accessible epitope of M(H)DM2 / 4 is within the terminal 60 amino acids at the C-terminus of HDM2 on the plasma membrane of the cancer cell. (Item 74) 67. The method of any one of paragraphs 43 to 50 and 54 to 66, wherein the antibody or fragment competes with the murine anti-HDM2 antibody OP145 for binding to M(H)DM2 / 4. (Item 75) 67. The method of any one of items 43 to 50 and 54 to 66, wherein the antibody or fragment competes with the murine anti-HDM2 antibody 965 (SMP14) for binding to M(H)DM2 / 4. (Item 76) 67. The method of any one of items 43 to 50 and 54 to 66, wherein the antibody or fragment competes with rabbit anti-HDM2 antibody sc-813(N-20) for binding to M(H)DM2 / 4. (Item 77) 67. The method of any one of items 43 to 50 and 54 to 66, wherein the antibody or fragment competes with rabbit anti-HDM2 antibody sc-812 (C-18) for binding to M(H)DM2 / 4. (Item 78) 67. The method of any one of items 43 to 50 and 54 to 66, wherein the antibody or fragment competes with murine anti-HDM2 antibody M01, clone 1A7, for binding to M(H)DM2 / 4. (Item 79) 79. The method of any one of items 43 to 50 and 54 to 78, wherein the M(H)DM2 / 4 is an HDM2 variant lacking a nuclear localization signal domain, an HDM2 variant lacking the sequence of amino acids 179 to 185 of SEQ ID NO: 4, and / or an HDM2 variant lacking the sequence of amino acids 464 to 471 of SEQ ID NO: 4. (Item 80) 80. The method of any one of items 43 to 50 and 54 to 79, wherein the antibody or fragment is purified. (Item 81) 50. The method of any one of items 43, 45, 47, and 49, comprising administering to the subject an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, conjugated to a cytotoxic drug, wherein the antibody or fragment thereof is not conjugated to a cell-penetrating peptide. (Item 82) 81. The method of any one of items 54 to 80, wherein the antibody or fragment is not conjugated to a cytotoxic drug. (Item 83) 83. The method of any one of items 43 to 82, wherein the cancer is a type of cancer known to metastasize. (Item 84) 84. The method of any one of items 43 to 83, wherein the cancer is an advanced stage cancer. (Item 85) 85. The method of any one of items 43 to 84, wherein the cancer is a metastatic cancer. (Item 86) 86. The method of any one of items 43 to 85, wherein the cancer is a solid cancer. (Item 87) 87. The method of item 86, wherein the cancer is lung cancer, cervical cancer, endometrial cancer, ovarian cancer, pancreatic cancer, melanoma, breast cancer, colon cancer, bladder cancer, astrocytic neoplasm, glioblastoma, or childhood rhabdomyosarcoma. (Item 88) Item 88. The method of item 87, wherein the cancer is pancreatic cancer, lung cancer, or colon cancer. (Item 89) 86. The method of any one of items 43 to 85, wherein the cancer is a non-solid cancer. (Item 90) Item 89. The method of item 89, wherein the cancer is leukemia or lymphoma. (Item 91) 91. The method of any one of items 43 to 90, wherein the antibody or fragment is administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or intratumorally. (Item 92) 92. The method of any one of items 43 to 91, further comprising administering to the subject a cancer therapy different from the antibody or fragment or antibody-drug conjugate. (Item 93) Item 93. The method of item 92, wherein the cancer therapy is chemotherapy. (Item 94) 94. The method of item 93, wherein the chemotherapy is gemcitabine. (Item 95) Item 94. The method of item 93, wherein the chemotherapy is nab-paclitaxel. (Item 96) Item 94. The method of item 93, wherein the chemotherapy is cisplatin. (Item 97) 94. The method of item 93, wherein the chemotherapy is 5-FU. (Item 98) Item 94. The method of item 93, wherein the chemotherapy is paclitaxel. (Item 99) Item 94. The method of item 93, wherein the cancer is pancreatic cancer and the chemotherapy is a combination of gemcitabine and nab-paclitaxel. (Item 100) 100. The method of claim 99, wherein the gemcitabine and nab-paclitaxel are administered at a dose lower than that used when the gemcitabine and nab-paclitaxel are administered without combination with an anti-cancer antibody. (Item 101) 99. The method of claim 98, wherein the subject is a human, the gemcitabine is administered at a dose equal to or less than 1,000 mg / m2, and the nab-paclitaxel is administered at a dose equal to or less than 125 mg / m2. (Item 102) Item 99. The method of item 99, wherein the subject is a human, gemcitabine is administered at a dose equal to or less than 500 mg / m2, and the nab-paclitaxel is administered at a dose equal to or less than 62.5 mg / m2. (Item 103) 103. The method of any one of items 99 to 102, wherein the combination of gemcitabine and nab-paclitaxel is administered once every two weeks or less frequently. (Item 104) 101. The method of any one of items 43 to 100, wherein the subject is a human. (Item 105) Item 93. The method of item 92, wherein the cancer therapy is immunotherapy. (Item 106) 106. The method of claim 105, wherein the immunotherapy is an inhibitor of one or more inhibitory checkpoint molecules. (Item 107) 107. The method of claim 106, wherein the one or more inhibitory checkpoint molecules are selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, OX40, and LAG-3. (Item 108) 1. A method for selecting and treating a subject with cancer, comprising: identifying a subject with cancer, wherein an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 binds to the surface of intact cells of said cancer; administering to the subject (i) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, the antibody or fragment thereof not conjugated to a cell-penetrating peptide, or an antibody-drug conjugate comprising the antibody or fragment conjugated to a cytotoxic drug, (ii) an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, the antibody or fragment thereof not conjugated to a cytotoxic moiety, (iii) the antibody or fragment of any one of Items 1 to 40, (iv) the antibody-drug conjugate of Item 41, or (v) the pharmaceutical composition of Item 42; A method comprising: (Item 109) 109. The method of claim 108, further comprising, prior to step (b), determining whether the antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 binds to the surface of intact cells of the cancer, wherein the antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 is the antibody or fragment of any one of claims 1 to 40. (Item 110) 110. The method of claim 109, further comprising the step of obtaining intact cells of the cancer prior to the determining step. (Item 111) 1. A method of diagnosing cancer in a subject, comprising: (a) detecting whether the antibody or fragment according to any one of items 1 to 40 binds to the surface of an intact cell of the subject; (b) diagnosing the subject with cancer if binding is detected in step (a); A method comprising: (Item 112) 112. The method according to item 111, which is an ex vivo method. (Item 113) 112. The method of claim 111, further comprising, prior to step (a), obtaining intact cells from the subject. (Item 114) 112. The method of claim 111, further comprising administering the antibody or fragment to the subject prior to the detecting in step (a), wherein the detecting is carried out by in vivo imaging of the subject. (Item 115) 115. The method of any one of items 108 to 114, wherein in step (a) the antibody or fragment is labeled. (Item 116) 115. The method of any one of items 108 to 114, wherein the subject is a human. (Item 117) 5. The antibody or fragment thereof of any one of items 1 to 4, wherein the antibody or fragment thereof specifically binds to an extracellularly accessible epitope of HDM2. (Item 118) 51. The method of any one of items 43 to 50, comprising administering to the subject an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2.

[0072] 5.1 Terminology As used herein, the term "HDM2" refers to the protein product of the human E3 ubiquitin-protein ligase (i.e., full-length HDM2 protein) of UniProt Accession No. Q00987 (SEQ ID NO: 4), or any splice variant of the full-length HDM2 protein known in the art or described herein. The amino acid sequences of exemplary splice variants of the full-length HDM2 protein are set forth as SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.

[0073] As used herein, the term "MDM2" refers to the protein product of the mouse E3 ubiquitin-protein ligase of UniProt Accession No. P23804 (SEQ ID NO: 5) (i.e., the full-length MDM2 protein), or any splice variant of the full-length MDM2 protein known in the art or described herein.

[0074] As used herein, the term "M(H)DM2" refers to HDM2, MDM2, or an E3 ubiquitin-protein ligase from a species other than human and mouse that is a homolog of HDM2 or MDM2.

[0075] As used herein, the term "HDM4" refers to the protein product of the human protein of UniProt Accession No. O15151 (SEQ ID NO: 6) (i.e., the full-length HDM4 protein), or any splice variant of the full-length HDM4 protein known in the art or described herein.

[0076] As used herein, the term "MDM4" refers to the protein product of the mouse protein of UniProt accession number 035618 (i.e., the full-length MDM4 protein), or any splice variant of the full-length MDM4 protein known in the art or described herein. The amino acid sequence of an exemplary splice variant of the full-length MDM4 protein is set forth as SEQ ID NO: 6. Other splice variants of the full-length MDM4 protein known in the art include, but are not limited to, MDM4-S, MDM4-A, MDM4-G, MDM4-XALT1 / XALT2, and MDM4-211.

[0077] As used herein, the term "M(H)DM4" refers to HDM4 (also called HDMX), MDM4 (also called MDMX), or proteins from species other than human and mouse that are homologs of HDM4 or MDM4.

[0078] As used herein, the term "M(H)DM2 / 4" refers to HDM2, MDM2, HDM4, MDM4, or proteins from species other than human and mouse that are homologs of HDM2, MDM2, HDM4, or MDM4.

[0079] As used herein, the term "about," when used to modify a numerical value, indicates that a deviation of up to 10% above and below that numerical value is included within the intended meaning of the recited value.

[0080] As used herein, the term "intact" with respect to cells refers to cells that are viable or cells that have been fixed but not permeabilized.

[0081] As used herein, the term "extracellularly accessible" in reference to an epitope of M(H)DM2 / 4 refers to an epitope of M(H)DM2 / 4 that is available for binding to an antibody extracellularly (without requiring intracellular transport of the antibody) when M(H)DM2 / 4 is expressed by an intact cell. An antibody or fragment thereof can be determined to bind to an extracellularly accessible epitope of M(H)DM2 / 4 if the antibody binds extracellularly to M(H)DM2 / 4 expressed by an intact cell.

[0082] As used herein, the term "VL" refers to the light chain variable region of an antibody.

[0083] As used herein, the term "VH" refers to the heavy chain variable region of an antibody.

[0084] As used herein, the term "percent (%) amino acid sequence identity" or "percent sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished by various means known in the art, for example, using publicly available computer software such as BLASTp, BLAST-2, ALIGN (e.g., ALIGN-2), or Megalign (DNASTAR) software. [Brief explanation of the drawings]

[0085] [Figures 1A-1D] Figures 1A-D show that monoclonal antibodies NMC-103, NMC-204, and NMC-303 specifically bound to the NMC-P1 (SEQ ID NO: 1), NMC-P2 (SEQ ID NO: 2), and NMC-P3 (SEQ ID NO: 3) peptide antigens, respectively, in peptide ELISA experiments. (A) NMC-103 bound to the NMC-P1 peptide, but NMC-204 showed no binding to NMC-P1. (B) NMC-204 bound to the NMC-P2 peptide, but NMC-103 did not bind to NMC-P2. (C) NMC-303 bound to the NMC-P3 peptide, but NMC-204 did not bind to NMC-P3. (D) NMC-103 bound to NMC-P1, but preincubation of the NMC-P1 peptide with NMC-103 abolished the binding of NMC-103 to the NMC-P1 peptide on the plate. In contrast, preincubation of NMC-103 with either NMC-P2 or NMC-P3 did not affect the binding of NMC-103 to NMC-P1.

[0086] [Figure 2]FIG. 2 shows that monoclonal antibodies NMC-103, NMC-204, and NMC-303 bound to HDM2 recombinant protein.

[0087] [Figure 3A-3B] 3A-B show that monoclonal antibody NMC-103 bound to an extracellularly accessible epitope of HDM2 on intact human (A) and mouse (B) cancer cells.

[0088] [Figure 4A-4B] 4A-B show that monoclonal antibody NMC-204 bound to an extracellularly accessible epitope of HDM2 on intact human (A) and mouse (B) cancer cells.

[0089] [Figure 5A-5B] 5A-B show that monoclonal antibody NMC-303 bound to an extracellularly accessible epitope of HDM2 on intact human (A) and mouse (B) cancer cells.

[0090] [Figure 6] Figure 6 shows that monoclonal antibody NMC-204 bound to an extracellularly accessible epitope of HDM2 on intact human cancer cells but not on intact normal human peripheral blood mononuclear cells.

[0091] [Figures 7A-7B] 7A-B show binding curves for monoclonal antibodies NMC-103 (A) and NMC-204 (B) binding to intact MIAPaCa-2 cells.

[0092] [Figures 8A-8C]Figures 8A-C show that the binding of monoclonal antibody NMC-103 to its extracellularly accessible epitope of HDM2 on the plasma membrane of intact human pancreatic cancer MIAPaCa-2 cells was competed by the NMC-P1 peptide (A), the binding of monoclonal antibody NMC-204 to its extracellularly accessible epitope of HDM2 on the plasma membrane of intact human pancreatic cancer MIAPaCa-2 cells was competed by the NMC-P2 peptide (B), and the binding of monoclonal antibody NMC-303 to its extracellularly accessible epitope of HDM2 on the plasma membrane of intact human pancreatic cancer MIAPaCa-2 cells was competed by the NMC-P3 peptide (C).

[0093] [Figure 9] FIG. 9 shows that the binding of monoclonal antibody NMC-103 to its extracellularly accessible epitope of HDM2 on the plasma membrane of intact human pancreatic cancer MIAPaCa-2 cells was competed for by full-length recombinant HDM2 protein.

[0094] [Figure 10] FIG. 10 shows the cell-ELISA binding results of monoclonal antibody NMC-103, monoclonal antibody NMC-204, an antibody against E-cadherin, and an antibody against cytochrome C to intact human pancreatic cancer MiaPaCa-2 cells.

[0095] [Figures 11A-11C] 11A-C show flow cytometry data on % cells stained with monoclonal antibodies NMC-103 (A), NMC-204 (B), and anti-Na+ / K+ ATPase alpha-1 (C), respectively.

[0096] [Figures 12A-12C]Figures 12A-D show that monoclonal antibodies NMC-103 (A and D) and NMC-204 (B and D), but not anti-cytochrome C antibody (C and D), inhibited cell proliferation of intact human pancreatic cancer MIAPaCa-2 cells. [Figure 12D] Figures 12A-D show that monoclonal antibodies NMC-103 (A and D) and NMC-204 (B and D), but not anti-cytochrome C antibody (C and D), inhibited cell proliferation of intact human pancreatic cancer MIAPaCa-2 cells.

[0097] [Figures 13A-13B] Figures 13A-C show that monoclonal antibody NMC-103 in the presence of normal human serum (B and C) induces complement-mediated cytotoxicity against human pancreatic cancer MIAPaCa-2 cells compared to cells treated with normal human serum in the absence of any antibody (A). [Figure 13C] Figures 13A-C show that monoclonal antibody NMC-103 in the presence of normal human serum (B and C) induces complement-mediated cytotoxicity against human pancreatic cancer MIAPaCa-2 cells compared to cells treated with normal human serum in the absence of any antibody (A).

[0098] [Figures 14A-14B] Figures 14A-B show that many commercially available monoclonal antibodies do not bind to either NMC-P1 (A) or NMC-P2 (B).

[0099] [Figure 15A]Figures 15A-B show that the anti-HDM2 antibody designated "MDM2 Monoclonal Antibody (M01), Clone 1A7" (Abnova, Catalog No. H00004193-M01) reacted with intact cancer cells (A), whereas the anti-HDM2 antibody designated "Anti-MDM2 (Ab-4) Mouse mAb (2A9C1.18)" (EMD Millipore, Catalog No. OP144) and the anti-HDM2 antibody designated "Anti-MDM2 (Ab-1) Mouse mAb (IF2)" (EMD Millipore, Catalog No. OP46) did not react with intact cancer cells (B). [Figure 15B] Figures 15A-B show that the anti-HDM2 antibody designated "MDM2 Monoclonal Antibody (M01), Clone 1A7" (Abnova, Catalog No. H00004193-M01) reacted with intact cancer cells (A), whereas the anti-HDM2 antibody designated "Anti-MDM2 (Ab-4) Mouse mAb (2A9C1.18)" (EMD Millipore, Catalog No. OP144) and the anti-HDM2 antibody designated "Anti-MDM2 (Ab-1) Mouse mAb (IF2)" (EMD Millipore, Catalog No. OP46) did not react with intact cancer cells (B).

[0100] [Figure 16] FIG. 16 shows the effect of monoclonal antibody NMC-204 on tumor volume in an LL / 2 syngeneic mouse lung cancer model.

[0101] [Figures 17A-17B] 17A-B show the effect of monoclonal antibody NMC-103 on tumor volume (A) and tumor cell proliferation (B) in the MC-38 syngeneic mouse colon cancer model.

[0102] [Figures 18A-18B] 18A-B show the effect of monoclonal antibody NMC-204 on tumor volume (A) and tumor cell proliferation (B) in the MC-38 syngeneic murine colon cancer model.

[0103] [Figure 19]Figure 19 shows the effects of monoclonal antibody NMC-103 alone (2 mg / kg), the combination of low-dose gemcitabine (25 mg / kg) and nab-paclitaxel (5 mg / kg), the combination of low-dose gemcitabine (25 mg / kg), nab-paclitaxel (5 mg / kg), and NMC-103 (2 mg / kg), and isotype control mouse IgG1 (2 mg / kg) on ​​tumor volume in the Panc-2 syngeneic mouse pancreatic cancer model. Treatment began when mouse tumors reached approximately 70 mm3.

[0104] [Figure 20] FIG. 20 shows the DNA and protein sequences of the heavy and light chain variable regions, respectively, of monoclonal antibody NMC-103.

[0105] [Figure 21] FIG. 21 shows the DNA and protein sequences of the heavy and light chain variable regions, respectively, of monoclonal antibody NMC-204.

[0106] [Figure 22] Figure 22 shows the DNA and protein sequences of the heavy and light chain variable regions of monoclonal antibody NMC-303. The leader sequences preceding the DNA and protein sequences of the heavy and light chain variable regions are shown in bold (not underlined).

[0107] [Figure 23] Figure 23 shows tumor size in mice treated with the anti-HDM2 antibody designated "MDM2 Monoclonal Antibody (M01), clone 1A7" (Abnova, catalog number H00004193-M01), tumor size in mice treated with NMC-103, tumor size in mice treated with NMC-204, and tumor size in mice treated with an isotype control.

[0108] [Figures 24A-24B]In Figures 24A-F, anti-HDM2-specific antibodies stain the surface of cancer cells but not normal cells. Intact cells released with either EDTA or trypsin were blocked with 5% human serum albumin. Next, the cells were incubated with either the polyclonal N-20 M(H)DM2-specific antibody (sc-813, N-20, rabbit IgG; Santa Cruz; "N-20") or the monoclonal M(H)DM2-specific OP145 antibody (OP145, mouse IgG1; Calbiochem; "OP145") on ice for 90 minutes. Another set of cells, prepared under the same conditions, was incubated with the same antibodies preincubated with their corresponding blocking peptides prior to incubation with the cells. After primary antibody incubation, the cells were washed three times with ice-cold PBS and then incubated with FITC-secondary antibodies for 60 minutes. The cells were then washed three times with PBS and subjected to FACS analysis. Human melanoma cells (24A, 24B, and 24C), primary cultured human ovarian cancer cells (24D and 24E), and normal mouse splenocytes (24F). Figure 24A: Area under the curve #1 represents cells incubated with goat anti-rabbit secondary antibody alone; area under the curve #2 represents cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by incubation with goat anti-rabbit secondary antibody; area under the curve #3 represents cells incubated with anti-HDM2 polyclonal antibody N-20 followed by incubation with goat anti-rabbit secondary antibody. Figure 24B: Area under the curve #1 represents cells incubated with goat anti-mouse secondary antibody alone; area under the curve #2 represents cells incubated with anti-HDM2 monoclonal antibody OP145 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under the curve #3 represents cells incubated with anti-HDM2 monoclonal antibody OP145 followed by goat anti-mouse secondary antibody.Figure 24C: Area under the curve #1 represents cells incubated with goat anti-rabbit secondary antibody alone; area under the curve #2 represents trypsin-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody; area under the curve #3 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under the curve #4 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody. Figure 24D&E: Area under the curve #1 represents cells incubated with goat anti-rabbit secondary antibody alone; area under the curve #2 represents cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under the curve #3 represents cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody. Figure 24F: Area under the curve #1 represents cells incubated with goat anti-rabbit secondary antibody alone; area under the curve #2 represents trypsin-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody; area under the curve #3 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under the curve #4 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody. [Figures 24C-24D]In Figures 24A-F, anti-HDM2-specific antibodies stain the surface of cancer cells but not normal cells. Intact cells released with either EDTA or trypsin were blocked with 5% human serum albumin. Next, the cells were incubated with either the polyclonal N-20 M(H)DM2-specific antibody (sc-813, N-20, rabbit IgG; Santa Cruz; "N-20") or the monoclonal M(H)DM2-specific OP145 antibody (OP145, mouse IgG1; Calbiochem; "OP145") on ice for 90 minutes. Another set of cells, prepared under the same conditions, was incubated with the same antibodies preincubated with their corresponding blocking peptides prior to incubation with the cells. After primary antibody incubation, the cells were washed three times with ice-cold PBS and then incubated with FITC-secondary antibodies for 60 minutes. The cells were then washed three times with PBS and subjected to FACS analysis. Human melanoma cells (24A, 24B, and 24C), primary cultured human ovarian cancer cells (24D and 24E), and normal mouse splenocytes (24F). Figure 24A: Area under the curve #1 represents cells incubated with goat anti-rabbit secondary antibody alone; area under the curve #2 represents cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by incubation with goat anti-rabbit secondary antibody; area under the curve #3 represents cells incubated with anti-HDM2 polyclonal antibody N-20 followed by incubation with goat anti-rabbit secondary antibody. Figure 24B: Area under the curve #1 represents cells incubated with goat anti-mouse secondary antibody alone; area under the curve #2 represents cells incubated with anti-HDM2 monoclonal antibody OP145 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under the curve #3 represents cells incubated with anti-HDM2 monoclonal antibody OP145 followed by goat anti-mouse secondary antibody.Figure 24C: Area under the curve #1 represents cells incubated with goat anti-rabbit secondary antibody alone; area under the curve #2 represents trypsin-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody; area under the curve #3 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under the curve #4 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody. Figure 24D&E: Area under the curve #1 represents cells incubated with goat anti-rabbit secondary antibody alone; area under the curve #2 represents cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under the curve #3 represents cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody. Figure 24F: Area under the curve #1 represents cells incubated with goat anti-rabbit secondary antibody alone; area under the curve #2 represents trypsin-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody; area under the curve #3 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under the curve #4 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody. [Figures 24E-24F]In Figures 24A-F, anti-HDM2-specific antibodies stain the surface of cancer cells but not normal cells. Intact cells released with either EDTA or trypsin were blocked with 5% human serum albumin. Next, the cells were incubated with either the polyclonal N-20 M(H)DM2-specific antibody (sc-813, N-20, rabbit IgG; Santa Cruz; "N-20") or the monoclonal M(H)DM2-specific OP145 antibody (OP145, mouse IgG1; Calbiochem; "OP145") on ice for 90 minutes. Another set of cells, prepared under the same conditions, was incubated with the same antibodies preincubated with their corresponding blocking peptides prior to incubation with the cells. After primary antibody incubation, the cells were washed three times with ice-cold PBS and then incubated with FITC-secondary antibodies for 60 minutes. The cells were then washed three times with PBS and subjected to FACS analysis. Human melanoma cells (24A, 24B, and 24C), primary cultured human ovarian cancer cells (24D and 24E), and normal mouse splenocytes (24F). Figure 24A: Area under the curve #1 represents cells incubated with goat anti-rabbit secondary antibody alone; area under the curve #2 represents cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by incubation with goat anti-rabbit secondary antibody; area under the curve #3 represents cells incubated with anti-HDM2 polyclonal antibody N-20 followed by incubation with goat anti-rabbit secondary antibody. Figure 24B: Area under the curve #1 represents cells incubated with goat anti-mouse secondary antibody alone; area under the curve #2 represents cells incubated with anti-HDM2 monoclonal antibody OP145 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under the curve #3 represents cells incubated with anti-HDM2 monoclonal antibody OP145 followed by goat anti-mouse secondary antibody.Figure 24C: Area under the curve #1 represents cells incubated with goat anti-rabbit secondary antibody alone; area under the curve #2 represents trypsin-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody; area under the curve #3 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under the curve #4 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody. Figure 24D&E: Area under the curve #1 represents cells incubated with goat anti-rabbit secondary antibody alone; area under the curve #2 represents cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under the curve #3 represents cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody. Figure 24F: Area under the curve #1 represents cells incubated with goat anti-rabbit secondary antibody alone; area under the curve #2 represents trypsin-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody; area under the curve #3 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under the curve #4 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody.

[0109] [Figure 25A]Figures 25A-C. (A) Human pancreatic or ovarian cancer cells and normal human fibroblasts were treated with normal human serum (NHS) alone, NHS plus anti-HDM2 OP145 monoclonal antibody (mouse IgG1, Calbiochem, "OP145"), or control antibody (NHS plus cytochrome C). When cancer cells were treated with OP145 antibody in the presence of NHS (see panels b and e), widespread cell death was observed as evidenced by propidium iodide (PI) staining, whereas the same antibody had no effect on the viability of normal human fibroblasts (see panel g). A control antibody against cytochrome C showed no cytotoxicity (see panel c) beyond that observed in untreated cells (see panel a). The lack of cell death is evident by little to no PI staining in panels a, c, d, f, and g. The cell death marker PI was visualized using an Olympus FluoView FV1000 confocal laser scanning biological microscope on an Olympus IX81 inverted microscope. (B) Rodent pancreatic cancer cells were treated with anti-HDM2 antibody and a control cytochrome C antibody. When cells were treated with anti-HDM2 antibodies, N-20 (polyclonal antibody, sc-813 N-20, rabbit IgG, Santa Cruz, "N-20"), or C-18 (polyclonal, sc-812 C-18, rabbit IgG; Santa Cruz; "C-18") (see panels b and e), extensive cell death was observed as evidenced by propidium iodide (PI) staining, whereas no cytotoxicity was observed when cells were treated with anti-HDM2 monoclonal OP46 antibody (OP46 (Ab-1); mouse IgG1; Calbiochem; "OP46") (see panel d) or a control cytochrome C antibody (see panel e). (C) M(H)DM2-specific antibodies are cytotoxic to pancreatic cancer MiaPaCa-2 cells in the presence of NHS. Quantitative representation of M(H)DM2-specific antibody-dependent complement cytotoxicity against human pancreatic cancer cells.Cells treated with anti-M(H)DM2(C-18) antibody in the presence of NHS exhibited cytotoxicity for 15–30 min after treatment, whereas anti-HDM2 OP46 did not exhibit a cytotoxic effect beyond that observed when cells were treated with a control anti-cytochrome C antibody or when cells were treated with NHS in the absence of anti-M(H)DM2 antibody. [Figure 25B]Figures 25A-C. (A) Human pancreatic or ovarian cancer cells and normal human fibroblasts were treated with normal human serum (NHS) alone, NHS plus anti-HDM2 OP145 monoclonal antibody (mouse IgG1, Calbiochem, "OP145"), or control antibody (NHS plus cytochrome C). When cancer cells were treated with OP145 antibody in the presence of NHS (see panels b and e), widespread cell death was observed as evidenced by propidium iodide (PI) staining, whereas the same antibody had no effect on the viability of normal human fibroblasts (see panel g). A control antibody against cytochrome C showed no cytotoxicity (see panel c) beyond that observed in untreated cells (see panel a). The lack of cell death is evident by little to no PI staining in panels a, c, d, f, and g. The cell death marker PI was visualized using an Olympus FluoView FV1000 confocal laser scanning biological microscope on an Olympus IX81 inverted microscope. (B) Rodent pancreatic cancer cells were treated with anti-HDM2 antibody and a control cytochrome C antibody. When cells were treated with anti-HDM2 antibodies, N-20 (polyclonal antibody, sc-813 N-20, rabbit IgG, Santa Cruz, "N-20"), or C-18 (polyclonal, sc-812 C-18, rabbit IgG; Santa Cruz; "C-18") (see panels b and e), extensive cell death was observed as evidenced by propidium iodide (PI) staining, whereas no cytotoxicity was observed when cells were treated with anti-HDM2 monoclonal OP46 antibody (OP46 (Ab-1); mouse IgG1; Calbiochem; "OP46") (see panel d) or a control cytochrome C antibody (see panel e). (C) M(H)DM2-specific antibodies are cytotoxic to pancreatic cancer MiaPaCa-2 cells in the presence of NHS. Quantitative representation of M(H)DM2-specific antibody-dependent complement cytotoxicity against human pancreatic cancer cells.Cells treated with anti-M(H)DM2(C-18) antibody in the presence of NHS exhibited cytotoxicity for 15–30 min after treatment, whereas anti-HDM2 OP46 did not exhibit a cytotoxic effect beyond that observed when cells were treated with a control anti-cytochrome C antibody or when cells were treated with NHS in the absence of anti-M(H)DM2 antibody. [Figure 25C]Figures 25A-C. (A) Human pancreatic or ovarian cancer cells and normal human fibroblasts were treated with normal human serum (NHS) alone, NHS plus anti-HDM2 OP145 monoclonal antibody (mouse IgG1, Calbiochem, "OP145"), or control antibody (NHS plus cytochrome C). When cancer cells were treated with OP145 antibody in the presence of NHS (see panels b and e), widespread cell death was observed as evidenced by propidium iodide (PI) staining, whereas the same antibody had no effect on the viability of normal human fibroblasts (see panel g). A control antibody against cytochrome C showed no cytotoxicity (see panel c) beyond that observed in untreated cells (see panel a). The lack of cell death is evident by little to no PI staining in panels a, c, d, f, and g. The cell death marker PI was visualized using an Olympus FluoView FV1000 confocal laser scanning biological microscope on an Olympus IX81 inverted microscope. (B) Rodent pancreatic cancer cells were treated with anti-HDM2 antibody and a control cytochrome C antibody. When cells were treated with anti-HDM2 antibodies, N-20 (polyclonal antibody, sc-813 N-20, rabbit IgG, Santa Cruz, "N-20"), or C-18 (polyclonal, sc-812 C-18, rabbit IgG; Santa Cruz; "C-18") (see panels b and e), extensive cell death was observed as evidenced by propidium iodide (PI) staining, whereas no cytotoxicity was observed when cells were treated with anti-HDM2 monoclonal OP46 antibody (OP46 (Ab-1); mouse IgG1; Calbiochem; "OP46") (see panel d) or a control cytochrome C antibody (see panel e). (C) M(H)DM2-specific antibodies are cytotoxic to pancreatic cancer MiaPaCa-2 cells in the presence of NHS. Quantitative representation of M(H)DM2-specific antibody-dependent complement cytotoxicity against human pancreatic cancer cells.Cells treated with anti-M(H)DM2(C-18) antibody in the presence of NHS exhibited cytotoxicity for 15–30 min after treatment, whereas anti-HDM2 OP46 did not exhibit a cytotoxic effect beyond that observed when cells were treated with a control anti-cytochrome C antibody or when cells were treated with NHS in the absence of anti-M(H)DM2 antibody.

[0110] [Figure 26] Figure 26 shows tumor size in mice treated with anti-HDM2 antibody OP145 and PBS control (in the Panc02 syngeneic mouse pancreatic cancer model). The x-axis indicates the number of days after tumor cell injection into the mice. The y-axis indicates tumor volume in mm. The arrow indicates the day treatment began.

[0111] [Figure 27] Figure 27 shows the effects of monoclonal antibody NMC-103 alone (10 mg / kg), the combination of low-dose gemcitabine (25 mg / kg) and nab-paclitaxel (5 mg / kg), the combination of low-dose gemcitabine (25 mg / kg), nab-paclitaxel (5 mg / kg), and NMC-103 (10 mg / kg), and isotype control mouse IgG1 (10 mg / kg) on ​​tumor volume in the Panc-2 syngeneic mouse pancreatic cancer model (number of mice / group = 8). Treatment in this study began when the tumors in the mice reached approximately 80-100 mm3.

[0112] [Figure 28] FIG. 28 depicts a Kaplan / Meier survival analysis demonstrating a survival benefit in mice administered NMC-103 alone or in combination with chemotherapy compared to chemotherapy alone or a control antibody in the experimental conditions described in FIG. 27.

[0113] [Figure 29]Figure 29 shows that mice previously treated with NMC-103, as described in Figure 27, are immune to tumor rechallenge after drug discontinuation. To assess the long-term antitumor effects of NMC-103, mice previously treated with the G+nP combination, group C mice (mice previously treated with NMC-103), and group D mice (mice previously treated with the NMC-103+G+nP combination) were rechallenged with a second inoculation of Panc-2 (2 x 10 cells / mouse subcutaneously) in the left dorsal flank 62 days after the start of the study. Tumor growth was monitored for 10 days, at which time 90 mm tumors were measured in group B mice. No tumors were observed in the two groups (groups C and D) previously treated with the NMC-103 antibody.

[0114] [Figure 30] Figure 30 shows the effect of adding a single dose of monoclonal antibody NMC-103 (10 mg / kg) or isotype control mouse IgG1 (10 mg / kg) to a treatment regimen combining low-dose gemcitabine (25 mg / kg) and nab-paclitaxel (5 mg / kg) on ​​tumor volume in the Panc-2 syngeneic mouse pancreatic cancer model for the treatment of large tumors (i.e., advanced cancer). Mice were treated with standard pancreatic cancer therapy (gemcitabine (25 mg / kg) + nab-paclitaxel (5 mg / kg)) for 19 days, at which time tumor size reached approximately 450 mm3. Mice were then randomized into two groups and administered a single dose of isotype control mouse IgG1 (10 mg / kg) or NMC-103 (10 mg / kg). As shown in this figure, a single intraperitoneal injection of NMC-103 nearly halved tumor size (from 438 mm 3 to 233 mm 3 ) 6 days after treatment.

[0115] [Figure 31]Figure 31 shows the effect of monoclonal antibody NMC-103 on tumor volume in the MC-38 syngeneic mouse colon cancer model. As shown in this figure, mice treated with 10 mg / kg of NMC-103 twice a week for 2 weeks reached an average tumor size of 210 mm, while mice treated with 10 mg / kg of isotype control antibody experienced rapid growth, reaching 1168 mm by day 12. Compared with mice treated with 0.4 mg / kg of NMC-103 (Figure 17), these data support the dose-dependent antitumor effect of the NMC-103 antibody.

[0116] [Figure 32A] Figures 32A and 32B show the effects of the chimeric form of monoclonal antibody NMC-303. Mouse NMC-303 was isotype-class switched to convert mouse IgM to chimeric IgG1. The mouse heavy and light chain variable regions were cloned with human Ig gamma-1 and kappa chains as constant regions. A total of eight BALB / c mice were subcutaneously injected with CT-26. The mice were then divided into two groups (n=4) and administered A) a control antibody (10 mg / kg) or B) the chimeric form of NMC-303 antibody (10 mg / kg) twice weekly for three weeks. Figure 32A shows that by 24 days after tumor inoculation, mice treated with the chimeric form of NMC-303 (10 mg / kg) had reached an average tumor size of 726 mm3, while mice treated with the control antibody (10 mg / kg) had an average tumor size of 1746 mm3. Additionally, Figure 32B shows individual mouse tumor sizes at 24 days post tumor inoculation. [Figure 32B]Figures 32A and 32B show the effects of the chimeric form of monoclonal antibody NMC-303. Mouse NMC-303 was isotype-class switched to convert mouse IgM to chimeric IgG1. The mouse heavy and light chain variable regions were cloned with human Ig gamma-1 and kappa chains as constant regions. A total of eight BALB / c mice were subcutaneously injected with CT-26. The mice were then divided into two groups (n=4) and administered A) a control antibody (10 mg / kg) or B) the chimeric form of NMC-303 antibody (10 mg / kg) twice weekly for three weeks. Figure 32A shows that by 24 days after tumor inoculation, mice treated with the chimeric form of NMC-303 (10 mg / kg) had reached an average tumor size of 726 mm3, while mice treated with the control antibody (10 mg / kg) had an average tumor size of 1746 mm3. Additionally, Figure 32B shows individual mouse tumor sizes at 24 days post tumor inoculation. DETAILED DESCRIPTION OF THE INVENTION

[0117] 7. Detailed Description Provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2). In certain embodiments, the extracellularly accessible epitope is contained within SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. The antibodies provided herein are described in Section 5.1, below. Also provided herein are antibody-drug conjugates comprising an antibody or fragment that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2) described herein, linked (e.g., covalently) to a cytotoxic drug. Also provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2), where the antibody or antigen-binding fragment is not linked to a cytotoxic moiety.

[0118] Also provided herein are pharmaceutical compositions comprising an antibody or fragment that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2) described herein. In certain embodiments, such pharmaceutical compositions comprise a therapeutically effective amount of such an antibody or fragment (i.e., an amount that can be used to treat cancer in a subject, e.g., by achieving one or more anti-tumor effects described herein).

[0119] Also provided herein are nucleic acids encoding the antibodies and antigen-binding fragments described herein. In certain embodiments, provided herein are vectors and cells comprising nucleic acids encoding such antibodies or antigen-binding fragments thereof. Also provided are cells that recombinantly produce the antibodies or antigen-binding fragments described herein.

[0120] Chimeric antigen receptors (CARs) are engineered receptors that provide both antigen-binding and immune cell activation functions (Sadelain et al., 2013, Cancer Discovery 3:388-398). A linker is used to bind intracellular T cell activation domains, such as CD3 zeta domains. Also provided herein is a CAR comprising a single-chain variable fragment (scFv) that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4, e.g., an scFv comprising the VH and VL of an anti-M(H)DM2 / 4 antibody described herein, fused to a transmembrane domain (e.g., of CD3 zeta), optionally further fused to a costimulatory domain (e.g., the CD28 intracellular domain). T cells expressing such a CAR are also provided.

[0121] Also provided herein are peptides having the amino acid sequence MCNTNMSVPTDGAVT (SEQ ID NO: 1), TTSQIPASEQE (SEQ ID NO: 2), or CPVCRQPIQMIVLTYFP (SEQ ID NO: 3). The peptides can be, for example, synthetic or recombinant. In some embodiments, the peptides are purified. In some embodiments, the peptides are labeled with a detectable marker (e.g., a fluorescent marker or an isotope). In some embodiments, the peptides are tagged (e.g., with a GST, His, Strep, myc, FLAG, or HA tag). In some embodiments, a cysteine ​​is added to one of the ends of the peptide (which may allow for linkage to a carrier protein). In some embodiments, the peptides are linked to a carrier protein (e.g., linked to keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, thyroglobulin, tetanus toxoid, or diphtheria toxin). Nucleic acids encoding the peptides described herein are also provided herein. Also provided herein are vectors and cells containing nucleic acids encoding the peptides described herein. Also provided are cells that recombinantly produce the peptides described herein. Also provided herein is the use of the peptides described herein as immunogens. The peptides of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 described herein contain extracellularly accessible epitopes of MDM2 and HDM2. Specifically, provided herein are methods for producing anti-M(H)DM2 / 4 antibodies (antibodies that specifically bind to M(H)DM2 / 4) by immunizing animals (e.g., mice or rabbits) with the peptides described herein. Also provided herein are methods for screening antibodies for binding to one or more of the peptides described herein (e.g., using ELISA with plate-bound peptides).Also provided herein is a method for identifying an anti-M(H)DM2 / 4 antibody that is suitable for therapeutic use in treating or preventing metastasis of cancer, or suitable for use in diagnosing cancer, by contacting the antibody with a peptide described herein under conditions suitable for binding between the antibody and the peptide and detecting or measuring binding that occurs between the antibody and the peptide, wherein detection of binding between the antibody and the peptide indicates that the antibody is suitable for therapeutic or diagnostic use. For example, provided herein is a method for identifying an anti-M(H)DM2 / 4 antibody that is suitable for therapeutic use by contacting the anti-M(H)DM2 / 4 antibody with a peptide described herein under conditions suitable for binding between the antibody and the peptide and detecting or measuring binding that occurs between the antibody and the peptide, wherein if binding between the antibody and the peptide is detected, the antibody is used in a method for treating cancer described herein. In another example, provided herein is a method for identifying an anti-M(H)DM2 / 4 antibody suitable for diagnostic use by contacting an anti-M(H)DM2 / 4 antibody with a peptide described herein under conditions suitable for binding between the antibody and the peptide, and detecting or measuring binding that occurs between the antibody and the peptide, wherein if binding between the antibody and the peptide is detected, the antibody is used in a method for diagnosing cancer described herein.

[0122] Also provided herein are methods for treating cancer, inhibiting tumor growth or proliferation, inhibiting tumor progression, and / or preventing metastasis in a subject by administering to the subject an anti-M(H)DM2 / 4 antibody or fragment described herein, particularly an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (particularly a region exposed on the plasma membrane surface of cancer cells). Preferably, the antibody or fragment thereof specifically binds to an extracellularly accessible epitope of HDM2 (particularly a region exposed on the plasma membrane surface of cancer cells). In certain embodiments, the antibody or fragment (e.g., for use in the methods described herein) does not bind, or only minimally binds, to the plasma membrane surface of normal cells of the tissue type from which the cancer in the subject originates.

[0123] In certain embodiments, when the subject to be treated is a human, the antibody or fragment thereof used herein specifically binds to the extracellularly accessible epitope (region exposed on the plasma membrane surface of cancer cells) of HDM2 and / or HDM4.In one embodiment of treating a human, the antibody or fragment thereof used herein specifically binds to the extracellularly accessible epitope (region exposed on the plasma membrane surface of cancer cells) of HDM2 (optionally, an antibody or fragment that does not bind to HDM4).In one embodiment of treating a human, the antibody or fragment thereof used herein specifically binds to the extracellularly accessible epitope (region exposed on the plasma membrane surface of cancer cells) of HDM4 (optionally, an antibody or fragment that does not bind to HDM2).

[0124] When the subject is a non-human animal (e.g., a mammal such as a dog or cat), the antibody or fragment thereof used herein binds to an extracellularly accessible epitope of M(H)DM2 / 4 (a region exposed on the plasma membrane surface of cancer cells), where M(H)DM2 / 4 is a homolog of HDM2 and / or HDM4 expressed in such an animal. In one embodiment, the antibody or fragment thereof used herein binds to an extracellularly accessible epitope of M(H)DM2 (a region exposed on the plasma membrane surface of cancer cells), where M(H)DM2 is a homolog of HDM2 expressed in such an animal (optionally, such an antibody or fragment does not bind to M(H)DM4). In one embodiment, the antibody or fragment thereof used herein binds to an extracellularly accessible portion of M(H)DM4 (a region exposed on the plasma membrane surface of cancer cells), which is a homolog of HDM4 expressed in such animals (optionally, such an antibody or fragment does not bind to M(H)DM2).

[0125] It should be understood that the following description of the invention is primarily directed to HDM2, and antibodies and antibody fragments thereto, for use in treating humans; however, it will be apparent to those skilled in the art that these descriptions should also be considered applicable to (i) HDM4, and antibodies and antibody fragments thereto, and their use for treating humans (unless expressly indicated otherwise or indicated by context), and (ii) M(H)DM2 / 4, and antibodies and antibody fragments thereto, and their use for treating non-human animals, e.g., mammals (unless expressly indicated otherwise or indicated by context). In preferred embodiments, the patients and subjects treated using the methods described herein are human.

[0126] In preferred embodiments, the anti-HDM2 antibodies or fragments thereof used in accordance with the methods described herein mediate complement-dependent cytotoxicity (CDC), mediate antibody-dependent cell-mediated cytotoxicity (ADCC), and / or are conjugated to a cytotoxic drug(s) (e.g., are antibody-drug conjugates). In preferred embodiments, the invention provides for the use of antibodies that mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).

[0127] In certain embodiments, the anti-HDM2 antibody or fragment thereof used in accordance with the methods described herein is not conjugated to a cell-penetrating peptide. Cell-penetrating peptides can insert into the plasma membrane of a cell and transport molecules bound to them into the cell. Such cell-penetrating peptides include, but are not limited to, membrane-resident peptides (MRPs), the membrane transduction domain of antennapedia, transactivating transcription activator (TAT), and penetratin peptides. In certain embodiments, the anti-HDM2 antibody or fragment thereof used in accordance with the methods described herein is not conjugated to a membrane-resident peptide (MRPs), the membrane transduction domain of antennapedia, TAT, and / or penetratin peptide. In certain embodiments, the anti-HDM2 antibody or fragment thereof used in accordance with the methods described herein is not conjugated to any peptide sequence that can insert into the lipid bilayer of the plasma membrane of a cell. In one embodiment, the anti-HDM2 antibody or fragment thereof used in accordance with the methods described herein is not conjugated to an MRP. In one embodiment, the anti-HDM2 antibody or fragment thereof used in accordance with the methods described herein is not conjugated to a penetratin peptide.

[0128] The examples described herein demonstrate that antibodies targeting HDM-2 alone are selectively cytotoxic to cancer cells. Furthermore, as described in the examples herein, it has been demonstrated that the extracellularly accessible epitope of HDM2 is a suitable therapeutic target for anti-HDM2 antibodies, and that cancer cells expressing HDM2 on their surface can be successfully targeted and destroyed by antibodies directed against such extracellular domains of HDM2. Specifically, the data presented in the examples demonstrate that selective HDM2-specific antibodies can bind to the extracellularly accessible sequence of HDM2 on the surface membrane of intact cancer cells, but exhibit negligible binding to the surface membrane of normal human blood mononuclear cells. In addition, the data presented in the examples demonstrate that such HDM2-specific antibodies can inhibit the growth of cancer cells in vitro and in vivo, strongly suggesting that they can be used as therapeutic agents in vivo. Furthermore, data presented in the Examples demonstrate that such HDM2-specific antibodies can have synergistic anti-tumor effects when combined with chemotherapeutic agents.

[0129] 7.1 Antibodies Provided herein are antibodies or antigen-binding fragments thereof that (immuno)specifically bind to an extracellularly accessible epitope (a region exposed on the plasma membrane surface of a cell) of M(H)DM2 / 4 (e.g., HDM2). As used herein, the term "specifically binds" does not exclude cross-reactivity of the antibody or antigen-binding fragment; thus, for example, an antibody or antigen-binding fragment thereof that (immuno)specifically binds to an extracellularly accessible epitope of HDM2 that is exposed on the plasma membrane surface of a cell may also specifically bind to (cross-react with) MDM2. Specifically, provided herein are anti-M(H)DM2 / 4 antibodies and fragments thereof that (immuno)specifically bind to an extracellularly accessible epitope of M(H)DM2 / 4 and have anti-tumor activity (e.g., inhibit tumor growth in vivo). In certain embodiments, the antibody or antigen-binding fragment thereof specifically binds to an epitope of M(H)DM2 / 4 that is extracellularly accessible on cancer cells but not on non-cancerous cells (e.g., non-cancerous cells of the same organ or tissue type as the cancer cells). In other specific embodiments, the antibody or antigen-binding fragment thereof specifically binds to an epitope of M(H)DM2 / 4, and the exposure or accessibility of this epitope on the plasma membrane surface of cancer cells is increased compared to its exposure or accessibility on the plasma membrane surface of non-cancerous cells (e.g., non-cancerous cells of the host organ or tissue). Also provided herein are antibodies or antigen-binding fragments thereof that (immuno)specifically bind to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2), which is not linked to a cell-penetrating peptide (e.g., a membrane-resident peptide).

[0130] Also provided herein is an antibody or antigen-binding fragment thereof that (immuno)specifically binds to M(H)DM2 / 4 (e.g., HDM2), specifically to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or fragment specifically binds to a peptide consisting of the sequence MCNTNMSVPTDGAVT (SEQ ID NO: 1), TTSQIPASEQE (SEQ ID NO: 2), or CPVCRQPIQMIVLTYFP (SEQ ID NO: 3). In certain embodiments, also provided herein is an antibody or antigen-binding fragment thereof that (immuno)specifically binds to M(H)DM2 / 4 (e.g., HDM2), specifically to an extracellularly accessible epitope of M(H)DM2 / 4, wherein the antibody or antigen-binding fragment specifically binds to a peptide, the peptide consisting of the sequence MCNTNMSVPTDGAVT (SEQ ID NO: 1), TTSQIPASEQE (SEQ ID NO: 2), or CPVCRQPIQMIVLTYFP (SEQ ID NO: 3), wherein such antibody or fragment has an anti-tumor effect in vivo, and / or wherein such antibody or fragment is not conjugated to a cell-penetrating peptide.

[0131] Also provided herein are anti-M(H)DM2 / 4 antibodies and fragments having the heavy and / or light chain variable regions described herein (e.g., having the sequences of the heavy and / or light chain variable regions of antibodies NMC-103, NMC-204, and NMC-303 provided herein; see, e.g., Section 8 and Figures 20-22). Also provided herein are anti-M(H)DM2 / 4 antibodies and fragments having one or more complementarity-determining regions (CDRs) described herein (see, e.g., the CDRs provided in Tables 4-9 and Figures 20-22).

[0132] CDRs are defined in various ways in the art, including Kabat, Chothia, AbM, Contact, and IMGT. In certain embodiments, antibody CDRs can be defined according to the Kabat system, which is based on sequence variability (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391; Kabat See EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242. In certain embodiments, for the Kabat system: (i) VH CDR1 is located at amino acid positions 31-35 of the heavy chain, (ii) VH CDR2 is located at amino acid positions 50-68 or 50-66 of the heavy chain, and (iii) VH CDR3 is located at amino acid positions 101-105, 99-104, or 99-106 of the heavy chain. In a specific embodiment, with respect to the Kabat system, (i) VL CDR1 is located at amino acid positions 24-39 or 24-34 of the light chain, (ii) VH CDR2 is located at amino acid positions 55-61 or 50-56 of the light chain, and (iii) VH CDR3 is located at amino acid positions 94-102 or 89-97 of the light chain. As is well known to those skilled in the art, with respect to the Kabat system, the actual linear amino acid sequence of an antibody variable domain may contain fewer or additional amino acids due to shortening or elongation of framework regions (FRs) and / or CDRs, and therefore the Kabat numbering of an amino acid is not necessarily the same as its linear amino acid numbering. Kabat CDR positions may vary depending on the antibody and can be determined according to methods known in the art. In certain embodiments, the CDRs of the antibodies described herein are determined using the Kabat system.

[0133] In certain embodiments, the CDRs of an antibody can be defined according to the Chothia system, which is based on the locations of the structural loop regions of an immunoglobulin (e.g., Chothia (See C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226.) The term "R" and similar terms are art-recognized and refer to the C of an antibody as determined according to the method of Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917. (ii) VH CDR2 is located at amino acid positions 52-59 or 52-57 of the heavy chain, and (iii) VH CDR3 is located at amino acid positions 101-105, or 99-104, or 99-106 of the heavy chain. In certain embodiments, for the Chothia system, using the Kabat numbering system for numbering amino acid residues in the VL region, (i) VL CDR1 is located at amino acids 24-39 or 24-34 of the light chain, (ii) VL CDR2 is located at amino acids 55-61 or 50-56 of the light chain, and (iii) VL CDR3 is located at amino acids 94-102 or 89-97 of the light chain. Chothia CDR positions can vary depending on the antibody and can be determined according to methods known in the art. In certain embodiments, the CDRs of the antibodies described herein are determined using the Chothia system.

[0134] In certain aspects, antibody CDRs can be defined according to the AbM system, which is based on AbM hypervariable regions that represent a compromise between Kabat CDRs and Chothia structural loops, and the CDRs are determined using Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In certain embodiments, for the AbM system, using the Kabat numbering system for numbering amino acid residues in the VH region, (i) VH CDR1 is located at amino acids 26-35 of the heavy chain, (ii) VH CDR2 is located at amino acids 50-61 or 50-59 of the heavy chain, and (iii) VH CDR3 is located at amino acids 101-105, 99-104, or 99-106 of the heavy chain. In certain embodiments, for the AbM system, using the Kabat numbering system for numbering amino acid residues in the VL region, (i) VL CDR1 is located at amino acids 24-39 or 24-34 of the light chain, (ii) VH CDR2 is located at amino acids 55-61 or 50-56 of the light chain, and (iii) VH CDR3 is located at amino acids 94-102 or 89-97 of the light chain. The AbM CDR positions may vary depending on the antibody and can be determined according to methods known in the art. In certain embodiments, the CDRs of the antibodies described herein are determined using the AbM numbering system.

[0135] In certain embodiments, the CDRs of an antibody can be defined according to the IMGT system (see IMGT®, the international ImMunoGeneTics information system® website imgt.org, founder and director: Marie-Paule Lefranc, Montpellier, France; see, e.g., Lefranc, M.-P., 1999, The Immunologist, 7:132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212). (See, for example, the following references, all of which are incorporated herein by reference in their entireties.) In certain embodiments, with respect to the IMGT system, (i) the VH CDR1 is located at amino acid positions 27-33 or 26-33 of the heavy chain, (ii) the VH CDR2 is located at amino acid positions 51-60 or 51-58 of the heavy chain, and (iii) the VH CDR3 is located at amino acid positions 99-105 or 97-103 of the heavy chain. In certain embodiments, with respect to the IMGT system, (i) the VL CDR1 is located at amino acid positions 27-37 of the light chain, (ii) the VH CDR2 is located at amino acid positions 55-57 of the light chain, and (iii) the VH CDR3 is located at amino acid positions 94-102 of the light chain. The CDR positions for IMGT can vary depending on the antibody and can be determined according to methods known in the art. In certain embodiments, the CDRs of the antibodies described herein are determined using the IMGT system.

[0136] In certain embodiments, antibody CDRs can be defined according to the Contact system. Contact definitions are based on analysis of available complex crystal structures (bioinf.org.uk / abs) (see MacCallum RM et al., (1996) J Mol Biol 5: 732-745). For more information, see, for example, Martin A. "Protein Sequence and Structure Analysis of (See also "Antibody Variable Domains," in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001)). In certain embodiments, for the Contact system, using the Kabat numbering system for numbering amino acid residues in the VH region, (i) VH CDR1 is located at amino acid positions 30-35 of the heavy chain, (ii) VH CDR2 is located at amino acid positions 47-61 or 47-59 of the heavy chain, and (iii) VH CDR3 is located at amino acid positions 99-104, 97-103, or 97-105 of the heavy chain. In certain embodiments, for the Contact system, using the Kabat numbering system for numbering amino acid residues in the VL region, (i) VL CDR1 is located at amino acid positions 30-41 or 30-36 of the light chain, and (ii) VH CDR3 is located at amino acid positions 99-104, 97-103, or 97-105 of the heavy chain. (iii) CDR2 is located at amino acids 51-60 or 46-55 of the light chain, and (iv) VH CDR3 is located at amino acids 94-101 or 89-96 of the light chain. Contact CDR positions may vary depending on the antibody and can be determined according to methods known in the art. In certain embodiments, the CDRs of the antibodies described herein are determined using the Contact system.

[0137] In specific embodiments, provided herein is an antibody or fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., HDM2) and comprises the CDRs of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303), wherein the CDRs are defined according to any of the above-described systems.

[0138] In certain embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2) and comprises a heavy chain variable region (VH) having one, two, or all three VH CDRs (preferably all three VH CDRs) of any anti-HDM2 antibody described herein (e.g., NMC-103, NMC-204, or NMC-303). As known in the art, a VH contains VH CDRs surrounded by framework regions (the CDR and FR sequences appear in the following order in a VH: FR1-VH CDR1-FR2-VH CDR2-FR3-VH CDR3-FR4), and optionally, the framework regions are human. In certain embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2) and comprises a heavy chain variable region (VH) having one, two, or all three VH CDRs of a VH having the amino acid sequence of SEQ ID NO: 36 (which is the VH of NMC-103). In certain embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2) and comprises a heavy chain variable region (VH) having one, two, or all three VH CDRs of a VH having the amino acid sequence of SEQ ID NO: 38 (which is the VH of NMC-204). In certain embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2) and comprises a heavy chain variable region (VH) having one, two, or all three VH CDRs of a VH having the amino acid sequence of SEQ ID NO: 40 (which is the VH of NMC-303). In certain embodiments, such an antibody or fragment is a humanized antibody or fragment.

[0139] In certain embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2) and comprises a light chain variable region (VL) having one, two, or all three VL CDRs (preferably all three VL CDRs) of any anti-HDM2 antibody described herein (e.g., NMC-103, NMC-204, or NMC-303). As known in the art, the VL contains the VL CDR surrounded by framework regions (the CDR and FR sequences appear in the following order in the VL: FR1-VL CDR1-FR2-VL CDR2-FR3-VL CDR3-FR4), and optionally the framework regions are human. In certain embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2) and comprises a light chain variable region (VL) having one, two, or all three VL CDRs of a VL having the amino acid sequence of SEQ ID NO: 37 (which is the VL of NMC-103). In certain embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2) and comprises a light chain variable region (VL) having one, two, or all three VL CDRs of a VL having the amino acid sequence of SEQ ID NO: 39 (which is the VL of NMC-204). In certain embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2) and comprises a light chain variable region (VL) having one, two, or all three VL CDRs of a VL having the amino acid sequence of SEQ ID NO: 40 (which is the VL of NMC-303). In certain embodiments, such an antibody or fragment is a humanized antibody or fragment.

[0140] In certain embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 (e.g., HDM2) and comprises a heavy chain variable region (VH) having one, two, or all three VH CDRs (preferably, all three VH CDRs) of any anti-HDM2 antibody described herein (e.g., NMC-103, NMC-204, or NMC-303), and a light chain variable region (VL) having one, two, or all three VL CDRs (preferably, all three VL CDRs) of such an anti-HDM2 antibody.

[0141] In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, or all three VH CDRs identified in Table 4 (which provides the VH CDRs of NMC-103). In one embodiment, provided herein is an antibody or fragment thereof that specifically binds to HDM2 and comprises a VH CDR3 ("CDR-H3") identified in Table 4. In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two, or all three VL CDRs identified in Table 5 (which provides the VL CDRs of NMC-103). In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, or all three VH CDRs identified in Table 4, and a light chain variable region (VL) having one, two, or all three VL CDRs identified in Table 5.

[0142] In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, or all three VH CDRs identified in Table 6 (which provides the VH CDRs of NMC-204). In one embodiment, provided herein is an antibody or fragment thereof that specifically binds to HDM2 and comprises a VH CDR3 ("CDR-H3") identified in Table 6. In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two, or all three VL CDRs identified in Table 7 (which provides the VL CDRs of NMC-204). In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, or all three VH CDRs identified in Table 6, and a light chain variable region (VL) having one, two, or all three VL CDRs identified in Table 7.

[0143] In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, or all three VH CDRs identified in Table 8 (which provides the VH CDRs of NMC-303). In one embodiment, provided herein is an antibody or fragment thereof that specifically binds to HDM2 and comprises a VH CDR3 ("CDR-H3") identified in Table 8. In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two, or all three VL CDRs identified in Table 9 (which provides the VL CDRs of NMC-303). In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, or all three VH CDRs identified in Table 8, and a light chain variable region (VL) having one, two, or all three VL CDRs identified in Table 9.

[0144] In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2, comprising the VH of any antibody described herein, e.g., the VH of any antibody provided in Section 8 or Figures 20-22 (e.g., the VH of NMC-103, the VH of NMC-204, or the VH of NMC-303), or a VH with at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto. In certain embodiments, provided herein are antibodies or fragments thereof that specifically bind to HDM2, comprising the VL of any antibody described herein, e.g., the VL of any antibody provided in Section 8 or Figures 20-22 (e.g., the VL of NMC-103, the VL of NMC-204, or the VL of NMC-303), or a VL having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., the FRs).

[0145] In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2, comprising the VH and VL of any antibody described herein, e.g., the VH and VL of any antibody provided in Section 8 or Figures 20-22 (e.g., the VH and VL of NMC-103, the VH and VL of NMC-204, or the VH and VL of NMC-303), or a VH and VL having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto.

[0146] In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2, wherein the antibody or fragment thereof comprises: (i) a VH having the amino acid sequence of SEQ ID NO: 36, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto; and / or (ii) a VL having the amino acid sequence of SEQ ID NO: 37, or a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto.

[0147] In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2, wherein the antibody or fragment thereof comprises: (i) a VH having the amino acid sequence of SEQ ID NO: 38, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto; and / or (ii) a VL having the amino acid sequence of SEQ ID NO: 39, or a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto.

[0148] In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2, wherein the antibody or fragment thereof comprises: (i) a VH having the amino acid sequence of SEQ ID NO: 40, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto; and / or (ii) a VL having the amino acid sequence of SEQ ID NO: 41, or a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto.

[0149] In certain aspects, provided herein is an antibody or fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., HDM2) and comprises one or more Kabat VL CDRs of the VL of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303) and / or one or more Kabat VH CDRs of the VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided herein is an antibody or fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., HDM2) and comprises Kabat VH CDR3 of the VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided herein is an antibody or fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., HDM2) and comprises the three Kabat VL CDRs of the VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and / or the three Kabat VH CDRs of the VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).

[0150] In certain aspects, provided herein is an antibody or fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., HDM2) and comprises one or more VL CDRs of Chothia in the VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and / or one or more VH CDRs of Chothia in the VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided herein is an antibody or fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., HDM2) and comprises VH CDR3 of Chothia in the VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided herein is an antibody or fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., HDM2) and comprises the three Chothia VL CDRs of the VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and / or the three Chothia VH CDRs of the VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).

[0151] In certain aspects, provided herein is an antibody or fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., HDM2) and comprises the VL CDR of one or more AbM of the VL of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303) and / or the VH CDR of one or more AbM of the VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided herein is an antibody or fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., HDM2) and comprises the VH CDR 3 of the AbM of the VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided herein is an antibody or fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., HDM2) and comprises the VL CDRs of the three AbMs of the VL of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303) and / or the VH CDRs of the three AbMs of the VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).

[0152] In certain aspects, provided herein is an antibody or fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., HDM2) and comprises one or more VL CDRs of Contact in the VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and / or one or more VH CDRs of Contact in the VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided herein is an antibody or fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., HDM2) and comprises VH CDR 3 of Contact in the VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided herein is an antibody or fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., HDM2) and comprises the VL CDRs of three Contacts in the VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and / or the VH CDRs of three Contacts in the VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).

[0153] In certain embodiments, the VL of one or more IMGTs specifically binds to M(H)DM2 / 4 (e.g., HDM2) and is a VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303). Provided herein is an antibody or fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., HDM2) and that comprises VH CDR 3 of IMGT of the VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided herein is an antibody or fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., HDM2) and comprises the VL CDRs of the three IMGTs in the VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and / or the VH CDRs of the three IMGTs in the VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).

[0154] In certain embodiments, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2 / 4 (e.g., HDM2) and comprise a combination of Kabat CDRs, Chothia CDRs, AbM CDRs, IMGT CDRs, and Contact CDRs (or a combination of CDRs defined by any two, three, four, or five of these CDR definition systems).

[0155] In certain embodiments, the position of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of an antibody described herein may be varied by one, two, three, four, five, or six amino acid positions, as long as immunospecific binding to M(H)DM2 / 4 (e.g., HDM2) is maintained (e.g., substantially, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% maintained). For example, in one embodiment, the positions defining the CDRs of any of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303) may be varied by shifting the N-terminal and / or C-terminal borders of the CDR by one, two, three, four, five, or six amino acids, as long as immunospecific binding to M(H)DM2 / 4 (e.g., HDM2) is maintained (e.g., substantially, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In another embodiment, the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of an antibody described herein may be varied (e.g., shortened or lengthened) by one, two, three, four, five, or more amino acids, so long as immunospecific binding to M(H)DM2 / 4 (e.g., HDM2) is maintained (e.g., substantially, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% maintained).

[0156] In certain embodiments, the anti-M(H)DM2 / 4 (e.g., HDM2) antibodies described herein comprise a VH antibody comprising each of the three VH domains of any of the antibodies described herein (any one of the murine antibodies NMC-103, NMC-204, and NMC-303). VH CDR1, VH CDR2, VH CDR3, and VH CDR4. Also provided by the present invention are humanized immunoglobulins (e.g., IgG) comprising human or human-derived constant regions (VL CDR1, VL CDR2, and VL CDR3), human or human-derived framework regions, and human or human-derived constant regions, and antigen-binding fragments of such humanized antibodies. Non-limiting examples of human framework regions are described in the art, e.g., Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of (See National Institute of Health and Human Services, NIH Publication No. 91-3242) In certain embodiments, a humanized anti-M(H)DM2 / 4 (e.g., HDM2) antibody or antigen-binding fragment thereof comprises a VH in which the VH CDR1, VH CDR2, and VH CDR3 described herein (e.g., those of MNC-103, NMC-204, or NMC-303) are surrounded by VH framework regions that are or are derived from human framework regions. In certain embodiments, an anti-M(H)DM2 / 4 (e.g., HDM2) antibody or antigen-binding fragment thereof comprises a VL in which the VL CDR1, VL CDR2, and VL CDR3 described herein (e.g., those of MNC-103, NMC-204, or NMC-303) are surrounded by VL framework regions that are or are derived from human framework regions. In certain embodiments, an anti-M(H)DM2 / 4 (e.g., HDM2) antibody or antigen-binding fragment thereof comprises (i) a VH in which the VH CDR1, VH CDR2, and VH CDR3 described herein (e.g., those of MNC-103, NMC-204, or NMC-303) are surrounded by VH framework regions that are or are derived from human framework regions, and (ii) a VL in which the VL CDR1, VL CDR2, and VL CDR3 described herein (e.g., those of MNC-103, NMC-204, or NMC-303) are surrounded by VL framework regions that are or are derived from human framework regions.

[0157] Human framework regions that can be used include: (i) those derived from the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); (ii) framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992), and and Presta et al. J. Immunol., 151:2623 (1993)), (iii) human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)), and (iv) framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)). For a list of human framework regions, see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998).

[0158] In certain embodiments, routine recombinant DNA techniques can be used to insert one or more of the CDRs of an anti-M(H)DM2 / 4 (e.g., HDM2) or antigen-binding fragment thereof described herein into known framework regions, which may be naturally occurring or consensus framework regions, preferably human framework regions.

[0159] In certain embodiments, described herein are polynucleotides comprising a combination of framework regions and CDRs that encode an anti-M(H)DM2 / 4 (e.g., HDM2) or antigen-binding fragment thereof that specifically binds to M(H)DM2 / 4 (e.g., HDM2). One or more (e.g., one, two, or three) amino acid substitutions may be made within the framework regions, preferably one or more (e.g., one, two, or three) amino acid substitutions that improve binding of the antibody to M(H)DM2 / 4 (e.g., HDM2).

[0160] In alternative embodiments where the antibody or fragment thereof is not humanized, the framework regions in the variable domains may be those of the original (eg, murine) antibody).

[0161] The antibodies provided herein include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules (i.e., molecules having an antigen-binding site) that specifically bind to extracellular regions (epitopes) of M(H)DM2 / 4 that are accessible on the plasma membrane surface of cancer cells (e.g., epitopes that are expressed or exposed at higher levels on the plasma membrane of cancer cells than on non-cancerous cells (e.g., when such cancerous and non-cancerous cells originate from the same tissue)).

[0162] In preferred embodiments, the anti-M(H)DM2 / 4 antibodies described herein are monoclonal antibodies or fragments thereof. The antibodies and fragments described herein are preferably human, humanized, or chimeric. Human antibodies may be human immunoglobulins, which may be isolated from a human immunoglobulin library or from mice or other animals expressing antibodies derived from human genes. In one embodiment, the antibodies provided herein are human (or fragments of human antibodies). In one embodiment, the antibodies provided herein are humanized (or fragments of humanized antibodies). In one embodiment, the antibodies provided herein are chimeric (or fragments of chimeric antibodies) (chimeric antibodies are antibodies having variable regions from one species (e.g., mouse) and constant regions from another species (e.g., human)). Preferably, the antibodies provided herein are human, humanized, or chimeric monoclonal antibodies (which are particularly suitable for treating human subjects). In one embodiment, the antibodies provided herein are synthetic antibodies. In one embodiment, the antibody provided herein is a multispecific antibody (e.g., a bispecific antibody). In one embodiment, the antibody provided herein is a single-chain antibody, e.g., a single-chain Fv (scFv). In one embodiment, an antigen-binding fragment of an anti-M(H)DM2 / 4 antibody is provided herein, which may be, but is not limited to, an Fv fragment, a Fab fragment, a F(ab') fragment, a F(ab')2 fragment, or a disulfide-linked Fv (sdFv). In one embodiment, the antigen-binding fragment provided herein is an Fv fragment. In one embodiment, the antigen-binding fragment provided herein is a Fab fragment. In one embodiment, the antigen-binding fragment provided herein is a F(ab')2 fragment. In one embodiment, the antigen-binding fragment provided herein is a F(ab')2 fragment.

[0163] In certain embodiments, the antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies) that specifically bind to an extracellularly accessible epitope of M(H)DM2 / 4 exposed on the plasma membrane surface of cancer cells and specifically bind to a second antigen, allowing effector cells to be retargeted to tumor cells (for an example of such an engineered bispecific antibody directed to different targets, see Chames et al., 2009, MAbs 1:539-547, which describes the antibody designated catumaxomab, a T cell targeting agent). In one embodiment, the antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies) that specifically bind to an extracellularly accessible epitope of M(H)DM2 / 4 exposed on the plasma membrane surface of cancer cells and also bind to an antigen exposed on the plasma membrane surface of effector cells. Effector cells include, but are not limited to, T cells, natural killer cells, neutrophils, macrophages, dendritic cells, and B lymphocytes. In one embodiment, an antibody provided herein is a multispecific antibody (e.g., a bispecific antibody) that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 exposed on the plasma membrane surface of a cancer cell and also specifically binds to an antigen exposed on the surface of a T cell (e.g., a cytotoxic T cell). In one embodiment, an antibody provided herein is a multispecific antibody (e.g., a bispecific antibody) that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 exposed on the plasma membrane surface of a cancer cell and also specifically binds to CD3. In certain embodiments, an antibody provided herein is a multispecific antibody (e.g., a bispecific antibody) that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 exposed on the plasma membrane surface of a cancer cell and also specifically binds to an antigen exposed on the surface of a natural killer cell, neutrophil, macrophage, dendritic cell, and / or B lymphocyte.In certain embodiments, the antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies) that specifically bind to an extracellularly accessible epitope of M(H)DM2 / 4 exposed on the plasma membrane surface of cancer cells and also specifically bind to an antigen exposed on the surface of neutrophils, macrophages, dendritic cells, and / or B lymphocytes. In certain embodiments, the antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies) that specifically bind to an extracellularly accessible epitope of M(H)DM2 / 4 exposed on the plasma membrane surface of cancer cells and also specifically bind to an antigen exposed on the surface of natural killer cells, macrophages, and / or dendritic cells.

[0164] In preferred embodiments in which human subjects are treated, the antibodies used are monoclonal antibodies or antigen-binding fragments thereof that are human, humanized, or chimeric.

[0165] For example, in certain embodiments in which non-human subjects, e.g., cats, dogs, cows, and other domestic, livestock, and wild animals, are treated, the antibody can be an antibody or fragment appropriate for use in the species being treated (i.e., of that species). The antibodies described herein can be derived from any animal species, e.g., mammalian (e.g., mouse, donkey, sheep, rabbit, goat, guinea pig, camel, horse, dog, cat), or avian (e.g., chicken).

[0166] In certain embodiments in which the antibody is an immunoglobulin, the immunoglobulin molecule that can be used is of any type (e.g., IgG, IgE, IgM, IgD, IgA, IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), or subclass of immunoglobulin molecule. In a preferred embodiment, the antibody is an immunoglobulin, specifically an IgG. In another embodiment, the antibody is an IgM.

[0167] In preferred embodiments, the anti-M(H)DM2 / 4 antibodies or fragments described herein are antibodies or fragments that mediate complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or cytotoxicity due to a cytotoxic drug attached to the antibody or fragment.

[0168] In certain embodiments, the anti-M(H)DM2 / 4 antibodies or fragments described herein are antibodies or fragments capable of inducing cytotoxicity against cancer cells targeted by such antibodies or fragments, where the cytotoxicity may be due to complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or the cytotoxicity of a drug bound to the antibody (when the antibody used is in the form of an antibody-drug conjugate).

[0169] In certain embodiments, the anti-M(H)DM2 / 4 antibodies or fragments described herein are antibodies or fragments that mediate complement-dependent cytotoxicity (CDC). Methods for generating antibodies with CDC function are known in the art. In some embodiments in which CDC activity is desired, the Fc region of the antibodies described herein is of human IgG (e.g., IgG1, IgG2, IgG3, IgG4) type or human IgM type. In some embodiments in which CDC activity is desired, the Fc region of the antibodies described herein is of mouse IgG (e.g., IgG1, IgG2a, IgG2b, IgG3) or mouse IgM type. In one embodiment, the Fc region of the antibodies described herein is of the human IgG1 isotype. In one embodiment, the Fc region of the antibodies described herein is of the human IgG3 isotype. In one embodiment, the Fc region of the antibodies described herein is of the human IgG2 isotype. In one embodiment, the Fc region of the antibodies described herein has been bioengineered (e.g., mutated) to increase its CDC activity. In one embodiment, the antibody or fragment is a bispecific antibody or fragment that specifically binds to two different extracellular epitopes on M(H)DM2 / 4 (e.g., this may result in amplified complement activation, increased deposition of fragments (C3b, iC3b, C3d, C3g, C4b) on cancer cell surface membranes, and / or increased cancer cell killing by MAC). In another embodiment, the antibody or fragment is a bispecific antibody or fragment that specifically binds to an extracellular epitope of M(H)DM2 / 4 and an extracellular epitope of a complement regulatory protein (CRP) (e.g., this may prevent degradation of newly deposited immunologically active fragments (C3b, iC3b, C3d, C3g, C4b) by CRP, may amplify activation of the complement cascade, and / or may amplify MAC-induced cancer cell lysis). Such bispecific anti-M(H)DM2 / 4 antibodies or fragments may increase CDC activity, may increase ADCC activity, may increase antibody-dependent cellular phagocytosis (ADCP) by neutrophils and macrophages, or may increase CDC, ADCC, and ADCP.

[0170] In certain embodiments, the anti-M(H)DM2 / 4 antibodies or fragments described herein are antibodies or fragments that mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP).

[0171] Methods for generating antibodies with ADCC function are known in the art. Methods for generating antibodies with ADCP function are known in the art. Generally, the Fc region of an antibody mediates its binding to Fc receptors, FcR, on neutrophils, macrophages, natural killer cells, eosinophils, and mast cells, thereby triggering ADCC and producing ADCP on neutrophils, macrophages, and dendritic cells. In some embodiments where ADCC and / or ADCP activity is desired, the Fc region of the antibodies described herein is of the human IgG (e.g., IgG1, IgG2, IgG3) type or human IgE type. In one embodiment, the Fc region of the antibodies described herein is of the human IgG1 isotype.

[0172] In one embodiment, the Fc region of an antibody described herein has been bioengineered (e.g., by cross-linking, disulfide bond formation, oligosaccharide addition, or mutation) to increase its ADCC and / or ADCP activity. In one embodiment, the Fc region of an antibody described herein has been mutated to increase the lifespan of the intact antibody (e.g., by the methods described in Vaccaro et al., 2005, Nat Biotechnol. 23:1283-8128, the disclosure of which is incorporated herein by reference). In another embodiment, amino acid substitutions in the Fc CH2 and CH3 domains can be used to direct potency toward ADCC / ADCP rather than CDC, or to increase the efficiency of all three cytotoxic activities (i.e., ADCC, ADCP, and CDC). In another embodiment, the CH2 and / or CH3 domains of the Fc region of the antibodies described herein may be modified to improve ADCC and / or ADCP function, e.g., by modifying the CH2 and / or CH3 domains of the Fc region of the antibodies described herein, e.g., as described in Satoh et al., 2006, Expert Opin Biol Ther. 6:1161-1173 and / or Liu et al., 2015, Ca Immunol Res. 3:173-183 Their glycosylation sites have been modified to remove / reduce fucose residues according to the methods described, the disclosures of which are incorporated herein by reference).

[0173] In certain embodiments, anti-M(H)DM2 / 4 antibodies described herein having an IgG Fc region have had their Fc region bioengineered to alter the N-glycan structures at their glycosylation sites to the G0 glycan type, terminating in GlcNAc (N-acetylglucosamine), and to lack fucose and sialic acid residues (which can, for example, lead to activation of both the classical and alternative complement pathways and increased binding to lectins, including mannose-binding lectin, secreted during inflammatory responses).

[0174] In one embodiment, the Fc region of an antibody described herein is of the human IgG1 isotype and has an alanine substitution at position 333 in its CH2 domain. In one embodiment, the Fc region of an antibody described herein is of the human IgG1 isotype and has the triple mutation S239D / I332E / A330L, which confers higher affinity for FcγRIIIa and lower affinity for FcγRIIb, resulting in enhanced ADCC (such Fc modifications are described, for example, in Lazar et al., 2006, PNAS 103:4005-4010). The Fc regions of the antibodies described herein are of the human IgG1 isotype and contain the triple mutation S239D / I332E / G236A, which results in improved FcγRIIIa affinity and an improved FcγRIIa / FcγRIIb ratio, and mediates enhanced phagocytosis of target cells by macrophages (such Fc modifications can be made, for example, according to the methods described in Richards et al., 2008, Mol. Cancer Ther. 7:2517-27). can).

[0175] In certain embodiments, the anti-M(H)DM2 / 4 antibodies or fragments described herein are antibodies or fragments that mediate both complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). In certain embodiments, the anti-M(H)DM2 / 4 antibodies or fragments described herein are antibodies or fragments that mediate complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). In other embodiments, antibodies or fragments that mediate only CDC activity or only ADCC activity are contemplated herein. The CDC and ADCC functions of the antibodies described herein can be tested by any in vitro and / or in vivo cytotoxicity assay known in the art. In certain embodiments, the antibodies or fragments used herein comprise one or more amino acid mutations or substitutions in the Fc region that improve their CDC or ADCC activity (e.g., any of the mutations or substitutions described herein or known in the art (see, e.g., Idusogie et al., 2001. J Immunol. 166(4):2571-5; Strohl, 2009, Curr Opin Biotechnol. 20(6):685-91; Lazar et al., 2006, PNAS 103(11): 4005-4010, the disclosures of which are incorporated herein by reference)). Included in the area.

[0176] In certain embodiments, the anti-M(H)DM2 / 4 antibody or fragment is unconjugated, eg, not conjugated to a cytotoxic drug.

[0177] In certain embodiments, the anti-M(H)DM2 / 4 antibodies or fragments described herein are not linked (e.g., conjugated) to a drug (e.g., to a cytotoxic drug). In some of these embodiments, the anti-M(H)DM2 / 4 antibodies or fragments mediate CDC and / or ADCC.

[0178] In one embodiment, the anti-M(H)DM2 / 4 antibody or fragment thereof specifically binds to an extracellularly accessible segment (i.e., epitope) within amino acids 1-15, 15-25, or 475-491 of HDM2 (SEQ ID NO: 4).

[0179] In one embodiment, the anti-HDM2 antibody or fragment thereof specifically binds to HDM2 within amino acids of SEQ ID NO: 1 (amino acids 1-15 of HDM2 (SEQ ID NO: 4)). Amino acids of SEQ ID NO: 1 (amino acids 1-15 of SEQ ID NO: 4) are included in the extracellularly accessible epitope of HDM2.

[0180] In one embodiment, the anti-HDM2 antibody or fragment thereof specifically binds to HDM2 within amino acids SEQ ID NO:2 (amino acids 15-25 of HDM2 (SEQ ID NO:4)). Amino acids SEQ ID NO:2 (amino acids 15-25 of SEQ ID NO:4) are included in the extracellularly accessible epitope of HDM2.

[0181] In one embodiment, the anti-HDM2 antibody or fragment thereof specifically binds to HDM2 within the amino acids of SEQ ID NO: 3 (amino acids 475-491 of HDM2 (SEQ ID NO: 4)). The amino acids of SEQ ID NO: 3 (amino acids 475-491 of SEQ ID NO: 4) are included in the extracellularly accessible epitope of HDM2.

[0182] In one embodiment, the anti-M(H)DM2 / 4 antibody or fragment thereof specifically binds to an extracellularly accessible epitope of HDM2 within amino acids 50-60 of M(H)DM2 / 4 (SEQ ID NO: 4 or SEQ ID NO: 6).

[0183] In one embodiment, the anti-M(H)DM2 / 4 antibody or fragment thereof specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 within amino acids 100-110 of M(H)DM2 / 4 (SEQ ID NO: 4 or SEQ ID NO: 6).

[0184] In one embodiment, the anti-M(H)DM2 / 4 antibody or fragment thereof specifically binds to an extracellular epitope of M(H)DM2 / 4 within amino acids 1-126 of M(H)DM2 / 4 (SEQ ID NO: 4 or SEQ ID NO: 6).

[0185] In one embodiment, the anti-M(H)DM2 / 4 antibody or fragment thereof specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 within amino acids 436 to 482 of M(H)DM2 / 4 (SEQ ID NO: 4 or SEQ ID NO: 6).

[0186] In one embodiment, the anti-M(H)DM2 / 4 antibody or fragment thereof specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 within the terminal 100 amino acids at the C-terminus of M(H)DM2 / 4 (e.g., an M(H)DM2 / 4 protein variant (splice variant) known or predicted to be expressed on the plasma membrane of cells of the cancer type being treated, or an M(H)DM2 / 4 protein variant (splice variant) determined to be expressed on the plasma membrane of cancer cells of the subject being treated).

[0187] The present invention also provides anti-M(H)DM2 / 4 antibodies or fragments thereof that compete for binding to HDM2 with an antibody that specifically binds to HDM2 within the amino acid sequence of SEQ ID NO: 1 (e.g., the NMC-103 antibody described herein, or any antibody or fragment having the VH of NMC-103 (i.e., the VH of SEQ ID NO: 36) and the VL of NMC-103 (i.e., the VL of SEQ ID NO: 37), or any antibody or fragment having the VH and VL CDRs of NMC-103). The present invention also provides anti-M(H)DM2 / 4 antibodies or fragments thereof that compete for binding to HDM2 with an antibody that specifically binds to HDM2 within the amino acid sequence of SEQ ID NO:2 (e.g., an NMC-204 antibody described herein, or any antibody or fragment having the VH of NMC-204 (i.e., the VH of SEQ ID NO:38) and the VL of NMC-204 (i.e., the VL of SEQ ID NO:39), or any antibody or fragment having the VH and VL CDRs of NMC-204). The present invention also provides anti-M(H)DM2 / 4 antibodies or fragments thereof that compete for binding to HDM2 with an antibody that specifically binds to HDM2 within the amino acid sequence of SEQ ID NO: 3 (e.g., the NMC-303 antibody described herein, or any antibody or fragment having the VH of NMC-303 (i.e., the VH of SEQ ID NO: 40) and the VL of NMC-303 (i.e., the VL of SEQ ID NO: 41), or any antibody or fragment having the VH and VL CDRs of NMC-303).

[0188] Any competition assay known in the art can be used to identify antibodies that compete with the antibodies described herein for binding to M(H)DM2 / 4 (Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring (See, e.g., Harvard Medical School, Cold Spring Harbor, NY). In the assay, immobilized M(H)DM2 / 4 (e.g., immobilized on a microplate or well) is incubated in a solution containing a first, labeled antibody that binds to M(H)DM2 / 4 and a second, unlabeled antibody that is tested for its ability to compete with the first antibody for binding to M(H)DM2 / 4. As a control, immobilized M(H)DM2 / 4 may be incubated in a solution containing the first, labeled antibody but not the second, unlabeled antibody. After incubation, excess unbound antibody is removed, and the amount of label associated with immobilized M(H)DM2 / 4 is measured. A substantial reduction in the amount of label in the test sample compared to the control sample indicates that the second antibody competes with the first antibody for binding to M(H)DM2 / 4.

[0189] In certain embodiments, an antibody that competes with an antibody described herein (e.g., an antibody having the VH and VL of NMC-103, NMC-204, or NMC-303) for binding to M(H)DM2 / 4 also binds to the same peptide from M(H)DM2 / 4 to which such an antibody binds (e.g., the peptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3). In certain embodiments, an antibody that competes with an antibody described herein (e.g., an antibody having the VH and VL of NMC-103, NMC-204, or NMC-303) for binding to M(H)DM2 / 4 also binds to the same epitope in M(H)DM2 / 4 to which such an antibody binds.

[0190] In one embodiment, the anti-M(H)DM2 / 4 antibody or fragment thereof binds to the same epitope of M(H)DM2 / 4 as an antibody or fragment having a VH of SEQ ID NO: 36 and a VL of SEQ ID NO: 37. In one embodiment, the anti-M(H)DM2 / 4 antibody or fragment thereof described herein binds to the same epitope of M(H)DM2 / 4 as an antibody or fragment having a VH of SEQ ID NO: 38 and a VL of SEQ ID NO: 39. In one embodiment, the anti-M(H)DM2 / 4 antibody or fragment thereof described herein binds to the same epitope of M(H)DM2 / 4 as an antibody or fragment having a VH of SEQ ID NO: 40 and a VL of SEQ ID NO: 41.

[0191] In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within amino acids 19-50 of HDM2 (SEQ ID NO: 4).

[0192] In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within amino acids 19-108 of HDM2 (SEQ ID NO: 4).

[0193] In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within amino acids 154-167 of HDM2 (SEQ ID NO: 4).

[0194] In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within amino acids 1-60 of HDM2 (SEQ ID NO: 4).

[0195] In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within amino acids 1-100 of HDM2 (SEQ ID NO: 4).

[0196] In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within amino acids 1-108 of HDM2 (SEQ ID NO: 4).

[0197] In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within amino acids 26-60 of HDM2 (SEQ ID NO: 4).

[0198] In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within the terminal 60 amino acids at the C-terminus of HDM2 (e.g., an HDM2 protein variant (splice variant) known or predicted to be expressed on the plasma membrane of cells of the cancer type being treated, or an HDM2 protein variant (splice variant) determined to be expressed on the plasma membrane of cancer cells of the subject being treated).

[0199] In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within the terminal 100 amino acids at the C-terminus of HDM2 (e.g., an HDM2 protein variant (splice variant) known or predicted to be expressed on the plasma membrane of cells of the cancer type being treated, or an HDM2 protein variant (splice variant) determined to be expressed on the plasma membrane of cancer cells of the subject being treated).

[0200] In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within amino acids 101-200 of HDM2 (SEQ ID NO: 4).

[0201] In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein competes with antibody OP145 (a monoclonal antibody commercially available from Calbiochem, catalog number OP145-100UG; see Table 10 below for further details regarding OP145) for binding to HDM2. In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein competes with antibody 965 (SMP14) (a monoclonal antibody commercially available from Santa Cruz, catalog number Sc-965; see Tables 3 and 10 below for further details regarding 965 (SMP14)) for binding to HDM2. In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein competes with antibody sc-813(N-20) (a polyclonal antibody commercially available from Santa Cruz, catalog number Sc-813, see Table 10 below for further details regarding sc-813(N-20)). In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein competes with antibody sc-812(C-18) (a polyclonal antibody commercially available from Santa Cruz, catalog number Sc-812, see Table 10 below for further details regarding sc-812(C-18)) for binding to HDM2. In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein competes with antibody M01, clone 1A7 (commercially available monoclonal antibody from Abnova, catalog number H00004193-M01, see Table 3 below for further details regarding M01, clone 1A7) for binding to HDM2.

[0202] Any competition assay known in the art can be used to identify antibodies that compete with antibody OP145, SMP14, N-20, C-18, or M01, clone 1A7, for binding to M(H)DM2 / 4 (e.g., HDM2) (see Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)).

[0203] In certain embodiments, antibodies that compete with antibody OP145, SMP14, N-20, C-18, or M01, clone 1A7 for binding to M(H)DM2 / 4 also bind to the same epitope to which such antibodies bind.

[0204] In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein binds to the same epitope of HDM2 as antibody OP145 (see Table 10 below for further details regarding OP145). In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein binds to the same epitope of HDM2 as antibody 965 (SMP14) (see Tables 3 and 10 below for further details regarding 965 (SMP14)). In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein binds to one of the same epitopes of HDM2 as polyclonal antibody sc-813 (N-20) (see Table 10 below for further details regarding 813 (N-20)). In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein binds to one of the same epitopes of HDM2 as polyclonal antibody sc-812(C-18) (see Table 10 below for further details regarding sc-812(C-18)). In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein binds to the same epitope of HDM2 as antibody M01, clone 1A7 (i.e., a monoclonal antibody commercially available from Abnova, catalog number H00004193-M01; see Table 3 below for further details regarding M01, clone 1A7).

[0205] In certain embodiments, the anti-M(H)DM2 / 4 antibodies or fragments thereof described herein are purified. In certain embodiments, the antibodies or fragments are purified to greater than 95% or 99% purity, as determined, for example, by electrophoretic methods (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic methods (e.g., ion exchange or reverse-phase HPLC) (see, e.g., Flatman et al., J. Chromatogr. B, for a discussion of methods for assessing antibody purity). 848:79-87 (2007).

[0206] The anti-M(H)DM2 / 4 antibodies or fragments described herein may be fused or conjugated (e.g., covalently or non-covalently linked) to a detectable label or substance. Such labeled antibodies or fragments can be used to detect M(H)DM2 / 4 on the plasma membrane surface of a cell.

[0207] Examples of detectable labels or substances include enzyme labels, radioisotopes (e.g., iodine, carbon, sulfur, tritium, indium, and technetium), luminescent labels, fluorescent labels, and biotin. This technique can be used to determine whether cells of a particular cancer (e.g., cancer cells in a patient) express M(H)DM2 / 4 or a particular splice variant of M(H)DM2 / 4 on the plasma membrane, and detection of M(H)DM2 / 4 using an antibody (or fragment) can indicate that the antibody (or fragment) (with or without a detectable label or substance) can be used to diagnose and treat cancer or prevent cancer metastasis.

[0208] 7.2 Antibody-drug conjugates In certain embodiments, the invention provides antibody-drug conjugates comprising an anti-M(H)DM2 / 4 antibody or fragment described herein linked (e.g., covalently linked) to a cytotoxic drug. In such embodiments, the antibody-drug conjugate is intended to mediate cytotoxicity by delivery of the cytotoxic drug to cells of the cancer.

[0209] Thus, the anti-M(H)DM2 / 4 antibodies or antigen-binding fragments described herein can be linked or conjugated to one or more cytotoxic agents. A cytotoxic agent can be any agent that inhibits or prevents biological cell function (e.g., cell division) and / or causes cell death or destruction. Cytotoxic agents that can be linked or conjugated to anti-M(H)DM2 / 4 antibodies or fragments include, but are not limited to, chemotherapeutic agents (e.g., any chemotherapeutic agent known in the art or described herein), toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), radioactive isotopes, growth inhibitory agents, and nucleases. Antibody-drug conjugates and methods for making them (including antibodies that can be used in such conjugates, drugs that can be used in such conjugates, and types of linkers that can be used to link antibodies to drugs) are known in the art (see, e.g., Peters & Brown, 2015, Biosci. Rep. 35, e00225, doi:10.1042 / BSR20150089).

[0210] Examples of cytotoxic agents that can be conjugated to the anti-M(H)DM2 / 4 antibodies or fragments described herein include, but are not limited to, anthracyclines, doxorubicin, methotrexate, antimetabolites, antifolates, auristatins (e.g., MMAE or MMAF), maytansine, calicheamicin, duocarymucin, and pyrrolobenzodiazepine (PBD) dimers.

[0211] In certain embodiments, the M(H)DM2 / 4 antibody or fragment described herein is conjugated to one or more of the following drugs: maytansinoids, auristatins (e.g., monomethylauristatin drug moieties DE and DF (MMAE and MMAF)), dolastatins, calicheamicin or derivatives thereof, anthracyclines (e.g., daunomycin or doxorubicin), methotrexate, vindesine, taxanes (e.g., docetaxel), paclitaxel, larotaxel, tesetaxel, ortataxel, and trichothecenes.

[0212] In another embodiment, the M(H)DM2 / 4 antibody or fragment described herein is capable of binding to a toxin or fragment thereof (e.g., diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca ) Phytolacca americana protein, Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, or a trichothecene.

[0213] In another embodiment, the M(H)DM2 / 4 antibodies or fragments described herein can be administered with a radioisotope (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , or a radioactive isotope of Lu).

[0214] In another embodiment, the M(H)DM2 / 4 antibodies or fragments thereof described herein are conjugated to nanoparticles or other targeting tools to facilitate delivery and retention of the antibodies at tumor sites.

[0215] 7.3 Antibody production The anti-M(H)DM2 / 4 antibodies or fragments described herein can be produced by any method known in the art.

[0216] Methods for producing polyclonal antibodies are known in the art (see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York).

[0217] Methods for producing monoclonal antibodies are also known in the art and include the use of hybridomas, recombinant and phage display technologies, and the use of humanized mice. For example, monoclonal antibodies can be produced by, for example, the methods described in Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988), Hammerling GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981), or Kohler G & Milstein C (1975) Nature 256: 495. In another example, human monoclonal antibodies can be produced using hybridoma techniques, for example, as taught in Laffleur et al., 2012, Methods Mol. Biol. 901:149-59. These can be produced using modified mice.

[0218] Methods for producing specific antibodies using hybridoma technology are conventional and well known in the art. Specifically, mice or another suitable host animal can be immunized to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the target protein used in immunization (i.e., the extracellular region of M(H)DM2 / 4). The lymphocytes are then fused with myeloma cells to form hybridoma cells (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986); see also Kozbor D (1984) J Immunol 133: 3001-5; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987). The hybridoma cells are then grown in a suitable culture medium, and can be assayed for the production of monoclonal antibodies against M(H)DM2 / 4. The binding specificity of monoclonal anti-M(H)DM2 / 4 antibodies produced by this method can be determined by methods known in the art, such as immunoprecipitation or in vitro assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Hybridoma clones selected in this manner are then grown by standard methods (Goding et al., 2004). See JW (Ed), Monoclonal Antibodies: Principles and Practice (supra). The monoclonal antibodies can then be isolated from the culture medium and purified.

[0219] Furthermore, the antibodies or fragments described herein can also be generated using various phage display techniques known in the art (Brinkman U et al., (1995) J Immunol Methods 182: 41-50; Ames RS et al., (1995) J Immunol Methods 184: 177-186; Kettleborough CA et al., (1994) Eur J Immunol 24: 952-958; Persic L et al., (1997) Gene 187: 9-18; and Burton DR & Barbas CF (1994) Advan Immunol 57: 191-280).

[0220] Methods for producing chimeric antibodies (i.e., antibodies having variable regions from one species (e.g., mouse) and constant regions from another species (e.g., human)) are known in the art (Morrison SL (1985) Science 229: 1202-7, Oi VT & Morrison SL (1986) BioTechniques 4: 214-221, Gillies SD et al., (1989) J Immunol Methods 125: 191-202, and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415).

[0221] Methods of producing antibody fragments are known in the art. For example, Fab and F(ab')2 fragments can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).

[0222] Methods for producing humanized antibodies are also known in the art (International Publication No. WO91 / 09967, Padlan EA (1991) Mol Immunol 28(4 / 5): 489-498, Studnicka GM et al., (1994) Prot Engineering 7(6): 805-814, and Roguska MA et al., (1994) PNAS 91: 969-973, International Publication No. WO93 / 17105, Tan P et al., (2002) J Immunol 169: 1119-25, Caldas C et al., (2000) Protein Eng. 13(5): 353-60, Morea V et al., (2000), Methods 20(3): 267-79, Baca M et al., (1997) J Biol Chem 272(16): 10678-84, Roguska MA et al., (1996) Protein Eng 9(10): 895 904, Couto JR et al., (1995) Cancer Res. 55 (23 Supp): 5973s-5977s, Couto JR et al., (1995) Cancer Res 55(8): 1717-22, Sandhu JS (1994) Gene 150(2): 409-10, and Pedersen JT et al., (1994) J Mol Biol 235(3): 959-73. In one embodiment, a humanized antibody is produced by CDR grafting.

[0223] Methods of making human antibodies are known in the art and include phage display methods using antibody libraries derived from human immunoglobulin sequences (see U.S. Pat. Nos. 4,444,887, 4,716,111, and 5,885,793, and International Publication Nos. WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741). In some embodiments, human antibodies can be produced using mouse-human hybridomas (see Shinmoto H et al., (2004) Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14: 27-31).

[0224] Methods for generating single domain antibodies, e.g., antibodies lacking light chains, are also known in the art (Riechmann L & Muyldermans S (1999) J Immunol 231: 25-38, Nuttall SD et al., (2000) Curr Pharm Biotechnol 1(3): 253-263 , Muyldermans S, (2001) J Biotechnol 74(4): 277-302).

[0225] Methods for producing single-chain Fv (scFv) antibodies are also known in the art (see Ahmad et al., 2012, Clinical and Developmental Immunology, doi:10.1155 / 2012 / 980250; Wang et al., 2006, Anal. Chem. 78, 997-1004; Pansri et al., 2009, BMC Biotechnology 9:6). For example, scFv antibodies can be constructed by fusing the variable domains of immunoglobulin heavy and light chains via a short polypeptide linker (using recombinant expression techniques), and scFv antibodies with the desired antigen-binding properties can be selected by phage display technology.

[0226] Methods for producing bispecific antibodies are well known in the art (Konterman, 2012, MAbs 4:182-197; Gramer et al., 2013, MAbs 5:962-973).

[0227] Methods for conferring CDC or ADCC activity to antibodies (e.g., antibodies that do not initially have CDC or ADCC activity) are known in the art (Kellner et al., 2014, Methods 65:105-113, International Publication No. WO2012010562, Natsume et al. al., 2009, Drug Design, Development and Therapy 3(3):7-16. Such methods include, but are not limited to, Fc region isotype shuffling, amino acid mutations in the Fc region that confer enhanced or optimized CDC and / or ADCC activity, and altered Fc region glycosylation profiles that confer enhanced or optimized CDC and / or ADCC activity.

[0228] 7.4 Antibody Selection If a candidate antibody or fragment for use in the therapeutic and diagnostic methods provided herein is not yet known or has not yet been shown to bind to a region of M(H)DM2 / 4 that is exposed on the surface of cancer cells, the antibody or fragment can optionally be tested by any of the following methods.

[0229] In certain aspects, provided herein are methods for identifying an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein (e.g., for diagnosing cancer, treating cancer, or preventing metastasis), comprising: (a) contacting intact cancer cells (e.g., cancer cells predicted, known, or determined to express M(H)DM2 / 4) with an anti-M(H)DM2 / 4 antibody or fragment thereof; and (b) determining whether the antibody or fragment binds to the intact cancer cells, particularly to the extracellular surface of the cancer cells (e.g., compared to intact cancer cells not contacted with the antibody or fragment), wherein binding of the anti-M(H)DM2 / 4 antibody or fragment to intact cancer cells contacted with such antibody or fragment indicates that the antibody or fragment is suitable for use in the methods described herein. In certain embodiments, the cancer cells are derived from a patient proposed to be treated with the antibody or fragment thereof.

[0230] In certain aspects, provided herein are methods for identifying an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein (e.g., for diagnosing cancer, treating cancer, or preventing metastasis), the methods comprising: (a) contacting intact cancer cells (e.g., cancer cells predicted, known, or determined to express M(H)DM2 / 4) with an anti-M(H)DM2 / 4 antibody or fragment thereof; and (b) determining whether the antibody or fragment binds to the intact cancer cells (particularly on the extracellular surface of the cancer cells) at an increased level relative to binding to intact normal cells (e.g., non-cancerous cells of the same tissue or organ type as the cancer cells), wherein increased binding of the anti-M(H)DM2 / 4 antibody or fragment to intact cancer cells relative to normal cells indicates that the antibody or fragment is suitable for use in the methods described herein.

[0231] In certain aspects, a method for identifying an anti-M(H)DM2 / 4 antibody or fragment thereof for use in a method described herein (e.g., for treating cancer or preventing metastasis) includes the steps of: (a) contacting an intact cancer cell (e.g., a cancer cell known, predicted, or determined to express M(H)DM2 / 4) with an anti-M(H)DM2 / 4 antibody or fragment thereof; and (b) contacting the intact cancer cell with an anti-M(H)DM2 / 4 antibody or fragment thereof; and (c) detecting cell death or destruction (e.g., by staining with a cell death marker, e.g., propidium iodide) of the intact cancer cell. and determining whether the antibody or fragment causes an increase in the amount of cell death or destruction (as determined by the antibody or fragment's ability to kill or destroy intact cancer cells) compared to the amount of death or destruction of intact cancer cells not contacted with the antibody or fragment and / or compared to the amount of death or destruction of intact normal cells (e.g., non-cancerous cells of the same tissue or organ as the cancer cells) contacted with the antibody or fragment, wherein an increase in the amount of cell death or destruction of intact cancer cells contacted with the antibody or fragment indicates that the antibody or fragment is suitable for use in the methods described herein.

[0232] In certain aspects, methods for identifying an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein (e.g., for treating cancer or preventing metastasis) include: (a) contacting intact cancer cells (e.g., cells known, predicted, or determined to express M(H)DM2 / 4) with an anti-M(H)DM2 / 4 antibody or fragment; and (b) the contacting step comprises measuring the activity of the antibody or fragment against the intact cancer cells by complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) (using one or more cytotoxicity assays). and determining whether the antibody or fragment results in an increase in the level of CDC or ADCC (as determined by) compared to the level of CDC or ADCC on intact cancer cells not contacted with the antibody or fragment and / or compared to the level of CDC or ADCC on intact normal cells (e.g., non-cancerous cells of the same tissue or organ as the cancer cells) contacted with the antibody or fragment, wherein an increase in the level of CDC or ADCC on intact cancer cells indicates that the antibody or fragment is suitable for use in the methods described herein.

[0233] In certain aspects, provided herein are methods for identifying an anti-M(H)DM2 / 4 antibody or fragment thereof for use in the methods described herein (e.g., for treating cancer or preventing metastasis), comprising: (a) contacting intact cancer cells (e.g., cells known, predicted, or determined to express M(H)DM2 / 4) with an anti-M(H)DM2 / 4 antibody or fragment thereof; and (b) determining whether the contacting step results in increased inhibition of growth and proliferation of the intact cancer cells compared to the inhibition of growth and proliferation of intact cancer cells not contacted with the antibody or fragment and / or compared to the inhibition of growth and proliferation of intact normal cells (e.g., non-cancerous cells of the same tissue or organ as the cancer cells) contacted with the antibody or fragment, wherein an increased inhibition of growth and proliferation of intact cancer cells contacted with the antibody or fragment indicates that the antibody or fragment is suitable for use in the methods described herein.

[0234] 7.5 Pharmaceutical Compositions Provided herein is a pharmaceutical composition comprising an anti-M(H)DM2 / 4 antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier. Also provided herein is a pharmaceutical composition comprising an antibody-drug conjugate described herein and a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers, including but not limited to excipients and stabilizers, are known in the art (see, for example, Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). The anti-M(H)DM2 / 4 antibody or fragment or antibody-drug conjugate in the pharmaceutical compositions described herein may be purified.

[0235] Pharmaceutically acceptable carriers can include isotonicity agents, buffers, suspending agents, dispersing agents, emulsifying agents, wetting agents, sequestering or chelating agents, pH buffers, solubility enhancers, antioxidants, anesthetics, and / or antimicrobial agents.Suitable excipients include, but are not limited to, water, saline, glycerol, ethanol, starch, lactose, sucrose, gelatin, malt, propylene, silica gel, sodium stearate, cream bases, and dextrose.For parenteral administration, suitable pharmaceutically acceptable carriers can include saline or phosphate buffered saline (PBS), and solutions containing drugs or other drugs such as glucose, polyethylene glycol, polypropylene glycol, etc.

[0236] In certain embodiments, the pharmaceutical compositions provided herein comprise an anti-M(H)DM2 / 4 antibody or antigen-binding fragment thereof, or antibody-drug conjugate described herein, and optionally one or more other therapeutic (e.g., anti-cancer) agents, in a pharmaceutically acceptable carrier.

[0237] Pharmaceutical compositions can be formulated for any route of administration to a subject. Injectable formulations can be prepared as liquid solutions, suspensions, emulsions, or solid forms suitable for solution or suspension in water prior to injection.

[0238] The anti-M(H)DM2 / 4 antibody or fragment thereof or antibody-drug conjugate may be used or may be present in the pharmaceutical composition in a therapeutically effective amount. A therapeutically effective amount of the antibody or conjugate can be determined by standard clinical techniques.

[0239] In certain embodiments, pharmaceutical compositions contemplated for use in the methods of treatment described herein comprise an anti-M(H)DM2 / 4 antibody or antigen-binding fragment thereof, or antibody-drug conjugate described herein, without any additional anti-cancer agent or therapy. In another specific embodiment, pharmaceutical compositions contemplated for use in the methods of treatment described herein comprise an anti-M(H)DM2 / 4 antibody or antigen-binding fragment thereof, or antibody-drug conjugate described herein, and further comprise an additional anti-cancer agent or therapy (e.g., any one, two, or more of the additional anti-cancer agents or therapies described herein).

[0240] 7.6 Treatment method In one aspect, the present invention provides treating cancer (e.g., inhibiting cancer growth, inhibiting cancer progression) in a subject in need thereof, comprising administering to the subject any of the anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibodies or fragments described herein. In certain embodiments, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4. In another specific embodiment, the present invention provides a method of preventing metastasis in a subject in need thereof, comprising administering to the subject an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4. In certain embodiments of the methods described herein, the antibody or fragment is not conjugated to a cell-penetrating peptide.

[0241] In one aspect, the present invention provides for treating cancer (e.g., inhibiting cancer growth, inhibiting cancer progression) in a subject in need thereof, comprising administering to the subject an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2 exposed on the surface of cancer cells (e.g., when cells of the type of cancer being treated are known or predicted to have such an extracellular region of HDM2 exposed on their plasma membrane surface), preferably wherein the antibody or fragment is not conjugated to a cell-penetrating peptide. In one embodiment, the method of treating cancer includes preventing cancer metastasis. In one embodiment, the method of treating cancer is for reducing tumor size (e.g., as measured by tumor volume or diameter), inhibiting tumor growth, reducing tumor growth, or eradicating tumors.

[0242] In another aspect, the invention provides for preventing metastasis in a subject having cancer, comprising administering to the subject any anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibody or fragment described herein. In another aspect, the invention provides for preventing metastasis in a subject having cancer, comprising administering to the subject an antibody or fragment thereof that specifically binds to an extracellular region of HDM2 exposed on the surface of cancer cells (e.g., when cells of the type of cancer being treated are known or predicted to have such an extracellular region of HDM2 exposed on their plasma membrane surface).

[0243] In another aspect, the invention provides for reducing the number, size, or invasiveness of metastases or eradicating metastases in a subject with metastatic cancer, comprising administering to the subject any anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibody or fragment described herein. In another aspect, the invention provides for reducing the number, size, or invasiveness of metastases or eradicating metastases in a subject with metastatic cancer, comprising administering to the subject an antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2 exposed on the surface of cancer cells (e.g., where cells of the type of cancer being treated are known or predicted to have such an extracellularly accessible epitope of HDM2 exposed on their plasma membrane surface).

[0244] In one aspect, the invention provides for treating a cancer that is resistant to another cancer therapy(ies) (e.g., a vaccine, a targeted therapy (e.g., a small molecule targeted therapy), chemotherapy, radiation therapy, or immunotherapy (e.g., treatment with another monoclonal antibody)). In one embodiment, the invention provides for treating a cancer that is resistant to chemotherapy (i.e., resistant to one or more chemotherapeutic agents). In one embodiment, the invention provides for treating a cancer that is resistant to treatment with another monoclonal antibody(ies). In one embodiment, the invention provides for treating a cancer that is resistant to radiation therapy. In one embodiment, the invention provides for treating a cancer that is resistant to a small molecule targeted therapy.

[0245] Disclosed herein is the therapeutic use of an anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibody or an antigen-binding fragment thereof in patients with cancer (e.g., diagnosed with cancer). In a preferred embodiment, disclosed herein is the therapeutic use of an anti-HDM2 antibody (or a fragment thereof) in patients with cancer known to metastasize (i.e., a type of cancer that is generally known to become metastatic). In some embodiments, the patient being treated has metastatic cancer. In other embodiments, a patient with a cancer that has not metastasized is treated according to the methods described herein to prevent cancer metastasis. In some embodiments, the patient being treated has previously been treated with another cancer therapy (e.g., a vaccine, targeted therapy, chemotherapy, immunotherapy). In certain embodiments, a patient with a metastatic cancer is treated according to the methods described herein to reduce, delay, or stop metastasis, or to reduce the number or size of cancer metastases.

[0246] The methods described herein are suitable for treating cancers that are predicted, known, or determined to express anti-M(H)DM2 / 4 (e.g., HDM2) on the surface of the cancer cells. The HDM2 on the surface of cancer cells targeted by the methods described herein can be one of the splice variants of the HDM2 protein known in the art or described herein. While not intending to be bound by the mechanism of action, HDM2 on the surface of cancer cells is typically thought to be a splice variant of the HDM2 protein that lacks at least one or all nuclear localization signals (e.g., a nuclear localization sequence in the N-terminal portion of HDM2, a nuclear localization signal in the C-terminal portion of HDM2, or both nuclear localization signals). In certain embodiments, HDM2 on the surface of cancer cells is a splice variant of the HDM2 protein that lacks at least one or all nuclear localization signals and further lacks a nuclear export signal. In one embodiment, the HDM2 on the surface of the cancer cell is HDM2 lacking the nuclear localization signal at amino acids 179-185 of SEQ ID NO: 4 (i.e., amino acids 179-185 of SEQ ID NO: 4 are missing). In one embodiment, the HDM2 on the surface of the cancer cell is HDM2 lacking the nuclear localization signal at amino acids 466-473 of SEQ ID NO: 4 (i.e., amino acids 466-473 of SEQ ID NO: 4 are missing). In one embodiment, the HDM2 on the surface of the cancer cell is HDM2 lacking the nuclear localization signal at amino acids 179-185 and 466-473 of SEQ ID NO: 4 (i.e., amino acids 179-185 and 466-473 of SEQ ID NO: 4 are missing). In some embodiments, the HDM2 on the surface of the cancer cells is HDM2 that lacks a nuclear export signal, for example, a nuclear export signal at amino acids 190-202 of SEQ ID NO: 4 (i.e., amino acids 190-202 of SEQ ID NO: 4 are missing).

[0247] In certain embodiments, HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein, e.g., one of the splice variants known in the art or described herein (see Tables 1 and 2 and Section 8 for a list of HDM2 variants). In some embodiments, HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-A (SEQ ID NO: 8), which lacks amino acids 28-222 of SEQ ID NO: 4. In some embodiments, HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-A1 (SEQ ID NO: 9), which lacks amino acids 28-222 and 275-300 of SEQ ID NO: 4. In some embodiments, HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-B (SEQ ID NO: 10), which lacks amino acids 28-300 of SEQ ID NO: 4. In some embodiments, HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-C (SEQ ID NO: 11), which lacks amino acids 53-222 of SEQ ID NO: 4. In some embodiments, HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-D (SEQ ID NO: 12), which lacks amino acids 30-388 of SEQ ID NO: 4. In some embodiments, the HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-E (SEQ ID NO: 13), which lacks amino acids 76-102 and 103-491 of SEQ ID NO: 4.In some embodiments, HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-F (SEQ ID NO: 14), which lacks amino acids 53-97 of SEQ ID NO: 4. In some embodiments, HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-G (SEQ ID NO: 15), which lacks amino acids 115-169 of SEQ ID NO: 4. In some embodiments, HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-11 (SEQ ID NO: 16), in which amino acid M is substituted for M in SEQ ID NO: 4 with MVRSRQM. In some embodiments, the HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-KB2 (SEQ ID NO: 17), which lacks amino acids 157-248 of SEQ ID NO: 4. In some embodiments, the methods described herein target splice variants of M(H)DM4 on the surface of cancer cells, for example, targeting one or more of the following splice variants: MDMX-S, MDM4-S, MDM4-A, MDM4-G, MDM4-XALT1 / XALT2, and MDM4-211 (or the human equivalents of the listed splice variants).

[0248] In certain embodiments, an anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibody or fragment thereof for use in the methods described herein has been tested to determine that it is predicted to bind to HDM2 exposed on the surface of cells of the cancer to be treated. This binding to HDM2 can be demonstrated, for example, by the ability of the anti-HDM2 antibody or fragment to bind to intact cancer cells (which may, but need not, be derived from the subject to be treated) of a tissue of the tissue type from which the cancer to be treated originates. In one embodiment, prior to administering the antibody or fragment to a subject, the anti-HDM2 antibody or fragment thereof has been tested to determine that it is predicted to bind to intact cells of the subject's cancer. This testing can be performed, for example, by demonstrating binding of the anti-HDM2 antibody or fragment to the surface of intact cancer cells obtained by biopsy or to a cancer cell line of the appropriate tissue type.

[0249] If desired, cancer cells of a prospective patient to be treated can be tested for expression of M(H)DM2 / 4 (e.g., HDM2) on the surface of the cancer cells using techniques known in the art to determine whether the subject is an appropriate candidate for an anti-HDM2 therapy described herein, although such would not typically be considered necessary if the patient has a cancer of a histological type known or predicted to have an extracellularly accessible epitope of M(H)DM2 / 4. In certain embodiments, the cancer cells in the subject to be treated have been tested and determined to have an extracellularly accessible epitope of HDM2 (targeted by an anti-HDM2 antibody or fragment thereof) exposed on their plasma membrane surface (e.g., determined to express a variant of HDM2 known to have this extracellular region exposed on the plasma membrane surface). In certain embodiments, the cancer treated using the methods described herein is one that is known or determined to express a splice variant of HDM2 (e.g., MDM2-A (SEQ ID NO: 8), MDM2-A1 (SEQ ID NO: 9), MDM2-B (SEQ ID NO: 10), MDM2-C (SEQ ID NO: 11), MDM2-D (SEQ ID NO: 12), MDM2-E (SEQ ID NO: 13), MDM2-F (SEQ ID NO: 14), MDM2-G (SEQ ID NO: 15), MDM2-11 (SEQ ID NO: 16), or MDM-KB2 (SEQ ID NO: 17)) on the plasma membrane surface of its cells. Such can be detected using techniques known in the art (e.g., RT-PCR on a nucleic acid sample derived from a cancer biopsy).

[0250] In certain embodiments, administration of an anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibody or antigen-binding fragment thereof according to the methods described herein may be carried out to achieve or may be found to result in the achievement of at least one, two, three, four, or more of the following effects (e.g., in a subject having a cancerous tumor): (i) a decrease in tumor size (e.g., volume or diameter), (ii) a reduction in tumor growth, (iii) an inhibition of tumor growth progression, (iv) tumor regression, (v) an inhibition of tumor recurrence, (vi) a decrease in tumor size (e.g., volume or diameter), (vii) a decrease in tumor growth (e.g., volume or diameter), (viii) a decrease in tumor growth (e.g., volume or diameter), (viiii) a decrease in tumor size (e.g., volume or diameter), (viiii ... (xiii) inhibiting the development or onset of one or more symptoms associated with cancer; (x) enhancing or improving the therapeutic efficacy of another therapy; (xi) reducing the subject's hospitalization (e.g., length of hospitalization); (xii) improving the subject's quality of life; (xiii) reducing mortality; or (xiv) increasing the length of recurrence-free survival or remission in a subject. Any of these effects can be assessed by any method known in the art. For example, tumor size can be assessed using magnetic resonance imaging (MRI), dynamic contrast-enhanced MRI (DCE-MRI), X-ray, computed tomography (CT) scan, or positron emission tomography (PET) scan.

[0251] In certain embodiments, administration of an anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibody or antigen-binding fragment thereof according to the methods described herein, when used alone or in combination with another therapy, is effective to treat cancer in a subject (e.g., reduce tumor volume or diameter, reduce tumor growth, reduce tumor proliferation, eradicate tumor, or ameliorate one or more symptoms of cancer). In certain embodiments, administration of an anti-HDM2 antibody or antigen-binding fragment thereof according to the methods described herein, when used alone or in combination with another therapy, is effective to prevent metastasis in a subject with cancer. In certain embodiments, administration of an anti-HDM2 antibody or antigen-binding fragment thereof according to the methods described herein, when used alone or in combination with another therapy, is effective to treat metastatic cancer (e.g., reduce the number, size, or invasiveness of metastases, or eradicate metastases).

[0252] In specific embodiments, administration of an anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibody or antigen-binding fragment thereof according to the methods described herein is effective to treat cancer or prevent metastasis in a subject when used alone (i.e., without additional therapies). In other specific embodiments, administration of an anti-HDM2 antibody or antigen-binding fragment thereof according to the methods described herein is effective to treat cancer or prevent metastasis in a subject when used in combination with one or more of the additional therapies described herein.

[0253] The effectiveness of the therapeutic methods described herein can be assessed by evaluating a parameter (e.g., tumor size) before and after administration of the therapeutic methods described herein to the subject being treated. Alternatively, the effectiveness of the therapeutic methods can be assessed by evaluating a parameter (e.g., tumor size) before and after administration of the therapeutic methods described herein to an animal model (e.g., in an animal model such as a mouse model, rat model, or hamster model of the cancer being treated). Any assay known in the art can be used to evaluate the therapeutic effectiveness of the therapeutic methods described herein.

[0254] It will be understood that in the methods of treatment described herein using anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibodies or antigen-binding fragments thereof, the antibody-drug conjugates described herein can alternatively be used.

[0255] 7.6.1 Cancer to be treated Examples of cancers that can be treated according to the methods described herein include breast cancer, cervical cancer, ovarian cancer, endometrial cancer, uterine cancer, pancreatic cancer, skin cancer (e.g., melanoma), prostate cancer (e.g., hormone-refractory, e.g., castration-resistant, prostate cancer), lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), colorectal cancer (e.g., colon cancer or rectal cancer), gastrointestinal cancer, stomach cancer, small intestine cancer, appendix cancer, esophageal cancer, gastric cancer, renal cancer, bladder cancer, gallbladder cancer, kidney cancer (and others). For example, renal cell carcinoma, or Wilms' tumor), liver cancer (e.g., hepatoma or hepatocellular carcinoma), cancer of the central nervous system (e.g., brain cancer), cancer of the peripheral nervous system, bronchial cancer, cancer of the oral cavity or pharynx (e.g., oropharyngeal cancer, laryngeal cancer), thyroid cancer, bile duct cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, vulvar cancer, testicular cancer, urethral cancer, vaginal cancer, penile cancer, bone cancer, eye cancer (e.g., retinoblastoma or uveal melanoma), and head and neck cancer (e.g., head and neck squamous cell carcinoma). In certain embodiments, the cancer is cervical cancer, endometrial cancer, ovarian cancer, pancreatic cancer, melanoma, breast cancer, or colon cancer. In one embodiment, the cancer is pancreatic cancer. In one embodiment, the cancer is melanoma. In one embodiment, the cancer is breast cancer. In one embodiment, the cancer is ovarian cancer.

[0256] In certain embodiments, a cancer that can be treated according to the methods described herein is resistant to another cancer therapy(ies) (e.g., a vaccine, a targeted therapy (e.g., a small molecule targeted therapy), chemotherapy, radiation therapy, or immunotherapy (e.g., treatment with another monoclonal antibody)). In one embodiment, a cancer that can be treated according to the methods described herein is resistant to chemotherapy. In one embodiment, a cancer that can be treated according to the methods described herein is resistant to treatment with another monoclonal antibody(ies). In one embodiment, a cancer that can be treated according to the methods described herein is resistant to radiation. In one embodiment, a cancer that can be treated according to the methods described herein is resistant to a small molecule targeted therapy.

[0257] In certain embodiments, the cancer treated according to the methods described herein is a solid cancer. In another specific embodiment, the cancer treated according to the methods described herein is a non-solid cancer (e.g., a hematopoietic cancer).

[0258] In certain embodiments, the cancer treated in accordance with the present invention is a leukemia (e.g., an acute leukemia (e.g., acute lymphocytic leukemia or acute myelocytic leukemia), a chronic leukemia (e.g., chronic myelocytic leukemia or chronic lymphocytic leukemia), or hairy cell leukemia), a lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, lymphoblastic lymphoma, mantle cell lymphoma, or T-cell lymphoma).

[0259] In certain embodiments, the cancer treated in accordance with the present invention is a carcinoma (e.g., adenocarcinoma, basal cell carcinoma, renal cell carcinoma, squamous cell carcinoma, bone cancer, thyoma / thymic carcinoma, or choriomas), blastoma, sarcoma (e.g., soft tissue sarcoma, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, or synovial sarcoma), lymphoma, leukemia, germ cell tumor, myeloma (e.g., multiple myeloma), squamous cell carcinoma, mesothelioma, glioblastoma (e.g., glioblastoma multiforme), glioma, neuroblastoma, melanoma, astrocytoma, medulloblastoma, hepatocellular carcinoma, seminoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, neuroma, oligodendroglioma, meningioma, or retinoblastoma.

[0260] In certain embodiments, the cancer treated in accordance with the methods described herein is a sarcoma or carcinoma, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangioendothelial cell sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, endometrial stromal sarcoma, mast cell sarcoma, adult soft tissue sarcoma, uterine sarcoma, Kaposi's sarcoma, Merkel cell carcinoma, urothelial carcinoma, colon carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, cystadenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, cholangiocarcinoma, embryonal carcinoma, lung cancer (e.g., small cell lung carcinoma), bladder carcinoma, or epithelial carcinoma.

[0261] In certain embodiments, the cancer treated according to the methods of the present invention is metastatic. In some embodiments, the cancer treated according to the methods described herein is metastatic melanoma, metastatic ovarian cancer, metastatic cervical cancer, metastatic endometrial cancer, metastatic pancreatic cancer, metastatic breast cancer, metastatic colon cancer, or metastatic brain cancer.

[0262] 7.6.2 Method of administration Anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibodies or fragments described herein (and pharmaceutical compositions comprising such antibodies) can be administered to a subject by any suitable means, including, but not limited to, parenteral (e.g., intravenous, intraarterial, intramuscular, intraosseous, intracerebral, intraventricular, intrathecal, subcutaneous), intraperitoneal, intratumoral, intrapulmonary, intradermal, transdermal, conjunctival, intraocular, intranasal, intratracheal, oral, and local intralesional routes of administration. In certain embodiments, an anti-HDM2 antibody or fragment described herein is administered intravenously, intraarterially, intramuscularly, intraperitoneally, intratumorally, or subcutaneously. In one embodiment, an anti-HDM2 antibody or fragment described herein is administered intravenously. In one embodiment, an anti-HDM2 antibody or fragment described herein is administered intraperitoneally. In one embodiment, an anti-HDM2 antibody or fragment described herein is administered intramuscularly. In one embodiment, an anti-HDM2 antibody or fragment described herein is administered subcutaneously. In one embodiment, the anti-HDM2 antibodies or fragments described herein are administered intratumorally (e.g., by injection into the tumor of the cancer being treated). In a specific embodiment, the anti-HDM2 antibodies or fragments described herein are administered intravenously, intraperitoneally, or intratumorally.

[0263] In certain embodiments, nanoparticles coated with the anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibodies or fragments thereof described herein are used to target and treat tumors (for antibody-coated nanoparticles, see, e.g., Cardoso et al., 2012, Curr. Med. Chem. 19(19):3103-27 and Arruebo et al., 2009, J. of Nanomater. 2009:Article ID 439389). In one embodiment, provided herein is a method for treating cancer or preventing metastasis in a subject having cancer, the method comprising administering to the subject nanoparticles coated with an anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibody or fragment thereof.

[0264] Various dosing schedules for the anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibodies or fragments described herein (and pharmaceutical compositions comprising such antibodies) are contemplated, including a single administration or multiple administrations over a period of time. Methods of administration include, but are not limited to, bolus administration, pulse infusion, and continuous infusion.

[0265] Treatment regimens for use in the methods described herein can include administration of an anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibody or fragment thereof (and compositions comprising such an antibody) once per week, once per two weeks, once per three weeks, once per four weeks, once per six weeks, once per eight weeks, or once per twelve weeks (e.g., resulting in a subject receiving at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, or at least 10 doses of the antibody, or between 2 and 20 doses of the antibody). In certain embodiments, the anti-HDM2 antibody or fragment thereof (and compositions comprising such an antibody) is administered daily, every other day, or two, three, or four times per week (e.g., for a period of time, e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, or 3 months). Treatment regimens contemplated herein include regimens in which an initial higher dose of antibody can be followed by one or more lower doses, or an initial lower dose of antibody followed by one or more higher doses. Exemplary treatment courses (in which anti-HDM2 antibodies or fragments are administered) can last for 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or several years.

[0266] In some embodiments, an initial treatment period (in which the antibody is administered, e.g., once a month, once every two weeks, once a week, twice a week, or three times a week) is followed by a rest period (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, 6 months, or 1 year) in which the antibody is not administered, and then a second treatment period (in which the antibody is administered, e.g., once a month, once every two weeks, once a week, twice a week, or three times a week). The duration of such initial treatment and such second treatment can last, e.g., 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, or 6 months (in which case the initial treatment period can be the same as or different from the second treatment period). This course of treatment (having an initial treatment period, a washout period, and a second treatment period) may be repeated two, three, four, five, six, ten, or more than ten times.

[0267] In some embodiments, two or more antibodies or fragments thereof with different binding specificities for M(H)DM2 / 4 (e.g., HDM2) are administered simultaneously or sequentially to the subject being treated.

[0268] Appropriate dosages of anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibodies or fragments for use in the methods described herein will depend on the type of antibody used, the type of cancer being treated, the severity of the cancer being treated, the route of administration, the target site, the condition of the patient (e.g., age, weight, health), the patient's responsiveness to the antibody, other medications the patient is taking, and other factors considered in the judgment of the treating medical professional.

[0269] In certain embodiments, the dosage of an anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibody or fragment described herein administered to a subject can be about 1 μg to 200 mg per kg of patient body weight. In certain embodiments, the dosage of an anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibody or fragment described herein administered to a subject can be about 1 μg to 100 mg per kg of patient body weight (e.g., about 0.01 mg / kg to about 100 mg / kg, about 0.05 mg / kg to about 100 mg / kg, or about 0.5 mg / kg to about 100 mg / kg). In certain embodiments, the dosage of an anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibody or fragment described herein administered to a subject can be about 1 mg to 200 mg per kg of patient body weight. In one embodiment, the dosage of an anti-HDM2 antibody or fragment described herein administered to a subject is 0.025 mg / kg to about 5 mg / kg. In one embodiment, the dosage of an anti-HDM2 antibody or fragment described herein administered to a subject is 0.05 mg / kg to about 2 mg / kg. In one embodiment, the dosage of an anti-HDM2 antibody or fragment described herein administered to a subject is 5 mg / kg to about 30 mg / kg. In certain embodiments, a dose (e.g., one or more doses) of about 0.025 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 7.5 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 75 mg / kg, or 100 mg / kg of an anti-HDM2 antibody or fragment described herein may be administered to a subject to be treated. In one embodiment, a dose (e.g., one or more doses) of about 0.1 mg / kg of an anti-HDM2 antibody or fragment described herein may be administered to a subject to be treated (e.g., when the antibody is administered intratumorally).

[0270] 7.7 Diagnostic, Companion Diagnostic, and Prognostic Methods In one aspect, provided herein is a method of diagnosing cancer in a subject (e.g., a human), the method comprising: (a) detecting whether an antibody or fragment thereof (e.g., a labeled antibody or fragment) that specifically binds to M(H)DM2 / 4 (e.g., HDM2) binds to the surface of intact cells of the subject, where the antibody or fragment is any anti-M(H)DM2 / 4 antibody or fragment described herein (specifically, any antibody or fragment that specifically binds to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3); and (b) diagnosing the subject with cancer if binding is detected in step (a). In one embodiment, the method of diagnosis further comprises, prior to the detection in step (a), obtaining intact cells from the subject, and then performing the detection, e.g., by determining whether the labeled antibody or fragment binds to the intact cells obtained from the subject, e.g., using FACS or cell-based ELISA analysis. In one embodiment, the method of diagnosis comprises, prior to the detection in step (a), administering the antibody or fragment to the subject, and the detection is performed by in vivo imaging of the subject.

[0271] In one aspect, a patient is selected for treatment using an anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibody or antibody fragment described herein based on detection of binding of such an antibody or fragment to the surface of intact cancer cells obtained from the patient. In certain embodiments, provided herein are methods of selecting a patient having cancer for treatment with an antibody or fragment that specifically binds to an extracellularly accessible epitope of HDM2, comprising obtaining intact cancer cells from the patient (e.g., by biopsy of a cancerous tumor in the patient or by obtaining a blood sample from the patient having circulating cancer cells) and determining whether the antibody or fragment binds to the surface of the patient's intact cancer cells (using any method known in the art or described herein, e.g., using cell-based ELISA or FACS analysis), wherein detection of binding indicates that the patient can be treated with the antibody or fragment. In certain embodiments, provided herein are methods for selecting patients having cancer for treatment with an antibody or fragment that specifically binds to an extracellularly accessible epitope of HDM2, the methods comprising obtaining intact cancer cells from the patient (e.g., by biopsy of a cancerous tumor in the patient or by obtaining a blood sample having circulating cancer cells from the patient), determining whether the antibody or fragment binds to the surface of the patient's intact cancer cells (using any method known in the art or described herein, e.g., using cell-based ELISA or FACS analysis), and if binding is detected, administering the antibody or fragment to the patient. The antibody or fragment administered to the patient may be the same as or different from the antibody or fragment used to select patients to be treated.In one embodiment, provided herein is a method for selecting a patient with ovarian cancer for treatment with an antibody or fragment that specifically binds to an extracellularly accessible epitope of HDM2, the method comprising obtaining intact ovarian cancer cells from the patient, determining whether the antibody or fragment binds to the surface of the patient's intact cancer cells, and administering the antibody or fragment to the patient if binding is detected. In one embodiment, any antibody or fragment thereof that specifically binds to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 is used to select and / or treat such patients. In one embodiment, any antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 that is not bound to a cell-penetrating peptide is used to select and / or treat such patients. In one embodiment, any antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2 / 4 that is not bound to a cell-penetrating peptide is used to select such patients, and any antibody or fragment thereof that specifically binds to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 is used to treat such patients.

[0272] Non-limiting exemplary samples that can be used in the diagnostic or patient selection methods using an anti-M(H)DM2 / 4 antibody (e.g., an anti-HDM2 antibody) or fragment thereof described herein include tissue biopsies, intact cells obtained from malignant tissue, and circulating cancer cells isolated from blood. For example, a tissue sample can be obtained from a patient, and immunohistochemistry can be performed to detect whether a labeled anti-M(H)DM2 / 4 antibody (e.g., an anti-HDM2 antibody) or fragment thereof binds to the tissue sample. Alternatively, intact cells (known or suspected to be malignant) can be isolated from a patient, and FACS or cell-based ELISA analysis can be performed to detect whether a labeled anti-M(H)DM2 / 4 antibody (e.g., an anti-HDM2 antibody) or fragment thereof binds to such cells. In yet another example, a blood sample containing circulating cancer cells can be obtained from a patient, and FACS or cell-based ELISA analysis can be performed to detect whether a labeled anti-M(H)DM2 / 4 antibody (e.g., an anti-HDM2 antibody) or fragment thereof binds to such cells.

[0273] In one embodiment, the duration and / or dosage of treatment of an anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibody or antibody fragment described herein to be used in treating a patient is determined based on detecting binding of such antibody or fragment to the surface of intact cancer cells obtained from the patient. In certain embodiments, provided herein are methods for determining whether to continue treatment of a patient having cancer with an antibody or fragment that specifically binds to an extracellularly accessible epitope of HDM2, the methods comprising administering the antibody or fragment to the patient for a first period of time (e.g., the patient has been selected for treatment as described above), obtaining intact cancer cells from the patient (e.g., by biopsy of a cancerous tumor in the patient or by obtaining a blood sample having circulating cancer cells from the patient), and determining whether the antibody or fragment binds to the surface of the patient's intact cancer cells (using any method known in the art or described herein, e.g., using cell-based ELISA or FACS analysis), and if binding is detected, continuing to administer the antibody or fragment to the patient for a second period of time (however, e.g., if binding is not detected, treatment is discontinued).In certain embodiments, a method of determining whether to increase the dose of an antibody or fragment that specifically binds to an extracellularly accessible epitope of HDM2 for use in treating a patient with cancer includes administering a dose of the antibody or fragment to the patient for a period of time (e.g., the patient has been selected for treatment as described above), obtaining intact cancer cells from the patient (e.g., by biopsy of a cancerous tumor in the patient or by obtaining a blood sample from the patient having circulating cancer cells), and determining whether the antibody or fragment binds to the surface of the patient's intact cancer cells. Provided herein are methods comprising determining (using any method known in the art or described herein, e.g., using cell-based ELISA or FACS analysis) whether binding is detected, and if binding is detected, administering to the patient a dose of the antibody or fragment for a second period of time, wherein the dose administered during the second period is higher (e.g., 2-fold or 3-fold higher) than the dose administered during the first period of time (however, e.g., if binding is not detected, then treatment is discontinued or a lower dose of the antibody or fragment is administered during the second period of time).

[0274] In another specific embodiment, treatment with an anti-M(H)DM2 / 4 (e.g., anti-HDM2) antibody or antibody fragment described herein is monitored in a patient by determining the amount of HDM2 expressed on the surface of cancer cells obtained from the patient before and after treatment, and a decrease in the amount is a positive prognosis.

[0275] 7.8 Patient Population The patient or subject to be treated according to the methods described herein includes, but is not limited to, human and non-human vertebrate animals.In certain embodiments, the subject to be treated is a mammal, such as a human, a dog, a cat, a monkey, a rabbit, a cow, a horse, a goat, a sheep, or a pig.In a preferred embodiment, the subject to be treated is a human.

[0276] In certain embodiments, the subject being treated according to the methods described herein has been diagnosed with cancer (e.g., using biopsy or any other method known in the art). In specific embodiments, the subject being treated has been diagnosed with early stage cancer. In other embodiments, the subject being treated has been diagnosed with advanced stage cancer. In specific embodiments, the subject being treated has been diagnosed with a high-grade tumor. In other embodiments, the subject being treated has been diagnosed with a low-grade tumor. In certain embodiments, the subject being treated has been diagnosed with a cancer that can metastasize. In certain embodiments, the subject being treated has been diagnosed with metastatic cancer.

[0277] In certain embodiments, a subject treated according to the methods described herein has been diagnosed with cervical cancer, endometrial cancer, ovarian cancer, pancreatic cancer, melanoma, breast cancer, colorectal cancer (e.g., colon cancer), bladder cancer, astrocytic neoplasm, glioblastoma, pediatric rhabdomyosarcoma, or lung cancer (e.g., non-small cell lung cancer). In certain embodiments, a subject treated according to the methods described herein has been diagnosed with melanoma, pancreatic cancer, breast cancer, or ovarian cancer. In one embodiment, a subject treated according to the methods described herein has been diagnosed with melanoma. In one embodiment, a subject treated according to the methods described herein has been diagnosed with pancreatic cancer. In one embodiment, a subject treated according to the methods described herein has been diagnosed with breast cancer. In one embodiment, a subject treated according to the methods described herein has been diagnosed with ovarian cancer. In one embodiment, a subject treated according to the methods described herein has been diagnosed with lung cancer.

[0278] In certain embodiments, the subject being treated has previously received one or more other cancer therapies (e.g., a vaccine, a targeted therapy (e.g., a small molecule targeted therapy), chemotherapy, radiation therapy, or immunotherapy (e.g., treatment with another monoclonal antibody)), and the subject's cancer has developed resistance to the one or more other cancer therapies. In one embodiment, the subject being treated is resistant to chemotherapy. In one embodiment, the subject being treated is resistant to radiation therapy. In one embodiment, the subject being treated is resistant to small molecule targeted therapy. In one embodiment, the subject being treated is resistant to treatment with another monoclonal antibody.

[0279] In certain embodiments, the subject being treated has a type of cancer that is known or predicted to have M(H)DM2 / 4 (e.g., HDM2) on the surface of the cancer cells. In certain embodiments, the subject being treated has a type of cancer whose cells express one or more splice variants of HDM2 on their cell surface, such as (but not limited to) one or more of the following splice variants: MDM2-A (SEQ ID NO: 8), MDM2-A1 (SEQ ID NO: 9), MDM2-B (SEQ ID NO: 10), MDM2-C (SEQ ID NO: 11), MDM2-D (SEQ ID NO: 12), MDM2-E (SEQ ID NO: 13), MDM2-F (SEQ ID NO: 14), MDM2-G (SEQ ID NO: 15), MDM2-11 (SEQ ID NO: 16), or MDM-KB2 (SEQ ID NO: 17).

[0280] In certain embodiments, the subject to be treated has a cancer whose cells have been tested and determined (using any assay known in the art) to have M(H)DM2 / 4 (e.g., HDM2) on the plasma membrane of the cancer's cells. In a specific embodiment, the subject to be treated has a cancer whose cells have been tested and determined (by any method known in the art) to have exposed on their plasma membrane an extracellular region of HDM2 that can be targeted by an anti-HDM2 antibody or fragment (and such an antibody can then be administered to the subject). In certain embodiments, the subject being treated has a cancer whose cells have been tested and determined (using any method known in the art) to express on their cell surface one or more splice variants of HDM2, such as (but not limited to) one or more of the following splice variants: MDM2-A (SEQ ID NO: 8), MDM2-A1 (SEQ ID NO: 9), MDM2-B (SEQ ID NO: 10), MDM2-C (SEQ ID NO: 11), MDM2-D (SEQ ID NO: 12), MDM2-E (SEQ ID NO: 13), MDM2-F (SEQ ID NO: 14), MDM2-G (SEQ ID NO: 15), MDM2-11 (SEQ ID NO: 16), or MDM-KB2 (SEQ ID NO: 17).

[0281] 7.9 Combination Therapies and Kits In certain embodiments, an anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject in combination with one or more anti-cancer therapies different from the antibody or fragment, such as chemotherapy, surgery, radiation therapy, another antibody with anti-cancer activity, a cytokine, T-cell therapy, a vaccine (e.g., a cellular vaccine), a small molecule with anti-cancer activity, an anti-hormonal agent, or any other anti-cancer therapy known in the art.

[0282] In certain embodiments, the anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject in combination with chemotherapy. Examples of types of chemotherapeutic agents that can be used in the methods described herein include, but are not limited to, alkylating agents, nitrosoureas, antimetabolites, topoisomerase inhibitors, aromatase inhibitors, antitumor antibiotics, plant-derived alkaloids, hormone antagonists, P-glycoprotein inhibitors, and platinum complex derivatives. Specific examples of chemotherapeutic agents that can be used in the methods described herein include taxol, paclitaxel, nab-paclitaxel, 5-fluorouracil (5-FU), gemcitabine, doxorubicin, daunorubicin, colchicine, mitoxantrone, tamoxifen, cyclophosphamide, mechlorethamine, melphalan, chlorambucil, busulfan, uramustine, mustalgen, ifosfamide, benzamidine, benzodiazepine, benzodiazepine, benzophenone ... Damustine, carmustine, lomustine, semustine, fotemustine, streptozocin, thiotepa, mitomycin, diaziquone, tetrazine, altretamine, dacarbazine, mitozolomide, temozolomide, procarbazine, hexamethylmelamine, altretamine, hexalen, trofosfamide, estramustine, treosulfan, mannosulfan, triaziquone, carboquone, nimustine, ranimustine, a Zathioprine, sulfanilamide, fluoropyrimidine, thiopurine, thioguanine, mercaptopurine, cladribine, capecitabine, pemetrexed, fludarabine, methotrexate, hydroxyurea, nelarabine or clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, thioguanine, azacitidine, cladribine, pentostatin, mercaptopurine, imatinib, dactinomycin, Cerubicin, bleomycin, actinomycin, luteomycin, epirubicin, idarubicin, plicamycin, vincristine, vinblastine, vinorelbine, vindesine, vinflunine, paclitaxel, docetaxel, etoposide, teniposide, periwinkle, vinca, taxane, irinotecan, topotecan, camptothecin, teniposide, pirarubicin, novobiocin, mervalone, aclarubicin, amsacrine,Antiandrogens, antiestrogens, bicalutamide, medroxyprogesterone, fluoxymesterone, diethylstilbestrol, estrose, octreotide, megestrol, raloxifene, toremifene, fulvestrant, prednisone, flutamide, leuprolide, goserelin, aminoglutethimide, testolactone, anastrozole, letrozole, exemestane, vorozole, formestane, fadrozole, androstene, resveratrol In certain embodiments, the anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject in combination with one or more of the following chemotherapeutic agents: gemcitabine, nab-paclitaxel, capecitabine, irinotecan, and celecoxib. In certain embodiments, an anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject in combination with one or more of the following chemotherapeutic agents: gemcitabine, nab-paclitaxel, cisplatin, 5-FU, and paclitaxel (e.g., paclitaxel formulated as albumin-bound particles, e.g., ABRAXANE®). In certain embodiments, the cancer treated using the combination therapy described herein is pancreatic cancer, breast cancer, lung cancer, or ovarian cancer.

[0283] In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject in combination with gemcitabine (e.g., for the treatment of non-small cell lung cancer, pancreatic cancer, or ovarian cancer). In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject in combination with capecitabine. In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject in combination with irinotecan. In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject in combination with celecoxib. In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject in combination with paclitaxel (e.g., paclitaxel formulated as albumin-bound particles, e.g., ABRAXANE®) (e.g., for the treatment of metastatic breast cancer). In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject in combination with nab-paclitaxel. In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject in combination with cisplatin. In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject in combination with 5-FU (e.g., for the treatment of colorectal cancer, e.g., colon cancer). In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject in combination with carboplatin. In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject in combination with gemcitabine and nab-paclitaxel (e.g., for the treatment of pancreatic cancer). In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject in combination with gemcitabine and carboplatin (e.g., for the treatment of ovarian cancer).In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject in combination with paclitaxel (e.g., paclitaxel formulated as albumin-bound particles, e.g., ABRAXANE®) and gemcitabine (e.g., for the treatment of breast cancer, or pancreatic cancer, e.g., adenocarcinoma of the pancreas or metastatic adenocarcinoma of the pancreas). In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject in combination with gemcitabine and cisplatin (e.g., for the treatment of non-small cell lung cancer). In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject in combination with gemcitabine and 5-FU. In one embodiment, an anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject in combination with paclitaxel (e.g., paclitaxel formulated as albumin-bound particles, e.g., ABRAXANE®) and carboplatin (e.g., for the treatment of non-small cell lung cancer).

[0284] In certain embodiments in which the subject is a human, gemcitabine is administered at a dose of 1,500 mg / m 2 In certain embodiments where the subject is a human, nab-paclitaxel is administered at a dose of 300 mg / m 2 In certain embodiments where the subject is a human, gemcitabine is administered at a dose of 1,000 mg / m 2 In certain embodiments where the subject is a human, nab-paclitaxel is administered at a dose of 125 mg / m 2 is administered at a dose of

[0285] In certain embodiments, gemcitabine and / or nab-paclitaxel are administered at a dose lower than that used when gemcitabine and / or nab-paclitaxel are administered without combination with an anti-cancer antibody (e.g., an anti-M(H)DM2 / 4 antibody or fragment described herein). In certain embodiments where the subject is a human, gemcitabine is administered at a dose of 1,500 mg / m 2and / or nab-paclitaxel is administered at a dose lower than 300 mg / m 2 In certain embodiments where the subject is a human, gemcitabine is administered at a dose lower than 1,000 mg / m 2 and / or nab-paclitaxel is administered at a dose lower than 125 mg / m 2 In one embodiment, in which the subject is a human, gemcitabine is administered at a dose lower than 500 mg / m 2 , 400 mg / m 2 , 300 mg / m 2 , or 200 mg / m 2 and / or nab-paclitaxel is administered at a dose equal to or lower than 62.5 mg / m 2 , 50 mg / m 2 , 40 mg / m 2 , 30 mg / m 2 , or 20 mg / m 2 In one embodiment, in which the subject is a human, gemcitabine is administered at a dose equal to or lower than 900 mg / m 2 , 800 mg / m 2 , 700 mg / m 2 , or 600 mg / m 2 and / or nab-paclitaxel is administered at a dose equal to or lower than 110 mg / m 2 , 100 mg / m 2 , 90 mg / m 2 , 80 mg / m 2 , or 70 mg / m 2In certain embodiments, gemcitabine and / or nab-paclitaxel are administered at a dose equal to or lower than 100 mg / kg / day. In certain embodiments, gemcitabine and / or nab-paclitaxel are administered once every two weeks or less (e.g., once every three weeks, once every four weeks, once every six weeks, or once every eight weeks, or less). In certain embodiments, gemcitabine is administered once a day, four times a week, three times a week, twice a week, or once a week. In certain embodiments, nab-paclitaxel is administered once a day, four times a week, three times a week, twice a week, or once a week. In certain embodiments, gemcitabine and nab-paclitaxel are administered once a day, four times a week, three times a week, twice a week, or once a week. In one embodiment, gemcitabine and / or nab-paclitaxel are administered once a week. In certain embodiments, the total duration of treatment with gemcitabine and / or nab-paclitaxel is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more.

[0286] In certain embodiments, an anti-M(H)DM2 / 4 antibody or fragment thereof described herein is administered to a subject with cancer in combination with a chemotherapeutic agent indicated for said cancer, optionally at a dosage and / or administration regimen indicated for said cancer. Non-limiting examples of chemotherapeutic agents and their dosages and administration regimens indicated for various cancers are provided below.

[0287] Ovarian cancer: The following information applies to Gemzar® (injectable gemcitabine), Eli Lilly and Company, http: / / pi.lilly.com / us / gemzar.pdf 201 Retrieved from Highlights of Prescribing Information, revised March 2017 (last accessed July 27, 2017). Gemcitabine in combination with carboplatin (Gemzar®) is indicated for the treatment of patients with advanced ovarian cancer that has recurred at least 6 months after completion of platinum-based therapy. The recommended dose of Gemzar® is 1000 mg / m as an intravenous infusion over 30 minutes on days 1 and 8 of each 21-day cycle. 2 which is in combination with intravenous carboplatin (AUC 4) following administration of Gemzar® on day 1 of each 21-day cycle.

[0288] Breast Cancer: The following information is based on 2017 data from Gemzar® (injectable gemcitabine), Eli Lilly and Company, http: / / pi.lilly.com / us / gemzar.pdf Retrieved from Highlights of Prescribing Information, revised March 2017 (last accessed July 27, 2017). Gemcitabine in combination with paclitaxel (Gemzar®) is indicated for the first-line treatment of patients with metastatic breast cancer after failure of prior anthracycline-containing adjuvant chemotherapy, unless the anthracycline is clinically contraindicated. The recommended dose of Gemzar® is 1250 mg / m 2 intravenously over 30 minutes on days 1 and 8 of each 21-day cycle that includes paclitaxel. 2 Paclitaxel may be administered at 175 mg / m2 on Day 1 as a 3-hour intravenous infusion prior to administration of Gemzar®.

[0289] Non-Small Cell Lung Cancer: The following information is from Gemzar® (Gemcitabine for Injection), Eli Lilly and Company, http: / / pi.lilly.com / us / gemzar.pdf Highlights of Prescribing revised in March 2017 Retrieved from http: / / www.ncbi.nlm.nih.gov / pubmed / 1024747. Information (last accessed July 27, 2017). Gemcitabine (Gemzar®), in combination with cisplatin, is indicated for the first-line treatment of patients with uncontrolled locally advanced (stage IIIA or IIIB) or metastatic (stage IV) non-small cell lung cancer. Every 4-week schedule: The recommended dose of Gemzar® is 1000 mg / m 2 intravenously over 30 minutes on days 1, 8, and 15 in combination with cisplatin therapy. 2 and cisplatin was administered at 100 mg / m on day 1 after Gemzar® infusion. 2 Every 3 weeks schedule: The recommended dose of Gemzar® is 1250 mg / m2 intravenously over 30 minutes on days 1 and 8 in combination with cisplatin therapy. 2 and cisplatin was administered at 100 mg / m on day 1 after Gemzar® infusion. 2 It can be administered intravenously at 100 mg / kg.

[0290] Pancreatic cancer: The following information applies to Gemzar® (injectable gemcitabine), Eli Lilly and Company, http: / / pi.lilly.com / us / gemzar.pdf 201 Retrieved from Highlights of Prescribing Information, revised March 2017 (last accessed July 27, 2017). Gemcitabine (Gemzar®) is indicated as first-line treatment for patients with locally advanced (unresectable Stage II or Stage III) or metastatic (Stage IV) adenocarcinoma of the pancreas. Gemzar® is indicated for patients previously treated with 5-FU. The recommended dose of Gemzar® is 1000 mg / m IV intravenously over 30 minutes. 2The recommended treatment schedule is as follows: Weeks 1-8 - weekly dosing for the first 7 weeks, followed by a 1-week rest period; Weeks 8 and beyond - weekly dosing on days 1, 8, and 15 of a 28-day cycle.

[0291] Metastatic Breast Cancer: The following information is for ABRAXANE® (paclitaxel-protein-bound particles for injectable suspension, albumin-bound), Celgene Corporation, http: / / www.abraxane.com / wp-content / pi / prescribing-info.html Retrieved from Highlights of Prescribing Information, revised July '15 (last accessed July 27, 2017). ABRAXANE® is indicated for the treatment of breast cancer after failure of combination chemotherapy for metastatic disease or recurrence within 6 months of adjuvant chemotherapy. Previous therapy should have included an anthracycline unless clinically contraindicated. The recommended regimen for ABRAXANE® after failure of combination chemotherapy for metastatic breast cancer or recurrence within 6 months of adjuvant chemotherapy is 260 mg / m 2 intravenously over 30 minutes every 3 weeks. 2 is administered.

[0292] Non-Small Cell Lung Cancer: The following information pertains to ABRAXANE® (paclitaxel protein-bound particles for injectable suspension, albumin-bound), Celgene Corporation, http: / / www.abraxane.com / wp-content / pi / prescribing-info.html Retriev...

Claims

[Claim 1] The invention described in the specification.

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