US20210032364A1 - Antibodies to m(h)dm2/4 and their use in diagnosing and treating cancer - Google Patents

Antibodies to m(h)dm2/4 and their use in diagnosing and treating cancer Download PDF

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US20210032364A1
US20210032364A1 US16/634,022 US201816634022A US2021032364A1 US 20210032364 A1 US20210032364 A1 US 20210032364A1 US 201816634022 A US201816634022 A US 201816634022A US 2021032364 A1 US2021032364 A1 US 2021032364A1
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antibody
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Ehsun Sarafraz-Yazdi
Brad R. Evans
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Nomocan Pharmaceuticals LLC
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
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    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5748—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncogenic proteins
    • A—HUMAN NECESSITIES
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]

Definitions

  • the present 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 metastases.
  • the MDM2 (MDM2 is a mouse homologue of HDM2) protein is composed 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 website, at UniProt Accession No. P23804).
  • Its human homologue, HDM2 is composed 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 website, at UniProt Accession No. Q00987).
  • Mouse protein MDM4 (also identified as MDMX) is a homologue of the MDM2 protein (see UniProt website, at UniProt Accession No. 035618), 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 human homologue of MDM4 (see UniProt website, at UniProt Accession No. 015151).
  • M(H)DM2 and M(H)DM4 proteins The most conserved domain within all M(H)DM2 and M(H)DM4 proteins is the RING domain which is responsible for ubiquitination of its target proteins, including p53 protein, and heterodimerization between M(H)DM2 and M(H)DM4. M(H)DM4 is required for M(H)DM2-mediated polyubiquitination of p53.
  • a distinctive feature of M(H)DM2 and M(H)DM4 are their very complex expression pattern.
  • M(H)DM2 variants have been shown to be expressed in a variety of tumors such as human ovarian, bladder, breast and astrocytic neoplasms, glioblastomas, leukemia and pediatric Rhabdomyosarcoma tumors (reviewed by Iwakuma & Lozano 2003; Rosso et al., 2014, Subcell Biochem. 85:247-61 (“Rosso 2014”)). Most interestingly they have been found to be more frequent in tumors of advanced stage (Bartel 2002).
  • the multiple-sized M(H)DM2 transcripts that have been shown to be splice variants forms of the M(H)DM2 mRNA have been reported to be expressed more frequently in tumor cells than in normal cells (Bartel et al., 2004, Mol. Cancer Res. 2:29 (“Bartel 2004”)). It has been proposed that a mRNA surveillance system exists in untransformed cells, which degrades spliced transcripts and protects the cells from errors of transcription, mRNA processing, or mRNA transport whereas in transformed cells this system may not be functioning correctly (Bartel 2004).
  • MDM2-A and MDM2-B are common to several tumor types (Sigalas 1996). Others have only been found in specific tumors, for example MDM2-FB25 and MDM2-FB26 in pediatric rhabdomyosarcoma.
  • MDM2-FB25 and MDM2-FB26 are common to several tumor types.
  • M(H)DM2 variants were aberrantly localized to the cytoplasm (Evans et al., 2001, Oncogene 20:4041-4049). This result led to the discovery that the cytoplasmic compartmentalization of the full-length M(H)DM2 was due to binding and sequestration by an alternative-spliced M(H)DM2 product (HDM2ALT1).
  • HDM2ALT1 alternative-spliced M(H)DM2 product
  • MDM2-D150-230 localized to the cytoplasm of U20S cells (Schuster et al., 2007, Mol. Cancer Res. 5:403-412 (“Schuster 2007”)).
  • M(H)DM4 protein variants have also been characterized, including splicing variant MDMX-S(Lenos and Jochemsen, 2011, J. Biomed Biotechnol, doi:10.1155/2011/876173).
  • HDM2 was found to be expressed in the plasma membrane of cancer cells (Sarafraz-Yazdi et al., 2010, PNAS 107:1918-1923 (“Sarafraz-Yazdi 2010”). Further, anti-cancer peptides, PNC-27 and PNC-28, which bind to HDM2 expressed in the cancer cell membranes and kill cancer cells by inducing necrosis, have been developed (Sarafraz-Yazdi 2010; Davitt et al., 2014, Annals Clin. Lab. Sci. 44:241248) (the amino acid sequences of PNC-27 and PNC-28 are provided in Table I of U.S. Patent Application Publication No. 2012/0177566).
  • PNC-27 and PNC-28 peptides which comprise a membrane resident peptide (“MRP”) and a p53 sequence
  • MRP membrane resident peptide
  • p53 sequence component the cargo (i.e., the p53 sequence component) is attached to the MRP component so as to form a cytotoxic structure (Kanovsky et al, 2001, PNAS 98:12438-12443 (“Kanovsky 2001”); Bowne et al., 2008, Ann Surg Oncol. 15:3588-3600 “Bowne 2008”)).
  • the MRP component or the HDM-2 targeting component i.e.
  • U.S. Pat. No. 9,765,117 discloses HDM2 targeting peptides and fusion peptides comprising an HDM2 targeting peptide and a transmembrane penetrating sequence, such as MRP; it is disclosed that MRP is required for induction of cell necrosis (see col. 4, lines 27-28).
  • U.S. Pat. No. 9,765,117 indicates that expression of the p53 HDM2 targeting sequence in the absence of the MRP in cancer cells causes p53-dependent apoptosis and not tumor necrosis (see col. 4, lines 29-32).
  • CPPs The cell-penetrating peptides
  • MRPs such as MRPs, Membrane Transduction Domain of Antennapedia, trans-activating transcriptional activator (“TAT”) and Penetratin peptides
  • TAT trans-activating transcriptional activator
  • Penetratin peptides enable cellular membrane delivery of the peptides, and molecules attached to the peptides, to plasma membrane lipid bilayers, including those of normal healthy cells.
  • TAT trans-activating transcriptional activator
  • Penetratin peptides enable cellular membrane delivery of the peptides, and molecules attached to the peptides, to plasma membrane lipid bilayers, including those of normal healthy cells.
  • TAT trans-activating transcriptional activator
  • Penetratin peptides enable cellular membrane delivery of the peptides, and molecules attached to the peptides, to plasma membrane lipid bilayers, including those of normal healthy cells.
  • CPPs such as MRPs, Membrane Transduction Domain of Antennapedia, TAT and Penetratin peptides
  • cargos such as other peptides, DNA, RNA, small molecules, antibodies or fragments thereof
  • PNC-27 and PNC-28 peptides are examples of Penetratin-/MRP-cargo conjugates that exhibit a cytotoxic function that is dependent on the attachment and linkage of their cargo to the MRP, which is required for the formation of their membrane active structure, and hence, cytotoxic function (Kanovsky 2001; Rosal 2005; Bowne 2008).
  • described herein is an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4.
  • antibodies or fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibodies or fragments inhibit tumor growth in vivo (or inhibit tumor cell proliferation in vivo).
  • the antibodies or fragments described herein are not bound to a cell-penetrating peptide (e.g., a membrane resident peptide).
  • the antibodies or fragments described herein are not bound to a cytotoxic component (i.e., not bound to a cytotoxic agent).
  • antibodies or fragments thereof that specifically bind 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 MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3).
  • M(H)DM2/4 e.g., HDM2
  • the antibody or fragment 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 MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3).
  • the antibody or fragment 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 is MCNTNMSVPTDGAVT (SEQ ID NO:1). In one embodiment, the antibody or fragment 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 is TTSQIPASEQE (SEQ ID NO:2).
  • the antibody or fragment 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 is CPVCRQPIQMIVLTYFP (SEQ ID NO:3).
  • described herein is an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 contained within a peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
  • described herein is an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 contained within a peptide of SEQ ID NO:1.
  • described herein is an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 contained within a peptide of SEQ ID NO:2. In one embodiment, described herein is an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 contained within a peptide of SEQ ID NO:3.
  • a humanized antibody or a fragment thereof that specifically binds to HDM2 said antibody or fragment comprising: (i) a heavy chain variable region (VH) comprising VH complementarity determining region (“CDR”) 1, VH CDR 2, and VH CDR3; said VH CDR 1, VH CDR 2 and VH CDR 3 being the CDRs of a VH that has an amino acid sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:38, and SED ID NO:40, or (ii) a light chain variable region (VL) comprising VL CDR 1, VL CDR 2 and VL CDR 3 being the CDRs of a VL that has an amino acid sequence selected from the group consisting of SEQ ID NO:37, SEQ ID NO:39, and SEQ ID NO:41.
  • VH heavy chain variable region
  • CDR VH complementarity determining region
  • VH CDR 2 and VH CDR 3 being the CDRs of a VH that has an amino acid
  • the humanized antibody or a fragment that specifically binds to HDM2 comprises a VH wherein VH CDR 1, VH CDR 2 and VH CDR 3 are of a VH having the amino acid sequence of SEQ ID NO:36. In one embodiment, the humanized antibody or a fragment that specifically binds to HDM2 comprises a VH wherein VH CDR 1, VH CDR 2 and VH CDR 3 are of a VH having the amino acid sequence of SEQ ID NO:38. In one embodiment, the humanized antibody or a fragment that specifically binds to HDM2 comprises a VH wherein VH CDR 1, VH CDR 2 and VH CDR 3 are of a VH having the amino acid sequence of SEQ ID NO:40.
  • the humanized antibody or a fragment that specifically binds to HDM2 comprises a VL wherein VL CDR 1, VL CDR 2 and VL CDR 3 are of a VL having the amino acid sequence of SEQ ID NO:37 (and, optionally, a VH wherein VH CDR 1, VH CDR 2 and VH CDR 3 are of a VH having the amino acid sequence of SEQ ID NO:36).
  • the humanized antibody or a fragment that specifically binds to HDM2 comprises a VL wherein VL CDR 1, VL CDR 2 and VL CDR 3 are of a VL having the amino acid sequence of SEQ ID NO:39 (and, optionally, a VH wherein VH CDR 1, VH CDR 2 and VH CDR 3 are of a VH having the amino acid sequence of SEQ ID NO:38).
  • the humanized antibody or a fragment that specifically binds to HDM2 comprises a VL wherein VL CDR 1, VL CDR 2 and VL CDR 3 are of a VL having the amino acid sequence of SEQ ID NO:41 (and, optionally, the a VH wherein VH CDR 1, VH CDR 2 and VH CDR 3 are of a VH having the amino acid sequence of SEQ ID NO:40).
  • VH heavy chain variable region
  • CDR VH complementarity determining region
  • 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);
  • 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);
  • 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);
  • the VH CDR 1 has the amino acid sequence THYYMS (SEQ ID NO:18)
  • an antibody or a fragment thereof that specifically binds to M(H)DM2/4 e.g., HDM2
  • said antibody or fragment comprising a heavy chain variable region (VH) comprising VH complementarity determining region (“CDR”) 1, VH CDR 2, and VH CDR 3, wherein:
  • 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);
  • the VH CDR 1 has the amino acid sequence GDTLSGSWIVIR (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);
  • 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);
  • the VH CDR 1 has the amino acid sequence SGSWMH (SEQ ID NO:24)
  • an antibody or a fragment thereof that specifically binds to M(H)DM2/4 e.g., HDM2
  • said antibody or fragment comprising a heavy chain variable region (VH) comprising VH complementarity determining region (“CDR”) 1, VH CDR 2, and VH CDR 3, wherein:
  • 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 the amino acid sequence 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
  • VH heavy chain variable region
  • CDR VH complementarity determining region
  • 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);
  • 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);
  • 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);
  • the VH CDR 1 has the amino acid sequence THYYMS (SEQ ID NO:18)
  • a humanized antibody or a fragment thereof that specifically binds to HDM2 said antibody or fragment comprising a VH, wherein the VH comprises VH CDR1, VH CDR2, and VH CDR 3, wherein:
  • VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (ii) the VH CDR 3 has the amino acid sequence ARDIGD (SEQ ID NO:48); or (iii) the VH CDR 3 has the amino acid sequence ARDIGDN (SEQ ID NO:146).
  • VL light chain variable region
  • CDR VL complementarity determining region
  • 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).
  • a humanized antibody or a fragment thereof that specifically binds to HDM2 said antibody or fragment comprising:
  • VH heavy chain variable region
  • CDR VH complementarity determining region
  • VH CDR 1 has the amino acid sequence GFTFTHY (SEQ ID NO:18)
  • VH CDR 2 has the amino acid sequence RNKAKGYT (SEQ ID NO:19)
  • VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20)
  • 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)
  • the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20)
  • 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
  • VH heavy chain variable region
  • CDR VH complementarity determining region
  • 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);
  • 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);
  • 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);
  • the VH CDR 1 has the amino acid sequence SGSWMH (SEQ ID NO:54
  • an antibody or a fragment thereof that specifically binds to HDM2 said antibody or fragment comprising a VH, wherein the VH comprises VH CDR1, VH CDR2, and VH CDR 3, wherein:
  • VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO:26); or (ii) the VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO:58).
  • VL light chain variable region
  • CDR VL complementarity determining region
  • 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).
  • an antibody or a fragment thereof that specifically binds to HDM2 said antibody or fragment comprising:
  • VH heavy chain variable region
  • CDR VH complementarity determining region
  • VH CDR 1 has the amino acid sequence GDTLSGS (SEQ ID NO:24)
  • VH CDR 2 has the amino acid sequence HLNRGT (SEQ ID NO:25)
  • the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO:26)
  • 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)
  • the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO:26)
  • 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)
  • VH CDR 3 has the amino acid sequence EIHLNRGTTNYNEKFKG (SEQ ID NO:56
  • VH heavy chain variable region
  • CDR VH complementarity determining region
  • 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 the amino acid sequence 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
  • an antibody or a fragment thereof that specifically binds to HDM2 said antibody or fragment comprising a VH, wherein the VH comprises VH CDR1, VH CDR2, and VH CDR 3, wherein:
  • VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO:32); or (ii) the VH CDR 3 has the amino acid sequence TRSGYYAMD (SEQ ID NO:68).
  • an antibody or fragment further comprises a light chain variable region (VL) comprising VL complementarity determining region (“CDR”) 1, VL CDR 2, and VL CDR 3, wherein:
  • 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 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).
  • an antibody or a fragment thereof that specifically binds to HDM2 said antibody or fragment comprising:
  • VH heavy chain variable region
  • CDR VH complementarity determining region
  • VH CDR 1 has the amino acid sequence GYTFTSY (SEQ ID NO:30)
  • VH CDR 2 has the amino acid sequence NPRNGG (SEQ ID NO:31)
  • the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO:32)
  • 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)
  • the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO:32)
  • 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:31)
  • an antibody or fragment thereof that specifically binds to HDM2 comprising 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.
  • an antibody or fragment thereof that specifically binds to HDM2 comprising 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, comprising 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).
  • an antibody or fragment thereof that specifically binds to HDM2 comprising 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.
  • an antibody or fragment thereof that specifically binds to HDM2 comprising 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, comprising 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).
  • an antibody or fragment thereof that specifically binds to HDM2 comprising 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.
  • an antibody or fragment thereof that specifically binds to HDM2 comprising 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, comprising 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).
  • the anti-M(H)DM2/4 antibody described herein is a monoclonal antibody. In certain embodiments, the anti-M(H)DM2/4 antibody described herein is a human, humanized, or a chimeric antibody (e.g., a human, humanized or chimeric monoclonal antibody). In one embodiment, the anti-M(H)DM2/4 antibody described herein is a human antibody. In one embodiment, the anti-M(H)DM2/4 antibody described herein is a humanized antibody. In one embodiment, the anti-M(H)DM2/4 antibody described herein is a chimeric antibody.
  • the anti-M(H)DM2/4 antibody described herein is a purified antibody.
  • the anti-M(H)DM2/4 antibody described herein is an immunoglobulin (e.g., IgG or IgM). In one embodiment, the immunoglobulin is an IgG. In one embodiment, the immunoglobulin is an IgM. In certain embodiments, the immunoglobulin is of IgG1 isotype. In other embodiments, the immunoglobulin is of IgG3 isotype. In other embodiments, the immunoglobulin is of IgG2 isotype. In certain embodiments, the anti-M(H)DM2/4 antibody described herein comprises an Fc region, wherein the Fc region is a human IgG Fc region or a human IgM Fc region.
  • the anti-M(H)DM2/4 antibody described herein comprises an Fc region, which 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 antibody described herein comprises a human IgG1 Fc region. In one embodiment, the anti-M(H)DM2/4 antibody described herein comprises a human IgG3 Fc region. In one embodiment, the anti-M(H)DM2/4 antibody described herein comprises a human IgG2 Fc region. In one embodiment, the anti-M(H)DM2/4 antibody described herein comprises a human IgM Fc region. In one embodiment, the anti-M(H)DM2/4 antibody described herein comprises a human IgE Fc region.
  • the anti-M(H)DM2/4 antibody or fragment described herein is an antigen-binding fragment of an anti-M(H)DM2/4 antibody.
  • the antibody or fragment described herein 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).
  • the antibody or fragment described herein is an Fv fragment.
  • the antibody or fragment described herein is a Fab fragment.
  • the antibody or fragment described herein is a Fab′ fragment.
  • the antibody or fragment described herein is a F(ab′)2 fragment.
  • the antibody or fragment described herein is a single chain antibody molecule.
  • the antibody or fragment described herein is a single chain Fv (scFv).
  • the anti-M(H)DM2/4 antibody or antigen-binding fragment described herein mediates complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytoxicity (ADCC). In one embodiment, the anti-M(H)DM2/4 antibody or antigen-binding fragment mediates complement-dependent cytotoxicity (CDC).
  • the anti-M(H)DM2/4 antibody described herein is a bispecific antibody that also specifically binds 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).
  • an effector cell e.g., a T cell, a B lymphocyte, a neutrophil, a macrophage, a natural killer cell, or a dendritic cell.
  • the antibody or fragment described herein specifically binds to an extracellularly accessible epitope of M(H)DM2 (e.g., HDM2) and does not bind to M(H)DM4 (e.g., HDM4). In another embodiment, the antibody or fragment described herein specifically binds to extracellularly accessible epitopes of both M(H)DM2 (e.g., HDM2) and M(H)DM4 (e.g., HDM4). In one embodiment, the antibody or fragment described herein specifically binds to an extracellularly accessible epitope of HDM2, and optionally, may also bind to an extracellularly accessible epitope of MDM2.
  • the anti-HDM2 antibody or fragment described herein specifically binds HDM2 within amino acids of SEQ ID NO: 1 (which are amino acids 1 to 15 of HDM2 (SEQ ID NO:4)). In another specific embodiment, the anti-HDM2 antibody described herein specifically binds HDM2 within amino acids of SEQ ID NO: 2 (which are amino acids 15 to 25 of HDM2 (SEQ ID NO:4)). In another specific embodiment, the anti-HDM2 antibody described herein specifically binds HDM2 within 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 antibody described herein specifically binds within amino acids 19 to 50 of SEQ ID NO: 4.
  • the anti-HDM2 antibody described herein specifically binds within amino acids 154 to 167 of SEQ ID NO: 4. In yet another specific embodiment, the anti-HDM2 antibody described herein specifically binds within amino acids 1 to 60 of SEQ ID NO: 4. In yet another specific embodiment, the anti-HDM2 antibody described herein specifically binds within amino acids 1 to 100 of SEQ ID NO: 4. In another specific embodiment, the anti-HDM2 antibody described herein specifically binds within amino acids 100 to 110 of SEQ ID NO: 4. In another specific embodiment, the anti-HDM2 antibody described herein specifically binds within amino acids 50 to 60 of SEQ ID NO: 4.
  • the anti-HDM2 antibody described herein specifically binds within amino acids 1 to 109 of SEQ ID NO: 4. In another specific embodiment, the anti-HDM2 antibody described herein specifically binds within amino acids 26 to 60 of SEQ ID NO: 4. In one specific embodiment, the anti-HDM2 antibody described herein specifically binds within the terminal 60 amino acids at the C-terminus of the HDM2 on the plasma membrane of the cancer cells. In another specific embodiment, the anti-HDM2 antibody described herein specifically binds within the terminal 100 amino acids at the C-terminus of the HDM2 on the plasma membrane of the cancer cells.
  • the anti-M(H)DM2/4 antibody described herein does not bind within amino acids 101 to 200 of SEQ ID NO:4. In one embodiment, the anti-M(H)DM2/4 antibody described herein does not bind to the epitope of HDM2 or MDM2 to which “MDM2 monoclonal antibody (M01), clone 1A7” (Abnova, Cat. No. H00004193-M01) binds. In one embodiment, the anti-M(H)DM2/4 antibody described herein does not compete with “MDM2 monoclonal antibody (M01), clone 1A7” (Abnova, Cat. No. H00004193-M01) for binding to HDM2.
  • the anti-M(H)DM2/4 antibody described herein binds within amino acids 101 to 200 of SEQ ID NO:4. In one embodiment, the anti-M(H)DM2/4 antibody described herein binds to the epitope of HDM2 or MDM2 to which “MDM2 monoclonal antibody (M01), clone 1A7” (Abnova, Cat. No. H00004193-M01) binds. In one embodiment, the anti-M(H)DM2/4 antibody described herein competes with “MDM2 monoclonal antibody (M01), clone 1A7” (Abnova, Cat. No. H00004193-M01) for binding to HDM2.
  • the anti-M(H)DM2/4 antibody described herein does not bind within amino acids 153 to 222 of SEQ ID NO:4. In one embodiment, the anti-M(H)DM2/4 antibody described herein does not bind within amino acids 26 to 169 of SEQ ID NO:4. In a specific embodiment, the anti-M(H)DM2/4 antibody described herein does not bind within amino acids 26 to 222 of SEQ ID NO:4.
  • the M(H)DM2/4 exposed on the surface of cancer cells being targeted by the antibodies or fragments described herein is an M(H)DM2/4 variant that lacks one or more nuclear localization signal domains.
  • the HDM2 exposed on the surface of cancer cells being targeted by the antibodies or fragments described herein is an HDM2 variant that lacks the sequence of amino acids 179 to 185 of SEQ ID NO: 4 and/or the sequence of amino acids 464 to 471 of SEQ ID NO: 4.
  • the HDM2 exposed on the surface of cancer cells being targeted by the antibodies or fragments described herein is an HDM2 variant that lacks the sequence of amino acids 181 to 185 of SEQ ID NO: 4.
  • antibodies or fragments thereof that compete for binding to M(H)DM2/4 with an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment thereof described herein.
  • anti-M(H)DM2/4 e.g., anti-HDM2
  • such antibodies or fragments that compete for binding are monoclonal antibodies or fragments thereof.
  • antibodies or fragments thereof that compete 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.
  • 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 (
  • antibodies or fragments thereof that: (i) compete for binding to a peptide of sequence SEQ ID NO:1 with a mouse 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; or (ii) compete for binding to a peptide of SEQ ID NO:2 with a mouse 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; or (iii) compete for binding to a peptide of SEQ ID NO:3 with a mouse 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.
  • compositions comprising a therapeutically effective amount of any antibody or fragment described herein.
  • kits 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 a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide); or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein.
  • any anti-M(H)DM2/4 antibody described herein comprising administering to the subject: (i) any anti-M(H)DM2/4 antibody described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H
  • provided herein are methods for treating cancer in a subject in need thereof, said method comprising administering to the subject: an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a cytotoxic component.
  • an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a cytotoxic component.
  • methods 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 a fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein.
  • a cell-penetrating peptide e.g., a membrane resident peptide
  • an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug,
  • the method comprises administering to the subject an antibody-drug conjugate comprising the antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide.
  • 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, wherein the sequence of the peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3).
  • provided herein is a method for treating cancer in a subject in need thereof, said 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 a peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
  • 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 a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein.
  • a cell-penetrating peptide e.g., a membrane resident peptide
  • an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating
  • provided herein are methods for inhibiting tumor growth in a subject in need thereof, said method comprising administering to the subject: an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a cytotoxic component.
  • an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a cytotoxic component.
  • 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 a fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetraitng peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein.
  • a cell-penetrating peptide e.g., a membrane resident peptide
  • an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetraitng peptide) bound to a
  • 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, wherein the sequence of the peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3).
  • provided herein is a method for inhibiting tumor growth in a subject in need thereof, said 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 a peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
  • provided herein are methods for inhibiting tumor progression in a subject in need thereof, said method comprising administering to the subject: (i) any anti-M(H)DM2/4 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein.
  • a cell-penetrating peptide e.g., a membrane resident peptide
  • an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-
  • provided herein are methods for inhibiting tumor progression in a subject in need thereof, said method comprising administering to the subject: an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a cytotoxic component.
  • an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a cytotoxic component.
  • 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 a fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein.
  • a cell-penetrating peptide e.g., a membrane resident peptide
  • an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a
  • the method comprises administering to the subject an antibody-drug conjugate comprising the antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide.
  • 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 antibody or fragment specifically binds to a peptide, wherein the sequence of the peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3).
  • a method for preventing cancer recurrence or relapse 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 a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein.
  • any anti-M(H)DM2/4 antibody or fragment described herein comprising administering to the subject: (i) any anti-M(H)DM2/4 antibody or fragment described herein; (ii) an antibody or a fragment thereof
  • a method for preventing cancer recurrence or relapse in a subject in need thereof comprising administering to the subject: an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a cytotoxic component.
  • an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 e.g., HDM2
  • a method for preventing cancer recurrence or relapse in a subject in need thereof comprising administering to the subject: (i) any anti-HDM2 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein.
  • any anti-HDM2 antibody or fragment described herein comprising administering to the subject: (i) any anti-HDM2 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of HD
  • a method for preventing cancer recurrence or relapse 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, wherein the sequence of the peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3).
  • a method for increasing survival in a subject having a cancer comprising administering to the subject: (i) any anti-M(H)DM2/4 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein.
  • a cell-penetrating peptide e.g., a membrane resident peptide
  • an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptid
  • a method for increasing survival in a subject a cancer comprising administering to the subject: (i) any anti-HDM2 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein.
  • any anti-HDM2 antibody or fragment described herein comprising administering to the subject: (i) any anti-HDM2 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, wherein said antibody or fragment is not
  • the method comprises administering to the subject an antibody-drug conjugate comprising the antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide.
  • kits for preventing metastasis in a subject in need thereof comprising administering to the subject: an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a cytotoxic component.
  • an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 e.g., HDM2
  • the method comprises administering to the subject an antibody-drug conjugate comprising the antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide.
  • a method for preventing metastasis in a subject having a 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 specifically binds to a peptide, wherein the sequence of the peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3).
  • a method for preventing metastasis in a subject having a 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 a peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
  • provided herein are methods for inhibiting metastasis in a subject in need thereof, said method comprising administering to the subject: an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a cytotoxic component.
  • an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a cytotoxic component.
  • a method for inhibiting metastasis comprising administering to the subject: (i) any anti-HDM2 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein.
  • any anti-HDM2 antibody or fragment described herein comprising administering to the subject: (i) any anti-HDM2 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, wherein said antibody or fragment is not bound to
  • the method comprises administering to the subject an antibody-drug conjugate comprising the antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide.
  • the antibody or fragment thereof in step (b) can be the same or different from the antibody or fragment thereof in step (a).
  • methods that further comprise, before step (b), a step of determining whether the antibody or fragment binds to the surface of the intact cell of the cancer e.g., using FACS or cell-based ELISA analysis) (using any anti-M(H)DM2/4 antibody described herein).
  • methods that further comprise, before the determining step, the step of obtaining intact cells of the cancer e.g., by biopsy of the cancerous tumor in the subject, or by obtaining a blood sample with circulating cancer cells from the subject).
  • a subject e.g., a human
  • methods for selecting a subject having a cancer for treatment comprising: (a) selecting a subject having a cancer for treatment by: (i) obtaining an intact cancer cell from the subject (e.g., by biopsy of the cancerous tumor in the subject, or by obtaining a blood sample with circulating cancer cells from the subject), and (ii) determining whether an antibody or a fragment thereof (e.g., a labeled antibody or fragment) 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, such as any anti-M(H)DM2/4 antibody or fragment described herein) binds to the surface of the intact cancer cell obtained from the subject (e.g., using FACS or cell-based ELISA analysis), and (b) if the binding is detected in step (a), administering to the subject said antibody or fragment, wherein said antibody
  • a subject for treatment and treating cancer in the subject comprising: (a) selecting a subject having a cancer for treatment by: (i) obtaining an intact cancer cell from the subject (e.g., by biopsy of the cancerous tumor in the subject, or by obtaining a blood sample with circulating cancer cells from the subject), and (ii) determining whether an antibody or a fragment thereof that specifically binds to HDM2 (e.g., an antibody or fragment that specifically binds to an extracellularly accessible epitope of HDM2, such as any anti-M(H)DM2/4 antibody or fragment described herein) binds to the surface of the intact cancer cell obtained from the subject (e.g., using FACS or cell-based ELISA analysis), and (b) if the binding is detected in step (a), administering to the subject said antibody or fragment, wherein said antibody or fragment is not bound to a cell-penetrating peptide.
  • HDM2 e.g., an antibody or fragment that specifically binds to an extracellularly accessible epitop
  • the cancer treated in accordance with the methods described herein is a type of cancer that is known to metastasize.
  • the cancer treated in accordance with the methods described herein is an advanced stage cancer.
  • the cancer treated in accordance with the methods described herein is an early stage cancer.
  • the cancer treated in accordance with the methods described herein is a metastatic cancer.
  • the cancer being treated can be a solid cancer or a non-solid cancer (e.g., leukemia).
  • the subject treated in accordance with the methods described herein can be a human or a non-human animal (such as a mammal). In a preferred embodiment, the subject is a human.
  • 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.
  • the subject being treated in accordance with the methods described herein is further administered chemotherapy, wherein the chemotherapy is gemcitabine (e.g., where the cancer being treated is a pancreatic cancer).
  • the subject being treated in accordance with the methods described herein is further administered chemotherapy, wherein the chemotherapy is nab-paclitaxel (e.g., where the cancer being treated is a pancreatic cancer).
  • the cancer is a pancreatic cancer, and the subject being treated in accordance with the methods described herein is further administered chemotherapy, wherein the chemotherapy is a combination of gemcitabine and nab-paclitaxel.
  • gemcitabine is administered in a dose that is equal to or less than 1,000 mg/m 2 and/or the nab-paclitaxel is administered in a dose that is equal to or less than 125 mg/m 2 . In one embodiment, wherein the subject is human, gemcitabine is administered in a dose that is equal to or less than 500 mg/m 2 and/or the nab-paclitaxel is administered in a dose that is equal to or less than 62.5 mg/m 2 . In certain embodiments, the combination of gemcitabine and nab-paclitaxel is administered with a frequency of every 2 weeks or less.
  • the anti-M(H)DM2/4 antibody used in the methods described herein specifically binds within amino acids 1 to 109 of SEQ ID NO:4. In another specific embodiment, the anti-M(H)DM2/4 antibody used in the methods described herein specifically binds within amino acids 26 to 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 the 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 the 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 to 200 of SEQ ID NO:4.
  • the anti-M(H)DM2/4 antibody or fragment used in the methods described herein competes for binding to M(H)DM2/4 with mouse anti-HDM2 antibody OP145 (which is described herein, see, e.g., Table 10).
  • the anti-M(H)DM2/4 antibody or fragment used in the methods described herein competes for binding to M(H)DM2/4 with mouse anti-HDM2 antibody 965 (SMP14) (which is described herein, see, e.g., Tables 3 and 10).
  • the anti-M(H)DM2/4 antibody or fragment used in the methods described herein competes for binding to M(H)DM2/4 with rabbit anti-HDM2 antibody sc-813 (N-20) (which is described herein, see, e.g., Table 10).
  • the anti-M(H)DM2/4 antibody or fragment used in the methods described herein competes for binding to M(H)DM2/4 with rabbit anti-HDM2 antibody sc-812 (C-18) (which is described herein, see, e.g., Table 10).
  • kits for treating cancer or preventing metastases in a subject in need thereof comprising administering to the subject any anti-M(H)DM2/4 antibody described herein (such as an antibody that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 on the surface of cells of said cancer), wherein the antibody comprises a human IgG Fc region that mediates complement-dependent cytotoxicity (CDC) and/or antibody-dependent cell-mediated cytotoxicity (ADCC).
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • 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 of SEQ ID NO: 1 (which are amino acids 1 to 15 of SEQ ID NO:4), amino acids of SEQ ID NO: 2 (which are amino acids 15 to 25 of SEQ ID NO:4), amino acids of SEQ ID NO: 3 (which are amino acids 475 to 491 of SEQ ID NO:4), amino acids 19 to 50 of SEQ ID NO: 4, amino acids 50 to 60 of SEQ ID NO: 4, amino acids 100 to 110 of SEQ ID NO: 4, amino acids 154 to 167 of SEQ ID NO: 4, amino acids 1 to 60 of SEQ ID NO: 4, or the terminal 60 amino acids at the C-terminus of the HDM2 on the plasma membrane of the cancer cells.
  • the cancer is a leukemia, a lung cancer, a colon cancer, a melanoma, a pancreatic cancer,
  • the method of diagnosing is an ex vivo method. In one embodiment, the method of diagnosing further comprises, before step (a), obtaining intact cells from the subject. In one embodiment, the method of diagnosing comprises administering the antibody or fragment to the subject before the detecting in step (a), and wherein the detecting is performed by in vivo imaging of the subject.
  • M(H)DM2 refers to HDM2, MDM2, or an E3 ubiquitin-protein ligase from species other than human and mouse that is a homolog of HDM2 or MDM2.
  • MDM4 refers to the mouse protein of UniProt Accession Number O35618 (i.e., full-length MDM4 protein) or a protein product of 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 shown as SEQ ID NO:6.
  • Other splice variants of the full length MDM4 protein known in the art include, without limitation MDM4-S, MDM4-A, MDM4-G, MDM4-XALT1/XALT2 and MDM4-211.
  • M(H)DM4 refers to HDM4 (also called HDMX), MDM4 (also called MDMX), or a protein from a species other than human and mouse that is a homolog of HDM4 or MDM4.
  • the term “about,” when used to modify a numeric value, indicate that deviations of up to 10% above and below the numeric value remain within the intended meaning of the recited value.
  • the term “intact” with reference to a cell refers to a cell that is viable or fixed but not permeabilized.
  • extracellularly accessible with reference to an epitope of M(H)DM2/4, refers to an epitope of M(H)DM2/4 that, when the M(H)DM2/4 is expressed by an intact cell, the epitope is available for binding with an extracellular antibody (without a need for intracellular transport of the antibody).
  • An antibody or a fragment thereof can be determined to bind to an extracellularly accessible epitope of M(H)DM2/4, when the antibody, when extracellular, binds to M(H)DM2/4 expressed by an intact cell.
  • VL refers to the light chain variable region of an antibody.
  • VH refers to the heavy chain variable region of an antibody.
  • 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 with the 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 achieved in various ways that are known in the art, for instance, using publicly available computer software such as BLASTp, BLAST-2, ALIGN (e.g., ALIGN-2) or Megalign (DNASTAR) software.
  • FIGS. 1A-D show that monoclonal antibodies NMC-103, NMC-204 and NMC-303 specifically bound to 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.
  • C NMC-303 bound to NMC-P3 peptide while NMC-204 did not bind to NMC-P3.
  • FIG. 2 shows that monoclonal antibodies NMC-103, NMC-204, NMC-303 bound to HDM2 recombinant protein.
  • FIGS. 3A-B show that monoclonal antibody NMC-103 bound to an extracellularly accessible epitope of HDM2 on intact human (A) and murine (B) cancer cells.
  • FIGS. 4A-B show that monoclonal antibody NMC-204 bound to an extracellularly accessible epitope of HDM2 on intact human (A) and murine (B) cancer cells.
  • FIGS. 5A-B show that monoclonal antibody NMC-303 bound to an extracellularly accessible epitope of HDM2 on intact human (A) and murine (B) cancer cells.
  • FIG. 6 shows that monoclonal antibody NMC-204 bound to an extracellularly accessible epitope of HDM2 on intact human cancer cells but did not bind to intact normal human peripheral blood mononuclear cells.
  • FIGS. 7A-B depict the binding curves of the binding of monoclonal antibodies NMC-103 (A) and NMC-204 (B) to intact MIA PaCa-2 cells.
  • FIGS. 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 MIA PaCa-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 MIA PaCa-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 MIA PaCa-2 cells was competed by the NMC-P3 peptide (C).
  • 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 MIA PaCa-2 cells was competed by the full-length recombinant HDM2 protein.
  • 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 MiaPaCa-2 cells.
  • FIGS. 11A-C present flow cytometry data on % cells stained with monoclonal antibodies NMC-103 (A), NMC-204 (B), and anti-Na+/K+ ATPase ⁇ -1 (C), respectively.
  • FIGS. 12A-D show that monoclonal antibodies NMC-103 (A and D) and NMC-204 (B and D), but not an anti-Cytochrome-C antibody (C and D), inhibited cell proliferation of intact human pancreatic MIAPaCa-2 cells.
  • FIGS. 13A-C show that monoclonal antibody NMC-103 (B and C) in the presence of normal human serum induces complement-mediated cytotoxicity against human pancreatic MIAPaCa-2 cells as compared with cells treated with normal human serum in the absence of any antibody (A).
  • FIGS. 14A-B show the lack of binding of many commercially available monoclonal antibodies to either NMC-P1 (A) or NMC-P2 (B).
  • FIGS. 15A-B show that an anti-HDM2 antibody termed “MDM2 monoclonal antibody (M01), clone 1A7” (Abnova, Cat. No. H00004193-M01) reacted with intact cancer cells (A), but an anti-HDM2 antibody termed “Anti-MDM2 (Ab-4) Mouse mAb (2A9C1.18)” (EMD Millipore, Cat. No. OP144) and an anti-HDM2 antibody termed “Anti-MDM2 (Ab-1) Mouse mAb (IF2)” (EMD Millipore, Cat. No. OP46) did not react with intact cancer cells (B).
  • MDM2 monoclonal antibody M01
  • clone 1A7 Abnova, Cat. No. H00004193-M01
  • Anti-MDM2 Ab-4 Mouse mAb (2A9C1.18)
  • Anti-M2 antibody termed “Anti-MDM2 (Ab-1) Mouse mAb (IF2)” EMD Millipore, Cat.
  • FIG. 16 depicts the effect of monoclonal antibody NMC-204 on tumor volume of the LL/2 syngeneic mouse model of lung cancer.
  • FIGS. 17A-B depict the effect of monoclonal antibody NMC-103 on tumor volume (A) and tumor cell proliferation (B) of the MC-38 syngeneic mouse model of colon cancer.
  • FIGS. 18A-B depict the effect of monoclonal antibody NMC-204 on tumor volume (A) and tumor cell proliferation (B) of the MC-38 syngeneic mouse model of colon cancer.
  • FIG. 19 depicts the effect of monoclonal antibody NMC-103 alone (2 mg/kg), a combination of low dose Gemcitabine (25 mg/kg) and nab-Paclitaxel (5 mg/kg), a 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), respectively, on tumor volume of the Panc-2 syngeneic mouse model of pancreatic cancer. Treatment started when tumors in mice reached approximately 70 mm 3 .
  • FIG. 20 depicts the DNA sequence and protein sequence of the heavy chain variable region and the light chain variable region, respectively, of monoclonal antibody NMC-103.
  • FIG. 21 depicts the DNA sequence and protein sequence of the heavy chain variable region and the light chain variable region, respectively, of monoclonal antibody NMC-204.
  • FIG. 22 depicts the DNA sequence and protein sequence of the heavy chain variable region and the light chain variable region, respectively, of monoclonal antibody NMC-303.
  • the leader sequence before the DNA and protein sequences of the heavy chain variable region and the light chain variable region is in bold (but not underlined).
  • FIG. 23 shows the tumor size of mice treated with the anti-HDM2 antibody termed “MDM2 monoclonal antibody (M01), clone 1A7” (Abnova, Cat. No. H00004193-M01), the tumor size of mice treated with NMC-103, the tumor size of mice treated with NMC-204, and the tumor size of mice treated with isotype control.
  • MDM2 monoclonal antibody (M01), clone 1A7” Abnova, Cat. No. H00004193-M01
  • FIGS. 24A-F Anti-HDM2-specific antibodies stain the surface of cancer cells but not normal cells. Intact cells released either with either EDTA or Trypsin were blocked with 5% human serum albumin. Cells were then incubated with either polyclonal N-20 M(H)DM2-specific antibody (sc-813, N-20, rabbit IgG; from Santa Cruz; “N-20”) or monoclonal M(H)DM2-specific OP145 antibody (0P145, mouse IgG1; from Calbiochem; “OP145”) for 90 min. on ice. Another set of cells prepared under the same conditions were incubated with the same antibodies that were pre-incubated with their corresponding blocking peptides before incubation with cells.
  • polyclonal N-20 M(H)DM2-specific antibody sc-813, N-20, rabbit IgG; from Santa Cruz; “N-20”
  • monoclonal M(H)DM2-specific OP145 antibody (0P145, mouse IgG1; from Calbiochem; “OP145”
  • area under curve #1 represents cells incubated with goat anti-rabbit secondary antibody only; area under 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 curve #3 represents cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody.
  • FIG. 24A area under curve #1 represents cells incubated with goat anti-rabbit secondary antibody only; area under 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 curve #3 represents cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody.
  • area under curve #1 represents cells incubated with goat anti-mouse secondary antibody only; area under 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 curve #3 represents cells incubated with anti-HDM2 monoclonal antibody OP145 followed by goat anti-mouse secondary antibody.
  • FIG. 24B area under curve #1 represents cells incubated with goat anti-mouse secondary antibody only; area under 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 curve #3 represents cells incubated with anti-HDM2 monoclonal antibody OP145 followed by goat anti-mouse secondary antibody.
  • area under curve #1 represents cells incubated with goat anti-rabbit secondary antibody only; area under curve #2 represents trypsin-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody; area under 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 curve #4 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody.
  • area under curve #1 represents cells incubated with goat anti-rabbit secondary antibody only; area under 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 curve #3 represents cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody.
  • FIG. 24D & E area under curve #1 represents cells incubated with goat anti-rabbit secondary antibody only; area under 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 curve #3 represents cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody.
  • area under curve #1 represents cells incubated with goat anti-rabbit secondary antibody only; area under curve #2 represents trypsin-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody; area under 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 curve #4 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody.
  • FIGS. 25A-C Human pancreatic or ovarian cancer cells and normal human fibroblasts were treated with normal human serum (NHS) alone, NHS+anti-HDM2 OP145 monoclonal antibody (mouse IgG1, from Calbiochem, “OP145”) or control antibody (NHS+Cytochrome C). Extensive cell death as evident by Propidium Iodide (PI) staining was observed when cancer cells were treated with the OP145 antibody (see panels b and e) in the presence of NHS, whereas the same antibody had no effect on the viability of normal human fibroblasts (see panel g).
  • NHS normal human serum
  • OP145 normal human serum
  • NHS+Cytochrome C control antibody
  • PI Propidium Iodide
  • Control antibody to Cytochrome C shows no cytotoxicity (see panel c) beyond that observed in untreated cells (see panel a). Lack of cell death is manifested by no or little PI staining in panels a, c, d, f and g.
  • the cell death marker PI was visualized using Olympus FluoView FV1000 Confocal Laser Scanning Biological Microscope built on the Olympus IX81 Inverted Microscope.
  • M(H)DM2-specific antibodies are cytotoxic to pancreatic cancer MiaPaCa-2 cells in the presence of NHS. Quantitative representations 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 demonstrated cytotoxicity over 15-30 min. post-treatment, whereas anti-HDM2 OP46 shows no cytotoxic effect beyond that observed when cells were treated with control anti-Cytochrome C antibody or when cells were treated with NHS in the absence of anti-M(H)DM2 antibodies.
  • FIG. 26 shows the tumor size of mice treated with anti-HDM2 antibody OP145 and the tumor size of mice treated with PBS control (in Panc02 syngeneic mouse model of pancreatic cancer).
  • the x axis shows days after tumor cell injection into the mice.
  • the y axis shows tumor volume in mm 3 .
  • the arrow shows the day on which the treatment was started.
  • FIG. 28 depicts a Kaplan Meier survival analysis demonstrating survival benefit in mice that received NMC-103 alone or in combination with chemotherapy when compared to chemotherapy alone or control antibody under the experimental conditions described in FIG. 27 .
  • FIG. 29 shows that mice previously treated with NMC-103 as described in FIG. 27 , become immune to tumor re-challenging after drug withdrawal.
  • mice that had previously received with a combination of G+nP mice in group C (mice that had been previously treated with NMC-103) and group D (mice that had been previously treated with a combination of NMC-103+G+nP) were re-challenged by a second round of Panc-2 inoculation (subcutaneous injection of 2 ⁇ 10 6 cells/mouse), on the left dorsal flank. Tumor growth was monitored for 10 days at which point, a tumor of 90 mm 3 was measured in the mice from group B. No tumor was observed in mice from the two groups that had previously received NMC-103 antibody (Groups C and D).
  • FIG. 30 depicts the effect of a single dose of monoclonal antibody NMC-103 (10 mg/kg) or isotype control mouse IgG1 (10 mg/kg), when added to a treatment regimen of a combination of low dose Gemcitabine (25 mg/kg) and nab-Paclitaxel (5 mg/kg), in the treatment of large size tumors (i.e. advanced cancers) on tumor volume of the Panc-2 syngeneic mouse model of pancreatic cancer. Mice were treated with pancreatic cancer standard of care (Gemcitabine (25 mg/kg)+nab-Paclitaxel (5 mg/kg)) for 19 days at which point they reached a tumor size of approximately 450 mm 3 .
  • mice were then randomly divided in 2 groups that received a single dose of an isotype control mouse IgG1 (10 mg/kg) or NMC-103 (10 mg/kg). As shown in this figure, a single i.p. injection of NMC-103 reduced the tumor size by almost half 6 days post treatment (from 438 mm 3 to 233 mm 3 ).
  • FIG. 31 depicts the effect of monoclonal antibody NMC-103 on tumor volume of the MC-38 syngeneic mouse model of colon cancer.
  • mice treated with NMC-103 at 10 mg/kg, 2 times per week for 2 weeks reached an average tumor size of 210 mm 3
  • mice in the group that were treated with isotype control antibody at 10 mg/kg grew rapidly and reached 1168 mm 3 by day 12.
  • FIG. 17 When compared to mice treated with NMC-103 at 0.4 mg/kg ( FIG. 17 ), these data support the dose-dependent anti-tumor effect of NMC-103 antibody.
  • FIGS. 32A and 32B depict the effect of a chimeric version of monoclonal antibody NMC-303.
  • Isotype class-switching was performed on a mouse NMC-303 to convert it from a mouse IgM to a chimeric IgG1.
  • the mouse Heavy and Light chain variable regions were cloned into a human Ig gamma-1 chain and human Ig kappa chain as constant region.
  • FIG. 32A shows that by day 24 post tumor inoculation, mice treated with chimeric version of NMC-303 (10 mg/kg) reached an average tumor size of 726 mm3, while mice treated with control antibody (10 mg/kg) had an average tumor size of 1746 mm3. Furthermore, FIG. 32B shows the individual mouse tumor sizes on day 24 post tumor inoculation.
  • antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2).
  • the extracellularly accessible epitope is contained within SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
  • Antibodies provided herein are described in Section 5.1, below.
  • 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 bound (e.g., covalently) to a cytotoxic drug.
  • antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibodies or fragments are not bound to a cytotoxic component.
  • nucleic acids encoding the antibodies and antigen-binding fragments described herein.
  • vectors and cells comprising nucleic acids encoding such antibodies or antigen-binding fragments thereof.
  • Cells recombinantly producing the antibodies or antigen-binding fragments thereof described herein are also provided.
  • Chimeric antigen receptors are engineered receptors that provide both antigen binding and immune cell activation functions (Sadelain et al., 2013, Cancer Discovery 3:388-398).
  • CARs comprising a single-chain variable fragment (scFv) that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, such as a scFv comprising the VH and VL of an anti-M(H)DM2/4 antibody described herein, fused via a linker to a transmembrane domain (e.g., of CD3 zeta) fused to an intracellular T cell activation domain such as CD3 zeta intracellular domain, optionally further fused to a co-stimulatory domain (e.g., CD28 intracellular domain).
  • T cells expressing such CARs are also provided.
  • a peptide the amino acid sequence of which is MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3).
  • the peptide can be, for example, synthetic or recombinant.
  • the peptide is purified.
  • the peptide islabeled with a detectable marker (e.g., a fluorescent marker or an isotope).
  • the peptide is tagged (e.g., with a GST, His, Strep, myc, FLAG, or HA tag).
  • a cysteine is added at one of the ends of the peptide(which may allow for linkage to a carrier protein).
  • the peptide islinked to a carrier protein (e.g., linked to Keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, thyroglobulin, tetanus toxoid, or diphtheria toxoid).
  • KLH Keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • ovalbumin ovalbumin
  • thyroglobulin thyroglobulin
  • tetanus toxoid tetanus toxoid
  • diphtheria toxoid diphtheria toxoid.
  • nucleic acids encoding a peptide described herein.
  • vectors and cells comprising a nucleic acid encoding a peptide described herein. Cells recombinantly producing a
  • 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.
  • an anti-M(H)DM2/4 antibody e.g., an antibody that specifically binds to M(H)DM2/4
  • an animal e.g., a mouse or a rabbit
  • methods of screening antibodies for binding to one or more of thepeptides described herein for example, using ELISA with a plate-bound peptide.
  • an anti-M(H)DM2/4 antibody suitable for therapeutic 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 between the antibody and the peptide that occurs, and, if the binding between the antibody and the peptide is detected, using the antibody in the methods of treating cancer described herein.
  • provided herein are methods of 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 between the antibody and the peptide that occurs, and, if the binding between the antibody and the peptide is detected, using the antibody in the methods of diagnosing cancer described herein.
  • an anti-M(H)DM2/4 antibody or fragment described herein in particular, an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (in particular, a region exposed on the plasma membrane surface of cancer cells).
  • the antibody or fragment thereof specifically binds to an extracellularly accessible epitope of HDM2 (in particular, a region exposed on the plasma membrane surface of cancer cells).
  • the antibody or fragment 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.
  • an antibody or a fragment thereof used herein specifically binds to an extracellularly accessible epitope of HDM4 (a region exposed on the plasma membrane surface of cancer cells) (optionally, such antibody or fragment that does not bind to HDM2).
  • an antibody or a 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 the M(H)DM2/4 is a homologue of HDM2 and/or HDM4 expressed in such animal.
  • an antibody or a 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 the M(H)DM2 is a homologue of HDM2 expressed in such animal (optionally, such antibody or fragment does not bind to M(H)DM4).
  • an antibody or a fragment thereof used herein binds to an extracellularly accessible of M(H)DM4 (a region exposed on the plasma membrane surface of cancer cells), where the M(H)DM4 is a homologue of HDM4 expressed in such animal (optionally, such antibody or fragment does not bind to M(H)DM2).
  • the anti-HDM2 antibody or a fragment thereof used in accordance with the methods described herein mediates complement-dependent cytotoxicity (CDC), mediates antibody-dependent cell-mediated cytotoxicity (ADCC), and/or is bound to a cytotoxic drug or drugs (e.g., is an antibody-drug conjugate).
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the invention provides for the use of antibodies that mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the anti-HDM2 antibody or a fragment thereof used in accordance with the methods described herein is not bound to a cell-penetrating peptide.
  • Cell penetrating peptides can insert into a cell plasma membrane and transport molecules to which they are attached into the cell.
  • Such cell-penetrating peptides include, without limitation, a membrane resident peptide (MRP), Membrane Transduction Domain of Antennapedia, trans-activating transcriptional activator (TAT), and a Penetratin peptide.
  • the anti-HDM2 antibody or a fragment thereof used in accordance with the methods described herein is not attached to a membrane resident peptide (MRP), Membrane Transduction Domain of Antennapedia, TAT, and/or a Penetratin peptide.
  • MRP membrane resident peptide
  • the anti-HDM2 antibody or a fragment thereof used in accordance with the methods described herein is not attached to any peptide sequence that can insert into the lipid bilayer of the plasma membrane of cells.
  • the anti-HDM2 antibody or a fragment thereof used in accordance with the methods described herein is not attached to an MRP.
  • the anti-HDM2 antibody or a fragment thereof used in accordance with the methods described herein is not attached to a Penetratin peptide.
  • HDM-2 targeting antibodies alone are selectively cytotoxic to cancer cells.
  • extracellularly accessible epitopes of HDM2 are appropriate therapeutic targets for anti-HDM2 antibodies, and that cancer cells expressing HDM2 on their surface can be successfully targeted and destroyed with antibodies to such extracellular regions of HDM2.
  • select HDM2-specific antibodies can bind to the extracellularly accessible sequences of HDM2 on the surface membrane of intact cancer cells, while exhibiting minimal binding to the surface membrane of normal human blood mononuclear cells.
  • 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. Further, the data presented in the examples show that such HDM2-specific antibodies can have a synergistic anti-tumor effect when combined with chemotherapeutic drugs.
  • antibodies or antigen-binding fragments thereof that (immuno) specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) (a region exposed on the plasma membrane surface of cells).
  • bind[s]/binding does not exclude cross-reactivity of the antibody or antigen-binding fragment; thus, for example, antibodies or antigen-binding fragments thereof that (immuno) specifically bind to an extracellularly accessible epitope of HDM2 exposed on the plasma membrane surface of cells may also specifically bind to (cross-react with) MDM2.
  • anti-M(H)DM2/4 antibodies and fragments thereof that (immuno) specifically bind to an extracellularly accessible epitope of M(H)DM2/4 and that have an anti-tumor effect (e.g., inhibit tumor growth in vivo).
  • an antibody or an antigen-binding fragment thereof specifically binds an epitope of M(H)DM2/4 that is extracellularly accessible on cancer cells but not on non-cancer cells (e.g., non-cancerous cells of the same organ type or tissue type as the cancer cells).
  • an antibody or an antigen-binding fragment thereof specifically binds an epitope of M(H)DM2/4, exposure or accessibility of which on the plasma membrane surface of cancer cells is increased relative to its exposure or accessibility on the plasma membrane surface of non-cancer cells (e.g., non-cancerous cells of the organ or tissues of the host).
  • antibodies or antigen-binding fragments thereof that (immuno) specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), which are not bound to a cell-penetrating peptide (e.g., a membrane resident peptide).
  • antibodies or antigen-binding fragments thereof that (immuno) specifically bind to M(H)DM2/4 (e.g., HDM2), in particular to an extracellularly accessible epitope of M(H)DM2/4, wherein the antibody or fragment specifically binds to a peptide the sequence of which peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3).
  • M(H)DM2/4 e.g., HDM2
  • the antibody or fragment specifically binds to a peptide the sequence of which peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3).
  • antibodies or antigen-binding fragments thereof that (immuno) specifically bind to M(H)DM2/4 (e.g., HDM2), in particular to an extracellularly accessible epitope of M(H)DM2/4 wherein the antibody or fragment specifically binds to a peptide the sequence of which peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3); and wherein such antibodies or fragments have an anti-tumor effect in vivo, and/or wherein such antibodies or fragments are not bound to a cell-penetrating peptide.
  • M(H)DM2/4 e.g., HDM2
  • the antibody or fragment specifically binds to a peptide the sequence of which peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO
  • anti-M(H)DM2/4 antibodies and fragments having heavy chain variable regions and/or light chain variable regions described herein see, e.g., having sequences of heavy chain variable regions and/or light chain variable regions of antibodies NMC-103, NMC-204 and NMC-303 provided herein, see, e.g., Section 8 and FIGS. 20-22 ).
  • anti-M(H)DM2/4 antibodies and fragments having one or more complementarity determining regions (CDRs) described herein see, e.g., CDRs provided in Tables 4-9 and FIGS. 20-22 ).
  • CDRs are defined in various ways in the art, including the Kabat, Chothia, AbM, Contact, and IMGT.
  • the CDRs of an antibody can be defined according to the Kabat system, which is based on sequence variability (see, e.g., Kabat E A & Wu T T (1971) Ann NY Acad Sci 190: 382-391; Kabat E A et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242.
  • the VH CDR1 is present at amino acid positions 31 to 35 of the heavy chain; (ii) the VH CDR2 is present at amino acid positions 50 to 68 or 50 to 66 of the heavy chain; and (iii) the VH CDR3 is present at amino acid positions 101 to 105 or 99 to 104 or 99 to 106 of the heavy chain.
  • the VL CDR1 is present at amino acid positions 24 to 39 or 24 to 34 of the light chain;
  • the VH CDR2 is present at amino acid positions 55 to 61 or 50 to 56 of the light chain;
  • the VH CDR3 is present at amino acid positions 94 to 102 or 89 to 97 of the light chain.
  • the actual linear amino acid sequence of the antibody variable domain can contain fewer or additional amino acids due to a shortening or lengthening of a framework region (FR) and/or CDR and, as such, an amino acid's Kabat number is not necessarily the same as its linear amino acid number.
  • the Kabat CDR positions may vary depending on the antibody, and may be determined according to methods known in the art.
  • the CDRs of the antibodies described herein are determined using the Kabat system.
  • the CDRs of an antibody can be defined according to the Chothia system, which is based on the location of immunoglobulin structural loop regions (see, e.g., Chothia C & Lesk A M, (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. Pat. No. 7,709,226).
  • Chothia CDRs and like terms are recognized in the art and refer to antibody CDR sequences as determined according to the method of Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917, which will be referred to herein as the “Chothia CDRs” (see also, e.g., U.S. Pat. No. 7,709,226 and Martin, A., “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Dilbel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001)).
  • the VH CDR1 is present at amino acid positions 26 to 32 of the heavy chain;
  • the VH CDR2 is present at amino acid positions 52 to 59 or 52 to 57 of the heavy chain; and
  • the VH CDR3 is present at amino acid positions 101 to 105 or 99 to 104 or 99 to 106 of the heavy chain.
  • the VL CDR1 is present at amino acid positions 24 to 39 or 24 to 34 of the light chain;
  • the VL CDR2 is present at amino acid positions 55 to 61 or 50 to 56 of the light chain;
  • the VL CDR3 is present at amino acid positions 94 to 102 or 89 to 97 of the light chain.
  • the Chothia CDR positions may vary depending on the antibody, and may be determined according to methods known in the art.
  • the CDRs of the antibodies described herein are determined using the Chothia system.
  • the CDRs of an antibody can be defined according to the AbM system, which is based on AbM hypervariable regions that represent a compromise between the Kabat CDRs and Chothia structural loops, and where CDRs are determined using Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.).
  • the VH CDR1 is present at amino acid positions 26 to 35 of the heavy chain;
  • the VH CDR2 is present at amino acid positions 50 to 61 or 50 to 59 of the heavy chain; and
  • the VH CDR3 is present at amino acid positions 101 to 105 or 99 to 104 or 99 to 106 of the heavy chain.
  • the VL CDR1 is present at amino acid positions 24 to 39 or 24 to 34 of the light chain;
  • the VH CDR2 is present at amino acid positions 55 to 61 or 50 to 56 of the light chain;
  • the VH CDR3 is present at amino acid positions 94 to 102 or 89 to 97 of the light chain.
  • the AbM CDR positions may vary depending on the antibody, and may be determined according to methods known in the art.
  • the CDRs of the antibodies described herein are determined using the AbM numbering system.
  • 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, adjoin, 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, both of which are incorporated herein by reference in their entirety).
  • IMGT® the international ImMunoGeneTics information System® website imgt.org, founder and director: Marie-Paule Lefranc, adjoin, 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, both of which are incorporated herein by reference in their
  • the VH CDR1 is present at amino acid positions 27 to 33 or 26 to 33 of the heavy chain;
  • the VH CDR2 is present at amino acid positions 51 to 60 or 51 to 58 of the heavy chain;
  • the VH CDR3 is present at amino acid positions 99 to 105 or 97 to 103 of the heavy chain.
  • the VL CDR1 is present at amino acid positions 27 to 37 of the light chain;
  • the VH CDR2 is present at amino acid positions 55 to 57 of the light chain; and
  • the VH CDR3 is present at amino acid positions 94 to 102 of the light chain.
  • the IMGT CDR positions may vary depending on the antibody, and may be determined according to methods known in the art. In a specific embodiment, the CDRs of the antibodies described herein are determined using the IMGT system.
  • the CDRs of an antibody can be defined according to the Contact system.
  • the Contact definition is based on an analysis of the available complex crystal structures (bioinf.org.uk/abs) (see MacCallum R M et al., (1996) J Mol Biol 5: 732-745; see also, e.g., Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering , Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001)).
  • the VH CDR1 is present at amino acid positions 30 to 35 of the heavy chain;
  • the VH CDR2 is present at amino acid positions 47 to 61 or 47 to 59 of the heavy chain; and
  • the VH CDR3 is present at amino acid positions 99 to 104 or 97 to 103 or 97 to 105 of the heavy chain.
  • the VL CDR1 is present at amino acid positions 30 to 41 or 30 to 36 of the light chain;
  • the VH CDR2 is present at amino acid positions 51 to 60 or 46 to 55 of the light chain;
  • the VH CDR3 is present at amino acid positions 94 to 101 or 89 to 96 of the light chain.
  • the Contact CDR positions may vary depending on the antibody, and may be determined according to methods known in the art. In a specific embodiment, the CDRs of the antibodies described herein are determined using the Contact system.
  • antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise CDRs of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303), which are defined according to any of the above-described systems.
  • antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise 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 (such as NMC-103, NMC-204, or NMC-303).
  • VHs contain VH CDRs surrounded by framework regions (the CDR and FR sequences appear in the following sequence in the VH: FR1-VH CDR 1-FR2-VH CDR 2-FR3-VH CDR 3-FR4), optionally the framework regions are human framework regions.
  • antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise 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).
  • VH heavy chain variable region
  • antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise 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).
  • VH heavy chain variable region
  • antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise 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).
  • VH heavy chain variable region
  • such antibody or fragment is a humanized antibody or fragment.
  • antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise 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 (such as NMC-103, NMC-204, or NMC-303).
  • VLs contain VL CDRs surrounded by framework regions (the CDR and FR sequences appear in the following sequence in the VL: FR1-VL CDR 1-FR2-VL CDR 2-FR3-VL CDR 3-FR4); optionally the framework regions are human framework regions.
  • antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise 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).
  • VL light chain variable region
  • antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise 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).
  • VL light chain variable region
  • antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise 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).
  • VL light chain variable region
  • such antibody or fragment is a humanized antibody or fragment.
  • antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise 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 (such as NMC-103, NMC-204, or NMC-303) and comprise a light chain variable region (VL) having one, two or all three VL CDRs (preferably all three VL CDRs) of such anti-HDM2 antibody.
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H3 VH CDR3
  • provided herein is an antibody or a 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 (providing VL CDRs of NMC-103).
  • an antibody or a fragment thereof that specifically binds to HDM2 and comprises a haeavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 4; and comprises a light chain variable region (VL) having one, two or all threeVL CDRs identified in Table 5.
  • VH haeavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H3 VH CDR3
  • provided herein is an antibody or a 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 (providing VL CDRs of NMC-204).
  • an antibody or a 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 comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 7.
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H3 VH CDR3
  • provided herein is an antibody or a 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 (providing VL CDRs of NMC-303).
  • an antibody or a 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 comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 9.
  • VH heavy chain variable region
  • VL light chain variable region
  • an antibody or fragment thereof that specifically binds to HDM2 comprising a VH of any antibody described herein, such as a VH of any antibody provided in Section 8 or FIGS. 20-22 (e.g., the VH of NMC-103, the VH of NMC-204, or the VH of NMC-303), or a VH having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto.
  • a VH of any antibody described herein such as a VH of any antibody provided in Section 8 or FIGS.
  • VL of NMC-103, the VL of NMC-204, or the VL of NMC-303 a VL having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto.
  • substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the FRs).
  • an antibody or fragment thereof that specifically binds to HDM2 comprising a VH and a VL of any antibody described herein, such as a VH and VL of any antibody provided in Section 8 or FIGS. 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 VHand VL having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto.
  • an antibody or fragment thereof that specifically binds to HDM2 comprising: (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.
  • an antibody or fragment thereof that specifically binds to HDM2 comprising: (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.
  • antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise Kabat VH CDR 3 of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).
  • antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise three Kabat VL CDRs of a VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and/or three Kabat VH CDRs of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).
  • antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise Chothia VH CDR 3 of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).
  • antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise AbM VH CDR 3 of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).
  • antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more Contact VL CDRs of a VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and/or one or more Contact VH CDRs of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).
  • antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise Contact VH CDR 3 of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).
  • antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise three Contact VL CDRs of a VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and/or three Contact VH CDRs of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).
  • antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more IMGT VL CDRs of a VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and/or one or more IMGT VH CDRs of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).
  • M(H)DM2/4 e.g., HDM2
  • IMGT VL CDRs of a VL of any one of the antibodies described herein any one of NMC-103, NMC-204, and NMC-303
  • IMGT VH CDRs of a VH of any one of the antibodies described herein any one of antibodies NMC-103, NMC-204, and NMC-303.
  • antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise IMGT VH CDR 3 of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).
  • antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise three IMGT VL CDRs of a VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and/or three IMGT VH CDRs of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).
  • antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise combinations 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 defining systems).
  • 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) region of an antibody described herein may vary by one, two, three, four, five, or six amino acid positions so long as immunospecific binding to M(H)DM2/4 (e.g., HDM2) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).
  • M(H)DM2/4 e.g., HDM2
  • the position defining a CDR of any of antibody described herein may vary by shifting the N-terminal and/or C-terminal boundary of the CDR by one, two, three, four, five, or six amino acids, so long as immunospecific binding to M(H)DM2/4 (e.g., HDM2) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).
  • M(H)DM2/4 e.g., HDM2
  • 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) region of an antibody described herein may vary (e.g., be shorter or longer) 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 maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).
  • M(H)DM2/4 e.g., HDM2
  • an anti-M(H)DM2/4 (e.g., HDM2) antibody described herein is a humanized immunoglobulin (e.g., an IgG) that comprises the 3 VH CDRs and the 3 VL CDRs (i.e., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) of any of the antibodies described herein (any one of murine antibodies NMC-103, NMC-204, and NMC-303), respectively, human or human-derived framework regions, and human or human-derived constant regions; antigen-binding fragments of such humanized antibodies are also provided by the present invention.
  • a humanizedanti-M(H)DM2/4 (e.g., HDM2) antibody or antigen-binding fragment thereof comprises a VH with VH CDR1, VH CDR2, and VH CDR3 as described herein (e.g., those of MNC-103, NMC-204, or NMC-303), surrounded by VH framework regions that are human framework regions or derived from human framework regions.
  • an anti-M(H)DM2/4 (e.g., HDM2) antibody or antigen-binding fragment thereof comprises a VL with VL CDR1, VL CDR2, and VL CDR3 as described herein (e.g., those of MNC-103, NMC-204, or NMC-303), surrounded by VL framework regions that are human framework regions or derived from human framework regions.
  • an anti-M(H)DM2/4 (e.g., HDM2) antibody or antigen-binding fragment thereof comprises (i) a VH with VH CDR1, VH CDR2, and VH CDR3 as described herein (e.g., those of MNC-103, NMC-204, or NMC-303), surrounded by VH framework regions that are human framework regions or derived from human framework regions; and (ii) a VL with VL CDR1, VL CDR2, and VL CDR3 as described herein (e.g., those of MNC-103, NMC-204, or NMC-303), surrounded by VL framework regions that are human framework regions or derived from human framework regions.
  • a VH with VH CDR1, VH CDR2, and VH CDR3 as described herein (e.g., those of MNC-103, NMC-204, or NMC-303), surrounded by VL framework regions that are human framework regions or derived from human framework regions.
  • Human framework regions that may be used include, without limitation: (i) framework regions selected using 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 (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); 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.
  • framework regions derived from screening 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)). See, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions.
  • one or more of the CDRs of an anti-M(H)DM2/4 may be inserted within known framework regions.
  • the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions.
  • polynucleotides comprising combinations of the framework regions and CDRs that encode an anti-M(H)DM2/4 (e.g., HDM2) or antigen-binding fragment thereof that specifically binds M(H)DM2/4 (e.g., HDM2).
  • an anti-M(H)DM2/4 e.g., HDM2
  • antigen-binding fragment thereof that specifically binds M(H)DM2/4 (e.g., HDM2).
  • One or more (e.g., one or two or three) amino acid substitutions may be made within the framework regions, preferably, one or more (e.g, one or two or three) amino acid substitutions may be made that improve binding of the antibody to M(H)DM2/4 (e.g., HDM2).
  • the framework regions in the variable domains can be those of the native (e.g., murine) antibody).
  • Antibodies provided herein include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules (i.e., molecules that possess an antigen-binding site) that specifically bind to an extracellular region (epitope) of M(H)DM2/4 accessible on the plasma membrane surface of cancer cells (for example, an epitope that is expressed or exposed on the plasma membrane of cancer cells at greater levels than on non-cancer cells (e.g., when such cancer and non-cancer cells originated from the same tissue)).
  • immunoglobulin molecules i.e., molecules that possess an antigen-binding site
  • epitope that is expressed or exposed on the plasma membrane of cancer cells at greater levels than on non-cancer cells (e.g., when such cancer and non-cancer cells originated from the same tissue)
  • an antigen-binding fragment of an anti-M(H)DM2/4 antibody is provided herein wherein the fragment can be, without limitation, an Fv fragment, a Fab fragment, a F(ab′) fragment, a F(ab′) 2 fragment, or a disulfide-linked Fv (sdFv).
  • an antigen-binding fragment provided herein is an Fv fragment.
  • an antigen-binding fragment provided herein is a Fab fragment.
  • an antigen-binding fragment provided herein is a F(ab′) 2 fragment.
  • an antigen-binding fragment provided herein is a F(ab′) fragment.
  • an antibody provided herein is a multispecific antibody (such as a bi-specific antibody) that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 exposed on the plasma membrane surface of cancer cells and specifically binds to a second antigen, wherein such binding allows re-targeting of effector cells towards tumor cells (as an example of such engineered bi-specific antibodies directed to a different target see Chames et al., 2009, MAbs 1:539-547, describing an antibody termed catumaxomab, a T-cell targeting agent).
  • a multispecific antibody such as a bi-specific antibody
  • an antibody provided herein is a multispecific antibody (e.g., a bi-specific antibody) that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 exposed on the plasma membrane surface of cancer cells, and also specifically binds to an antigen exposed on the surface of T cells (e.g., cytotoxic T cells).
  • an antibody provided herein is a multispecific antibody (e.g., a bi-specific antibody) that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 exposed on the plasma membrane surface of cancer cells, and also specifically binds to CD3.
  • an antibody provided herein is a multispecific antibody (e.g., a bi-specific antibody) that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 exposed on the plasma membrane surface of cancer cells, and also specifically binds to an antigen exposed on the surface of natural killer cells, neutrophils, macrophages, dendritic cells, and/or B-lymphocytes.
  • a multispecific antibody e.g., a bi-specific antibody
  • the antibody used is a monoclonal antibody or an antigen-binding fragment thereof that is human, humanized or chimeric.
  • the antibody can be an antibody or fragment appropriate for use in the treated species (i.e., of that species).
  • the antibodies described herein can be from any animal species, such as mammals (e.g., mouse, donkey, sheep, rabbit, goat, guinea pig, camel, horse, dog, cat) or birds (e.g., chicken).
  • the immunoglobulin molecules that can be used are 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.
  • the antibody is an immunoglobulin, and, in particular, an IgG.
  • the antibody is an IgM.
  • 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 cytoxicity (ADCC), and/or cytotoxicity due to a cytotoxic drug bound to the antibody or fragment.
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytoxicity
  • cytotoxicity due to a cytotoxic drug bound to the antibody or fragment are antibodies or fragments that mediate complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytoxicity (ADCC), and/or cytotoxicity due to a cytotoxic drug bound to the antibody or fragment.
  • the anti-M(H)DM2/4 antibodies or fragments described herein are antibodies or fragments that are capable of inducing cytotoxicity against the cancer cells being targeted by such antibodies or fragments, where the cytotoxicity can be due to complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or due to cytotoxicity of a drug bound to the antibody (where the antibody used is in a form of an antibody-drug conjugate).
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • cytotoxicity of a drug bound to the antibody where the antibody used is in a form of an antibody-drug conjugate.
  • the anti-M(H)DM2/4 antibodies or fragments described herein are antibodies or fragments that mediate complement-dependent cytotoxicity (CDC).
  • CDC complement-dependent cytotoxicity
  • the Fc region of the antibody described herein is of a human IgG (e.g., IgG1, IgG2, IgG3, IgG4) type or a human IgM type.
  • the Fc region of the antibody described herein is of a mouse IgG (e.g., IgG1, IgG2a, IgG2b, IgG3) or mouse IgM type.
  • the Fc region of the antibody described herein is of a human IgG1 isotype. In one embodiment, the Fc region of the antibody described herein is of a human IgG3 isotype. In one embodiment, the Fc region of the antibody described herein is of a human IgG2 isotype. In one embodiment, the Fc region of the antibody described herein is bioengineered (e.g., mutated) to increase its CDC activity.
  • the antibody or fragment is a bispecific antibody or fragment that specifically binds to an extracellular epitope of M(H)DM2/4 and specifically binds to an extracellular epitope of a complement regulatory protein (CRP) (which may, e.g., prevent the degradation of the freshly deposited immunologically active fragments (C3b, iC3b, C3d, C3g, C4b) by CRPs, amplify the activation of the complement cascade, and/or amplify MAC induced cancer cell lysis).
  • CRP complement regulatory protein
  • Such bispecific anti-M(H)DM2/4 antibodies or fragments may increase CDC activity, increase ADCC activity, increase antibody-dependent cellular phagocytosis (ADCP) by neutrophils and macrophages, or increase CDC, ADCC and ADCP.
  • ADCP antibody-dependent cellular phagocytosis
  • the Fc region of the antibody mediates its binding to an Fc receptor, FcR, on neutrophils, macrophages, natural killer cells, eosinophils and mast cells, which leads to ADCC and on neutrophils, macrophages and dendritic cells resulting in ADCP.
  • FcR Fc receptor
  • the Fc region of the antibody described herein is of a human IgG (e.g., IgG1, IgG2, IgG3) type or a human IgE type.
  • the Fc region of the antibody described herein is of a human IgG1 isotype.
  • the anti-M(H)DM2/4 antibodies described herein having an IgG Fc region are bioengineered at their Fc region to change the N-glycan structure at their glycosylation site to the GO glycan type terminating in GlcNAc (N-acetylglucosamine), and without fucose and sialic acid residues (which may result in, e.g., the activation of both the classic and alternate pathway of complement pathways and in increased binding to lectins including the mannose-binding lectins secreted during inflammatory responses).
  • GlcNAc N-acetylglucosamine
  • the Fc region of the antibody described herein is of a human IgG1 isotype and has alanine substitution at position 333 of its CH2 domain.
  • the Fc region of the antibody described herein is of a human IgG1 isotype and has a triple mutation S239D/I332E/A330L (which leads to a higher affinity for Fc ⁇ RIIIa and a lower affinity for Fc ⁇ RIIb resulting in enhanced ADCC) (such Fc modification can be made, e.g., in accordance with the methods described in Lazar et al., 2006, PNAS 103:4005-4010).
  • 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 cytoxicity (ADCC).
  • 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 cytoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP).
  • contemplated herein is an antibody or fragment that mediates only CDC or only ADCC activity.
  • the anti-M(H)DM2/4 antibodies or fragments are unconjugated, for example, are not conjugated to a cytotoxic drug).
  • the anti-M(H)DM2/4 antibodies or fragments described herein are not bound (e.g., not 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.
  • an anti-HDM2 antibody or fragment thereof specifically binds to HDM2 within amino acids of SEQ ID NO:1 (which are amino acids 1 to 15 of HDM2 (SEQ ID NO:4)). Amino acids of SEQ ID NO:1 (which are amino acids 1 to 15 of SEQ ID NO:4) are in an extracellularly accessible epitope of HDM2.
  • an anti-HDM2 antibody or fragment thereof specifically binds to HDM2 within amino acids of SEQ ID NO:2 (which are amino acids 15 to 25 of HDM2 (SEQ ID NO:4)). Amino acids of SEQ ID NO:2 (which are amino acids 15 to 25 of SEQ ID NO:4) are in an extracellularly accessible epitope of HDM2.
  • anti-M(H)DM2/4 antibodies or fragments thereof specifically bind to an extracellularly accessible epitope of M(H)DM2/4 within amino acids 100 to 110 of M(H)DM2/4 (SEQ ID NO:4 or SEQ ID NO:6).
  • anti-M(H)DM2/4 antibodies or fragments thereof specifically bind 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).
  • anti-M(H)DM2/4 antibodies or fragments thereof specifically bind to an extracellularly accessible epitope of M(H)DM2/4 within the terminal 100 amino acids at the C-terminus of the M(H)DM2/4 (e.g., M(H)DM2/4 protein variant (splice variant) known or expected to be expressed on the plasma membrane of cells of the cancer type being treated, or M(H)DM2/4 protein variant (splice variant) determined to be expressed on the plasma membrane of cancer cells of the subject being treated).
  • M(H)DM2/4 protein variant splice variant
  • the 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., 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 NMC204).
  • an antibody that specifically binds to HDM2 within the amino acid sequence of SEQ ID NO:2 (e.g., 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
  • the invention also provides anti-M(H)DM2/4 antibodies or fragments thereof that compete for binding to HDM2 with an antibodythat specifically binds to HDM2 within the amino acid sequence of SEQ ID NO:3 (e.g., 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).
  • 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).
  • any competition assay known in the art can be used to identify an antibody that competes with an antibody described herein for binding to M(H)DM2/4 (see Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)).
  • immobilized M(H)DM2/4 e.g., immobilized on a microtiter plate or well
  • a solution comprising a first labeled antibody that binds to M(H)DM2/4 and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to M(H)DM2/4.
  • immobilized M(H)DM2/4 can be incubated in a solution comprising the first labeled antibody but without 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. The substantial reduction of the amount of label in the test sample relative to the control sample indicates that the second antibody is competing with the first antibody for binding to M(H)DM2/4.
  • an antibody that competes with an antibody described herein e.g., antibodies 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 derived from M(H)DM2/4 that is bound by such antibody (e.g., the peptide of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3).
  • an antibody that competes with an antibody described herein e.g., antibodies 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 that is bound by such antibody.
  • 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 to 108 of HDM2 (SEQ ID NO:4).
  • 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 to 167 of HDM2 (SEQ ID NO:4).
  • 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 to 60 of HDM2 (SEQ ID NO:4).
  • 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 to 100 of HDM2 (SEQ ID NO:4).
  • 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 to 108 of HDM2 (SEQ ID NO:4).
  • 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 to 60 of HDM2 (SEQ ID NO:4).
  • 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 the HDM2 (e.g., HDM2 protein variant (splice variant) known or expected to be expressed on the plasma membrane of cells of the cancer type being treated, or HDM2 protein variant (splice variant) determined to be expressed on the plasma membrane of cancer cells of the subject being treated).
  • HDM2 protein variant splice variant known or expected to be expressed on the plasma membrane of cells of the cancer type being treated
  • HDM2 protein variant splice variant
  • 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 to 200 of HDM2 (SEQ ID NO:4).
  • an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein competes for binding to HDM2 with antibody OP145 (monoclonal antibody commercially available from Calbiochem, Catalogue No. OP145-100UG; see Table 10, below, for further details regarding OP145).
  • an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein competes for binding to HDM2 with antibody 965 (SMP14) (monoclonal antibody commercially available from Santa Cruz, Catalogue No. Sc-965; see Tables 3 and 10, below, for further details regarding 965 (SMP14)).
  • an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein competes for binding to HDM2 with antibody sc-813 (N-20) (polyclonal antibody commercially available from Santa Cruz, Catalogue No. Sc-813; see Table 10, below, for further details regarding sc-813 (N-20)).
  • an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein competes for binding to HDM2 with antibody sc-812 (C-18) (polyclonal antibody commercially available from Santa Cruz, Catalogue No. Sc-812; see Table 10, below, for further details regarding sc-812 (C-18)).
  • an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein competes for binding to HDM2 with antibody M01, clone 1A7 (monoclonal antibody commercially available from Abnova, Catalogue No. H00004193-M01; see Table 3, below, for further details regarding M01, clone 1A7).
  • an antibody that competes with antibody OP145, SMP14, N-20, C-18, or M01, clone 1A7 for binding to M(H)DM2/4 also binds to the same epitope that is bound by such antibodies.
  • 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)).
  • the anti-M(H)DM2/4 antibody or fragment thereof described herein is purified.
  • an antibody or fragment is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods (see Flatman et al., J. Chromatogr. B 848:79-87 (2007) for review of methods for assessment of antibody purity).
  • electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatographic e.g., ion exchange or reverse phase HPLC
  • the anti-M(H)DM2/4 antibody or fragment described herein can be fused or conjugated (e.g., covalently or non-covalently linked) to a detectable label or substance.
  • a detectable label or substance e.g., covalently or non-covalently linked
  • Such labeled antibodies or fragments can be used to detect M(H)DM2/4 on the plasma membrane surface of cells.
  • detectable labels or substances examples include enzyme labels, radioisotopes (e.g., iodine, carbon, sulfur, tritium, indium, and technetium), luminescent labels, fluorescent labels, and biotin.
  • This methodology can be used to determine whether cells of a certain cancer (e.g., cells of cancer in a patient) express M(H)DM2/4, or a certain splice variant of M(H)DM2/4, on the plasma membrane, where the detection of M(H)DM2/4 using the antibody (or fragment) may indicate that the antibody (or fragment) (with or without the detectable label or substance) can be used in the diagnosis and treatment of the cancer or preventing metastases of the cancer.
  • the invention provides antibody-drug conjugates comprising an anti-M(H)DM2/4 antibody or fragment described herein bound (e.g., covalently bound) to a cytotoxic drug.
  • the antibody-drug conjugates are intended to mediate cytotoxicity by delivery of a cytotoxic drug to the cells of the cancer.
  • 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 e.g., radioactive isotopes, growth inhibitory agents, and nucleolytic enzymes.
  • an M(H)DM2/4 antibody or fragment described herein is conjugated to one or more of the following drugs: a maytansinoid, an auristatin (such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF)), a dolastatin, a calicheamicin or derivative thereof, an anthracycline (such as daunomycin or doxorubicin), methotrexate, vindesine, a taxane (such as docetaxel), paclitaxel, larotaxel, tesetaxel, ortataxel, and a trichothecene.
  • a maytansinoid such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF)
  • a dolastatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF)
  • a calicheamicin or derivative thereof an anthracycline (such as daun
  • an M(H)DM2/4 antibody or fragment described herein is conjugated to a toxin or a fragment thereof (e.g., diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins, Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, or a tricothecene).
  • a toxin or a fragment thereof e.g., diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites for
  • an M(H)DM2/4 antibody or fragment described herein is conjugated to a radioactive isotope (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).
  • a radioactive isotope 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.
  • monoclonal antibodies are also known in the art, and include the use of hybridoma, recombinant and phage display technologies, and the use of humanized mice.
  • monoclonal antibodies can be produced using hybridoma techniques as taught, for example, in Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling G J et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981), or in Kohler G & Milstein C (1975) Nature 256: 495.
  • human monoclonal antibodies can be produced using humanized mice as taught, for example, in Laffleur et al., 2012, Methods Mol. Biol. 901:149-59.
  • a mouse or another appropriate host animal can be immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the target protein (i.e., extracellular region of M(H)DM2/4) used for immunization. Lymphocytes then are fused with myeloma cells to form a hybridoma cell (see Goding J W (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.
  • the hybridoma cells are then grown in a suitable culture medium, which can be assayed for production of monoclonal antibodies directed 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, e.g., immunoprecipitation or an in vitro assay, such as radioimmunoassay (MA) or enzyme-linked immunoabsorbent assay (ELISA).
  • MA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the antibodies or fragments described herein can also be made using various phage display technologies known in the art (see Brinkman U et al., (1995) J Immunol Methods 182: 41-50; Ames R S et al., (1995) J Immunol Methods 184: 177-186; Kettleborough C A et al., (1994) Eur J Immunol 24: 952-958; Persic L et al., (1997) Gene 187: 9-18; and Burton D R & Barbas C F (1994) Advan Immunol 57: 191-280).
  • chimeric antibodies i.e., antibody with a variable region of one species (e.g., murine) and a constant region of another species (e.g., human)
  • a variable region of one species e.g., murine
  • a constant region of another species e.g., human
  • Methods for producing chimeric antibodies are known in the art (see Morrison S L (1985) Science 229: 1202-7; Oi V T & Morrison S L (1986) BioTechniques 4: 214-221; Gillies S D et al., (1989) J Immunol Methods 125: 191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415).
  • 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).
  • 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).
  • 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).
  • scFv antibodies can be constructed by fusing variable domains of heavy and light chains of immunoglobulins via short polypeptide linkers (using recombinant expression techniques), and scFv antibodies having desired antigen-binding properties can be selected by phage display technology.
  • Methods of conferring CDC or ADCC activity on an antibody are known in the art (see Kellner et al., 2014, Methods 65:105-113; International Publication No. WO 2012010562; Natsume et al., 2009, Drug Design, Development and Therapy 3(3):7-16).
  • Such methods include, without limitation, Fc region isotype shuffling, amino acid mutations in the Fc region conferring enhanced or optimized CDC and/or ADCC activity, and changes in the Fc region glycosylation profile conferring enhanced or optimized CDC and/or ADCC activity).
  • the antibody or fragment may optionally be tested by any of the following methods:
  • a method for identifying an anti-M(H)DM2/4 antibody or a fragment thereof for use in the methods described herein comprising: (a) contacting intact cancer cells (e.g., cancer cells expected, known, or determined to express M(H)DM2/4) with an anti-M(H)DM2/4 antibody or a fragment thereof; and (b) determining whether the antibody or fragment binds to the intact cancer cells, in particular on the extracellular surface of the cancer cells (e.g., relative to intact cancer cells not contacted by said antibody or fragment), wherein the binding of the anti-M(H)DM2/4 antibody or fragment to the intact cancer cells contacted with such antibody or fragment indicates that said antibody or fragment is suitable for use in the methods described herein.
  • the cancer cells are from the patient proposed to be treated with the antibody or fragment thereof.
  • a method for identifying an anti-M(H)DM2/4 antibody or a fragment thereof for use in the methods described herein comprising: (a) contacting intact cancer cells (e.g., cancer cells expected, known, or determined to express M(H)DM2/4) with an anti-M(H)DM2/4 antibody or a fragment thereof; and (b) determining whether the antibody or fragment binds to the intact cancer cells (in particular 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 the intact cancer cells relative to normal cells indicates that said antibody or fragment is suitable for use in the methods described herein.
  • intact cancer cells e.g., cancer cells expected, known, or determined to express M(H)DM2/4
  • an anti-M(H)DM2/4 antibody or a fragment thereof e.g., for diagnosis of cancer, treating cancer or for
  • a method for identifying an anti-M(H)DM2/4 antibody or a fragment thereof for use in the methods described herein comprising: (a) contacting intact cancer cells (e.g., cancer cells known, expected or determined to express M(H)DM2/4) with an anti-M(H)DM2/4 antibody or a fragment thereof and (b) determining whether the contacting step results in an increased amount of cell death or destruction of the intact cancer cells (e.g., as determined by cell-death markers such as Propidium Iodide staining) relative to the amount of death or destruction of intact cancer cells not contacted by said antibody or fragment and/or relative 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 by said antibody or fragment, wherein increased amount of cell death or destruction of the intact cancer cells contacted with the antibody or fragment indicates that said antibody or fragment is suitable for
  • a method for identifying an anti-M(H)DM2/4 antibody or a fragment thereof for use in the methods described herein comprising: (a) contacting intact cancer cells (e.g., cells known, expected or determined to express M(H)DM2/4) with an anti-M(H)DM2/4 antibody or fragment; and (b) determining whether the contacting step results in an increased level of complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) (as determined by one or more cytotoxicity assays) towards the intact cancer cells relative to the level of CDC or ADCC towards intact cancer cells not contacted by said antibody or fragment and/or relative to the level of CDC or ADCC towards intact normal cells (e.g., non-cancerous cells of the same tissue or organ as the cancer cells) contacted by said antibody or fragment, wherein increased level of CDC or ADCC towards the intact cancer cells indicates that said antibody or fragment is suitable
  • a method for identifying an anti-M(H)DM2/4 antibody or a fragment thereof for use in the methods described herein comprising: (a) contacting intact cancer cells (e.g., cells known, expected or determined to express M(H)DM2/4) with an anti-M(H)DM2/4 antibody or a fragment thereof; and (b) determining whether the contacting step results in increased inhibition of growth and proliferation of the intact cancer cells relative to the inhibition of growth and proliferation of intact cancer cells not contacted by said antibody or fragment and/or relative 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 by said antibody or fragment, wherein increased inhibition of growth and proliferation of the intact cancer cells contacted with the antibody or fragment indicates that said antibody or fragment is suitable for use in the methods described herein.
  • intact cancer cells e.g., cells known, expected or determined to express M(H)DM2/4
  • an anti-M(H)DM2/4 antibody or a fragment thereof comprising:
  • compositions comprising an anti-M(H)DM2/4 antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier.
  • pharmaceutical compositions comprising an antibody-drug conjugate as described herein and a pharmaceutically acceptable carrier.
  • Appropriate pharmaceutically acceptable carriers including, but not limited to, excipients and stabilizers) are known in the art (see, e.g., 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 can be purified.
  • Pharmaceutically acceptable carriers may include an isotonic agent, a buffer, a suspending agent, a dispersing agent, an emulsifying agent, a wetting agent, a sequestering or chelating agent, a pH buffering agent, a solubility enhancer, an antioxidant, an anesthetic, and/or an antimicrobial agent.
  • Suitable excipients include, without limitation, water, saline, glycerol, ethanol, starch, lactose, sucrose, gelatin, malt, propylene, silica gel, sodium stearate, base cream and dextrose.
  • suitable pharmaceutically acceptable carriers may include physiological saline or phosphate buffered saline (PBS), and solutions containing such agents as glucose, polyethylene glycol, polypropylene glycol or other agents.
  • PBS physiological saline or phosphate buffered saline
  • compositions provided herein comprise an anti-M(H)DM2/4 antibody or an 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.
  • therapeutic agents e.g., anti-cancer
  • a pharmaceutical composition may be formulated for any route of administration to a subject.
  • Formulations for injections can be prepared as liquid solutions, suspensions, emulsions, or solid forms suitable for making into a solution or suspension prior to injection.
  • the anti-M(H)DM2/4 antibody or fragment thereof, or antibody-drug conjugate can be used or present in the pharmaceutical composition in a therapeutically effective amount.
  • the therapeutically effective amount of the antibody or conjugate can be determined by standard clinical techniques.
  • the invention provides for treating cancer (e.g., inhibiting cancer proliferation, inhibiting cancer progression) in a subject in need thereof comprising administering to the subject any anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein.
  • the invention provides a method of treating cancer in a subject in need thereof, said method comprising administering to the subject: an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4.
  • the invention provides a method of preventing metastasis in a subject in need thereof, said method comprising administering to the subject: an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4.
  • the antibody or fragment is not bound to a cell-penetrating peptide.
  • the invention provides for treating cancer (e.g., inhibiting cancer proliferation, inhibiting cancer progression) in a subject in need thereof comprising administering to the subject an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2 exposed on the surface of cancer cells (e.g., where the cells of the type of cancer being treated are known or expected to have such extracellular region of HDM2 exposed on their plasma membrane surface), preferably wherein the antibody or fragment is not bound to a cell-penetrating peptide.
  • the method of treating cancer encompasses preventing metastasis of the cancer.
  • the method of treating cancer is a method for reducing tumor size (as measured, e.g., by tumor volume or diameter), inhibiting the growth of the tumor, reducing the growth of the tumor, or eradicating the tumor.
  • the invention provides for preventing metastases in a subject that has cancer comprising administering to the subject any anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein.
  • the invention provides for preventing metastases in a subject that has cancer comprising administering to the subject an antibody or a fragment thereof that specifically binds to an extracellular region of HDM2 exposed on the surface of cancer cells (e.g., where the cells of the type of cancer being treated are known or expected to have such extracellular region of HDM2 exposed on their plasma membrane surface).
  • the invention provides for reducing the number, size or invasiveness of metastases, or eradicating metastases, in a subject that has a metastatic cancer comprising administering to the subject any anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein.
  • anti-M(H)DM2/4 e.g., anti-HDM2
  • the invention provides for reducing the number, size or invasiveness of metastases, or eradicating metastases, in a subject that has a metastatic cancer comprising administering to the subject an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2 exposed on the surface of cancer cells (e.g., where the cells of the type of cancer being treated are known or expected to have such extracellularly accessible epitope of HDM2 exposed on their plasma membrane surface).
  • the invention provides for treatment of a cancer that is resistant to another cancer therapy or therapies (e.g., vaccine, targeted therapy (such as small molecule targeted therapy), chemotherapy, radiotherapy, or immunotherapy (such as treatment with another monoclonal antibody)).
  • another cancer therapy or therapies e.g., vaccine, targeted therapy (such as small molecule targeted therapy), chemotherapy, radiotherapy, or immunotherapy (such as treatment with another monoclonal antibody)).
  • the invention provides for treating a cancer resistant to chemotherapy (i.e., resistant to one or more chemotherapeutic drugs).
  • the invention provides for treating a cancer resistant to treatment with another monoclonal antibody or antibodies.
  • the invention provides for treating a cancer resistant to radiotherapy.
  • the invention provides for treating a cancer resistant to small molecule targeted therapy or therapies.
  • an anti-M(H)DM2/4 e.g., anti-HDM2
  • an anti-HDM2 antibody or antigen-binding fragment thereof in a patient who has cancer (e.g., has been diagnosed with cancer).
  • therapeutic use of an anti-HDM2 antibody (or a fragment thereof) in a patient who has cancer that is known to metastasize i.e., is a type of cancer that is commonly known to become metastatic cancer.
  • the patient being treated has metastatic cancer.
  • a patient with a cancer that has not metastasized is treated in accordance with a method described herein in order to prevent metastasis of the cancer.
  • the patient being treated has been previously treated with other cancer therapies (e.g. vaccine, targeted therapy, chemotherapy, immunotherapy).
  • other cancer therapies e.g. vaccine, targeted therapy, chemotherapy, immunotherapy.
  • the patient with a cancer that has metastasized is treated in accordance with a method described herein in order to reduce, slow down or stop metastases, or decrease the number or size of metastases of the cancer.
  • the methods described herein are suitable for treating cancers that are expected, known or determined to express anti-M(H)DM2/4 (e.g., HDM2) on the surface of their cells.
  • the HDM2 on the surface of cancer cells targeted by the methods described herein can be one of the splice variants of HDM2 protein known in the art or described herein. Without intending to be bound by a mechanism of action, it is believed that HDM2 on the surface of cancer cells is usually a splice variant of the HDM2 protein that lacks at least one or all nuclear localization signals (e.g., the nuclear localization sequence at the N-terminal portion of HDM2, the nuclear localization signal at the C-terminal portion of HDM2, or both nuclear localization signals).
  • nuclear localization signals e.g., the nuclear localization sequence at the N-terminal portion of HDM2, the nuclear localization signal at the C-terminal portion of HDM2, or both nuclear localization signals.
  • 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.
  • the HDM2 on the surface of cancer cells is HDM2 that lacks the nuclear localization signal at amino acids 179-185 of SEQ ID NO: 4 (i.e., lacks amino acids 179 to 185 of SEQ ID NO: 4).
  • the HDM2 on the surface of cancer cells is HDM2 that lacks the nuclear localization signal at amino acids 466-473 of SEQ ID NO: 4 (i.e., lacks amino acids 466 to 473 of SEQ ID NO: 4).
  • the HDM2 on the surface of cancer cells is HDM2 that lacks the nuclear localization signals at amino acids 179-185 and amino acids 466-473 of SEQ ID NO: 4 (i.e., lacks amino acids 179 to 185 and 466 to 473 of SEQ ID NO: 4).
  • the HDM2 on the surface of cancer cells is HDM2 that lacks the nuclear export signal, such as the nuclear export signal at amino acids 190-202 of SEQ ID NO: 4 (i.e., lacks amino acids 190 to 202 of SEQ ID NO: 4).
  • the HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein, such as one of the splice variants known in the art or described herein (see Table 1 and Table 2, and Section 8, for the list of HDM2 variants).
  • 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-A (SEQ ID NO: 8), which lacks amino acids 28-222 of SEQ ID NO: 4.
  • 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-A1 (SEQ ID NO: 9), which lacks amino acids 28-222 and 275-300 of SEQ ID NO: 4.
  • 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-B (SEQ ID NO: 10), which lacks amino acids 28-300 of SEQ ID NO: 4.
  • 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-C(SEQ ID NO: 11), which lacks amino acids 53-222 of SEQ ID NO: 4.
  • 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-D (SEQ ID NO:12), which lacks amino acids 30-388 of SEQ ID NO: 4.
  • 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.
  • 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-F (SEQ ID NO: 14), which lacks amino acids 53-97 of SEQ ID NO: 4.
  • 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-G (SEQ ID NO: 15), which lacks amino acids 115-169 of SEQ ID NO: 4.
  • the HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-11 (SEQ ID NO: 16), in which amino acid M has been substituted with M MVRSRQM in SEQ ID NO: 4.
  • 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.
  • the methods described herein target a splice variant of M(H)DM4 on the surface of cancer cells, for example, target one or more of the following splice variants: MDMX-S, MDM4-S, MDM4-A, MDM4-G, MDM4-XALT1/XALT2 and MDM4-211 (or a human equivalent of the listed splice variants).
  • the anti-M(H)DM2/4 e.g., anti-HDM2
  • anti-HDM2 anti-HDM2
  • This binding to HDM2 can be shown by, for example, the ability of the anti-HDM2 antibody or fragment to bind to an intact cancer cell of the tissue type of the tissue of origin of the cancer being treated (which can be but does not need to be from the subject being treated).
  • an anti-HDM2 antibody or a fragment thereof is tested and determined to be expected to bind to the intact cells of the cancer of a subject, before administering the antibody or fragment to the subject. This testing can be done, e.g., by showing 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.
  • the cancer cells of the prospective patient to be treated can be tested for expression of M(H)DM2/4 (e.g., HDM2) on their surface using techniques known in the art in order to determine whether the subject is an appropriate candidate for anti-HDM2 therapy described herein; however, such ordinarily would not be deemed necessary if the patient has a cancer of a tissue type that is known or expected to have an extracellularly accessible epitope of M(H)DM2/4.
  • M(H)DM2/4 e.g., HDM2
  • the cancer being treated using the methods described herein is a cancer that is known or determined to express a splice variant of HDM2 (for example, 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.
  • a splice variant of HDM2 for example, 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
  • the tumor size can be assessed using magnetic resonance imaging (MM), dynamic contrast-enhanced MM (DCE-MRI), X-ray, computed tomography (CT) scan, or positron emission tomography (PET) scan.
  • MM magnetic resonance imaging
  • DCE-MRI dynamic contrast-enhanced MM
  • CT computed tomography
  • PET positron emission tomography
  • an anti-M(H)DM2/4 e.g., anti-HDM2
  • an antigen-binding fragment thereof in accordance with the methods described herein is effective to treat cancer in a subject (e.g., reduces tumor volume or diameter, reduces tumor growth, reduce tumor proliferation, eradicates the tumor, or improves one or more symptoms of cancer), when used alone or in combination with another therapy.
  • the administration of an anti-HDM2 antibody or an antigen-binding fragment thereof in accordance with the methods described herein is effective to prevent metastases in a subject that has cancer, when used alone or in combination with another therapy.
  • an anti-HDM2 antibody or an antigen-binding fragment thereof in accordance with the methods described herein is effective to treat a metastatic cancer (e.g., reduces the number, size or invasiveness of metastases, or eradicates metastases), when used alone or in combination with another therapy.
  • an anti-M(H)DM2/4 e.g., anti-HDM2
  • an antigen-binding fragment thereof in accordance with the methods described herein is effective to treat cancer or prevent metastasis in a subject when used alone (i.e., without an additional therapy).
  • the administration of an anti-HDM2 antibody or an antigen-binding fragment thereof in accordance with 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.
  • the effectiveness of therapies described herein can be assessed by evaluating a parameter (e.g., tumor size) before and after administration of the therapies described herein to the subject being treated.
  • a parameter e.g., tumor size
  • the effectiveness of therapy can be assessed by evaluating a parameter (e.g., tumor size) before and after administration of the therapies described herein to an animal model (e.g., in an animal model, such as a mouse model, a rat model, or a hamster model, of the cancer being treated).
  • an animal model e.g., in an animal model, such as a mouse model, a rat model, or a hamster model, of the cancer being treated.
  • Any assay known in the art can be used to evaluate the therapeutic effectiveness of the therapies described herein.
  • cancers that can be treated in accordance with the methods described herein include, but are not limited to, breast cancer, cervical cancer, ovarian cancer, endometrial cancer, uterine cancer, pancreatic cancer, skin cancer (e.g., melanoma), prostate cancer (e.g., hormone refractory, such as 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 bowel cancer, appendix cancer, esophageal cancer, gastric cancer, renal cancer, bladder cancer, gallbladder cancer, kidney cancer (e.g., renal cell carcinoma, or Wilms tumor)), liver cancer (e.g., hepatic carcinoma, or hepatoma), central nervous system cancer (e.g., brain cancer), peripheral nervous system cancer, bronchial cancer, cancer of the oral cavity or pharynx (e.g., orophary
  • the cancer is cervical cancer, endometrial cancer, ovarian cancer, pancreatic cancer, melanoma, breast cancer, or colon cancer.
  • the cancer is a pancreatic cancer.
  • the cancer is a melanoma.
  • the cancer is a breast cancer.
  • the cancer is an ovarian cancer.
  • the cancer that can be treated in accordance with the methods described herein is resistant to another cancer therapy or therapies (e.g., vaccine, targeted therapy (such as small molecule targeted therapy), chemotherapy, radiotherapy, or immunotherapy (such as treatment with another monoclonal antibody)).
  • the cancer that can be treated in accordance with the methods described herein is resistant to chemotherapy.
  • the cancer that can be treated in accordance with the methods described herein is resistant to treatment with another monoclonal antibody or antibodies.
  • the cancer that can be treated in accordance with the methods described herein is resistant to radiation.
  • the cancer that can be treated in accordance with the methods described herein is resistant to small molecule targeted therapy.
  • the cancer treated in accordance with the methods described herein is a solid cancer.
  • the cancer treated in accordance with the methods described herein is a non-solid cancer (e.g., hematologic cancer).
  • the cancer treated in accordance with the invention is leukemia (e.g., acute leukemia (such as acute lymphocytic leukemia or acute myelocytic leukemia), chronic leukemia (such as chronic myelocytic leukemia or chronic lymphocytic leukemia), or hairy cell leukemia), 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).
  • acute leukemia such as acute lymphocytic leukemia or acute myelocytic leukemia
  • chronic leukemia such as chronic myelocytic leukemia or chronic lymphocytic leukemia
  • hairy cell leukemia e.g., lymphoma (e.g., Hodgkin's lymph
  • the cancer treated in accordance with the invention is carcinoma (e.g., adenocarcinoma, basal cell carcinoma, renal cell carcinoma, squamous cell carcinoma, osteocarcinoma, thyoma/thymic carcinoma, or choriocarcinoma), blastoma, sarcoma (e.g., soft tissue sarcoma, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, or synovia sarcoma), lymphoma, leukemia, a germ cell tumor, myeloma (e.g., multiple myeloma), squamous cell cancer, mesothelioma, glioblastoma (e.g., glioblastoma multiforme), glioma, neuroblastoma, melanoma, astrocytoma, medulloblastoma, hepatoma, seminoma,
  • carcinoma
  • the cancer treated in accordance with the methods described herein is a sarcoma or carcinoma, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, endometrial stromal sarcoma, mast cell sarcoma, adult soft tissue sarcoma, uterine sarcoma, Kaposi sarcoma, merkel cell carcinoma, urothelial carcinoma, colon carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary
  • the cancer treated in accordance with the methods of the invention is metastatic.
  • the cancer treated in accordance with the methods described herein is a metastatic melanoma, a metastatic ovarian cancer, a metastatic cervical cancer, a metastatic endometrial cancer, a metastatic pancreatic cancer, a metastatic breast cancer, a metastatic colon cancer, or a metastatic brain cancer.
  • anti-M(H)DM2/4 e.g., anti-HDM2
  • antibodies or fragments described herein can be administered to a subject by any suitable means which include, but are not limited to, parenteral (e.g., intravenous, intraarterial, intramuscular, intraosseous, intracerebral, intracerebroventricular, intrathecal, subcutaneous), intraperitoneal, intratumoral, intrapulmonary, intradermal, transdermal, conjunctival, intraocular, intranasal, intratracheal, oral and local intralesional routes of administration.
  • parenteral e.g., intravenous, intraarterial, intramuscular, intraosseous, intracerebral, intracerebroventricular, intrathecal, subcutaneous
  • intraperitoneal intratumoral
  • intrapulmonary intradermal
  • transdermal transdermal
  • conjunctival intraocular
  • intranasal intratracheal
  • oral and local intralesional routes of administration e.g., parent
  • the anti-HDM2 antibodies or fragments described herein are administered intravenously, intraarterially, intramuscularly, intraperitoneally, intratumorally, or subcutaneously. In one embodiment, the anti-HDM2 antibodies or fragments described herein are administered intravenously. In one embodiment, the anti-HDM2 antibodies or fragments described herein are administered intraperitoneally. In one embodiment, the anti-HDM2 antibodies or fragments described herein are administered intramuscularly. In one embodiment, the anti-HDM2 antibodies or fragments described herein are administered subcutaneously. In one embodiment, the anti-HDM2 antibodies or fragments described herein are administered intratumorally (such as by an injection into the tumor of the cancer being treated). In particular embodiments, the anti-HDM2 antibodies or fragments described herein are administered intravenously, intraperitoneally, or intratumorally.
  • nano-particles coated with an anti-M(H)DM2/4 e.g., anti-HDM2
  • an anti-M(H)DM2/4 e.g., anti-HDM2
  • nano-particles coated with an anti-M(H)DM2/4 e.g., anti-HDM2
  • an anti-M(H)DM2/4 e.g., anti-HDM2
  • Arruebo et al. 2009, J. of Nanomater. 2009: Article ID 439389, regarding nano-particle coating with antibodies.
  • provided herein are methods for treating cancer or preventing metastasis in a subject having a cancer comprising administering to the subject nano-particles coated with an an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or a fragment thereof.
  • an anti-M(H)DM2/4 e.g., anti-HDM2
  • Various dosing schedules of the anti-M(H)DM2/4 e.g., anti-HDM2
  • methods of administration include, without limitation, bolus administration, pulse infusions, and continuous infusions.
  • the therapeutic regimen for use in the methods described herein may include administration of anti-M(H)DM2/4 (e.g., anti-HDM2) antibodies or fragments thereof (and compositions comprising such antibodies) once every week, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every eight weeks or once every twelve weeks (e.g., such that the subject receives from at least two, at least three, at least four, at least five, at least six, at least eight, or at least ten doses of the antibody, or from two to twenty doses of the antibody).
  • anti-M(H)DM2/4 e.g., anti-HDM2
  • compositions comprising such antibodies once every week, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every eight weeks or once every twelve weeks (e.g., such that the subject receives from at least two, at least three, at least four, at least five, at least six, at least eight, or at least ten doses of the antibody, or from
  • anti-HDM2 antibodies or fragments thereof are administered daily, every other day, or two, three, or four times a week (e.g., for a period of time, such as one week, two weeks, three weeks, four weeks, six weeks, two months or three months).
  • the treatment regimens contemplated herein include regimens wherein the initial higher dose of the antibody may be followed by one or more lower doses, or wherein the initial lower dose of the antibody is followed by one or more higher doses.
  • An exemplary treatment course in which the anti-HDM2 antibody or fragment is administered) may last for one week, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, five months, six months, one year, or over several years.
  • the initial treatment period (where the antibody is administered, e.g., once a month, once in two weeks, once a week, twice a week or three times a week) is followed by a withdrawal period in which the antibody is not administered (for, e.g., a week, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, six months or one year), and then followed by a second treatment period (where the antibody is administered, e.g., once a month, once in two weeks, once a week, twice a week or three times a week).
  • Such initial treatment and such second treatment periods can last, for example, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, or six months (where the initial treatment period can be the same or different from the second treatment period).
  • This course of treatment (having the initial treatment period, a withdrawal period and a second treatment period) can be repeated twice, three times, four times, five times, six times, ten times or more than ten times.
  • two or more antibodies or fragments thereof with different binding specificities for M(H)DM2/4 are administered simultaneously or sequentially to the subject being treated.
  • anti-M(H)DM2/4 e.g., anti-HDM2
  • the appropriate dosage 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, body weight, health), the responsiveness of the patient to the antibody, other medications used by the patient, and other factors to be considered at the discretion of the medical practitioner performing the treatment.
  • the dosage of an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein which is administered to the subject can be from about 1 ⁇ g/kg to 200 mg/kg of the patient's body weight. In certain embodiments, the dosage of an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein which is administered to the subject can be from about 1 ⁇ g/kg to 100 mg/kg of the patient's body weight (e.g., from about 0.01 mg/kg to about 100 mg/kg, from about 0.05 mg/kg to about 100 mg/kg, or from about 0.5 mg/kg to about 100 mg/kg).
  • the dosage of an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein which is administered to the subject can be from about 1 mg/kg to 200 mg/kg of the patient's body weight. In one embodiment, the dosage of an anti-HDM2 antibody or fragment described herein which is administered to the subject is from 0.025 mg/kg to about 5 mg/kg. In one embodiment, the dosage of an anti-HDM2 antibody or fragment described herein which is administered to the subject is from 0.05 mg/kg to about 2 mg/kg. In one embodiment, the dosage of an anti-HDM2 antibody or fragment described herein which is administered to the subject is from 5 mg/kg to about 30 mg/kg.
  • an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein which is administered to the subject can be from about 1 mg/kg to 200 mg/kg of the patient's body weight. In one embodiment, the dosage of an anti-HDM2 antibody or fragment described herein which is administered to the
  • doses 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 can be administered to the subject being treated.
  • a dose e.g., one or more doses
  • a dose e.g., one or more doses
  • 0.1 mg/kg of an anti-HDM2 antibody or fragment described herein can be administered to the subject being treated (e.g., when the antibody is administered intratumorally).
  • a subject e.g., a human
  • methods of diagnosing cancer in a subject comprising: (a) detecting whether an antibody or a 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 an intact cell of the subject, wherein the antibody or fragment is any anti-M(H)DM2/4 antibody or fragment described herein (in particular, 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 the binding is detected in step (a).
  • an antibody or a fragment thereof e.g., a labeled antibody or fragment
  • M(H)DM2/4 e.g., HDM2
  • the method of diagnosing further comprises, before the detecting in step (a), obtaining the intact cell from the subject, and then performing the detecting by, e.g., determining whether a labeled antibody or fragment binds to the intact cell from the subject using, e.g., FACS or cell-based ELISA analysis.
  • the method of diagnosing comprises administering the antibody or fragment to the subject before the detecting in step (a), and wherein the detecting is performed by in vivo imaging of the subject.
  • 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 the detection of binding of such antibody or fragment to the surface of intact cancer cells obtained from the patient.
  • an anti-M(H)DM2/4 e.g., anti-HDM2
  • a method of selecting a patient having a cancer for treatment with an antibody or fragment that specifically binds to an extracellularly accessible epitope of HDM2 comprising: obtaining an intact cancer cell from the patient (e.g., by biopsy of the cancerous tumor in the patient, or by obtaining a blood sample with circulating cancer cells from the patient), and determining whether the antibody or fragment binds to the surface of the intact cancer cell of the patient (using any method known in the art or described herein, e.g., using cell-based ELISA or FACS analysis), wherein the detection of binding indicates that the patient can be treated with the antibody or fragment.
  • a method of selecting a patient having a cancer for treatment with an antibody or fragment that specifically binds to an extracellularly accessible epitope of HDM2 comprising: obtaining an intact cancer cell from the patient (e.g., by biopsy of the cancerous tumor in the patient, or by obtaining a blood sample with circulating cancer cells from the patient), determining whether the antibody or fragment binds to the surface of the intact cancer cell of the patient (using any method known in the art or described herein, e.g., using cell-based ELISA or FACS analysis), and, if the binding is detected, administering the antibody or fragment to the patient.
  • the antibody or fragment administered to the patient can be the same or different from the antibody or fragment used for selection of the patient to be treated.
  • a method of selecting a patient having an ovarian cancer for treatment with an antibody or fragment that specifically binds to an extracellularly accessible epitope of HDM2 comprising: obtaining an intact ovarian cancer cell from the patient, determining whether the antibody or fragment binds to the surface of the intact cancer cell of the patient, and, if the binding is detected, administering the antibody or fragment to the patient.
  • any antibody or a fragment thereof that specifically binds to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 is used for such patient selection and/or treatment.
  • any antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, which is not bound to a cell-penetrating peptide is used for such patient selection and/or treatment.
  • any antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, which is not bound to a cell-penetrating peptide is used for such patient selection, and any antibody or a fragment thereof that specifically binds to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 is used for the treatment of the patient.
  • Non-limiting exemplary samples that can be used for in diagnostic or patient selection methods using an anti-M(H)DM2/4 antibody (e.g., anti-HDM2 antibody) or fragment thereof described herein include: tissue biopsies, intact cells obtained from malignant tissues, and circulating cancer cells isolated from blood.
  • 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., anti-HDM2 antibody) or fragment thereof binds to the tissue sample.
  • intact cells 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., anti-HDM2 antibody) or fragment thereof binds to such cell.
  • a blood sample with 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., anti-HDM2 antibody) or fragment thereof binds to such cells.
  • the duration of treatment and/or dosage of an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or antibody fragment described herein to be used in the treatment of a patient is determined based on the detection of binding of such antibody or fragment to the surface of intact cancer cells obtained from the patient.
  • a method of determining whether to continue the treatment of a patient having a cancer with an antibody or fragment that specifically binds to an extracellularly accessible epitope of HDM2 comprising: administering the antibody or fragment to the patient for a first period of time (e.g., where the patient had been selected for treatment as described above), obtaining an intact cancer cell from the patient (e.g., by biopsy of the cancerous tumor in the patient, or by obtaining a blood sample with circulating cancer cells from the patient), and determining whether the antibody or fragment binds to the surface of the intact cancer cell of the patient (using any method known in the art or described herein, e.g., using cell-based ELISA or FACS analysis), and, if the binding is detected, continuing administering the antibody or fragment to the patient for a second period of time (but, e.g., if the binding is not detected, discontinuing the treatment).
  • a first period of time e.g., where the patient had been selected for treatment as described above
  • 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 the treatment of a patient having a cancer comprising: administering a dose the antibody or fragment to the patient for a period of time (e.g., where the patient had been selected for treatment as described above), obtaining an intact cancer cell from the patient (e.g., by biopsy of the cancerous tumor in the patient, or by obtaining a blood sample with circulating cancer cells from the patient), and determining whether the antibody or fragment binds to the surface of the intact cancer cell of the patient (using any method known in the art or described herein, e.g., using cell-based ELISA or FACS analysis), and, if the binding is detected, administering a dose of the antibody or fragment to the patient for a second period of time, wherein the dose administered during the second period of time is higher than the dose administered during the first period of time (e.g., two times, or three
  • treatment with an anti-M(H)DM2/4 e.g., anti-HDM2
  • an anti-M(H)DM2/4 e.g., anti-HDM2
  • an 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, wherein a decrease in the amount is a positive prognosis.
  • the patients or subjects being treated in accordance with the methods described herein include, but are not limited to, humans and non-human vertebrates.
  • the subject being treated is a mammal, e.g., a human, a dog, a cat, a monkey, a rabbit, a cow, a horse, a goat, a sheep, or a pig.
  • the subject being treated is a human.
  • the subject being treated in accordance with the methods described herein has been diagnosed with a cancer (e.g., using a biopsy or any another method known in the art).
  • the subject being treated has been diagnosed with an early stage cancer.
  • the subject being treated has been diagnosed with an advanced stage cancer.
  • the subject being treated has been diagnosed with a high-grade tumor.
  • the subject being treated has been diagnosed with a low-grade tumor.
  • the subject being treated has been diagnosed with a cancer that can metastasize.
  • the subject being treated has been diagnosed with a metastatic cancer.
  • the subject being treated in accordance with the methods described herein has been diagnosed with a cervical cancer, an endometrial cancer, an ovarian cancer, a pancreatic cancer, a melanoma, a breast cancer, a colorectal cancer (e.g., a colon cancer), a bladder cancer, an astrocytic neoplasm, a glioblastoma, a pediatric Rhabdomyosarcoma, or a lung cancer (e.g., non-small cell lung carcinoma).
  • the subject being treated in accordance with the methods described herein has been diagnosed with a melanoma, a pancreatic cancer, a breast cancer, or an ovarian cancer.
  • the subject being treated in accordance with the methods described herein has been diagnosed with a melanoma. In one embodiment, the subject being treated in accordance with the methods described herein has been diagnosed with a pancreatic cancer. In one embodiment, the subject being treated in accordance with the methods described herein has been diagnosed with a breast cancer. In one embodiment, the subject being treated in accordance with the methods described herein has been diagnosed with an ovarian cancer. In one embodiment, the subject being treated in accordance with the methods described herein has been diagnosed with a lung cancer.
  • the subject being treated has previously undergone one or more other cancer therapies (e.g., vaccine, targeted therapy (such as small molecule targeted therapy), chemotherapy, radiotherapy, or immunotherapy (such as treatment with another monoclonal antibody)), and the subject's cancer has developed resistance to the one or more other cancer therapies.
  • the subject being treated is resistant to chemotherapy.
  • the subject being treated is resistant to radiotherapy.
  • the subject being treated is resistant to a small molecule targeted therapy.
  • the subject being treated is resistant to treatment with another monoclonal antibody.
  • the subject being treated has a type of a cancer that is known or expected to have M(H)DM2/4 (e.g., HDM2) on the surface of its cells.
  • the subject being treated has a type of cancer, the cells of which express one or more of splice variants of HDM2 on their cell surface, for example (and without limitation), 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).
  • the subject being treated has a cancer that has been tested and determined (using any assay known in the art) to carry M(H)DM2/4 (e.g., HDM2) on the plasma membrane of its cells.
  • M(H)DM2/4 e.g., HDM2
  • the subject being treated has a cancer, the cells of which have been tested and determined (by any method known in the art) to expose on their plasma membrane surface an extracellular region of HDM2 that can be targeted by an anti-HDM2 antibody or fragment (and such antibody can then be administered to the subject).
  • the subject being treated has a cancer, the cells of which have been tested and determined (by any method known in the art) to express one or more of splice variants of HDM2 on their cell surface, for example (and without limitation), 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).
  • MDM2-A SEQ ID NO: 8
  • MDM2-A1 SEQ ID NO: 9
  • MDM2-B SEQ ID NO: 10
  • MDM2-D SEQ ID NO:12
  • 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 said antibody or fragment, e.g., a chemotherapy, a surgery, a radiation therapy, another antibody with an anti-cancer activity, a cytokine, a T cell therapy, a vaccine (e.g., a cellular vaccine), a small molecule with an anti-cancer activity, an anti-hormonal agent, or any other anti-cancer therapy known in the art.
  • a chemotherapy e.g., a surgery, a radiation therapy, another antibody with an anti-cancer activity, a cytokine, a T cell therapy, a vaccine (e.g., a cellular vaccine), a small molecule with an anti-cancer activity, an anti-hormonal agent, or any other anti-cancer therapy known in the art.
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with chemotherapy.
  • chemotherapeutic agents include, without limitation, an alkylating agent, a nitrosourea agent, an antimetabolite, a topoisomerase inhibitor, an aromatase inhibitor, an antitumor antibiotic, an alkaloid derived from a plant, a hormone antagonist, a P-glycoprotein inhibitor, and a platimum complex derivative.
  • chemotherapeutic drugs that can be used in the methods described herein include, without limitation, taxol, paclitaxel, nab-paclitaxel, 5-fluorouracil (5-FU), gemcitabine, doxorubicin, daunorubicin, colchicin, mitoxantrone, tamoxifen, cyclophosphamide, mechlorethamine, melphalan, chlorambucil, busulfan, uramustine, mustargen, ifosamide, bendamustine, carmustine, lomustine, semustine, fotemustine, streptozocin, thiotepa, mitomycin, diaziquone, tetrazine, altretamine, dacarbazine, mitozolomide, temozolomide, procarbazine, hexamethylmelamine, altretamine, hexalen, trofosfamide, estramustine, treosul
  • 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, capecitabine, irinotecan, and celecoxib.
  • 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 such as ABRAXANE®).
  • the cancer treated using a combination therapy described herein is a pancreatic cancer, a breast cancer, a lung cancer or an ovarian cancer.
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with gemcitabine (e.g., for treatment of a non-small cell lung cancer, a pancreatic cancer or an ovarian cancer).
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with capecitabine.
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with irinotecan.
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with celecoxib.
  • 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 such as ABRAXANE®) (e.g., for treatment of a metasatic breast cancer).
  • paclitaxel e.g., paclitaxel formulated as albumin-bound particles such as ABRAXANE®
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with nab-paclitaxel.
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with cisplatin.
  • 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 treatment of a colorectal cancer such as a colon cancer).
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with carboplatin.
  • 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 treatment of a pancreatic cancer).
  • 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 treatment of an ovarian cancer).
  • 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 such as ABRAXANE®) and gemcitabine (e.g., for treatment of a breast cancer, or a pancreatic cancer such as adenocarcinoma of the pancreas or metastatic adenocarcinoma of the pancreas).
  • paclitaxel e.g., paclitaxel formulated as albumin-bound particles such as ABRAXANE®
  • gemcitabine e.g., for treatment of a breast cancer, or a pancreatic cancer such as adenocarcinoma of the pancreas or metastatic adenocarcino
  • 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 treatment of anon-small cell lung cancer).
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with gemcitabine and 5-FU.
  • 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 such as ABRAXANE®) and carboplatin (e.g., for treatment of a non-small cell lung cancer).
  • paclitaxel e.g., paclitaxel formulated as albumin-bound particles such as ABRAXANE®
  • carboplatin e.g., for treatment of a non-small cell lung cancer.
  • gemcitabine is administered in a dose of 1,500 mg/m 2 . In certain embodiments, wherein the subject is human, nab-paclitaxel is administered in a dose of 300 mg/m 2 . In certain embodiments, wherein the subject is human, gemcitabine is administered in a dose of 1,000 mg/m 2 . In certain embodiments, wherein the subject is human, nab-paclitaxel is administered in a dose of 125 mg/m 2 .
  • the gemcitabine and/or nab-paclitaxel are administered in doses that are lower than doses used when gemcitabine and/or nab-paclitaxel are administered not in combination with an anti-cancer antibody (such as an anti-M(H)DM2/4 antibody or fragment described herein).
  • an anti-cancer antibody such as an anti-M(H)DM2/4 antibody or fragment described herein.
  • gemcitabine is administered in a dose that is less than 1,500 mg/m 2
  • nab-paclitaxel is administered in a dose that is less than 300 mg/m 2 .
  • gemcitabine is administered in a dose that is less than 1,000 mg/m 2
  • nab-paclitaxel is administered in a dose that is less than 125 mg/m 2
  • gemcitabine is administered in a dose that is equal to or less than 500 mg/m 2 , 400 mg/m 2 , 300 mg/m 2 or 200 mg/m 2
  • the nab-paclitaxel is administered in a dose that is equal to or less than 62.5 mg/m 2 , 50 mg/m 2 , 40 mg/m 2 , 30 mg/m 2 , or 20 mg/m 2 .
  • gemcitabine is administered in a dose that is equal to or less than 900 mg/m 2 , 800 mg/m 2 , 700 mg/m 2 or 600 mg/m 2
  • the nab-paclitaxel is administered in a dose that is equal to or less than 110 mg/m 2 , 100 mg/m 2 , 90 mg/m 2 , 80 mg/m 2 , or 70 mg/m 2
  • gemcitabine and/or nab-paclitaxel are administered with a frequency of every 2 weeks or less (e.g., every 3 weeks, every 4 weeks, every 6 weeks, or every 8 weeks, or less).
  • gemcitabine is administered with a frequency of once a day, 4 times per week, 3 times per week, 2 times per week, or once per week.
  • nab-paclitaxel is administered with a frequency of once a day, 4 times per week, 3 times per week, 2 times per week, or once per week.
  • gemcitabine and nab-paclitaxel are administered with a frequency of once a day, 4 times per week, 3 times per week, 2 times per week, or once per week.
  • gemcitabine and/or nab-paclitazel is administered once a week.
  • the total duration of treatment with gemcitabine and/or nab-paclitaxel is, or is more than, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks.
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject with a cancer in combination with the chemotherapy drug(s) indicated for said cancer, which chemotherapy drug(s) can be optionally administered in the dosage and/or regime of administration indicated for said cancer.
  • chemotherapy drugs as well as their dosage and regime of administration indicated for various cancers are provided below.
  • Ovarian Cancer The following information is taken from Gemzar® (gemcitabine for injection), Eli Lilly and Company, Highlights of Prescribing Information, revised March 2017, http://pi.lilly.com/us/gemzar.pdf (last accessed on Jul. 27, 2017).
  • Gemcitabine (Gemzar®) in combination with carboplatin is indicated for the treatment of patients with advanced ovarian cancer that has relapsed at least 6 months after completion of platinum-based therapy.
  • the recommended dose of Gemzar® is 1000 mg/m 2 as an intravenous infusion over 30 minutes on Days 1 and 8 of each 21-day cycle, in combination with carboplatin (AUC 4) intravenously after Gemzar® administration on Day 1 of each 21-day cycle.
  • Gemzar® gemcitabine for injection
  • Eli Lilly and Company Highlights of Prescribing Information, revised March 2017, http://pi.lilly.com/us/gemzar.pdf (last accessed on Jul. 27, 2017).
  • Gemcitabine (Gemzar®) in combination with paclitaxel is indicated for the first-line treatment of patients with metastatic breast cancer after failure of prior anthracycline-containing adjuvant chemotherapy, unless anthracyclines were 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.
  • Paclitaxel can be administered at 175 mg/m2 on Day 1 as a 3 hour intravenous infusion before Gemzar® administration.
  • Non-Small Cell Lung Cancer The following information is taken from Gemzar® (gemcitabine for injection), Eli Lilly and Company, Highlights of Prescribing Information, revised March 2017, http://pi.lilly.com/us/gemzar.pdf (last accessed on Jul. 27, 2017).
  • Gemcitabine (Gemzar®) is indicated in combination with cisplatin for the first-line treatment of patients with inoperable, locally advanced (Stage IIIA or or metastatic (Stage IV) non-small cell lung cancer.
  • 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; cisplatin can be administered intravenously at 100 mg/m 2 on Day 1 after the infusion of Gemzar®.
  • the recommended dose of Gemzar® is 1250 mg/m 2 intravenously over 30 minutes on Days 1 and 8 in combination with cisplatin therapy; cisplatin can be administered intravenously at 100 mg/m 2 on Day 1 after the infusion of Gemzar®.
  • Pancreatic Cancer The following information is taken from Gemzar® (gemcitabine for injection), Eli Lilly and Company, Highlights of Prescribing Information, revised March 2017, http://pi.lilly.com/us/gemzar.pdf (last accessed on Jul. 27, 2017).
  • Gemcitabine (Gemzar®) is indicated as first-line treatment for patients with locally advanced (nonresectable 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 2 over 30 minutes intravenously.
  • the recommended treatment schedule is as follows: weeks 1-8—weekly dosing for the first 7 weeks followed by one week rest; after week 8—weekly dosing on Days 1, 8, and 15 of 28-day cycles.
  • Metastatic Breast Cancer The following information is taken from ABRAXANE® (paclitaxel protein-bound particles for injectable suspension, albumin-bound), Celgene Corporation, Highlights of Prescribing Information, revised July 2015, http://www.abraxane.com/wp-content/pi/prescribing-info.html (last accessed Jul. 27, 2017).
  • ABRAXANE® is indicated for the treatment of breast cancer after failure of combination chemotherapy for metastatic disease or relapse within 6 months of adjuvant chemotherapy. Prior therapy should have included an anthracycline unless clinically contraindicated. After failure of combination chemotherapy for metastatic breast cancer or relapse within 6 months of adjuvant chemotherapy, the recommended regimen for ABRAXANE® is 260 mg/m 2 administered intravenously over 30 minutes every 3 weeks.
  • Non-Small Cell Lung Cancer The following information is taken from ABRAXANE® (paclitaxel protein-bound particles for injectable suspension, albumin-bound), Celgene Corporation, Highlights of Prescribing Information, revised July 2015, http://www.abraxane.com/wp-content/pi/prescribing-info.html (last accessed Jul. 27, 2017).
  • ABRAXANE® is indicated for the first-line treatment of locally advanced or metastatic non-small cell lung cancer, in combination with carboplatin, in patients who are not candidates for curative surgery or radiation therapy.
  • the recommended dose of ABRAXANE® is 100 mg/m 2 administered as an intravenous infusion over 30 minutes on Days 1, 8, and 15 of each 21-day cycle. Carboplatin can be administered on Day 1 of each 21 day cycle immediately after ABRAXANE®.
  • Adenocarcinoma of the Pancreas The following information is taken from ABRAXANE® (paclitaxel protein-bound particles for injectable suspension, albumin-bound), Celgene Corporation, Highlights of Prescribing Information, revised July 2015, http://www.abraxane.com/wp-content/pi/prescribing-info.html (last accessed Jul. 27, 2017).
  • ABRAXANE® is indicated for the first-line treatment of patients with metastatic adenocarcinoma of the pancreas, in combination with gemcitabine.
  • the recommended dose of ABRAXANE® is 125 mg/m 2 administered as an intravenous infusion over 30-40 minutes on Days 1, 8 and 15 of each 28-day cycle.
  • Gemcitabine can be administered immediately after ABRAXANE® on Days 1, 8 and 15 of each 28-day cycle.
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with an immunomodulator (e.g., a cytokine, an antigen, or a checkpoint targeting agent).
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with a checkpoint targeting agent such as, without limitation, an antagonist of PD-1, an antagonist of PD-L1, an antagonist of PD-L2, an antagonist of CTLA-4, an antagonist of TIM-3, an antagonist of GITR, an antagonist of OX40, an antagonist of LAG-3 (e.g., the antagonist of any of the above-mentioned checkpoint molecules can be an antibody, such as an inhibitory antibody to these molecules, an antibody fragment, or a small molecule).
  • a checkpoint targeting agent such as, without limitation, an antagonist of PD-1, an antagonist of PD-L1, an antagonist of PD-L2, an antagonist of CTLA-4, an antagonist of TIM-3, an antagonist of
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with an inhibitor of PD-1, an inhibitor of PD-L1, or an inhibitor of CTLA-4 (where the inhibitor can be an antagonistic antibody, an antibody fragment, or a small molecule).
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with surgery (such as a surgery to remove part or all of the cancerous tumor being treated).
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with a Treg-inhibitory agent.
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with a T-cell therapy.
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with a tumor vaccine.
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with EGFR inhibitor (e.g., erlotinib).
  • EGFR inhibitor e.g., erlotinib
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is used to treat a subject that is not treated with a cell cycle inhibitor (i.e., the additional therapy is not an agent that inhibits cell cycle).
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is used to treat a subject that is not concurrently (during the same treatment period) treated with a cell cycle inhibitor (i.e., the subject is not treated with an anti-M(H)DM2/4 antibody or fragment thereof and a cell cycle inhibitor during the same period of time, e.g., day or week).
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein is used to treat a subject that has not been previously treated and is not concurrently treated with a cell cycle inhibitor.
  • an anti-M(H)DM2/4 antibody or fragment thereof described herein can be used before, during, or after the second therapy (e.g., a chemotherapy, a radiation therapy, a surgery, or any other therapy described herein or known in the art).
  • the second therapy e.g., a chemotherapy, a radiation therapy, a surgery, or any other therapy described herein or known in the art.
  • the subject being treated in accordance with the methods described herein has not received an anti-cancer therapy prior to the administration of an anti-M(H)DM2/4 antibody or fragment thereof.
  • an anti-M(H)DM2/4 antibody or fragment thereof is administered to a subject that has received an anti-cancer therapy prior to administration of the antibody or fragment.
  • anti-M(H)DM2/4 antibody or fragment thereof is administered to a subject recovering from or receiving an immunosuppressive therapy.
  • kits comprising an anti-M(H)DM2/4 antibody or a fragment thereof, and one or more additional anti-cancer agents.
  • kits comprising (i) an anti-M(H)DM2/4 antibody or a fragment thereof (e.g., in a therapeutically effective amount), and (ii) one or more of chemotherapeutic drugs, for example, gemcitabine, paclitaxel, or gemcitabine and nab-paclitaxel (e.g., in therapeutically effective amounts, such as any amounts described herein, which may be less than the therapeutically effective amount of the drug or drugs when the drug or drugs are used without the anti-M(H)DM2/4 antibody or fragment).
  • chemotherapeutic drugs for example, gemcitabine, paclitaxel, or gemcitabine and nab-paclitaxel
  • HDM2 segment(s) of HDM2 are extracellularly accessible on the plasma membrane surface of intact (i.e., viable and non-permeabilized) cancer cells.
  • Three (3) different extracellularly accessible epitopes have been identified.
  • Specific segments of HDM2 that are extracellularly accessible include but are not limited to epitopes present in the NMC-P1, NMC-P2 and NMC-P3 peptide sequences (SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively). These extracellularly accessible sequences are appropriate therapeutic and diagnostic targets for anti-HDM2 antibodies. Therefore, cancer cells expressing HDM2 on their surface membrane can be targeted with antibodies to HDM2 for diagnostic and therapeutic (i.e., anti-tumor cytotoxic and inhibitory effect) purposes.
  • HDM2-specific antibodies bound to the extracellularly accessible sequences of M(H)DM2/4 on the surface membrane of intact cells of several rodent and human cancer cell lines as well as primary tumor cells from human patients.
  • the same HDM2-specific antibodies exhibited minimal binding to the surface membrane of normal human blood mononuclear cells. It was found that these HDM2-specific antibodies selectively bound to various cancer cells such as: intact human melanoma, uveal melanoma, pancreatic, breast, colon, lung and ovarian cancer cells in vitro.
  • HDM2-specific antibodies inhibited the growth of cancer cells in vitro and in vivo, strongly indicating that they can be used as therapeutic agents in vivo.
  • Data from in vivo studies and described herein showed that select HDM2-specific antibodies inhibited tumor growth and were cytotoxic against tumors in rodent tumor models.
  • select HDM2-specific antibodies were not only cytotoxic to tumor cells but also inhibited tumor growth in mouse models of pancreatic cancer, lung cancer and colon cancer.
  • Data herein further demonstrated that only select antibodies recognized extracellularly accessible epitopes of HDM2.
  • Anti-HDM2 antibodies that specifically bind to (i) NMC-P1, i.e., the peptide of SEQ ID NO:1 (“NMC-100s series of monoclonal antibodies)”, (ii) NMC-P2, i.e., the peptide of SEQ ID NO:2 (“NMC-200s series of monoclonal antibodies”), and (iii) NMC-P3, i.e., the peptide of SEQ ID NO:3 (“NMC-300s series of monoclonal antibodies”) were generated using the hybridoma approach.
  • NMC-P1, NMC-P2 and NMC-P3 peptides were conjugated to Keyhole limpet hemocyanin (KLH) using Sulfo-SMCC method (Thermo Scientific, Cat. No. 22122). Briefly, Protein-NH2 was made in Conjugation Buffer (provided by manufacturer). Twenty-fold molar excess of crosslinker was added to the protein solution and the reaction mixture was incubated for 30 minutes at room temperature. Excess cross linker was then removed using desalting column equilibrated with Conjugation Buffer. Protein-SH and desalted Protein-NH2 were then combined, mixed and incubated for 30 minutes at room temperature.
  • Conjugation Buffer provided by manufacturer. Twenty-fold molar excess of crosslinker was added to the protein solution and the reaction mixture was incubated for 30 minutes at room temperature. Excess cross linker was then removed using desalting column equilibrated with Conjugation Buffer. Protein-SH and desalted Protein-NH2 were then combined,
  • mice BALB/c female
  • mice BALB/c female
  • Boosters were injected 5-7 times to provoke immune response.
  • Specific-antibody production was then evaluated by peptide-ELISA of the mice serum for antibody titration. Spleens of mice with high antibody titer were then harvested from each mice and single cell suspension of splenocytes were prepared.
  • Splenocytes were then fused with SP2/0 myeloma cells (1:5 ratio). Briefly, 250 ⁇ l of EDTA was added to the mixture of splenocytes and myeloma cells. Cells were then span down and supernatant was removed. Cell pellet was then loosened and 1 mL of PEG was dispensed alongside the tube slowly over 1 min and the mixture was incubated for 5 min in 37° C. water bath. 1 mL of 100 ⁇ FBS and 10 mL of IMDM medium (10% FBS) was then added and kept in the incubator for 1 hour. Cells were then centrifuged and supernatant was removed.
  • IMDM medium 10% FBS
  • Monoclonal antibody NMC-103 is an antibody that binds to NMC-P1 (SEQ ID NO:1) (it is one of the NMC-100s series of antibodies).
  • Monoclonal antibody NMC-204 is an antibody that binds to NMC-P2 (SEQ ID NO:2) (it is one of the NMC-200s series of antibodies).
  • Monoclonal antibody NMC-303 is an antibody that binds to NMC-P3 (SEQ ID NO:3) (it is one of the NMC-300s series of antibodies).
  • the heavy chain/light chain frame work region sequences, complementarity determining region (CDR) sequences, and variable region sequences of these antibodies are listed in Section 8, below.
  • anti-HDM2 antibodies were used in the experiments described in Examples 1-9: (i) purified NMC-103 mouse monoclonal antibody (mAb) of the IgG1 isotype (NMC-103 mAbs produced by single-cell cloned hybridoma cells were purified on protein G/A columns), (ii) purified NMC-204 mouse mAb of the IgG3 isotype (NMC-204 mAbs produced by single-cell cloned hybridoma cells were purified on protein G/A columns); (iii) purified NMC-303 mouse mAb of the IgM isotype (NMC-303 mAbs produced by single-cell cloned hybridoma cells were purified on protein G/A columns); (iv) an anti-HDM2 antibody termed “MDM2 monoclonal antibody (M01), clone 1A7” (Abnova, Cat.
  • MDM2 Antibody (D-7) an anti-HDM2 antibody termed “MDM2 Antibody (D-7)” (Santa Cruz, Cat. No. sc-13161);
  • SMP14 an anti-HDM2 antibody termed “MDM2 Antibody (SMP14)” (Santa Cruz, Cat. No.
  • MDM2 Antibody (C-18): sc-812” (Santa Cruz, polyclonal, Cat No. sc-812); and n anti-HDM2 antibody termed “MDM2 Antibody (N-20): sc-813” (Santa Cruz, polyclonal, Cat No. sc-813).
  • mAb NMC-103 specifically bound to peptide NMC-P1 (SEQ ID NO:1) corresponding to amino acids 1-15 of HDM2, while mAb NMC-204 specifically bound to NMC-P2 peptide (SEQ ID NO:2) corresponding to amino acids 15-25 of HDM2, and mAb NMC-303 specifically bound to NMC-P3 peptide (SEQ ID NO:3) corresponding to amino acids 475-491 of HDM2.
  • immunoblot analysis presented here shows that mAbs NMC-103, NMC-204 and NMC-303 recognized the full-length recombinant HDM2 protein.
  • Peptide-ELISA Methodology 5 ⁇ g/ml of NMC-P1, NMC-P2 or NMC-P3 peptide antigen was dried onto a 96-well ELISA plate overnight. Plates were then blocked with 5% BSA in 1 ⁇ phosphate-buffered saline (PBS) (100 ⁇ l/well) for 2 hours at room temperature. Microplate wells were then washed 5 times with 300 ⁇ l of ice cold lx PBS.
  • PBS phosphate-buffered saline
  • peptide antigen binding competition experiments As for peptide antigen binding competition experiments, 1 ⁇ l/ml of mAb NMC-103 was pre-incubated with 10 ⁇ l/ml of NMC-P1, NMC-P2 or NMC-P3 for 1 hour at room temperature. MAb NMC-103 was then incubated with ELISA plates coated with NMC-P1 as described above.
  • the membrane was then washed ⁇ 3 times (10 minutes each) and incubated for 1 hour with corresponding HRP-conjugated secondary antibody (Goat anti-Mouse IgG (H&L), F(ab′)2 Frag Cross-adsorbed HRP (HRP-GaM F(ab′)2), ThermoFisher, Cat. No. A24524) diluted 1:5000 in 1% BSA-PBS.
  • the membrane was then washed ⁇ 3 times and incubated with Pierce ECL Plus Western Blotting Substrate (ThermoFisher, Cat. No. #32132) for 10 min before developing on a LICOR Scanner.
  • FIG. 1 The peptide-ELISA experiments shown in FIG. 1 demonstrated selective and specific binding of monoclonal antibodies (mAb) NMC-103, NMC-204 and NMC-303 to their corresponding peptide antigens.
  • FIG. 1A shows that NMC-103 bound to NMC-P1 peptide while NMC-204 did not bind to NMC-P1.
  • FIG. 1B shows that mAb NMC-204 bound to NMC-P2 peptide while mAb NMC-103 did not bind to NMC-P2.
  • FIG. 1C shows that mAb NMC-303 bound to NMC-P3 peptide while NMC-204 did not bind to NMC-P3.
  • the results of the immunoblot experiments in FIG. 2 demonstrated that mAbs NMC-103, NMC-204 and NMC-303 reacted with the recombinant full-length HDM2 protein.
  • the recombinant protein has a GST tag at its N-terminal which brings the molecular weight to approximately 83 kD.
  • Lane 2 of the immunoblot shows the reactivity of a commercially available antibody raised against amino acids 100-320 of MDM2 of human origin (MDM2 (D-7); Santa Cruz, Cat. No. sc-13161) to the full-length recombinant HDM-2 protein.
  • Cell-ELISA Methodology 8,000-10,000 cells/well of a 96-well microplate were grown overnight. The next day, unbound cells were washed off with sterile lx PBS. The cells in each well were fixed with freshly prepared 4% buffered paraformaldehyde (pH 7.2) for 1 hour followed by 3 washes with lx PBS. The wells were then blocked with 5% BSA in PBS (100 ⁇ l/well) for 2 hours at room temperature. Microplate wells were then washed 5 times with 300 ⁇ l of ice cold lx PBS.
  • MAbs NMC-103, NMC-204 or NMC-303 at 1 ⁇ g/mL in 1% BSA/PBS were then incubated with various cancer or normal untransformed cells for 2 hours at room temperature.
  • Wells were then washed with 300 ⁇ l of ice-cold lx PBS for 5 times and corresponding secondary antibody HRP-GaM F(ab′)2 diluted 1:2500 or 1:5000 in PBS with 1% BSA were added at 100 ⁇ l/well for 1 hour at room temperature followed by washing 5 times with 300 ⁇ l of ice-cold lx PBS.
  • TMB Substrate Solution (1-StepTM Ultra TMB-ELISA, ThermoFisher, Cat. No.
  • FIGS. 3-5 show the relative binding of mAbs NMC-103, NMC-204 and NMC-303 to intact cells of different types of human (A) and rodent (B) cancers.
  • FIG. 3 shows reactivity of mAb NMC-103 to human breast cancer MCF-7 cells, human triple negative breast cancer HCC1806 cells, human pancreatic cancer MIA PaCa-2 cells, human ovarian cancer OVCAR-3 cells that are resistant to adriamycin, melphalan, and cisplatin, primary patient-derived human ovarian cancer OVCA4 cells, human melanoma A2058 cells, human uveal melanoma 92.1 cells, mouse colon cancer MC-38 cells, mouse Lewis Lung LL/2 cells and mouse pancreatic Panc02 cells.
  • FIG. 4 presents the reactivity of NMC-204 monoclonal antibody to human breast cancer MCF-7 cells, human triple negative breast cancer HCC1806 cells, human pancreatic cancer MIA PaCa-2 cells, human ovarian cancer OVCAR-3 cells, primary patient-derived human ovarian cancer OVCA.4 cells, human melanoma A2058 cells, human uveal melanoma 92.1 cells, mouse colon cancer MC-38 cells, mouse Lewis Lung LL/2 cells and mouse pancreatic Panc02 cells.
  • FIG. 4 presents the reactivity of NMC-204 monoclonal antibody to human breast cancer MCF-7 cells, human triple negative breast cancer HCC1806 cells, human pancreatic cancer MIA PaCa-2 cells, human ovarian cancer OVCAR-3 cells, primary patient-derived human ovarian cancer OVCA.4 cells, human melanoma A2058 cells, human uveal melanoma 92.1 cells, mouse colon cancer MC-38 cells, mouse Lewis Lung LL/2 cells and mouse
  • FIG. 5 shows reactivity of NMC-303 monoclonal antibody to human breast cancer MCF-7 cells, human triple negative breast cancer HCC1806 cells, human pancreatic cancer MIA PaCa-2 cells, human ovarian cancer OVCAR-3 cells, primary patient-derived human ovarian cancer OVCA.4 cells, human melanoma A2058 cells, human uveal melanoma 92.1 cells, mouse colon cancer MC-38 cells, mouse Lewis Lung LL/2 cells and mouse pancreatic Panc02 cells.
  • FIG. 6 demonstrates that NMC-204 did not react with normal human PBMCs. In contrast to cancer cells, neither mAb NMC-103, NMC-204 nor NMC-303 bound to normal intact cells (the data for NMC-103 and NMC-303 is not shown). Data presented herein show that, while mAb NMC-204 reacted with human pancreatic cancer MIA PaCa-2 cells, no binding was seen above the background when mAb NMC-204 was incubated with freshly isolated normal human peripheral blood mononuclear cells (PBMCs). Moreover, FIG. 6 demonstrates that while mAb NMC-204 did not react with normal human PBMCs ( FIG. 6 left; white bar graph), these cells showed strong reactivity with an mAb against CD3e, a cell surface marker for T cells ( FIG. 6 left; shaded bar graph).
  • PBMCs peripheral blood mononuclear cells
  • FIG. 7 depicts mAb NMC-103 and NMC-204 saturation curves.
  • FIG. 7A shows that cell-ELISA binding of mAb NMC-103 to intact MIA PaCa-2 cells increased as the concentration of the antibody increased. However, this binding reached a plateau at concentrations of above 50 ⁇ g/mL of mAb NMC-103, demonstrating the saturation of antigen binding sites by the specific antibody NMC-103.
  • FIG. 7B depicts the binding saturation curve of mAb NMC-204 to intact MIA PaCa-2 cells.
  • Example 3 mAb NMC-103.
  • NMC-204 and NMC-303 were Specific for Extracellularly Accessible NMC-P1.
  • FIG. 8 shows the binding of mAb NMC-103 to intact human pancreatic cancer MIA PaCa-2 cells (left, solid bar).
  • the binding of monoclonal antibody NMC-103 to its epitope of HDM2 that is accessible on the surface plasma membrane of MIA PaCa-2 cells was completely abolished when mAb NMC-103 was competed with soluble NMC-P1 (middle column, solid bar).
  • competition with NMC-P2 did not have any effect on the reactivity of NMC-103 with the epitope of HDM2 to which it binds and which is accessible on the cell surface of the MIA PaCa-2 cells (solid, filled bar).
  • mAb NMC-204's reactivity with intact MIA PaCa-2 cells was competed with NMC-P2 peptide and not with NMC-P1, demonstrating epitope specificity for another extracellularly accessible epitope/peptide of HDM2 expressed on the plasma membrane of MIA PaCa-2 cells.
  • MAbs NMC-103, NMC-204 and NMC-303 were raised against amino acids 1-15 (NMC-P1), 15-25 (NMC-P2) and 475-491 (NMC-P3) of HDM2, respectively, and were highly specific for HDM2.
  • NMC-P1 amino acids 1-15
  • NMC-P2 15-25
  • NMC-P3 475-491
  • all three mAbs NMC-103, NMC-204 and NMC-303 recognized and bound to purified HDM2 protein, providing evidence that the unique antigenic epitopes recognized by the three mAbs in NMC-P1, NMC-P2 and NMC-P3, respectively, are de facto structures of intact HDM2.
  • Another set of the same cells was released with either EDTA or Trypsin and fixed with 4% buffered paraformaldehyde as above, then washed and blocked with 5% human serum albumin in PBS, and was treated separately with Triton X-100 (0.1% in PBS) (International Biotechnologies Inc. 07100) for 5 minutes at room temperature for membrane permeabilization. After washing off the Triton X-100 solution with PBS the cells were adjusted to 10 6 cells/ml and stored in ice-cold PBS. Cells (10 6 /ml) from each preparation were then incubated either with 5 ⁇ g/ml of mAb to Na+/K+ ATPase ⁇ -1 (Abcam, Cat. No.
  • FIG. 11 panels A, B and C present flow cytometry data on % cells stained with mAbs NMC-103, NMC-204 and anti-Na+/K+ ATPase ⁇ -1, respectively.
  • Each antibody was reacted with cells from four treatment conditions: EDTA-treated intact cells, EDTA-treated permeabilized cells, trypsin-treated intact cells, trypsin-treated permeabilized cells.
  • EDTA-treated intact cells showed staining with mAb NMC-103 (70.4%), NMC-204 (51.2%) and anti-Na+/K+ ATPase ⁇ -1 antibody (29.9%).
  • NMC-103 70.4%
  • NMC-204 51.2%)
  • anti-Na+/K+ ATPase ⁇ -1 antibody 29.9%.
  • staining with NMC-103, NMC-204 and anti-Na+/K+ ATPase ⁇ -1 increased to 75.8%, 52.85 and 63.8%, respectively.
  • FIG. 12 (A-D) demonstrates that human pancreatic cancer MIAPaCa-2 cells that were treated with mAb NMC-103 or NMC-204 exhibited a concentration-dependent growth inhibition, while an antibody against an intracellular marker (i.e., Cytochrome C) had no effect on the growth of these cancer cells.
  • MIAPaCa-2 cells treated with 1 ⁇ g/mL (top panel, left image), 5 ⁇ g/mL (to panel, middle image) and 10 ⁇ g/mL (top panel, left image) mAb NMC-103 showed an increasing effect in inhibition of their growth.
  • FIG. 12A shows that human pancreatic cancer MIAPaCa-2 cells that were treated with mAb NMC-103 or NMC-204 exhibited a concentration-dependent growth inhibition, while an antibody against an intracellular marker (i.e., Cytochrome C) had no effect on the growth of these cancer cells.
  • FIG. 12A MIAPaCa-2 cells treated with 1 ⁇ g/mL (top panel,
  • FIG. 12B presents images of the cells treated with 1, 10 and 20 ⁇ g/mL of mAb NMC-204, showing a concentration-dependent inhibition of cell growth.
  • FIG. 12C demonstrates that treatment of MIAPaCa-2 cells with an antibody against Cytochrome-C, which was used as an intracellular marker control, had no effect on the growth of these cells at any of the 1, 10 and 20 ⁇ g/mL concentration.
  • FIG. 12C demonstrates that treatment of MIAPaCa-2 cells with an antibody against Cytochrome-C, which was used as an intracellular marker control, had no effect on the growth of these cells at any of the 1, 10 and 20 ⁇ g/mL concentration.
  • 12D quantifies the growth inhibitory effect of mAb NMC-103 at 1 ⁇ g/mL (65%), 5 ⁇ g/mL (84%) and 10 ⁇ g/mL (91%) (solid black line) and of NMC-204 at 1 ⁇ g/mL (65%), 5 ⁇ g/mL (73%) and 10 ⁇ g/mL (77%) (dashed black line).
  • treatment with an antibody to Cytochrome C an intracellular target; Santa Cruz, Cat. No. sc-13156
  • the data presented herein show the ability of HDM2-specific antibodies to not only bind to the extracellularly accessible sequences of HDM2 on the surface membrane of cancer cells but also to initiate a potent cytotoxic effect in the presence of fresh normal human serum (NHS).
  • the cytotoxic effect was measured in pancreatic cancer cells as well as in normal human fibroblasts.
  • Data presented herein demonstrated one such cytotoxic effect when human pancreatic cancer MIAPaCa-2 cells were treated with mAb NMC-103 in the presence of NHS.
  • FIG. 13 demonstrates the cytotoxic effect of HDM2-specific mAb NMC-103 against human pancreatic cells.
  • mAb NMC-103 in the presence of NHS triggered complement-mediated cytotoxicity in cancer cells, resulting in the death of the cancer cells as evident by the nuclear uptake of the cell-death marker Propidium Iodide (PI).
  • FIG. 13C provides a quantitative representation of HDM2-specific antibody complement-dependent-cytotoxicity (CDC) against human pancreatic cancer cells.
  • Cells treated with mAb NMC-103 ( FIG. 13B ) in the presence of NHS demonstrated cytotoxicity over 2 hours post-treatment as compared with cells treated with NHS in the absence of any antibody ( FIG. 13A ). Control experiement was performed with anti-Cytochrome C antibody or with cells left untreated in the presence of NHS.
  • Example 8 Evaluation of Other Anti-HDM2 mAb Antibodies in their Binding to NMC-P1, NMC-P2 and NMC-P3 and to Intact Cancer Cells
  • HDM2-specific monoclonal antibodies that are commercially available.
  • mAbs utilizing peptide- and cell-ELISA, we tested a number of such mAb for their binding to intact cancer cells.
  • These mAbs included antibodies that were raised against various regions from the N-terminus or C-terminus, or segments in the middle of the HDM2 protein, and were tested for their binding to newly identified extracellularly accessible NMC-P1 and NMC-P2 sequences as well as to intact cancer cells.
  • Table 3 summarizes the commercially available mAbs that were tested, the companies that generated these mAbs, and amino acid residues of HDM2 against which they had been raised.
  • FIGS. 14A and 14B demonstrated the lack of binding of any of these mAbs to either NMC-P1 or NMC-P2.
  • NMC-103 and NMC-204 showed strong binding to NMC-P1 and NMC-P2, respectively.
  • HDM-2 binding components such as peptides, for example PNC-27, interfered with the binding of mAb antibodies to NMC-P1 and NMC-P2.
  • HDM2-binding component of PNC-27 and PNC-28 peptides binds on HDM2
  • it has been reported to bind within amino acids 25-109 of HDM2 Do et al., 2003, Oncogene 22(10):1431-1444 (“Do 2003”); Chesse, 2003, Nat. Rev. Cancer 3(2):102-109).
  • HDM-2 binding components of PNC-27 and PNC-28 have no anti-cancer activity by themselves and are only active when attached to a membrane resident peptide (MRP or Penetratin sequence) (see Kanovsky 2001, Do 2003, and Bowne 2008).
  • MDM2 monoclonal antibody (M01), clone 1A7 was raised against amino acid 101 to 200 of full-length HDM2.
  • the data demonstrate that epitopes of HDM2 other than NMC-P1, NMC-P2 and NMC-P3 may be extracellularly accessible on cancer cells for binding.
  • two HDM2-specific monoclonal antibodies that did not react with intact cancer cells were identified ( FIG. 15B ).
  • One mAb was an anti-HDM2 antibody termed “Anti-MDM2 (Ab-4) Mouse mAb (2A9C1.18)” (EMD Millipore, Cat. No.
  • HDM2 which is raised against amino acid 153 to 222 of full length HDM2 (SEQ ID NO:4) that includes one Nuclear Localization Signal (NLS) and one Nuclear Export Signal (NES).
  • Another HDM2-specific mAb that did not react with intact cancer cell membrane was an anti-HDM2 antibody termed “Anti-MDM2 (Ab-1) Mouse mAb (IF2)” (EMD Millipore, Cat. No. OP46), which is raised against amino acids 26-169 of HDM2 ( FIG. 15B ).
  • HDM2 there are select epitopes of HDM2 that are extracellularly accessible on cancer cells. Described herein are three (3) such extracellularly accessible segments of HDM2, namely NMC-P1 (SEQ ID NO:1), NMC-P2 (SEQ ID NO: 2) and NMC-P3 (SEQ ID NO: 3). Data herein demonstrated that select antibodies raised against these 3 segments selectively and specifically bound to various types of cancers but not normal health cells ( FIGS. 3-9 ). Moreover, results presented herein demonstrated the extracellular accessibility of these 3 segments of HDM2 on cancer cells ( FIGS. 10 and 11 ). Furthermore, data herein show that HDM2-specific antibodies had both growth inhibitory and cytotoxic effect against cancer cells in vitro. Finally, the anti-tumor activity of HDM2-specific antibodies was evaluated in vivo. As an example, efficacy of mAb NMC-103 and NMC-204 on lung, colon and pancreatic cancer was tested.
  • mice were micro-chipped and registered following tumor implantation. Tumor volume measurements and body weights were recorded and mice were randomized into groups that received: A) mAb NMC-103 at 0.4 mg/kg (3 times a week for 3 weeks), 2 mg/kg (2 times a week for 3 weeks), or 4 mg/kg (2 times a week for 3 weeks); B) NMC-204 at 0.4 mg/kg (3 times a week for 3 weeks); C) isotype control mouse IgG1 (Abcam, Cat. No. ab18447) or IgG3 (Abcam, Cat. No.
  • FIG. 16 demonstrates the anti-tumor efficacy of mAb NMC-204 (0.4 mg/kg 3 times a week for 3 weeks; dashed line), in mice that were simultaneously inoculated with LL/2 Lewis Lung cancer cells.
  • treatment with NMC-204 reduced tumor volume (1446 mm 3 ) as compared with treatment with isotype control mouse IgG3 (2138 mm 3 ).
  • mAb NMC-204 an anti-HDM2-specific antibody that targets extracellularly accessible epitopes of HDM2 on lung cancer cells.
  • the dosing and method of administration can be further expanded, e.g., to oral or intravenous delivery of the drug for optimum effect.
  • FIGS. 17 and 18 demonstrate the efficacy of mAb NMC-103 and NMC-204 against MC-38 syngeneic mouse model of colon cancer, respectively.
  • mice treated for 18 days with mAb NMC-103 0.4 mg/kg; 3 times a week for 2.5 weeks; dashed line
  • mice treated with isotype control mouse IgG1 reached a tumor volume of 2205 mm 3 (solid line).
  • immunohistochemical staining for Ki67 protein a well-established cell proliferation marker (Li et al., 2015, Mol. Med. Rep.
  • FIG. 18A tumor volume in mice treated with mAb NMC-204 (0.4 mg/kg; 3 times a week for 3 weeks) grew to 1670 mm 3 while tumor in the control antibody-treated group reached 2555 mm 3 in volume. Furthermore, FIG. 18B shows that mice treated with NMC-204 had 30% of their tumor cells stained positively for Ki67 while mice in the control group had approximately 80% of their tumor cells staining positively for Ki67, demonstrating the anti-proliferative effect of NMC-204 treatment on tumor growth.
  • mice in groups that received either isotype control IgG (group A) or a combination of low dose Gemcitabine and nab-Paclitaxel (group B) reached an average of 2175 mm 3 (open triangle) and 2314 mm 3 (open circle), respectively. Both of these groups had reached morbidity criteria and were terminated.
  • tumors in mice of group C that received mAb NMC-103 (2 mg/kg) only reached on average 1523 mm 3 (filled triangle)
  • tumors in mice of group D that had received a combination of NMC-103 (2 mg/kg), Gemcitabine and nab-Paclitaxel were measured at an average of 797 mm 3 (filled circle).
  • mice were then treated with a combination of NMC-103 (10 mg/kg), Gemcitabine and nab-Paclitaxel two times a week for 2 weeks. As demonstrated in FIG. 19 , within 2 weeks tumors in both groups (C and D) reached the point where no measurable tumor was found.
  • mice in group A that had received isotype control antibody reached an average tumor size of 2028 mm 3 and were terminated.
  • a Kaplan Meier survival analysis demonstrated significant survival benefit in mice that received NMC-103 alone or in combination with chemotherapy when compared to chemotherapy alone or control antibody under the experimental conditions described in FIG. 27 .
  • This study confirms the observations discussed above in connection with FIG. 19 and the ability of NMC-103 antibody to be used as single arm or in combination with chemotherapy.
  • mice that had previously received a combination of G+nP, mice in group C (mice that had previously been treated with NMC-103) and group D (mice that had been treated with a combination of NMC-103+G+nP), as described in FIG. 27 were re-challenged by a second round of Panc-2 inoculation (subcutaneous injection of 2 ⁇ 10 6 cells/mouse), on the left dorsal flank. Tumor growth was monitored for 10 days at which point, a tumor of 90 mm 3 ( FIG. 29 ) was measured in the mice from group B. In contrast, as shown in FIG.
  • mice were treated with pancreatic cancer standard of care (Gemcitabine (25 mg/kg)+nab-Paclitaxel (5 mg/kg)) for 19 days at which point the tumor reached a size of approximately 450 mm 3 .
  • pancreatic cancer standard of care Gemcitabine (25 mg/kg)+nab-Paclitaxel (5 mg/kg)
  • the mice were then randomly divided in 2 groups that received a single dose of: A) isotype control mouse IgG1 (10 mg/kg) or B) NMC-103 (10 mg/kg).
  • a single i.p. injection of NMC-103 reduced the tumor size by almost half 6 days post treatment (from 438 mm 3 to 233 mm 3 ).
  • FIG. 31 mice treated with NMC-103 at 10 mg/kg, two times per week for three weeks resulted in a greater tumor reduction when compared to mice treated with NMC-103 at 0.4 mg/kg ( FIG. 17 ).
  • mice The mouse Heavy and Light chain variable regions (SEQ ID NO:40 and SEQ ID NO:41, respectively) were cloned into a human Ig gamma-1 chain and human Ig kappa chain as constant region.
  • FIG. 32A shows that by day 24 post tumor inoculation, mice treated with the chimeric version of monoclonal antibody NMC-303 (10 mg/kg) reached an average tumor size of 726 mm 3 , while mice treated with control antibody (10 mg/kg) had an average tumor size of 1746 mm 3 . Furthermore, FIG. 32B shows the individual mouse tumor sizes on day 24 post tumor inoculation. Due to the human constant region of the chimeric version of the monoclonal antibody NMC-303, the anti-tumor efficacy of this antibody might be improved if tested in mouse models with human immune background. The above in vivo results further support the anti-tumor efficacy of antibodies raised against NMC-P3 immunogen that target the extracellularly accessible epitopes of the M(H)DM2/4 on cancer cells.
  • M(H)DM2/4-specific antibodies that recognize extracellularly accessible segments of M(H)DM2/4 in cancers cell have growth inhibitory and cytotoxic effect against a variety of cancers while sparing normal untransformed cells.
  • the selective anti-tumor effect of these antibodies is believed to be due at least in part to their recognition of extracellularly accessible epitopes of M(H)DM2/4 protein variants that are expressed on the surface of cancer cells while the expression levels of these M(H)DM2/4 variants on the cell surface of normal cells are low or absent.
  • Examples 10-12 below describe data obtained using other anti-HDM2 antibodies that bind to segments of HDM2 that are extracellularly accessible on cancer cells.
  • anti-HDM2 antibodies were used in the experiments described in Examples 10-12: (i) polyclonal sc-813, N-20, rabbit IgG, from Santa Cruz (abbreviated throughout the specification as “N-20” or “sc-813 (N-20)”); (ii) monoclonal OP145, mouse IgG1, from Calbiochem (abbreviated throughout the specification as “OP145”); (iii) monoclonal OP46 (Ab-1), mouse IgG1, from Calbiochem (abbreviated throughout the specification as “OP46”); (iv) monoclonal OP144 (Ab-4), mouse IgG1, from Calbiochem (abbreviated throughout the specification as “OP144”); (v) polyclonal sc-812, C-18, rabbit IgG, from Santa Cruz (abbreviated throughout the specification as “C-18” or “sc-812 (C-18)”); and (vi) monoclonal 965 (SMP14), mouse IgG1, from Santa Cruz (abbrevi
  • Table 10 provides information regarding the HDM2 recognition sites of these antibodies (i.e., amino acids of HDM2 recognized by these antibodies), and whether or not these antibodies are cytotoxic to cancer cells.
  • OP145, N-20, C-18 and SMP14 were cytotoxic to cancer cells tested, and OP46 and OP144 were not cytotoxic to cancer cells tested.
  • FACS Fluorescence-activated cell sorting
  • FIG. 24 presents results of the FACS analysis of human melanoma, primary ovarian cancer, and normal mouse spleenocytes.
  • FIG. 24A area under curve #1 represents cells incubated with goat anti-rabbit secondary antibody only; area under 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 curve #3 represents cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody.
  • FIG. 24A area under curve #1 represents cells incubated with goat anti-rabbit secondary antibody only; area under 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 curve #3 represents cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody.
  • area under curve #1 represents cells incubated with goat anti-mouse secondary antibody only; area under 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 curve #3 represents cells incubated with anti-HDM2 monoclonal antibody OP145 followed by goat anti-mouse secondary antibody.
  • FIG. 24B area under curve #1 represents cells incubated with goat anti-mouse secondary antibody only; area under 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 curve #3 represents cells incubated with anti-HDM2 monoclonal antibody OP145 followed by goat anti-mouse secondary antibody.
  • area under curve #1 represents cells incubated with goat anti-rabbit secondary antibody only; area under 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 curve #3 represents cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody.
  • FIG. 24D & E area under curve #1 represents cells incubated with goat anti-rabbit secondary antibody only; area under 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 curve #3 represents cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody.
  • area under curve #1 represents cells incubated with goat anti-rabbit secondary antibody only; area under curve #2 represents trypsin-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody; area under 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 curve #4 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody.
  • the data presented herein show the ability of select HDM2-specific antibodies to not only bind to the surface membrane of cancer cells but also to initiate a cytotoxic effect in the presence of fresh normal human serum (NHS).
  • the cytotoxic effect was measured in various cancer cells such as human melanoma, pancreatic, breast and ovarian cancer cells as well as in normal human fibroblasts and blood cells.
  • FIG. 25 demonstrates the cytotoxic effect of HDM2-specific antibodies against human pancreatic and ovarian cancer cells ( FIG. 25A ), and rodent pancreatic cells ( FIG. 25B ).
  • OP145, N-20 and C-18 antibodies in the presence of NHS trigger complement-mediated cytotoxicity in cancer cells incubated with such antibodies, resulting in the death of the cancer cells as evident by the nuclear uptake of the cell-death marker Propidium Iodide (PI) (see FIG. 25A , panels b and e, for ovarian cancer cells incubated with OP145 and FIG. 25B , panels b and c, for pancreatic cancer cells incubated with N-20 and C-18, respectively).
  • PI Propidium Iodide
  • cancer cell death is similar to that of control NHS alone, without any antibodies (see FIG. 25A , panels a and d, and FIG. 25B , panel a) or normal cells (Fibroblasts, FIG. 25A , panel g) treated with HDM2-specific antibodies (i.e., there is no or minimal cell death as indicated by lack of PI staining, se FIG. 25A , panels a, c, d, f and g, and FIG. 25B , panel 1).
  • control antibodies i.e., anti-cytochrome C antibody, see FIG. 25A , panel c, and FIG. 25B , panel e
  • FIG. 25C provides quantitative representations of HDM2-specific antibody-dependent complement cytotoxicity against human pancreatic cancer cells.
  • Cells treated with anti-HDM2 (C-18) antibody in the presence of NHS demonstrated cytotoxicity over 15-30 min. post-treatment, whereas anti-HDM2 OP46 shows no cytotoxic effect beyond that observed when cells were treated with control anti-Cytochrome C antibody or when cells were left untreated in the presence of NHS.
  • HDM2-specific antibodies in the presence of heat-inactivated human serum did not have any cytotoxic effect on cancer cells (i.e., did not result in the death of the cancer cells as was evident by PI staining), demonstrating that the cytotoxic effect is due to complement activity.
  • Table 10 lists antibodies tested by the inventors and summarizes results obtained relating to the in vitro cytotoxic effects of various anti-HDM2 antibodies against cancer cells.
  • Tumor measurements from the first 10 days post treatment with OP145 antibody versus PBS were made, and results are represented in FIG. 26 .
  • mice administered the OP145 antibody had a size of 260 mm 3 at 21 days post tumor cell injection, whereas subcutaneous tumors in mice administered PBS had a size of 375 mm 3 (see FIG. 26 ).
  • mAb NMC-103 and NMC-204 given to mice at the same dose demonstrated a significant (p ⁇ 0.005) reduction in tumor volume with lasting anti-tumor activity ( FIG. 19 ; open circle and open square, respectively; also see FIGS. 17A and 18A ).
  • This finding indicates that although mAb M01 had strong binding to cells in vitro, the anti-tumor effect of NMC-103 and NMC-204 was superior to that of mAb M01 ( FIG. 23 ).
  • SEQ ID NO: 1 MCNTNMSVPTDGAVT SEQ ID NO: 2 (NMC-P2) TTSQIPASEQE SEQ ID NO: 3 (NMC-P3) CPVCRQPIQMIVLTYFP
  • SEQ ID NO: 4 Human HDM2 Protein: MCNTNMSVPT DGAVTTSQIP ASEQETLVRP KPLLLKLLKS VGAQKDTYTM KEVLFYLGQY IMTKRLYDEK QQHIVYCSND LLGDLFGVPS FSVKEHRKIY TMIYRNLVVV NQQESSDSGT SVSENRCHLE GGSDQKDLVQ ELQEEKPSSS HLVSRPSTSS RRRAISETEE NSDELSGERQ RKRHKSDSIS LSFDESLALC VIREICCERS SSSESTGTPS NPDLDAGVSE HSGDWLDQDS VSDQFSVEFE VESLDSEDYS LSEEGQELSD EDDEVYQVTV YQAGESDT

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