WO2022133492A1 - Compositions et méthodes permettant de générer une réponse immunitaire anti-tumorale - Google Patents

Compositions et méthodes permettant de générer une réponse immunitaire anti-tumorale Download PDF

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WO2022133492A1
WO2022133492A1 PCT/US2021/073007 US2021073007W WO2022133492A1 WO 2022133492 A1 WO2022133492 A1 WO 2022133492A1 US 2021073007 W US2021073007 W US 2021073007W WO 2022133492 A1 WO2022133492 A1 WO 2022133492A1
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methylene
substituted
seq
imidazol
dione
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PCT/US2021/073007
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English (en)
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Lan Huang
James R. Tonra
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Beyondspring Pharmaceuticals, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • 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
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific

Definitions

  • the present disclosure relates to the field of chemistry and medicine. More particularly, the present disclosure relates to dehydrophenylahistin analogs, compositions including dehydrophenylahistin analogs, and their use in treatment.
  • TAM tumor associated macrophages
  • CD47 is a cell surface receptor comprised of an extracellular IgV set domain, a 5 transmembrane domain, and a cytoplasmic tail that is alternatively spliced. Two ligands bind CD47: signal inhibitory receptor protein a (SIRPa) and thrombospondin- 1 (TSP1). CD47 expression and/or activity have been implicated in a number of diseases and disorders. [0006] Tumor cells hijack the immunosuppressive mechanism by overexpressing CD47, which efficiently helps them to escape immune surveillance and killing by innate immune cells. (Majeti R, Ch et al., CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells, Cell. 2009 Jul.
  • CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis, Cell. 2009 Jul. 23; 138(2):271- 85).
  • CD47 expression is upregulated in most human cancers (e.g., NHL, AML, breast, colon, glioblastoma, glioma, ovarian, bladder and prostate cancers) and increased levels of CD47 expression clearly correlate with aggressive disease and poor survival.
  • CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors, Proc Natl Acad Sci USA. 2012 Apr. 24; 109(17):6662-7; Chao M P, et al., Therapeutic antibody targeting of CD47 eliminates human acute lymphoblastic leukemia, Cancer Res. 2011 Feb. 15; 71(4): 1374-84).
  • Some aspects of the disclosure relate to a method of treating a subject with cancer.
  • the method comprises administering a therapeutic dose of a compound of Formula (I): wherein
  • Ri, R4, and Re are each separately selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, and saturated Ci,-C24 alkyl, unsaturated Ci- C24 alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy, — CO — O — R7, cyano, alkylthio, halogenated alkyl including polyhalogenated alkyl, halogenated carbonyl, and carbonyl — CH2CO — R7, wherein R7 is selected from a hydrogen atom, a halogen atom, and saturated C1-C24 alkyl, unsaturated C1-C24 alkenyl, cycloalky
  • Ri' and Ri" are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, and saturated C1-C24 alkyl, unsaturated Ci- C24 alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy, — CO — O — R7, cyano, alkylthio, halogenated alkyl including polyhalogenated alkyl, halogenated carbonyl, and carbonyl — CH2CO — R7, wherein R7 is selected from a hydrogen atom, a halogen atom, and saturated C1-C24 alkyl, unsaturated C1-C24 alkenyl, cycloalkyl
  • R, Ri' and Ri are either covalently bound to one another or are not covalently bound to one another;
  • R2, R3, and R5 are each separately selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, and saturated C1-C12 alkyl, unsaturated Ci- C12 alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl and substituted sulfonyl groups; m is an integer equal to zero, one or two;
  • Xi and X2 are separately selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom, and
  • Y is selected from the group consisting of a nitrogen atom, a substituted nitrogen atom with a R5 group from above, an oxygen atom, a sulfur atom, a oxidized sulfur atom, a methylene group and a substituted methylene group; Z, for each separate n, if non-zero, and Zi, Z2, Za and Z4 are each separately selected from a carbon atom, a sulfur atom, a nitrogen atom or an oxygen atom; and the dashed bonds may be either single or double bonds an anti-CD47 agent.
  • the anti-CD47 agent is an antibody or antigenbinding fragment that specifically binds to CD47.
  • the antibody that specifically binds to CD47 is an isolated monoclonal antibody.
  • the anti-CD47 agent is an antibody or antigen-binding fragment that specifically binds to SIRPa.
  • the antibody that specifically binds to SIRPa is an isolated monoclonal antibody.
  • the antibody promotes macrophage-mediated phagocytosis of a CD47-expressing cell.
  • the anti-CD47 agent is a soluble CD47 binding SIRPa fragment.
  • the anti-CD47 agent prevents interaction between CD47 and SIRPa.
  • the anti-CD47 agent is an IgG isotype selected from the group consisting of IgG 1 isotype, IgG2 isotype, IgG3 isotype, IgG4 isotype, IG-G1-N297Q, IG4-S228P, and IG64 PE.
  • the cancer is selected from non-Hodgkin lymphoma, acute lymphoblastic leukemia (ALL), T-ALL, B- ALL, acute myelogenous leukemia (AML), B -lymphoblastic leukemia/lymphoma; diffuse large B cell lymphoma (DLBCL); B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, chronic lymphocytic leukemia (CLL), chronic myelocytic leukemia (CML), Burkitt's lymphoma, follicular lymphoma, SLL, marginal zone lymphoma, CNS lymphoma, Richter's Syndrome, multiple myeloma, myelofibrosis, polycythemia vera, cutaneous T-cell lymphoma, MGUS, myelodysplastic syndrome (MDS), immunoblastic large cell lymphoma, precursor B -lymphoblastic lymphoma and anaplastic large cell lymphom
  • ALL acute
  • the compound of formula (I) is administered at a dose from about 5 mg/m 2 to 150 mg/m 2 .
  • the compound of formula (I) is administered orally, sublingually, buccally, subcutaneously, intravenously, intranasally, intratumorally, topically, transdermally, intradermally, intraperitoneally, intramuscularly, intrapulmonarilly, vaginally, rectally, or intraocularly.
  • the compound of formula (I) is administered in combination with radiation.
  • the compound of formula (I) is administered once a week.
  • the compound of formula (I) is administered once on each of day 1 and day 8 of a three- week (21 day) treatment cycle.
  • the cancer is non-Hodgkin’s lymphoma.
  • the compound of Formula (I) includes plinabulin, (3Z,6Z)-3-(phenyl-2,3,4,5,6-d5)-methylene-6-((5-(tert-butyl)-lH-imidazol-4- yl)methylene)piperazine-2, 5-dione; (3Z, 6Z)-3-(phenyl-2, 3,4,5, 6-d5)-methylene-d-6-((5-(tert- butyl)-lH-imidazol-4-yl)methylene)piperazine-2, 5-dione; (3Z,6Z)-3-(phenylmethylene-d)-6- ((5-(tert-butyl)-lH-imidazol-4-yl)methylene-d)piperazine-2, 5-dione; (3Z,6Z)-3-(phenyl- 2,3,4,5,6-d5)-methylene-6-((5-(tert-butyl)-lH-imidazol-4-yl)m
  • the method includes administering a therapeutic dose of plinabulin and an anti-CD47 agent.
  • the anti-CD47 agent is an antibody or antigen-binding fragment that specifically binds to CD47.
  • the antibody that specifically binds to CD47 is an isolated monoclonal antibody.
  • the anti-CD47 agent is an antibody or antigen-binding fragment that specifically binds to SIRPa.
  • the antibody that specifically binds to SIRPa is an isolated monoclonal antibody.
  • the antibody promotes macrophage-mediated phagocytosis of a CD47-expressing cell.
  • the anti-CD47 agent is a soluble CD47 binding SIRPa fragment. In some embodiments, the anti-CD47 agent prevents interaction between CD47 and SIRPa. In some embodiments, the anti-CD47 agent is an IgG isotype selected from the group consisting of IgGl isotype, IgG2 isotype, IgG3 isotype, IgG4 isotype, IG-G1-N297Q, IG4-S228P, and IG64 PE.
  • the cancer is selected from non-Hodgkin lymphoma, acute lymphoblastic leukemia (ALL), T-ALL, B-ALL, acute myelogenous leukemia (AML), B- lymphoblastic leukemia/lymphoma; diffuse large B cell lymphoma (DLBCL); B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, chronic lymphocytic leukemia (CLL), chronic myelocytic leukemia (CML), Burkitt's lymphoma, follicular lymphoma, SLL, marginal zone lymphoma, CNS lymphoma, Richter's Syndrome, multiple myeloma, myelofibrosis, polycythemia vera, cutaneous T-cell lymphoma, MGUS, myelodysplastic syndrome (MDS), immunoblastic large cell lymphoma, precursor B -lymphoblastic lymphoma and anaplastic large cell lymphoma.
  • ALL acute
  • the plinabulin is administered at a dose from about 5 mg/m 2 to 150 mg/m 2 .
  • the plinabulin is administered orally, sublingually, buccally, subcutaneously, intravenously, intranasally, intratumorally, topically, transdermally, intradermally, intraperitoneally, intramuscularly, intrapulmonarilly, vaginally, rectally, or intraocularly.
  • the plinabulin is administered in combination with radiation.
  • the plinabulin is administered once a week.
  • the plinabulin is administered once on each of day 1 and day 8 of a three- week (21 day) treatment cycle.
  • the cancer is non-Hodgkin’ s lymphoma.
  • the method includes administering to the subject an effective amount of one or more anti-CD47 agent and plinabulin.
  • the one or more anti-CD47 agent is a monoclonal antibody that inhibits the interaction between human CD47 and SIRPa.
  • the monoclonal antibody is a human antibody.
  • the monoclonal antibody comprises a human kappa constant region.
  • the monoclonal antibody is administered prior to the administration of plinabulin.
  • the monoclonal antibody is administered after the administration of plinabulin.
  • the monoclonal antibody is an optimized antibody molecule.
  • the one or more anti-CD47 agent is selected from rituximab or daratumumab.
  • the cancer is selected from acute lymphoblastic leukemia (ALL), T-ALL, B-ALL, acute myelogenous leukemia (AML), Non-Hodgkin lymphoma, B -lymphoblastic leukemia/lymphoma; diffuse large B cell lymphoma (DLBCL); B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, chronic lymphocytic leukemia (CLL), chronic myelocytic leukemia (CML), Burkitt's lymphoma, follicular lymphoma, SLL, marginal zone lymphoma, CNS lymphoma, Richter's Syndrome, multiple myeloma, myelofibrosis, polycythemia vera, cutaneous T-cell lympho
  • ALL acute lymphoblastic leuk
  • the cancer is a cancer of a tissue selected from the group consisting of: lung, pancreas, breast, liver, ovary, testicle, kidney, bladder, spine, brain, cervix, endometrium, colon/rectum, anus, esophagus, gallbladder, gastrointestinal tract, skin, prostate, pituitary, stomach, uterus, vagina, and thyroid.
  • the one or more anti-CD47 agent is administered in combination with a pharmaceutically acceptable carrier or diluent.
  • the one or more anti-CD47 agent is administered subcutaneously.
  • the one or more anti-CD47 agent is administered intravenously.
  • the anti- CD47 agent is an antibody or antigen -binding fragment that specifically binds to CD47.
  • the antibody that specifically binds to CD47 is an isolated monoclonal antibody.
  • the anti-CD47 agent is an antibody or antigen-binding fragment that specifically binds to SIRPa.
  • the antibody that specifically binds to SIRPa is an isolated monoclonal antibody.
  • the antibody promotes macrophage-mediated phagocytosis of a CD47-expressing cell.
  • the anti-CD47 agent is a soluble CD-47 binding SIRPa fragment. In some embodiments, the anti-CD47 agent prevents interaction between CD47 and SIRPa. In some embodiments, the anti-CD47 agent is an IgG isotype selected from the group consisting of IgGl isotype, IgG2 isotype, IgG3 isotype, IgG4 isotype, IG-G1-N297Q, IG4-S228P, and IG64 PE. In some embodiments, the plinabulin is in an amount from about 5 mg to 150 mg. [0013]
  • the method comprises detecting tumor cell expression of CD-47 in a subject, and providing the subject a therapeutic amount of plinabulin and an anti-CD47 agent.
  • detecting a tumor cell expression of CD-47 comprises at least one of immunocytochemistry, proteomics, mRNA quantification, or a combination thereof, used to detect tumor cell expression of CD-47 in a subject.
  • FIG. 1A illustrates two scatterplot graphs depicting Ml polarization of intratumoral TAM cells from C57BV6 mice treated with plinabulin and M1/M2 ratio (macrophages).
  • FIG. IB illustrates a line graph depicting survival of C57BI/6 and Rag -/- mice treated with plinabulin.
  • FIG. 2A illustrates bar graphs (left) and histrograms (right) depicting M2 to Ml polarization of macrophages derived from murine tumors.
  • FIG. 2B illustrates bar graphs (top) and histograms (bottom) depicting murine bone marrow and human monocytes following ex vivo treatment with plinabulin.
  • FIG. 3 illustrates density plots depicting increased phagocytosis following treatment with plinabulin.
  • FIG. 4 illustrates line graphs depicting plinabulin induces an increase of Ml markers.
  • FIG. 5A illustrates a graph depicting mice bearing MC38 tumors (50-100 mm ⁇ ) were i e ft untreated or treated with plinabulin (7 mg/kg) administered peri-tumoral on days 0, 1, 2, 4, 7, 9, 11 post treatment start (first dose administered 18 days post MC38 cell innoculation).
  • FIG. 5B illustrates a bar graph depicting tumor volume (left) and percentage of live CD45" cells out of total cells (i.e. tumor cells, right) 7 days post plinabulin treatment start in plinabulin-treated or control mice.
  • FIG. 5C illustrates a line graph depicting Kaplan-Meier survival to humane endpoint curve of plinabulin-treated vs control mice (log-rank test p-value is shown).
  • FIG. 5D illustrates a graph depicting mice bearing MC38 tumors (50-100 mm3) were left untreated or treated with plinabulin (7.5 mg/kg) administered intra-peritoneal twice a day on days 0, 2, 4, post treatment start (first dose administered 18 days post MC38 cell innoculation). Tumors were collected for flow cytometry analyses on day 7 post treatment start.
  • FIG. 5E illustrates a bar graph depicting the percentage of intratumoral CD4 + (left) and CD8 + (right) T cells out of total live CD45 + CD3 + cells in plinabulin-treated and untreated MC38 tumors.
  • FIG. 5F illustrates a bar graph depicting the percentage of intratumoral Tregs (left) and CD8+ T cells to Treg ratio (right) in plinabulin treated and untreated MC38 tumors.
  • FIG. 5G illustrates a bar graph depicting the percentage of IFNy + CD4 + and IFNy " CD8 + cells after ex vivo anti-CD3 and anti-CD28 mAb re-stimulation of tumor infiltrating immune cells from plinabulin-treated or untreated MC38 tumors.
  • FIG. 5H illustrates a line graph depicting Kaplan-Meier survival curve of plinabulin-treated vs untreated mice at the experiment endpoint in wild type C57BL/6 (WT) or T- and B-cell deficient C57BL/6 RAG /_ mice.
  • FIG. 51 illustrates a bar graph depicting frequency of CDl lb + CDl lc + positive DCs (of all CD45+ cells, left) and F4/80 + Ly6C“ Ly6G“ TAMs (of CDl lb + population, right) in MC38-tumor bearing mice treated or untreated with plinabulin.
  • FIG. 5 J illustrates a bar graph depicting M1/M2 ratio (defined as the ratio between CD80+ and CD206+ TAMs) in plinabulin-treated MC38-tumor bearing mice and in untreated animals.
  • FIG. 6A illustrates a graph depicting MC38 tumors (400-600 mm3) from C57BL/6 mice were isolated (day -1). Tumor infiltrating TAMs were sorted by FACS and treated with plinabulin or relevant controls (day 0). Macrophage polarization was assessed by flow cytometry (day 2).
  • FIG. 6B illustrates Left: Histograms depicting expression of CD80 in murine TAMs treated ex vivo for 48 hours with plinabulin (1000 nM or 200 nM), IL-4 (25 ng/mL) or LPS (20 ng/mL) and IFN-y (50 ng/mL) combination and in untreated control. Right: gMFI of CD80 expression of murine TAMs in different treatment conditions (as indicated) after 48 hours.
  • FIG. 6C illustrates density plots depicting CD80 and CD206 expression in murine TAMs treated with plinabulin or in control conditions for 48 hours.
  • FIG. 6D illustrates a bar depicting quantification of M1/M2 ratio in murine TAMs treated with plinabulin or in control conditions for 48 hours.
  • FIG. 6E illustrates a graph depicting murine BMDMs were generated by culturing murine bine marrow cells with M-CSF (20 ng/mL) and IL-4 (25 ng/mL) for 7 days.
  • BMDMs were treated with plinabulin or control conditions (concentrations as if Fig. 5B) for 2 days prior to assessment with flow cytometry.
  • FIG. 6F illustrates bar graphs depicting frequency of CD80+ (left) or CD206+ (right) cells out of F4/80+ BMDMs, treated with plinabulin or control conditions.
  • FIG. 6G illustrates a bar graph depicting quantification of M1/M2 ratio in murine BMDMs treated with plinabulin or control conditions for 48 hours.
  • FIG. 6H illustrates a graph depicting experimental outline of macrophage generation with M-CSF (50 ng/mL) and IL-4 (25 ng/mL) from healthy donor PBMCs and treatment with plinabulin at indicated doses or controls (LPS at 25 ng/mL and IFN-y at 50 ng/mL).
  • FIG. 61 illustrates bar graphs depicting frequency of CD80 + (left) or CD206+ (right) cells out of CDl lb + CD 14+ human macrophages, treated with plinabulin or control conditions.
  • FIG. 6J illustrates a bar graph depicting quantification of M1/M2 ratio in human CD 14+ derived macrophages treated with plinabulin or control conditions for 48 hours.
  • FIG. 7A illustrates a graph depicting experimental outline of macrophage generation from healthy donor PBMCs, CTV labelling and treatment with plinabulin or controls prior to analysis by flow cytometry and multiplex cytokine analysis.
  • FIG. 7B illustrates histograms depicting CTV expression i.e. proliferation of CD86 + (left) or CD206+ (right) human macrophages treated for 48 hours with plinabulin (1000 nM or 200 nM), IL-4 (25 ng/mL), LPS (25 ng/mL), and IFN-y (50 ng/mL), combination or untreated.
  • plinabulin 1000 nM or 200 nM
  • IL-4 25 ng/mL
  • LPS 25 ng/mL
  • IFN-y 50 ng/mL
  • FIG. 7C illustrates bar graphs depicting quantification of CTV signal as gMFI in CD86+ (left) or CD206+ (right) human macrophages treated for 48 hours with plinabulin or control treatments.
  • FIG. 7D illustrates bar graphs depicting percentage of Annexin V + cells (left) and gMFI of AnnexinV (right) in human macrophages treated for 48 hours with plinabulin or control conditions.
  • FIG. 7E illustrates line graphs depicting quantification of pro- inflammatory cytokinesIL-ip IL-6 and IL12p40 in the supernatant of human macrophages from four healthy donors treated for 0, 24 or 48 hours with plinabulin (top) or LPS (25 ng/mL), and IFN-y (50 ng/mL), combination treatment (bottom).
  • FIG. 7F illustrates line graphs depicting quantification of iNOS mRNA expression by qPCR in human macrophages after 4 or 8 hours of treatment with plinabulin or LPS (25 ng/mL), and IFN-y (50 ng/mL), combination.
  • FIG. 8A illustrates a graph depicting experimental outline of macrophage generation from healthy donor PBMCs and treatment with plinabulin at indicated doses or controls in the presence of a JNK inhibitor SP600125 (iJNK, 20
  • FIG. 8B illustrates bar graphs depicting percentage of CD86+ (left) or CD163+ (right) cells out of CDl lb + CD 14+ human macrophages, treated with plinabulin or control conditions in the presence or absence of a JNK inhibitor.
  • FIG. 8C illustrates bar graphs depicting CD86+ (left) or CD163+ (right) events out of CDl lb+ CD 14+ human macrophages, treated with plinabulin or control conditions in the presence or absence of a JNK inhibitor, calculated using counting beads on flow cytometry.
  • FIG. 8D illustrates a bar graph depicting the percentage of live cells (cells negative for the live cell exclusion dye) out of total human macrophages, treated with plinabulin or control conditions, measured by flow cytometry.
  • FIG. 9A illustrates a graph depicting experimental outline of macrophage generation from healthy donor PBMCs, treatment with plinabulin or controls and co-culture with CTV-labelled HuT 78 tumor cells for 48 hours.
  • FIG. 9C illustrates histograms depicting CTV signal i.e. proliferation of HuT 78 tumor cells after co-culture with human macrophages pre-treated with plinabulin or control treatments at a 5:1 (left) or 10:1 (right) E:T ratio.
  • Left Frequency of Fas + HuT 78 tumor cells after co-culture with human macrophages that were pre-treated with plinabulin or control conditions.
  • Right Frequency of Fas-L + Ml macrophages (CD86+) treated with plinabulin or control conditions.
  • FIG. 9D illustrates bar graphs depicting quantification of CTV signal as gMFI in HuT 78 tumor cells after co-culture with human macrophages pre-treated with plinabulin or control treatments at a 5:1 (left) or 10:1 (right) E:T ratio.
  • FIG. 9E illustrates a bar graph depicting an increase in Fas+ tumor cells in co-culture with plinabulin-treated macrophages, compared to untreated cells and positive control.
  • FIG. 9F illustrates a bar graph depicting a dose-dependent increase in Fas- L expression on human macrophages treated with plinabulin.
  • FIG. 10A illustrates a graph depiciting experimental outline of TAM isolation from ovarian patient tumor digests, CTV labelling and treatment with plinabulin or controls.
  • FIG. 10B illustrates bar graphs depicting frequency of CD86+ (left) or CD206+ (right) cells out of CDl lb + CD 14+ human tumor infiltrating macrophages, treated with plinabulin or control conditions for 48 hours.
  • FIG. 10C illustrates histograms depicting CTV signal i.e. proliferation of human ovarian TAMs treated ex vivo for 48 hours with plinabulin (1000 nM or 200 nM) or control treatments.
  • FIG. 10D illustrates bar graphs depicting quantification of CTV signal as gMFI in human ovarian TAMs treated ex vivo for 48 hours with plinabulin (1000 nM or 200 nM) or control treatments.
  • FIG. 11A illustrates line graphs depicting EMT-6 tumor growth in untreated (black lines) or plinabulin-treated animals (blue lines; used at the dose of 15 mg/kg) over time.
  • FIG. 11B illustrates line graphs depicting Kaplan-Meier survival to humane end-point curve of EMT-6 tumour bearing, plinabulin-treated vs untreated mice. Statistical significance was determined by log-rank Mantel-Cox test with p value indicated on the graph.
  • FIG. 11C illustrates bar graphs depicting the percentage of TNFoc + CD4 + and TNFoc + CD8 + cells after ex vivo anti-CD3 and anti-CD28 mAb re-stimulation of intratumoral CD4+ and CD8+ T cells from plinabulin-treated or untreated MC38 tumors.
  • FIG. 12A illustrates a bar graph depicting the percentage live murine BMDMs following treatment with Plinabulin or controls as per schematic in Fig. 6E.
  • FIG. 12B illustrates a bar graph depicting frequency of CD86+ cells out of F4/80+ BMDMs, treated with plinabulin or control treatments.
  • FIG. 12C illustrates a bar graph depicting quantification of M1/M2 ratio in BMDMs treated with plinabulin or control treatments.
  • FIG. 12D illustrates a bar graph depicting frequency of CD163+ cells out of CD1 lb + CD14+ human macrophages, treated with plinabulin or control treatments.
  • FIG. 12E illustrates a line graph depicting frequency of CD86 + cells out of CDl lb+ CD14+ human macrophages after 48, 72 or 86 hours of treatment with plinabulin or control treatments.
  • FIG. 12F illustrates a line graph depicting frequency of CD206+ cells out of CDl lb + CD14+ human macrophages after 48, 72 or 86 hours of treatment with plinabulin or control treatments.
  • FIG. 13 A illustrates a line graph depicting quantification of IL lb mRNA expression by qPCR in human macrophages after 6 or 18 hours of treatment with plinabulin or LPS and IFN-y combination.
  • FIG. 13B illustrates a line graph depicting Quantification of Tgfb, Egr2, 114 and Ccll7 mRNA expression by qPCR in human macrophages after 4, 8 or 20 hours of treatment with plinabulin or LPS and IFN-y combination.
  • FIG. 14 illustrates bar graphs depicting the frequency of plinabulin or control-treated live human macrophages after co-culture with Fas + HuT 78 tumor cells at 5:1 (left) or 10:1 (right) E:T ratio.
  • FIG. 15 illustrates bar graphs depicting frequency of CD80+ and CD163+ cells in TAMs isolated from untreated human ovarian tumor digest.
  • FIG. 16 illustrate a bar graph depicting the percentage of phagocytosis index of Plinabulin.
  • FIG. 17 illustrate a bar graph depicting the percentage of phagocytosis index of anti-CD47.
  • Methods and compositions provided herein are useful in treating, delaying the progression of, preventing relapse of or alleviating a symptom of a cancer or other neoplastic condition using a synergistic combination of agents targeting CD47 and a compound of Formula (I).
  • the compound of Formula (I) is plinabulin.
  • Plinabulin, (3Z,6Z)-3-Benzylidene-6- ⁇ [5-(2-methyl-2-propanyl)-l/Z-imidazol-4- yl]methylene]-2,5-piperazinedione is a synthetic analog of the natural compound phenylahistin.
  • Plinabulin can be readily prepared according to methods and procedures detailed in U.S. Patent Nos.
  • the agents targeting CD47 include one or more monoclonal antibody or an antigen-binding fragment.
  • the one or more monoclonal antibody or the antigen-binding fragment are capable of modulating, e.g., blocking, inhibiting, reducing, antagonizing, neutralizing or otherwise interfering with CD47 expression, activity and/or signaling.
  • the one or more anti-CD47 agent is an antibody or antigen-binding fragment that specifically binds to CD47.
  • Methods recited herein may be carried out in any order of the recited events which is logically possible, as well as the recited order of events.
  • agent is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.
  • antibody or “antibody moiety” is intended to include any polypeptide chain-containing molecular structure with a specific shape that fits to and recognizes an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope.
  • Antibodies utilized in the present disclosure may be polyclonal antibodies or monoclonal antibodies. Antibodies also include free antibodies and antigen binding fragments derived therefrom, and conjugates, e.g. pegylated antibodies, drug, radioisotope, or toxin conjugates, and the like. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the targeting and/or depletion of cellular populations expressing the marker.
  • Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al. Cell, 96:737-49 (1999)). These techniques allow for the screening of particular populations of cells; in immunohistochemistry of biopsy samples; in detecting the presence of markers shed by cancer cells into the blood and other biologic fluids, and the like. Humanized versions of such antibodies are also within the scope of this disclosure. Humanized antibodies are especially useful for in vivo applications in humans due to their low antigenicity.
  • cancer neoplasm
  • tumor tumor
  • cancer tumor-associated fibroblast
  • cancer neoplasm
  • tumor tumor-associated plasm
  • tumor tumor-associated plasm
  • tumor tumor-associated plasm
  • CD47 refers a protein encoded by the CD47 gene. CD47 is overexpressed on the surface of many types of cancer cells. CD47 forms a signaling complex with signal-regulatory protein a (SIRPa), enabling the escape of these cancer cells from macrophage-mediated phagocytosis.
  • SIRPa signal-regulatory protein a
  • CD47 polypeptides denotes the three transcript variants of human CD47 (variant 1, NM 001777; variant 2, NM 198793; and variant 3, NM 001025079) encode three isoforms of CD47 polypeptide.
  • CD47 isoform 1 (NP 001768), the longest of the three isoforms, is 323 amino acids long.
  • CD47 isoform 2 (NP 942088) is 305 amino acid long.
  • CD47 isoform 3 is 312 amino acids long.
  • the three isoforms are identical in sequence in the first 303 amino acids.
  • Amino acids 1-8 comprise the signal sequence
  • amino acids 9-142 comprise the CD47 immunoglobulin like domain, which is the soluble fragment
  • amino acids 143-300 is the transmembrane domain.
  • the first 142 amino acids of CD47 polypeptide comprise the extracellular region of CD47 (SEQ ID NO: 1).
  • the three isoforms have identical amino acid sequence in the extracellular region, and thus any of the isoforms are can be used to generate soluble CD47.
  • “Soluble CD47” is a CD47 protein that lacks the transmembrane domain. Soluble CD47 is secreted out of the cell expressing it, instead of being localized at the cell surface.
  • a “fusion” polypeptide denotes a polypeptide comprising a polypeptide or portion (e.g., one or more domains) thereof fused or bonded to heterologous polypeptide.
  • a fusion soluble CD47 protein for example, will share at least one biological property in common with a native sequence soluble CD47 polypeptide.
  • Examples of fusion polypeptides include immunoadhesins, as described above, which combine a portion of the CD47 polypeptide with an immunoglobulin sequence, and epitope tagged polypeptides, which comprise a soluble CD47 polypeptide or portion thereof fused to a “tag polypeptide”.
  • the tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with biological activity of the CD47 polypeptide.
  • Suitable tag polypeptides generally have at least six amino acid residues and usually between about 6-60 amino acid residues.
  • immunological binding refers to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific.
  • the strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity.
  • Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigenbinding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions.
  • both the “on rate constant” (k on ) and the “off rate constant” (k O ff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)).
  • the ratio of k O ff/k O n enables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant Kd. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473).
  • An antibody of the present invention is said to specifically bind to CD47, when the equilibrium binding constant (Kd) is ⁇ 1 pM, preferably ⁇ 100 nM, more preferably ⁇ 10 nM, and most preferably ⁇ 100 pM to about 1 pM, as measured by assays such as radioligand binding assays, surface plasmon resonance (SPR), flow cytometry binding assay, or similar assays known to those skilled in the art.
  • Kd equilibrium binding constant
  • isolated polynucleotide shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the “isolated polynucleotide” (1) is not associated with all or a portion of a polynucleotide in which the “isolated polynucleotide” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.
  • isolated protein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin, or source of derivation, the “isolated protein” (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, e.g., free of marine proteins, (3) is expressed by a cell from a different species, or (4) does not occur in nature.
  • polypeptide is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence. Hence, native protein fragments, and analogs are species of the polypeptide genus.
  • immunospecifically binds are analogous terms in the context of antibodies and refer to molecules that bind to an antigen/epitope as such binding is understood by one skilled in the art.
  • a molecule e.g., an antibody
  • that specifically binds to an antigen may bind to other peptides or polypeptides, generally with lower affinity as determined by, e.g., immunoassays, surface plasmon resonance assays, for example, BiacoreTM KinExA platform (Sapidyne Instruments, Boise, Id.), or other assays known in the art.
  • molecules that specifically bind to an antigen bind to the antigen with a K a that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater than the Kawhen the molecules bind to another antigen.
  • molecules that specifically bind to an antigen do not cross react with other proteins.
  • molecules that specifically bind to an antigen do not cross react with other non- CD47 proteins.
  • a monoclonal antibody as used herein is a well-known term of art that refers to an antibody obtained from a population of homogenous or substantially homogeneous antibodies.
  • the term “monoclonal” is not limited to any particular method for making the antibody.
  • a population of monoclonal antibodies can be generated by cells, a population of cells, or a cell line.
  • a “monoclonal antibody,” as used herein is an antibody produced by a single cell or cell line wherein the antibody immunospecifically binds to a CD47 epitope as determined, e.g., by ELISA or other antigenbinding or competitive binding assay known in the art or in the Examples provided herein.
  • a monoclonal antibody can be a chimeric antibody or a humanized antibody.
  • a monoclonal antibody is a monovalent antibody or multivalent (e.g., bivalent) antibody.
  • non-natural amino acid refers to an amino acid that is not a proteinogenic amino acid, or a post-translationally modified variant thereof.
  • the term refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine, or post-translationally modified variants thereof.
  • polyclonal antibodies refers to an antibody population that includes a variety of different antibodies that immunospecifically bind to the same and/or to different epitopes within an antigen or antigens.
  • variable region or “variable domain” as used herein refer to a portion of an antibody, generally, a portion of an antibody light or heavy chain, typically about the amino-terminal 110 to 120 amino acids in a mature heavy chain and about the amino-terminal 90 to 100 amino acids in a mature light chain.
  • Variable regions comprise complementarity determining regions (CDRs) flanked by framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • spacial orientation of CDRs and FRs are as follows, in an N-terminal to C- terminal direction: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • variable region is a human variable region.
  • variable region comprises murine (e.g., mouse or rat) CDRs and human framework regions (FRs).
  • variable region is a primate (e.g., human or non-human primate) variable region.
  • variable region comprises murine (e.g., mouse or rat) CDRs and primate (e.g., human or non-human primate) framework regions (FRs).
  • FRs framework regions
  • a variable region described herein is obtained from assembling two or more fragments of human sequences into a composite human sequence.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. In addition, various adjuvants such as are commonly used in the art may be included. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Gilman et al. (Eds.) (1990); Goodman and Gilman’s: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press, which is incorporated herein by reference in its entirety.
  • the pharmaceutically acceptable excipient can be a monosaccharide or monosaccharide derivative.
  • subject means a human or a non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate.
  • a non-human mammal e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate.
  • mammal is used in its usual biological sense. Thus, it specifically includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, or the like.
  • primates including simians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, or the like.
  • an effective amount or a “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent that is effective to relieve, to some extent, or to reduce the likelihood of onset of, one or more of the symptoms of a disease or condition, and can include curing a disease or condition.
  • the terms “treat,” “treatment,” or “treating,” as used herein refers to administering a compound or pharmaceutical composition to a subject for prophylactic and/or therapeutic purposes.
  • the term “prophylactic treatment” refers to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition.
  • therapeutic treatment refers to administering treatment to a subject already suffering from a disease or condition.
  • chemotherapeutic agent refers to an agent that reduces, prevents, mitigates, limits, and/or delays the growth of metastases or neoplasms, or kills neoplastic cells directly by necrosis or apoptosis of neoplasms or any other mechanism, or that can be otherwise used, in a pharmaceutically-effective amount, to reduce, prevent, mitigate, limit, and/or delay the growth of metastases or neoplasms in a subject with neoplastic disease.
  • Chemotherapeutic agents include but are not limited to, for example, fluoropyrimidines; pyrimidine nucleosides; purine nucleosides; anti-folates, platinum-based agents; anthracyclines/anthracenediones; epipodophyllotoxins; camptothecins; hormones; hormonal complexes; antihormonals; enzymes, proteins, peptides and polyclonal and/or monoclonal antibodies; vinca alkaloids; taxanes; epothilones; antimicrotubule agents; alkylating agents; antimetabolites; topoisomerase inhibitors; antivirals; and various other cytotoxic and cytostatic agents.
  • the compounds described herein are a dehydrophenylahistin represented by Formula (I):
  • Ri, R4, and Re are each separately selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, and saturated Ci,-C24 alkyl, unsaturated C1-C24 alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy, — CO — O — R7, cyano, alkylthio, halogenated alkyl including polyhalogenated alkyl, halogenated carbonyl, and carbonyl — CH2CO — R7, wherein R7 is selected from a hydrogen atom, a halogen atom, and saturated C1-C24 alkyl, unsaturated C
  • Ri' and Ri" are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, and saturated C1-C24 alkyl, unsaturated C1-C24 alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy, — CO — O — R7, cyano, alkylthio, halogenated alkyl including polyhalogenated alkyl, halogenated carbonyl, and carbonyl — CH2CO — R7, wherein R?
  • phenyl is selected from a hydrogen atom, a halogen atom, and saturated C1-C24 alkyl, unsaturated C1-C24 alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups.
  • R, Ri' and Ri are either covalently bound to one another or are not covalently bound to one another;
  • R2, R3, and R5 are each separately selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, and saturated C1-C12 alkyl, unsaturated Ci-C 12 alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl and substituted sulfonyl groups.
  • m is an integer equal to zero, one or two.
  • Xi and X2 are separately selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom.
  • Y is selected from the group consisting of a nitrogen atom, a substituted nitrogen atom with a R5 group from above, an oxygen atom, a sulfur atom, a oxidized sulfur atom, a methylene group and a substituted methylene group.
  • Z for each separate n, if non-zero, and Zi, Z2, Za and Z4 are each separately selected from a carbon atom, a sulfur atom, a nitrogen atom or an oxygen atom; and the dashed bonds may be either single or double bonds.
  • the compounds described herein are a dehydrophenylahistin represented by Formula (II):
  • R2 and Ra arc each separately selected from the group consisting of a hydrogen atom; a halogen atom; mono-substituted; poly-substituted or unsubstituted, straight or branched chain variants of the following residues: C1-C12 alkyl, Ci-C 12 alkenyl, acyl, and alkoxy; and mono-substituted, polysubstituted or unsubstituted variants of the following residues: cycloalkyl, cycloalkoxy, aryl, heteroaryl, amino, nitro, and sulfonyl; or R2 is a bond to Ar.
  • R4 and Re are each separately selected from the group consisting of hydrogen; halogen; hydroxyl; mono-substituted, polysubstituted or unsubstituted, straight or branched chain variants of the following residues: Ci- C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, alkoxy, acyl, arylalkyl, heteroarylalkyl, alkyloxycarbonyloxy, ester, arylalkoxy, alkoxy, and alkylthio; mono-substituted, polysubstituted or unsubstituted variants of the following residues: acyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, cycloalkoxy, aryl, heteroaryl, aryloxy, arylcarbonyl, heterocycloalkyl, carbonyl, amino, aminocarbonyl
  • Xi and X2 are separately selected from the group consisting of an oxygen atom, a sulfur atom, and a nitrogen atom substituted with a R5 group;
  • Rs is selected from the group consisting of a hydrogen atom, a halogen atom, and saturated C1-C12 alkyl, unsaturated Ci- C12 alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl and substituted sulfonyl groups.
  • Y is selected from the group consisting of a nitrogen atom substituted with R5, an oxygen atom, a sulfur atom, a oxidized sulfur atom, a methylene group, and a substituted methylene group.
  • n is 0, 1, 2, 3, or 4.
  • Ar is a cyclic or polycyclic aryl or heteroaryl ring system comprising between one and three rings.
  • each ring in said system is separately a 5, 6, 7, or 8 membered ring.
  • each ring in said system separately comprises 0, 1, 2, 3, or 4 heteroatoms selected from the group consisting of oxygen, sulfur, and nitrogen.
  • each ring in the system is optionally substituted with one or more subtituents selected from the group consisting of hydrogen; halogen; hydroxyl; mono-substituted, poly-substituted or unsubstituted, straight or branched chain variants of the following residues: C1-C24 alkyl, C2-C24 alkenyl, C2- C24 alkynyl, alkoxy, acyl, arylalkyl, heteroarylalkyl, alkyloxycarbonyloxy, ester, arylalkoxy, alkoxy, and alkylthio; mono-substituted, poly-substituted or unsubstituted variants of the following residues: acyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, cycloalkoxy, aryl, heteroaryl, aryloxy, arylcarbonyl, heterocycloalkyl, carbonyloxy,
  • a compound of Formula (I) is selected from plinabulin, (3Z,6Z)-3-(phenyl-2,3,4,5,6-d5)-methylene-6-((5-(tert-butyl)-lH-imidazol-4- yl)methylene)piperazine-2, 5-dione; (3Z, 6Z)-3-(phenyl-2, 3,4,5, 6-d5)-methylene-d-6-((5-(tert- butyl)-lH-imidazol-4-yl)methylene)piperazine-2, 5-dione; (3Z,6Z)-3-(phenylmethylene-d)-6- ((5-(tert-butyl)-lH-imidazol-4-yl)methylene-d)piperazine-2, 5-dione; (3Z,6Z)-3-(phenyl- 2,3,4,5,6-d5)-methylene-6-((5-(tert-butyl)-lH
  • a compound of Formula (I) is plinabulin.
  • antibodies which specifically bind to CD47 comprise modifications in one or more amino acid residues. For example in the heavy chain variable region.
  • the CD47 antibodies inhibit SIRPa interaction with CD47.
  • the CD47 antibodies have low or no Fc effector function.
  • Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, murine antibodies (e.g., mouse or rat antibodies), chimeric antibodies, synthetic antibodies, and tetrameric antibodies comprising two heavy chain and two light chain molecules.
  • antibodies can include, but are not limited to an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain- antibody heavy chain pair, intrabodies, heteroconjugate antibodies, single domain antibodies, and monovalent antibodies.
  • antibodies can include antigen-binding fragments or epitope binding fragments such as, but not limited to, single chain antibodies or single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies, affybodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, and disulfide-linked Fvs (sdFv).
  • scFv single chain antibodies or single-chain Fvs
  • sdFv single-chain Fvs
  • antibodies described herein refer to polyclonal antibody populations.
  • an anti-CD47 antibody or antibodies is an anti-CD47 agent.
  • an anti-CD47 agent does not include an anti-CD
  • Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class, (e.g., IgGl, IgG2, IgG3, IgG4, IgAl or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule.
  • antibodies described herein are IgG antibodies, or a class (e.g., human IgGl, IgG2, IgG3 or IgG4) or subclass thereof.
  • antibodies described herein are IgGl antibodies (e.g., human IgGl) or a subclass thereof.
  • IgGl antibodies described herein comprise one or more amino acid substitutions and/or deletions in the constant region.
  • antibodies described herein are IgG4 antibodies (e.g., human IgG4) or a subclass thereof.
  • IgG4 antibodies described herein comprise one or more amino acid substitutions and/or deletions in the constant region.
  • the CD47 antibodies of the disclosure are monoclonal antibodies.
  • Monoclonal antibodies that modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with CD47- and/or CD47/SIRPa-mediated cell signaling are generated, e.g., by immunizing an animal with membrane bound and/or soluble CD47, such as, for example, human CD47 or an immunogenic fragment, derivative or variant thereof.
  • the animal is immunized with cells transfected with a vector containing a nucleic acid molecule encoding CD47 such that CD47 is expressed and associated with the surface of the transfected cells.
  • the antibodies are obtained by screening a library that contains antibody or antigen binding domain sequences for binding to CD47.
  • This library is prepared, e.g., in bacteriophage as protein or peptide fusions to a bacteriophage coat protein that is expressed on the surface of assembled phage particles and the encoding DNA sequences contained within the phage particles (i.e., “phage displayed library”).
  • phage displayed library Hybridomas resulting from myeloma/B cell fusions are then screened for reactivity to CD47.
  • the monoclonal antibodies of the disclosure that bind CD47, as well as fragments of these monospecific antibodies that are immunologically active and still bind CD47 include the exemplary antibodies described herein, e.g., the 5A3 antibody, the 5A3M4 antibody, the 5A3M3 antibody, the 5A3M5 antibody, the KE8 antibody, the KE8-P6H5 antibody (also referred to herein as KE8H5), the KE8-P3B2 antibody (also referred to herein as KE8B2), the KE8-P2A2 antibody (also referred to herein as KE8A25), the KE8F2 antibody, the KE8G2 antibody, the KE84G9 antibody, the KE81G9 antibody, the KE81A3 antibody, the KE8E8 antibody, the KE8G6 antibody, the KE8H3 antibody, the KE8C7 antibody, the KE8A4 antibody, the KE8A
  • the one or more anti-CD47 agent includes isolated bispecific antibodies having a first arm that includes a first amino acid sequence that binds CD47 and a second arm that includes a second amino acid sequence that does not bind CD47, wherein the bispecific antibody inhibits interaction between CD47 and signal- regulatory protein alpha (SIRPa).
  • SIRPa signal- regulatory protein alpha
  • the second amino acid sequence binds a tumor associated antigen (TAA).
  • TAA tumor associated antigen
  • the bispecific antibody inhibits interaction between human CD47 and human SIRPa.
  • the one or more anti-CD47 agents may include one or more bispecific antibodies.
  • the bispecific antibodies of the disclosure allow for simultaneous binding of the two antibody arms to two antigens on the surface of the cell (termed co -engagement), which results in additive or synergistic increase of affinity due to avidity mechanism. As a consequence, co-engagement confers high selectivity towards cells expressing both antigens as compared to cells that express just one single antigen.
  • the affinities of the two arms of a bispecific antibody to their respective targets can be set up in a way that binding to target cells is principally driven by one of the antibody arms.
  • the bispecific antibody includes a first arm that binds CD47 and a second arm that binds a tumor associated antigen (TAA), where the second arm binds to the TAA with high affinity, and the first arm binds to CD47 with low affinity, i.e., an affinity that is sufficient to inhibit CD47/SIRPa upon TAA co-engagement.
  • TAA tumor associated antigen
  • This design allows the bispecific antibodies of the invention to preferentially inhibit CD47 in cancer versus normal cells.
  • a bispecific antibody with a first arm that binds CD47 with low affinity and a second arm that binds CD 19 with high affinity (termed a CD47xCD19 bispecific) allow preferential inhibition of CD47 in cancer versus normal cells.
  • the CD47 x TAA bispecific antibody requires a functional Fc portion to recruit macrophages and/or other immune effector cells.
  • a fully human bispecific IgG format (such as the KA-body format described herein) is well suited for the generation of dual targeting CD47 x TAA bispecific antibodies.
  • the ability of dual targeting bispecific antibodies to coengage CD47 and CD 19 results in a significant increase in the affinity of binding to CD 19- positive cells and in CD19-dependent neutralization of the CD47-SIRPa interaction.
  • the bispecific antibody inhibits interaction between human CD47 and human SIRPa at a level that is at least ten times more potent than a corresponding level of inhibition of human CD47/human SIRPa interaction exhibited by a monovalent anti-CD47 antibody that includes the first amino acid sequence that binds CD47 and a second amino acid sequence that does not bind a human protein.
  • the bispecific antibody inhibits interaction between human CD47 and human SIRPa at a level that is at least 100 times more potent than a corresponding level of inhibition of human CD47/human SIRPa interaction exhibited by a monovalent anti-CD47 antibody that includes the first amino acid sequence that binds CD47 and a second amino acid sequence that does not bind a human protein.
  • the bispecific antibody inhibits interaction between human CD47 and human SIRPa at a level that is at least 1,000 times more potent than a corresponding level of inhibition of human CD47/human SIRPa interaction exhibited by a monovalent anti-CD47 antibody that includes the first amino acid sequence that binds CD47 and a second amino acid sequence that does not bind a human protein.
  • the antibody comprises light chain and heavy chain segments.
  • the light chain of an antibody described herein is a kappa (K) light chain.
  • the light chain of an antibody described herein is a lambda ( ) light chain.
  • light chain is a mixed sequence, e.g., the variable portion of the light chain comprises kappa light chain sequences and the constant region of the light chain comprises lambda light chain sequences, or vice versa.
  • the light chain of an antibody described herein is a human kappa light chain or a human lambda light chain.
  • the bispecific antibody includes two copies of a single heavy chain polypeptide and a first light chain and a second light chain, wherein the first and second light chains are different.
  • the an antibody e.g. a monoclonal antibody, which specifically binds to human CD47, wherein such an anti-CD47 antibody is a variant of a parental anti-CD47 antibody, wherein the anti-CD47 antibody, when produced using a cell- free (CF) expression system, has a higher antibody expression titer or yield compared to that of the parental anti-CD47 antibody when expressed in the CF system, and wherein the anti- CD47 antibody comprises one or more amino acid modifications, for example, 1-15 amino acid modifications, relative to the parental anti-CD47 antibody. In some embodiments, one or more amino acid modifications are within the heavy chain or VH.
  • the anti-CD47 antibody provided herein may be a variant of a parental anti-CD47 antibody comprising one or more CDRs of the parental anti-CD47 antibody.
  • an antibody e.g. a monoclonal antibody, which specifically binds to human CD47, wherein such an anti-CD47 antibody is a variant of a parental anti-CD47 antibody, wherein the anti-CD47 antibody, when produced using a cell-free (CF) expression system, has a higher antibody expression titer or yield compared to that of the parental anti-CD47 antibody when expressed in the CF system, and wherein the anti-CD47 antibody comprising one or more amino acid modifications, for example, 1-15 amino acid modifications, relative to the parental anti-CD47 antibody.
  • the one or more amino acid modifications for example, 5 or 14 amino acid modifications, are within the heavy chain or VH.
  • the one or more amino acid modifications are within the framework region of a VH.
  • the one or more amino acid modifications are within the framework region of a VH (e.g., SEQ ID NO: 1).
  • the anti-CD47 antibody provided herein which is a variant of a parental anti-CD47 antibody comprising the CDRs (e.g., Kabat CDRs) of the parental anti-CD47 antibody.
  • such anti-CD47 antibody is an IgGl, IgG2, IgG3, or IgG4 isotype antibody.
  • such anti-CD47 antibody is an IgGl isotype antibody. In certain aspects, such anti-CD47 antibody is an IgGl Z allotype isotype antibody. In certain aspects, such anti-CD47 antibody is an IgG4, such as an IgG4P or IgG4PE, isotype antibody.
  • an antibody e.g. a monoclonal antibody, which specifically binds to human CD47, wherein such an anti-CD47 antibody is a variant of a parental anti-CD47 antibody, wherein the anti-CD47 antibody, when produced using a cell- free (CF) expression system, has a higher antibody expression titer or yield compared to that of the parental anti-CD47 antibody when expressed in the CF system.
  • the parental anti-CD47 antibody is antibody AB6.12 (see, e.g., U.S. Application Publication No. US 2014/0140989 Al or U.S. Application Publication No. US 2021/0054070, which is incorporated herein by reference in its entirety).
  • the anti-CD47 antibody provided herein is a variant of parental antibody AB6.12, and comprises the CDRs (e.g., Kabat CDRs) of parental antibody AB6.12.
  • such anti-CD47 antibody is an IgGl, IgG2, IgG3, or IgG4 isotype antibody.
  • such anti-CD47 antibody is an IgGl isotype antibody.
  • such anti- CD47 antibody is an IgGl Z allotype isotype antibody.
  • such anti- CD47 antibody is an IgG4, such as an IgG4P or IgG4PE, isotype antibody.
  • the monoclonal antibodies may include fully human antibodies or humanized antibodies. In some embodiments, the monoclonal antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.
  • Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567, which is incorporated herein by reference in its entirety.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • Monoclonal antibodies of the disclosure may have the ability to bind CD47, to inhibit the binding of SIRPa to CD47, decrease CD47-SIRPa-mediated signaling, promote phagocytosis, and to inhibit tumor growth and/or migration.
  • Exemplary antibodies of the invention include the 2A1 antibody, the chimeric version of 2A1, and humanized variants of 2A1.
  • Exemplary antibodies of the invention include an antibody having a variable heavy (VH) chain selected from SEQ ID NOs: 305-330, and having a variable light (VL) chain selected from SEQ ID NOs: 331-347.
  • exemplary antibodies include those provided in Table 1.
  • anti-CD47 antibodies monospecific anti-CD47 antibodies, monovalent anti-CD47 antibodies, and/or bispecific antibodies in which at least one binding site is specific for CD47 contain a variable heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 114 and a variable light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from SEQ ID NO: 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166
  • the disclosure also provides monovalent antibodies that bind CD47. These antibodies are collectively referred to herein as anti-CD47 monovalent antibodies or anti-CD47 mono mAbs.
  • the monovalent antibodies include one arm that specific recognizes CD47, and a second arm referred to herein as a dummy arm.
  • the dummy arm includes an amino acid sequence that does not bind or otherwise cross-react with a human protein.
  • the dummy arm includes an amino acid sequence that does not bind or otherwise cross-react with a human protein that is found in whole blood.
  • human proteins found in the blood are a proxy that represent all, or substantially all, antigens present in system circulation.
  • the dummy arm includes an amino acid sequence that does not bind or otherwise cross-react with a human protein that is found in solid tissue.
  • the monovalent antibodies are specific for at least human CD47.
  • the monovalent antibodies that recognize human CD47 are also cross -reactive for at least one other non-human CD47 protein, such as, by way of non-limiting example, non-human primate CD47, e.g., cynomolgus monkey CD47, and/or rodent CD47.
  • the one or more anti-CD47 agent includes an immunizing agent.
  • an immunizing agent may include a protein antigen, a fragment thereof or a fusion protein thereof.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT)
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT -deficient cells.
  • an immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibodyproducing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of monoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).
  • antibodies that bind to the same epitope as the CD47 antibodies described herein specifically bind to an epitope that includes one or more amino acid residues on human CD47 (see e.g., GenBank Accession No. Q08722.1).
  • the amino acid sequence of an exemplary human CD47 is provided below (GenBank Accession No. Q08722.1 (GI: 1171879), incorporated herein by reference)
  • amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%.
  • conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains.
  • amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine.
  • the hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine.
  • the hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine.
  • Other families of amino acids include (i) serine and threonine, which are the aliphatic -hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family.
  • Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases.
  • computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. Bowie et al. Science 253:164 (1991).
  • sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the invention.
  • Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such analogs.
  • Analogs can include various muteins of a sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally-occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts.
  • a conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence).
  • Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991).
  • Antibodies are purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).
  • Screening of monoclonal antibodies can be carried out, e.g., by measuring CD47- and/or CD47/SIRPa- mediated signaling, and determining whether the test monoclonal antibody is able to modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with CD47- and/or CD47/SIRPa-mediated signaling.
  • These assays can include competitive binding assays. Additionally, these assays can measure a biologic readout, for example the ability to promote phagocytosis of a CD47 expressing cell by a macrophage.
  • CD47 antibodies are identified using a modified RIMMS (Repetitive Immunization Multiple Sites) immunization strategy in mice and subsequent hybridoma generation.
  • Human monoclonal antibodies can be also prepared by using the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized and may be produced by using human hybridomas (see Cote, et al., 1983.
  • Antibodies may also be produced by Chinese hamster ovary (CHO) cells (see Dhara, et ah, 2018. BioDrugs 32, 571- 584)
  • An alternative method for determining whether a monoclonal antibody has the specificity of monoclonal antibody is to pre-incubate the monoclonal antibody of the invention with soluble CD47 protein (with which it is normally reactive), and then add the monoclonal antibody being tested to determine if the monoclonal antibody being tested is inhibited in its ability to bind CD47. If the monoclonal antibody being tested is inhibited then, in all likelihood, it has the same, or functionally equivalent, epitope specificity as the monoclonal antibody.
  • a CD47 antibody is developed, for example, using phage-display methods using antibodies containing only human sequences. Such approaches are well-known in the art, e.g., in W092/01047 and U.S. Pat. No. 6,521,404, which are hereby incorporated by reference. In this approach, a combinatorial library of phage carrying random pairs of light and heavy chains are screened using natural or recombinant source of cd47 or fragments thereof. In another approach, a CD47 antibody can be produced by a process wherein at least one step of the process includes immunizing a transgenic, non-human animal with human CD47 protein.
  • the present disclosure also provides isolated, recombinant and/or synthetic anti-CD47 human, primate, rodent, mammalian, chimeric, humanized and/or CDR- grafted antibodies as well as compositions and encoding nucleic acid molecules comprising at least one polynucleotide encoding at least a portion of one anti-CD47 antibody molecule.
  • the present disclosure further includes, but is not limited to, methods of making and using such nucleic acids and antibodies including diagnostic and therapeutic compositions, methods and devices.
  • An exemplary anti-CD47 monovalent and bispecific antibodies include a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ
  • the 5A3 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 4) encoded by the nucleic acid sequence shown in SEQ ID NO: 3.
  • SEQ ID NO: 2 a common heavy chain encoded by the nucleic acid sequence shown in SEQ ID NO: 1
  • SEQ ID NO: 4 encoded by the nucleic acid sequence shown in SEQ ID NO: 3.
  • 5A3-LC-NT SEQ ID NO: 3
  • the 5A3 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 96) encoded by the nucleic acid sequence shown in SEQ ID NO: 95.
  • SEQ ID NO: 114 encoded by the nucleic acid sequence shown in SEQ ID NO: 113
  • SEQ ID NO: 96 kappa variable light domain
  • the 5A3-M4 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 6) encoded by the nucleic acid sequence shown in SEQ ID NO: 5.
  • the 5A3-M4 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 98) encoded by the nucleic acid sequence shown in SEQ ID NO: 97.
  • SEQ ID NO: 114 encoded by the nucleic acid sequence shown in SEQ ID NO: 113
  • SEQ ID NO: 98 kappa variable light domain
  • the 5A3-M3 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 8) encoded by the nucleic acid sequence shown in SEQ ID NO: 7.
  • the 5A3-M3 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 100) encoded by the nucleic acid sequence shown in SEQ ID NO: 99.
  • SEQ ID NO: 114 encoded by the nucleic acid sequence shown in SEQ ID NO: 113
  • SEQ ID NO: 100 kappa variable light domain
  • the 5A3-M5 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 10) encoded by the nucleic acid sequence shown in SEQ ID NO: 9.
  • the 5A3-M5 antibody includes a common variable heavy domain (SEQ ID NO: 1]
  • SEQ ID NO: 114 encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 102) encoded by the nucleic acid sequence shown in SEQ ID NO: 101.
  • the Ke8 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 12) encoded by the nucleic acid sequence shown in SEQ ID NO: 11.
  • the Ke8 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 104) encoded by the nucleic acid sequence shown in SEQ ID NO: 103.
  • the Ke8H5 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 14) encoded by the nucleic acid sequence shown in SEQ ID NO: 13.
  • KE8H5-LC-AA (SEQ ID NO: 14) DIQMTQSPSSLSASVGDRVTITCRASQSIARYLNWYQQKPGKAPKLLIYA ASSLQSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFHKRAPQTFG QGTKVEIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC
  • the Ke8H5 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 106) encoded by the nucleic acid sequence shown in SEQ ID NO: 105.
  • SEQ ID NO: 114 encoded by the nucleic acid sequence shown in SEQ ID NO: 113
  • SEQ ID NO: 106 encoded by the nucleic acid sequence shown in SEQ ID NO: 105.
  • the Ke8B2 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 16) encoded by the nucleic acid sequence shown in SEQ ID NO: 15.
  • Ke8B2 antibody includes a common variable heavy domain (SEQ ID NO:
  • the Ke8A2 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 18) encoded by the nucleic acid sequence shown in SEQ ID NO: 17.
  • Ke8A2 antibody includes a common variable heavy domain (SEQ ID NO: 1
  • SEQ ID NO: 114 encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 110) encoded by the nucleic acid sequence shown in SEQ ID NO: 109.
  • the Ke8E8 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 20) encoded by the nucleic acid sequence shown in SEQ ID NO: 19.
  • the Ke8E8 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 112) encoded by the nucleic acid sequence shown in SEQ ID NO: 111.
  • the Ke8H3 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 22) encoded by the nucleic acid sequence shown in SEQ ID NO: 21.
  • the Ke8H3 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113.
  • SEQ ID NO: 113 a common variable heavy domain encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113.
  • the Ke8G6 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 24) encoded by the nucleic acid sequence shown in SEQ ID NO: 23.
  • the Ke8G6 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 116) encoded by the nucleic acid sequence shown in SEQ ID NO: 115.
  • the Ke8A3 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 26) encoded by the nucleic acid sequence shown in SEQ ID NO: 25.
  • the Ke8A3 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 118) encoded by the nucleic acid sequence shown in SEQ ID NO: 117.
  • the Ke81A3 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 28) encoded by the nucleic acid sequence shown in SEQ ID NO: 27.
  • the Ke81A3 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 120) encoded by the nucleic acid sequence shown in SEQ ID NO: 119.
  • KE81A3-VL-AA (SEQ ID NO: 120) DIQMTQSPSSLSASVGDRVTITCQASQDINRYLNWYQQKPGKAPKLLIYA ASSLQSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRHPRAPRTFG QGTKVEIK
  • the Ke8A8 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 30) encoded by the nucleic acid sequence shown in SEQ ID NO: 29.
  • the Ke8A8 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 122) encoded by the nucleic acid sequence shown in SEQ ID NO: 121.
  • the Ke8C7 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 32) encoded by the nucleic acid sequence shown in SEQ ID NO: 31.
  • the Ke8C7 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 124) encoded by the nucleic acid sequence shown in SEQ ID NO: 123.
  • the Ke8G2 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 34) encoded by the nucleic acid sequence shown in SEQ ID NO: 33.
  • SEQ ID NO: 2 a common heavy chain encoded by the nucleic acid sequence shown in SEQ ID NO: 1
  • SEQ ID NO: 34 a kappa light chain encoded by the nucleic acid sequence shown in SEQ ID NO: 33.
  • the Ke8G2 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 126) encoded by the nucleic acid sequence shown in SEQ ID NO: 125.
  • the Ke81G9 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 36) encoded by the nucleic acid sequence shown in SEQ ID NO: 35.
  • the Ke81G9 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 128) encoded by the nucleic acid sequence shown in SEQ ID NO: 127.
  • the Ke8F2 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 38) encoded by the nucleic acid sequence shown in SEQ ID NO: 37.
  • the Ke8F2 antibody includes a common variable heavy domain (SEQ ID NO: 1]
  • the Ke8B7 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 40) encoded by the nucleic acid sequence shown in SEQ ID NO: 39.
  • the Ke8B7 antibody includes a common variable heavy domain (SEQ ID NO: 1]
  • SEQ ID NO: 114 encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 132) encoded by the nucleic acid sequence shown in SEQ ID NO: 131.
  • the Ke8C4 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 42) encoded by the nucleic acid sequence shown in SEQ ID NO: 41.
  • the Ke8C4 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 134) encoded by the nucleic acid sequence shown in SEQ ID NO: 133.
  • the Ke8Fl antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 44) encoded by the nucleic acid sequence shown in SEQ ID NO: 43.
  • KE8F1-LC-AA (SEQ ID NO: 44) DIQMTQSPSSLSASVGDRVTITCRASQSIASYVNWYQQKPGKAPKLLIYA ASGLQSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFHKRRPQTFG QGTKVEIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC
  • the Ke8Fl antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 136) encoded by the nucleic acid sequence shown in SEQ ID NO: 135.
  • the Ke8Gll antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 46) encoded by the nucleic acid sequence shown in SEQ ID NO: 45.
  • the Ke8Gll antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 138) encoded by the nucleic acid sequence shown in SEQ ID NO: 137.
  • the Ke8H6 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 48) encoded by the nucleic acid sequence shown in SEQ ID NO: 47.
  • the Ke8H6 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 140) encoded by the nucleic acid sequence shown in SEQ ID NO: 139.
  • the Ke84G9 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 50) encoded by the nucleic acid sequence shown in SEQ ID NO: 49.
  • the Ke84G9 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 142) encoded by the nucleic acid sequence shown in SEQ ID NO: 141.
  • SEQ ID NO: 114 encoded by the nucleic acid sequence shown in SEQ ID NO: 113
  • SEQ ID NO: 142 a kappa variable light domain encoded by the nucleic acid sequence shown in SEQ ID NO: 141.
  • the Ke8A4 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 52) encoded by the nucleic acid sequence shown in SEQ ID NO: 51.
  • the Ke8A4 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 144) encoded by the nucleic acid sequence shown in SEQ ID NO: 143.
  • the Ke86G9 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 54) encoded by the nucleic acid sequence shown in SEQ ID NO: 53.
  • the Ke86G9 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 146) encoded by the nucleic acid sequence shown in SEQ ID NO: 145.
  • the Ka3 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 56) encoded by the nucleic acid sequence shown in SEQ ID NO: 55.
  • the Ka3 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 148) encoded by the nucleic acid sequence shown in SEQ ID NO: 147.
  • KA3-VL-AA (SEQ ID NO: 148) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA ASSLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQMHPRAPKTFG QGTKVEIK
  • the Ka3A2 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 58) encoded by the nucleic acid sequence shown in SEQ ID NO: 57.
  • KA3A2-LC-NT (SEQ ID NO: 57)
  • the Ka3A2 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 150) encoded by the nucleic acid sequence shown in SEQ ID NO: 149.
  • SEQ ID NO: 114 encoded by the nucleic acid sequence shown in SEQ ID NO: 113
  • SEQ ID NO: 150 kappa variable light domain
  • the Ka3H3 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 60) encoded by the nucleic acid sequence shown in SEQ ID NO: 59.
  • the Ka3H3 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 152) encoded by the nucleic acid sequence shown in SEQ ID NO: 151.
  • the Ka3A3 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 62) encoded by the nucleic acid sequence shown in SEQ ID NO: 61.
  • the Ka3A3 antibody includes a common variable heavy domain (SEQ ID NO: 1]
  • the Ka3H8 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 64) encoded by the nucleic acid sequence shown in SEQ ID NO: 63.
  • SEQ ID NO: 2 a common heavy chain encoded by the nucleic acid sequence shown in SEQ ID NO: 1
  • SEQ ID NO: 64 kappa light chain encoded by the nucleic acid sequence shown in SEQ ID NO: 63.
  • the Ka3H8 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 156) encoded by the nucleic acid sequence shown in SEQ ID NO: 155.
  • the Ka3B2 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 66) encoded by the nucleic acid sequence shown in SEQ ID NO: 65.
  • the Ka3B2 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 158) encoded by the nucleic acid sequence shown in SEQ ID NO: 157.
  • the Ka3C5 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 68) encoded by the nucleic acid sequence shown in SEQ ID NO: 67.
  • KA3C5-LC-NT (SEQ ID NO: 67)
  • the Ka3C5 antibody includes a common variable heavy domain (SEQ ID NO: 1]
  • the Ka3G2 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 70) encoded by the nucleic acid sequence shown in SEQ ID NO: 69.
  • the Ka3G2 antibody includes a common variable heavy domain (SEQ ID NO: 1]
  • SEQ ID NO: 114 encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 162) encoded by the nucleic acid sequence shown in SEQ ID NO: 161.
  • the Ka3D3 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a kappa light chain (SEQ ID NO: 72) encoded by the nucleic acid sequence shown in SEQ ID NO: 71.
  • the Ka3D3 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a kappa variable light domain (SEQ ID NO: 164) encoded by the nucleic acid sequence shown in SEQ ID NO: 163.
  • the Kc4 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a lambda light chain (SEQ ID NO: 74) encoded by the nucleic acid sequence shown in SEQ ID NO: 73.
  • KC4-LC-AA (SEQ ID NO: 74) QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMI YEVSNRP SGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDWWFRPK VFGGGTKLTVLGQ PKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVT VAWKADSSPVKAGVETTTPS KQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECS
  • the Kc4 antibody includes a common variable heavy domain (SEQ ID NO: 1]
  • the Kc4Gll antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a lambda light chain (SEQ ID NO: 76) encoded by the nucleic acid sequence shown in SEQ ID NO: 75.
  • the Kc4Gll antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a lambda variable light domain (SEQ ID NO: 168) encoded by the nucleic acid sequence shown in SEQ ID NO: 167.
  • the Kc4Cll antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a lambda light chain (SEQ ID NO: 78) encoded by the nucleic acid sequence shown in SEQ ID NO: 77.
  • the Kc4Cll antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a lambda variable light domain (SEQ ID NO: 170) encoded by the nucleic acid sequence shown in SEQ ID NO: 169.
  • the Kc4Al antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a lambda light chain (SEQ ID NO: 80) encoded by the nucleic acid sequence shown in SEQ ID NO: 79.
  • the Kc4Al antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a lambda variable light domain (SEQ ID NO: 172) encoded by the nucleic acid sequence shown in SEQ ID NO: 171.
  • the Kc4A4 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a lambda light chain (SEQ ID NO: 82) encoded by the nucleic acid sequence shown in SEQ ID NO: 81.
  • the Kc4A4 antibody includes a common variable heavy domain (SEQ ID NO:
  • the Kc4E10 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a lambda light chain (SEQ ID NO: 84) encoded by the nucleic acid sequence shown in SEQ ID NO: 83.
  • the Kc4E10 antibody includes a common variable heavy domain (SEQ ID NO: 1]
  • SEQ ID NO: 1134 encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a lambda variable light domain (SEQ ID NO: 176) encoded by the nucleic acid sequence shown in SEQ ID NO: 175.
  • the Kc4G9 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a lambda light chain (SEQ ID NO: 86) encoded by the nucleic acid sequence shown in SEQ ID NO: 85.
  • the Kc4G9 antibody includes a common variable heavy domain (SEQ ID NO:
  • the Kc4C3 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a lambda light chain (SEQ ID NO: 88) encoded by the nucleic acid sequence shown in SEQ ID NO: 87.
  • the Kc4C3 antibody includes a common variable heavy domain (SEQ ID NO: 1]
  • KC4C3-VL-AA (SEQ ID NO: 180) QSALTQPASVSGSPGQSITISCTGTSSDVRAANYVSWYQQHPGKAPKLMI YKNSTRP SGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDWWFRPK VFGGGTKLTVL
  • the Kc4F4 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a lambda light chain (SEQ ID NO: 90) encoded by the nucleic acid sequence shown in SEQ ID NO: 89.
  • the Kc4F4 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a lambda variable light domain (SEQ ID NO: 182) encoded by the nucleic acid sequence shown in SEQ ID NO: 181.
  • the Kc4Bl antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a lambda light chain (SEQ ID NO: 92) encoded by the nucleic acid sequence shown in SEQ ID NO: 91.
  • the Kc4B 1 antibody includes a common variable heavy domain (SEQ ID NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a lambda variable light domain (SEQ ID NO: 184) encoded by the nucleic acid sequence shown in SEQ ID NO: 183.
  • the Kc4E2 antibody includes a common heavy chain (SEQ ID NO: 2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 and includes a lambda light chain (SEQ ID NO: 94) encoded by the nucleic acid sequence shown in SEQ ID NO: 93.
  • NO: 114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113 and includes a lambda variable light domain (SEQ ID NO: 186) encoded by the nucleic acid sequence shown in SEQ ID NO: 185.
  • Exemplary monoclonal antibodies of the disclosure include, for example, humanized antibodies having a variable heavy chain (VH) and/or variable light (VL) chain region shown in the sequences below.
  • VH variable heavy chain
  • VL variable light
  • SEQ ID NO: 187 EVQLQQSGAELVRSGASVKLSCTASQPN1KDYYLHWVKQRPEQGLEWIGWIDPDNG DTEFAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNAAYGSSSYPMDYWG QGTSVTV
  • variable light (VL) chain regions of some CD47 antibodies are provided below:
  • the anti-CD47 antibody is SEQ ID NO: 230.
  • the anti-CD47 antibody is SEQ ID NO: 231.
  • the anti-CD47 antibody is Hu5f9-g4. SEQ ID NO: 232.
  • the anti-CD47 antibody is STI-6643.
  • STI-6643 is described in U.S. Application Publication No. US 2014/0140989 Al, which is hereby incorporated by reference in its entirety.
  • the anti-CD47 antibody is Rituximab. SEQ ID NO:
  • the anti-CD47 antibody is Obinutuzumab. SEQ ID NO:
  • the anti-CD47 antibody is Ofatumumab. SEQ ID NO: 236.
  • IVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIP ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR
  • the anti-CD47 antibody is Daratumumab. SEQ ID NO: 237.
  • IVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIP ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
  • the anti-CD47 antibody is CC-90002.
  • CC-90002 s another humanized IgG4 anti-CD47 antibody that inhibits CD47-SIRPa interaction and enabled phagocytosis in cancer cell lines, including hematological cancer cell lines, as well as in solid tumor xenografts.
  • CC-90002 is described in Eladl et al, “Role of CD47 in Hematological Malignancies,” Journal of Hematology & Oncology, 13, 96 (2020), which is hereby incorporated by reference in its entirety.
  • the anti-CD47 antibody is SRF231.
  • SRF231 is a fully humanized monoclonal anti-CD47 antibody produced by phage technology. Data presented in an abstract showed it selectively blocks CD47- SIRP a, promoting phagocytosis of cancer cells and sparing T-cells and RBCs in vitro. SRF231 is described in Eladl et al, “Role of CD47 in Hematological Malignancies,” Journal of Hematology & Oncology, 13, 96 (2020), which is hereby incorporated by reference in its entirety.
  • the anti-CD47 antibody is ALX148.
  • ALX148 is generated by fusing an inactivated human IgGl Fc with a modified SIRPa DI domain. It blocks the CD47-SIRPa interaction and stimulates both innate and acquired immune responses, promoting DCs, macrophage, and T-cell responses.
  • ALX148 is described in Eladl et al, “Role of CD47 in Hematological Malignancies,” Journal of Hematology & Oncology, 13, 96 (2020), which is hereby incorporated by reference in its entirety.
  • the anti-CD47 antibody is KWAR23.
  • KWAR23 is a blocking antibody to human SIRPa, and is inert when administered on its own, but enhances the effect of rituximab.
  • KWAR23 is described in Eladl et al, “Role of CD47 in Hematological Malignancies,” Journal of Hematology & Oncology, 13, 96 (2020), which is hereby incorporated by reference in its entirety.
  • the anti-CD47 antibody is C47B 116. In some embodiment, the anti-CD47 antibody is C47B91. In some embodiments the anti-CD47 antibody is C47B222.
  • the antibodies C47B 116, C47B91, and C47B222 are described in Pietsch, et al, “Anti-leukemic activity and tolerability of anti-human CD47 monoclonal antibodies,” Blood Cancer Journal 7, e536 (2017), which is hereby incorporated by reference in its entirety.
  • the anti-CD47 antibody is TJC4.
  • TJC4 is a fully human anti-CD47 IgG4 antibody that shares a similar binding affinity to human and cynomolgus monkey CD47. Like other anti-CD47 antibodies, TJC4 blocks the interaction of CD47 and SIRPa, leading to the enhanced macrophage phagocytosis of various CD47+ tumor cell lines and primary AML cells. Mono-treatment of TJC4 completely eradicated tumor cells in a Raji cell xenograft model and significantly extended the overall survival of treated mice in an AML model. When combined with Rituximab, TJC4 showed a superior efficacy in a DLBCL model over the mono-treatment group.
  • TJC4 is described in Meng et al, “TJC4, a Differentiated Anti-CD47 Antibody with Novel Epitope and RBC Sparing Properties,” Blood (2019), 134 (Supplement- 1): 4063, which is hereby incorporated by reference in its entirety.
  • an antibody e.g. a monoclonal antibody, which specifically binds to human CD47, wherein such an anti-CD47 antibody is a variant of a parental anti-CD47 antibody, wherein the anti-CD47 antibody, when produced using a cell- free (CF) expression system, has a higher antibody expression titer or yield compared to that of the parental anti-CD47 antibody when expressed in the CF system, and wherein the anti- CD47 antibody comprises a VH comprising SEQ ID NO: 22.
  • such anti- CD47 antibody is an IgGl, IgG2, IgG3, or IgG4 isotype antibody.
  • such anti-CD47 antibody is an IgGl isotype antibody. In certain aspects, such anti-CD47 antibody is an IgGl Z allotype isotype antibody. In certain aspects, such anti-CD47 antibody is an IgG4, such as an IgG4P or IgG4PE, isotype antibody.
  • an anti-CD47 antibody (IgGl- 13m) provided herein comprises an IgGl heavy chain comprising the amino acid sequence as set forth below:
  • an anti-CD47 antibody (IgGl-13mZ) provided herein comprises an IgGl-Z allotype heavy chain comprising the amino acid sequence as set forth below:
  • an anti-CD47 antibody (IgGl -5m) provided herein comprises an IgGl heavy chain comprising the amino acid sequence as set forth below:
  • an anti-CD47 antibody (IgG4P-13m) provided herein comprises an IgG4P antibody comprising the amino acid sequence as set forth below:
  • an anti-CD47 antibody (IgG4P-5m) provided herein comprises an IgG4P heavy chain comprising the amino acid sequence as set forth below:
  • an anti-CD47 antibody (IgG4PE-13m) provided herein comprises an IgG4PE heavy chain comprising the amino acid sequence as set forth below:
  • an anti-CD47 antibody (IgG4PE-5m) provided herein comprises an IgG4PE heavy chain comprising the amino acid sequence as set forth below:
  • an antibody e.g. a monoclonal antibody, which specifically binds to human CD47, wherein such an anti-CD47 antibody is a variant of a parental anti-CD47 antibody, wherein the anti-CD47 antibody, when produced using a cell-free (CF) expression system, has a higher antibody expression titer or yield compared to that of the parental anti-CD47 antibody when expressed in the CF system, and wherein the anti-CD47 antibody comprises a light chain comprising a kappa or lambda light chain constant region (e.g., human kappa or lambda light chain constant region), for example SEQ ID NO: 507.
  • CF cell-free
  • an antibody e.g. a monoclonal antibody, which specifically binds to human CD47, wherein such an anti-CD47 antibody is a variant of a parental anti-CD47 antibody, wherein the anti-CD47 antibody, when produced using a cell-free (CF) expression system, has a higher antibody expression titer or yield compared to that of the parental anti-CD47 antibody when expressed in the CF system, and wherein the anti-CD47 antibody comprises (i) a VH described herein or a heavy chain described herein, and (ii) a light chain comprising a kappa or lambda light chain constant region (e.g., human kappa or lambda light chain constant region), for example SEQ ID NO: 507, e.g., as set forth below (anti-CD47 antibody light chain (Ig K)), or SEQ ID NO: 507 without the amino acid M at the N-terminus:
  • CF cell-free
  • an anti-CD47 described herein is an anti- CD47 antibody described in U.S. Application Publication No. US 2014/0140989 Al, which is hereby incorporated by reference in its entirety, for example any one of anti- CD47 antibodies in Table 1 of the publication (e.g., anti-CD47 antibody 2A1, AB2.03, AB2.04, AB2.05, AB2.06, AB2.07, AB2.08, AB2.09, AB2.13, AB3.09, AB6.12, AB6.13, AB6.14, AB6.17, AB10.13, AB10.14, AB11.05, AB 12.05, AB15.05, AB16.05, AB 17.05, AB22.05, AB23.05, AB24.05, and AB25.05), or any antibody comprising any of SEQ ID NOS: 5-30 of the publication.
  • the human IgG4 constant region is modified within the hinge region to prevent or reduce strand exchange, e.g., Ser228Pro (S228P).
  • the human IgG4 constant region is modified at amino acid 235 to alter Fc receptor interactions, e.g., Leu235Glu (L235E).
  • the human IgG4 constant region is modified within the hinge and at amino acid 235, e.g., Ser228Pro and Leu235Glu (S228P/L235E).
  • the human IgG4 constant region is modified at amino acid Asn297 (Kabat Numbering) to prevent to glycosylation of the antibody, e.g., Asn297Ala (N297A).
  • the human IgG4 constant region is modified at amino acid positions Ser228, Leu235, and Asn297 (e.g., S228P/L235E/N297A). (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).
  • the antibody is of human IgG4 subclass and lacks glycosylation.
  • the glycosylation can be eliminated by mutation at position 297 (Kabat numbering), for example N297A.
  • the glycosylation can be eliminated by production of the antibody in a host cell that lacks the ability for post-translational glycosylation, for example a bacterial or yeast derived system or a modified mammalian cell expression system.
  • the human IgG constant region is modified to alter antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), e.g., the amino acid modifications described in Natsume et al., 2008 Cancer Res, 68(10): 3863-72; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Moore et al., 2010 mAbs, 2(2): 181-189; Lazar et al., 2006 PNAS, 103(11): 4005-4010, Shields et al., 2001 JBC, 276(9): 6591-6604; Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48: 152-164; Alegre et al, 1992 J Immunol, 148: 3461- 3468; Reviewed in Kaneko and Ni
  • anti-CD47 antibodies provided herein exhibit one or more desirable characteristics, such as, by way of non-limiting example, blocking of the interaction between CD47 and its ligand SIRPa and/or promoting (e.g., inducing or increasing) phagocytosis, without promoting (e.g., inducing or increasing) hemagglutination of erythrocytes, as well as anti-tumor activity.
  • desirable characteristics such as, by way of non-limiting example, blocking of the interaction between CD47 and its ligand SIRPa and/or promoting (e.g., inducing or increasing) phagocytosis, without promoting (e.g., inducing or increasing) hemagglutination of erythrocytes, as well as anti-tumor activity.
  • anti-CD47 antibodies provided herein block at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, or at least 99% of the interaction between CD47 and SIRPa as compared to the level of interaction between CD47 and SIRPa in the absence of the anti-CD47 antibody described herein.
  • anti-CD47 antibodies described herein promote (e.g., induce or increase) phagocytosis of cells, e.g., CD47-expressing cells (e.g., CCRF-CEM cells), for example, by macrophages.
  • the level of phagocytosois in the presence of anti-CD47 antibodies described herein is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, at least 150%, at least 200%, compared to the level of phagocytosis in the presence of anti-CD47 antibodies described herein.
  • anti-CD47 antibodies described herein do not promote (e.g, induce or increase), or cause a significant level of, agglutination of cells, e.g., anti- CD47 antibodies described herein do not promote (e.g, induce or increase), or cause a significant level of, hemagglutination of red blood cells.
  • the level of agglutination in the presence of anti-CD47 antibodies described herein is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% compared to the level of agglutination in the presence of anti-CD47 antibodies known to induce agglutination, such as MCA911 mouse anti-human CD47 antibody (BRIC126).
  • anti-CD47 antibodies known to induce agglutination such as MCA911 mouse anti-human CD47 antibody (BRIC126).
  • anti-CD47 antibodies described herein do not promote (e.g., induce or increase), or cause a significant level of, agglutination if the level of agglutination in the presence of anti-CD47 antibodies described herein is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% compared to the level of agglutination in the presence of existing anti-CD47 antibodies known to induce agglutination, such as MCA911 mouse anti-human CD47 antibody (BRIC126).
  • BRIC126 MCA911 mouse anti-human CD47 antibody
  • compositions comprising: (a) a safe and therapeutically effective amount of a compound of Formula (I) or pharmaceutically acceptable salts thereof; (b) one or more monoclonal antibodies that bind to CD47 or an immunologically active fragment of one or more monoclonal antibodies that bind to CD47 and (c) a pharmaceutically acceptable carrier, diluent, excipient or combination thereof.
  • a compound of Formula (I) is selected from plinabulin, (3Z,6Z)-3-(phenyl-2, 3,4,5, 6-d5)-methylene-6-((5-(tert-butyl)-lH- imidazol-4-yl)methylene)piperazine-2, 5-dione; (3Z, 6Z)-3-(phenyl-2, 3,4,5, 6-ds)-methylene-d- 6-((5-(tert-butyl)-lH-imidazol-4-yl)methylene)piperazine-2, 5-dione; (3Z,6Z)-3-
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.
  • various adjuvants such as are commonly used in the art may be included. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Gilman et al. (Eds.) (1990); Goodman and Gilman’s: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press, which is incorporated herein by reference in its entirety.
  • substances which can serve as pharmaceutically- acceptable carriers or components thereof, are sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the TWEENS; wetting agents, such sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives;
  • methods for treating, managing, reducing, ameliorating, or preventing a disease or condition are provided herein. Some embodiments relate to a method for treating, managing, reducing, ameliorating, or preventing a disease or condition by coadministering a compound of Formula (I) and one or more anti-CD47 agents to a subject in need thereof.
  • coadministering does not restrict the order in which therapies are administered.
  • the therapies may be administered, e.g., serially, sequentially, concurrently, or concomitantly.
  • the terms “manage,” “managing,” and “management” refer to the beneficial effects that a subject derives from a therapy (e.g., a prophylactic or therapeutic agent), which does not result in a cure of a condition associated with CD47.
  • a subject is administered one or more therapies (e.g., prophylactic or therapeutic agents, such as an antibody described herein) to “manage” a condition or disorder described herein, one or more symptoms thereof, so as to prevent the progression or worsening of the condition or disorder.
  • the terms “reduce” or “reducing” in the context of a condition or disorder provided herein refer to the total or partial inhibition (e.g., less than 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%) or blockage of the development, recurrence, onset or spread of a condition or disorder provided herein (e.g., cancer, metastasis, or angiogenesis) and/or symptom related thereto, resulting from the administration of a therapy or combination of therapies provided herein (e.g., a combination of prophylactic or therapeutic agents, such as an antibody described herein).
  • a therapy or combination of therapies provided herein (e.g., a combination of prophylactic or therapeutic agents, such as an antibody described herein).
  • Some embodiments relate to a method for treating cancer using the pharmaceutical composition described herein to a subject in need thereof. Some embodiments relate to a method for treating cancer, comprising co-administering a compound of Formula (I) and one or more anti-CD47 agents.
  • the compound of Formula (I) is selected from plinabulin, (3Z, 6Z)-3-(phenyl-2, 3,4,5, 6-ds)- methylene-6-((5-(tert-butyl)-lH-imidazol-4-yl)methylene)piperazine-2, 5-dione; (3Z,6Z)-3- (phenyl-2,3,4,5,6-d5)-methylene-d-6-((5-(tert-butyl)-lH-imidazol-4-yl)methylene)piperazine- 2, 5-dione; (3Z,6Z)-3-(phenylmethylene-d)-6-((5-(tert-butyl)-lH-imidazol-4-yl)methylene- d)piperazine-2, 5-dione; (3Z,6Z)-3-(phenyl-2,3,4,5,6-d5)-methylene-6-((5-(tert-butyl)-lH- imid
  • the one or more anti-CD47 agents include one or more monoclonal antibodies that bind to CD47 to a subject in need thereof.
  • the subject can be an animal, e.g., a mammal, a human. In some embodiments, the subject is a human.
  • the monoclonal antibody specifically binds to CD47 is an isolated monoclonal antibody.
  • the one or more anti-CD47 agent is an antibody or antigen-binding fragment that specifically binds to SIRPa. In some embodiments, the monoclonal antibody that specifically binds to SIRPa is an isolated monoclonal antibody. In some embodiments, the monoclonal antibody promotes macrophage-mediated phagocytosis of a CD47- expressing cell. In some embodiments, the one or more anti-CD47 agents is a soluble CD-47 binding SIRPa fragment.
  • the one or more anti-CD47 agents prevents interaction between CD47 and SIRPa.
  • the anti-CD47 agent is an IgG isotype selected from the group consisting of IgG 1 isotype, IgG2 isotype, IgG3 isotype, IgG4 isotype, IG-G1-N297Q, IG4-S228P, and IG64 PE.
  • the cancer is selected from non-Hodgkin lymphoma.
  • plinabulin is administered at a dose from about 5 mg/m 2 to 150 mg/m 2 .
  • the plinabulin is administered orally, sublingually, buccally, subcutaneously, intravenously, intranasally, intratumorally, topically, transdermally, intradermally, intraperitoneally, intramuscularly, intrapulmonarilly, vaginally, rectally, or intraocularly.
  • plinabulin is administered in combination with radiation.
  • plinabulin is administering in combination with a chemotherapy agent.
  • the plinabulin is administered once a week.
  • the plinabulin is administered once on each of day 1 and day 8 of a three-week (21 day) treatment cycle.
  • Some aspects of the disclosure provide for a method of treating a subject with a cancer cell expressing CD-47.
  • the method comprises detecting tumor cell expression of CD-47 in a subject, and providing the subject a therapeutic amount of plinabulin and an anti-CD47 agent.
  • detecting a tumor cell expression of CD-47 comprises at least one of immunocytochemistry, proteomics, mRNA quantification, or a combination thereof, used to detect tumor cell expression of CD-47 in a subject.
  • Diseases or disorders related to aberrant CD47 expression, activity and/or signaling include, by way of non-limiting example, hematological cancer and/or solid tumors.
  • Hematological cancers include, e.g., leukemia, lymphoma and myeloma.
  • Certain forms of leukemia include, by way of non-limiting example, acute lymphocytic leukemia (ALL); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); chronic myelogenous leukemia (CML); Myeloproliferative disorder/neoplasm (MPDS); and myelodysplasia syndrome.
  • Certain forms of lymphoma include, by way of non-limiting example, Hodgkin's lymphoma, both indolent and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma (small cell and large cell).
  • myeloma include, by way of non-limiting example, multiple myeloma (MM), giant cell myeloma, heavy-chain myeloma, and light chain or Bence-Jones myeloma.
  • Solid tumors include, e.g., breast tumors, ovarian tumors, lung tumors, pancreatic tumors, prostate tumors, melanoma tumors, colorectal tumors, lung tumors, head and neck tumors, bladder tumors, esophageal tumors, liver tumors, and kidney tumors.
  • Symptoms associated with cancers and other neoplastic disorders include, for example, inflammation, fever, general malaise, fever, pain, often localized to the inflamed area, loss of appetite, weight loss, edema, headache, fatigue, rash, anemia, muscle weakness, muscle fatigue and abdominal symptoms such as, for example, abdominal pain, diarrhea or constipation.
  • a method for treating, delaying the progression of, impeding, or alleviating a symptom of cancer in a subject in need thereof by coadministering a compound of Formula (I) and an anti-CD47 antibody to the subject in need thereof by coadministering a compound of Formula (I) and an anti-CD47 antibody to the subject in need thereof.
  • the CD47 antibodies described herein are useful in treating hematological malignancies and/or tumors, e.g., hematological malignancies and/or tumors.
  • the CD47 antibodies described herein are useful in treating CD47+ tumors.
  • the CD47 antibodies described herein are useful in treating non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, leiomyoma, leiomyosarcoma, glioma, glioblastoma, and so on.
  • NHL non-Hodgkin's lymphoma
  • ALL acute lymphocytic leukemia
  • AML acute myeloid leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • MM multiple myeloma
  • breast cancer ovarian cancer
  • head and neck cancer bladder cancer
  • melanoma
  • Solid tumors include, e.g., breast tumors, ovarian tumors, lung tumors (e.g., NSCLC), pancreatic tumors, prostate tumors, melanoma tumors, colorectal tumors, lung tumors, head and neck tumors, bladder tumors, esophageal tumors, liver tumors (e.g., hepatocellular carcinoma), sarcoma, and kidney tumors.
  • lung tumors e.g., NSCLC
  • pancreatic tumors e.g., prostate tumors, melanoma tumors, colorectal tumors, lung tumors, head and neck tumors, bladder tumors, esophageal tumors, liver tumors (e.g., hepatocellular carcinoma), sarcoma, and kidney tumors.
  • liver tumors e.g., hepatocellular carcinoma
  • sarcoma e.g., hepatocellular carcinoma
  • a method of treating cancer in a subject comprises administering (e.g., administering concurrently or sequentially) to a subject in need thereof (i) an anti-CD47 antibody described herein or antigen-binding fragment thereof which specifically binds to CD47 such as human CD47, (ii) a compound of Formula (I); and (iii) another anti-cancer agent.
  • the anti-cancer agent is a chermotherapeutic agent (e.g., microtubule disassembly blocker, antimetabolite, topisomerase inhibitor, and DNA crosslinker or damaging agent).
  • the anti-cancer agent is a tyrosine kinase inhibitor (e.g., GLEEVEC® (imatinib mesylate) or SUTENT® (SU11248 or Sunitinib)).
  • tyrosine kinse inhibitors include 706 and AMNI07 (nilotinib).
  • RADOOI PKC412, gefitinib (IRESSATM), erlotinib (TARCEVA®), sorafenib (NEXAVAR®), pazopanib (VOTRIENTTM), axitinib, bosutinib, cediranib (RECENTIN®), SPRYCEL® (dasatinib), lapatinib (TYKERB®), lestaurtinib, neratinib, nilotinib (TASIGNA®), semaxanib, toceranib (PALLADIATM), vandetanib (ZACTIMATM), and vatalanib.
  • a method of treating cancer e.g., a hematological disorder/cancer or solid cancer
  • administering e.g., administering concurrently or sequentially
  • an anti- CD47 antibody described herein or antigen-binding fragment thereof which specifically binds to CD47 such as human CD47
  • a compound of Formula (I) e.g., a compound of Formula (I)
  • radiation therapy e.g., radiation therapy, radiation therapy.
  • a method of reducing tumor volume comprising contacting an effective amount of an anti-CD47 antibody described herein which specifically binds to human CD47 with the tumor and a compound of Formula (I).
  • Also provided herein is a method of reducing tumor volume in a subject in need thereof (e.g., a subject with a tumor, such as a CD47 expressing tumor), comprising administering to the subject an effective amount of an anti-CD47 antibody described herein which specifically binds to human CD47 and a compound of Formula (I).
  • a subject in need thereof e.g., a subject with a tumor, such as a CD47 expressing tumor
  • a method of inhibiting cancer cell growth or proliferation comprising contacting an effective amount of an anti-CD47 antibody described herein which specifically binds to human CD47 with cancer cells and a compound of Formula (I). Also provided herein is a method of inhibiting cancer cell growth or proliferation in a subject in need thereof (e.g., a subject with cancer cells, such as CD47 expressing cancer cells), comprising administering to the subject an effective amount of an anti-CD47 antibody described herein which specifically binds to human CD47 and a compound of Formula (I).
  • plinabulin and an anti-CD47 agent are administered in the same formulation.
  • Other embodiments include co-administering a compound of Formula (I) and one or more monoclonal antibodies that bind to CD47 in separate compositions.
  • some embodiments include a first pharmaceutical compositions comprising: (a) a safe and therapeutically effective amount of a compound of Formula (I) or pharmaceutically acceptable salts thereof and (b) a pharmaceutically acceptable carrier, diluent, excipient or combination thereof; and a second pharmaceutical composition comprising: (a) one or more monoclonal antibodies that bind to CD47 and (b) a pharmaceutically acceptable carrier, diluent, excipient or combination thereof.
  • cancer is head and neck cancer, lung cancer, stomach cancer, colon cancer, pancreatic cancer, prostate cancer, breast cancer, kidney cancer, bladder cancer, ovary cancer, cervical cancer, melanoma, glioblastoma, myeloma, lymphoma, hematological cancer, or leukemia.
  • the cancer is renal cell carcinoma, malignant melanoma, non-small cell lung cancer (NSCLC), ovarian cancer, Hodgkin's lymphoma or squamous cell carcinoma.
  • NSCLC non-small cell lung cancer
  • the cancer is selected from breast cancer, colon cancer, rectal cancer, lung cancer, prostate cancer, melanoma, leukemia, ovarian cancer, gastric cancer, renal cell carcinoma, liver cancer, pancreatic cancer, lymphomas and myeloma.
  • the cancer is a solid tumor or hematological cancer.
  • Some embodiments include using the compositions described herein to diagnose, prognose, monitor, treat, alleviate, and/or prevent a disease or pathology associated with aberrant CD47 expression, activity and/or signaling in a subject.
  • a therapeutic regimen is carried out by identifying a subject, e.g., a human patient suffering from (or at risk of developing) a disease or disorder associated with aberrant CD47 expression, activity and/or signaling, e.g., a cancer or other neoplastic disorder, using standard methods.
  • the diseases or disorders related to aberrant CD47 expression, activity and/or signaling include, by way of non-limiting example, hematological cancer and/or solid tumors.
  • Hematological cancers include, e.g., leukemia, lymphoma and myeloma.
  • leukemia include, by way of non-limiting example, acute lymphocytic leukemia (ALL); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); chronic myelogenous leukemia (CML); Myeloproliferative disorder/neoplasm (MPDS); and myelodysplasia syndrome.
  • ALL acute lymphocytic leukemia
  • AML acute myeloid leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • MPDS Myeloproliferative disorder/neoplasm
  • myelodysplasia syndrome myelodysplasia syndrome.
  • lymphoma include, by way of non-limiting example, Hodgkin's lymphoma, both indolent and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma (small cell and large cell).
  • Certain forms of myeloma include, by way of non-limiting example, multiple myeloma (MM), giant cell myeloma, heavy-chain myeloma, and light chain or Bence-Jones myeloma.
  • Solid tumors include, e.g., breast tumors, ovarian tumors, lung tumors, pancreatic tumors, prostate tumors, melanoma tumors, colorectal tumors, lung tumors, head and neck tumors, bladder tumors, esophageal tumors, liver tumors, and kidney tumors.
  • Symptoms associated with cancers and other neoplastic disorders include, for example, inflammation, fever, general malaise, fever, pain, often localized to the inflamed area, loss of appetite, weight loss, edema, headache, fatigue, rash, anemia, muscle weakness, muscle fatigue and abdominal symptoms such as, for example, abdominal pain, diarrhea or constipation.
  • a therapeutically effective amount of a composition of the invention relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target.
  • the amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered.
  • Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 100 mg/kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week.
  • Administration of the pharmaceutical compositions described herein can be via any of the accepted modes of administration for agents that serve similar utilities including, but not limited to, orally, sublingually, buccally, subcutaneously, intravenously, intranasally, intratumorally, topically, transdermally, intradermally, intraperitoneally, intramuscularly, intrapulmonarilly, vaginally, rectally, or intraocularly.
  • Oral and parenteral administrations are customary in treating the indications that are the subject of the preferred embodiments.
  • compositions described herein may be provided in unit dosage form.
  • a "unit dosage form” is a composition containing an amount of a compound or composition that is suitable for administration to an animal, preferably mammal subject, in a single dose, according to good medical practice.
  • the preparation of a single or unit dosage form does not imply that the dosage form is administered once per day or once per course of therapy.
  • Such dosage forms are contemplated to be administered once, twice, thrice or more per day and may be administered as infusion over a period of time (e.g., from about 30 minutes to about 2-6 hours), or administered as a continuous infusion, and may be given more than once during a course of therapy, although a single administration is not specifically excluded.
  • the skilled artisan will recognize that the formulation does not specifically contemplate the entire course of therapy and such decisions are left for those skilled in the art of treatment rather than formulation.
  • compositions useful as described above may be in any of a variety of suitable forms for a variety of routes for administration, for example, for oral, sublingual, buccal, nasal, rectal, topical (including transdermal and intradermal), ocular, intracerebral, intracranial, intrathecal, intra-arterial, intravenous, intramuscular, or other parental routes of administration.
  • routes for administration for example, for oral, sublingual, buccal, nasal, rectal, topical (including transdermal and intradermal), ocular, intracerebral, intracranial, intrathecal, intra-arterial, intravenous, intramuscular, or other parental routes of administration.
  • oral and nasal compositions include compositions that are administered by inhalation, and made using available methodologies.
  • a variety of pharmaceutically- acceptable carriers well-known in the art may be used.
  • Pharmaceutically-acceptable carriers include, for example, solid or liquid fillers, diluents, hydrotropies, surface-active agents, and encapsulating substances.
  • Optional pharmaceutically-active materials may be included, which do not substantially interfere with the inhibitory activity of the compound or composition.
  • the amount of carrier employed in conjunction with the compound or composition is sufficient to provide a practical quantity of material for administration per unit dose of the compound.
  • Various oral dosage forms can be used, including such solid forms as tablets, capsules (e.g. solid gel capsules and liquid gel capsules), granules and bulk powders. Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed, containing suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents.
  • Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules, containing suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, melting agents, coloring agents and flavoring agents.
  • the pharmaceutically-acceptable carriers suitable for the preparation of unit dosage forms for peroral administration is well-known in the art.
  • Tablets typically comprise conventional pharmaceutically-compatible adjuvants as inert diluents, such as calcium carbonate, sodium carbonate, mannitol, lactose and cellulose; binders such as starch, gelatin and sucrose; disintegrants such as starch, alginic acid and croscarmelose; lubricants such as magnesium stearate, stearic acid and talc.
  • Glidants such as silicon dioxide can be used to improve flow characteristics of the powder mixture.
  • Coloring agents such as the FD&C dyes, can be added for appearance.
  • Sweeteners and flavoring agents such as aspartame, saccharin, menthol, peppermint, and fruit flavors, are useful adjuvants for chewable tablets.
  • Capsules typically comprise one or more solid diluents disclosed above. The selection of carrier components depends on secondary considerations like taste, cost, and shelf stability, which are not critical, and can be readily made by a person skilled in the art.
  • Peroral compositions also include liquid solutions, emulsions, suspensions, and the like.
  • the pharmaceutically-acceptable carriers suitable for preparation of such compositions are well known in the art.
  • Typical components of carriers for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water.
  • typical suspending agents include methyl cellulose, sodium carboxymethyl cellulose, AVICEL RC-591, tragacanth and sodium alginate;
  • typical wetting agents include lecithin and polysorbate 80; and typical preservatives include methyl paraben and sodium benzoate.
  • Peroral liquid compositions may also contain one or more components such as sweeteners, flavoring agents and colorants disclosed above.
  • compositions may also be coated by conventional methods, typically with pH or time-dependent coatings, such that the subject composition is released in the gastrointestinal tract in the vicinity of the desired topical application, or at various times to extend the desired action.
  • dosage forms typically include, but are not limited to, one or more of cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, Eudragit coatings, waxes and shellac.
  • compositions described herein may optionally include additional drug actives.
  • compositions useful for attaining systemic delivery of the subject compounds include sublingual, buccal and nasal dosage forms.
  • Such compositions typically comprise one or more of soluble filler substances such as sucrose, sorbitol and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose and hydroxypropyl methyl cellulose. Glidants, lubricants, sweeteners, colorants, antioxidants and flavoring agents disclosed above may also be included.
  • a liquid composition, which is formulated for topical ophthalmic use is formulated such that it can be administered topically to the eye. The comfort may be maximized as much as possible, although sometimes formulation considerations (e.g. drug stability) may necessitate less than optimal comfort.
  • the liquid may be formulated such that the liquid is tolerable to the patient for topical ophthalmic use.
  • an ophthalmically acceptable liquid may either be packaged for single use, or contain a preservative to prevent contamination over multiple uses.
  • solutions or medicaments are often prepared using a physiological saline solution as a major vehicle.
  • Ophthalmic solutions may preferably be maintained at a comfortable pH with an appropriate buffer system.
  • the formulations may also contain conventional, pharmaceutically acceptable preservatives, stabilizers and surfactants.
  • Preservatives that may be used in the pharmaceutical compositions disclosed herein include, but are not limited to, benzalkonium chloride, PHMB, chlorobutanol, thimerosal, phenylmercuric, acetate and phenylmercuric nitrate.
  • a useful surfactant is, for example, Tween 80.
  • various useful vehicles may be used in the ophthalmic preparations disclosed herein. These vehicles include, but are not limited to, polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose, poloxamers, carboxymethyl cellulose, hydroxyethyl cellulose and purified water.
  • Tonicity adjustors may be added as needed or convenient. They include, but are not limited to, salts, particularly sodium chloride, potassium chloride, mannitol and glycerin, or any other suitable ophthalmically acceptable tonicity adjustor.
  • buffers include acetate buffers, citrate buffers, phosphate buffers and borate buffers. Acids or bases may be used to adjust the pH of these formulations as needed.
  • Ophthalmically acceptable antioxidants include, but are not limited to, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole and butylated hydroxy toluene.
  • Other excipient components which may be included in the ophthalmic preparations, are chelating agents.
  • a useful chelating agent is edetate disodium, although other chelating agents may also be used in place or in conjunction with it.
  • Topical formulations may generally be comprised of a pharmaceutical carrier, co-solvent, emulsifier, penetration enhancer, preservative system, and emollient.
  • compositions described herein may be dissolved or dispersed in a pharmaceutically acceptable diluent, such as a saline or dextrose solution.
  • a pharmaceutically acceptable diluent such as a saline or dextrose solution.
  • Suitable excipients may be included to achieve the desired pH, including but not limited to NaOH, sodium carbonate, sodium acetate, HC1, and citric acid.
  • the pH of the final composition ranges from 2 to 8, or preferably from 4 to 7.
  • Antioxidant excipients may include sodium bisulfite, acetone sodium bisulfite, sodium formaldehyde, sulfoxylate, thiourea, and EDTA.
  • excipients found in the final intravenous composition may include sodium or potassium phosphates, citric acid, tartaric acid, gelatin, and carbohydrates such as dextrose, mannitol, and dextran. Further acceptable excipients are described in Powell, et al., Compendium of Excipients for Parenteral Formulations, PDA J Pharm Sci and Tech 1998, 52 238-311 and Nema et al., Excipients and Their Role in Approved Injectable Products: Current Usage and Future Directions, PDA J Pharm Sci and Tech 2011, 65 287-332, both of which are incorporated herein by reference in their entirety.
  • Antimicrobial agents may also be included to achieve a bacteriostatic or fungistatic solution, including but not limited to phenylmercuric nitrate, thimerosal, benzethonium chloride, benzalkonium chloride, phenol, cresol, and chlorobutanol.
  • compositions for intravenous administration may be provided to caregivers in the form of one more solids that are reconstituted with a suitable diluent such as sterile water, saline or dextrose in water shortly prior to administration.
  • a suitable diluent such as sterile water, saline or dextrose in water shortly prior to administration.
  • the compositions are provided in solution ready to administer parenterally.
  • the compositions are provided in a solution that is further diluted prior to administration.
  • the combination may be provided to caregivers as a mixture, or the caregivers may mix the two agents prior to administration, or the two agents may be administered separately.
  • the plinabulin composition and the anti-CD47 composition are administered using the same route of administration. In other embodiments, the plinabulin composition and the anti-CD47 composition are administered using different routes of administration. In some embodiments, both the plinabulin and anti-CD47 compositions are administered intravenously. In some embodiments, both the plinabulin and anti-CD47 compositions are administered orally. In some embodiments, both the plinabulin and anti-CD47 compositions are administered intratumorally. In some embodiments, the plinabulin composition is administered intravenously and the anti-CD47 composition is administered orally.
  • the plinabulin composition is administered intravenously and the anti-CD47 composition is administered intratumorally. In some embodiments, the plinabulin composition is administered orally and the anti-CD47 composition is administered intravenously. In some embodiments, the plinabulin composition is administered orally and the anti-CD47 composition is administered intratumorally. In some embodiments, the plinabulin composition is administered intratumorally and the anti-CD47 composition is administered intravenously. In some embodiments, the plinabulin composition is administered intratumorally and the anti-CD47 composition is administered orally.
  • plinabulin may be administered at a dose in the range of about 1 mg/m 2 to about 50 mg/m 2 . In some embodiments, the plinabulin is administered at a dose in the range of about 1-50 mg/m 2 of the body surface area.
  • the plinabulin is administered at a dose in the range of about 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-13.75, 1-14, 1-15, 1- 16, 1-17, 1-18, 1-19, 1-20, 1-22.5, 1-25, 1-27.5, 1-30, 1.5-2, 1.5-3, 1.5-4, 1.5-5, 1.5-6, 1.5-7,
  • the plinabulin is administered at a dose of about 0.5, 1,
  • the plinabulin is administered at a dose less than about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,
  • the plinabulin is administered at a dose greater than about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 32, 33, 34, 35, 36, 37,
  • the plinabulin dose is about 5 mg - 300 mg, 5 mg - 200 mg, 7.5 mg - 200 mg, 10 mg - 100 mg, 15 mg - 100 mg, 20 mg - 100 mg, 30 mg - 100 mg, 40 mg - 100 mg, 10 mg - 80 mg, 15 mg - 80 mg, 20 mg - 80 mg, 30 mg - 80 mg, 40 mg - 80 mg, 10 mg - 60 mg, 15 mg - 60 mg, 20 mg - 60 mg, 30 mg - 60 mg, or about 40 mg - 60 mg.
  • the plinabulin administered is about 20 mg - 60 mg, 27 mg - 60 mg, 20 mg - 45 mg, or 27 mg - 45 mg.
  • the plinabulin administered is about 5 mg-7.5 mg, 5 mg-9 mg, 5 mg-10 mg, 5 mg-12mg, 5mg-14mg, 5mg-15 mg, 5 mg-16 mg, 5 mg- 18 mg, 5 mg-20 mg, 5 mg-22 mg, 5 mg-24 mg, 5 mg-26 mg, 5 mg-28mg, 5mg- 30mg, 5mg-32mg, 5mg-34mg, 5mg-36mg, 5mg-38mg, 5mg-40mg, 5mg-42mg, 5mg-44mg, 5mg-46mg, 5mg-48mg, 5mg-50mg, 5mg-52mg, 5mg-54mg, 5mg-56mg, 5mg-58mg, 5mg- 60mg, 7 mg-7.7 mg, 7 mg-9 mg, 7 mg-10 mg, 7 mg-12mg, 7mg-14mg, 7mg-15 mg, 7 mg-16 mg, 7
  • the plinabulin dose is greater than about 5 mg, about 10 mg, about 12.5 mg, about 13.5 mg, about 15 mg, about 17.5 mg, about 20 mg, about 22.5 mg, about 25 mg, about 27 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about 150mg, or about 200 mg.
  • the plinabulin dose is about less than about 5 mg, about 10 mg, about 12.5 mg, about 13.5 mg, about 15 mg, about 17.5 mg, about 20 mg, about 22.5 mg, about 25 mg, about 27 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about 150mg, or about 200 mg.
  • a daily dose of one or more monoclonal antibodies that bind to CD47 may be from about 100 pg/kg to about 100 mg/kg or more of body weight, from about 0.5 pg/kg or less to about 1 mg/kg, from about 1.0 mg/kg to about 100 mg/kg of body weight, or from about 1.0 mg/kg to about 50 mg/kg of body weight.
  • a daily dose of a one or more monoclonal antibodies that bind to CD47 may be from about 0.1 mg to about 150 mg per dose, from about 0.5 mg or less to about 100 mg, from about 1.0 mg to about 75 mg per dose, or from about 35 mg to about 50 mg per dose.
  • anti-CD47 agent may be administered at a dose in the range of about 1 mg/kg to about 50 mg/kg. In some embodiments, the anti-CD47 is administered at a dose in the range of about 1-50 mg/kg. In some embodiments, the anti- CD47 agent is administered at a dose in the range of about 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,
  • the anti-CD47 agent is administered at a dose of about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,
  • the anti-CD47 agent is administered at a dose less than about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16,
  • the anti-CD47 agent is administered at a dose greater than about 0.5, 1,
  • the anti-CD47 agent dose is about 0.5 mg - 3000 mg, 0.5 mg - 2500 mg, 0.5 mg - 2000 mg, 0.5 mg - 1500 mg, 0.5 mg - 1000 mg, 0.5 mg - 500 mg, 0.5 mg -200 mg, 0.75 mg - 200 mg, 1.0 mg - 100 mg, 1.5 mg - 100 mg, 2.0 mg - 100 mg, 3.0 mg - 100 mg, 4.0 mg - 100 mg, 1.0 mg - 80 mg, 1.5 mg - 80 mg, 2.0 mg - 80 mg, 3.0 mg - 80 mg, 4.0 mg - 80 mg, 1.0 mg - 60 mg, 1.5 mg - 60 mg, 2.0 mg - 60 mg, 3.0 mg - 60 mg, or about 4.0 mg - 60 mg.
  • the anti-CD47 agent administered is about 20 mg - 60 mg, 27 mg - 60 mg, 20 mg - 45 mg, or 27 mg - 45 mg. In some embodiments, the anti-CD47 agent administered is about 5 mg-7.5 mg, 5 mg-9 mg, 5 mg- 10 mg, 5 mg-12mg, 5mg-14mg, 5mg-15 mg, 5 mg-16 mg, 5 mg-18 mg, 5 mg-20 mg, 5 mg-22 mg, 5 mg-24 mg, 5 mg-26 mg, 5 mg-28mg, 5mg-30mg, 5mg-32mg, 5mg-34mg, 5mg-36mg, 5mg-38mg, 5mg-40mg, 5mg-42mg, 5mg-44mg, 5mg-46mg, 5mg-48mg, 5mg-50mg, 5mg- 52mg, 5mg-54mg, 5mg-56mg, 5mg-58mg, 5mg-60mg,
  • the anti- CD47 agent dose is greater than about 5 mg, about 10 mg, about 12.5 mg, about 13.5 mg, about 15 mg, about 17.5 mg, about 20 mg, about 22.5 mg, about 25 mg, about 27 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about 150mg, or about 200 mg.
  • the anti- CD47 agent dose is about less than about 5 mg, about 10 mg, about 12.5 mg, about 13.5 mg, about 15 mg, about 17.5 mg, about 20 mg, about 22.5 mg, about 25 mg, about 27 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about 150mg, or about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 1000 mg, about 2000 mg, or about 3000 mg.
  • the initial dose of the anti-CD47 agent is 1 mg/kg on day 1 followed by a weekly dose of 45 mg/kg.
  • the plinabulin is administered prior to the administration of the anti-CD47 agent or anti-CD47 antibody composition. In some embodiments, the plinabulin is administered concurrently with the anti-CD47 agent or anti- CD47 antibody composition. In some embodiments, the plinabulin is administered after the anti-CD47 agent or anti-CD47 antibody composition.
  • the plinabulin is administered about 1 min, 5min, 10 min, 15 min, 20 min, 25 min, 30 min, Ih, 1.5h, 2h, 2.5h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, lOh, l lh, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 24h, 30h, 36h, 40h, or 48h after the administration of the anti-CD47 agent or anti-CD47 antibody composition.
  • the plinabulin is administered in less than about 1 min, 5min, 10 min, 15 min, 20 min, 25 min, 30 min, Ih, 1.5h, 2h, 2.5h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, lOh, l lh, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 30h, 36h, 40h, or 48h after the administration of a the anti-CD47 agent or anti-CD47 antibody composition.
  • the plinabulin is administered in more than about 1 min, 5min, 10 min, 15 min, 20 min, 25 min, 30 min, Ih, 1.5h, 2h, 2.5h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, lOh, l lh, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h30h, 36h, 40h, or 48h after the administration of the anti-CD47 agent or anti-CD47 antibody composition.
  • the plinabulin is administered in about lmin-5min, Imin-lOmin, lmin-15min, lmin-20min, 1 min-25min, 1 min-30min, 0.25h-0.5h, 0.25-0.75h, 0.25-lh,0.5h-lh, 0.5h-2h, 0.5h-2.5h, lh-2h, lh-3h, lh-5h, lh-24h, lmin-24h, or 1 min-2h, 1 day- 2days, Iday - 3days, 1 day-4 days, 1 day-5 days, or 1 day-6 days after the administration of the anti-CD47 agent or anti-CD47 antibody composition.
  • the plinabulin when plinabulin is administered prior to the anti- CD47 agent or anti-CD47 antibody composition administration, the plinabulin is administered about lmin-5min, Imin-lOmin, lmin-15min, lmin-20min, 1 min-25min, 1 min- 30min, 0.25h-0.5h, 0.25-0.75h, 0.25-lh,0.5h-lh, 0.5h-2h, 0.5h-2.5h, lh-2h, lh-3h, lh-5h, Ih- 24h, Imin-lh, lmin-2h, lmin-5h, lmin-24h, 1 day- 2days, Iday - 3days, 1 day-4 days, 1 day- 5 days, or 1 day-6 days before the administration of the anti-CD47 agent or anti-CD47 antibody composition.
  • the plinabulin is administered about 1 min, 5min, 10 min, 15 min, 20 min, 25 min, 30 min, Ih, 1.5h, 2h, 2.5h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, lOh, l lh, 12h, 30h, 36h, 40h, 48h, 4 days, 5 days, 6 days, or 7 days before the administration of the anti-CD47 agent or anti-CD47 antibody composition.
  • the plinabulin is administered in less than about 1 min, 5min, 10 min, 15 min, 20 min, 25 min, 30 min, Ih, 1.5h, 2h, 2.5h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, lOh, l lh, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 30h, 36h, 40h, 48h, 4 days, 5 days, 6 days, or 7 days before the administration of the anti-CD47 agent or anti-CD47 antibody composition.
  • the plinabulin is administered in more than about 1 min, 5min, 10 min, 15 min, 20 min, 25 min, 30 min, Ih, 1.5h, 2h, 2.5h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, lOh, l lh, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 30h, 36h, 40h, 48h, 3 days, 4 days, 5 days, 6 days, or 7 days before the administration of the anti-CD47 agent or anti-CD47 antibody composition.
  • the treatment schedule includes co-administration of the anti-CD47 agent or anti-CD47 antibody composition and plinabulin once every 3 weeks. In some embodiments, the treatment schedule includes co-administration of the anti- CD47 agent or anti-CD47 antibody composition and plinabulin once every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In some embodiments, the treatment schedule includes co-administration of the anti-CD47 agent or anti-CD47 antibody and plinabulin two times every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks.
  • the treatment schedule includes co-administration of the anti-CD47 agent or anti-CD47 antibody composition and plinabulin once every 1 week in a treatment cycle of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks.
  • the treatment schedule includes co-administration of the anti- CD47 agent or anti-CD47 antibody composition and plinabulin twice every 1 week in a treatment cycle of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks.
  • the treatment schedule includes co-administration of the anti- CD47 agent or anti-CD47 antibody composition and plinabulin on day 1, day 8, and day 15 of a 21 -day treatment cycle.
  • co-administration of the anti-CD47 agent or anti-CD47 antibody composition and plinabulin includes administering the anti-CD47 agent or anti-CD47 antibody prior to administering plinabulin. In some embodiments, co- administration of the anti-CD47 agent or anti-CD47 antibody composition and plinabulin includes administering the anti-CD47 agent or anti-CD47 antibody after administering plinabulin. In some embodiments, co-administration of the anti-CD47 agent or anti-CD47 antibody composition and plinabulin includes administering the anti-CD47 agent or anti- CD47 antibody concurrently with plinabulin.
  • the anti-CD47 agent or anti-CD47 antibody composition described in this paragraph can independently be a first, second, third, fourth, fifth, sixth, seventh, or eighth anti-CD47 agent or anti-CD47 antibody.
  • the treatment schedule includes co-administration of the anti-CD47 agent or anti-CD47 antibody composition and plinabulin every day of the week for a week.
  • the treatment schedule includes co-administration of the anti-CD47 agent or anti-CD47 antibody composition and plinabulin every day of the week for 2 weeks, 3 weeks, or 4 weeks.
  • the treatment schedule includes co- administration of the anti-CD47 agent or anti-CD47 antibody composition and plinabulin on day 1 in weekly treatment.
  • the treatment schedule includes co- administration of the anti-CD47 agent or anti-CD47 antibody composition and plinabulin on day 1 and day 2 in weekly treatment. In some embodiments, the treatment schedule includes co-administration of the anti-CD47 agent or anti-CD47 antibody composition and plinabulin on day 1, day 2, and day 3 in weekly treatment. In some embodiments, the treatment schedule includes co-administration of the anti-CD47 agent or anti-CD47 antibody composition and plinabulin on day 1, day 2, day 3 in weekly treatment. In some embodiments, the treatment schedule includes co-administration of the anti-CD47 agent or anti-CD47 antibody composition and plinabulin on day 1, day 2, day 3, and day 4 in weekly treatment.
  • the treatment schedule includes co-administration of the anti-CD47 agent or anti-CD47 antibody composition and plinabulin on day 1, day 2, day 3, day 4, and day 5 in weekly treatment. In some embodiments, the treatment schedule includes co-administration of the anti-CD47 agent or anti-CD47 antibody composition and plinabulin on day 1, day 2, day 3, day 4, day 5, and day 6 in weekly treatment. In some embodiments, the treatment schedule includes co-administration of the anti-CD47 agent or anti-CD47 antibody composition and plinabulin on day 1, day 3, and day 5 in weekly treatment. In some embodiments, the anti-CD47 agent or anti-CD47 antibody used on each administration day can be the same or different.
  • the anti-CD47 agent or anti-CD47 antibody used on the first administration day is different from the anti-CD47 agent or anti- CD47 antibody used on the rest of the administration days. In some embodiments, the anti- CD47 agent or anti-CD47 antibody used on the first administration day is the same as or different from the anti-CD47 agent or anti-CD47 antibody used on the second administration day. In some embodiments, the anti-CD47 agent or anti-CD47 antibody used on the first administration day is the same as or different from the anti-CD47 agent or anti-CD47 antibody composition used on the third administration day.
  • the anti- CD47 agent or anti-CD47 antibody composition used on the first administration day is the same as or different from the anti-CD47 agent or anti-CD47 antibody composition used on the fourth administration day. In some embodiments, the anti-CD47 agent or anti-CD47 antibody composition used on the first administration day is the same as or different from the anti-CD47 agent or anti-CD47 antibody composition used on the fifth administration day. In some embodiments, the anti-CD47 agent or anti-CD47 antibody used on the first administration day is the same as or different from the anti-CD47 agent or anti-CD47 antibody composition used on the sixth administration day.
  • the anti- CD47 agent or anti-CD47 antibody composition used on the first administration day is the same as or different from the anti-CD47 agent or anti-CD47 antibody composition used on the seventh administration day.
  • the treatment schedule includes administration of the anti-CD47 agent or anti-CD47 antibody composition (e.g., the first, the second, the third, the fourth, the fifth, the sixth, the seventh, or the eighth) once every 3 weeks.
  • the treatment schedule includes administration of the anti-CD47 agent or anti- CD47 antibody composition once every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks.
  • the treatment schedule includes administration of the anti-CD47 agent or anti-CD47 antibody composition two times every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In some embodiments, the treatment schedule includes administration of the anti-CD47 agent or anti- CD47 antibody composition once every 1 week in a treatment cycle of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In some embodiments, the treatment schedule includes administration of the anti-CD47 agent or anti-CD47 antibody composition twice every 1 week in a treatment cycle of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks.
  • the treatment schedule includes administration of the anti-CD47 agent or anti-CD47 antibody composition three times (e.g., day 1, 2, 3, or day 1, 3, 5) every week in a treatment cycle of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks.
  • the treatment schedule includes administration of the anti-CD47 agent or anti-CD47 antibody composition day 1, day 8, and day 15 of a 21-day treatment cycle.
  • the anti-CD47 agent or anti-CD47 antibody composition described in this paragraph can independently be the first, second, third, fourth, fifth, sixth, seventh, or eighth the anti-CD47 agent or anti-CD47 antibody.

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Abstract

Sont divulguées des compositions qui comprennent des analogues de la déhydrophénylahistine tels que la plinabuline et un agent anti-CD47 pour le traitement du cancer. Sont également divulguées des méthodes de traitement du cancer par co-administration de l'analogue de la déhydrophénylahistine et d'un ou plusieurs agents anti-CD47 à un sujet en ayant besoin. Dans certains modes de réalisation, le ou les agents anti-CD47 sont une molécule d'anticorps anti-CD47 capable de se lier à CD47, ainsi que des fragments de ces anticorps monospécifiques qui sont immunologiquement actifs et se lient encore à CD47.
PCT/US2021/073007 2020-12-18 2021-12-17 Compositions et méthodes permettant de générer une réponse immunitaire anti-tumorale WO2022133492A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014160183A1 (fr) * 2013-03-13 2014-10-02 The United States Of America,As Represented By The Secretary,Department Of Health And Human Services Procédés de modulation de la cytotoxicité chimiothérapeutique
WO2016130839A1 (fr) * 2015-02-12 2016-08-18 Beyondspring Pharmaceuticals, Inc. Utilisation de plinabuline en combinaison avec des inhibiteurs du point de contrôle immunitaire
WO2018169887A1 (fr) * 2017-03-13 2018-09-20 Beyondspring Pharmaceuticals, Inc. Compositions de plinabuline et leur utilisation
WO2019152530A1 (fr) * 2018-02-01 2019-08-08 Beyondspring Pharmaceuticals, Inc. Composition et méthode pour réduire la neutropénie induite par chimiothérapie par l'administration de plinabuline et de l'agent g-csf
WO2021225908A1 (fr) * 2020-05-04 2021-11-11 Beyondspring Pharmaceuticals, Inc. Trithérapie pour améliorer la destruction de cellules cancéreuses dans des cancers à faible immunogénicité

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014160183A1 (fr) * 2013-03-13 2014-10-02 The United States Of America,As Represented By The Secretary,Department Of Health And Human Services Procédés de modulation de la cytotoxicité chimiothérapeutique
WO2016130839A1 (fr) * 2015-02-12 2016-08-18 Beyondspring Pharmaceuticals, Inc. Utilisation de plinabuline en combinaison avec des inhibiteurs du point de contrôle immunitaire
WO2018169887A1 (fr) * 2017-03-13 2018-09-20 Beyondspring Pharmaceuticals, Inc. Compositions de plinabuline et leur utilisation
WO2019152530A1 (fr) * 2018-02-01 2019-08-08 Beyondspring Pharmaceuticals, Inc. Composition et méthode pour réduire la neutropénie induite par chimiothérapie par l'administration de plinabuline et de l'agent g-csf
WO2021225908A1 (fr) * 2020-05-04 2021-11-11 Beyondspring Pharmaceuticals, Inc. Trithérapie pour améliorer la destruction de cellules cancéreuses dans des cancers à faible immunogénicité

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