WO2019200357A1

WO2019200357A1 - Biomarker for cd47 targeting therapeutics and uses therefor

Info

Publication number: WO2019200357A1
Application number: PCT/US2019/027386
Authority: WO
Inventors: Alison Paterson; Marisa O. PELUSO; Benjamin H. LEE
Original assignee: Surface Oncology, Inc.
Priority date: 2018-04-12
Filing date: 2019-04-12
Publication date: 2019-10-17

Abstract

The present disclosure relates to agents that bind CD47 and comprise an Fc domain, and methods of identifying patients likely to respond to said agents. The disclosure also relates to methods for treating or ameliorating one or more symptoms of a disease, such as cancer, by administering the agent.

Description

BIOMARKER FOR CD47 TARGETING THERAPEUTICS AND USES THEREFOR

REUATED APPUI CATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 62/656,911, filed on April 12, 2018. The content of this application is hereby incorporated by reference in its entirety.

BACKGROUND

CD47 (Cluster of Differentiation 47) is a cell surface transmembrane protein involved in numerous cellular processes in humans including proliferation, adhesion, migration, fusion, activation, death, and phagocytosis. For example, CD47 is known to modulate phagocytosis by ligation to signal regulatory protein alpha (SIRPa) expressed on phagocytes (Oldenborg (2013) ISRN Hematol 2013:614619, and references contained therein). CD47 also serves as a receptor for the extracellular matrix glycoprotein thrombospondin- 1 (TSP-l), wherein the interaction between CD47 and TSP-l can induce cell death of the CD47 -expressing cell (Manna & Frazier (2004) Cancer Res 64(3): 1026- 1036).

CD47 is expressed by virtually all normal cells in the human body (Oldenborg (2013) ISRN Hematol 2013:614619). CD47 is highly expressed in multiple human cancer types, such as acute and chronic myeloid leukaemia, non-Hodgkin's lymphomas and multiple myeloma, leiomyosarcoma, glioblastoma, mesothelioma, and various carcinomas including bladder, ovarian, hepatocellular, prostate, breast and colon (Schurch et ah, (2018) Oncoimmunology 7(1) el373235, and references therein). Overexpression of CD47 on cancer cells is known to contribute to tumor immune evasion, in part, by blocking phagocytic uptake. For example, certain human leukemias upregulate CD47 to evade macrophage killing (Jaiswal et ah, (2009) Cell 138(2):271-285). Increased CD47 expression also predicted poor overall survival in adult patients with acute myelogenous leukemia (Majeti et ah, (2009) Cell l38(2):286-299) Similarly, high CD47 expression has been observed in solid tumors such as small cell lung cancer (Weiskopf et ah, (2016) J Clin Investigation 124(7):2610-2620). Accordingly, targeting CD47 is a potential strategy for the treatment of cancer. Molecules that target CD47 and/or block the CD47-SIRPa interaction can restore phagocytic uptake of CD47+ target cells and lower the threshold for macrophage activation, thereby leading to cancer cell destruction and elimination of tumors in various mouse cancer models. Molecules that serve as CD47 ligands can also induce CD47-mediated cell death of CD47+ target cells.

Despite the significant advancement in the treatment of cancer, improved therapies and diagnostic methods are still being sought. Although several therapeutic molecules and agents that target CD47 have been developed, understanding the mechanisms and mediators through which these molecules exert their effect is critical for the identification of patients that are likely to effectively respond to treatment. Determining whether a patient is or would be responsive to therapeutic agents that target CD47 will enable decisions of whether treatment should be initiated, continued, or altered.

SUMMARY OF THE DISCLOSURE

Disclosed herein are methods and kits for identifying a subject likely to respond to treatment with an agent that specifically binds to CD47 (Cluster of Differentiation 47), wherein the agent comprises an antibody or polypeptide comprising an Fc domain. In some aspects, the disclosure provides methods and kits for identifying a tumor susceptible to treatment with an agent that specifically binds to CD47 (Cluster of Differentiation 47), wherein the agent comprises an antibody or polypeptide comprising an Fc domain. The agents disclosed herein can be used (alone or in combination with other therapeutic agents or procedures) to treat, prevent and/or diagnose disorders, including immune disorders and cancer.

In some aspects, the disclosure provides a method of identifying a subject likely to respond to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: detecting the presence of CD32a in a sample obtained from the subject, wherein the presence of CD32a indicates the subject is likely to respond to treatment with the agent. In some embodiments, the sample is from a subject having, or at risk of having a cancer. In some embodiments, the sample is from a subject having cancer. In some embodiments, the sample is a tumor sample. In some embodiments, detecting the presence of CD32a comprises determining an amount of CD32a in the sample, wherein the amount of CD32a indicates the subject is likely to respond to treatment with the agent. In some embodiments, determining the amount of CD32a comprises comparing the amount of CD32a in the sample relative to a reference amount of CD32a. In some embodiments, when the amount of CD32a in the sample is increased relative to the reference amount of CD32a indicates the patient is likely to respond to treatment with the agent.

In some aspects, the disclosure provides a method of identifying a subject likely to respond to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, wherein the Fc domain comprises a human IgG4 wild-type Fc domain or a IgG4 mutant Fc domain, the method comprising: detecting the presence of CD32a in a sample obtained from the subject, wherein the presence of CD32a indicates the subject is likely to respond to treatment with the agent. In some embodiments, the sample is from a subject having, or at risk of having a cancer. In some embodiments, the sample is from a subject having cancer. In some embodiments, the sample is a tumor sample. In some embodiments, detecting the presence of CD32a comprises determining an amount of CD32a in the sample, wherein the amount of CD32a indicates the subject is likely to respond to treatment with the agent. In some embodiments, determining the amount of CD32a comprises comparing the amount of CD32a in the sample relative to a reference amount of CD32a. In some embodiments, when the amount of CD32a in the sample is increased relative to the reference amount of CD32a indicates the patient is likely to respond to treatment with the agent.

In some embodiments, the method further comprising detecting the presence of CD47 in the sample, wherein the presence of CD47 indicates the subject is likely to respond to treatment with the agent. In some embodiments, detecting the presence of CD47 comprises determining an amount of CD47 in the sample, wherein the amount of CD47 indicates the subject is likely to respond to treatment with the agent. In some embodiments, determining the amount of CD47 comprises comparing the amount of CD47 in the sample relative to a reference amount of CD47. In some embodiments, when the amount of CD47 in the sample is increased relative to the reference amount of CD47 indicates the patient is likely to respond to treatment with the agent.

In some embodiments, the method further comprising detecting the presence of CD32b in the sample. In some embodiments, detecting the presence of CD32b comprises determining an amount of CD32b in the sample. In some embodiments, determining the amount of CD32b comprises comparing the amount of CD32b in the sample relative to a reference amount of CD32b. In some embodiments, when the amount of CD32b in the sample is decreased relative to the reference amount of CD32b indicates the patient is likely to respond to treatment with the agent. In some embodiments, when the amount of CD32b in the sample is decreased relative to the amount of CD32a in the sample indicates the patient is likely to respond to treatment with the agent.

In some embodiments, detecting the presence of CD32a comprises determining an expression level of CD32a in the sample, wherein the expression level of CD32a indicates the subject is likely to respond to treatment with the agent. In some embodiments, determining the expression level of CD32a comprises comparing the expression level of CD32a in the sample relative to a reference expression level of CD32a. In some embodiments, when the expression level of CD32a in the sample is increased relative to the reference expression level of CD32a indicates the subject is likely to respond to treatment with the agent.

In some embodiments, detecting the presence of CD47 comprises determining an expression level of CD47 in the sample, wherein the expression level of CD47 indicates the subject is likely to respond to treatment with the agent. In some embodiments, determining the expression level of CD47 comprises comparing the expression level of CD47 in the sample relative to a reference expression level of CD47. In some embodiments, when the expression level of CD47 in the sample is increased relative to the reference expression level of CD47 indicates the patient is likely to respond to treatment with the agent.

In some embodiments, detecting the presence of CD32b comprises determining an expression level of CD32b in the sample. In some embodiments, determining the expression level of CD32b comprises comparing the expression level of CD32b relative to a reference expression level of CD32b. In some embodiments, when the expression level of CD32b in the sample is decreased relative to the reference expression level of CD32b indicates the patient is likely to respond to treatment with the agent. In some embodiments, when the expression level of CD32b in the sample is decreased relative to the expression level of CD32a in the sample indicates the patient is likely to respond to treatment with the agent.

In some embodiments, the method further comprising detecting the presence of one or more polypeptides in the sample selected from the group consisting of: CDl6a, CDl6b, CD32c, CD64, CD68, CD163, PD-l, PD-L1, SIRPa, and a combination thereof. In some embodiments, the polypeptide is CDl6a, CDl6b or a combination thereof. In some embodiments, the polypeptide is CD64. In some embodiments, the polypeptide is SIRPa.

In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: contacting a tumor sample from the patient with an antibody that specifically binds to CD32a, or antigen-binding fragment thereof, thereby forming an antibody-CD32a complex; and detecting the presence of the antibody-CD32a complex, wherein the presence of the complex indicates the patient is likely to respond to treatment. In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, wherein the Fc domain comprises a human IgG4 wild-type Fc domain or a IgG4 mutant Fc domain, the method comprising: contacting a tumor sample from the patient with an antibody that specifically binds to CD32a, or antigen-binding fragment thereof, thereby forming an antibody-CD32a complex; and detecting the presence of the antibody-CD32a complex, wherein the presence of the complex indicates the patient is likely to respond to treatment.

In some embodiments, detecting the presence of the antibody-CD32a complex comprises determining an amount of antibody-CD32a complex in the sample, wherein the amount of antibody-CD32a complex relative to a control indicates the subject is likely to respond to treatment with the agent. In some embodiments, when the amount of antibody-CD32a complex in the sample is increased relative to the control indicates the patient is likely to respond to treatment with the agent.

In some embodiments, the method further comprising detecting the presence of CD47 in the sample, wherein the presence of CD47 indicates the subject is likely to respond to treatment with the agent. In some embodiments, detecting the presence of CD47 comprises determining an amount or expression level of CD47 in the sample, wherein the amount or expression level of CD47 in the sample, relative to a reference amount or reference expression level of CD47, indicates the subject is likely to respond to treatment with the agent. In some embodiments, when the amount or expression level of CD47 in the sample is increased relative to the reference amount or reference expression level of CD47 indicates the patient is likely to respond to treatment with the agent. In some embodiments, the method further comprising contacting the sample with an antibody that specifically binds CD47, or antigen-binding fragment thereof, thereby forming an antibody-CD47 complex; and detecting the presence of the antibody-CD47 complex, wherein the presence of the complex indicates the patient is likely to respond to treatment. In some embodiments, when the amount antibody-CD47 complex in the sample is increased relative to a control indicates the patient is likely to respond to treatment with the agent.

In some embodiments, the method further comprising detecting the presence of CD32b in the sample. In some embodiments, detecting the presence of CD32b comprises determining an amount or expression level of CD32b in the sample relative to a reference amount or reference expression level. In some embodiments, when the amount or expression level of CD32b in the sample is decreased relative to the reference amount or reference expression level of CD32b indicates the patient is likely to respond to treatment with the agent. In some embodiments, when the amount or expression level of CD32b in the sample is decreased relative to the amount antibody-CD32a complex indicates the patient is likely to respond to treatment with the agent.

In some embodiments, the method further comprising contacting the sample with an antibody that specifically binds CD32b, or antigen-binding fragment thereof, thereby forming an antibody-CD32b complex; and detecting the presence of the antibody-CD32b complex. In some embodiments, when the amount of antibody-CD32b complex in the sample is decreased relative to the amount of antibody-CD32a complex in the sample indicates the patient is likely to respond to treatment with the agent.

In some embodiments, the method further comprising detecting the presence of one or more polypeptides in the tumor sample selected from the group consisting of: CDl6a, CDl6b, CD32c, CD64, CD68, CD163, SIRPa, PD-l, PD-L1, and a combination thereof. In some embodiments, the polypeptide is CDl6a, CDl6b or a combination thereof. In some embodiments, the polypeptide is CD64. In some embodiments, the polypeptide is SIRPa.

In some aspects, the disclosure provides a method of detecting a tumor susceptible to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: determining an expression level of a panel of polypeptides comprising CD32a, CD32b, and CD47 in a tumor sample, wherein the expression level of the panel polypeptides in the tumor sample indicates the tumor is susceptible to treatment with the agent. In some aspects, the disclosure provides a method of detecting a tumor susceptible to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, wherein the Fc domain comprises a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain, the method comprising: determining an expression level of a panel of polypeptides comprising CD32a, CD32b, and CD47 in a tumor sample, wherein the expression level of the panel polypeptides in the tumor sample indicates the tumor is susceptible to treatment with the agent.

In some embodiments, determining an expression level of the panel of polypeptides comprises comparing the expression levels of the panel of polypeptides in the tumor sample to an expression level of the panel of polypeptides in a reference sample. In some embodiments, when the expression level of CD32a in the tumor sample is increased relative to the reference sample indicates the tumor is susceptible to treatment with the agent. In some embodiments, when the expression level of CD47 in the tumor sample is increased relative to the reference sample indicates the tumor is susceptible to treatment with the agent. In some embodiments, when the expression level of CD32b in the tumor sample is decreased relative to the reference sample indicates the tumor is susceptible to treatment with the agent. In some embodiments, when the expression level of CD32a and CD47 in the tumor sample is increased relative to the reference sample indicates the tumor is susceptible to treatment with the agent. In some embodiments, when the expression level of CD32a and CD47 in the tumor sample is increased and the expression level of CD32b is decreased relative to the reference sample indicates the tumor is susceptible to treatment with the agent. In some embodiments, the method further comprising determining an expression level of one or more polypeptides in the tumor sample selected from the group consisting of: CDl6a, CDl6b, CD32c, CD64, CD68, CD163, SIRPa, PD-l, PD-L1, and a combination thereof. In some embodiments, the polypeptide is CDl6a, CDl6b or a combination thereof. In some embodiments, the polypeptide is CD64. In some embodiments, the polypeptide is SIRPa.

In some embodiments of the methods provided by the disclosure, the sample comprises tumor-infiltrating immune cells, tumor cells, stromal cells, or a combination thereof. In some embodiments, the tumor-infiltrating immune cells comprise macrophages, monocytes, neutrophils, dendritic cells, NK cells, innate lymphoid cells, B cells , or any combination thereof. In some embodiments, the sample comprises a fresh tissue. In some embodiments, the sample comprises a fixed tissue. In some embodiments, the sample is frozen or is formalin-fixed and embedded in paraffin wax (FFPE). In some embodiments, the detecting or determining is by IHC, IF, flow cytometry, ELISA, or immunoblotting. In some embodiments, the detecting or determining is by IHC staining. In some embodiments, the IHC staining is determined on the same sample. In some embodiments, the IHC staining is determined on separate tissue sections of the same sample. In some embodiments of the methods provided by the disclosure, the antibody is covalently linked to a chromogenic molecule or a fluorescent molecule.

In some embodiments of the methods provided by the disclosure, the complex is contacted with a secondary antibody, or antigen-binding fragment thereof, covalently linked to a chromogenic molecule or a fluorescent molecule.

In some embodiments of the methods provided by the disclosure, the cancer is selected from multiple myeloma (MM), acute myeloid leukemia (AML)/ myelodysplastic syndrome (MDS), T cell lymphoma (TCL), non-Hodgkin’s lymphoma (NHL), chronic lymphocytic leukemia (CLL), ovarian carcinoma. In some embodiments, the cancer is selected from colorectal cancer, gastric cancer, head and neck cancer, renal cell carcinoma, urothelial/bladder carcinoma, ovarian carcinoma, myeloma, melanoma, lung cancer, squamous cell carcinoma, classical Hodgkin's lymphoma, breast cancer, small cell lung cancer, salivary gland carcinoma, vulvar carcinoma, thyroid carcinoma, anal canal carcinoma, biliary carcinoma, mesothelioma, cervical carcinoma, sarcoma, glioblastoma, and neuroendocrine carcinoma. In some

embodiments of the methods provided by the disclosure, the cancer is lung cancer. In some embodiments of the methods provided by the disclosure, the cancer is lung adenocarcinoma. In some embodiments of the methods provided by the disclosure, the cancer is squamous cell lung cancer. In some embodiments of the methods provided by the disclosure, the cancer is ovarian cancer. In some embodiments of the methods provided by the disclosure, the cancer is glioblastoma. In some embodiments of the methods provided by the disclosure, the cancer is acute myeloid leukemia. In some embodiments of the methods provided by the disclosure, the cancer is mesothelioma. In some embodiments of the methods provided by the disclosure, the cancer is sarcoma.

In some embodiments of the methods provided by the disclosure, the methods further comprising selecting a treatment for the subject or patient. In some embodiments, the methods further comprising administering a treatment to the subject or patient. In some aspects, the treatment comprises an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain. In some aspects, the agent inhibits the interaction between CD47 and SIRPa. In some aspects, the agent provides an anti-tumor activity. In some embodiments, the anti-tumor activity is selected from:

i. an induction of phagocytosis of tumor cells; ii. an induction of cell death of tumor cells; and

iii. a combination of (i) and (ii).

In some embodiments, the anti-tumor activity comprises an induction of phagocytosis of tumor cells and an induction of cell death of tumor cells.

In some embodiments of the methods provided by the disclosure, the agent specifically binds human CD47 expressed on tumor cells. In some embodiments, the agent specifically binds human CD32a expressed on tumor-infiltrating immune cells. In some embodiments, the tumor- infiltrating immune cells comprise macrophages, monocytes, neutrophils, dendritic cells, NK cells, innate lymphoid cells, B cells, or any combination thereof. In some embodiments, binding of the agent to human CD32a induces phagocytosis of tumor cells expressing CD47.

In some embodiments of the methods provided by the disclosure, the agent is a monoclonal antibody that specifically binds human CD47, or antigen binding fragment thereof. In some embodiments, the agent is a SIRPa-Fc fusion protein. In some embodiments, the Fc domain is selected from the group consisting of IgGl, IgG2, IgG3 and IgG4. In some embodiments, the Fc domain is a mutant or wild-type IgG4 Fc domain. In some embodiments, the Fc domain is mutated to increase binding to CD32a or to decrease binding to CD32b.

In some embodiments of the methods provided by the disclosure, CD32a contains a polymorphism that affects binding to an Fc domain.

In some embodiments provided by the disclosure, the agent that specifically binds to CD47 is an antibody comprising:

a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 5;

a heavy chain complementarity determining region 1 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 6;

a heavy chain complementarity determining region 1 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 7;

a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 8;

a light chain complementarity determining region 1 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 9; a light chain complementarity determining region 1 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 10.

In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 13; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 16.

In some aspects, the disclosure provides a kit comprising a container comprising one or more reagents for detecting the presence of CD32a, optionally CD47 and/or CD32b in a tumor sample, and a package insert comprising instructions for determining an amount or expression level of CD32a, optionally CD47 and/or CD32b in the tumor sample. In some embodiments, the kit comprises a reagent for detecting the presence of CD47 in the sample and instructions for determining an amount or expression level of CD47 in the tumor sample. In some embodiments, the kit comprises a reagent for detecting the presence of CD32b in the sample and instructions for determining an amount or expression level of CD32b in the tumor sample.

In some embodiments, the kit further comprises a reagent for detecting the presence of one or more polypeptides in the sample selected from the group consisting of CDl6a, CDl6b, CD32a, CD32b, CD32c, CD47, CD64, CD68, CD163, PD-l, PD-L1, SIRPa, or a combination thereof, and instructions for determining an amount or expression level of the one or more polypeptides in the tumor sample. In some embodiments, determining an amount or expression level of CD32a, optionally CD47 and/or CD32b, or one or more polypeptides selected from the group consisting of CDl6a, CDl6b, CD32a, CD32b, CD32c, CD47, CD64, CD68, CD163, PD-l, PD-L1, SIRPa, or a combination thereof comprises comparing the amount or expression level of CD32a, optionally CD47 and/or CD32b, or the one or more polypeptides in a tumor sample relative to the amount or expression level in a reference sample. In some embodiments, the reagent is an antibody specifically reactive with CD32a, optionally an antibody specifically reactive with CD47 and/or an antibody specifically reactive with CD32b, or an antibody specifically reactive with one more polypeptides selected from the group consisting of CDl6a, CDl6b, CD32a, CD32b, CD32c, CD47, CD64, CD68, CD163, PD-l, PD-L1, SIRPa, or a combination thereof. In some embodiments, the one or more antibodies is covalently attached to a label. In some embodiments, the label is a fluorescent molecule or a chromogenic molecule.

In some embodiments, the kit further comprises a reagent for detecting a complex formed upon binding of the one or more antibodies to CD32a, optionally CD47 and/or CD32a, or one more polypeptides selected from the group consisting of CDl6a, CDl6b, CD32a, CD32b, CD32c, CD47, CD64, CD68, CD163, PD-l, PD-L1, SIRPa, or a combination thereof, and instructions for determining an amount of one or more complexes in the sample.

In some aspects, the disclosure provides a method of identifying a subject likely to respond to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: detecting the presence of the FcyRIIa H131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a) in a sample obtained from the subject, wherein the presence of the FcyRIIa H131 allelic variant indicates the subject is likely to respond to treatment with the agent.

In some aspects, the disclosure provides a method of identifying a subject likely to respond to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: detecting the presence of the FcyRIIa R131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a) in a sample obtained from the subject, wherein the presence of the FcyRIIa R131 allelic variant indicates the subject is likely to respond to treatment with the agent.

In some aspects, the disclosure provides a method of identifying a subject likely to respond to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: detecting the presence of the Q57X SNP (rs759550223, also known as Q13X) of the FCGR2C gene encoding Fey R lie (CD32c) in a sample obtained from the subject, wherein the presence of the Q57X SNP indicates the subject is likely to respond to treatment with the agent.

In some aspects, the disclosure provides a method of identifying a subject likely to respond to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: detecting the presence of the F158 allotype of the FCGR3A gene encoding FcyRIIIa (CDl6a) in a sample obtained from the subject, wherein the presence of the F158 allotype indicates the subject is likely to respond to treatment with the agent.

In some aspects, the disclosure provides a method of identifying a subject likely to respond to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: detecting the presence of the VI 58 allotype of the FCGR3A gene encoding FcyRIIIa (CDl6a) in a sample obtained from the subject, wherein the presence of the VI 58 allotype indicates the subject is likely to respond to treatment with the agent.

In some aspects, the disclosure provides a method for treating cancer in a subject in need thereof, comprising detecting the presence and/or expression level of CD32a in a sample obtained from the subject; and administering to the subject a monoclonal antibody that specifically binds human CD47, or antigen binding fragment thereof, wherein the antibody comprises: a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 5; a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 6; a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 7; a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 8; a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 9; a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 10.

In some aspects, the disclosure provides a method for treating cancer in a subject in need thereof, comprising detecting the presence and/or expression level of CD32a in a sample obtained from the subject; and administering to the subject a monoclonal antibody that specifically binds human CD47, or antigen binding fragment thereof, wherein the antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 4.

In some aspects, the disclosure provides a method for treating cancer in a subject in need thereof, comprising detecting the presence and/or expression level of CD32a in a sample obtained from the subject; and administering to the subject a monoclonal antibody that specifically binds human CD47, or antigen binding fragment thereof, wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16.

In some aspects, the disclosure provides a method for treating cancer in a subject in need thereof, comprising detecting the presence and/or expression level of CD32a in a sample obtained from the subject; and administering to the subject a monoclonal antibody that specifically binds human CD47, or antigen binding fragment thereof, wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 13; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 16.

In some embodiments, the CD32a comprises a 131R single nucleotide polymorphism (SNP). In some emboidments, the CD32a comprises a 131H SNP.

In some embodiments, the method of any of the foregoing aspects, further comprises detecting the presence of CD47 in the sample.

In some aspects, the disclosure provides a method for treating cancer in a subject in need thereof, comprising administering to the subject a monoclonal antibody that specifically binds human CD47, or antigen binding fragment thereof, wherein the antibody comprises: a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 5; a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 6; a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 7; a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 8; a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 9; a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 10, wherein cells in a tumor sample from the subject express CD32a.

In some embodiments, the cells in the tumor sample are myeloid cells. In some embodiments, the cells in the tumor sample are macrophages. In some embodiments, the cells in the tumor sample are immune cells. In some embodiments, the cells in the tumor sample are monocytes. In some aspects, the disclosure provides a method for treating cancer in a subject in need thereof, comprising administering to the subject a monoclonal antibody that specifically binds human CD47, or antigen binding fragment thereof, wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16, and wherein cells in a tumor sample from the subject express CD32a.

In some aspects, the disclosure provides a method for treating cancer in a subject in need thereof, comprising administering to the subject a monoclonal antibody that specifically binds human CD47, or antigen binding fragment thereof, wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 13; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 16, and wherein cells in a tumor sample from the subject express CD32a.

In some embodiments of the foregoing aspects, the cancer cells in the tumor sample express CD47.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1A provides a graph of flow cytometric data showing the extent of phagocytosis of Jurkat cells in co-culture with macrophages in the presence of increasing amounts of 2.3D11 antibody, 2.3D11 F(ab’)2 antibody fragment or control antibody as indicated.

Figure IB provides a graph of flow cytometric data showing the binding of increasing amounts of 2.3D11 antibody to Jurkat cells.

Figure 2A provides an image of a phospho-immunoreceptor array showing the extent of phosphorylation of immunoreceptors in lysates of Jurkat cells and macrophages treated with 2.3D11 or a control antibody.

Figure 2B provides a graph depicting the quantification of the phosphorylation signal of CD32a generated from the phospho-immunoreceptor array in Figure 2A.

Figure 2C provides a graph depicting the quantification of the phosphorylation signal of CD32a provided by a phospho-immunoreceptor microarray from lysates of cells incubated in the presence of 2.3D11 antibody, 2.3D11 F(ab’)2 antibody fragment or control antibody as indicated. Figure 3 provides a graph of flow cytometric data depicting CD32a surface expression on primary human macrophages, Jurkat and U937 cells, as indicated.

Figure 4A provides a graph of flow cytometric data depicting CD47 surface expression on Jurkat cells and Jurkat CD47^_/ knockout cells treated with 2.3D11 antibody or control antibody.

Figure 4B provides a graph depicting the quantification of the phosphorylation signal of CD32a provided by a phospho-immunoreceptor array from lysates of cells described in Figure 4A.

Figure 4C provides a graph of flow cytometric data showing the extent of phagocytosis of Jurkat cells and Jurkat CD47^_/ knockout cells in the presence of 2.3D 11 antibody or control antibody.

Figure 5A provides a graph of flow cytometric data showing the extent of phagocytosis of Jurkat cells in the presence of 2.3D 11 antibody in combination with anti-CD32a antibody, or control antibodies, as indicated.

Figure 5B provides a graph of flow cytometric data showing the extent of phagocytosis of Jurkat cells in the presence of anti-CD47 monoclonal antibody (“mAb A”) in combination with anti-CD32a antibody, or control antibodies, as indicated.

Figure 6A provides a graph of flow cytometric data showing the percentage of Jurkat cells that are undergoing apoptosis or programmed cell death (AnnexinV+/PI-) or are necrotic or post- apoptotic (AnnexinV+/PI+) in the presence of soluble anti-CD47 antibodies, or control antibody, as indicated.

Figure 6B provides scatterplots of flow cytometric data used to generate the graphs shown in Figure 6A.

Figure 7A provides a graph of flow cytometric data showing the extent phagocytosis of Jurkat cells in co-culture with macrophages in the presence of 2.3D 11 antibody, anti-CD47 monoclonal antibody (“mAb B”) or control antibodies as indicated.

Figure 7B provides a graph of flow cytometric data showing the percentage of dead, non- phagocytosed Jurkat cells in co-culture with macrophages in the presence 2.3D 11 antibody, anti- CD47 mAb B antibody, or control antibodies as indicated.

Figure 7C provides a graph of flow cytometric data showing the extent of non- phagocytosed Jurkat cells that have undergone programmed cell death or are necrotic in co-culture with macrophages in the presence of 2.3D 11 antibody, anti-CD47 mAb A antibody, anti-CD47 mAbB antibody or control antibodies as indicated.

Figure 8A provides a graph of flow cytometric data showing the extent of phagocytosis of of Jurkat cells in co-culture with macrophages in the bottom well of a transwell system following treatment with 2.3D 11 antibody, anti-CD47 mAb B antibody or control antibodies, as indicated.

Figure 8B provides a graph of flow cytometric data showing the percentage of dead, non- phagocytosed Jurkat cells either in co-culture with macrophages in the bottom well of a transwell system or culture alone in the top well following treatment with 2.3D11 antibody, anti-CD47 mAb B antibody or control antibodies, as indicated.

Figure 9A provides a graph of flow cytometric data showing the extent of phagocytosis of Jurkat cells in co-culture with macrophages following treatment with 2.3D11 antibody or control antibody and in the presence of phagocytosis inhibitors.

Figure 9B provides a graph of flow cytometric data showing the percentage of dead, non- phagocytosed Jurkat cells in co-culture with macrophages following treatment with 2.3D 11 antibody or control antibody and in the presence of phagocytosis inhibitors.

Figure 10A provides a graph of flow cytometric data showing the extent of phagocytosis of Jurkat cells in co-culture with macrophages pretreated with anti-CD32a blocking antibody (IV.3), followed by treatment with 2.3D11 antibody or control antibody, as indicated.

Figure 10B provides a graph of flow cytometric data showing the percentage of dead, non- phagocytosed Jurkat cells in co-culture with macrophages pretreated with anti-CD32a blocking antibody (IV.3), followed by treatment with 2.3D 11 antibody or control antibody, as indicated.

Figure 11A provides a graph of flow cytometric data showing the percentage of Jurkat cells that are undergoing programmed cell death (AnnexinV+/PI-) or are necrotic or post-apoptotic (AnnexinV+/PI+) in co-culture with macrophages in the presence of Protein-G immobilized 2.3D 11 antibody, anti-CD47 mAb B antibody, anti-CD47 mAb A antibody or control antibodies at various dosages as indicated.

Figure 11B provides a graph of flow cytometric data showing the percentage of U937 cells that are undergoing programmed cell death (AnnexinV+/PI-) or are necrotic or post-apoptotic (AnnexinV+/PI+) in co-culture with macrophages in the presence of Protein-G immobilized 2.3D 11 antibody, anti-CD47 mAb B antibody, anti-CD47 mAb A antibody or control antibodies at various dosages as indicated. Figure 12 provides a graph of flow cytometric data showing the extent of phagocytosis of Jurkat cells in co-culture with macrophages pretreated with anti-CDl6 blocking antibody (3G8), followed by treatment with 2.3D11 antibody or control antibodies, as indicated.

Figure 13A provides a graph depicting the percentage area within a region of interest labeled with macrophage-specific anti-F4/80 antibody from a tumor treated with 2.3D11 antibody or control antibody at various dosages, as indicated.

Figures 13B and 13C provide images of a tumor sample treated with control antibody (13B) or 2.3D11 antibody (13C) and subsequently stained with macrophage specific anti-F4/80 antibody, as determined by immunohistochemistry analysis.

Figure 14 provides a graph depicting the expression of CD47 and CD32a in multiple human sarcoma tumor samples, as determined by immunohistochemistry analysis.

Figure 15 graphically depicts the difference in the ability of several Fc variants of antibody 2.3D11 to induce phagocytosis.

Figure 16A graphically depicts the abrogation of antibody 2.3D11 IgG4 WT and antibody 2.3D11 IgG4 double mutant mediated phagocytosis by CD32a antibody blockade.

Figure 16B graphically depicts the abrogation of antibody 2.3D11 IgG4 WT and antibody 2.3D11 IgG4 double mutant mediated cell death by CD32a antibody blockade.

Figure 17 graphically depicts the minimal impact of CD64 blockade on 2.3D11 IgG4 WT mediated phagocytosis.

Figure 18 depicts the co-expression of CD32a and CD47 us multiple tumor types.

DETAILED DESCRIPTION

The present disclosure is based, in part, on the discovery that certain anti-tumor agents that specifically bind CD47 and inhibit the interaction with SIRPa, concomitantly effect Fc receptor and CD47-mediated cellular responses. It was discovered that agents that specifically bind CD47 and comprise an Fc domain (e.g., anti-CD47 antibodies) induce phagocytosis of tumor cells in a manner that is influenced by (i) disrupting the interaction between CD47 expressed on tumor cells and SIRPa expressed on the surface of phagocytes (e.g., macrophages); and (ii) enhancing the interaction between the Fc domain and activating Fey receptors, in particular CD32a.

In particular, it was discovered that both the induction of macrophage-mediated tumor cell phagocytosis and cell death by an anti-CD47 antibody, 2.3D11, a fully human anti-CD47 IgG4 antibody is dependent on the interaction with CD32a. It has also been discovered that blockade of the 2.3D 11 interaction with CD32a (as well as a 2.3D 11 IgG4 variant having Fc mutations) abrogated 2.3D 11 mediated cell death and phagocytosis but not cell death and phagocytosis mediated by a 2.3D11 IgGl isotype variant. In contrast, blockade of the 2.3D11 interaction with CD64 did not significantly impact 2.3D 11 mediated phagocytosis. These data demonstrate that 2.3D11, a human IgG4 antibody, has a differential dependence on CD32a compared to the human IgGl variant of 2.3D11, and that antibodies and Fc-bearing proteins targeting CD47 that have an IgG4 Fc (or mutant IgG4 Fc) are uniquely dependent upon CD32a engagement.

Without being bound by theory, it is believed that these anti-tumor agents engage in a multi-domain binding or“scaffolding” phenomenon, whereby the agent specifically binds to CD47 on a target cell (e.g., a tumor cell) and inhibits interaction with SIRPa, and the Fc domain binds activating Fey receptors (e.g., CD32a) on a phagocyte (e.g., a macrophage), resulting in signaling which leads to phagocytosis of target cells and signaling which leads to cell death of target cells that are not phagocytosed, but are nonetheless in contact with the phagocyte. It is hypothesized that when CD47 on target cells (e.g., tumor cells) is bound by an agent which inhibits the interaction with SIRPa and comprises an Fc domain (e.g., an anti-CD47 antibody), scaffolding occurs between CD47 and Fc domains engaged with Fey receptors on immune effector cells (e.g., macrophages). Furthermore, binding of multiple antibody molecules in scaffolded clusters may enhance the affinity for Fey receptors (e.g., CD32a) and result in activating signals to both effector cells (e.g., antibody-dependent cellular phagocytosis (ADCP)) and target cells (e.g., cell death). Thus,“scaffolding” of an anti-tumor agent, such as an anti-CD47 antibody, between CD47 on the tumor cell and CD32a on the phagocyte (e.g., macrophage) may overcome what is a relatively low affinity interaction between an Fc domain (e.g., an IgG4 Fc domain) and an Fey receptor (e.g., CD32a) on a 1: 1 basis.

Accordingly, the presence of CD32a on immune cells infiltrating a tumor may positively influence the treatment outcome with an anti-tumor agent described herein, and a patient selection strategy that assesses the presence (e.g., abundance) of CD32a in a tumor sample from the patient may help inform treatment decisions, and may influence patient outcomes. Based on this reasoning, expression levels of both CD47 and CD32a in patient tumors may be relevant metrics to consider prior to, during, or following treatment with an anti-tumor agent as described herein. Likewise, as CD32b is the inhibitory partner of CD32a, and such paired receptors have been suggested to regulate immune cell activation via the relative abundance of the inhibitory (e.g., CD32b) and activating (e.g., CD32a) molecules on immune cells, the relative expression of CD32a and CD32b within a patient’s tumor may be assessed to help inform treatment decisions with an anti-tumor agent as described herein.

Accordingly, the present disclosure provides methods and kits for identifying a subject with a tumor (e.g., a cancer patient) that is likely to respond to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, as described herein. The disclosure also provides biomarkers useful for the identification of a subject with a tumor (e.g., a cancer patient) that is likely to respond to treatment with an agent described herein. The disclosure further provides compositions and methods for treating a subject with a tumor (e.g., a cancer patient) with an agent described herein.

Methods and Uses of Biomarkers

Provided herein are diagnostic and therapeutic methods and uses of biomarkers described herein. In particular, the disclosure provides methods of identifying subjects likely to respond to treatment with an anti-tumor agent as described herein by detecting the presence of one or more biomarkers (e.g., amount or expression level) in a sample obtained from the subject, wherein the presence of the biomarker indicates the subject is likely to respond to treatment with the agent.

CD32a (Fc RIIa)

As used herein, the term“CD32a” refers to the low affinity immunoglobulin gamma Fc region receptor Ila, a transmembrane Fc receptor encoded by the FCGR2A gene in humans. Alternative names for CD32A include Fc fragment of IgG receptor Ila, Immunoglobulin G Fc Receptor Ila, IgG Fc Receptor Il-a, Fc-Gamma-RIIa, FcyRIIa, FCGR2A, CD32A, CDw32, FCG2, FCGR2, FCGR2A1, FcGR, and IGFR2. An amino acid sequence of an exemplary human CD32a (isoform 1) protein is provided in SEQ ID NO: 31 (NCBI Reference Sequence: NP_00l 129691.1). An nucleic acid sequence of an exemplary human CD32a (isoform 1) mRNA is provided in SEQ ID NO: 38 (NCBI Reference Sequence: NM_00l 136219.1). An amino acid sequence of an exemplary human CD32a (isoform 2) protein is provided in SEQ ID NO: 39 (NCBI Reference Sequence: NP_067674.2). An nucleic acid sequence of an exemplary human CD32a (isoform 2) mRNA is provided in SEQ ID NO: 40 (NCBI Reference Sequence: NM_02l642.3). In some aspects, the disclosure provides methods for detecting the presence of or determining an amount or expression level of CD32a (FcyRIIa) in a sample from a subject (e.g., a tumor sample from cancer patient). CD32a is a member of a family of activating Fey receptors (FcyRs) that bind IgG and stimulate immune cell effector mechanisms, in particular antibody- dependent cell-mediated phagocytosis (ADCP), which facilitates antibody-mediated tumor cell killing (Nimmerjahn and Ravetch (2008) Nat Rev Immunol 8:34-47; Hogarth and Pietersz (2012) Nat Rev Drug Discov 11:311-331). CD32a is expressed on granulocytes, monocytes and monocyte-derived cells such as macrophages and DCs. It has been observed that CD32a exhibits a low affinity for the Fc domain of IgG and that functional engagement is only expected to occur upon binding of antibody complexed through antigen engagement (Bruhns et al., (2016) Blood 113: 16; Stewart et al., (2014) J Immunother Cancer 2:29). Two allelic variants of CD32a have been identified, FcyRIIa H131 and FcyRIIa R131, conferring differential affinities for various IgG subclasses (Hogarth et al., (2012) Nat Rev Drug Discovery l l(4):311-331).

Accordingly, in some aspects the disclosure provides methods of identifying subjects likely to respond to treatment with an anti-tumor agent as described herein by detecting the presence of CD32a (e.g., an amount or expression level) in a sample obtained from the subject, wherein the presence of CD32a indicates the subject is likely to respond to treatment with the agent. In some aspects, the amount or expression level of CD32a in a sample is determined and compared to a reference amount or reference expression level of CD32a. In some aspects, when the amount or expression level of CD32a in the sample is increased relative to the reference amount or reference expression level of CD32a, then the patient is likely to respond to treatment with an anti-tumor agent as disclosed herein.

In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent as described herein (e.g., an anti-CD47 antibody) in which a tumor sample from the patient is contacted with an antibody that specifically binds to CD32a, or antigen-binding fragment thereof, thereby forming an antibody-CD32a complex, and the presence of the antibody-CD32a complex is detected, wherein the presence of the complex indicates the patient is likely to respond to treatment. In some aspects, the amount of antibody- CD32a complex in the sample is compared to a control. In some aspects, when the amount of antibody-CD32a complex in the sample is increased relative to the control then the patient is likely to respond to treatment with the agent. In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent as described herein (e.g., an anti-CD47 antibody) in which a tumor sample from the patient is contacted with a nucleic acid probe that hybridizes to a complementary target sequence in CD32a DNA or RNA, thereby forming a hybridization complex between the nucleic acid probe and the target CD32a DNA or RNA. To detect hybridization of the probe to the target CD32a DNA or RNA sequence, the probe is labeled with a molecular marker; for example, a radioactive marker, a fluorescent marker, an enzymatic marker, or digoxigenin. In some aspects, the presence of the probe-CD32a target complex indicates the patient is likely to respond to treatment. In some aspects, the amount of probe- CD32a target complex in the sample is compared to a control. In some aspects, when the amount of probe-CD32a target complex in the sample is increased relative to the control then the patient is likely to respond to treatment with the agent (e.g., an anti-CD47 antibody).

CD47

As used herein, the term“CD47” refers to a multi-spanning transmembrane receptor belonging to the immunoglobulin superfamily and encoded by the CD47 gene in humans. Alternative names for CD47 include integrin-associated protein (IAP), ovarian cancer antigen OA3, Rh-related antigen and MER6. An amino acid sequence of an exemplary human CD47 (isoform 1) protein is provided in SEQ ID NO: 1 (NCBI Reference Sequence: NP_00l768. l). An nucleic acid sequence of an exemplary human CD47 (isoform 1) mRNA is provided in SEQ ID NO: 2 (NCBI Reference Sequence: NM_00l777.3). An amino acid sequence of an exemplary human CD47 (isoform 2) protein is provided in SEQ ID NO: 41 (NCBI Reference Sequence: NP_942088.l). An nucleic acid sequence of an exemplary human CD47 (isoform 2) mRNA is provided in SEQ ID NO: 42 (NCBI Reference Sequence: NM_l98793.2).

In some aspects, the disclosure provides methods for detecting the presence of or determining an amount or expression level of CD47 in a sample from a subject (e.g., a tumor sample from cancer patient). CD47 is ubiquitously expressed on hematopoietic and non- hematopoietic cells and is involved in diverse cellular functions. CD47 has been characterized as a marker of“self’ in that it provides a“don’t-eat-me” signal to phagocytes (Reinhold et ah, (1995) J Cell Sci 108:3419-3425; Oldenborg et ah, (2000) Science 288(5473):2051-2054). This“don’t- eat-me” signal is mediated through the interaction between CD47 and signal regulatory protein alpha (SIRPa) expressed on phagocytes (e.g., macrophages), wherein the interaction results in the inhibition of phagocytosis of the CD47-expressing cell.

Accordingly, in some aspects the disclosure provides methods of identifying subjects likely to respond to treatment with an anti-tumor agent as described herein by detecting the presence of CD32a and the presence of CD47 (e.g., an amount or expression level) in a sample obtained from the subject, wherein the presence of CD32a and CD47 indicates the subject is likely to respond to treatment with the agent. In some aspects, the amount or expression level of CD47 in a sample is determined and compared to a reference amount or reference expression level of CD47. In some aspects, when the amount or expression level of CD47 in the sample is increased relative to the reference amount or reference expression level of CD47, then the patient is likely to respond to treatment with an anti-tumor agent as disclosed herein.

In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent as described herein (e.g., an anti-CD47 antibody) in which a tumor sample from the patient is contacted with an antibody that specifically binds to CD47, or antigen-binding fragment thereof, thereby forming an antibody-CD47 complex, and the presence of the antibody-CD47 complex is detected, wherein the presence of the complex indicates the patient is likely to respond to treatment. In some aspects, the amount of antibody-CD47 complex in the sample is compared to a control. In some aspects, when the amount of antibody-CD47 complex in the sample is increased relative to the control then the patient is likely to respond to treatment with the agent.

In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent as described herein (e.g., an anti-CD47 antibody) in which a tumor sample from the patient is contacted with a nucleic acid probe that hybridizes to a complementary target sequence in CD47 DNA or RNA, thereby forming a hybridization complex between the nucleic acid probe and the target CD47 DNA or RNA. To detect hybridization of the probe to the target CD47 DNA or RNA sequence, the probe is labeled with a molecular marker; for example, a radioactive marker, a fluorescent marker, an enzymatic marker, or digoxigenin. In some aspects, the presence of the probe-CD47 target complex indicates the patient is likely to respond to treatment. In some aspects, the amount of probe-CD47 target complex in the sample is compared to a control. In some aspects, when the amount of probe- CD47 target complex in the sample is increased relative to the control then the patient is likely to respond to treatment with the agent (e.g., an anti-CD47 antibody).

CD32b (FcyRIIb)

As used herein, the term“CD32b” refers to the low affinity immunoglobulin gamma Fc region receptor lib, a transmembrane Fc receptor encoded by the FCGR2B gene in humans. An amino acid sequence of an exemplary human CD32b protein is provided in SEQ ID NO: 32 (NCBI Reference Sequence: NP_003992.3). An nucleic acid sequence of an exemplary human CD32b mRNA is provided in SEQ ID NO: 45 (NCBI Reference Sequence: NM_00400l.4).

In some aspects, the disclosure provides methods for detecting the presence of or determining an amount or expression level of CD32b (FcyRIIb) in a sample from a subject (e.g., a tumor sample from cancer patient). In some embodiments, the amounts, expression levels, or presence of CD32a relative to CD32b in a tumor sample is determined. Both human and mice possess a single inhibitory FcyR, FcyRIIb (CD32b), which is expressed on B cells, DCs and basophils in both species, and found additionally on monocytes, macrophages and all granulocyte populations in mice. FcyRIIb (CD32b) is the inhibitory partner of FcyRIIa (CD32a). Both CD32a and CD32b share a highly homologous extracellular domain, but differ in their intracellular domains and downstream signaling capacities: CD32b is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing molecule with well described inhibitory functions, while CD32a contains an immunoreceptor tyrosine-based activation motif (IT AM) and is therefore an activating receptor. Such paired receptors have been postulated to regulate immune cell activation via the relative abundance of the inhibitory (CD32b) and activating (CD32a) molecules expressed on the cell surface. Macrophages are among the cell types of the immune system that can co express CD32a and CD32b.

Accordingly, in some aspects the disclosure provides methods of identifying subjects likely to respond to treatment with an anti-tumor agent as described herein by detecting the presence of CD32a and the presence of CD32b, and optionally, CD47 (e.g., an amount or expression level) in a sample obtained from the subject, wherein the presence of CD32a relative to CD32b indicates the subject is likely to respond to treatment with the agent. In some aspects, the amount or expression level of CD32b in a sample is determined and compared to a reference amount or reference expression level of CD32b. In some aspects, when the amount or expression level of CD32b in the sample is decreased relative to the amount or expression level of CD32a, then the patient is likely to respond to treatment with an anti-tumor agent as disclosed herein.

In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent as described herein (e.g., an anti-CD47 antibody) in which a tumor sample from the patient is contacted with an antibody that specifically binds to CD32b, or antigen-binding fragment thereof, thereby forming an antibody-CD32b complex, and the presence of the antibody-CD32b complex is detected, wherein the presence of the complex indicates the patient is likely to respond to treatment. In some aspects, the amount of antibody-CD32b complex in the sample is compared to a control. In some aspects, when the amount of antibody-CD32b complex in the sample is decreased relative to the amount of CD32a in the sample, then the patient is likely to respond to treatment with the agent.

CD32c (Fc RIIc)

As used herein, the term“CD32c” refers to the low affinity immunoglobulin gamma Fc region receptor lie, a transmembrane receptor encoded by the FCGR2C gene in humans. An amino acid sequence of an exemplary human CD32c protein is provided in SEQ ID NO: 33 (NCBI Reference Sequence: NP_963857.3). An nucleic acid sequence of an exemplary human CD32c mRNA is provided in SEQ ID NO: 46 (NCBI Reference Sequence: NM_20l563.5). In some aspects, the disclosure provides methods for detecting the presence of or determining an amount or expression level of CD32c (FcyRIIc) in a sample from a subject (e.g., a tumor sample from cancer patient). CD32c is expressed in approximately 20% of people and is closely related to, but expressed more restrictedly than, FcyRIIa (CD32a). The Q57X SNP (rs759550223, also known as Q13X) of the FCGR2C gene encoding FcyRIIc (CD32c) determines functional expression of this gene. This SNP results in either an open reading frame or the more common stop codon, leading to a truncated, non-functional protein. CD32c arose from an unequal crossover event between the CD32a and CD32b genes, resulting in an additional ITAM-containing family member. Expression of CD32c is active in NK cells and therefore, this SNP determines whether or not NK cells express a CD32 protein.

Accordingly, in some aspects the disclosure provides methods of identifying subjects likely to respond to treatment with an anti-tumor agent as described herein by detecting the presence of CD32a and the presence of CD32c, and, optionally CD32b and/or CD47 (e.g., an amount or expression level) in a sample obtained from the subject, wherein the presence of CD32c indicates the subject is likely to respond to treatment with the agent. In some aspects, the amount or expression level of CD32c in a sample is determined and compared to a reference amount or reference expression level of CD32c. In some aspects, when the amount or expression level of CD32c in the sample is increased relative to the reference amount or reference expression level, then the patient is likely to respond to treatment with an anti-tumor agent as disclosed herein. CD32c protein may be detected on NK cells within whole blood using a flow cytometry-based method wherein a CD32B/C specific antibody will only react with NK cells expressing full-length CD32C as CD32B is not expressed on NK cells. Additionally, CD32C polymorphisms may be detected at the DNA or transcript level.

In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent as described herein (e.g., an anti-CD47 antibody) in which a tumor sample from the patient is contacted with an antibody that specifically binds to CD32c, or antigen-binding fragment thereof, thereby forming an antibody-CD32c complex, and the presence of the antibody-CD32c complex is detected, wherein the presence of the complex indicates the patient is likely to respond to treatment. In some aspects, the amount of antibody-CD32c complex in the sample is compared to a control. In some aspects, when the amount of antibody-CD32c complex in the sample is increased relative to the control then the patient is likely to respond to treatment with the agent.

In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent as described herein (e.g., an anti-CD47 antibody) in which a tumor sample from the patient is contacted with a nucleic acid probe that hybridizes to a complementary target sequence in CD32c DNA or RNA, thereby forming a hybridization complex between the nucleic acid probe and the target CD32c DNA or RNA. To detect hybridization of the probe to the target CD32c DNA or RNA sequence, the probe is labeled with a molecular marker; for example, a radioactive marker, a fluorescent marker, an enzymatic marker, or digoxigenin. In some aspects, the presence of the probe-CD32c target complex indicates the patient is likely to respond to treatment. In some aspects, the amount of probe- CD32c target complex in the sample is compared to a control. In some aspects, when the amount of probe-CD32c target complex in the sample is increased relative to the control then the patient is likely to respond to treatment with the agent (e.g., an anti-CD47 antibody).

CD16a (Fc RIIIa) and CD16b (Fc RIIIb)

As used herein, the term“CDl6a” refers to the Fc fragment of IgG, low affinity Ilia, receptor, a transmembrane Fc receptor encoded by the FCGR3A gene. An amino acid sequence of an exemplary human CDl6a protein is provided in SEQ ID NO: 29 (NCBI Reference Sequence: NP_000560.6). A nucleic acid sequence of an exemplary human CDl6a mRNA is provided in SEQ ID NO: 43 (NCBI Reference Sequence: NM_000569.7)

As used herein, the term“CDl6b” refers to the Fc fragment of IgG, low affinity Mb, receptor, a transmembrane Fc receptor encoded by the FCGR3B gene. An amino acid sequence of an exemplary human CDl6b protein is provided in SEQ ID NO: 30 (NCBI Reference Sequence: NP_00l231682.1). An nucleic acid sequence of an exemplary human CDl6b mRNA is provided in SEQ ID NO: 44 (NCBI Reference Sequence: NM_00l244753.l).

In some aspects, the disclosure provides methods for detecting the presence of or determining an amount or expression level of CDl6a (FcyRIIIa) in a sample from a subject (e.g., a tumor sample from cancer patient). CDl6a is expressed primarily on NK cells, but is also found on monocytes and macrophages under some circumstances. FcyRIIIa (CDl6a) exists as two allotypic variants with differing binding affinity for IgG. For FcyRIIIa the more common F158 allotype has a lower affinity than the VI 58 allotype.

In some aspects, the disclosure provides methods for detecting the presence of or determining an amount or expression level of CDl6b (FcyRIIIb) in a sample from a subject (e.g., a tumor sample from cancer patient). CDl6b (FcyRIIIb) is a GPI linked Fc receptor expressed mainly on the surface of neutrophils.

Accordingly, in some aspects the disclosure provides methods of identifying subjects likely to respond to treatment with an anti-tumor agent as described herein by detecting the presence of CD32a and the presence of CDl6a and/or CDl6b, and optionally CD47 and/or CD32b and or CD32c (e.g., an amount or expression level) in a sample obtained from the subject, wherein the presence of CD32a and CDl6a and/or CDl6b indicates the subject is likely to respond to treatment with the agent. In some aspects, the amount or expression level of CDl6a and/or CDl6b in a sample is determined and compared to a reference amount or reference expression level of CDl6a and/or CDl6b. In some aspects, when the amount or expression level of CDl6a and/or CDl6b in the sample is increased relative to the reference amount or reference expression level of CDl6a and/or CDl6b, then the patient is likely to respond to treatment with an anti-tumor agent as disclosed herein.

In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent as described herein (e.g., an anti-CD47 antibody) in which a tumor sample from the patient is contacted with an antibody that specifically binds to CDl6a and/or CDl6b, or antigen-binding fragment thereof, thereby forming an antibody-CDl6a and/or CDl6b complex, and the presence of the antibody-CDl6a and/or CDl6b complex is detected, wherein the presence of the complex indicates the patient is likely to respond to treatment. In some aspects, the amount of antibody-CDl6a and/or CDl6b complex in the sample is compared to a control. In some aspects, when the amount of antibody-CDl6a and/or CDl6b complex in the sample is increased relative to the control then the patient is likely to respond to treatment with the agent.

In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent as described herein (e.g., an anti-CD47 antibody) in which a tumor sample from the patient is contacted with a nucleic acid probe that hybridizes to a complementary target sequence in CDl6a and/or CDl6b DNA or RNA, thereby forming a hybridization complex between the nucleic acid probe and the target CDl6a and/or CDl6b DNA or RNA. To detect hybridization of the probe to the target CDl6a and/or CDl6b DNA or RNA sequence, the probe is labeled with a molecular marker; for example, a radioactive marker, a fluorescent marker, an enzymatic marker, or digoxigenin. In some aspects, the presence of the probe-CDl6a and/or CDl6b target complex indicates the patient is likely to respond to treatment. In some aspects, the amount of probe-CDl6a and/or CDl6b target complex in the sample is compared to a control. In some aspects, when the amount of probe-CDl6a and/or CDl6b target complex in the sample is increased relative to the control then the patient is likely to respond to treatment with the agent (e.g., an anti-CD47 antibody).

CD64 (Fc RI)

As used herein, the term“CD64” refers to a high affinity immunoglobulin gamma Fc region receptor I, a transmembrane Fc receptor. There are three distinct (but highly similar) genes in humans for CD64 called FcyRIA (CD64A), FcyRIB (CD64B), and FcyRIC (CD64C). An amino acid sequence of an exemplary human CD64A protein is provided in SEQ ID NO: 34 (NCBI Reference Sequence: NP_000557.l). An nucleic acid sequence of an exemplary human CD64 mRNA is provided in SEQ ID NO: 47 (NCBI Reference Sequence: NM_000566.3).

In some aspects, the disclosure provides methods for detecting the presence of or determining an amount or expression level of CD64 (FcyRI) in a sample from a subject (e.g., a tumor sample from cancer patient). Humans and mice possess two classes of FcyRs, activating and inhibitory receptors. In both species FcyRI (CD64) is an activating receptor with high affinity for IgG, and is expressed on monocytic DCs and on monocytes/macrophages broadly in humans but in select locations in mice. FcyRI (CD64) is the only FcyR with a functionally meaningful binding affinity for monomeric IgG, the remaining FcyRs all exhibit such low affinity for the Fc region of IgG that functional engagement is only expected to occur upon binding of antibody complexed through antigen engagement.

Accordingly, in some aspects the disclosure provides methods of identifying subjects likely to respond to treatment with an anti-tumor agent as described herein by detecting the presence of CD32a and the presence of CD64, and optionally CD47, CD32b, CD32c, CDl6a and/or CDl6b (e.g., an amount or expression level) in a sample obtained from the subject, wherein the presence of CD32a and CD64 indicates the subject is likely to respond to treatment with the agent. In some aspects, the amount or expression level of CD64 in a sample is determined and compared to a reference amount or reference expression level of CD64. In some aspects, when the amount or expression level of CD64 in the sample is increased relative to the reference amount or reference expression level of CD64, then the patient is likely to respond to treatment with an anti-tumor agent as disclosed herein.

In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent as described herein (e.g., an anti-CD47 antibody) in which a tumor sample from the patient is contacted with an antibody that specifically binds to CD64, or antigen-binding fragment thereof, thereby forming an antibody-CD64 complex, and the presence of the antibody-CD64 complex is detected, wherein the presence of the complex indicates the patient is likely to respond to treatment. In some aspects, the amount of antibody-CD64 complex in the sample is compared to a control. In some aspects, when the amount of antibody-CD64 complex in the sample is increased relative to the control then the patient is likely to respond to treatment with the agent.

In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent as described herein (e.g., an anti-CD47 antibody) in which a tumor sample from the patient is contacted with a nucleic acid probe that hybridizes to a complementary target sequence in CD64 DNA or RNA, thereby forming a hybridization complex between the nucleic acid probe and the target CD64 DNA or RNA. To detect hybridization of the probe to the target CD64 DNA or RNA sequence, the probe is labeled with a molecular marker; for example, a radioactive marker, a fluorescent marker, an enzymatic marker, or digoxigenin. In some aspects, the presence of the probe-CD64 target complex indicates the patient is likely to respond to treatment. In some aspects, the amount of probe-CD64 target complex in the sample is compared to a control. In some aspects, when the amount of probe- CD64 target complex in the sample is increased relative to the control then the patient is likely to respond to treatment with the agent (e.g., an anti-CD47 antibody).

CD68

As used herein, the term“CD68” refers to a transmembrane glycoprotein encoded by the CD68 gene in humans. Other names or aliases for this gene in humans and other animals include: CD68 Molecule, CD68 Antigen, GP110, Macrosialin, Scavenger Receptor Class D, Member 1, SCARD1, and LAMP4. An amino acid sequence of an exemplary human CD68 protein is provided in SEQ ID NO: 35 (NCBI Reference Sequence: NP_00l242.2). An nucleic acid sequence of an exemplary human CD68 mRNA is provided in SEQ ID NO: 48 (NCBI Reference Sequence: NM _.001251.3).

In some aspects, the disclosure provides methods for detecting the presence of or determining an amount or expression level of CD68 in a sample from a subject (e.g., a tumor sample from cancer patient). CD68 is a protein highly expressed by cells in the monocyte lineage (e.g., monocytic phagocytes, osteoclasts), by circulating macrophages, and by tissue macrophages (e.g., Kupffer cells, microglia). Functionally, the CD68 protein binds to tissue- and organ- specific lectins or selectins, allowing macrophages to home in on particular targets. Immunohistochemistry (IHC) can be used to identify the presence of CD68, which is found in the cytoplasmic granules of a range of different blood cells. It is particularly useful as a marker for the various cells of the macrophage lineage, including monocytes, histiocytes, giant cells, Kupffer cells, and osteoclasts. This allows it to be used to distinguish diseases of otherwise similar appearance, such as the monocyte/macrophage and lymphoid forms of leukaemia (the latter being CD68 negative). Its presence in macrophages also makes it useful in diagnostic methods related to, for example, the infiltration of macrophages into tumors.

Accordingly, in some aspects the disclosure provides methods of identifying subjects likely to respond to treatment with an anti-tumor agent as described herein by detecting the presence of CD32a and the presence of CD68, and optionally CD47, CD32b, CD32c, CDl6a, CDl6b, and/or CD64 (e.g., an amount or expression level) in a sample obtained from the subject, wherein the presence of CD32a and CD68 indicates the subject is likely to respond to treatment with the agent. In some aspects, the amount or expression level of CD68 in a sample is determined and compared to a reference amount or reference expression level of CD68. In some aspects, when the amount or expression level of CD68 in the sample is increased relative to the reference amount or reference expression level of CD68, then the patient is likely to respond to treatment with an anti-tumor agent as disclosed herein.

In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent as described herein (e.g., an anti-CD47 antibody) in which a tumor sample from the patient is contacted with an antibody that specifically binds to CD68, or antigen-binding fragment thereof, thereby forming an antibody-CD68 complex, and the presence of the antibody-CD68 complex is detected, wherein the presence of the complex indicates the patient is likely to respond to treatment. In some aspects, the amount of antibody-CD68 complex in the sample is compared to a control. In some aspects, when the amount of antibody-CD68 complex in the sample is increased relative to the control then the patient is likely to respond to treatment with the agent.

In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent as described herein (e.g., an anti-CD47 antibody) in which a tumor sample from the patient is contacted with a nucleic acid probe that hybridizes to a complementary target sequence in CD68 DNA or RNA, thereby forming a hybridization complex between the nucleic acid probe and the target CD68 DNA or RNA. To detect hybridization of the probe to the target CD68 DNA or RNA sequence, the probe is labeled with a molecular marker; for example, a radioactive marker, a fluorescent marker, an enzymatic marker, or digoxigenin. In some aspects, the presence of the probe-CD68 target complex indicates the patient is likely to respond to treatment. In some aspects, the amount of probe-CD68 target complex in the sample is compared to a control. In some aspects, when the amount of probe- CD68 target complex in the sample is increased relative to the control then the patient is likely to respond to treatment with the agent (e.g., an anti-CD47 antibody).

CD163

As used herein, the term“CD 163” refers to a transmembrane protein encoded by the CD163 gene in humans. Other names or aliases for this gene in humans and other animals include: M130, MM130, SCARI1. An amino acid sequence of an exemplary human CD163 protein is provided in SEQ ID NO: 36 (NCBI Reference Sequence: NP_004235.4). An nucleic acid sequence of an exemplary human CD163 mRNA is provided in SEQ ID NO: 49 (NCBI Reference Sequence: NM_004244.5).

In some aspects, the disclosure provides methods for detecting the presence of or determining an amount or expression level of CD163 in a sample from a subject (e.g., a tumor sample from a cancer patient). CD 163 is a l30-kDa membrane protein with a large ectodomain consisting of nine scavenger receptor cysteine-rich scavenger receptor class B domains and functions as a hemoglobin (Hb) scavenger receptor. Its expression is restricted to the monocyte- macrophage lineage with relatively low expression on monocytes and high expression in tissue macrophages (e.g., splenic red pulp macrophages, Kupffer cells in the liver, etc.) Immunohistochemistry (IHC) can be used to identify the presence of CD 163 and it is a particularly useful marker for tissue macrophages making it useful in diagnostic methods related to, for example, the infiltration of macrophages into tumors.

Accordingly, in some aspects the disclosure provides methods of identifying subjects likely to respond to treatment with an anti-tumor agent as described herein by detecting the presence of CD32a and the presence of CD163, and optionally CD47, CD32b, CD32c, CDl6a, CDl6b, CD64, and/or CD68 (e.g., an amount or expression level) in a sample obtained from the subject, wherein the presence of CD32a and CD 163 indicates the subject is likely to respond to treatment with the agent. In some aspects, the amount or expression level of CD163 in a sample is determined and compared to a reference amount or reference expression level of CD 163. In some aspects, when the amount or expression level of CD 163 in the sample is increased relative to the reference amount or reference expression level of CD 163, then the patient is likely to respond to treatment with an anti-tumor agent as disclosed herein.

In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent as described herein (e.g., an anti-CD47 antibody) in which a tumor sample from the patient is contacted with an antibody that specifically binds to CD 163, or antigen -binding fragment thereof, thereby forming an antibody-CDl63 complex, and the presence of the antibody-CDl63 complex is detected, wherein the presence of the complex indicates the patient is likely to respond to treatment. In some aspects, the amount of antibody-CDl63 complex in the sample is compared to a control. In some aspects, when the amount of antibody-CDl63 complex in the sample is increased relative to the control then the patient is likely to respond to treatment with the agent.

In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent as described herein (e.g., an anti-CD47 antibody) in which a tumor sample from the patient is contacted with a nucleic acid probe that hybridizes to a complementary target sequence in CD163 DNA or RNA, thereby forming a hybridization complex between the nucleic acid probe and the target CD163 DNA or RNA. To detect hybridization of the probe to the target CD 163 DNA or RNA sequence, the probe is labeled with a molecular marker; for example, a radioactive marker, a fluorescent marker, an enzymatic marker, or digoxigenin. In some aspects, the presence of the probe-CDl63 target complex indicates the patient is likely to respond to treatment. In some aspects, the amount of probe- CD163 target complex in the sample is compared to a control. In some aspects, when the amount of probe-CDl63 target complex in the sample is increased relative to the control then the patient is likely to respond to treatment with the agent (e.g., an anti-CD47 antibody).

SIRPa

As used herein, the term“SIRPa” refers to the signal regulatory protein alpha, a member of the immunoglobulin superfamily that is encoded by the SIRPA gene in humans. SIRPa is also known as brain-immunoglobulin-like molecule with tyrosine -based activation motifs (BIT), CD 172 antigen-like family member A (CD172A), macrophage fusion receptor (MFR), myd-l antigen (MYD-l), P84, protein tyrosine phosphatase non-receptor type substrate 1 (PTPNS 1), and SHPS-l. An amino acid sequence of an exemplary human SIRPa protein is provided in SEQ ID NO: 37 (NCBI Reference Sequence: NP_00l317657.1). A nucleic acid sequence of an exemplary human SIRPa mRNA is provided in SEQ ID NO: 50 (NCBI Reference Sequence: NM_00l330728. l).

In some aspects, the disclosure provides methods for detecting the presence of or determining an amount or expression level of SIRPa in a sample from a subject (e.g., a tumor sample from cancer patient). SIRPa is a regulatory membrane glycoprotein from SIRP family expressed mainly by myeloid cells. SIRP family members are receptor-type transmembrane glycoproteins known to be involved in the negative regulation of receptor tyrosine kinase-coupled signaling processes. This protein can be phosphorylated by tyrosine kinases and its phospho- tyrosine residues have been shown to recruit SH2 domain containing tyrosine phosphatases (PTP), and serve as substrates of PTPs. This protein was found to participate in signal transduction mediated by various growth factor receptors. CD47 has been demonstrated to be a ligand for this receptor protein.

SIRPa acts as an inhibitory receptor and interacts with a broadly expressed transmembrane protein CD47 also called the "donT eat me" signal. This interaction negatively controls effector function of innate immune cells such as host cell phagocytosis. SIRPa diffuses laterally on the macrophage membrane and accumulates at a phagocytic synapse to bind CD47 and signal 'self, which inhibits the cyto skeleton-intensive process of phagocytosis by the macrophage. The extracellular domain of SIRPa binds to CD47 and transmits intracellular signals through its cytoplasmic domain. CD47-binding is mediated through the NH2-terminal V-like domain of SIRPa. The cytoplasmic region contains four ITIMs that become phosphorylated after binding of ligand. The phosphorylation mediates activation of tyrosine kinase SHP2. SIRPa has been shown to bind also phosphatase SHP1, adaptor protein SCAP2 and FYN-binding protein. Recruitment of SHP phosphatases to the membrane leads to the inhibition of myosin accumulation at the cell surface and results in the inhibition of phagocytosis. Cancer cells that express CD47 can activate SIRPa and inhibit macrophage-mediated anti-tumor activity.

Accordingly, in some aspects the disclosure provides methods of identifying subjects likely to respond to treatment with an anti-tumor agent as described herein by detecting the presence of CD32a and the presence of SIRPa, and optionally CD47, CD32b, CD32c, CDl6a, CDl6b, CD64, CD68, and/or CD163 (e.g., an amount or expression level) in a sample obtained from the subject, wherein the presence of CD32a and SIRPa indicates the subject is likely to respond to treatment with the agent. In some aspects, the amount or expression level of SIRPa in a sample is determined and compared to a reference amount or reference expression level of SIRPa. In some aspects, when the amount or expression level of SIRPa in the sample is increased relative to the reference amount or reference expression level of SIRPa, then the patient is likely to respond to treatment with an anti-tumor agent as disclosed herein.

In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent as described herein (e.g., an anti-CD47 antibody) in which a tumor sample from the patient is contacted with an antibody that specifically binds to SIRPa, or antigen-binding fragment thereof, thereby forming an antibody- SIRPa complex, and the presence of the antibody- SIRPa complex is detected, wherein the presence of the complex indicates the patient is likely to respond to treatment. In some aspects, the amount of antibody-SIRPa complex in the sample is compared to a control. In some aspects, when the amount of antibody-SIRPa complex in the sample is increased relative to the control then the patient is likely to respond to treatment with the agent.

In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent as described herein (e.g., an anti-CD47 antibody) in which a tumor sample from the patient is contacted with a nucleic acid probe that hybridizes to a complementary target sequence in SIRPa DNA or RNA, thereby forming a hybridization complex between the nucleic acid probe and the target SIRPa DNA or RNA. To detect hybridization of the probe to the target SIRPoc DNA or RNA sequence, the probe is labeled with a molecular marker; for example, a radioactive marker, a fluorescent marker, an enzymatic marker, or digoxigenin. In some aspects, the presence of the probe-SIRPa target complex indicates the patient is likely to respond to treatment. In some aspects, the amount of probe- SIRPa target complex in the sample is compared to a control. In some aspects, when the amount of probe-SIRPa target complex in the sample is increased relative to the control then the patient is likely to respond to treatment with the agent (e.g., an anti-CD47 antibody).

PD-1/PD-L1

PD-l is known as an immune-inhibitory protein that negatively regulates TCR signals (Ishida, Y. et al. (1992) EMBO J. 11:3887-3895; Blank, C. et al. (Epub 2006 Dec. 29) Immunol. Immunother. 56(5):739-745). The interaction between PD-l and PD-L1 can act as an immune checkpoint, which can lead to a decrease in T-cell receptor mediated proliferation (Dong et al. (2003) J. Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immune suppression can be reversed by inhibiting the local interaction of PD-l with PD-L1 or PD-L2; the effect is additive when the interaction of PD-l with PD-L2 is blocked as well (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66).

For several cancers, tumor survival and proliferation is sustained by tumor-mediated immune checkpoint modulation. This modulation can result in the disruption of anti-cancer immune system functions. For example, recent studies have indicated that the expression of immune checkpoint receptors ligands, such as PD-L1 or PD-L2, by tumor cells can downregulate immune system activity in the tumor microenvironment and promote cancer immune evasion particularly by suppressing T cells. PD-L1 is abundantly expressed by a variety of human cancers (Dong et al., (2002) Nat Med 8:787-789). The receptor for PD-L1, PD-l, is expressed on lymphocytes (e.g., activated T cells) and is normally involved in down-regulating the immune system and promoting self-tolerance, particularly by suppressing T cells. However, when PD-l receptors expressed on T cells bind to cognate PD-L1 ligands on tumor cells, the resulting T cell suppression contributes to an impaired immune response against the tumor (e.g., a decrease in tumor infiltrating lymphocytes or the establishment of immune evasion by cancer cells). In large sample sets of e.g. ovarian, renal, colorectal, pancreatic, liver cancers and melanoma, it was shown that PD-L1 expression correlated with poor prognosis and reduced overall survival irrespective of subsequent treatment (see e.g., Dong et al., (2002) Nat Med 8(8):793-800; Yang et al., (2008) Invest Ophthalmol Vis Sci 49(6):25l8-2525; Ghebeh et al., (2006) Neoplasia 8: 190-198; Hamanishi et al., (2007) Proc Nat Acad Sci USA 104:3360-3365; Thompson et al., (2006) Clin Genitourin Cancer 5:206-211; Nomi et al., (2005) Clin Cancer Res 11:2947-2953; Inman et al., (2007) Cancer 109: 1499-1505; Shimauchi et al., (2007) Int J Cancer 121:2585-2590; Gao et al., (2009) Clin Cancer Res 15:971-979; Nakanishi et al., (2007) Cancer Immunol Immunother 56: 1173-1182; Hino et al., (2010) Cancer 116(7): 1757-1766). Similarly, PD-l expression on tumor lymphocytes was found to mark dysfunctional T cells in breast cancer (Kitano et al., (2017) ESMO Open 2(2):e000l50) and melanoma (Kleffel et al., (2015) Cell 162(6): 1242- 1256). PD-l antagonists, such as those that affect the function of the PD-1/PD-L1/PD-L2 signaling axis and/or disrupt the interaction between PD-l and PD-L1 and/or PD-L2, for example, have been developed and represent a novel class of anti-tumor inhibitors that function via modulation of immune cell-tumor cell interaction.

Accordingly, in some aspects the disclosure provides methods of identifying subjects likely to respond to treatment with an anti-tumor agent as described herein by detecting the presence of CD32a and the presence of PD-L1 and/or PD-l, and optionally CD47, CD32b, CD32c, CDl6a, CDl6b, CD64, CD68, and/or CD163 (e.g., an amount or expression level) in a sample obtained from the subject, wherein the presence of CD32a and PD-L1 indicates the subject is likely to respond to treatment with the agent. In some aspects, the amount or expression level of PD-L1 in a sample is determined and compared to a reference amount or reference expression level of PD- Ll. In some aspects, when the amount or expression level of PD-L1 in the sample is increased relative to the reference amount or reference expression level of PD-L1, then the patient is likely to respond to treatment with an anti-tumor agent as disclosed herein.

In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent as described herein (e.g., an anti-CD47 antibody) in which a tumor sample from the patient is contacted with an antibody that specifically binds to PD-L1, or antigen -binding fragment thereof, thereby forming an antibody-PD-Ll complex, and the presence of the antibody-PD-Ll complex is detected, wherein the presence of the complex indicates the patient is likely to respond to treatment. In some aspects, the amount of antibody-PD-Ll complex in the sample is compared to a control. In some aspects, when the amount of antibody-PD-Ll complex in the sample is increased relative to the control then the patient is likely to respond to treatment with the agent.

In some aspects, the disclosure provides a method of identifying a cancer patient likely to respond to treatment with an agent as described herein (e.g., an anti-CD47 antibody) in which a tumor sample from the patient is contacted with a nucleic acid probe that hybridizes to a complementary target sequence in PD-l DNA or RNA, or PD-L1 DNA or RNA, thereby forming a hybridization complex between the nucleic acid probe and the target PD-l DNA or RNA, or target PD-L1 DNA or RNA. To detect hybridization of the probe to the target PD-l DNA or RNA sequence, or target PD-L1 DNA or RNA sequence, the probe is labeled with a molecular marker; for example, a radioactive marker, a fluorescent marker, an enzymatic marker, or digoxigenin. In some aspects, the presence of the probe-PD-l or probe-PD-Ll target complex indicates the patient is likely to respond to treatment. In some aspects, the amount of probe-PD- 1 or probe-PD-Ll target complex in the sample is compared to a control. In some aspects, when the amount of probe-PD-l or probe-PD-Ll target complex in the sample is increased relative to the control then the patient is likely to respond to treatment with the agent (e.g., an anti-CD47 antibody).

In some embodiments, the compositions, methods, uses and kits described herein can be used in combination with a diagnostic assay or test for PD-1/PD-L1. Diagnostic assays and tests for PD-L1 are described in Udall et ah, (2018) Diagn Pathol 13: 12, and Scheerens et ah, (2017) Clin Transl Sci (10:84-92, both of which are incorporated herein by reference in their entirety. Exemplary PD-1/PD-L1 diagnostic assays include, but are not limited to, the PD-L1 IHC 22C3 pharmDx assay, PD-L1 IHC 28-8 pharmDx assay, VENTANA PD-L1 (SP142) assay, and the VENT ANA PD-L1 (SP263) assay.

Diagnostic Methods

The disclosure provides methods related to detecting and/or quantifying one or more biomarkers described herein (e.g., CD32a, and optionally CD47, CD32b, CD32c, CDl6a, CDl6b, CD64, CD68, CD163, PD-l, PD-L1, and/or SIRPa), in one or more samples, wherein the detection and/or quantification of the one or more biomarkers individually or in combination, will indicate a likelihood that an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, will provide a therapeutic effect or benefit.

Provided herein are methods of identifying a cancer patient likely to respond to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: determining an amount of CD32a in a tumor sample obtained from the patient relative to a reference amount of CD32a, wherein the amount of CD32a in the tumor sample relative to the reference amount of CD32a indicates a likelihood the patient will respond to treatment. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of identifying a cancer patient likely to respond to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: contacting a tumor sample from the patient with an antibody that specifically binds to CD32a, or antigen-binding fragment thereof, thereby forming a complex; and determining an amount of the complex in the tumor sample relative to a reference amount of complex, wherein an amount of the complex in the tumor sample relative to the reference amount of complex indicates a likelihood the patient will respond to treatment. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of identifying a cancer patient likely to respond to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: determining an amount of CD32a in a tumor sample obtained from the patient; and comparing the amount of CD32a in a tumor sample to a reference amount of CD32a, wherein the patient is likely to respond to treatment when the amount of CD32a in the tumor sample is equal to or greater than the reference amount of CD32a. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of identifying a cancer patient likely to respond to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: contacting a tumor sample from the patient with an antibody that specifically binds to CD32a, or antigen-binding fragment thereof, thereby forming a complex; determining an amount of the complex in the tumor sample; and comparing the amount of the complex in the tumor sample to a reference amount of complex, wherein the patient is likely to respond to treatment when the amount of complex in the tumor sample is equal to or greater than the reference amount of the complex. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of evaluating a tumor as to select a therapy comprising an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: determining the expression levels of a panel of biomarkers in a sample of the tumor, wherein the panel comprises CD32a, CD32b, and CD47; comparing the expression levels of the panel in the sample to the expression levels of the panel in a control, and selecting a therapy comprising the agent, wherein a) the expression level of CD32a and CD47 in the sample is increased relative to the control; b) the expression level of CD32b in the sample is decreased relative to the control; or c) a combination of (a) and (b). In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of evaluating a tumor as to select a therapy comprising an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: determining the expression levels of a panel of biomarkers in a sample of the tumor, wherein the panel comprises CD32a, the FcyRIIa H131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a), the FcyRIIa R131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a), CD32b, and CD47; comparing the expression levels of the panel in the sample to the expression levels of the panel in a control, and selecting a therapy comprising the agent, wherein a) the expression level of CD32a and CD47 in the sample is increased relative to the control; b) the expression level of CD32b in the sample is decreased relative to the control; c) the expression level of , the FcyRIIa H131 allelic variant and CD47 in the sample is increased relative to the control; d) the expression level of the FcyRIIa R131 allelic variant and CD47 in the sample is increased relative to the control; or e) any combination of (a), (b), (c), and (d). In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 Fc domain or a mutant IgG4 Fc domain. Further provided herein are methods of detecting a tumor susceptible to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: contacting a tumor sample with at least one diagnostic antibody, or antigen-binding fragment thereof, that specifically binds to CD32a and, optionally, one or more additional biomarkers selected from the group consisting of: CD47, CD32b, CD32c, CDl6a, CDl6b, CD64, CD68, CD163, PD-l, PD-L1, and SIRPa, and a combination thereof, thereby forming a diagnostic antibody-biomarker complex; determining an amount of complex in the tumor sample relative to an amount of complex in a reference sample, wherein the amount of the complex in the tumor sample relative to the amount of the complex in the reference sample indicates a likelihood the tumor is susceptible treatment with the agent. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of detecting a tumor susceptible to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: contacting a tumor sample with at least one diagnostic antibody, or antigen-binding fragment thereof, that specifically binds to CD32a and CD47, thereby forming a diagnostic antibody-biomarker complex; determining an amount of complex in the tumor sample relative to an amount of complex in a reference sample, wherein the amount of the complex in the tumor sample relative to the amount of the complex in the reference sample indicates a likelihood the tumor is susceptible treatment with the agent. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of detecting a tumor susceptible to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: contacting a tumor sample with at least one diagnostic antibody, or antigen-binding fragment thereof, that specifically binds to the FcyRIIa H131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a) and, optionally, one or more additional biomarkers selected from the group consisting of: CD47, CD32b, CD32c, CDl6a, CDl6b, CD64, CD68, CD163, PD-l, PD-L1, and SIRPa, and a combination thereof, thereby forming a diagnostic antibody-biomarker complex; determining an amount of complex in the tumor sample relative to an amount of complex in a reference sample, wherein the amount of the complex in the tumor sample relative to the amount of the complex in the reference sample indicates a likelihood the tumor is susceptible treatment with the agent. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of detecting a tumor susceptible to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: contacting a tumor sample with at least one diagnostic antibody, or antigen-binding fragment thereof, that specifically binds to the FcyRIIa H131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a) and CD47, thereby forming a diagnostic antibody-biomarker complex; determining an amount of complex in the tumor sample relative to an amount of complex in a reference sample, wherein the amount of the complex in the tumor sample relative to the amount of the complex in the reference sample indicates a likelihood the tumor is susceptible treatment with the agent. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of detecting a tumor susceptible to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: contacting a tumor sample with at least one diagnostic antibody, or antigen-binding fragment thereof, that specifically binds to the FcyRIIa R131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a) and, optionally, one or more additional biomarkers selected from the group consisting of: CD47, CD32b, CD32c, CDl6a, CDl6b, CD64, CD68, CD163, PD-l, PD-L1, and SIRPa, and a combination thereof, thereby forming a diagnostic antibody-biomarker complex; determining an amount of complex in the tumor sample relative to an amount of complex in a reference sample, wherein the amount of the complex in the tumor sample relative to the amount of the complex in the reference sample indicates a likelihood the tumor is susceptible treatment with the agent. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of detecting a tumor susceptible to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: contacting a tumor sample with at least one diagnostic antibody, or antigen-binding fragment thereof, that specifically binds to the FcyRIIa R131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a) and CD47, thereby forming a diagnostic antibody-biomarker complex; determining an amount of complex in the tumor sample relative to an amount of complex in a reference sample, wherein the amount of the complex in the tumor sample relative to the amount of the complex in the reference sample indicates a likelihood the tumor is susceptible treatment with the agent. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of predicting a response of a tumor to an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: contacting a tumor sample with at least one diagnostic antibody, or antigen-binding fragment thereof, that specifically binds to CD32a and, optionally, one or more additional biomarkers selected from the group consisting of: CD47, CD32b, CD32c, CDl6a, CDl6b, CD64, CD68, CD163, PD-l, PD-L1, and SIRPa, and a combination thereof; determining an extent of binding of the at least one diagnostic antibody to the tumor sample; comparing the extent of binding of the at least one diagnostic antibody to the sample to an extent of binding of the at least one diagnostic antibody to a reference sample, wherein the extent of binding the tumor sample relative to the extent of binding to the reference sample indicates a likelihood the tumor will respond to the agent. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of predicting a response of a tumor to an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: contacting a tumor sample with at least one diagnostic antibody, or antigen-binding fragment thereof, that specifically binds to CD32a and CD47; determining an extent of binding of the at least one diagnostic antibody to the tumor sample; comparing the extent of binding of the at least one diagnostic antibody to the sample to an extent of binding of the at least one diagnostic antibody to a reference sample, wherein the extent of binding the tumor sample relative to the extent of binding to the reference sample indicates a likelihood the tumor will respond to the agent. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of predicting a response of a tumor to an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: contacting a tumor sample with at least one diagnostic antibody, or antigen -binding fragment thereof, that specifically binds to the FcyRIIa H131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a) and, optionally, one or more additional biomarkers selected from the group consisting of: CD47, CD32b, CD32c, CDl6a, CDl6b, CD64, CD68, CD163, PD-l, PD-L1, and SIRPa, and a combination thereof; determining an extent of binding of the at least one diagnostic antibody to the tumor sample; comparing the extent of binding of the at least one diagnostic antibody to the sample to an extent of binding of the at least one diagnostic antibody to a reference sample, wherein the extent of binding the tumor sample relative to the extent of binding to the reference sample indicates a likelihood the tumor will respond to the agent. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of predicting a response of a tumor to an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: contacting a tumor sample with at least one diagnostic antibody, or antigen -binding fragment thereof, that specifically binds to the FcyRIIa H131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a) and CD47, determining an extent of binding of the at least one diagnostic antibody to the tumor sample; comparing the extent of binding of the at least one diagnostic antibody to the sample to an extent of binding of the at least one diagnostic antibody to a reference sample, wherein the extent of binding the tumor sample relative to the extent of binding to the reference sample indicates a likelihood the tumor will respond to the agent. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of predicting a response of a tumor to an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: contacting a tumor sample with at least one diagnostic antibody, or antigen-binding fragment thereof, that specifically binds to the FcyRIIa R131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a) and, optionally, one or more additional biomarkers selected from the group consisting of: CD47, CD32b, CD32c, CDl6a, CDl6b, CD64, CD68, CD163, PD-l, PD-L1, and SIRPa, and a combination thereof; determining an extent of binding of the at least one diagnostic antibody to the tumor sample; comparing the extent of binding of the at least one diagnostic antibody to the sample to an extent of binding of the at least one diagnostic antibody to a reference sample, wherein the extent of binding the tumor sample relative to the extent of binding to the reference sample indicates a likelihood the tumor will respond to the agent. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of predicting a response of a tumor to an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: contacting a tumor sample with at least one diagnostic antibody, or antigen-binding fragment thereof, that specifically binds to the FcyRIIa R131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a) and CD47, determining an extent of binding of the at least one diagnostic antibody to the tumor sample; comparing the extent of binding of the at least one diagnostic antibody to the sample to an extent of binding of the at least one diagnostic antibody to a reference sample, wherein the extent of binding the tumor sample relative to the extent of binding to the reference sample indicates a likelihood the tumor will respond to the agent. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods for monitoring a response of a tumor treated with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: determining the expression level and/or activity of CD32a and, optionally, one or more additional biomarkers selected from: CD47, CD32b, CD32c, CDl6a, CDl6b, CD64, CD68, CD163, PD-l, PD-L1, and SIRPa, or a combination thereof, in a first tumor sample obtained prior to treatment with the agent; determining the expression level and/or activity of the at least one biomarker in a second tumor sample obtained after treatment with the agent; comparing the expression level and/or activity of the at least one biomarker in the second sample with the value of the indicator in the expression level and/or activity of the at least one biomarker in the first sample, wherein a change of the expression level and/or activity of the at least one biomarker in the second sample relative to the expression level and/or activity of the at least one biomarker in the first sample indicates a response of the tumor treated with the agent. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods for monitoring a response of a tumor treated with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: determining the expression level and/or activity of CD32a and CD47 in a first tumor sample obtained prior to treatment with the agent; determining the expression level and/or activity of the at least one biomarker in a second tumor sample obtained after treatment with the agent; comparing the expression level and/or activity of the at least one biomarker in the second sample with the value of the indicator in the expression level and/or activity of the at least one biomarker in the first sample, wherein a change of the expression level and/or activity of the at least one biomarker in the second sample relative to the expression level and/or activity of the at least one biomarker in the first sample indicates a response of the tumor treated with the agent. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods for monitoring a response of a tumor treated with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: determining the expression level and/or activity of the FcyRIIa H131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a) and, optionally, one or more additional biomarkers selected from: CD47, CD32b, CD32c, CDl6a, CDl6b, CD64, CD68, CD163, PD-l, PD-L1, and SIRPa, or a combination thereof, in a first tumor sample obtained prior to treatment with the agent; determining the expression level and/or activity of the at least one biomarker in a second tumor sample obtained after treatment with the agent; comparing the expression level and/or activity of the at least one biomarker in the second sample with the value of the indicator in the expression level and/or activity of the at least one biomarker in the first sample, wherein a change of the expression level and/or activity of the at least one biomarker in the second sample relative to the expression level and/or activity of the at least one biomarker in the first sample indicates a response of the tumor treated with the agent. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods for monitoring a response of a tumor treated with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: determining the expression level and/or activity of the FcyRIIa H131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a) and CD47, in a first tumor sample obtained prior to treatment with the agent; determining the expression level and/or activity of the at least one biomarker in a second tumor sample obtained after treatment with the agent; comparing the expression level and/or activity of the at least one biomarker in the second sample with the value of the indicator in the expression level and/or activity of the at least one biomarker in the first sample, wherein a change of the expression level and/or activity of the at least one biomarker in the second sample relative to the expression level and/or activity of the at least one biomarker in the first sample indicates a response of the tumor treated with the agent. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods for monitoring a response of a tumor treated with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: determining the expression level and/or activity of the FcyRIIa R131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a) and, optionally, one or more additional biomarkers selected from: CD47, CD32b, CD32c, CDl6a, CDl6b, CD64, CD68, CD163, PD-l, PD-L1, and SIRPa, or a combination thereof, in a first tumor sample obtained prior to treatment with the agent; determining the expression level and/or activity of the at least one biomarker in a second tumor sample obtained after treatment with the agent; comparing the expression level and/or activity of the at least one biomarker in the second sample with the value of the indicator in the expression level and/or activity of the at least one biomarker in the first sample, wherein a change of the expression level and/or activity of the at least one biomarker in the second sample relative to the expression level and/or activity of the at least one biomarker in the first sample indicates a response of the tumor treated with the agent. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain. Further provided herein are methods for monitoring a response of a tumor treated with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: determining the expression level and/or activity of the FcyRIIa R131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a) and CD47, in a first tumor sample obtained prior to treatment with the agent; determining the expression level and/or activity of the at least one biomarker in a second tumor sample obtained after treatment with the agent; comparing the expression level and/or activity of the at least one biomarker in the second sample with the value of the indicator in the expression level and/or activity of the at least one biomarker in the first sample, wherein a change of the expression level and/or activity of the at least one biomarker in the second sample relative to the expression level and/or activity of the at least one biomarker in the first sample indicates a response of the tumor treated with the agent. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of monitoring a response of a cancer patient receiving a therapy comprising an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: determining the expression level and/or activity of CD32a and, optionally, one or more additional biomarkers selected from: CD47, CD32b, CD32c, CDl6a, CDl6b, CD64, CD68, CD163, PD-l, PD-L1, and SIRPa, or a combination thereof, in a first tumor sample obtained from the patient prior to treatment with the agent; determining the expression level and/or activity of the at least one biomarker in a second tumor sample obtained from the patient after treatment with the agent; comparing the expression level and/or activity of the at least one biomarker in the second sample with the expression level and/or activity of the at least one biomarker in the first sample, wherein a change of the expression level and/or activity of the at least one biomarker in the second sample relative to the expression level and/or activity of the at least one biomarker in the first sample indicates a patient’ s response to treatment. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of monitoring a response of a cancer patient receiving a therapy comprising an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: determining the expression level and/or activity of CD32a and CD47, in a first tumor sample obtained from the patient prior to treatment with the agent; determining the expression level and/or activity of the at least one biomarker in a second tumor sample obtained from the patient after treatment with the agent; comparing the expression level and/or activity of the at least one biomarker in the second sample with the expression level and/or activity of the at least one biomarker in the first sample, wherein a change of the expression level and/or activity of the at least one biomarker in the second sample relative to the expression level and/or activity of the at least one biomarker in the first sample indicates a patient’s response to treatment. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild- type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of monitoring a response of a cancer patient receiving a therapy comprising an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: determining the expression level and/or activity of the FcyRIIa H131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a) and, optionally, one or more additional biomarkers selected from: CD47, CD32b, CD32c, CDl6a, CDl6b, CD64, CD68, CD163, PD-l, PD-L1, and SIRPa, or a combination thereof, in a first tumor sample obtained from the patient prior to treatment with the agent; determining the expression level and/or activity of the at least one biomarker in a second tumor sample obtained from the patient after treatment with the agent; comparing the expression level and/or activity of the at least one biomarker in the second sample with the expression level and/or activity of the at least one biomarker in the first sample, wherein a change of the expression level and/or activity of the at least one biomarker in the second sample relative to the expression level and/or activity of the at least one biomarker in the first sample indicates a patient’s response to treatment. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of monitoring a response of a cancer patient receiving a therapy comprising an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: determining the expression level and/or activity of the FcyRIIa H131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a) and CD47 in a first tumor sample obtained from the patient prior to treatment with the agent; determining the expression level and/or activity of the at least one biomarker in a second tumor sample obtained from the patient after treatment with the agent; comparing the expression level and/or activity of the at least one biomarker in the second sample with the expression level and/or activity of the at least one biomarker in the first sample, wherein a change of the expression level and/or activity of the at least one biomarker in the second sample relative to the expression level and/or activity of the at least one biomarker in the first sample indicates a patient’s response to treatment. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of monitoring a response of a cancer patient receiving a therapy comprising an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: determining the expression level and/or activity of the FcyRIIa R131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a) and, optionally, one or more additional biomarkers selected from: CD47, CD32b, CD32c, CDl6a, CDl6b, CD64, CD68, CD163, PD-l, PD-L1, and SIRPa, or a combination thereof, in a first tumor sample obtained from the patient prior to treatment with the agent; determining the expression level and/or activity of the at least one biomarker in a second tumor sample obtained from the patient after treatment with the agent; comparing the expression level and/or activity of the at least one biomarker in the second sample with the expression level and/or activity of the at least one biomarker in the first sample, wherein a change of the expression level and/or activity of the at least one biomarker in the second sample relative to the expression level and/or activity of the at least one biomarker in the first sample indicates a patient’s response to treatment. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Further provided herein are methods of monitoring a response of a cancer patient receiving a therapy comprising an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, the method comprising: determining the expression level and/or activity of the FcyRIIa R131 allelic variant of the FCGR2A gene encoding FcyRIIa (CD32a) and CD47 in a first tumor sample obtained from the patient prior to treatment with the agent; determining the expression level and/or activity of the at least one biomarker in a second tumor sample obtained from the patient after treatment with the agent; comparing the expression level and/or activity of the at least one biomarker in the second sample with the expression level and/or activity of the at least one biomarker in the first sample, wherein a change of the expression level and/or activity of the at least one biomarker in the second sample relative to the expression level and/or activity of the at least one biomarker in the first sample indicates a patient’s response to treatment. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain.

Determination of Biomarkers

The presence of, an amount or expression level of one or more biomarkers described herein in a sample can be detected or determined by a number of methodologies and techniques, which are known in the art and understood by the skilled artisan, including, but not limited to, immunohistochemistry (IHC), immunofluorescence (IF), Western blot analysis, immunoprecipitation, molecular binding assays, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofiltration assay (ELIFA), flow cytometry, MassARRAY, proteomics, quantitative blood based assays (e.g., Serum ELISA), biochemical enzymatic activity assays, in situ hybridization, fluorescence in situ hybridization (FISH), Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction (PCR) including quantitative real time PCR (qRT-PCR) and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like, RNA-Seq, microarray analysis, gene expression profiling, and/or serial analysis of gene expression (SAGE), as well as any one of the wide variety of assays that can be performed by protein, gene, and/or tissue array analysis. Typical protocols for evaluating the status of genes and gene products are found, for example in Ausubel et ah, eds., 1 995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Multiplexed immunoassays such as those available from Rules Based Medicine or Meso Scale Discovery ("MSD") may also be used. Diagnostic antibodies that bind the biomarkers of the disclosure are available from a variety of commercial sources, such as BD Biosciences, ebiosciences, BioLegend, Abeam, and the like.

Protein Biomarker Techniques

In some embodiments, the amount of a biomarker (e.g., CD32a) is measured by determining the protein expression level of the bio marker. There are a number of techniques that measure or determine protein expression levels known in the art and described herein that may be used in the methods provided by the disclosure. For example, in some embodiments, a protein expression level of a bio marker (e.g., CD32a) is determined using a method selected from the group consisting of flow cytometry (e.g., fluorescence-activated cell sorting (FACS™)), Western blot, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunohistochemistry (IHC), immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, and HPLC.

In some embodiments, a sample is contacted with an antibody that specifically binds to a biomarker (e.g., an anti-CD32a antibody) described herein under conditions permissive for binding of the biomarker, and the presence of a complex formed by the antibody and the biomarker is detected. In some embodiments, a sample is contacted with a combination of antibodies that specifically bind to a combination of biomarkers described herein. In some embodiments, the protein expression level of the biomarker (e.g., CD32a) is determined in tumor-infiltrating immune cells (e.g. tumor-infiltrating macrophages). In some embodiments, the protein expression level of a biomarker (e.g., CD47) is determined in tumor cells. In some embodiments, the protein expression level of the biomarker is determined in tumor-infiltrating immune cells and in tumor cells.

In some embodiments, the amount of a biomarker protein (e.g., CD32a) in a sample is determined using IHC and staining protocols. IHC staining of tissue sections has been shown to be a reliable method of determining or detecting the presence of proteins in a sample. In some embodiments, the biomarker is selected from CD47, CD32b, CD32c, CDl6a, CDl6b, CD64, CD68, CD163, PD-l, PD-L1, and SIRPa. In some embodiments, expression level of a biomarker is determined by performing IHC analysis of a sample (e.g., a tumor sample obtained from a patient) with an antibody. In some embodiments, IHC staining intensity of the biomarker in a sample is determined relative to a reference sample. In some embodiments, the reference sample is obtained from a control cell line or non-malignant cell line, a tissue sample from non-cancerous patient, or a tumor sample that does not express or contain a biomarker described herein.

Two general methods of IHC are available; direct and indirect assays. The direct assay uses a labeled reagent, such as a fluorescent tag or an enzyme-labeled primary antibody, which can be visualized without further antibody interaction. In contrast, an indirect assay includes an unconjugated primary antibody that binds to the antigen and a labeled secondary antibody that binds to the primary antibody. The secondary antibody is conjugated to an enzymatic label, a chromogenic or fluorogenic substrate to provide visualization of the antigen.

The primary and/or secondary antibody used for IHC typically will be labeled with a detectable moiety. Numerous labels are available which can be generally grouped into the following categories: (a) radioisotopes, such as 35S, 14C, 1251 , 3H, and 1311; (b) colloidal gold particles; (c) fluorescent labels including, but not limited to, rare earth chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, lissamine, umbelliferone, phycocrytherin, phycocyanin, or commercially- available fluorophores such as SPECTRUM ORANGE7 and SPECTRUM GREEN7 and/or derivatives of any one or more of the above; and various enzyme- substrate labels are available (see e.g. U.S. Patent No. 4,275,149 which is included herein by reference in its entirety). Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; see, e.g., U.S. Patent No. 4,737,456), luciferin, 2,3- dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, b-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), I acto peroxidase, microperoxidase, and the like.

Examples of enzyme- substrate combinations include, for example, horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate; alkaline phosphatase (AP) with para- Nitrophenyl phosphate as chromogenic substrate; and b-D-galactosidase (b-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-p-D-galactosidase) or fluorogenic substrate (e.g., 4- methylumbelliferyl-p- D-galactosidase). For a general review of these, see, for example, U.S. Patent Nos. 4,275,149 and 4,318,980.

In some embodiments, samples may be prepared for IHC analysis, for example, manually, or using an automated staining instrument (e.g., a Ventana BenchMark XT or Benchmark ULTRA instrument). Samples thus prepared may be mounted on slides and coverslipped. Slide evaluation is then determined, for example, using a microscope, and staining intensity criteria, routinely used in the art, may be employed. It is understood by one skilled in the art that when a sample from a tumor is analyzed using IHC, staining is generally determined or assessed in tumor cell(s) and/or tissue (as opposed to stromal or surrounding tissue that may be present in the sample). In some embodiments, when cells and/or tissue from a tumor are examined using IHC, staining includes determining or assessing in tumor-infiltrating immune cells, including intratumoral or peritumoral immune cells. In some embodiments, the presence of a biomarker (e.g., CD32a) is detected by IHC in >0% of the sample, for example, in at least 1 % of the sample, in at least 5% of the sample, in at least 10% of the sample, in at least 15% of the sample, in at least 15% of the sample, in at least 20% of the sample, in at least 25% of the sample, in at least 30% of the sample, in at least 35% of the sample, in at least 40% of the sample, in at least 45% of the sample, in at least 50% of the sample, in at least 55% of the sample, in at least 60% of the sample, in at least 65% of the sample, in at least 70% of the sample, in at least 75% of the sample, in at least 80% of the sample, in at least 85% of the sample, in at least 90% of the sample, in at least 95% of the sample, or more. Samples may be scored using any of the criteria described herein, for example, by a pathologist or automated image analysis.

In some embodiments, the amount of CD32a in a sample is determined using an anti- CD32a diagnostic antibody. In some embodiments, the anti-CD32a diagnostic antibody specifically binds human CD32a. In some embodiments, the anti-CD32a diagnostic antibody is a non-human antibody. In some embodiments, the anti-CD32a diagnostic antibody is a rat, mouse, or rabbit antibody. In some embodiments, the anti-CD32a diagnostic antibody is a monoclonal antibody. In some embodiments, the anti-CD32a diagnostic antibody is directly labeled. In other embodiments, the anti-CD32a diagnostic antibody is indirectly labeled.

In some embodiments, the amount of CD32b in a sample is determined using an anti- CD32b diagnostic antibody. In some embodiments, the anti-CD32b diagnostic antibody specifically binds human CD32b. In some embodiments, the anti-CD32b diagnostic antibody is a non-human antibody. In some embodiments, the anti-CD32b diagnostic antibody is a rat, mouse, or rabbit antibody. In some embodiments, the anti-CD32b diagnostic antibody is a monoclonal antibody. In some embodiments, the anti-CD32b diagnostic antibody is directly labeled. In other instances, the anti-CD32b diagnostic antibody is indirectly labeled.

In some embodiments, the amount of CD32c in a sample is determined using an anti- CD32c diagnostic antibody. In some embodiments, the anti-CD32c diagnostic antibody specifically binds human CD32c. In some embodiments, the anti-CD32c diagnostic antibody is a non-human antibody. In some embodiments, the anti-CD32c diagnostic antibody is a rat, mouse, or rabbit antibody. In some embodiments, the anti-CD32c diagnostic antibody is a monoclonal antibody. In some embodiments, the anti-CD32c diagnostic antibody is directly labeled. In other embodiments, the anti-CD32c diagnostic antibody is indirectly labeled.

In some embodiments, the amount of CD47 in a sample is determined using an anti-CD47 diagnostic antibody. In some embodiments, the anti-CD47 diagnostic antibody specifically binds human CD47. In some embodiments, the anti-CD47 diagnostic antibody is a non-human antibody. In some embodiments, the anti-CD47 diagnostic antibody is a rat, mouse, or rabbit antibody. In some embodiments, the anti-CD47 diagnostic antibody is a monoclonal antibody. In some embodiments, the anti-CD47 diagnostic antibody is directly labeled. In other embodiments, the anti-CD47 diagnostic antibody is indirectly labeled.

In some embodiments, the amount of CDl6a in a sample is determined using an anti- CD 16a diagnostic antibody. In some embodiments, the anti-CD 16a diagnostic antibody specifically binds human CDl6a. In some embodiments, the anti-CD 16a diagnostic antibody is a non-human antibody. In some embodiments, the anti-CD 16a diagnostic antibody is a rat, mouse, or rabbit antibody. In some embodiments, the anti-CD 16a diagnostic antibody is a monoclonal antibody. In some embodiments, the anti-CD 16a diagnostic antibody is directly labeled. In some embodiments, the anti-CD 16a diagnostic antibody is indirectly labeled.

In some embodiments, the amount of CDl6b in a sample is determined using an anti- CDl6b diagnostic antibody. In some embodiments, the anti-CDl6b diagnostic antibody specifically binds human CDl6b. In some embodiments, the anti-CDl6b diagnostic antibody is a non-human antibody. In some embodiments, the anti-CD 16b diagnostic antibody is a rat, mouse, or rabbit antibody. In some embodiments, the anti-CDl6b diagnostic antibody is a monoclonal antibody. In some embodiments, the anti-CD 16b diagnostic antibody is directly labeled. In some embodiments, the anti-CD 16b diagnostic antibody is indirectly labeled.

In some embodiments, the amount of CD64 in a sample is determined using an anti-CD64 diagnostic antibody. In some embodiments, the anti-CD64 diagnostic antibody specifically binds human CD64. In some embodiments, the anti-CD64 diagnostic antibody is a non-human antibody. In some embodiments, the anti-CD64 diagnostic antibody is a rat, mouse, or rabbit antibody. In some embodiments, the anti-CD64 diagnostic antibody is a monoclonal antibody. In some embodiments, the anti-CD64 diagnostic antibody is directly labeled. In other instances, the anti- CD64 diagnostic antibody is indirectly labeled. In some embodiments, the amount of CD68 in a sample is determined using an anti-CD68 diagnostic antibody. In some embodiments, the anti-CD68 diagnostic antibody specifically binds human CD68. In some embodiments, the anti-CD68 diagnostic antibody is a non-human antibody. In some embodiments, the anti-CD68 diagnostic antibody is a rat, mouse, or rabbit antibody. In some embodiments, the anti-CD68 diagnostic antibody is a monoclonal antibody. In some embodiments, the anti-CD68 diagnostic antibody is directly labeled. In other embodiments, the anti-CD68 diagnostic antibody is indirectly labeled.

In some embodiments, the amount of CD 163 in a sample is determined using an anti- CD163 diagnostic antibody. In some embodiments, the anti-CDl63 diagnostic antibody specifically binds human CD 163. In some embodiments, the anti-CD 163 diagnostic antibody is a non-human antibody. In some embodiments, the anti-CD l63diagnostic antibody is a rat, mouse, or rabbit antibody. In some embodiments, the anti-CDl63 diagnostic antibody is a monoclonal antibody. In some embodiments, the anti-CD 163 diagnostic antibody is directly labeled. In other embodiments, the anti-CD 163 diagnostic antibody is indirectly labeled.

In some embodiments, the amount of SIRPa in a sample is determined using an anti-SIRPa diagnostic antibody. In some embodiments, the anti-SIRPa diagnostic antibody specifically binds human SIRPa. In some embodiments, the anti-SIRPa diagnostic antibody is a non-human antibody. In some embodiments, the anti-SIRPa diagnostic antibody is a rat, mouse, or rabbit antibody. In some embodiments, the anti-SIRPa diagnostic antibody is a monoclonal antibody. In some embodiments, the anti-SIRPa diagnostic antibody is directly labeled. In other instances, the anti-SIRPa diagnostic antibody is indirectly labeled.

In some embodiments, the expression level of a biomarker described herein is detected in tumor-infiltrating immune cells, tumor cells, or combinations thereof using IHC. Tumor- infiltrating immune cells include, but are not limited to, intratumoral immune cells, peritumoral immune cells or any combinations thereof, and other tumor stroma cells (e.g., fibroblasts). Such tumor infiltrating immune cells may be T lymphocytes (such as CD8+ T lymphocytes and/or CD4+ T lymphocytes), B lymphocytes, or other bone marrow-lineage cells including granulocytes (neutrophils, eosinophils, basophils), monocytes, macrophages, dendritic cells (e.g., interdigitating dendritic cells), histiocytes, and natural killer cells. In some embodiments, the staining for a biomarker, described herein, is detected as membrane staining, cytoplasmic staining and combinations thereof. In some embodiments, the absence of a biomarker, described herein, is detected as absent or no staining in the sample.

Nucleic Acid Biomarker Techniques

In some embodiments, the expression level of a biomarker, described herein, may be a nucleic acid expression level. In some embodiments, the nucleic acid expression level is determined using qPCR, rtPCR, RNA-seq, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technique, or in situ hybridization (e.g., FISH). In some embodiments, the expression level of a biomarker (e.g., CD32a) is determined in tumor cells, tumor infiltrating immune cells, stromal cells, or combinations thereof. In some embodiments, the expression level of a biomarker described herein is determined in tumor-infiltrating immune cells. In some embodiments, the expression level of a biomarker described herein is determined in tumor cells.

Methods for the evaluation of mRNAs in cells are known in the art and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled riboprobes specific for the one or more genes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for one or more of the genes, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like). In addition, such methods can include one or more steps that allow one to determine the levels of target mRNA in a biological sample (e.g., by simultaneously examining the levels a comparative control mRNA sequence of a "housekeeping" gene such as an actin family member).

The nucleic acid sequences of the biomarkers of the disclosure are known in the art. For examples, the nucleic acid sequence of CD47 (isoform 1) is set forth in SEQ ID NO: 2; the nucleic acid sequence of CD47 (isoform 2) is set forth in SEQ ID NO: 42; the nucleic acid sequence of CD32a (isoform 1) is set forth in SEQ ID NO: 38; the nucleic acid sequence of CD32a (isoform 1) is set forth in SEQ ID NO: 40; the nucleic acid sequence of CD32b is set forth as SEQ ID NO: 45; the nucleic acid sequence of CD32c is set forth in SEQ ID NO: 46; the nucleic acid sequence of CDl6a is set forth in SEQ ID NO: 43; the nucleic acid sequence of CDl6b is set forth in SEQ ID NO: 44; the nucleic acid sequence of CD64 is set forth as SEQ ID NO: 47; the nucleic acid sequence of CD68 is set forth in SEQ ID NO: 48; the nucleic acid sequence of CD163 is set forth in SEQ ID NO: 49. In some embodiments, the sequence of the amplified target cDNA can be determined. Methods include protocols which examine or detect mRNAs, such as target mRNAs, in a tissue or cell sample by microarray technologies. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured such that the sequence and position of each member of the array is known. For example, a selection of genes whose expression correlates with increased or reduced clinical benefit of treatment comprising an agent that specifically binds to CD47, wherein the agent comprises an Fc domain, may be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene.

Inclusion of any of the diagnostic methods described herein as part of any method directed to methods for identifying patients likely to benefit from treatment as described herein (e.g., selection of a therapeutic treatment or intervention) or to the development of treatments (e.g., enrollment patients in clinical trials) provides an advantage over those methods that do not include the diagnostic methods, in that a patient population whose members are predicted to need and/or not need, benefit, or respond to treatment may be identified.

Accordingly, in some embodiments, the biomarker suitable for use in the present disclosure is a diagnostic biomarker. In some embodiments, the biomarker is a monitoring biomarker. In some embodiments, the biomarker is a predictive biomarker. In some embodiments, the biomarker is a pharmacodynamics/response biomarker.

Biomarkers can be a substance or a biological event whose detection indicates a particular physiological state (e.g., a diseased state). For example, the presence of an antibody in the serum of a patient may indicate an infection. Biomarkers measured in patients before treatment can be used to identify suitable patients for inclusion in a clinical trial. Biomarker changes after treatment may predict or identify safety problems related to a candidate drug, or reveal pharmacologic activity expected to predict an eventual benefit of treatment. Biomarkers may reduce uncertainty in drug development and evaluation by providing quantifiable predictions about drug performance, and they can contribute to dose selection. Composite biomarkers include several individual biomarkers in a stated algorithm which reaches a single interpretive readout when a single biomarker fails to provide all the relevant information required for assessment. A surrogate end point is a biomarker that is intended to substitute for a clinical end point and is expected, based on epidemiologic, therapeutic, pathophysiologic, or other scientific evidence, to predict clinical benefit.

Samples

In some embodiments of the methods provided by the disclosure, a sample includes any relevant biological sample (e.g., a tumor sample) suitable for use in the methods provided by the disclosure (e.g., sections of tissues such as biopsy or tissue removed during surgical or other procedures, autopsy samples, and frozen sections taken for histological purposes). Samples may be derived or obtained from blood and blood fractions or products (e.g., serum, buffy coat, plasma, platelets, red blood cells, peripheral blood mononuclear cells (PBMC)), sputum, cheek cells, cultured cells (e.g., primary cultures, explants, and transformed cells), stool, urine, other biological or bodily fluids (e.g., prostatic fluid, gastric fluid, intestinal fluid, renal fluid, lung fluid, cerebrospinal fluid, pleural and abdominal effusions, and the like), etc. A sample may be processed according to techniques understood by those in the art. A sample can be without limitation fresh, frozen or fixed. In some embodiments, a sample is obtained from a tumor or malignant tissue. In some embodiments, a sample comprises malignant and non-malignant cells. In some embodiments, a sample comprises formalin-fixed paraffin-embedded (FFPE) tissue or fresh frozen (FF) tissue. In some embodiments, a sample comprises cultured cells, including primary or immortalized cell lines derived from a subject. In some embodiments, a sample refer to an extract from a sample from a subject. For example, in some embodiments, a sample comprises DNA, RNA or protein extracted from a tissue or a bodily fluid. Many techniques and commercial kits are available for such purposes. In some embodiments, a fresh sample from the individual is treated with an agent to preserve RNA prior to further processing, e.g., cell lysis and extraction. Samples can include frozen samples collected for other purposes. In some embodiments, samples are associated with information such as age, gender, and clinical symptoms present in the subject from which the sample is obtained; source or location of the sample; and information regarding methods of collection, treatment, and/or storage of the sample.

In some embodiments, a sample is obtained from a biopsy. A biopsy comprises the process of removing a tissue sample for diagnostic or prognostic evaluation. Any biopsy technique known in the art can be applied to the methods provided by the disclosure. The biopsy technique applied can depend on the tissue type to be evaluated (e.g., colon, prostate, kidney, bladder, lymph node, liver, bone marrow, blood cell, lung, breast, etc.), the size and type of the tumor (e.g., solid or suspended, blood or ascites), among other factors. Representative biopsy techniques include, but are not limited to, excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy, liquid biopsy and bone marrow biopsy. In some embodiments, a sample is obtained from an“excisional biopsy”, which refers to the removal of an entire tumor mass with a small margin of normal tissue surrounding it. An“incisional biopsy” refers to the removal of a wedge of tissue that includes a cross-sectional diameter of the tumor. In some embodiments, a sample is obtained from a“core needle biopsy” of the tumor mass, or a“fine-needle aspiration biopsy” which generally obtains a suspension of cells from within the tumor mass. Biopsy techniques are discussed, for example, in Harrison's Principles of Internal Medicine, Kasper, et al., eds., l6th ed., 2005, Chapter 70, and throughout Part V.

In some embodiments, the amount of a biomarker, described herein, in a first sample is increased or elevated as compared to amount in a second sample. In some embodiments, the amount of a biomarker, described herein, in a first sample is decreased or reduced as compared to amount in a second sample. In some embodiments, the second sample is a reference sample. Additional disclosure for determining the amount of a biomarker are described herein.

In some embodiments, a reference sample is a single sample or a combination of multiple samples from the same subject that are obtained at one or more different time points than when the test sample is obtained. For example, a reference sample may be obtained at an earlier time point from the same subject than when the test sample is obtained. Such reference sample may be useful if the reference sample is obtained during initial diagnosis of cancer and the test sample is later obtained when the cancer has progressed (e.g., becomes metastatic).

In some embodiments, a reference sample is a combination of multiple samples from one or more healthy subjects who are not the subject being tested. In some embodiments, a reference sample is a combination of multiple samples from one or more subjects with a disease or disorder (e.g., cancer) who are not the subject being tested. In some embodiments, a reference sample is pooled RNA samples from normal tissues or pooled plasma or serum samples from one or more subjects who are not the subject being tested. In some embodiments, a reference sample is pooled RNA samples from tumor tissues or pooled plasma or serum samples from one or more subjects with a disease or disorder (e.g., cancer) who are not the subject being tested. In some embodiments, elevated or increased expression refers to an overall increase of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level of a biomarker (e.g., protein or nucleic acid (e.g., gene or mRNA)), detected by standard art-known methods such as those described herein, as compared to a reference sample. In some embodiments, the elevated expression refers to the increase in expression level/amount of a biomarker in the sample wherein the increase is at least about any of l.5x, l.75x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 25x, 50x, 75x, or lOOx the expression level/amount of the respective biomarker in a reference sample. In some embodiments, elevated expression refers to an overall increase of greater than about 1.5-fold, about 1.75-fold, about 2- fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold as compared to a reference sample.

In some embodiments, reduced expression refers to an overall reduction of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or greater, in the level of biomarker (e.g., protein or nucleic acid (e.g., gene or mRNA)) in a sample, as compared to a reference sample. In certain embodiments, reduced expression refers to the decrease in amount of a biomarker in the sample, wherein the decrease is about 0.9x, about 0.8x, about 0.7x, about 0.6x, about 0.5x, about 0.4x, about 0.3x, about 0.2x, about O.lx, about 0.05x, or about O.Olx the amount of a biomarker in the reference sample.

Therapeutic Methods

Following detection of a biomarker described herein, a treatment is selected and a subject (e.g., a cancer patient) is treated with an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain. Compositions comprising an agent that specifically binds CD47 and inhibits the interaction with SIRPa, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, are useful in, inter alia, methods for treating or reducing progression of a variety of cancers in a subject. In some embodiments, the disclosure provides a method for treating or reducing or delaying progression of a tumor in a subject in need thereof. In some embodiments the disclosure provides a method for reducing or inhibiting tumor growth in a subject in need thereof. In some embodiments the method comprises obtaining a sample of the tumor; transforming the sample by contacting the sample with an antibody, or antigen-binding fragment thereof, that specifically binds to one or more biomarkers described herein, thereby creating one or more antibody-biomarker complexes; detecting the one or more antibody-biomarker complexes; quantitating the amount of the one or more antibody- biomarker complexes; scoring the tumor, and administering to the subject an effective amount of the agent if the tumor is scored as susceptible to the agent. In some embodiments, the tumor is scored as susceptible to the agent if the total amount of the one or more antibody-biomarker complexes meets or exceeds a predetermined value that indicates a likelihood that the agent will provide an anti-tumor effect.

The compositions described herein can be administered to a subject, e.g., a human subject, using a variety of methods that depend, in part, on the route of administration. The route can be, e.g., oral, sublingual, sublabial, buccal, rectal, vaginal, intraocular, intranasal, intraotic, inhalation, cutaneous, topical, systemic, transdermal, epidural, intracerebral, intracerebroventricular, intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection, intramuscular injection (IM), or intrathecal injection (IT). The injection can be in a bolus or a continuous infusion.

Administration can be achieved by, e.g., local infusion, injection, or by means of an implant. The implant can be of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. The implant can be configured for sustained or periodic release of the composition to the subject. See, e.g., U.S. Patent Application Publication No. 20080241223; U.S. Patent Nos. 5,501,856; 4,863,457; and 3,710,795; EP488401; and EP 430539, the disclosures of each of which are incorporated herein by reference in their entirety. The composition can be delivered to the subject by way of an implantable device based on, e.g., diffusive, erodible, or convective systems, e.g., osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmosis systems, vapor pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion-based systems, or electromechanical systems.

In some embodiments, an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, is therapeutically delivered to a subject by way of local administration.

A suitable dose of an agent described herein, which dose is capable of treating or preventing cancer in a subject, can depend on a variety of factors including, e.g., the age, sex, and weight of a subject to be treated and the particular inhibitor compound used. For example, a different dose of an anti-CD47 antibody may be required to treat a subject with cancer as compared to the dose of an Fc domain-containing polypeptide that specifically binds to CD47 required to treat the same subject. Other factors affecting the dose administered to the subject include, e.g., the type or severity of the cancer. For example, a subject having metastatic melanoma may require administration of a different dosage of an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, than a subject with glioblastoma. Other factors can include, e.g., other medical disorders concurrently or previously affecting the subject, the general health of the subject, the genetic disposition of the subject, diet, time of administration, rate of excretion, drug combination, and any other additional therapeutics that are administered to the subject. It should also be understood that a specific dosage and treatment regimen for any particular subject will also depend upon the judgment of the treating medical practitioner (e.g., doctor or nurse). Suitable dosages are described herein.

A pharmaceutical composition can include a therapeutically effective amount of an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein. Such effective amounts can be readily determined by one of ordinary skill in the art based, in part, on the effect of the administered antibody, or the combinatorial effect of the antibody and one or more additional active agents, if more than one agent is used. A therapeutically effective amount of an antibody or fragment thereof described herein can also vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody (and one or more additional active agents) to elicit a desired response in the individual, e.g., reduction in tumor growth. For example, a therapeutically effective amount of an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, can inhibit (lessen the severity of or eliminate the occurrence of) and/or prevent a particular disorder, and/or any one of the symptoms of the particular disorder known in the art or described herein. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.

Suitable human doses of any of the agents described herein can further be evaluated in, e.g., Phase I dose escalation studies. See, e.g., van Gurp et al. (2008) Am J Transplantation 8(8):1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part l):523-53l; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10): 3499-3500. In some embodiments, the composition contains any of the agents described herein and one or more (e.g., two, three, four, five, six, seven, eight, nine, 10, or 11 or more) additional therapeutic agents such that the composition as a whole is therapeutically effective. For example, a combination therapy may include administration of an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain as described herein, and a chemotherapeutic agent, wherein the agent and chemotherapeutic agent are each at a concentration that when combined are therapeutically effective for treating or preventing a cancer in a subject.

Toxicity and therapeutic efficacy of such compositions can be determined by known pharmaceutical procedures in cell cultures or experimental animals (e.g., animal models of any of the cancers described herein). These procedures can be used, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. An antibody or agent described herein that exhibits a high therapeutic index is preferred. While compositions that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue and to minimize potential damage to normal cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such agents described herein lies generally within a range of circulating concentrations of the agents that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the antibody which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. In some embodiments, e.g., where local administration (e.g., to the eye or a joint) is desired, cell culture or animal modeling can be used to determine a dose required to achieve a therapeutically effective concentration within the local site. In some embodiments, the methods can be performed in conjunction with other therapies for cancer. For example, the composition can be administered to a subject at the same time, prior to, or after, radiation, surgery, targeted or cytotoxic chemotherapy, chemoradiotherapy, hormone therapy, immunotherapy, gene therapy, cell transplant therapy, precision medicine, genome editing therapy, or other pharmacotherapy.

As described above, the compositions described herein (e.g., comprising an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain) can be used to treat a variety of cancers such as but not limited to: Kaposi’s sarcoma, leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblasts promyelocyte myelomonocytic monocytic erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt’s lymphoma and marginal zone B cell lymphoma, polycythemia vera, lymphoma, Hodgkin’s disease, non-Hodgkin’ s disease, multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, solid tumors, sarcomas, and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chrondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangio sarcoma, lymphangioendothelio sarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm’s tumor, cervical cancer, uterine cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, non- small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system (CNS) cancer, cervical cancer, choriocarcinoma, colorectal cancers, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial neoplasm, kidney cancer, larynx cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma; oral cavity cancer (for example lip, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; cancer of the respiratory system, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, cancer of the urinary system, lung cancer, lung adenocarcinoma, squamous cell lung cancer, ovarian cancer, glioblastoma, acute myeloid leukemia, mesothelioma, and sarcoma.

In some embodiments, the compositions described herein (e.g., comprising an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain) can be used to treat lung cancer. In some embodiments, the compositions described herein (e.g., comprising an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain) can be used to treat lung adenocarcinoma. In some embodiments, the compositions described herein (e.g., comprising an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain) can be used to treat squamous cell lung cancer. In some embodiments, the compositions described herein (e.g., comprising an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain) can be used to treat ovarian cancer. In some embodiments, the compositions described herein (e.g., comprising an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain) can be used to treat glioblastoma. In some embodiments, the compositions described herein (e.g., comprising an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain) can be used to treat acute myeloid leukemia. In some embodiments, the compositions described herein (e.g., comprising an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain) can be used to treat mesothelioma. In some embodiments, the compositions described herein (e.g., comprising an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain) can be used to treat sarcoma.

In some embodiments, an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein can be administered to a subject as a combination therapy with another treatment, e.g., another treatment for a cancer. For example, the combination therapy can include administering to the subject (e.g., a human patient) one or more additional agents that provide a therapeutic benefit to a subject who has, or is at risk of developing, cancer. Chemotherapeutic agents suitable for co-administration with compositions of the present invention include, for example: taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrancindione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Further agents include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioTEPA, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlordiamine platinum (II)(DDP), procarbazine, altretamine, cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, or triplatin tetranitrate), anthracycline (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomcin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine) and temozolomide. In some embodiments, an anti-CD47 antibody and the one or more additional active agents are administered at the same time. In other embodiments, the agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, is administered first in time and the one or more additional active agents are administered second in time. In some embodiments, the one or more additional active agents are administered first in time and the agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, is administered second in time.

An agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein can replace or augment a previously or currently administered therapy. For example, upon treating with the agent, administration of one or more additional active agents can cease or diminish, e.g., be administered at lower levels. In some embodiments, administration of the previous therapy can be maintained. In some embodiments, a previous therapy will be maintained until the level of the agent reaches a level sufficient to provide a therapeutic effect. The two therapies can be administered in combination.

Monitoring a subject (e.g., a human patient) for an improvement in a cancer, as defined herein, means evaluating the subject for a change in a disease parameter, e.g., a reduction in tumor growth or prevention of tumor re-growth. In some embodiments, the evaluation is performed at least one (1) hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after an administration. The subject can be evaluated in one or more of the following periods: prior to beginning of treatment; during the treatment; or after one or more elements of the treatment have been administered. Evaluation can include evaluating the need for further treatment, e.g., evaluating whether a dosage, frequency of administration, or duration of treatment should be altered. It can also include evaluating the need to add or drop a selected therapeutic modality, e.g., adding or dropping any of the treatments for a cancer described herein.

In some embodiments, the disclosure provides methods for treating or delaying progression of a cancer in a subject in need thereof, comprising administering to the subject an effective amount of an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, and wherein the Fc domain binds CD32a.

In some embodiments, the disclosure provides methods for inhibiting tumor growth in a subject in need thereof, comprising administering to the subject an effective amount of an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, and wherein the Fc domain binds CD32a.

In some embodiments, the methods described herein further comprise determining the amount of CD47, CD32b, CD32c, CDl6a, CDl6b, CD64, CD68, CD163, PD-l, PD-F1, and/or SIRPa in a tumor sample as described supra.

Agents That Specifically Bind Human CD47

The present disclosure provides agents that specifically bind CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain. CD47, also known as integrin- associated protein (IAP), ovarian cancer antigen OA3, Rh-related antigen and MER6, is a multi- spanning transmembrane receptor belonging to the immunoglobulin superfamily. CD47 expression and/or activity has been implicated in a number of diseases and disorders, e.g., cancer. CD47 interacts with SIRPa (signal regulatory protein alpha) on macrophages and thereby inhibits phagocytosis.

In some embodiments, an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein, is capable of inhibiting, interfering with or blocking the interaction between CD47 and its cognate SIRPa ligand, without causing significant, or detectable, hemagglutination of erythrocytes, e.g., human erythrocytes. In some embodiments, the agent causes less hemagglutination of human erythrocytes than a reference agent (e.g. a reference anti-CD47 antibody), or causes less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% or less hemagglutination of human erythrocytes relative to a reference agent. Exemplary reference anti-CD47 antibodies include, but are not limited to, B6H12, MABL, BRIC126, and CC2C6. In some embodiments, the agent causes substantially no hemagglutination of erythrocytes, e.g., human erythrocytes, for example, the antibody causes less than 50%, 40%, 30%, 20%, or 10% or less hemagglutination of human erythrocytes than a reference agent, such as B6H12, MABL, BRIC126, and CC2C6, when tested under the same or similar conditions.

In some embodiments, an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein, causes a potent blocking of the interaction between CD47 and SIRPa without causing a significant level of hemagglutination of erythrocytes. For example, the agent blocks at least 40%, 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 90%, 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 agent. Optionally, the agent also causes less hemagglutination of human erythrocytes than a reference agent (e.g. a reference anti-CD47 antibody), or causes less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% or less hemagglutination of human erythrocytes relative to a reference agent. Exemplary reference agents include B6H12, MABL, BRIC126, and CC2C6.

In some embodiments, an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein, does not induce phagocytosis red blood cells to a significant or detectable level. In some embodiments, the agent has reduced (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) phagocytic activity towards red blood cells relative to a reference agent.

In some embodiments, an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein, enhances macrophage activity. For example, the agent enhances the phagocytic activity of a macrophage, e.g., an unpolarized macrophage, or an Ml or M2 polarized macrophage. In some embodiments, the phagocytic activity is enhanced, e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, relative to a macrophage in the absence of the agent. In some embodiments, an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein, enhances macrophage phagocytic activity towards a cancer cell, e.g., an AML cell. In some embodiments, the phagocytic activity is enhanced, e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, relative to a macrophage in the absence of the agent.

In some embodiments, an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein, binds CD47 with a K_D of 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM or lower, as measured using standard binding assays, for example, surface plasmon resonance or bio-layer interferometry.

Agents that specifically bind CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, may be characterized relative to a reference agent that binds CD47, for example, an antibody that specifically binds to CD47 (e.g. B6H12, 2D3, MABL, CC2C6, or BRIC126). Antibody B6H12 is described, for example, in U.S. Patent Nos. 5,057,604 and 9,017,675, and is commercially available from Abeam, PLC, Santa Cruz Biotechnology, Inc., and eBioscience, Inc. Antibody MABL is described, for example, in Uno S, Kinoshita Y, Azuma Y et al. (2007 ) ONCOL. REP. 17: 1189-94, and Kikuchi Y, Uno S, Yoshimura Y el al. (2004) BIOCHEM. BIOPHYS. RES . COMMUN. 315: 912-8. Antibody CC2C6 is described, for example, in Martina Seiffert et al. ( 1997) BLOOD 94(11): 3633-3643, and is commercially available from Santa Cruz Biotechnology, Inc. Antibody BRIC126 is described, for example, in Avent et al. ( 1988) BIOCHEM. J. 251: 499-505. Antibody 2D3 is commercially available from eBioscience, Inc., and unlike the other reference antibodies, does not interfere with the binding between CD47 and SIRPa.

In some embodiments, the agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, comprises: (a) an immunoglobulin heavy chain variable region comprising the structure HCDR1-HCDR2-HCDR3 and (b) an immunoglobulin light chain variable region, wherein the heavy chain variable region and the light chain variable region together define a single binding site for binding CD47. In some embodiments, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 5; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 6; and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7. The HCDR1, HCDR2, and HCDR3 sequences are interposed between immunoglobulin FR sequences.

In some embodiments, the agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, comprises: (a) an immunoglobulin light chain variable region comprising the structure FCDR1-FCDR2-FCDR3, and (b) an immunoglobulin heavy chain variable region, wherein the light chain variable region and the heavy chain variable region together define a single binding site for binding CD47. In some embodiments, the FCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; the FCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 9; and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10. The LCDR1, LCDR2, and LCDR3 sequences are interposed between immunoglobulin FR sequences.

In some embodiments, the agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, comprises: (a) an immunoglobulin heavy chain variable region comprising the structure HCDR1-HCDR2-HCDR3 and (b) an immunoglobulin light chain variable region comprising the structure LCDR1-LCDR2-LCDR3, wherein the heavy chain variable region and the light chain variable region together define a single binding site for binding CD47. In some embodiments, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 5; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 6; and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7. The HCDR1, HCDR2, and HCDR3 sequences are interposed between immunoglobulin FR sequences. In some embodiments, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 9; and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10. The LCDR1, LCDR2, and LCDR3 sequences are interposed between immunoglobulin FR sequences. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 wild-type Fc domain or an IgG4 mutant Fc domain.

In some embodiments, the agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, comprises an immunoglobulin heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL). In some embodiments, the agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, comprises an immunoglobulin light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 4, and an immunoglobulin heavy chain variable region (VH). In certain embodiments, the agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, comprises an immunoglobulin heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the agent that specifically binds to CD47 comprises an antibody or polypeptide comprising a human IgG4 Fc domain or a mutant IgG4 Fc domain.

In certain embodiments, the agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, comprises an immunoglobulin heavy chain variable region (VH) comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 3 and comprises an immunoglobulin light chain variable region (VL) comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 4.

In certain embodiments, the agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, comprises the antibodies described in U.S. Patent No. 9,045,541, including, for example, the antibodies referred to as 2A1, 2Al-xi, AB6.12, AB6.l2-IgGl, AB6.l2-IgG4P and AB6.l2-IgG4PE. For example antibody AB6.12 comprises the variable heavy chain sequence of SEQ ID NO: 11 and the variable light chain sequence of SEQ ID NO: 42 as set forth in Table 1 of U.S. Patent No. 9,045,541 (incorporated herein by reference). In certain embodiments, the agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, comprises a chimeric or humanized version of the monoclonal antibody 5F9G4 described in Liu et alJ 2016) PLoS ONE l0(9):e0l37345. Chimeric and humanized versions of 5F9G4, such as humanized 5F9G4 (hu5F9G4)) are disclosed in US9017675, incorporated herein by reference. In some embodiments, the agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, comprises the variable heavy and light chain sequences of antibody CC-90002 (Celgene). In some embodiments, the agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, comprises the variable heavy and light chain sequences of antibody TI-061 (Tioma Therapeutics). In some embodiments, the agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, comprises the variable heavy and light chain sequences of antibody INBRX-103 (CC-90002) (Inhibrx LP).

In certain embodiments, it is contemplated that a heavy chain variable region sequence, for example, the VH sequence set forth in SEQ ID NO: 3, may be covalently linked to a variety of heavy chain constant region sequences known in the art. Similarly, it is contemplated that a light chain variable region sequence, for example, the VL set forth in SEQ ID NO: 4, may be covalently linked to a variety of light chain constant region sequences known in the art. Similarly, it is contemplated that the heavy chain variable region sequences and/or the light chain variable region sequences of the anti-CD47 antibodies described in U.S. Patent No. 9,045,54land Liu et al. (2016) supra may be linked to a variety of heavy constant region sequences and/or light chain constant region sequences known in the art.

In some embodiments, the agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, may have a heavy chain constant region chosen from, e.g., the heavy chain constant regions of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE; particularly, chosen from, e.g., the (e.g., human) heavy chain constant regions of IgGl, IgG2, IgG3, and IgG4. In another embodiment, the antibody has a light chain constant region chosen from, e.g., the (e.g., human) light chain constant regions of kappa or lambda. The constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, and/or complement function). In one embodiment the antibody has effector function and can fix complement. In other embodiments the antibody does not recruit effector cells or fix complement. In another embodiment, the antibody has reduced or no ability to bind an Fc receptor. For example, it is an isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.

In certain embodiments, the constant region of the heavy chain of the antibody is a human IgGl isotype, having an amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the human IgGl constant region is modified at amino acid Asn297 to prevent to glycosylation of the antibody, for example Asn297Ala (N297A). In certain embodiments, the constant region of the antibody is modified at amino acid Leu235 to alter Fc receptor interactions, for example Leu235Glu (L235E) or Leu235Ala (L235A). In certain embodiments, the constant region of the antibody is modified at amino acid Leu234 to alter Fc receptor interactions, e.g., Leu234Ala (L234A). In certain embodiments, the constant region of the antibody is modified at amino acid Glu233, e.g., Glu233Pro (E233P). In some embodiments, the constant region of the antibody is altered at both amino acid 234 and 235, for example Leu234Ala and Leu235Ala (L234A/L235A). In certain embodiments, the constant region of the antibody is altered at amino acids 233, 234, and 234, for example, Glu233Pro, Leu234Ala, and Leu235Ala (E233P L234A/L235A) (Armour KL. et al. ( 1999) Eur J Immunol 29(8):26l3-24). All residue numbers are according to EU numbering (Kabat, E.A., et al., supra).

In certain embodiments, the constant region of the heavy chain of the antibody is a human IgG2 isotype, having an amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the human IgG2 constant region is modified at amino acid Asn297 to prevent to glycosylation of the antibody, e.g., Asn297Ala (N297A), where the residue numbers are according to EU numbering (Kabat, E.A., et al, supra).

In certain embodiments, the constant region of the heavy chain of the antibody is an human IgG3 isotype, having an amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the human IgG3 constant region is modified at amino acid Asn297 to prevent to glycosylation of the antibody, e.g., Asn297Ala (N297A). In some embodiments, the human IgG3 constant region is modified at amino acid Arg435 to extend the half-life, e.g., Arg435H (R435H). All residue numbers are according to EU numbering (Kabat, E.A., et al., supra).

In certain embodiments, the constant region of the heavy chain of the antibody is an human IgG4 isotype, having an amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the human IgG4 constant region is modified within the hinge region to prevent or reduce strand exchange, e.g., in some embodiments human IgG4 constant region is modified at Ser228, e.g. , Ser228Pro (S228P). In other embodiments, the human IgG4 constant region is modified at amino acid Leu235 to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In some embodiments, the human IgG4 constant region is modified at both 8er228 and Leu335, e.g., Ser228Pro and Leii235Glu (S228P/L235E), and comprises the amino acid sequence of SEQ ID NO: 21, hereafter referred to as IgG4mt2. In some embodiments, the human IgG4 constant region is modified at amino acid Asn297 to prevent to glycosylation of the antibody, e.g., Asn297Aia (N297A). All residue numbers are according to EU numbering (Kabat, E.A., et al., supra). In certain embodiments, the constant region of the heavy chain of the antibody is a human IgM isotype.

In certain embodiments, the human IgG constant region is modified to enhance Fc receptor binding. Examples of Fc mutations that enhance binding to Fc receptors are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E, respectively) (Dall’Acqua el al. (2006) J. BIOL. CHEM. 281(33): 23514-23524), or Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et «1.(2010) N ATURE BIOTECH. 28(2): 157-159). All residue numbers are according to EU numbering (Kabat, E.A., et al., supra).

In some embodiments, 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 a/. (2008) CANCER RES . 68(10): 3863-72; Idusogie ei«Z.(2Q01) J. IMMUNOL. 166(4): 2571-5; Moore et a . (2010) MABS 2(2): 181-189; Lazar et al. (2006) PROC. NATL. ACAD. SCI. USA 103(11): 4005-4010, Shields et a/. (2001) J. BIOL. CHEM. 276(9): 6591-6604; Stavenhagen et a/.(2007) CANCER RES. 67(18): 8882-8890; Stavenhagen et a . (2008) ADVAN. ENZYME REGUL. 48: 152-164; Alegre et al.( 1992) J. IMMUNOL. 148: 3461-3468.

In some embodiments, the human IgG constant region is modified to induce heterodimerization. For example, a heavy chain having an amino acid modification within the CH3 domain at Thr366, e.g., a substitution with a more bulky amino acid, e.g., Try (T366W), is able to preferentially pair with a second heavy chain having a CH3 domain having amino acid modifications to less bulky amino acids at positions Thr366, Leu368, and Tyr407, e.g., Ser, Ala and Yal, respectively (T366S/L368A/Y407V). Heterodimerization via CH3 modifications can be further stabilized by the introduction of a disulfide bond, for example by changing Ser354 to Cys (S354C) and Y349 to Cys (Y349C) on opposite CH3 domains (see, Carter (2001 ) J. IMMUNOL. METHODS 248: 7-15)

Accordingly, in certain embodiments, the agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, comprises an immunoglobulin heavy chain comprising an amino acid sequence selected from SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, and an immunoglobulin light chain. In some embodiments, the agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, comprises an immunoglobulin light chain comprising an amino acid sequence of SEQ ID NO: 16, and an immunoglobulin heavy chain. In certain embodiments, the agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, comprises an immunoglobulin heavy chain comprising an amino acid sequence selected from SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 16.

Exemplary nucleotide sequences encoding agents that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, disclosed herein include the nucleotide sequence provided in SEQ ID NO: 25, encoding an immunoglobulin heavy chain comprising a heavy chain variable domain of the 2.3D11 antibody and a human IgGl heavy chain constant domain (corresponding to the amino acid sequence depicted in SEQ ID NO: 12), the nucleotide sequence depicted in SEQ ID NO: 26, encoding an immunoglobulin heavy chain comprising a heavy chain variable domain of the 2.3D11 antibody and a human IgG4 heavy chain constant domain (corresponding to the amino acid sequence depicted in SEQ ID NO: 13), the nucleotide sequence depicted in SEQ ID NO: 27, encoding an immunoglobulin heavy chain comprising a heavy chain variable domain of the 2.3D11 antibody and a human IgG4 heavy chain constant domain with Ser228Pro and Leu235Glu substitutions (corresponding to the amino acid sequence depicted in SEQ ID NO: 14), and the nucleotide sequence depicted in SEQ ID NO: 28, encoding an immunoglobulin light chain comprising a light chain variable domain of the 2.3D 11 antibody and a human kappa constant domain (corresponding to the amino acid sequence depicted in SEQ ID NO: 16).

In some embodiments, an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, binds to the same epitope present in CD47 as that bound by a disclosed antibody, e.g., the 2.3D 11 antibody comprising an immunoglobulin heavy chain variable region referred to herein as 2.3DH-V_H and an immunoglobulin light chain variable region referred to herein as 2.3DH-V_L. In some embodiments, the invention provides agents that compete for binding to CD47 with a disclosed antibody, e.g., the 2.3D 11 antibody comprising an immunoglobulin heavy chain variable region referred to herein as 2.3DH-V_H and an immunoglobulin light chain variable region referred to herein as 2.3D1 l-V_L.

Competition assays for determining whether an agent (e.g. an antibody) binds to the same epitope as, or competes for binding with a disclosed antibody, e.g., the 2.3D 11 antibody, are known in the art. Exemplary competition assays include immunoassays (e.g., ELISA assays, RIA assays), surface plasmon resonance, (e.g., BIAcore analysis), bio-layer interferometry, and flow cytometry. Typically, a competition assay involves the use of an antigen (e.g., a human CD47 protein or fragment thereof) bound to a solid surface or expressed on a cell surface, a test CD47-binding agent and a reference agent (e.g., the 2.3D11 antibody). In some embodiments, the reference agent is labeled and the test antibody is unlabeled. In some embodiments, the reference agent is unlabeled and the test antibody is labeled. Competitive inhibition is measured by determining the amount of labeled reference agent bound to the solid surface or cells in the presence of the test agent. Usually the test agent is present in excess (e.g., lx, 5x, lOx, 20x or lOOx). Agent identified by competition assay ( i.e ., competing antibodies) include agents binding to the same epitope, or similar (e.g., overlapping) epitopes, as the reference agent, and agents binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference agent for steric hindrance to occur.

A competition assay can be conducted in both directions to ensure that the presence of the label does not interfere or otherwise inhibit binding. For example, in the first direction the reference agent is labeled and the test agent is unlabeled, and in the second direction, the test agent is labeled and the reference agent is unlabeled.

A test agent competes with the reference agent for specific binding to the antigen if an excess of one antibody (e.g., lx, 5x, lOx, 20x or lOOx) inhibits binding of the other agent, e.g., by at least 50%, 75%, 90%, 95% or 99% as measured in a competitive binding assay.

Two agents may be determined to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one agent reduce or eliminate binding of the other. Two agents may be determined to bind to overlapping epitopes if only a subset of the amino acid mutations that reduce or eliminate binding of one agent reduce or eliminate binding of the other.

The agents disclosed herein may be further optimized (e.g., affinity-matured) to improve biochemical characteristics including affinity and/or specificity, improve biophysical properties including aggregation, stability, precipitation and/or non-specific interactions, and/or to reduce immunogenicity. Affinity-maturation procedures are within ordinary skill in the art. For example, diversity can be introduced into an immunoglobulin heavy chain and/or an immunoglobulin light chain by DNA shuffling, chain shuffling, CDR shuffling, random mutagenesis and/or site-specific mutagenesis.

In some embodiments, an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein contains one or more somatic mutations. In these cases, the agent can be modified to a human germline sequence to optimize the antibody ( i.e ., a process referred to as germlining).

Generally, an optimized agent has at least the same, or substantially the same, affinity for the antigen as the non-optimized (or parental) agent from which it was derived. Preferably, an optimized agent has a higher affinity for the antigen when compared to the parental agent.

An agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein can be conjugated to an effector moiety such as a small molecule toxin or a radionuclide using standard in vitro conjugation chemistries. If the effector moiety is a polypeptide, the agent can be chemically conjugated to the effector or joined to the effector as a fusion protein. Construction of fusion proteins is within ordinary skill in the art.

Methods of Making Agents That Specifically Bind CD47

The disclosure also features methods for producing any of the agents that specifically bind CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, disclosed herein. In some embodiments, methods for preparing an agent described herein can include immunizing a subject (e.g., a non-human mammal) with an appropriate immunogen. Suitable immunogens for generating any of the antibodies described herein are set forth herein. For example, to generate an antibody that binds to CD47, a skilled artisan can immunize a suitable subject (e.g., a nonhuman mammal such as a rat, a mouse, a gerbil, a hamster, a dog, a cat, a pig, a goat, a horse, or a non-human primate) with a CD47 polypeptide (e.g., the CD47 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1).

A suitable subject (e.g., a non-human mammal) can be immunized with the appropriate antigen along with subsequent booster immunizations a number of times sufficient to elicit the production of an antibody by the mammal. The immunogen can be administered to a subject (e.g., a non-human mammal) with an adjuvant. Adjuvants useful in producing an antibody in a subject include, but are not limited to, protein adjuvants; bacterial adjuvants, e.g., whole bacteria (BCG, Corynebacterium parvum or Salmonella Minnesota ) and bacterial components including cell wall skeleton, trehalose dimycolate, monophosphoryl lipid A, methanol extractable residue (MER) of tubercle bacillus, complete or incomplete Freund’s adjuvant; viral adjuvants; chemical adjuvants, e.g., aluminum hydroxide, and iodoacetate and cholesteryl hemisuccinate. Other adjuvants that can be used in the methods for inducing an immune response include, e.g., cholera toxin and parapoxvirus proteins. See also Bieg et al. (1999) Autoimmunity 31(1): 15-24. See also, e.g., Lodmell et al. (2000) Vaccine 18: 1059-1066; Johnson et al. (1999) J Med Chem 42:4640-4649; Baldridge et al. (1999) Methods 19: 103-107; and Gupta et al. (1995) Vaccine 13(14): 1263-1276.

In some embodiments, the methods include preparing a hybridoma cell line that secretes a monoclonal antibody that binds to the immunogen. For example, a suitable mammal such as a laboratory mouse is immunized with a CD47 polypeptide as described above. Antibody-producing cells (e.g., B cells of the spleen) of the immunized mammal can be isolated two to four days after at least one booster immunization of the immunogen and then grown briefly in culture before fusion with cells of a suitable myeloma cell line. The cells can be fused in the presence of a fusion promoter such as, e.g., vaccinia virus or polyethylene glycol. The hybrid cells obtained in the fusion are cloned, and cell clones secreting the desired antibodies are selected. For example, spleen cells of Balb/c mice immunized with a suitable immunogen can be fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2/0-Ag 14. After the fusion, the cells are expanded in suitable culture medium, which is supplemented with a selection medium, for example HAT medium, at regular intervals in order to prevent normal myeloma cells from overgrowing the desired hybridoma cells. The obtained hybrid cells are then screened for secretion of the desired agent (e.g., an antibody that binds to human CD47 blocks interaction with SIRPoc).

In some embodiments, a skilled artisan can identify an antibody of interest from a non- immune biased library as described in, e.g., U.S. patent no. 6,300,064 (to Knappik et al.; Morphosys AG) and Schoonbroodt et al. (2005) Nucleic Acids Res 33(9):e8 l.

In some embodiments, the methods described herein can involve, or be used in conjunction with, e.g., phage display technologies, bacterial display, yeast surface display, eukaryotic viral display, mammalian cell display, and cell-free (e.g., ribosomal display) antibody screening techniques (see, e.g., Etz et al. (2001) J Bacteriol 183:6924-6935; Cornells (2000) Curr Opin Biotechnol 11:450-454; Klemm et al. (2000) Microbiology 146:3025-3032; Kieke et al. (1997) Protein Eng 10: 1303-1310; Yeung et al. (2002) Biotechnol Prog 18:212-220; Boder et al. (2000) Methods Enzymology 328:430-444; Grabherr et al. (2001) Comb Chem High Throughput Screen 4: 185-192; Michael et al. (1995) Gene Ther 2:660-668; Pereboev et al. (2001) J Virol 75:7107- 7113; Schaffitzel et al. (1999) J Immunol Methods 231: 119-135; and Hanes et al. (2000) Nat Biotechnol 18: 1287-1292). Methods for identifying antibodies using various phage display methods are known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. Such phage can be utilized to display antigen-binding domains of antibodies, such as Fab, Fv, or disulfide-bond stabilized Fv antibody fragments, expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage used in these methods are typically filamentous phage such as fd and M13. The antigen binding domains are expressed as a recombinantly fused protein to any of the phage coat proteins pill, pVIII, or pIX. See, e.g., Shi et al. (2010) JMB 397:385-396. Examples of phage display methods that can be used to make the immunoglobulins, or fragments thereof, described herein include those disclosed in Brinkman et al. (1995) J Immunol Methods 182:41-50; Ames et al. (1995) J Immunol Methods 184: 177-186; Kettleborough et al. (1994) Eur J Immunol 24:952- 958; Persic et al. (1997) Gene 187:9-18; Burton et al. (1994) Advances in Immunology 57: 191- 280; and PCT publication nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, and WO 95/20401. Suitable methods are also described in, e.g., U.S. patent nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

In some embodiments, the phage display antibody libraries can be generated using mRNA collected from B cells from the immunized mammals. For example, a splenic cell sample comprising B cells can be isolated from mice immunized with a CD47 polypeptide as described above. mRNA can be isolated from the cells and converted to cDNA using standard molecular biology techniques. See, e.g., Sambrook et al. (1989)“Molecular Cloning: A Laboratory Manual, 2nd Edition,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane (1988), supra; Benny K. C. Lo (2004), supra; and Borrebaek (1995), supra. The cDNA coding for the variable regions of the heavy chain and light chain polypeptides of immunoglobulins are used to construct the phage display library. Methods for generating such a library are described in, e.g., Merz et al. (1995) J Neurosci Methods 62(l-2):2l3-9; Di Niro et al. (2005) Biochem J 388(Pt 3):889-894; and Engberg et al. (1995) Methods Mol Biol 51:355-376.

In some embodiments, a combination of selection and screening can be employed to identify an antibody of interest from, e.g., a population of hybridoma-derived antibodies or a phage display antibody library. Suitable methods are known in the art and are described in, e.g., Hoogenboom (1997) Trends in Biotechnology 15:62-70; Brinkman et al. (1995), supra; Ames et al. (1995), supra; Kettleborough et al. (1994), supra; Persic et al. (1997), supra; and Burton et al. (1994), supra. For example, a plurality of phagemid vectors, each encoding a fusion protein of a bacteriophage coat protein (e.g., pill, pVIII, or pIX of M13 phage) and a different antigen combining region are produced using standard molecular biology techniques and then introduced into a population of bacteria (e.g., E. coli). Expression of the bacteriophage in bacteria can, in some embodiments, require use of a helper phage. In some embodiments, no helper phage is required (see, e.g., Chasteen et al., (2006) Nucleic Acids Res 34(2l):el45). Phage produced from the bacteria are recovered and then contacted to, e.g., a target antigen bound to a solid support (immobilized). Phage may also be contacted to antigen in solution, and the complex is subsequently bound to a solid support.

A subpopulation of antibodies screened using the above methods can be characterized for their specificity and binding affinity for a particular antigen (e.g., human CD47) using any immunological or biochemical based method known in the art. For example, specific binding of an antibody to CD47 may be determined for example using immunological or biochemical based methods such as, but not limited to, an ELISA assay, SPR assays, immunoprecipitation assay, affinity chromatography, and equilibrium dialysis as described above. Immunoassays which can be used to analyze immunospecific binding and cross -reactivity of the antibodies include, but are not limited to, competitive and noncompetitive assay systems using techniques such as Western blots, RIA, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routine and well known in the art.

In embodiments where the selected CDR amino acid sequences are short sequences (e.g., fewer than 10-15 amino acids in length), nucleic acids encoding the CDRs can be chemically synthesized as described in, e.g., Shiraishi et al. (2007) Nucleic Acids Symposium Series 51(1): 129- 130 and U.S. Patent No. 6,995,259. For a given nucleic acid sequence encoding an acceptor antibody, the region of the nucleic acid sequence encoding the CDRs can be replaced with the chemically synthesized nucleic acids using standard molecular biology techniques. The 5’ and 3’ ends of the chemically synthesized nucleic acids can be synthesized to comprise sticky end restriction enzyme sites for use in cloning the nucleic acids into the nucleic acid encoding the variable region of the donor antibody. In some embodiments, an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein comprises an altered heavy chain constant region that has reduced (or no) effector function relative to its corresponding unaltered constant region. Effector functions involving the constant region of the agent may be modulated by altering properties of the constant or Fc region. Altered effector functions include, for example, a modulation in one or more of the following activities: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc-receptors, and proinflammatory responses. Modulation refers to an increase, decrease, or elimination of an effector function activity exhibited by a subject antibody containing an altered constant region as compared to the activity of the unaltered form of the constant region. In particular embodiments, modulation includes situations in which an activity is abolished or completely absent.

An altered constant region with altered FcR binding affinity and/or ADCC activity and/or altered CDC activity is a polypeptide which has either an enhanced or diminished FcR binding activity and/or ADCC activity and/or CDC activity compared to the unaltered form of the constant region. An altered constant region which displays increased binding to an FcR binds at least one FcR with greater affinity than the unaltered polypeptide. An altered constant region which displays decreased binding to an FcR binds at least one FcR with lower affinity than the unaltered form of the constant region. Such variants which display decreased binding to an FcR may possess little or no appreciable binding to an FcR, e.g., 0 to 50% (e.g., less than 50, 49, 48, 47, 46, 45, 44, 43,

42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,

16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of the binding to the FcR as compared to the level of binding of a native sequence immunoglobulin constant or Fc region to the FcR. Similarly, an altered constant region that displays modulated ADCC and/or CDC activity may exhibit either increased or reduced ADCC and/or CDC activity compared to the unaltered constant region. For example, in some embodiments, an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein comprising an altered constant region can exhibit approximately 0 to 50% (e.g., less than 50, 49, 48, 47, 46, 45,

44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19,

18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of the ADCC and/or CDC activity of the unaltered form of the constant region. An agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein comprising an altered constant region displaying reduced ADCC and/or CDC may exhibit reduced or no ADCC and/or CDC activity.

In some embodiments, an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein exhibits reduced or no effector function. In some embodiments, an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein comprises a hybrid constant region, or a portion thereof, such as a G2/G4 hybrid constant region (see e.g., Burton et al. (1992) Adv Immun 51: 1-18; Canfield et al. (1991) J Exp Med 173: 1483-1491; and Mueller et al. (1997) Mol Immunol 34(6):44l-452). See above.

In some embodiments, an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein may contain an altered constant region exhibiting enhanced or reduced complement dependent cytotoxicity (CDC). Modulated CDC activity may be achieved by introducing one or more amino acid substitutions, insertions, or deletions in an Fc region of the agent. See, e.g., U.S. patent no. 6,194,551. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric agent thus generated may have improved or reduced internalization capability and/or increased or decreased complement-mediated cell killing. See, e.g., Caron et al. (1992) J Exp Med 176: 1191-1195 and Shopes (1992) Immunol 148:2918-2922; PCT publication nos. WO 99/51642 and WO 94/29351; Duncan and Winter (1988) Nature 322:738-40; and U.S. Patent Nos. 5,648,260 and 5,624,821.

Any of the agents that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein can be screened and/or tested for their ability to modulate any of the activities or functions ascribed to either CD47, either in vitro or in vivo, using any immunological or biochemical-based methods known in the art.

Recombinant Polypeptide Expression and Purification

The agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein can be produced using a variety of techniques known in the art of molecular biology and protein chemistry. For example, a nucleic acid encoding one or both of the heavy and light chain polypeptides of an antibody can be inserted into an expression vector that contains transcriptional and translational regulatory sequences, which include, e.g., promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcription terminator signals, polyadenylation signals, and enhancer or activator sequences. The regulatory sequences include a promoter and transcriptional start and stop sequences. In addition, the expression vector can include more than one replication system such that it can be maintained in two different organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.

Several possible vector systems are available for the expression of cloned heavy chain and light chain polypeptides from nucleic acids in mammalian cells. One class of vectors relies upon the integration of the desired gene sequences into the host cell genome. Cells which have stably integrated DNA can be selected by simultaneously introducing drug resistance genes such as E. coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78:2072) or Tn5 neo (Southern and Berg (1982) Mol Appl Genet 1:327). The selectable marker gene can be either linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16:77). A second class of vectors utilizes DNA elements which confer autonomously replicating capabilities to an extrachromosomal plasmid. These vectors can be derived from animal viruses, such as bovine papillomavirus (Sarver et al. (1982) Proc Natl Acad Sci USA, 79:7147), cytomegalovirus, polyoma virus (Deans et al. (1984) Proc Natl Acad Sci USA 81: 1292), or SV40 virus (Lusky and Botchan (1981) Nature 293:79).

The expression vectors can be introduced into cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is largely dictated by the targeted cell type, discussed below. Exemplary methods include CaP04 precipitation, liposome fusion, cationic liposomes, electroporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

Appropriate host cells for the expression of antibodies or antigen-binding fragments thereof include yeast, bacteria, insect, plant, and mammalian cells. Of particular interest are bacteria such as E. coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as SF9, mammalian cell lines (e.g., human cell lines), as well as primary cell lines.

In some embodiments, an antibody or fragment thereof can be expressed in, and purified from, transgenic animals (e.g., transgenic mammals). For example, an antibody can be produced in transgenic non-human mammals (e.g., rodents) and isolated from milk as described in, e.g., Houdebine (2002) Curr Opin Biotechnol l3(6):625-629; van Kuik- Romeijn et al. (2000) Transgenic Res 9(2): 155-159; and Pollock et al. (1999) J Immunol Methods 231(1-2): 147-157.

The antibodies and fragments thereof can be produced from the cells by culturing a host cell transformed with the expression vector containing nucleic acid encoding the antibodies or fragments, under conditions, and for an amount of time, sufficient to allow expression of the proteins. Such conditions for protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation. For example, antibodies expressed in E. coli can be refolded from inclusion bodies (see, e.g., Hou et al. (1998) Cytokine 10:319-30). Bacterial expression systems and methods for their use are well known in the art (see Current Protocols in Molecular Biology, Wiley & Sons, and Molecular Cloning— A Laboratory Manual—3^rd Ed., Cold Spring Harbor Laboratory Press, New York (2001)). The choice of codons, suitable expression vectors and suitable host cells will vary depending on a number of factors, and may be easily optimized as needed. An antibody (or fragment thereof) described herein can be expressed in mammalian cells or in other expression systems including but not limited to yeast, baculovirus, and in vitro expression systems (see, e.g., Kaszubska et al. (2000) Protein Expression and Purification 18:213-220).

Following expression, the antibodies and fragments thereof can be isolated. An antibody or fragment thereof can be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography. For example, an antibody can be purified using a standard anti-antibody column (e.g., a protein-A or protein-G column). Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. See, e.g., Scopes (1994)“Protein Purification, 3rd edition,” Springer- Verlag, New York City, New York. The degree of purification necessary will vary depending on the desired use. In some instances, no purification of the expressed antibody or fragments thereof will be necessary.

Methods for determining the yield or purity of a purified antibody or fragment thereof are known in the art and include, e.g., Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, amido black protein assay, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoretic methods (e.g., using a protein stain such as Coomassie Blue or colloidal silver stain).

Modification of the Agents

The agents that specifically bind CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein can be modified following their expression and purification. The modifications can be covalent or noncovalent modifications. Such modifications can be introduced into the antibodies or fragments by, e.g., reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Suitable sites for modification can be chosen using any of a variety of criteria including, e.g., structural analysis or amino acid sequence analysis of the antibodies or fragments.

In some embodiments, the agents that specifically bind CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein can be conjugated to a heterologous moiety. The heterologous moiety can be, e.g., a heterologous polypeptide, a therapeutic agent (e.g., a toxin or a drug), or a detectable label such as, but not limited to, a radioactive label, an enzymatic label, a fluorescent label, a heavy metal label, a luminescent label, or an affinity tag such as biotin or streptavidin. Suitable heterologous polypeptides include, e.g., an antigenic tag (e.g., FLAG (DYKDDDDK (SEQ ID NO: 22)), polyhistidine (6-His; HHHHHH (SEQ ID NO: 23), hemagglutinin (HA; YPYDVPDYA (SEQ ID NO: 24)), glutathione- S- transferase (GST), or maltose-binding protein (MBP)) for use in purifying the antibodies or fragments. Heterologous polypeptides also include polypeptides (e.g., enzymes) that are useful as diagnostic or detectable markers, for example, luciferase, a fluorescent protein (e.g., green fluorescent protein (GFP)), or chloramphenicol acetyl transferase (CAT). Suitable radioactive labels include, e.g., 32P, 33P, 14C, 1251, 1311, 35S, and 3H. Suitable fluorescent labels include, without limitation, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLight™ 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700, Cy5, allophycocyanin, and Cy7. Luminescent labels include, e.g., any of a variety of luminescent lanthanide (e.g., europium or terbium) chelates. For example, suitable europium chelates include the europium chelate of diethylene triamine pentaacetic acid (DTPA) or tetraazacyclododecane- l,4,7,l0-tetraacetic acid (DOT A). Enzymatic labels include, e.g., alkaline phosphatase, CAT, luciferase, and horseradish peroxidase.

Two proteins (e.g., an antibody and a heterologous moiety) can be crosslinked using any of a number of known chemical cross linkers. Examples of such cross linkers are those which link two amino acid residues via a linkage that includes a“hindered” disulfide bond. In these linkages, a disulfide bond within the cross-linking unit is protected (by hindering groups on either side of the disulfide bond) from reduction by the action, for example, of reduced glutathione or the enzyme disulfide reductase. One suitable reagent, 4-succinimidyloxycarbonyl-a-methyl-a(2-pyridyldithio) toluene (SMPT), forms such a linkage between two proteins utilizing a terminal lysine on one of the proteins and a terminal cysteine on the other. Heterobifunctional reagents that cross-link by a different coupling moiety on each protein can also be used. Other useful cross-linkers include, without limitation, reagents which link two amino groups (e.g., N-5-azido-2- nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., l,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g., mmaleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an amino group and a guanidinium group that is present in the side chain of arginine (e.g., p-azidophenyl glyoxal monohydrate).

In some embodiments, a radioactive label can be directly conjugated to the amino acid backbone of the agent. Alternatively, the radioactive label can be included as part of a larger molecule (e.g., 1251 in meta-[l25I]iodophenyl-N-hydroxysuccinimide ([l25I]mIPNHS) which binds to free amino groups to form meta-iodophenyl (mIP) derivatives of relevant proteins (see, e.g., Rogers et al. (1997) J Nucl Med 38: 1221-1229) or chelate (e.g., to DOTA or DTPA) which is in turn bound to the protein backbone. Methods of conjugating the radioactive labels or larger molecules/chelates containing them to the antibodies or polypeptides comprising an Fc domain described herein are known in the art. Such methods involve incubating the proteins with the radioactive label under conditions (e.g., pH, salt concentration, and/or temperature) that facilitate binding of the radioactive label or chelate to the protein (see, e.g., U.S. Patent No. 6,001,329).

Methods for conjugating a fluorescent label (sometimes referred to as a“fluorophore”) to a protein (e.g., an antibody) are known in the art of protein chemistry. For example, fluorophores can be conjugated to free amino groups (e.g., of lysines) or sulfhydryl groups (e.g., cysteines) of proteins using succinimidyl (NHS) ester or tetrafluorophenyl (TFP) ester moieties attached to the fluorophores. In some embodiments, the fluorophores can be conjugated to a heterobifunctional cross-linker moiety such as sulfo-SMCC. Suitable conjugation methods involve incubating an antibody protein, or fragment thereof, with the fluorophore under conditions that facilitate binding of the fluorophore to the protein. See, e.g., Welch and Redvanly (2003) “Handbook of Radiopharmaceuticals: Radiochemistry and Applications,” John Wiley and Sons (ISBN 0471495603).

In some embodiments, the agents that specifically bind CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein can be modified e.g., with a moiety that improves the stabilization and/or retention of the agent in circulation, e.g., in blood, serum, or other tissues. For example, the agent can be PEGylated as described in, e.g., Lee et al. (1999) Bioconjug Chem 10(6): 973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews 54:459-476 or HESylated (Fresenius Kabi, Germany; see, e.g., Pavisic et al. (2010) Int J Pharm 387(1-2): 110- 119). The stabilization moiety can improve the stability, or retention of, the agent by at least about 1.5 (e.g., at about least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold.

In some embodiments, the agents that specifically bind CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein can be glycosylated. In some embodiments, the agent can be subjected to enzymatic or chemical treatment, or produced from a cell, such that the agent has reduced or absent glycosylation. Methods for producing antibodies with reduced glycosylation are known in the art and described in, e.g., U.S. patent no. 6,933,368; Wright et al. (1991) EMBO J 10(10):2717-2723; and Co et al. (1993) Mol Immunol 30:1361.

Pharmaceutical Compositions and Formulations

In certain embodiments, the disclosure provides for a pharmaceutical composition comprising an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein with a pharmaceutically acceptable diluent, carrier, solubilizer, emulisifier, preservative and/or adjuvant.

In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation material(s) are for s.c. and/or I.V. administration. In certain embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt- forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company (1995). In certain embodiments, the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mM NAOAC, pH 5.2, 9% Sucrose. In certain embodiments, the optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and/or rate of in vivo clearance of an anti-CD47 antibody.

In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In certain embodiments, the saline comprises isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute therefore. In certain embodiments, a composition comprising an anti-CD47 antibody can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, in certain embodiments, a composition comprising an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein can be formulated as a lyophilizate using appropriate excipients such as sucrose.

In certain embodiments, the pharmaceutical composition can be selected for parenteral delivery. In certain embodiments, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art.

In certain embodiments, the formulation components are present in concentrations that are acceptable to the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.

In certain embodiments, when parenteral administration is contemplated, a therapeutic composition can be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein, in a pharmaceutically acceptable vehicle. In certain embodiments, a vehicle for parenteral injection is sterile distilled water in which an agent described herein is formulated as a sterile, isotonic solution, and properly preserved. In certain embodiments, the preparation can involve the formulation of the desired molecule with an additional substance, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection. In certain embodiments, hyaluronic acid can also be used, and can have the effect of promoting sustained duration in the circulation. In certain embodiments, implantable drug delivery devices can be used to introduce the desired molecule.

In certain embodiments, a pharmaceutical composition can be formulated for inhalation. In certain embodiments, an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein can be formulated as a dry powder for inhalation. In certain embodiments, an inhalation solution comprising an agent described herein can be formulated with a propellant for aerosol delivery. In certain embodiments, solutions can be nebulized. Pulmonary administration is further described in PCT application No. PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins.

In certain embodiments, it is contemplated that formulations can be administered orally. In certain embodiments, an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein that is administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. In certain embodiments, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. In certain embodiments, at least one additional agent can be included to facilitate absorption of an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein. In certain embodiments, diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.

In certain embodiments, a pharmaceutical composition can involve an effective quantity of an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein in a mixture with non-toxic excipients which are suitable for the manufacture of tablets. In certain embodiments, by dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit-dose form. In certain embodiments, suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilled in the art, including formulations involving an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein in sustained- or controlled delivery formulations. In certain embodiments, techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See for example, PCT Application No. PCT/US93/00829 which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. In certain embodiments, sustained-release preparations can include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP 058,481), copolymers of L- glutamic acid and gamma ethyl-L-glutamate (Sidman et ah, Biopolymers, 22:547-556 (1983)), poly (2-hydroxyethyl-methacrylate) (Langer et ah, J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem. Tech., 12:98- 105 (1982)), ethylene vinyl acetate (Langer et ah, supra) or poly- D(-)-3-hydroxybutyric acid (EP 133,988). In certain embodiments, sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Eppstein et al, Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.

The pharmaceutical composition to be used for in vivo administration typically is sterile. In certain embodiments, this can be accomplished by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization using this method can be conducted either prior to or following lyophilization and reconstitution. In certain embodiments, the composition for parenteral administration can be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

In certain embodiments, once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations can be stored either in a ready-to- use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

In certain embodiments, kits are provided for producing a single-dose administration unit. In certain embodiments, the kit can contain both a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments, kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes) are included. In certain embodiments, the effective amount of a pharmaceutical composition comprising an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, according to certain embodiments, will thus vary depending, in part, upon the molecule delivered, the indication for which an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient. In certain embodiments, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.

In certain embodiments, the frequency of dosing will take into account the pharmacokinetic parameters of an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein in the formulation used. In certain embodiments, a clinician will administer the composition until a dosage is reached that achieves the desired effect. In certain embodiments, the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. In certain embodiments, appropriate dosages can be ascertained through use of appropriate dose-response data.

In certain embodiments, the route of administration of the pharmaceutical composition is in accord with known methods, e.g. orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, subcutaneously, intra ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices. In certain embodiments, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device. In certain embodiments, individual elements of the combination therapy may be administered by different routes.

In certain embodiments, the composition can be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated. In certain embodiments, where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration. In certain embodiments, it can be desirable to use a pharmaceutical composition comprising an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein in an ex vivo manner. In such instances, cells, tissues and/or organs that have been removed from the patient are exposed to a pharmaceutical composition comprising an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein after which the cells, tissues and/or organs are subsequently implanted back into the patient.

In certain embodiments, an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptides. In certain embodiments, such cells can be animal or human cells, and can be autologous, heterologous, or xenogeneic. In certain embodiments, the cells can be immortalized. In certain embodiments, in order to decrease the chance of an immunological response, the cells can be encapsulated to avoid infiltration of surrounding tissues. In certain embodiments, the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.

Kits

The disclosure also provides kits comprising a container with one or more reagents for detecting the presence of CD32a, optionally CD47 and/or CD32b in a tumor sample, and a package insert comprising instructions for determining an amount or expression level of CD32a, optionally CD47 and/or CD32b in the tumor sample. In some aspects, the kit comprises a reagent for detecting the presence of CD47 in the sample and instructions for determining an amount or expression level of CD47 in the tumor sample. In some embodiments, the reagent for detecting the presence of CD47 in the sample is a diagnostic antibody or a nucleic acid probe. In some aspects, the kit comprises a reagent for detecting the presence of CD32a in the sample and instructions for determining an amount or expression level of CD32a in the tumor sample. In some embodiments, the reagent for detecting the presence of CD32a in the sample is a diagnostic antibody or a nucleic acid probe. In some aspects, the kit comprises a reagent for detecting the presence of CD32b in the sample and instructions for determining an amount or expression level of CD32b in the tumor sample. In some embodiments, the reagent for detecting the presence of CD32b in the sample is a diagnostic antibody or a nucleic acid probe. In other aspects, the kit comprises a reagent for detecting the presence of one or more polypeptides in the sample selected from the group consisting of CDl6a, CDl6b, CD32a, CD32b, CD32c, CD47, CD64, CD68, CD 163, SIRPa, or a combination thereof, and instructions for determining an amount or expression level of the one or more polypeptides in the tumor sample. In some embodiments, the reagent for detecting the presence of CDl6a, CDl6b, CD32a, CD32b, CD32c, CD47, CD64, CD68, CD163, or SIRPa in the sample is a diagnostic antibody or a nucleic acid probe.

In some aspects, the kits may comprise, in a suitable container, an agent that specifically binds CD32a, and, optionally, CDl6a, CDl6b, CD32b, CD32c, CD47, CD64, CD68, CD163, SIRPa, or a combination thereof, wherein the agent comprises an antibody or a nucleic acid probe, one or more controls, and various buffers, reagents, enzymes and other standard ingredients well known in the art. In some embodiments, a kit with an agent that specifically binds CD32a, wherein the agent comprises an antibody or a nucleic acid probe, includes instructions for determining an amount or expression level of the one or more polypeptides in the tumor sample. In other aspects, the kit comprises an anti-tumor agent as described herein and instructions for use to treat or delay progression of cancer in a subject following diagnosis. In some embodiments, a kit with an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, or a nucleic acid probe, described herein includes instructions for detecting expression of CD32a.

In some aspects, a kit includes a composition comprising an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein, and an optional pharmaceutically acceptable carrier, for use in treating or delaying progression of cancer in a subject comprising CD32a expressing malignant cells, wherein the treatment comprises administration of the agent. In some aspects, a kit described herein includes instructions for detecting expression of CD32a, diagnosing a patient and selecting a treatment. In some aspects, the disclosure provides a kit comprising a container which includes at least one vial, well, test tube, flask, bottle, syringe, or other container means, into which an agent that specifically binds CD32a, and, optionally, CDl6a, CDl6b, CD32b, CD32c, CD47, CD64, CD68, CD163, SIRPa, or a combination thereof, described herein may be placed, and in some instances, suitably aliquoted. Where an additional component is provided, the kit can contain additional containers into which this component may be placed. The kits can also include a means for containing an anti-tumor agent as described herein and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blowmolded plastic containers into which the desired vials are retained. Containers and/or kits can include labeling with instructions for use and/or warnings.

In some aspects, the disclosure provides a kit comprising a medicament comprising a composition comprising an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein, and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for detection of CD32a expressing malignant cells, and, optionally, CDl6a, CDl6b, CD32b, CD32c, CD47, CD64, CD68, CD163, SIRPa, or a combination thereof, and administration of the medicament, for treating or delaying progression of cancer in a subject.

In some aspects, the disclosure provides a kit comprising a container comprising a composition comprising an agent that specifically binds CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, described herein, and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for detection of CD32a expressing malignant cells and, optionally, CDl6a, CDl6b, CD32b, CD32c, CD47, CD64, CD68, CD163, SIRPa, or a combination thereof, and administration of the composition, for treating or delaying progression of cancer in a subject.

In some embodiments, the kits described herein comprise an assay for detecting CD32a expression on malignant cells.

Definitions

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

As used herein,“about” will be understood by persons of ordinary skill and will vary to some extent depending on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill given the context in which it is used,“about” will mean up to plus or minus 10% of the particular value.

As used herein, the term "agent" refers to a chemical compound, a small molecule, or a biological macromolecule (such as a nucleic acid, an antibody, an antibody fragment, a protein, a peptide, or a polypeptide). Agents may be identified as having a particular activity by screening assays described herein. The activity of such agents may render them suitable as a "therapeutic agent" which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject and thus may serve as a therapeutic intervention for a disease or condition. As used herein, the term "agent" is not limited to therapeutic agents and encompasses agents useful in other applications, including diagnostics, agriculture, research and manufacturing.

The term“ameliorating” refers to any therapeutically beneficial result in the treatment of a disease state, e.g., cancer, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.

As used herein, the term“amount” or“level” refers to a detectable quantity, level or abundance of a substance (e.g., a protein or a biomarker). When referring to a biomarker, such as those described herein, the terms“level of expression” or“expression level” in general are used interchangeably and generally refer to a detectable amount of a biomarker in a biological sample. In some aspects, a detectable amount or detectable level of a biomarker is associated with a likelihood of a response to an agent, such as those described herein.

As used herein, an“amino acid substitution” refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (an amino acid sequence of a starting polypeptide) with a second, different“replacement” amino acid residue. An“amino acid insertion” refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, larger“peptide insertions,” can also be made, e.g. insertion of about three to about five or even up to about ten, fifteen, or twenty amino acid residues. The inserted residue(s) may be naturally occurring or non- naturally occurring as disclosed above. An“amino acid deletion” refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

The polypeptides suitable for use in the methods disclosed herein may comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non- essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in a binding polypeptide is preferably replaced with another amino acid residue from the same side chain family. In certain embodiments, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members. Alternatively, in certain embodiments, mutations may be introduced randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resultant mutants can be incorporated into binding polypeptides and/or molecules of the invention and screened for their ability to bind to the desired target.

As used herein, the term “antibody” (Ab), which is synonymous with the term “immunoglobulin” (Ig), means a tetramer comprising two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa) inter-connected by disulfide bonds. There are two types of light chain: l and K. In humans they are similar, but only one type is present in each antibody. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgEl respectively. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Each heavy chain (herein sometimes referred to as H-chain or He) is comprised of a heavy chain variable domain (VH, or H-variable domain) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain (herein sometimes referred to as L-chain or Lc) is comprised of a light chain variable domain (VL, or L-variable domain) and a light chain constant region. The light chain constant region is comprised of one domain, CL. Within light and heavy chains, the variable and constant regions are joined by a“J” region of about 12 or more amino acids, with the heavy chain also including a“D” region of about 3 or more amino acids. The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (CDR), interspersed with regions that are more conserved, termed“framework regions” (FR). Each VH and VL is composed of three CDRs (H-CDR herein designates a CDR from the heavy chain; and L-CDR herein designates a CDR from the light chain) and four FRs, arranged from amino-terminus to carboxyl- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acids to each domain is in accordance with the definitions of Rabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)), or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et ah, Nature 342:878-883 (1989).

As used herein, the term“affinity” means a measure of the attraction between an antigen and an antibody.

As used herein, the term“antibody- antigen complex” or“immune complex” or“complex” refers to a molecular structure formed upon the specific binding of an antibody to an antigen (e.g., a biomarker). The bound antibody and antigen act as a unitary object. In some embodiments, a first antibody is bound to an antigen, thereby forming a complex. In some embodiments, the complex is detected by the binding of a second antibody to the first antibody, thereby indirectly detecting the antigen.

As used herein, the term“antigen-binding fragment" refers to one or more fragment of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Non limiting examples of binding fragments encompassed within the term“antigen-binding fragment” include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment which is obtained by cleaving a disulfide bond of the hinge region of the F(ab')2; (iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (vi) a dAb fragment (Ward et ah, (1989) Nature 341:544-546), which consists of a VH domain; (vii) an isolated complementarity determining region (CDR); and (viii) a dsFv, which consists of a VH::VL heterodimer stabilized by a disulfide bond. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)); see e.g., Bird et al. Science 242:423-426 (1988) and Huston et al. Proc. Natl. Acad. Sci. USA 85:5879- 5883 (1988)). Also within the scope of this disclosure are antigen-binding molecules comprising a VN and/or a VL, In the case of a VH, the molecule may also comprise one or more of a CH 1, hinge, CH2 or CH3 region. Such single chain antibodies are also intended to be encompassed within the term“antigen-binding fragment” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al. Structure 2: 1121- 1123 (1994)).

As used herein, the term“antibody fragment” also includes, e.g., single domain antibodies such as camelized single domain antibodies. See, e.g., Muyldermans et al. (2001) Trends Biochem Sci 26:230-235; Nuttall et al. (2000) Curr Pharm Biotech 1:253-263; Reichmann et al. (1999) J Immunol Meth 231:25-38; PCT application publication nos. WO94/04678 and WO 94/25591; and U.S. patent no. 6,005,079, all of which are incorporated herein by reference in their entireties. In some embodiments, the disclosure provides single domain antibodies comprising two VH domains with modifications such that single domain antibodies are formed.

As used herein, the term“apoptosis” refers to a process of programmed cell death characterized by distinct morphological characteristics. The two main pathways of apoptosis are the intrinsic apoptotic pathway and the extrinsic apoptotic pathway. A third apoptotic pathway mediated by cytotoxic T cells is the perforin/granzyme apoptotic pathway. Each pathway requires specific triggering signals to begin an energy-dependent cascade of molecular events that culminate in a programmed cell death phenotype. A hallmark characteristic of cells undergoing apoptosis is exposure of phosphatidyl serine (PS) on the outer membrane, which can be detected by staining cells with fluorescently labeled annexin V. Exposure of PS precedes the total loss of membrane integrity that occurs after all forms of cell death (post- apoptotic or necrotic) that leads to permeability to various dyes that distinguish between live and dead cells, such as propidium iodide (PI). Hence, a distinction can be made between cells undergoing apoptosis (Annexin V+PI- ) and cells that are post-apoptotic or necrotic (Annexin V+PI+). Each apoptosis pathway activates its own initiator caspase which in turn will activate the executioner caspase-3. The convergent execution pathway results in characteristic cytomorphological features including cell shrinkage, chromatin condensation, formation of cytoplasmic blebs and apoptotic bodies and finally phagocytosis of the apoptotic bodies by adjacent parenchymal cells, neoplastic cells or macrophages. Additional modes of cell death include autophagy or caspase-independent cell death (CICD) which may involve other proteases such as serine proteases, cathepsins or calpains (Elmore (2007) Toxicol Pathol 35(4):495-5l6) Tait (2008) Oncogene (27), 6452-61.

As used herein, the term“biomarker” refers to a characteristic (e.g., biological marker) that is measured as an indicator of normal biological processes, pathogenic processes, or responses to an exposure or intervention, including therapeutic interventions. Molecular, histologic, radiographic, or physiologic characteristics are types of biomarkers. A biomarker is not an assessment of how an individual feels, functions, or survives. Categories of biomarkers include: susceptibility/risk biomarker, diagnostic biomarker, monitoring biomarker, prognostic biomarker, predictive biomarker, pharmacodynamic/response biomarker, and safety biomarker.

As used herein, the term“bispecific” or“bifunctional antibody” refers to an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, (1990) Clin. Exp. Immunol. 79:315- 321; Kostelny et ah, (1992) J. Immunol. 148: 1547-1553.

Traditionally, the recombinant production of bispecific antibodies is based on the co expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chain/light-chain pairs have different specificities (Milstein and Cuello, (1983) Nature 305:537- 539). Antibody variable domains with the desired binding specificities (antibody- antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion of the heavy chain variable region is preferably with an immunoglobulin heavy-chain constant domain, including at least part of the hinge, CH2, and CH3 regions. For further details of illustrative currently known methods for generating bispecific antibodies see, e.g., Suresh et ah, (1986) Methods Enzymol. 121:210; PCT Publication No. WO 96/27011; Brennan et ah, (1985) Science 229:81; Shalaby et ah, J. Exp. Med. (1992) 175:217-225; Kostelny et ah, (1992) J. Immunol. 148(5): 1547-1553; Hollinger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Gruber et al., (1994) J. Immunol. 152:5368; and Tutt et al., (1991) J. Immunol. 147:60. Bispecific antibodies also include cross-linked or heteroconjugate antibodies. Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. See, e.g., Kostelny et al. (1992) J Immunol 148(5): 1547-1553. The leucine zipper peptides from the Fos and Jun proteins may be linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers may be reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al. (1993) Proc Natl Acad Sci USA 90:6444-6448 has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (scFv) dimers has also been reported. See, e.g., Gruber et al. (1994) J Immunol 152:5368. Alternatively, the antibodies can be “linear antibodies” as described in, e.g., Zapata et al. (1995) Protein Eng. 8( 10): 1057- 1062. Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

Antibodies with more than two valencies (e.g., trispecific antibodies) are contemplated and described in, e.g., Tutt et al. (1991) J Immunol 147:60.

The disclosure also embraces variant forms of multi- specific antibodies such as the dual variable domain immunoglobulin (DVD-Ig) molecules described in Wu et al. (2007) Nat Biotechnol 25(11): 1290-1297. The DVD-Ig molecules are designed such that two different light chain variable domains (VL) from two different parent antibodies are linked in tandem directly or via a short linker by recombinant DNA techniques, followed by the light chain constant domain. Similarly, the heavy chain comprises two different heavy chain variable domains (VH) linked in tandem, followed by the constant domain CH1 and Fc region. Methods for making DVD-Ig molecules from two parent antibodies are further described in, e.g., PCT Publication Nos. WO 08/024188 and WO 07/024715. In some embodiments, the bispecific antibody is a Fabs-in- Tandem immunoglobulin, in which the light chain variable region with a second specificity is fused to the heavy chain variable region of a whole antibody. Such antibodies are described in, e.g., International Patent Application Publication No. WO 2015/103072.

As used herein, the term“cancer” means cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth.

As used herein, the term“CDR” means a complementarity-determining region.

As used herein, the term“chimeric antibody” means a genetically engineered fusion of parts of an animal antibody, typically a mouse antibody, with parts of a human antibody. Chimeric antibodies are developed to reduce the human anti-animal antibody response elicited by animal antibodies, as they combine the specificity of the animal antibody with the efficient human immune system interaction of a human antibody.

As used herein, the term “chemotherapeutic agent” (alternatively “cytotoxic chemotherapeutic agent”) refers to a chemical or pharmacological agent that is known to be of use in the treatment of cancer. Furthermore, as used herein, the term connotes those pharmacological agents that are generally cytotoxic, non-specific intracellular poisons, especially those that function to inhibit the process of cell division known as mitosis, and excludes pharmacological agents that more selectively target cellular components known to cause or contribute to the formation, development and/or maintenance of cancer. Chemotherapeutic agents can induce one or more cell death modalities including immunogenic cell death.

As used herein, the term“chimeric antibody” means a genetically engineered fusion of parts of an animal antibody, typically a mouse antibody, with parts of a human antibody. Chimeric antibodies are developed to reduce a human anti-animal antibody response.

Throughout this specification and claims, the word“comprise,” or variations such as “comprises” or“comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Further, as used herein, the term“includes” means includes without limitation.

As used herein, the term“cross -reacts” refers to the ability of an antibody of the disclosure to bind to an antigen from a different species. For example, an antibody of the present disclosure which binds human CD47 may also bind another species of CD47. As used herein, cross-reactivity is measured by detecting a specific reactivity with purified antigen in binding assays (e.g., SPR, ELISA) or binding to, or otherwise functionally interacting with, cells physiologically expressing CD277. Methods for determining crossreactivity include standard binding assays as described herein, for example, by BiacoreTM surface plasmon resonance (SPR) analysis using a BiacoreTM 2000 SPR instrument (Biacore AB, Uppsala, Sweden), or flow cytometric techniques.

As used herein, the term“diagnostic antibody” denotes an antibody used for the detection and visualization of its target antigen (e.g., a biomarker). In some embodiments, diagnostic antibody is used e.g. in assay systems (e.g. ELISA), or for imaging techniques (e.g., IHC). A diagnostic antibody may e.g. be a labeled therapeutic antibody.

As used herein, the term“diagnostic biomarker” refers to a biomarker used to detect or confirm presence of a disease or condition of interest or to identify individuals with a subtype of the disease.

As used herein the terms "diagnose" or "diagnosis" or "diagnosing" refer to distinguishing or identifying a disease, syndrome or condition or distinguishing or identifying a person having a particular disease, syndrome or condition.

As used herein, the term“extent of binding” refers to the level of binding of an antibody (e.g., a diagnostic antibody) to an antigen (e.g., a biomarker) present in a sample. The extent of antibody binding to an antigen can be determined by any of the methods known in the art for determining binding levels of antibodies such as ELISA, Western Blotting, or FACS. The extent of binding may be determined using any detection system such as secondary immunoglobulins or fragments thereof linked to a detectable marker. Exemplary detectable markers include, without limitation radioactive groups, fluorescent or chromogenic molecules, an enzyme capable of catalyzing a reaction yielding a detectable product (such as a color reaction), and a biotin group capable of being detected by avidin.

As used herein, the term“effective amount” of an agent is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an“effective amount” depends upon the context in which it is being applied. For example, in the context of administering an agent that treats cancer, an effective amount of an agent is, for example, an amount sufficient to achieve treatment or delay progression of cancer, as compared to the response obtained without administration of the agent. In some embodiments, a therapeutically effective amount is an amount of an agent to be delivered (e.g., a monoclonal antibody) that is sufficient, when administered to a subject with cancer, to treat, improve symptoms of, prevent, and/or delay progression of disease and/or condition.

As used herein, the term“epitope” or“antigenic determinant” refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody ( i.e . , epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from the extracellular domain of the target of interest (e.g., CD47) are tested for reactivity with the given antibody. Methods of determining spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

Also encompassed by the present disclosure are antibodies that bind the same epitope and/or antibodies that compete for binding to a target of interest (e.g., human CD47) with the antibodies described herein. Antibodies that recognize the same epitope or compete for binding can be identified using routine techniques. Such techniques include, for example, an immunoassay, which shows the ability of one antibody to block the binding of another antibody to a target antigen, i.e., a competitive binding assay. Competitive binding is determined in an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common target antigen, such as CD47. Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct label RIA using 1-125 label (see Morel et al., Mol. Immunol. 25(l):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess. Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least about 50-55%, 55-60%, 60-65%, 65-70% 70-75% or more.

Other techniques include, for example, epitope mapping methods, such as, xray analyses of crystals of antigen: antibody complexes which provides atomic resolution of the epitope and mass spectrometry combined with hydrogen/deuterium (H/D) exchange which studies the conformation and dynamics of antigen: antibody interactions. Other methods monitor the binding of the antibody to antigen fragments or mutated variations of the antigen where loss of binding due to a modification of an amino acid residue within the antigen sequence is often considered an indication of an epitope component. In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. The peptides are then regarded as leads for the definition of the epitope corresponding to the antibody used to screen the peptide library. For epitope mapping, computational algorithms have also been developed which have been shown to map conformational discontinuous epitopes.

As used herein, the term“expression” generally refers to the process by which information contained within a gene is converted into the structures (e.g., a protein biomarker) present and operating in the cell. Therefore, as used herein,“expression” may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide). Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide) shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide, e.g., by proteolysis.“Expressed genes” include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs).“Elevated expression,”“elevated expression levels,” or“elevated levels” refers to an increased expression or increased levels of a substance within a sample relative to a control sample, such as an individual or individuals who are not suffering from the disease or disorder (e.g., cancer) or an internal control. In some embodiments, the elevated expression of a substance (e.g., a protein or a biomarker) in a sample refers to an increase in the amount of the substance of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the amount of the substance in a control sample, as determined by techniques known in the art (e.g., IHC).“Reduced expression,”“reduced expression levels,” or“reduced levels” refers to a decrease expression or decreased levels of a biomarker in an individual relative to a control, such as an individual or individuals who are not suffering from the disease or disorder (e.g., cancer) or an internal control. In some embodiments, reduced expression is little or no expression. In some embodiments, the reduced expression of a substance (e.g., a protein or a biomarker) in a sample refers to a decrease in the amount of the substance of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the amount of the substance in a control sample, as determined by techniques known in the art (e.g, IHC).

As used herein, the term“Fc domain” or“Fragment crystallizable region” or“Fc region” or“Fc” refers to a portion of an antibody constant region. Traditionally, the term Fc domain refers to a protease (e.g., papain) cleavage product encompassing the paired CH2, CH3 and hinge regions of an antibody. The Fc domain of antibody determines the antibody’s class effect. Classes of heavy chains in antibodies include alpha, gamma, delta epsilon, and mu, which define the antibody’s isotypes IgA, IgG, IgD, IgE, and IgM. In the context of this disclosure, the term Fc domain refers to any polypeptide (or nucleic acid encoding such a polypeptide), regardless of the means of production, that includes all or a portion of the CH2, CH3 and hinge regions of an immunoglobulin polypeptide. Fc domains bind to various cell receptors (e.g., Fc receptors) and complement proteins, which in turn mediate different physiological effects of antibodies including, without limitation: induction of phagocytosis, detection of opsonized particles, cell lysis, degranulation of immune cells, cytokine production and release, and cell activation.

As used herein, the term“Fc gamma receptor” (FcyR) refers to any one member of a family of immunoreceptors that recognize and bind to the Fc domain of IgG antibodies. Each IgG isotype has a different binding profile to the various FcyRs, and each FcyR has a different cellular expression pattern. These differences enable the breadth, flexibility and control of function required to mount an effective and regulated humoral response. Fey receptors (FcyR) belong to the immunoglobulin superfamily and are important for inducing phagocytosis of opsonized (marked) cells. This family includes several members, FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIB (CD32c), FcyRIIIA (CDl6a), FcyRIIIB (CDl6b), which differ in their affinity for Fc domains. Furthermore, Fey receptors must bind IgG molecules within an immune complex to be activated, making the level of expression of these receptors an important determinant in the phagocytic ability of phagocytes (e.g. macrophages), which underscores their suitability as biomarkers (e.g. to predict or determine the effect of therapeutic agents that comprise an Fc domain). Further description of Fc gamma receptors can be found in Stewart et ah, (2014) Journal for ImmunoTherapy of Cancer 2:29, which is incorporated herein by reference it its entirety.

Accordingly, in some aspects, a biomarker suitable for use in the present disclosure is an Fc gamma receptor (e.g., FcyRIIA (CD32a)). In some aspects, a biomarker is a combination of Fc gamma receptors (e.g., FcyRIIA (CD32a) and FcyRIIB (CD32b)). In some aspects, a biomarker is a combination of CD32a and CD47, and, optionally, CDl6a, CDl6b, CD32b, CD32c, CD64, CD68, CD163, SIRPa, or a combination thereof.

As used herein, the term“FR” means a framework region.

As used herein, the term“HCDR” means a heavy chain complementarity-determining region.

As used herein, the term“humanized antibody” means an antibody that has variable region framework and constant regions from a human antibody but retains the CDRs of the animal antibody.

As used herein, the term“indicates” or“indicating” refers to a relationship between the presence of a substance or substances (e.g., biomarkers) and one or more events (e.g., response to treatment). In some aspects, indicates refers to a positive relationship or positive correlation in which as one increases, the other increases as well. In some aspects, indicates refers to a negative relationship or negative correlation in which as one increases, the other decreases. The present disclosure provides biomarkers, the levels of which indicate or predict a likely outcome, such as presence, amount or level of a biomarker (e.g., CD32a) in a patient sample, and the likelihood a patient will respond to treatment with an agent. For example, the presence, amount or level of a biomarker may indicate a likelihood of a positive clinical outcome for the patient, such as an increased likelihood of long-term survival without recurrence and/or a positive response to treatment. Such a positive indication may be demonstrated statistically in various ways, e.g., by a low hazard ratio. In another example, the presence, amount or level of a biomarker may negatively indicate a likelihood of good clinical outcome for the patient. In this case, for example, the patient may have a decreased likelihood of a positive response to treatment. Such a negative indication may be demonstrated statistically in various ways, e.g., by a high hazard ratio.

As used herein, the terms“inhibits”,“blocks”, or“reduces” are used interchangeably and encompass both partial and complete inhibition/blocking as well as direct and allosteric inhibition/blocking. For example, the inhibition/blocking of CD47 or SIRPa reduces or alters the normal level or type of activity that occurs from CD47 or SIRPa in a given system in the absence of inhibition or blocking. As used herein,“inhibition”,“blocking”, or“reduces” are also intended to include any measurable decrease in biological function and/or activity of a target (e.g., CD47). For example, when an antibody, or an antigen-binding fragment thereof is in contact with the target as compared to the target not in contact with an antibody, an antigen-binding fragment. In some embodiments, an agent that targets CD47 inhibits or reduces CD47 function and/or activity in a given system by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, an agent that specifically binds CD47 and inhibits, blocks or reduces SIRPa function and/or activity in a given system by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

As used herein, the term“inhibits growth” (e.g., referring to cells) is intended to include any measurable decrease in the growth of a cell, e.g., the inhibition of growth of a cell by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

As used herein, the term“inhibits re-growth” (e.g., inhibits tumor re-growth) is intended to include any measurable decrease in the re-growth of a tumor, e.g., the inhibition of re-growth of a tumor by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

As used herein, a subject“in need of prevention,”“in need of treatment,” or“in need thereof,” refers to one, who by the judgment of an appropriate medical practitioner (e.g., a doctor, a nurse, or a nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals), would reasonably benefit from a given treatment (such as treatment with an agent that specifically binds human CD47).

The term "in vivo" refers to processes that occur in a living organism.

As used herein the term“KD” or“KD” refers to the equilibrium dissociation constant of a binding reaction between an antibody and an antigen. The value of KD is a numeric representation of the ratio of the antibody off-rate constant (kd) to the antibody on-rate constant (ka). The value of KD is inversely related to the binding affinity of an antibody to an antigen. The smaller the KD value the greater the affinity of the antibody for its antigen. Affinity is the strength of binding of a single molecule to its ligand and is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and rank order strengths of bimolecular interactions.

As used herein, the term“kd” or“k_d” (alternatively“koff’ or“k_0ff”) is intended to refer to the off-rate constant for the dissociation of an antibody from an antibody/antigen complex. The value of kd is a numeric representation of the fraction of complexes that decay or dissociate per second, and is expressed in units sec ¹.

As used herein, the term“ka” or“ka” (alternatively“kon” or“kon”) is intended to refer to the on-rate constant for the association of an antibody with an antigen. The value of ka is a numeric representation of the number of antibody /antigen complexes formed per second in a 1 molar (1M) solution of antibody and antigen, and is expressed in units M ¹ sec ¹. As used herein, the term“LCDR” means a light chain complementarity-determining region.

As used herein, the term“likelihood” generally refers to an increase in the probability of an event (e.g., a patient will respond to treatment) and may be expressed as a probability value, fraction, or percentage. The term“likelihood” may include the concept of“probability” as used in mathematics and by persons of ordinary skill in the art of statistics. The term“likelihood” when used in reference to a patient’s response to treatment, the term contemplates an increase in the probability that the patient responds positively to treatment. The term“likelihood” when used in reference to a tumor’ s response to treatment contemplates a change in the amount of one or more biomarkers present in the tumor or tumor sample that may evidence a progression in treating the tumor.

As used herein, the term“malignant cells” refers to a cell that has become abnormal and therefore divides without control. In some embodiments, a malignant cell can invade nearby tissue. Malignant cells can also spread to other parts of the body via the blood and lymph system. In some embodiments, a tumor comprises at least one malignant cell.

As used herein, the term“monitor,” carries its common usage and can refer to the surveillance or continual observation of a process or event (e.g., a response to treatment). For example, the term“monitoring a patient’s response to treatment” contemplates, for example, observation or surveillance of disease progression. The monitoring can be performed, for example, by following or determine the expression of biomarkers over time.

As used herein, the term“monitoring biomarker” refers to a biomarker measured serially for assessing status of a disease or medical conditions or for evidence of exposure to (or effect of) a medical product or agent.

As used herein, the term“myeloid cell” refers to cells derived from myeloid stem cells, and include erythrocytes, thrombocytes, neutrophils, monocytes, macrophages, eosinophils, basophils and mast cells. In some embodiments, myeloid populations of the tumor microenvironment prominently include monocytes, neutrophils, macrophages and dendritic cells.

As used herein, the term“nucleic acid probe” refers to a fragment of DNA or RNA that is labeled with a tag. The nucleic acid probe is added to a DNA or RNA sample to detect and/or recover nucleotide sequences that are complementary to the sequence in the probe. The probe hybridizes to a single-stranded nucleic acid (DNA or RNA) whose base sequence allows probe- target base pairing due to complementarity between the nucleic acid probe and target. Unless otherwise indicated, the term encompasses nucleic acid probes labeled with tags including radioactive labels, fluorophores, enzymes, or nucleotides modified with digoxygenin or biotin.

A nucleic acid is“operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to transcription regulatory sequences, operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. For switch sequences, operably linked indicates that the sequences are capable of effecting switch recombination.

As used herein, the term“panel of biomarkers” includes a group of markers, the quantity or activity of each member of which indicates responsiveness to treatment with an agent as described herein. The determination of a panel of biomarkers can provide more sensitive and specific information needed to evaluate a tumor or a patient. In some embodiments, the panel of biomarkers include one, two, three, four, five, six, seven, eight, or nine or more biomarkers. In some embodiments, the panel comprises CD32a, CD32b, and CD47. In some embodiments, the panel comprises CD32a and CD47, and, optionally, CDl6a, CDl6b, CD32b, CD32c, CD47, CD64, CD68, CD163, SIRPa, or a combination thereof.

As used herein, “parenteral administration,” “administered parenterally,” and other grammatically equivalent phrases, refer to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intranasal, intraocular, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid and intrasternal injection and infusion.

As used herein, the term“patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

As used herein, the term“Programmed Cell Death Protein 1” or“PD-l” refers to the Programmed Cell Death Protein 1 polypeptide, an immune-inhibitory receptor belonging to the CD28 family and is encoded by the PDCD1 gene in humans. Alternative names or synonyms for PD-l include: PDCD1, PD1, CD279 and SLEB2. PD-l is expressed predominantly on previously activated T cells, B cells, and myeloid cells in vivo , and binds to two ligands, PD-L1 and PD-L2. The term "PD-l" as used herein includes human PD-l (hPD-l), variants, isoforms, and species homologs of hPD-l, and analogs having at least one common epitope with hPD-l. The complete hPD-l sequence can be found under GenBank Accession No. AAC51773.

As used herein, the term "Programmed Death Ligand-l” or“PD-L1" is one of two cell surface glycoprotein ligands for PD-l (the other being PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-l. Alternative names and synonyms for PD-L1 include: PDCD1L1, PDL1, B7H1, B7-4, CD274 and B7-H. The term "PD-L1" as used herein includes human PD-L1 (hPD-Ll), variants, isoforms, and species homologs of hPD-Ll, and analogs having at least one common epitope with hPD-Ll. The complete hPD-Ll sequence can be found under GenBank Accession No. Q9NZQ7.

The term“percent identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the“percent identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et ah, infra).

One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et ah, J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website.

The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ( J . Mol. Biol. (48):444- 453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present disclosure can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(l7):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is“isolated” or“rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al, ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

The nucleic acid compositions of the present disclosure, while often in a native sequence (except for modified restriction sites and the like), from either cDNA, genomic or mixtures thereof may be mutated, in accordance with standard techniques to provide gene sequences. For coding sequences, these mutations, may affect amino acid sequence as desired. In particular, DNA sequences substantially homologous to or derived from native V, D, J, constant, switches and other such sequences described herein are contemplated (where "derived" indicates that a sequence is identical or modified from another sequence).

As used herein, the term "phagocytosis" refers to a process by which a particle (e.g., a cell) is engulfed or internalized by a host cell (e.g., a phagocyte, macrophage or neutrophil). Phagocytes mediate phagocytosis by multiple pathways, including at least: (i) direct cell surface receptors (for example, lectins, integrins and scavenger receptors), (ii) complement enhanced - using complement receptors (including CR1, receptor for C3b, CFG, CR4, CRIg) to bind and ingest complement opsonized pathogens, and (iii) antibody enhanced - using Fc receptors to bind antibody-opsonized particles which then become internalized and fuse with lysosomes to become phagolysosomes.

As generally used herein, “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

As used herein, a“pharmaceutically acceptable carrier” refers to, and includes, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The compositions can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see, e.g., Berge et al. (1977) J Pharm Sci 66: 1-19).

As used herein, the term “pharmacodynamics biomarker” (alternatively “response biomarker) refers to a biomarker used to show that a biological response has occurred in an individual who has been exposed to a medical product or agent.

As used herein, the terms“polypeptide”,“peptide”, and“protein” are used interchangeably to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

As used herein, the term“predictive biomarker” refers to a biomarker used to identify individuals who are more likely than similar individuals without the biomarker to experience a favorable or unfavorable effect from exposure to a medical product or agent.

As used herein, the term“preventing” when used in relation to a condition, refers to administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. As used herein, the term“prognostic biomarker” refers to a biomarker used to identify likelihood of a clinical event, disease recurrence or progression in patients who have the disease or medical condition of interest.

As used herein, the term“purified” or“isolated” as applied to any of the proteins (antibodies or fragments) described herein refers to a polypeptide that has been separated or purified from components (e.g., proteins or other naturally-occurring biological or organic molecules) which naturally accompany it, e.g., other proteins, lipids, and nucleic acid in a prokaryote expressing the proteins. Typically, a polypeptide is purified when it constitutes at least 60 (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99) %, by weight, of the total protein in a sample.

As used herein, the term“recombinant host cell” (or simply“host cell”) is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term“host cell” as used herein.

As used herein, the term“respond to treatment” refers to a beneficial clinical or therapeutic result or favorable reaction experienced by subject or patient at risk for, or suffering from, a disease or disorder, such as cancer. Responses can be assessed using any assay, metric or endpoint indicating a benefit to the patient or subject including, without limitation, (1) inhibition, to some extent, of disease progression (e.g., cancer progression), including a reduction in the rate of disease progression and complete arrest of disease progression; (2) a reduction in tumor size; (3) inhibition (i.e., reduction in the extent or rate or the complete arrest) of cancer cell infiltration into adjacent peripheral organs and/or tissues; (4) inhibition of metastasis; (5) relief, to some extent, of one or more symptoms associated with the disease or disorder (e.g., cancer); (6) increase or extension in the length of survival, including overall survival and progression free survival; and/or (9) decreased mortality at a given point of time following treatment. In one embodiment, a response to treatment includes any one or more of: extending survival (including overall survival and progression free survival); resulting in an objective response (including a complete response or a partial response); or improving signs or symptoms of cancer. As used herein, the term“safety biomarker” refers to a biomarker measured before or after an exposure to a medical product or agent to indicate the likelihood, presence, or extent of toxicity as an adverse effect.

As used herein, the term“sample” refers to a composition that is obtained or derived from a patient, subject or individual of interest that contains or may contain a cellular or molecular entity, signature or substance (e.g., a biomarker) that is to be detected, quantified, identified, or otherwise characterized, for example based on physical, biochemical, chemical, or physiological characteristics, or a combination thereof. A“tumor sample” refers to a sample taken from a subject having a tumor (e.g., a cancer patient), wherein the sample comprising tumor cells. In some embodiments, a tumor sample comprises malignant (tumor) and non-malignant cells, optionally, extracellular components. A“reference sample” (alternatively “control sample”) refers to a sample, cell, tissue, standard, or level that is used for comparison purposes. For example, in some embodiments a reference sample is a sample known to contain one or more biomarkers of interest. The amount or level of a biomarker in a reference sample is referred to as a“reference amount.” In certain embodiments of the disclosure, a reference sample is a sample obtained from a healthy and/or non-diseased part of the body of the same patient, subject or individual. For example, healthy and/or non-diseased cells or tissues adjacent to the diseased cells or tissues (e.g., cells or tissues adjacent to a tumor). In another embodiment, a reference sample is obtained from an untreated tissue and/or cell of the body of the same patient, subject or individual. In another embodiment, a reference sample is obtained from a healthy and/or non-diseased part of the body (e.g., tissues or cells) of an individual who is not the patient or subject. In another embodiment, a reference sample is obtained from an untreated tissue and/or cell of the body of an individual who is not the patient or subject. A sample may be obtained by any technique (e.g., biopsy) known to a person skilled in the art. In some embodiments, samples may comprise tissue (referred to as a “tissue sample”) or cells (referred to as a“cell sample”).

As used herein, the terms“specific binding,”“selective binding,”“selectively binds,” and “specifically binds,” refer to an antibody binding to an epitope on a predetermined antigen. Typically, the antibody binds with an equilibrium dissociation constant (Kd) of approximately less than 10 ⁶ M, such as approximately less than 10 ⁷, 10 ⁸ M, 10 ⁹ M or 10 ¹⁰ M or even lower when determined by surface plasmon resonance (SPR) technology in a BIACORE 2000 instrument. For example, when using the recombinant human CD47 extracellular domain as the analyte/predetermined antigen and an antibody that specifically binds human CD47 as the ligand, SPR can be used to measure binding of the ligand/antibody to the analyte/predetermined antigen with an affinity that is at least about two-fold greater than its affinity for binding to a non-specific antigen ( e.g ., BSA, casein) other than the predetermined antigen or a closely-related antigen. The phrases“an antibody recognizing an antigen” and“an antibody specific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

As used herein, the term“subject” includes any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat a subject with an immune disorder. The term“non-human animal” includes all vertebrates, e.g., mammals and non mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.

As used herein, the term“susceptible to treatment”, when used in the context of the present application refers to a tumor exhibiting a potential to be vulnerable to the anti-tumor effects provided by a treatment. In some embodiments, the potential can be determined by the measurement of e.g., one or more biomarkers that affect or effect the treatment.

As used herein, the term“susceptibility biomarker” (alternatively“risk biomarker”) refers to a biomarker that indicates the potential for developing a disease or medical condition in an individual who does not currently have clinically apparent disease or the medical condition.

As used herein, the term“therapeutic antibody” refers to an antibody, fragment of an antibody, or construct that is derived from an antibody, and can bind to a cell- surface antigen on a target cell to cause a therapeutic effect. Such antibodies can be chimeric, humanized or fully human antibodies. Methods are known in the art for producing such antibodies. Such antibodies include single chain Fc fragments of antibodies, minibodies and diabodies. Any of the therapeutic antibodies known in the art to be useful for cancer therapy can be used in combination therapy suitable for use in the methods disclosed herein. Therapeutic antibodies may be monoclonal antibodies or polyclonal antibodies.

As used herein, the terms“therapeutically effective amount” or“therapeutically effective dose,” or similar terms used herein are intended to mean an amount of an agent (e.g., an antibody that specifically binds human CD47) that will elicit the desired biological or medical response (e.g., an improvement in one or more symptoms of a cancer).

The terms“treat,”“treating,” and“treatment,” as used herein, refer to therapeutic or preventative measures described herein. The methods of“treatment” employ administration to a subject, in need of such treatment, a human antibody of the present disclosure, for example, a subject in need of an enhanced immune response against a particular antigen or a subject who ultimately may acquire such a disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

As used herein, the term“unrearranged” or“germline configuration” refers to a V segment refers to the configuration wherein the V segment is not recombined so as to be immediately adjacent to a D or J segment.

As used herein, the term“vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a“plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced ( e.g ., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply,“expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification,“plasmid” and“vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

As used herein, the term“VH” means a variable heavy domain.

As used herein, the term“VL” means a variable light domain.

Reference to particular amino acids may be made in respect of common 1- letter or 3 -letter codes as commonly understood by persons skilled in the art. Any reference to an“X” amino acid is reference to a variable amino acid. Amino acids can be modified as described herein.

Aspects of the invention will be illustrated in view of the following figures and examples. EXAMPLES

The disclosure will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

While the present disclosure has been described with reference to the specific

embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure.

In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the disclosure.

Example 1: Effect of whole IgG4 2.3D11 antibody and 2.3D11 F(ab’)2 Fragments on the Induction of Phagocytosis In Vitro

To understand the mechanism of 2.3D11 -induced phagocytosis, the ability of 2.3D11 formatted as whole IgG4 and 2.3D11 formatted as an F(ab’)2 fragment were tested for their ability to induce phagocytosis in an in vitro phagocytosis assay. Antibody 2.3D 11 comprises the 2.3D11 VH/VL sequences shown in SEQ ID NOs: 3 and 4 and, when formatted as a whole IgG4, has a human wild-type IgG4 Fc domain.

Briefly, the in vitro phagocytosis assay was performed using carboxyfluorescein succinimidyl ester (CFSE)-labeled Jurkat cells (cells were labeled according to manufacturer’s protocol - Life Technologies) co-cultured with primary human macrophages at a 2: 1 ratio in the presence of 2.3D11 antibody (2.3D11), a respective 2.3D11 F(ab’)2 fragment (2.3D11 F(ab)’2), or a human IgG4 isotype control antibody (hIgG4) for 2 h at 37°C. Cells were trypsinized and stained with macrophage specific marker (CD14-APC for human macrophages). Flow cytometry was performed followed by doublet exclusion analysis and phagocytosis reported as a percentage of CD14+ macrophages that were also CFSE+. As shown in Figure 1A, 2.3D11 is able to induce macrophage-mediated phagocytosis, whereas 2.3D 11 F(ab’)2 and hIgG4 are inactive.

To determine if 2.3D11 and 2.3D11 F(ab’)2 bind to Jurkat cells, Jurkat cells were incubated with varying amounts of unlabeled 2.3D11, 2.3D11 F(ab’)2, or hIgG4 for 30 min at 4°C. Cells were washed and incubated with a saturating dose of fluorescently-labeled 2.3D11- Alexa Fluor 647 (2.3D11-AF647) for an additional 30 min at 4°C. 2.3D1 l-Alexa Fluor 647 (AF647) conjugates were generated using Alexa Fluor® 647 Antibody Labeling Kit

(ThermoFisher) or custom ordered from BioLegend. Cells were washed again and 2.3D11- AF647 mean fluorescent intensity (MFI) was determined by flow cytometry. As shown in

Figure IB, treatment of Jurkat cells with increasing amounts of 2.3D11 or 2.3D11 F(ab’)2 corresponded with decrease 2.3D11-AF647 MFI, indicating that both 2.3D11 and 2.3D11 F(ab’)2 similarly bind to Jurkat cells.

Taken together, these data demonstrate that the whole IgG4 version of 2.3D11 is able to induce macrophage-mediated phagocytosis, whereas the F(ab’)2 fragment, which is capable of saturating CD47 binding sites on target cells, is insufficient for the induction of macrophage- mediated phagocytosis under these conditions. These results suggest that blockade of the CD47:SIRPa interaction alone, in a human in vitro system, is insufficient to drive phagocytosis and that additional pro-phagocytic signal(s) are necessary for the in vitro effects of the 2.3D11 antibody.

Example 2: Effect of whole IgG4 2.3D11 antibody and 2.3D11 F(ab’)2 Fragments on the Phosphorylation of CD32a

To search for additional signals that may be mediating the effects of 2.3D11,

phospho-immunoreceptor array screening was conducted within the context of the phagocytosis assay. Briefly, Jurkat cells (ATCC) were co-cultured with primary human macrophages in the presence of 10 pg/mL 2.3D11 antibody, 2.3D11 F(ab’)2 fragment, or hIgG4 for 10 min at 37°C. Following antibody treatment, Jurkat cells were separated from macrophages by gently pipetting, and each cellular fraction was assayed independently (according to the manufacturer’s instructions from the phospho-immunoreceptor array kit (R&D Systems, Bio-Techne

Corporation, Minneapolis, MN) to ascertain the cellular origin of any observed signaling event. Phospho-signals were detected by chemiluminescence and HLImage++ software was used to quantitate and normalize phospho-signals detected by chemiluminescence.

Figure 2A shows representative phospho-array images showing Jurkat and macrophage fractions, with corresponding quantitation of pCD32a mean pixel intensity shown in Figure 2B. In Figure 2C, data shown are from the Jurkat fraction from an independent experiment in which 2.3D11 whole IgG and 2.3D11 F(ab’)2 were run side by side.

This screening approach led to the identification of phospho-CD32a Fc receptor phosphorylation in both the Jurkat and macrophage fractions following a 10-minute treatment with 2.3D11 (Figure 2A and Figure 2B). This enhancement of CD32a phosphorylation was not observed with the 2.3D11 F(ab’)2 fragment (Figure 2C), indicating that the IgG portion of the antibody is required for CD32a phosphorylation and suggesting that phosphorylation of CD32a may be linked with the induction of phagocytosis. These data demonstrate that 2.3D11 antibody induces phosphorylation of CD32a within the context of phagocytosis in a manner dependent upon the whole IgG portion of the antibody.

The two cell types within the phagocytosis assay, human macrophages and Jurkat cells, were profiled for surface CD32a expression. Human myeloid (U937) cells were used as a positive control as they have been previously shown to express CD32a (Ierino et al., (1993) J Immunol 150(5):1794-1803). To determine the surface expression of CD32a, cells were stained with a fluorescent anti-CD32a antibody conjugated with fluorescein isothiocyanate (FITC)

(clone IV.3; StemCell Technologies) or a mouse IgG2B, kappa isotype control antibody, clone MFC- 1 1, FITC (Catalog #60072FI, SiemCeil Technologies) and then analyzed by flow cytometry. As expected, macrophages showed abundant surface expression of CD32a, while Jurkat cells were negative, as shown in Figure 3. Since the Jurkat fraction contained < 10% macrophage contamination (data not shown) in the phospho-array screening assays (Figures 2A- 2C), it is possible that the Jurkat cell fraction is enriched for a subset of“activated” macrophages that have already undergone conjugate formation with the Jurkat target cells in the presence of 2.3D11.

Taken together, these results suggest that 2.3Dl l-mediated macrophage phagocytosis (and potentially other macrophage effector functions triggered by 2.3D11) are mediated, at least in part, through Fc receptor signaling. Example 3: CD47 Expression on the Target Cell is a Requirement for the Induction of CD32a Phosphorylation and Phagocytosis

The requirement for CD47 expression on the target cell for both induction of CD32a phosphorylation (pCD32a) and phagocytosis induction by 2.3D 11 was evaluated using Jurkat wildtype (WT) (ATCC) and Jurkat CD47 knockout (KO) cells. The Jurkat CD47 KO cells were generated via CRISPR/CAS9 technology by Applied Biosystems. Jurkat wildtype (WT) cells and Jurkat CD47 (KO) cells were evaluated for CD47 surface expression by flow cytometry using an anti-CD47-AF647-labeled antibody (mAbB). As shown in Figure 4A, Jurkat WT cells express high levels of CD47 while CD47 expression is markedly reduced in the Jurkat CD47 KO cells.

To determine if the 2.3D1 l-mediated phosphorylation of CD32a observed in Figure 2 is dependent on CD47, Jurkat WT or CD47 KO cells were co-cultured with primary human macrophages ± 10 pg/mL antibody, as indicated, for 10 min at 37°C and pCD32a induction was evaluated in the Jurkat fraction using a phospho-immunoreceptor array kit (R&D Systems, Bio- Techne Corporation, Minneapolis, MN). Raw images were quantitated to obtain mean pixel intensity using HLImage++ Software. As shown in Figure 4B, CD47 expression on the target cell is required for pCD32a induction by 2.3D11.

To demonstrate the requirement for CD47 expression on target cells for the induction of phagocytosis by 2.3D11, wildtype and CD47 KO cells were CFSE-labeled and co-cultured with pre-seeded human macrophages at a 2: 1 ratio in the presence of isotype control or 2.3D11 antibody. Cells were incubated for 2h at 37°C and flow cytometry performed to determine phagocytosis induction which is reported as a percentage of CFSE+ target cells within the CD14+ macrophages. As shown in Figure 4C, 2.3D1 l-mediated induction of phagocytosis is not observed when target cells do not express CD47. These data demonstrate a requirement for CD47 expression on the target cell for both induction of CD32a phosphorylation (pCD32a) and phagocytosis induction by 2.3D11, as Jurkat CD47 KO cells are completely insensitive to 2.3D 11 in both assays.

Example 4: Effect of Anti-CD32a Blocking Antibody on 2.3D11-Induced Phagocytosis

To evaluate the functional significance of CD32a in the phagocytosis assay, an anti- CD32a antibody (clone IV.3; StemCell technologies) was used to block this receptor on macrophages prior to their co-culture with Jurkat targets. Human primary macrophages were pre- incubated with anti-CD32a blocking antibody or mIgG2b isotype control antibody prior to performing an in vitro phagocytosis assay essentially as described in Example 1 with increasing concentrations of either a human IgG4 version of 2.3D11 or an anti-CD47 comparator antibody (mAb A) that has a human IgG4 backbone comprising a single S228P mutation. A hIgG4 isotype control antibody was used as a negative control. Pre-incubation of macrophages with CD32a blocking antibody led to a complete abrogation of 2.3D 11 activity in the phagocytosis assay, as shown in Figure 5A. These data suggest that even though there is likely a relatively low affinity interaction, at the 1:1 basis, between the human IgG4 version of 2.3D 11 and CD32a, in this context, 2.3D11 can overcome an affinity threshold to engage CD32a and this event is required for the induction of phagocytosis.

A similar dependence on CD32a was seen when assays were performed in the presence of an anti-CD47 comparator antibody (mAb A) (Figure 5B) suggesting this phenomenon may be both target and class dependent as both antibodies are directed against CD47 and contain an hIgG4 Fc.

Example 5: Effect of 2.3D11 on the Induction of Target Cell Apoptosis

A central clinical hypothesis is that 2.3D 11 -mediated induction of tumor-cell

phagocytosis will lead to an anti-tumor effect, however 2.3D11 binding to CD47 may have other cellular outcomes in addition to the induction of phagocytosis. To determine if 2.3D11 has an effect on target cell apoptosis, Jurkat cells (ATCC) were incubated with 10 pg/mL of the following antibodies: 2.3D11, mAb A, mAb B, IgG4 isotype control and IgGl isotype control, in soluble form, for 24 h. mAb A and mAb B are anti-CD47 competitor antibodies. mAb A is described in Example 4 and mAb B is an mlgGl anti-hCD47 monoclonal antibody control. Cell death was assessed by staining the cultured cells with Annexin V/PI (Life Technologies) and subsequent analysis by flow cytometry. In a unicellular in vitro system, soluble 2.3D 11 is not able to induce apoptosis of Jurkat cells, whereas anti-CD47 comparator antibodies (mAbA and mAbB) are both able to induce apoptosis of Jurkat cells, as indicated by Annexin V+/PI- staining. The percentage of necrotic/dead cells, as indicated by Annexin V+/PI+ staining was unchanged across treatment conditions (Figure 6A). Representative flow images are also shown for each treatment group in Figure 6B. Similar results were obtained after 72 h of antibody treatment (data not shown). While soluble 2.3D11 does not induce cell death of Jurkat cells alone, as shown in

Figures 6A-6B, it remained a formal possibility that 2.3D11 may induce cell death of Jurkat cells in a Jurkakmacrophage co-culture. To test this possibility, CFSE-labeled Jurkat cells were co-culture with primary human macrophages in an in vitro phagocytosis assay as described in Example 1. The cells were cultured in the presence of increasing concentrations of the following antibodies: 2.3D11, mAbB, hIgG4, or mlgGl as indicated in Figures 7A and 7A. After 2 h of co-culture cells were stained with macrophage specific marker CD14-APC for human macrophages. For analysis of dead cells in this assay, a LIVE/DEAD dye (Life Technologies) was added to the CD14-APC staining cocktail Dead cells were identified in the non- phagocytosed target cell population. The results shown in Figure 7A show that both 2.3D11 and mAbB induce phagocytosis of Jurkat cells, consistent with previous findings. Notably, as shown in Figure 7B, the percent of CD14-CFSE+ (non-phagocytosed targets) that take up the

LIVE/DEAD dye (and are therefore not viable) increase in the presence of increasing concentrations of 2.3D11. Figure 7C shows Annexin V/propidium iodide (PI) (Life

Technologies) staining results on non-phagocytosed Jurkat cells treated with 10 pg/mL antibody from an independent experiment.

Taken together, these results demonstrate a 2.3D11 -dependent induction of apoptosis in a fraction of Jurkat cells that are not phagocytosed by macrophages (Figure 7B). This Jurkat cell death phenomenon is dependent on the presence of macrophages and correlates with

phagocytosis induction (Figure 7A). Although mAbA and mAbB also induced apoptosis under similar conditions (in part via apoptosis induction, Figure 7C), both mAbA and mAbB induce cell death of Jurkat cells alone. Taken together, these results demonstrate that the 2.3D11- mediated induction of Jurkat cell apoptosis is dependent on the presence of macrophages, whereas both mAbA and mAbB can induce Jurkat cell apoptosis in the absence of macrophages. These data demonstrate that the mechanism by which the 2.3D11 antibody induces apoptosis is necessarily distinct from the mechanism by which the mAbA and mAbB antibodies induce apoptosis.

To investigate whether the antibody-mediated induction of apoptosis required contact between the target Jurkat cells and the macrophages, a transwell system was used in which an in vitro phagocytosis assay (Jurkakmacrophage co-culture ± antibody) was set up in the bottom chamber while only Jurkat cells were seeded in the top chamber. The top and bottom chambers were separated by a porous membrane large enough to allow passage of cytokines between each chamber but small enough to prevent the passage of cells. In this transwell system, cell death in the Jurkat cells within the top chamber would indicate that cell death was being induced in a cell contact-independent manner. Cells were co-cultured in the presence of 10 pg/mL of 2.3D11, hIgG4 isotype control, mAb B and hlgGl isotype control for mAb B. After 2 h of co-culture cells on bottom wells were stained with CD 14 (to identify macrophages) and a LIVE/DEAD viability dye (Life Technologies) to identify dead cells, while cells in top wells were only stained for viability. Figure 8A shows phagocytosis of Jurkat cells is induced by the presence of both 2.3D11 and mAbB in the bottom wells, consistent with previous results. Figure 8B shows the percent of CD14-CFSE+ Jurkat cells that were non-viable on the top (grey) or bottom (black) wells.

As shown in Figure 8A, both 2.3D11 and mAbB induced phagocytosis on the bottom chamber of the transwell; however, 2.3D11 did not induce apoptosis of Jurkat cells within the top chamber indicating the 2.3D11 -dependent induction of Jurkat cell apoptosis within the context of the phagocytosis assay requires cell contact with macrophages. As a positive control, mAbB induced apoptosis in both the top and bottom chambers as expected (Figure 8B).

Example 6: 2.3D 11 -Mediated and Macrophage-Dependent Induction of Apoptosis Is Independent of the Phagocytosis Event

Since contact between the macrophage and the target cell is required for induction of Jurkat cell apoptosis, an experiment was conducted to determine whether the act of phagocytosis itself was required for the induction of apoptosis. Phagocytosis assays using Jurkat cell targets and primary human macrophage effectors were performed in the presence of 1 mM of small molecule inhibitors cytochalasin D (CytoD) or ibrutinib (IBR), and lOpg/ml antibody 2.3D 11 or hIgG4 isotype control. Dimethylsulfoxide (DMSO) was used as a control. Figure 9A shows the extent of Jurkat cell phagocytosis in the presence of antibody 2.3D11 and inhibitors. Figure 9B shows the extent of non-viable, non-phagocytosed Jurkat cells in the presence of antibody 2.3D11 and inhibitors.

Macrophage phagocytosis was inhibited using two different small molecule inhibitors, cytochalasin D and ibrutinib (IBR). Cytochalasin D is a potent inhibitor of actin polymerization (Brenner and Korn (1979) J Biol Chem 254(20):9982-9985, which is necessary for the cytoskeletal rearrangements that drive macrophage phagocytosis. In the presence of cytochalasin D, levels of phagocytosis were reduced in both isotype control- and 2.3D 11 -treated co-cultures, consistent with an overall inhibition of phagocytosis (Figure 9A). Ibrutinib (IBR), a Bruton's tyrosine kinase inhibitor, led to inhibition of 2.3Dl l-induced phagocytosis, without lowering the baseline levels ( i.e ., levels in the absence of 2.3D11) of phagocytosis. This inhibitory effect of IBR on macrophage-mediated phagocytosis has been reported previously (Borge et ah, (2015) Haematologica l00(4):el40; Chen et ah, (2017) Nature 544(765l):493-497). Regardless of the method of phagocytosis inhibition, the induction of cell death mediated by 2.3D11 was preserved, indicating that this phenomenon is independent from phagocytosis (Figure 9B). However, these data do not support the conclusion that phagocytosis is independent of apoptosis induction.

Example 7: Effect of CD32a Blockade on Macrophage-Dependent, 2.3D11-Mediated Induction of Target Cell Apoptosis

As shown in Example 6, 2.3D 11 -induced apoptosis of target cells does not require active phagocytosis. To determine if the macrophage-dependent, 2.3D 11 -mediated induction of target cell apoptosis requires CD32a engagement, phagocytosis assays were performed using Jurkat cell targets and primary human macrophage effectors. Macrophages were pre-incubated with 10pg/m 1 anti-CD32a blocking antibody (clone IV.3; StemCell Technologies) or DMSO, were co-cultured with Jurkat cells in the presence of 10 pg/mL of 2.3D11 or human IgG4 isotype control. The anti-CD32a blocking antibody (clone IV.3; StemCell Technologies) that had been previously shown to abrogate 2.3D 11 -mediated phagocytosis (Example 4), inhibited

phagocytosis (Figure 10A) and inhibited the induction of Jurkat cell apoptosis (Figure 10B). Jurkat cell apoptosis, measured as the percentage of non-viable, non-phagocytosed, was determined as in Example 5. These data demonstrate that engagement of CD32a is required for 2.3D11 -mediated induction of Jurkat cell apoptosis in co-culture with macrophages.

Example 8: Scaffolding of 2.3D11 Mimics the Presence of Macrophages and Induce Apoptosis

Taken together, the results provided by Example 7 suggest that Fc receptor (FcR) engagement of 2.3D 11 on macrophages immobilizes or scaffolds 2.3D 11 antibodies, providing a sufficient density and orientation that allows for antibody cross-linking of CD47 on target cells, and subsequent delivery of a cell death-induction signal downstream of CD47. To mimic this scaffolding and orientation event, 2.3D11 was immobilized on Protein G-coated plates. Protein G-coated plates were incubated overnight with 0.1, 1, 10, or 100 pg/mL of the antibodies indicated in Figures 11A-11B. Jurkat (Figure 11A) or U937 (Figure 11B) cells were then cultured in antibody-coated wells for 24 h. Cells were subsequently harvested and stained with Annexin V (AnnV) and Propidium Iodide (PI) to assess viability.

Protein-G bound 2.3D11 induced dose-dependent cell death in 2 different tumor cell lines, Jurkat (ATCC) (Figure 11A) and U937 (ATCC) (Figure 11B). These data further demonstrate that immobilization or scaffolding of 2.3D11 provides the mechanism by which a death signal(s) are transmitted to the target cells.

Example 9: 2.3D 11 -Induced Phagocytosis Partially Mediated by CD16 Engagement

Human macrophages express activating Fc receptors CD64, CD16 (isoforms a and b) & CD32a. Quantitative analyses have been reported on the relative abundance of these activating FcRs on macrophages (Richards et ah, (2008) Mol Cancer Ther 7(8):2517-2527). Although 2.3D11 -mediated phagocytosis was shown to depend on engagement with CD32a (Example 4), it remained a formal possibility that induction of phagocytosis could be mediated by engagement with either CD64 and/or CD16. Despite the lower abundance of CD64 relative to CD32a and CD16 in macrophages, CD64 has the highest affinity for hIgG4 (Hogarth et ah, (2012) Nat Rev Drug Discovery 11(4):311-331)

To determine if target cell phagocytosis induced by 2.3D11 is mediated by CDl6a engagement, phagocytosis assays were performed using Jurkat cell targets (ATCC) and primary human macrophage effectors. Human primary macrophages were pre-incubated with 10pg/ml anti-CDl6 (3G8) blocking antibody (Biolegend) or mlgGl isotype control prior to performing a phagocytosis assay using CFSE-labeled Jurkat cells co-cultured at a 2:1 ratio with the human macrophages for 2h at 37°C in the presence of increasing concentrations of 2.3D11. Flow cytometry was used evaluate phagocytosis which is reported as a percentage of CFSE+ target cells within the overall CD 14+ macrophage population (Figure 12). As shown in Figure 12, blocking CD16 using an anti-CDl6a blocking antibody (3G8) partially abrogated 2.3Dl l-induced phagocytosis. These data show that 2.3D 11 -induced phagocytosis is mediated, at least in part, through engagement of CDl6a.

Example 10: 2.3D11 Induces Macrophage Infiltration Into Tumors

In the tumor microenvironment, macrophages may provide an abundant source of effector cells that could mediate the anti-tumor effect of 2.3D11. To test if 2.3D11 could induce macrophage infiltration into tumors, subcutaneous ovarian cancer xenograft tumors were collected from mice treated with 2.3D11 at various doses (100 pg, 200 pg, or 400 pg) or treated with an isotype control antibody), fixed in formalin, and processed into formalin-fixed paraffin- embedded tissue blocks. Tissue blocks were subsequently sectioned (Leica RM2145 microtome) at approximately 4 microns, and stained with an F4/80 primary antibody (Bio-RAD) which recognizes murine macrophages. Glass slides were scanned using an Aperio AT2 whole slide scanner and quantitative image analysis was performed on the digital slide images using

Visiopharm software. Tumor tissue Regions of Interest (ROI) were outlined using a tissue detection algorithm and manually adjusted to ensure accurate ROIs. Tissue ROIs were processed using imaging filters that employ color deconvolution algorithms to separate positive F4/80 staining from counter staining. Processed images were classified using a thresholding method, where a threshold is established based on pixel values associated with positively stained F4/80 tissues (data not shown). Quantification of the amount of F4/80 was determined by analyzing the area of F4/80 labeled tissue divided by the total tissue area within the ROI.

Figure 13A shows the average amount (± SEM) of macrophage tumor infiltration observed within the different 2.3D11 treatment groups, as indicated. The images on the right show F4/80 (macrophage) staining in an isotype control animal (Figure 13B) vs. an 2.3D11 treated animal (200 pg; Figure 13C) where a significantly increased population of macrophages is observed within the tumor bed. These results demonstrate that treatment of mice with 2.3D11 induces macrophage infiltration into tumor xenografts. These results suggest that quantification of macrophage infiltration into tumors would be a relevant monitoring and/or response biomarker in patients treated with 2.3D11. Example 11: CD47 and CD32a are Co-Expressed in Sarcoma Tumors

The results provided in the preceding examples suggest that the expression of CD47 on target cells is required for the engagement of CD32a on macrophages by 2.3D11 and subsequent phagocytosis (Figure 10A) and cell death induction (Figure 10B). One or both of these may be driving the overall antitumor effect of 2.3D11. Based on this reasoning, levels of both CD47 and CD32a in patient tumors could be important metrics to consider prior to treatment with 2.3D11.

To determine the relative abundance of CD47 and CD32a in a tumor model, an immunohistochemical (IHC) analysis of CD47 and CD32a expression within multiple human sarcoma tumor samples was performed. Tissue microarrays comprised of multiple individual human sarcoma tumor cores were stained with antibodies directed against human CD47 (Spring Bioscience, CD47 rabbit monoclonal Ab clone SP279; 1:120 dilution) or CD32a (FCGR2A) (Origene, CD32A(FCGR2A) mouse monoclonal Ab, clone UMAB60; 1:150 dilution) by immunohistochemistry (IHC). CD47 protein expression in tumor cells for each sample was evaluated by a pathologist and given a histo-score (H-score; Y-axis, left). CD32a protein expression and positivity scores for individual tumor cores were determined through a digital image analysis algorithm (Aperio Positive Pixel Count; Y-axis, right). CD47 and CD32a expression scores for each individual tumor tissue core are displayed across the X-axis. CD47 and CD32a positive and negative expression thresholds for this analysis were set as indicated in Figure 14.

Figure 14 shows, in a sarcoma tissue microarray, that there is a subset of human tumors of this indication that have present within them, CD47 as well as CD32a protein. These results suggest that determining the relative expression level of biomarkers demonstrated to be involved in the function of 2.3D11 may identify the tumors, and thus patients, that may benefit most from treatment with 2.3D11.

Example 12: Fc Variants of 2.3D11 Have Varying Affinities for Fey Receptor Proteins

The affinity of several Fc variants of antibody 2.3D11 for Fey receptor proteins was evaluated. Specifically, the affinity of the following antibodies for Fey receptor proteins was evaluated: (1) antibody 2.3D11, which has a human wild-type IgG4 Fc domain, also referred to herein as“2.3D11 IgG4 WT” ; (2) a variant of 2.3D11 with the same variable regions as 2.3D11 but a human IgGl Fc domain,“2.3D11 IgGl WT”; and (3) a variant of 2.3D11 with the same variable regions as 2.3D11 but a modified human IgG4 Fc domain having S228P and L235E mutations (EU numbering),“2.3D11 IgG4 double mutant”. The S288P and L235E mutations prevent half antibody formation and diminish FcyR binding, respectively. All of the variants of antibody 2.3D11 comprise the 2.3D11 VH/VL sequences shown in SEQ ID NOs: 3 and 4.

Briefly, the affinities of the three antibodies for FcyR proteins were evaluated using Biacore Surface Plasmon Resonance (SPR) technology. The FcyRs evaluated were: FcyRIIa- 131R (CD32a), FcyRIIa- 131 H (CD32a), FcyRIIb (CD32b), FcyRIIIa- 158V (CDl6a), FcyRIIIa- 158F (CDl6a), and FcyRIIIb (CDl6b). Trastuzumab (Herceptin®; human IgGl anti-HER2 antibody) was used as an internal assay control. A direct binding methodology was utilized where the FcyR was immobilized onto a CM5 chip via amine coupling chemistry. Analyte (test antibodies) were injected over the chip and binding measured in response units for duplicate samples. Sensorgrams were produced for each sample and equilibrium dissociation constants (KDs) determined within Biacore Evaluation software. A lower Kd value corresponds with higher affinity.

Among the 2.3D11 Fc variants, 2.3D11 IgGl WT had the strongest affinity for all the FcyRs evaluated. 2.3D11 IgG4 WT had weak but measurable affinity for FcyRIIa/b (CD32a/b) but not Fey RHIa/b (CDl6a/b). Despite the L235E mutation present in the Fc domain of 2.3D11 IgG4 double mutant, low but measurable affinity for FcyRIIa/b, but not FcyRIIIa/b was also observed (Table 1). These data demonstrate that all of the variants of antibody 2.3D11 have a measurable affinity for CD32a as assessed by SPR.

Table 1

*Kd is outside of the concentration range reported

n.q. = no quantifiable data observed

p.f. = poor fit to binding model

Taken together, these data demonstrate that all of the variants of antibody 2.3D11 are capable of engaging CD32a and CD32b. In contrast, binding of the IgG4 and IgG4 double mutant variants to CD 16 is not detectable.

Example 13: Fc Variants of 2.3D11 Differ in Phagocytosis-Inducing Capacity

To evaluate the ability of Fc variants of 2.3D11 to induce phagocytosis, an in vitro phagocytosis assay was conducted with Jurkat target cells and human macrophages (as described above in Example 1). The following Fc variants of antibody 2.3D11 (described in Example 12) were tested for their ability to induce phagocytosis in the phagocytosis assay: 2.3D11 IgG4 WT, 2.3D11 IgG4 double mutant, 2.3D11 IgGl WT, and hlgG (human polyclonal IgG from

BioXCell) was used as a control.

As shown in Figure 15, all of the 2.3D11 Fc variants were able to induce phagocytosis. However, 2.3D11 IgG4 WT and 2.3D11 IgGl WT were able to induce phagocytosis to a greater extent than 2.3D11 IgG4 double mutant. These data demonstrate that enhanced potency of the Fc-variants in this assay correlates with enhanced affinity to CD32a as shown in Table 1.

Collectively, these data support the importance of CD32a in 2.3D11 IgG4 WT or 2.3D11 IgG4 double mutant mediated phagocytosis and the notion that tumors rich in both CD32a and CD47 expression have a higher likelihood of responding to anti-CD47 therapy. Example 14: 2.3D11 Complexed with CD47-Fc confers enhanced affinity to CD32a

(FCGR2A)

A cell microarray was used to evaluate the affinity of 2.3D11 complexed with CD47-Fc for approximately 4400 proteins, including CD32a (FCGR2A).

Briefly, a screen was performed with recombinant biotinylated CD47-Fc protein pre- complexed with either 2.3D11 IgG4 WT or hIgG4 isotype control at a ~2: 1 Antibody:rhCD47 molar ratio and incubated with fixed HEK293 cells overexpressing a library of surface proteins including various FcyR receptors: CD64a (FCGR1A), CD32a (FCGR2A), and CDl6a

(FCGR3A), in addition to IGHG3 (immunoglobulin heavy constant gamma 3). Streptavidin- AF647 detection was used to evaluate biotinylated CD47-Fc binding to HEK293 cells and the degree of binding was characterized as weak, medium, or strong.‘NG denotes no protein interaction above background.

As shown in Table 2, CD47-Fc pre-complexed with 2.3D11 IgG4 WT or a second anti- CD47 antibody“A” (mAb A) that has a human IgG4 Fc comprising a single S228P mutation (EU numbering), demonstrated a higher degree of binding to CD32a (FCGR2A) as compared to CD47-Fc pre-complexed with h!gG4 isotype control.

Table 2

These data support the hypothesis that 2.3D11 bound to CD47 results in antibody clustering/complex formation allowing the Fc portion of 2.3D 11 IgG4 WT to overcome an affinity threshold and engage with the CD32a FcyR. Of the Fc receptors evaluated in this assay, this phenomenon was only observed with the CD32a FcyR (FCGR2a) and not CD64 (FCGR1A) or CD16 (FCGR3A). In a cellular system, it is foreseeable that when the IgG4 and IgG4 double- mutant variants of antibody 2.3D11 are bound to target CD47, the strength of this interaction may be enhanced due to avidity effects. Levels of CD47 on the target cell may influence the ability of anti-CD47 antibodies to engage with FcyR (namely CD32a) to initiate subsequent tumor cell phagocytosis or to act as a scaffold for anti-CD47-mediated tumor cell death. These data support the data shown in Figure 4A-C demonstrating that that CD47 expression on the target cell is required for pCD32a induction by antibody 2.3D11.

Example 15: CD32a Blockade Abrogates 2.3D11 IgG4 WT and 2.3D11 IgG4 double mutant Mediated Phagocytosis and Cell Death

To evaluate the effect of CD32a on antibody 2.3D11 mediated phagocytosis and cell death, phagocytosis and cell death assays were performed with 2.3D 11 Fc variants (described in Example 12) following blockade of CD32a with a blocking antibody.

Briefly, human macrophages were pre-incubated with 1 Opg/mL CD32a blocking antibody (IV.3 Stem Cell Technologies Catalog #60012) or isotype control mIgG2b antibody for 20 minutes at 37°C prior to adding CFSE-labeled Jurkat target cells with and without 10pg/m L 2.3D11 antibodies (with different Fc variants) similar to that described in Example 4. After a 2 hour incubation at 37°C, phagocytosis and cell death readouts were evaluated by flow cytometry as described in Example 5.

As shown in Figure 16A and 16B, antibody blockade of CD32a (IV.3) abrogated 2.3D11 IgG4 WT and 2.3D11 IgG4 double mutant mediated cell death and phagocytosis but not 2.3D11 IgGl WT mediated cell death and phagocytosis. These data demonstrate that the human IgG4 variant of 2.3D11 has a differential dependence on CD32a compared to the human IgGl variant of 2.3D11 (2.3D11 IgGl WT ) and that antibodies and Fc-bearing proteins targeting CD47 that have an IgG4 backbone are uniquely dependent CD32a engagement.

Example 16: CD64 Blockade Minimally Impacts 2.3D11 Mediated Phagocytosis

The effect of CD64 on 2.3D11 mediated phagocytosis was evaluated by performing phagocytosis assays with 2.3D11 IgG4 WT following blockade of CD64 with a blocking antibody.

Briefly, human macrophages were pre-treated with l0pg/ml of anti-CD64 (Biolegend #305002) or anti-CD32a blocking antibody (IV.3, Stem Cell Technologies Catalog #60012) or isotype controls for 30 minutes at 37°C. Cell Trace Violet (CTV) labeled target Jurkat cells were then added with a dose response of hIgG4 isotype control or 2.3D11 IgG4 WT antibody. Cells were co-cultured for 2h at 37°C and phagocytosis was evaluated by flow cytometry as previously described.

As shown in Figure 17, anti-CD64 blocking antibody did not significantly impact 2.3D11 IgG4 WT mediated phagocytosis. This is in contrast to the dramatic impact observed when an anti-CD32a blocking antibody was used to abrogate 2.3D 11 IgG4 WT mediated phagocytosis (Figures 16 and 17, and Example 4).

Taken together, these data, in concert with the data from Figure 12, reinforce the dominance of the CD32a receptor over other Fc receptors in mediating the anti-tumor activity of antibody 2.3D11 IgG4 WT and 2.3D11 IgG4 double mutant.

Example 17: Co-Expression of CD32a and CD47 in Tumors Indicates Patients Likely to Respond to Therapy with Antibody 2.3D11

The expression of CD32a and CD47 was evaluated in multiple tumor types (Figure 18).

Briefly, gene expression values (in transcripts per million, or TPM) and annotations for patients and samples were obtained from the TCGA Pan-cancer Atlas

(https://gdc.cancer.gov/about-data/publications/panimmune). Expression values for the same gene symbol were averaged, as were values for the same sample type (e.g., primary, metastasis) in the occasional cases with multiple samples per patient. These values were then converted to log(2) scale. Normal samples were excluded. Data are reported as Mean CD32a expression ± SD (standard deviation vs. Mean CD47 expression ± SD.

Across multiple tumor types evaluated within this dataset, various tumor subtypes stand out as being enriched for both CD32a and CD47 transcript levels. In particular, lung, ovarian, glioblastoma, acute myeloid leukemia, mesothelioma and sarcoma are among cancer sub-types enriched in both CD32a and CD47. Therefore, CD32a and CD47 co-expression in these cancer sub-types, among others, may indicate patients likely to respond to 2.3D 11 therapy, as well as CD47 antagonists having human IgG4 isotype or mutant IgG4 isotype. SUMMARY OF SEQUENCES

Claims

CLAIMS What is claimed:

1. A method of identifying a subject likely to respond to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, wherein the Fc domain comprises a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain, the method comprising:

detecting the presence of CD32a in a sample obtained from the subject, wherein the presence of CD32a indicates the subject is likely to respond to treatment with the agent.

2. The method of claim 1, wherein the sample is from a subject having, or at risk of having a cancer.

3. The method of any one of claims 1 or 2, wherein the sample is from a subject having cancer.

4. The method of claim 3, wherein the sample is a tumor sample.

5. The method of any one of claims 1-4, wherein detecting the presence of CD32a comprises determining an amount of CD32a in the sample, wherein the amount of CD32a indicates the subject is likely to respond to treatment with the agent.

6. The method of claim 5, wherein determining the amount of CD32a comprises comparing the amount of CD32a in the sample relative to a reference amount of CD32a.

7. The method of claim 6, wherein when the amount of CD32a in the sample is increased relative to the reference amount of CD32a indicates the patient is likely to respond to treatment with the agent.

8. The method of any one of claims 1-7, further comprising detecting the presence of CD47 in the sample, wherein the presence of CD47 indicates the subject is likely to respond to treatment with the agent.

9. The method of claim 8, wherein detecting the presence of CD47 comprises determining an amount of CD47 in the sample, wherein the amount of CD47 indicates the subject is likely to respond to treatment with the agent.

10. The method of claim 8, wherein determining the amount of CD47 comprises comparing the amount of CD47 in the sample relative to a reference amount of CD47.

11. The method of claim 10, wherein when the amount of CD47 in the sample is increased relative to the reference amount of CD47 indicates the patient is likely to respond to treatment with the agent.

12. The method of any one of claims 1-11, further comprising detecting the presence of CD32b in the sample.

13. The method of claim 12, wherein detecting the presence of CD32b comprises determining an amount of CD32b in the sample.

14. The method of claim 13, wherein determining the amount of CD32b comprises comparing the amount of CD32b in the sample relative to a reference amount of CD32b.

15. The method of claim 14, wherein when the amount of CD32b in the sample is decreased relative to the reference amount of CD32b indicates the patient is likely to respond to treatment with the agent.

16. The method of claim 14, wherein when the amount of CD32b in the sample is decreased relative to the amount of CD32a in the sample indicates the patient is likely to respond to treatment with the agent.

17. The method of any one of claims 1-4, wherein detecting the presence of CD32a comprises determining an expression level of CD32a in the sample, wherein the expression level of CD32a indicates the subject is likely to respond to treatment with the agent.

18. The method of claim 17, wherein determining the expression level of CD32a comprises comparing the expression level of CD32a in the sample relative to a reference expression level of CD32a.

19. The method of claim 18, wherein when the expression level of CD32a in the sample is increased relative to the reference expression level of CD32a indicates the subject is likely to respond to treatment with the agent.

20. The method of any one of claims 17-19, further comprising detecting the presence of CD47 in the sample, wherein the presence of CD47 indicates the subject is likely to respond to treatment with the agent.

21. The method of claim 20, wherein detecting the presence of CD47 comprises determining an expression level of CD47 in the sample, wherein the expression level of CD47 indicates the subject is likely to respond to treatment with the agent.

22. The method of claim 21, wherein determining the expression level of CD47 comprises comparing the expression level of CD47 in the sample relative to a reference expression level of CD47.

23. The method of claim 22, wherein when the expression level of CD47 in the sample is increased relative to the reference expression level of CD47 indicates the patient is likely to respond to treatment with the agent.

24. The method of any one of claims 1-23, further comprising detecting the presence of CD32b in the sample.

25. The method of claim 24, wherein detecting the presence of CD32b comprises determining an expression level of CD32b in the sample.

26. The method of claim 25, wherein determining the expression level of CD32b comprises comparing the expression level of CD32b relative to a reference expression level of CD32b.

27. The method of claim 26, wherein when the expression level of CD32b in the sample is decreased relative to the reference expression level of CD32b indicates the patient is likely to respond to treatment with the agent.

28. The method of claim 26, wherein when the expression level of CD32b in the sample is decreased relative to the expression level of CD32a in the sample indicates the patient is likely to respond to treatment with the agent.

29. The method of any one of claims 1-28, further comprising detecting the presence of one or more polypeptides in the sample selected from the group consisting of: CDl6a, CDl6b, CD32c, CD64, CD68, CD163, PD-l, PD-L1, SIRPa, and a combination thereof.

30. The method of claim 29, wherein the polypeptide is CDl6a, CDl6b or a combination thereof.

31. The method of any one of claims 29 or 30, wherein the polypeptide is CD64.

32. The method of any one of claims 29-31, wherein the polypeptide is SIRPa.

33. A method of identifying a cancer patient likely to respond to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, wherein the Fc domain comprises a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain, the method comprising: contacting a tumor sample from the patient with an antibody that specifically binds to CD32a, or antigen-binding fragment thereof, thereby forming an antibody-CD32a complex; and detecting the presence of the antibody-CD32a complex, wherein the presence of the complex indicates the patient is likely to respond to treatment.

34. The method of claim 33, wherein detecting the presence of the antibody-CD32a complex comprises determining an amount of antibody-CD32a complex in the sample, wherein the amount of antibody-CD32a complex relative to a control indicates the subject is likely to respond to treatment with the agent.

35. The method of claim 34, wherein when the amount of antibody-CD32a complex in the sample is increased relative to the control indicates the patient is likely to respond to treatment with the agent.

36. The method of any one of claims 33-35, further comprising detecting the presence of CD47 in the sample, wherein the presence of CD47 indicates the subject is likely to respond to treatment with the agent.

37. The method of claim 36, wherein detecting the presence of CD47 comprises determining an amount or expression level of CD47 in the sample, wherein the amount or expression level of CD47 in the sample, relative to a reference amount or reference expression level of CD47, indicates the subject is likely to respond to treatment with the agent.

38. The method of claim 37, wherein when the amount or expression level of CD47 in the sample is increased relative to the reference amount or reference expression level of CD47 indicates the patient is likely to respond to treatment with the agent.

39. The method of any one of claims 33-38, further comprising contacting the sample with an antibody that specifically binds CD47, or antigen-binding fragment thereof, thereby forming an antibody-CD47 complex; and detecting the presence of the antibody-CD47 complex, wherein the presence of the complex indicates the patient is likely to respond to treatment.

40. The method of claim 39, wherein when the amount antibody-CD47 complex in the sample is increased relative to a control indicates the patient is likely to respond to treatment with the agent.

41. The method of any one of claims 33-40, further comprising detecting the presence of CD32b in the sample.

42. The method of claim 41, wherein detecting the presence of CD32b comprises

determining an amount or expression level of CD32b in the sample relative to a reference amount or reference expression level.

43. The method of claim 42, wherein when the amount or expression level of CD32b in the sample is decreased relative to the reference amount or reference expression level of CD32b indicates the patient is likely to respond to treatment with the agent.

44. The method of claim 42, wherein when the amount or expression level of CD32b in the sample is decreased relative to the amount of antibody-CD32a complex indicates the patient is likely to respond to treatment with the agent.

45. The method of any one of claims 33-40, further comprising contacting the sample with an antibody that specifically binds CD32b, or antigen-binding fragment thereof, thereby forming an antibody-CD32b complex; and detecting the presence of the antibody-CD32b complex.

46. The method of claim 45, wherein when the amount of antibody-CD32b complex in the sample is decreased relative to the amount of antibody-CD32a complex in the sample indicates the patient is likely to respond to treatment with the agent.

47. The method of any one of claims 33-46, further comprising detecting the presence of one or more polypeptides in the tumor sample selected from the group consisting of: CDl6a, CDl6b, CD32c, CD64, CD68, CD163, SIRPa, PD-l, PD-L1, and a combination thereof.

48. The method of claim 47, wherein the polypeptide is CDl6a, CDl6b or a combination thereof.

49. The method of any one of claims 47 or 48, wherein the polypeptide is CD64.

50. The method of any one of claims 47-49, wherein the polypeptide is SIRPa.

51. A method of detecting a tumor susceptible to treatment with an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain, wherein the Fc domain comprises a human IgG4 wild-type Fc domain or a human IgG4 mutant Fc domain, the method comprising:

determining an expression level of a panel of polypeptides comprising CD32a, CD32b, and CD47 in a tumor sample, wherein the expression level of the panel polypeptides in the tumor sample indicates the tumor is susceptible to treatment with the agent.

52. The method of claim 51, wherein determining an expression level of the panel of polypeptides comprises comparing the expression levels of the panel of polypeptides in the tumor sample to an expression level of the panel of polypeptides in a reference sample.

53. The method of any one of claims 51 or 52, wherein when the expression level of CD32a in the tumor sample is increased relative to the reference sample indicates the tumor is susceptible to treatment with the agent.

54. The method of any one of claims 51-53, wherein when the expression level of CD47 in the tumor sample is increased relative to the reference sample indicates the tumor is susceptible to treatment with the agent.

55. The method of any one of claims 51-54, wherein when the expression level of CD32b in the tumor sample is decreased relative to the reference sample indicates the tumor is susceptible to treatment with the agent.

56. The method of any one of claims 51 or 52, wherein when the expression level of CD32a and CD47 in the tumor sample is increased relative to the reference sample indicates the tumor is susceptible to treatment with the agent.

57. The method of any one of claims 51 or 52, wherein when the expression level of CD32a and CD47 in the tumor sample is increased and the expression level of CD32b is decreased relative to the reference sample indicates the tumor is susceptible to treatment with the agent.

58. The method of any one of claims 51-57, further comprising determining an expression level of one or more polypeptides in the tumor sample selected from the group consisting of: CDl6a, CDl6b, CD32c, CD64, CD68, CD163, SIRPa, PD-l, PD-L1, and a combination thereof.

59. The method of claim 58, wherein the polypeptide is CDl6a, CDl6b or a combination thereof.

60. The method of any one of claims 58 or 59, wherein the polypeptide is CD64.

61. The method of any one of claims 58-60, wherein the polypeptide is SIRPa.

62. The method of any one of claims 1-61, wherein the sample comprises tumor- infiltrating immune cells, tumor cells, stromal cells, or a combination thereof.

63. The method of claim 62, wherein the tumor- infiltrating immune cells comprise macrophages, monocytes, neutrophils, dendritic cells, NK cells, innate lymphoid cells, B cells , or any combination thereof.

64. The method of any one of claims 1-63, wherein the sample comprises a fresh tissue.

65. The method of any one of claims 1-63, wherein the sample comprises a fixed tissue.

66. The method of claim 65, wherein the sample is frozen or is formalin-fixed and embedded in paraffin wax (FFPE).

67. The method of any one of claims 1-66, wherein the detecting or determining is by IHC, IF, flow cytometry, ELISA, or immunoblotting.

68. The method of claim 67, wherein the detecting or determining is by IHC staining.

69. The method of claim 68, wherein the IHC staining is determined on the same sample.

70. The method of claim 68, wherein the IHC staining is determined on separate tissue sections of the same sample.

71. The method of any one of claims 33-50, where the antibody is covalently linked to a chromogenic molecule or a fluorescent molecule.

72. The method of any one of claims 33-50, wherein the complex is contacted with a secondary antibody, or antigen-binding fragment thereof, covalently linked to a chromogenic molecule or a fluorescent molecule.

73. The method of any one of claims 1-72, wherein the cancer is selected from multiple myeloma (MM), acute myeloid leukemia (AML)/ myelodysplastic syndrome (MDS), T cell lymphoma (TCL), non-Hodgkin’s lymphoma (NHL), chronic lymphocytic leukemia (CLL), ovarian carcinoma.

74. The method of any one of claims 1-72, wherein the cancer is selected from colorectal cancer, gastric cancer, head and neck cancer, renal cell carcinoma, urothelial/bladder carcinoma, ovarian carcinoma, myeloma, melanoma, lung cancer, squamous cell carcinoma, classical Hodgkin's lymphoma, breast cancer, small cell lung cancer, salivary gland carcinoma, vulvar carcinoma, thyroid carcinoma, anal canal carcinoma, biliary carcinoma, mesothelioma, cervical carcinoma, sarcoma, glioblastoma, and neuroendocrine carcinoma.

75. The method of any one of claims 1-74, further comprising selecting a treatment for the subject or patient.

76. The method of any one of claims 1-74, further comprising administering a treatment to the subject or patient.

77. The method of any one of claims 75 or 76, wherein the treatment comprises an agent that specifically binds to CD47, wherein the agent comprises an antibody or polypeptide comprising an Fc domain.

78. The method of claim 77, wherein agent inhibits the interaction between CD47 and SIRPa.

79. The method of any one of claims 77 and 78, wherein the agent provides an anti-tumor activity.

80. The method of any one of claims 77-79, wherein the anti-tumor activity is selected from:

(i) an induction of phagocytosis of tumor cells;

(ii) an induction of cell death of tumor cells; and

(iii) a combination of (i) and (ii).

81. The method of claim 79, wherein the anti-tumor activity comprises an induction of phagocytosis of tumor cells and an induction of cell death of tumor cells.

82. The method of any one of claims 77-81, wherein the agent specifically binds human CD47 expressed on tumor cells.

83. The method of any one of claims 77-82, wherein the agent specifically binds human CD32a expressed on tumor- infiltrating immune cells.

84. The method of claim 83, wherein the tumor-infiltrating immune cells comprise macrophages, monocytes, neutrophils, dendritic cells, NK cells, innate lymphoid cells, B cells, or any combination thereof

85. The method of any one of claims 83 or 84, wherein binding of the agent to human CD32a induces phagocytosis of tumor cells expressing CD47.

86. The method of any one of claims 77-85, wherein the agent is a monoclonal antibody that specifically binds human CD47, or antigen binding fragment thereof.

87. The method of any one of claims 77-85, wherein the agent is a SIRPa-Fc fusion protein.

88. The method of any one of claims claim 86 or 87, wherein the Fc domain is a human IgG4 wild-type Fc domain.

89. The method of any one of claims 86 or 87, wherein the Fc domain is a human IgG4 mutant Fc domain.

90. The method of any one of claims 77-89, wherein the Fc domain is mutated to increase binding to CD32a or to decrease binding to CD32b.

91. The method of any one of claims 77-89, wherein CD32a contains a polymorphism that affects binding to an Fc domain.

92. The method of any one of claims 86 or 88-91, wherein the antibody comprises:

a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 6;

a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 7; a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 8;

a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 9;

a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 10.

93. The method of any one of claims 86 or 88-91, wherein the antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 4.

94. The method of any one of claims 86 or 88-91, wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16.

95. The method of claim 94, wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 13; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 16.

96. A kit comprising a container comprising one or more reagents for detecting the presence of CD32a, optionally CD47 and/or CD32b in a tumor sample, and a package insert comprising instructions for determining an amount or expression level of CD32a, optionally CD47 and/or CD32b in the tumor sample.

97. The kit of claim 96, comprising a reagent for detecting the presence of CD47 in the sample and instructions for determining an amount or expression level of CD47 in the tumor sample

98. The kit of any one of claims 96 or 97, comprising a reagent for detecting the presence of CD32b in the sample and instructions for determining an amount or expression level of CD32b in the tumor sample.

99. The kit of any one of claims 96-98, further comprising a reagent for detecting the presence of one or more polypeptides in the sample selected from the group consisting of CDl6a, CDl6b, CD32a, CD32b, CD32c, CD47, CD64, CD68, CD163, PD-l, PD-L1, SIRPa, or a combination thereof, and instructions for determining an amount or expression level of the one or more polypeptides in the tumor sample.

100. The kit of any one of claims 96-99, wherein determining an amount or expression level of CD32a, optionally CD47 and/or CD32b, or one or more polypeptides selected from the group consisting of CDl6a, CDl6b, CD32a, CD32b, CD32c, CD47, CD64, CD68, CD163, PD-l, PD- Ll, SIRPa, or a combination thereof comprises comparing the amount or expression level of CD32a, optionally CD47 and/or CD32b, or the one or more polypeptides in a tumor sample relative to the amount or expression level in a reference sample.

101. The kit of any one of claims 96-100, wherein the reagent is an antibody specifically reactive with CD32a, optionally an antibody specifically reactive with CD47 and/or an antibody specifically reactive with CD32b, or an antibody specifically reactive with one more

polypeptides selected from the group consisting of CDl6a, CDl6b, CD32a, CD32b, CD32c, CD47, CD64, CD68, CD163, PD-l, PD-L1, SIRPa, or a combination thereof.

102. The kit of claim 101, wherein the one or more antibodies is covalently attached to a label.

103. The kit of claims 102, wherein the label is a fluorescent molecule or a chromogenic molecule.

104. The kit of any one of claims 102 or 103, further comprising a reagent for detecting a complex formed upon binding of the one or more antibodies to CD32a, optionally CD47 and/or CD32a, or one more polypeptides selected from the group consisting of CDl6a, CDl6b, CD32a, CD32b, CD32c, CD47, CD64, CD68, CD163, PD-l, PD-L1, SIRPa, or a combination thereof, and instructions for determining an amount of one or more complexes in the sample.

105. A method for treating cancer in a subject in need thereof, comprising

(i) detecting the presence and/or expression level of CD32a in a sample obtained from the subject; and

(ii) administering to the subject a monoclonal antibody that specifically binds human CD47, or antigen binding fragment thereof,

wherein the antibody comprises:

a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 7;

106. A method for treating cancer in a subject in need thereof, comprising

wherein the antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 4.

107. A method for treating cancer in a subject in need thereof, comprising

wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16.

108. A method for treating cancer in a subject in need thereof, comprising

wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 13; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 16.

109. The method of any one of claims 105-108, wherein the CD32a comprises a 131R single nucleotide polymorphism (SNP).

110. The method of any one of claims 105-108, wherein the CD32a comprises a 131H SNP.

111. The method of any one of claims 105-110, further comprising detecting the presence of CD47 in the sample.

112. A method for treating cancer in a subject in need thereof, comprising

administering to the subject a monoclonal antibody that specifically binds human CD47, or antigen binding fragment thereof, wherein the antibody comprises:

a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 5; a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 6;

a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 10,

wherein cells in a tumor sample from the subject express CD32a.

113. A method for treating cancer in a subject in need thereof, comprising

administering to the subject a monoclonal antibody that specifically binds human CD47, or antigen binding fragment thereof, wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16, and wherein cells in a tumor sample from the subject express CD32a.

114. A method for treating cancer in a subject in need thereof, comprising

administering to the subject a monoclonal antibody that specifically binds human CD47, or antigen binding fragment thereof, wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 13; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 16, and wherein cells in a tumor sample from the subject express CD32a.

115. The method of any one of claims 112-114, wherein cancer cells in the tumor sample express CD47.