WO2022037672A1

WO2022037672A1 - Anti-cd47 antibodies and uses thereof

Info

Publication number: WO2022037672A1
Application number: PCT/CN2021/113729
Authority: WO
Inventors: Yi Yang; Lei Chen; Yunyun CHEN; Yanan GUO; Yuelei SHEN
Original assignee: Doma Biopharmaceutical (Suzhou) Co., Ltd
Priority date: 2020-08-21
Filing date: 2021-08-20
Publication date: 2022-02-24

Abstract

Provided are anti-CD47 antibodies, antigen-binding fragments, and the uses thereof.

Description

ANTI-CD47 ANTIBODIES AND USES THEREOF

CLAIM OF PRIORITY

This application claims priority to PCT/CN2020/110554, filed on August 21, 2020. The entire contents of the foregoing are incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to anti-CD47 (integrin associated protein) antibodies, antigen-binding fragments, and the uses thereof.

BACKGROUND

Cancer is currently one of the diseases that have the highest human mortality. According to the World Health Organization statistical data, in 2012, the number of global cancer incidence and death cases reached 14 million and 8.2 million, respectively. In China, the newly diagnosed cancer cases are 3.07 million, and the death toll is 2.2 million.

Recent clinical and commercial success of anticancer antibodies has created great interest in antibody-based therapeutics. There is a need to develop anti-cancer antibodies for use in various antibody-based therapeutics to treat cancers.

SUMMARY

This disclosure relates to anti-CD47 (integrin associated protein; also known as “lAP” ) antibodies, antigen-binding fragment thereof, and the uses thereof.

In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof that binds to CD47 comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising

CDRs

1, 2, and 3, in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence; in some embodiments the selected

VH CDRs

1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:

(1) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;

(2) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7, 8, 9, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively;

(3) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13, 14, 15, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, 18, respectively;

(4) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19, 20, 21, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 23, 24, respectively;

(5) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 25, 26, 27, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 28, 29, 30, respectively;

(6) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 32, 33, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 34, 35, 36, respectively;

(7) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 37, 38, 39, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 40, 41, 42, respectively; and

(8) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 110, 3, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively.

In some embodiments, the VH comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3 respectively, and the VL comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.

In some embodiments, the VH comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and the VL comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.

In some embodiments, the VH comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the VL comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively.

In some embodiments, the VH comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, and the VL comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively.

In some embodiments, the VH comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively, and the VL comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively.

In some embodiments, the VH comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively, and the VL comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively.

In some embodiments, the VH comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively, and the VL comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively.

In some embodiments, the VH comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 110, and 3, respectively, and the VL comprises

CDRs

In some embodiments, the antibody or antigen-binding fragment specifically binds to human CD47. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) .

In one aspect, the disclosure is related to a nucleic acid comprising a polynucleotide encoding a polypeptide comprising:

(1) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 92, 93, 94, 95, or 97 binds to CD47;

(2) an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 89, 90, 91, or 96 binds to CD47;

(3) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising

CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 99 binds to CD47;

(4) an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 98 binds to CD47;

(5) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising

CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 101 binds to CD47;

(6) an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 100 binds to CD47;

(7) an immunoglobulin heavy chain or a fragment thereof comprising a

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20, 21, respectively, and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 103 binds to CD47;

(8) an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23, 24, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 102 binds to CD47;

(9) an immunoglobulin heavy chain or a fragment thereof comprising a

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 25, 26, 27, respectively, and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 105 binds to CD47;

(10) an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 29, 30, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 104 binds to CD47;

(11) an immunoglobulin heavy chain or a fragment thereof comprising a

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 31, 32, 33, respectively, and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 107 binds to CD47;

(12) an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 34, 35, 36, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 106 binds to CD47;

(13) an immunoglobulin heavy chain or a fragment thereof comprising a

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 37, 38, 39, respectively, and in some embodiments, the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 109 binds to CD47; or

(14) an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 40, 41, 42, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 108 binds to CD47.

In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively.

In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively.

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively.

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively.

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively.

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively.

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively.

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively.

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively.

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively.

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively.

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively.

In some embodiments, the VH when paired with a VL specifically binds to human CD47, or the VL when paired with a VH specifically binds to human CD47.

In some embodiments, the immunoglobulin heavy chain or the fragment thereof is a humanized immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or the fragment thereof is a humanized immunoglobulin light chain or a fragment thereof.

In some embodiments, the nucleic acid encodes a single-chain variable fragment (scFv) .

In some embodiments, the nucleic acid is cDNA.

In one aspect, the disclosure is related to a vector comprising one or more of the nucleic acids as described herein.

In one aspect, the disclosure is related to a vector comprising two of the nucleic acids described herein. In some embodiments, the vector encodes the VL region and the VH region that together bind to CD47.

In one aspect, the disclosure is related to a pair of vectors. In some embodiments, each vector comprises one of the nucleic acids as described herein. In some embodiments, together the pair of vectors encodes the VL region and the VH region that together bind to CD47.

In one aspect, the disclosure is related to a cell comprising the vector or the pair of vectors as described herein. In some embodiments, the cell is a CHO cell.

In one aspect, the disclosure is related to a cell comprising one or more of the nucleic acids as described herein.

In one aspect, the disclosure is related to a cell comprising two of the nucleic acids as described herein. In some embodiments, the two nucleic acids together encode the VL region and the VH region that together bind to CD47.

In one aspect, the disclosure is related to a method of producing an antibody or an antigen-binding fragment thereof, the method comprising (a) culturing the cell as described herein under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment; and (b) collecting the antibody or the antigen-binding fragment produced by the cell.

In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof that binds to CD47 comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%identical to a selected VL sequence, in some embodiments, the selected VH sequence and the selected VL sequence are one of the following:

(1) the selected VH sequence is SEQ ID NO: 89, 90, 91, or 96, and the selected VL sequence is SEQ ID NO: 92, 93, 94, 95, or 97;

(2) the selected VH sequence is SEQ ID NO: 98, and the selected VL sequence is SEQ ID NO: 99;

(3) the selected VH sequence is SEQ ID NO: 100, and the selected VL sequence is SEQ ID NO: 101;

(4) the selected VH sequence is SEQ ID NO: 102, and the selected VL sequence is SEQ ID NO: 103;

(5) the selected VH sequence is SEQ ID NO: 104, and the selected VL sequence is SEQ ID NO: 105;

(6) the selected VH sequence is SEQ ID NO: 106, and the selected VL sequence is SEQ ID NO: 107; and

(7) the selected VH sequence is SEQ ID NO: 108, and the selected VL sequence is SEQ ID NO: 109.

In some embodiments, the VH comprises the sequence of SEQ ID NO: 89 and the VL comprises the sequence of SEQ ID NO: 92. In some embodiments, the VH comprises the sequence of SEQ ID NO: 90 and the VL comprises the sequence of SEQ ID NO: 93. In some embodiments, the VH comprises the sequence of SEQ ID NO: 91 and the VL comprises the sequence of SEQ ID NO: 94. In some embodiments, the VH comprises the sequence of SEQ ID NO: 91 and the VL comprises the sequence of SEQ ID NO: 95.

In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof comprising the

VH CDRs

1, 2, 3, and the

VL CDRs

1, 2, 3 of the antibody or antigen-binding fragment thereof as described herein.

In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof as described herein.

In one aspect, the disclosure is related to an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof as described herein covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

In one aspect, the disclosure is related to a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof, or the antibody-drug conjugate as described herein, to the subject.

In some embodiments, the subject has a solid tumor.

In some embodiments, the cancer is hematologic malignancy, and/or relapsed or refractory hematologic malignancy. In some embodiments, the cancer is acute myeloid leukemia, non-Hodgkin's lymphoma, breast cancer, bladder cancer, ovarian cancer, and/or small cell lung cancer tumor.

In one aspect, the disclosure is related to a method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof, or the antibody-drug conjugate as described herein.

In one aspect, the disclosure is related to a method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof, or the antibody-drug conjugate as described herein.

In one aspect, the disclosure is related to a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described herein, and a pharmaceutically acceptable carrier.

In one aspect, the disclosure is related to a pharmaceutical composition comprising the antibody drug conjugate as described herein, and a pharmaceutically acceptable carrier.

In some embodiments, the antibody described herein is a IgG4 antibody. In some embodiments, the antibody described herein is a human IgG4 antibody.

In one aspect, the disclosure is related to an IgG4 antibody or antigen-binding fragment thereof that binds to CD47 comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising

CDRs

1, 2, and 3, in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence. In some embodiments, the selected

VH CDRs

1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are selected from one of the antibodies as set forth in FIG. 23 and FIG. 24.

In some embodiments, the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively.

In some embodiments, the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 110, and 3, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively.

In some embodiments, the antibody described herein is a human IgG4 antibody.

In one aspect, the disclosure is related to an IgG4 antibody or antigen-binding fragment thereof that binds to CD47 comprising: a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%identical to a selected VL sequence, in some embodiments, the selected VH sequence and the selected VL sequence are selected from one of the antibodies as set forth in FIG. 26 and FIG. 27.

In some embodiments, the selected VH sequence is SEQ ID NO: 89, and the selected VL sequence is SEQ ID NO: 92. In some embodiments, the antibody described herein is a human IgG4 antibody.

In some embodiments, Kabat numbering is used in the present disclosure. In some embodiments, Chothia numbering is used in the present disclosure.

As used herein, the term “cancer” refers to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, irrespective ofhistopathologic type or stage of invasiveness. Also included are malignancies of the various organ systems, such as respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal, and endocrine systems; as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, and cancer of the small intestine. Cancer that is “naturally arising” includes any cancer that is not experimentally induced by implantation of cancer cells into a subject, and includes, for example, spontaneously arising cancer, cancer caused by exposure of a patient to a carcinogen (s) , cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancer caused by infections, e.g., viral infections. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation. The term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin. A hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.

As used herein, the term “antibody” refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies) , single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody can contain an Fc region of a human antibody. The term antibody also includes derivatives, e.g., bi-specific antibodies, single-chain antibodies, diabodies, linear antibodies, and multi-specific antibodies formed from antibody fragments.

As used herein, the term “antigen-binding fragment” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of light chain) . Non-limiting examples of antibody fragments include, e.g., Fab, Fab', F (ab') ₂, and Fv fragments.

As used herein, the term “human antibody” refers to an antibody that is encoded by an endogenous nucleic acid (e.g., rearranged human immunoglobulin heavy or light chain locus) present in a human. In some embodiments, a human antibody is collected from a human or produced in a human cell culture (e.g., human hybridoma cells) . In some embodiments, a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line) . In some embodiments, a human antibody is produced in a bacterial or yeast cell. In some embodiments, a human antibody is produced in a transgenic non-human animal (e.g., a bovine) containing an unrearranged or rearranged human immunoglobulin locus (e.g., heavy or light chain human immunoglobulin locus) .

As used herein, the term “chimeric antibody” refers to an antibody that contains a sequence present in at least two different antibodies (e.g., antibodies from two different mammalian species such as a human and a mouse antibody) . A non-limiting example of a chimeric antibody is an antibody containing the variable domain sequences (e.g., all or part of a light chain and/or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and the constant domains of a human antibody. Additional examples of chimeric antibodies are described herein and are known in the art.

As used herein, the term “humanized antibody” refers to a non-human antibody which contains minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and contains sequences derived from a human immunoglobulin. In non-limiting examples, humanized antibodies are human antibodies (recipient antibody) in which hypervariable (e.g., CDR) region residues of the recipient antibody are replaced by hypervariable (e.g., CDR) region residues from a non-human antibody (e.g., a donor antibody) , e.g., a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capacity. In some embodiments, the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, humanized antibodies may contain residues which are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance. In some embodiments, the humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-human (e.g., mouse) immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin. The humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc) , typically, that of a human immunoglobulin. Humanized antibodies can be produced using molecular biology methods known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.

As used herein, the term “single-chain antibody” refers to a single polypeptide that contains at least two immunoglobulin variable domains (e.g., a variable domain of a mammalian immunoglobulin heavy chain or light chain) that is capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

As used herein, the term “multimeric antibody” refers to an antibody that contains four or more (e.g., six, eight, or ten) immunoglobulin variable domains. In some embodiments, the multimeric antibody is able to crosslink one target molecule (e.g., CD47) to at least one second target molecule (e.g., CD20) on the surface of a mammalian cell (e.g., a human cancer cell) .

As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated by the present invention. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old) . In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like) , rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits) , lagomorphs, swine (e.g., pig, miniature pig) , equine, canine, feline, bovine, and other domestic, farm, and zoo animals.

As used herein, when referring to an antibody, the phrases “specifically binding” and “specifically binds” mean that the antibody interacts with its target molecule (e.g., CD47) preferably to other molecules, because the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the target molecule; in other words, the reagent is recognizing and binding to molecules that include a specific structure rather than to all molecules in general. An antibody that specifically binds to the target molecule may be referred to as a target-specific antibody. For example, an antibody that specifically binds to a CD47 molecule may be referred to as a CD47 -specific antibody or an anti-CD47 antibody.

As used herein, the terms “polypeptide, ” “peptide, ” and “protein” are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.

As used herein, the terms “polynucleotide, ” “nucleic acid molecule, ” and “nucleic acid sequence” are used interchangeably herein to refer to polymers of nucleotides of any length of at least two nucleotides, and include, without limitation, DNA, RNA, DNA/RNA hybrids, and modifications thereof.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a flow chart showing the first part of an exemplary protocol of making anti-hCD47 antibodies.

FIG. 2 is a flow chart showing the second part of an exemplary protocol of making anti-hCD47 antibodies.

FIGS. 3A-3C are flow cytometry results showing that the anti-hCD47 antibodies block the binding between hCD47 and hSIRPα. PC and NC are positive and negative controls, respectively.

FIGS. 4A-4B are flow cytometry results showing the binding activity of anti-hCD47 antibodies with human CD47. NC stands for negative control.

FIGS. 5A-5C are flow cytometry results analyzing the anti-hCD47 antibodies' cross-reactivity with CHO-K 1 cells expressing mouse CD47 (CHO-K 1-mCD47) , monkey CD47 (CHO-K1-rCD47) , or human-mouse chimeric CD47 (CHO-K1-cCD47) . NC stands for negative control.

FIG. 6 shows the results of surface plasma resonance (SPR) using the chimeric anti-hCD47 antibody 1A5-mHvKv-IgG4 and human CD47.

FIG. 7 shows red blood cell agglutination induced by serially diluted anti-hCD47 antibodies. Boxed wells indicate a safe concentration of the antibodies.

FIGS. 8A-8B are flow cytometry results showing that the humanized anti-hCD47 antibodies block the binding between hCD47 and hSIRPα. NC stands for negative control. 1A5-mHvKv-IgG4 was used for comparison purpose.

FIGS. 9A-9B are flow cytometry results analyzing the humanized anti-hCD47 antibodies' cross-reactivity with CHO-K1 cells expressing mouse CD47 (CHO-K1-mCD47) , monkey CD47 (CHO-K1-rCD47) , or human-mouse chimeric CD47 (CHO-K1-cCD47) . NC stands for negative control (for both FIG. 9A and FIG. 9B) . 1A5-mHvKv-IgG4 was used for comparison purpose.

FIG. 10 shows the results of surface plasma resonance (SPR) using the humanized anti-hCD47 antibody 1A5-H1K1-IgG4 and human CD47.

FIG. 11 is a graph showing body weight over time of double humanized CD47/SIRPα mice (B-hSIRPα/hCD47 mice) injected with MC-38 tumor cells, and treated with anti-hCD47 antibodies 1A5-mHvKv-IgG4, or Hu5F9-IgG4.

FIG. 12 is a graph showing percentage change of body weight over time of B-hSIRPα/hCD47 mice injected with MC-38 tumor cells, and treated with anti-hCD47 antibodies1A5-mHvKv-IgG4, or Hu5F9-IgG4.

FIG. 13 is a graph showing tumor size over time of B-hSIRPα/hCD47 mice injected with MC-38 tumor cells, and treated with anti-hCD47 antibodies 1A5-mHvKv-IgG4, or Hu5F9-IgG4.

FIG. 14 is a graph showing body weight over time of CD132 knockout mice (B-NDG mice) injected with Raji tumor cells, and treated with antibodies 1A5-mHvKv-IgG4, Hu5F9-IgG4, or Rituximab-IgG1.

FIG. 15 is a graph showing percentage change of body weight over time of B-NDG mice injected with Raji-Luc tumor cells, and treated with antibodies 1A5-mHvKv-IgG4, Hu5F9-IgG4, or Rituximab-IgG1.

FIG. 16 is a graph showing tumor size over time of B-NDG mice injected with Raji-Luc tumor cells, and treated with antibodies 20-1A5-mHvKv-IgG4, Hu5F9-IgG4, or Rituximab-IgG1.

FIG. 17 is a graph showing body weight over time of CD132 knockout mice (B-NDG mice) injected with Raji-Luc tumor cells, and treated with anti-hCD47 antibodies 1A5-mHvKv-IgG4, 1G4-mHvKv-IgG4, 2C12-mHvKv-IgG4, 1D7-mHvKv-IgG4, 3G9-mHvKv-IgG4, 3H9-mHvKv-IgG4, or Hu5F9-IgG4.

FIG. 18 is a graph showing percentage change of body weight over time of B-NDG mice injected with Raji-Luc tumor cells, and treated with anti-hCD47 antibodies 1A5-mHvKv-IgG4, 1G4-mHvKv-IgG4, 2C12-mHvKv-IgG4, 1D7-mHvKv-IgG4, 3G9-mHvKv-IgG4, 3H9-mHvKv-IgG4, or Hu5F9-IgG4.

FIG. 19 is a graph showing tumor size over time of B-NDG/hSIRPα mice injected with Raji-Luc tumor cells, and treated with anti-hCD47 antibodies 1A5-mHvKv-IgG4, 1G4-mHvKv-IgG4, 2C12-mHvKv-IgG4, 1D7-mHvKv-IgG4, 3G9-mHvKv-IgG4, 3H9-mHvKv-IgG4, or Hu5F9-IgG4.

FIG. 20 is a graph showing body weight over time of B-NDG/hSIRPα mice injected with Raji-Luc tumor cells, and treated with anti-hCD47 antibodies 6F2-mHvKv-IgG4, 2B5-mHvKv-IgG4, 1D7-mHvKv-IgG4, 3G9-mHvKv-IgG4, 3H9-mHvKv-IgG4, 4E11-mHvKv-IgG4, or Hu5F9-IgG4.

FIG. 21 is a graph showing percentage change of body weight over time of B-NDG/hSIRPα mice injected with Raji-Luc tumor cells, and treated with anti-hCD47 antibodies 6F2-mHvKv-IgG4, 2B5-mHvKv-IgG4, 1D7-mHvKv-IgG4, 3G9-mHvKv-IgG4, 3H9-mHvKv-IgG4, 4E11-mHvKv-IgG4, or Hu5F9-IgG4.

FIG. 22 is a graph showing tumor size over time of B-NDG/hSIRPα mice injected with Raji-Luc tumor cells, and treated with anti-hCD47 antibodies 6F2-mHvKv-IgG4, 2B5-mHvKv-IgG4, 1D7-mHvKv-IgG4, 3G9-mHvKv-IgG4, 3H9-mHvKv-IgG4, 4E11-mHvKv-IgG4, or Hu5F9-IgG4.

FIG. 23 lists CDR sequences of several anti-hCD47 antibodies and CDR sequences of the humanized anti-hCD47 antibodies thereof as defined by Kabat numbering.

FIG. 24 lists CDR sequences of several anti-hCD47 antibodies and CDR sequences of humanized anti-hCD47 antibodies thereof as defined by Chothia numbering.

FIG. 25 lists amino acid sequences of human CD47 ( “hCD47” ) , mouse CD47 ( “mCD47” ) , monkey CD47 ( “rmCD47” or “rCD47” ) , and chimeric CD47 ( “chiCD47” or “cCD47” ) .

FIG. 26 lists amino acid sequences of heavy chain variable regions and light chain variable regions of humanized antibodies based on 1A5.

FIG. 27 lists the amino acid sequence of the heavy chain variable regions and light chain variable regions of several mouse anti-hCD47 antibodies.

DETAILED DESCRIPTION

The present disclosure provides examples of antibodies, antigen-binding fragments thereof, that bind to CD47 (integrin associated protein; also known as “IAP” ) .

CD47 and Cancer

CD47, also known as integrin associated protein (IAP) , is a transmembrane protein that in humans is encoded by the CD47 gene. CD47 belongs to the immunoglobulin superfamily and partners with membrane integrins and also binds the ligands thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRPα) . It is involved in a range of cellular processes, including apoptosis, proliferation, adhesion, and migration. CD47 is a ～50 kDa heavily glycosylated, ubiquitously expressed membrane protein of the immunoglobulin superfamily with a single IgV-like domain at its N-terminus, a highly hydrophobic stretch with five membrane-spanning segments and an alternatively spliced cytoplasmic C-terminus. Each of the four alternatively spliced cytoplasmic tails exists in vivo at different frequencies, but all lack a substantial signaling domain.

While CD47 was first identified as a membrane protein involved in β3 integrin-mediated signaling on leukocytes, it is now known to also interact with thrombospondin-1, signal regulatory protein-alpha (SIRPα, also known as SIRPA, Sirpα, Sirpa, or CD172A) , and others to regulate various cellular functions including cell migration, axon extension, cytokine production, and T cell activation.

Recent studies have focused most on CD47-SIRPα axis for its inhibitory role in phagocytosis. SIRPα, also known as Src homology 2 domain-containing protein tyrosine phosphatase substrate 1/brain Ig-like molecule with tyrosine-based activation motif/cluster of differentiation antigen-like family member A (SHPS-1/BIT/CD 172a) , is another membrane protein of the immunoglobulin superfamily that is particularly abundant in the myeloid-lineage hematopoietic cells such as macrophages and dendritic cells. The ligation of SIRPα on phagocytes by CD47 expressed on a neighboring cell results in phosphorylation of SIRPα cytoplasmic immunoreceptor tyrosine-based inhibition (ITIM) motifs, leading to the recruitment of SHP-1 and SHP-2 phosphatases. One resulting downstream effect is the prevention ofmyosin-IIA accumulation at the phagocytic synapse and consequently inhibition of phagocytosis. Thus, CD47-SIRPα interaction functions as a negative immune checkpoint to send a “don't eat me” signal to ensure that healthy autologous cells are not inappropriately phagocytosed.

Overexpression of CD47 has been found in nearly all types of tumors, some of which include acute myeloid leukemia, non-Hodgkin's lymphoma, bladder cancer, and breast cancer. While CD47 is implicated in the regulation of cancer cell invasion and metastasis, its most well-studied and important function related to tumor development is prevention ofphagocytosis via ligating with SIRPα on the surrounding phagocytes. Also, CD47 expression on cancer stem cells (CSCs) implies its role in cancer recurrence. It can increase the chance of CSC survival, which in turn could repopulate a new tumor mass and cause a tumor relapse.

CD47 down-regulation is also involved in the clearance of red blood cells (RBCs) and platelets by splenic macrophages, which may cause hemolytic anemia and idiopathic thrombocytopenic purpura, respectively. Thus, when CD47 antagonists are used as therapies, it is also very important to assess its toxicities.

CD47 provides a “do not eat” signal by binding to the N-terminus of signal regulatory protein alpha (SIRPα) on immune cells and suppresses phagocytosis, and it is ubiquitously expressed in human cells and has been found to be overexpressed in many different tumor cells. Thus, targeting CD47 is in the spotlight of cancer immunotherapy. Blocking CD47 triggers the recognition and elimination of cancer cells by the innate immunity. There are at least three CD47 antagonists in phase I clinical trials, including Hu5F9-G4, CC-90002, and TTI-621. These antibodies or binding agents can be used to treat various tumors and cancers, e.g., solid tumors, hematologic malignancies (e.g., relapsed or refractory hematologic malignancies) , acute myeloid leukemia, non-Hodgkin's lymphoma, breast cancer, bladder cancer, ovarian cancer, and small cell lung cancer tumors.

A detailed description of CD47 and its function can be found, e.g., in Liu, Xiaojuan, et al., "Is CD47 an innate immune checkpoint for tumor evasion? . " Journal of Hematology &Oncology 10.1 (2017) : 12; Huang et al., "Targeting CD47: the achievements and concerns of current studies on cancer immunotherapy. " Journal of thoracic disease 9.2 (2017) : E168; and Ansell et al., "A phase 1 study of TTI-621, a novel immune checkpoint inhibitor targeting CD47, in patients with relapsed or refractory hematologic malignancies. " (2016) : 1812-1812 each of which is incorporated by reference herein in its entirety.

The present disclosure provides anti-CD47 antibodies, antigen-binding fragments thereof, and methods of using these anti-CD47 antibodies and antigen-binding fragments to inhibit tumor growth and to treat cancers.

Antibodies and Antigen Binding Fragments

The present disclosure provides anti-CD47 antibodies and antigen-binding fragments thereof that comprise complementary determining regions (CDRs) , heavy chain variable regions, light chain variable regions, heavy chains, or light chains described herein.

In general, antibodies (also called immunoglobulins) are made up of two classes of polypeptide chains, light chains and heavy chains. A non-limiting antibody of the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgA, or IgD or subclasses including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain. An antibody can comprise two identical copies of a light chain and two identical copies of a heavy chain. The heavy chains, which each contain one variable domain (or variable region, V _H) and multiple constant domains (or constant regions) , bind to one another via disulfide bonding within their constant domains to form the “stem” of the antibody. The light chains, which each contain one variable domain (or variable region, V _L) and one constant domain (or constant region) , each bind to one heavy chain via disulfide binding. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bound. The variable regions of both the light chains and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR) .

These hypervariable regions, known as the complementary determining regions (CDRs) , form loops that comprise the principle antigen binding surface of the antibody. The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding region.

Methods for identifying the CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and a number of definitions of the CDRs are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described in, e.g., Martin, "Protein sequence and structure analysis of antibody variable domains, " Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan, et al. "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains, " Molecular immunology 45.14 (2008) : 3832-3839; Wu, T.T. and Kabat, E.A. (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991) ; Morea et al., Biophys Chem. 68 (1-3) : 9-16 (Oct. 1997) ; Morea et al., J Mol Biol. 275 (2) : 269-94 (Jan. 1998) ; Chothia et al., Nature 342 (6252) : 877-83 (Dec. 1989) ; Ponomarenko and Bourne, BMC Structural Biology 7: 64 (2007) ; each of which is incorporated herein by reference in its entirety.

The CDRs are important for recognizing an epitope of an antigen. As used herein, an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen binding domain of an antibody. The minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen’s primary structure, as the epitope may depend on an antigen’s three-dimensional configuration based on the antigen’s secondary and tertiary structure.

In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgD, IgE, IgA) . The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. The sequences and differences of the IgG subclasses are known in the art, and are described, e.g., in Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector functions. " Frontiers in immunology 5 (2014) ; Irani, et al. "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases. " Molecular immunology 67.2 (2015) : 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.

The antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, rat, camelid) . Antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, polyspecific antibodies, and chimeric antibodies that include an immunoglobulin binding domain fused to another polypeptide. The term “antigen binding domain” or “antigen binding fragment” is a portion of an antibody that retains specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody’s target molecule. It includes, e.g., Fab, Fab′, F (ab′) 2, and variants of these fragments. Thus, in some embodiments, an antibody or an antigen binding fragment thereof can be, e.g., a scFv, a Fv, a Fd, a dAb, a bispecific antibody, a bispecific scFv, a diabody, a linear antibody, a single-chain antibody molecule, a multi-specific antibody formed from antibody fragments, and any polypeptide that includes a binding domain which is, or is homologous to, an antibody binding domain. Non-limiting examples of antigen binding domains include, e.g., the heavy chain and/or light chain CDRs of an intact antibody, the heavy and/or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact antibody.

In some embodiments, the antigen binding fragment can form a part of a chimeric antigen receptor (CAR) . In some embodiments, the chimeric antigen receptor are fusions of single-chain variable fragments (scFv) as described herein, fused to CD3-zeta transmembrane-and endodomain. In some embodiments, the chimeric antigen receptor also comprises intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) . In some embodiments, the chimeric antigen receptor comprises multiple signaling domains, e.g., CD3z-CD28-41BB or CD3z-CD28-OX40, to increase potency. Thus, in one aspect, the disclosure further provides cells (e.g., T cells) that express the chimeric antigen receptors as described herein.

In some embodiments, the scFV has one heavy chain variable domain, and one light chain variable domain. In some embodiments, the scFV has two heavy chain variable domains, and two light chain variable domains. In some embodiments, the scFV has two antigen binding regions, and the two antigen binding regions can bind to the respective target antigens.

Anti-CD47 Antibodies and Antigen-Binding Fragments

The disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to CD47. In some embodiments, the antibodies and antigen-binding fragments described herein are capable of binding to CD47 and block the binding of CD47 and its ligands (e.g., SIRPα, thrombospondin, or integrin) . In some embodiments, the antibodies and antigen-binding fragments described herein are capable of binding to CD47 and block the binding of CD47 and SIRPα thereby promoting phagocytosis. These antibodies can be agonists or antagonists.

The disclosure provides e.g., mouse anti-CD47 antibodies 20-1A5 ( “1A5” ) , 23-1D7 ( “ID7” ) , 23-2B5 ( “2B5” ) , 24-3G9 ( “3G9” ) , 28-3H9 ( “3H9” ) , 29-4E11 ( “4E11” ) , and 18-10C10 ( “10C10” ) , the chimeric antibodies thereof, and the humanized antibodies thereof (e.g., some of the antibodies as shown in Table 1) .

The CDR sequences for 20-1A5, and 20-1A5 derived antibodies (e.g., humanized antibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs: 1-3, and CDRs of the light chain variable domain, SEQ ID NOs: 4-6 as defined by Kabat numbering. In some embodiments, the CDR sequences for 20-1A5, and 20-1A5 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NO: 1, SEQ ID NO: 110 (TISRGGTYTYYPDTVKG) , and SEQ ID NO: 3; and CDRs of the light chain variable domain, SEQ ID NOs: 4-6, as defined by Kabat numbering. The CDRs can also be defined by Chothia system. Under the Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 43-45 and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 46-48.

The CDR sequences for 23-1D7, and 23-1D7 derived antibodies (e.g., humanized antibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs: 7-9, and CDRs of the light chain variable domain, SEQ ID NOs: 10-12 as defined by Kabat numbering. The CDRs can also be defined by Chothia system. Under the Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 49-51 and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 52-54.

The CDR sequences for 23-2B5, and 23-2B5 derived antibodies (e.g., humanized antibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs: 13-15, and CDRs of the light chain variable domain, SEQ ID NOs: 16-18 as defined by Kabat numbering. The CDRs can also be defined by Chothia system. Under the Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 55-57 and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 58-60.

The CDR sequences for 24-3G9, and 24-3G9 derived antibodies (e.g., humanized antibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs: 19-21, and CDRs of the light chain variable domain, SEQ ID NOs: 22-24 as defined by Kabat numbering. The CDRs can also be defined by Chothia system. Under the Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 61-63 and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 64-66.

The CDR sequences for 28-3H9, and 28-3H9 derived antibodies (e.g., humanized antibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs: 25-27, and CDRs of the light chain variable domain, SEQ ID NOs: 28-30 as defined by Kabat numbering. The CDRs can also be defined by Chothia system. Under the Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 67-69 and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 70-72.

The CDR sequences for 29-4E11, and 29-4E11 derived antibodies (e.g., humanized antibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs: 31-33, and CDRs of the light chain variable domain, SEQ ID NOs: 34-36 as defined by Kabat numbering. The CDRs can also be defined by Chothia system. Under the Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 73-75 and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 76-78.

The CDR sequences for 18-10C10, and 18-10C10 derived antibodies (e.g., humanized antibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs: 37-39, and CDRs of the light chain variable domain, SEQ ID NOs: 40-42 as defined by Kabat numbering. The CDRs can also be defined by Chothia system. Under the Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 79-81 and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 82-84.

The amino acid sequences for heavy chain variable regions and light variable regions of the humanized antibodies are also provided. As there are different ways to humanize a mouse antibody (e.g., a sequence can be modified with different amino acid substitutions) , the heavy chain and the light chain of an antibody can have more than one version of humanized sequences. The amino acid sequences for the heavy chain variable regions of humanized 1A5 antibody are set forth in SEQ ID NOs: 89-91. The amino acid sequences for the light chain variable regions of humanized 1A5 antibody are set forth in SEQ ID NOs: 92-95. Any of these heavy chain variable region sequences (SEQ ID NO: 89-91) can be paired with any of these light chain variable region sequences (SEQ ID NO: 92-95) .

Some chimeric antibodies based on 23-1D7 ( “1D7” ) , 23-2B5 ( “2B5” ) , 24-3G9 ( “3G9” ) , 28-3H9 ( “3H9” ) , 29-4E11 ( “4E11” ) , and 18-10C10 ( “10C10” ) , are shown in Table 1.

Table 1

Humanization percentage means the percentage identity of the heavy chain or light chain variable region sequence as compared to human antibody sequences in International Immunogenetics Information System (IMGT) database. The top hit means that the heavy chain or light chain variable region sequence is closer to a particular species than to other species. For example, top hit to human means that the sequence is closer to human than to other species. Top hit to human and Macaca fascicularis means that the sequence has the same percentage identity to the human sequence and the Macaca fascicularis sequence, and these percentages identities are highest as compared to the sequences of other species. In some embodiments, humanization percentage is greater than 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. A detailed description regarding how to determine humanization percentage and how to determine top hits is known in the art, and is described, e.g., in Jones, et al., "The INNs and outs of antibody nonproprietary names. " MAbs. Vol. 8. No. 1. Taylor &Francis, 2016, which is incorporated herein by reference in its entirety. A high humanization percentage often has various advantages, e.g., more safe and more effective in humans, more likely to be tolerated by a human subject, and/or less likely to have side effects.

Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 1-3, SEQ ID NOs: 7-9, SEQ ID NOs: 13-15, SEQ ID NOs: 19-21, SEQ ID NOs: 25-27, SEQ ID NOs: 31-33, SEQ ID NOs: 37-39, SEQ ID NOs: 1, 110, and 3, SEQ ID NOs: 43-45, SEQ ID NOs: 49-51, SEQ ID NOs: 55-57, SEQ ID NOs: 61-63, SEQ ID NOs: 67-69, SEQ ID NOs: 73-75, and SEQ ID NOs: 79-81; and/or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 4-6, SEQ ID NOs: 10-12, SEQ ID NOs: 16-18, SEQ ID NOs: 22-24, SEQ ID NOs: 28-30, SEQ ID NOs: 34-36, SEQ ID NOs: 40-42, SEQ ID NOs: 46-48, SEQ ID NOs: 52-54, SEQ ID NOs: 58-60, SEQ ID NOs: 64-66, SEQ ID NOs: 70-72 SEQ ID NOs: 76-78, and SEQ ID NOs: 82-84.

In some embodiments, the antibodies can have a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR3 amino acid sequence, and a light chain variable region (VL) comprising CDRs 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR3 amino acid sequence. The selected

VH CDRs

1, 2, 3 amino acid sequences and the selected VL CDRs, 1, 2, 3 amino acid sequences are shown in FIG. 23 (Kabat CDR) and FIG. 24 (Chothia CDR) .

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 3 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 7 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 13 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 15 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 19 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 21 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 25 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 26 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 27 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 31 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 32 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 33 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 37 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 38 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 39 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 110 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 3 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 43 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 44 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 45 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 49 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 50 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 51 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 55 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 56 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 57 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 61 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 62 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 63 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 67 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 68 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 69 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 73 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 74 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 75 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 79 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 80 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 81 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 4 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 6 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 10 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 12 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 16 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 18 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 22 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 23 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 24 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 28 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 29 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 30 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 34 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 35 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 36 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 40 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 41 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 42 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 46 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 47 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 48 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 52 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 53 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 54 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 58 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 59 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 60 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 64 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 65 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 66 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 70 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 71 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 72 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 76 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 77 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 78 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 82 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 83 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 84 with zero, one or two amino acid insertions, deletions, or substitutions.

The insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.

The disclosure also provides antibodies or antigen-binding fragments thereof that bind to CD47. The antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 89, 90, 91, or 96, and the selected VL sequence is SEQ ID NO: 92, 93, 94, 95, or 97. In some embodiments, the selected VH sequence is SEQ ID NO: 98 and the selected VL sequence is SEQ ID NO: 99. In some embodiments, the selected VH sequence is SEQ ID NO: 100, and the selected VL sequence is SEQ ID NO: 101. In some embodiments, the selected VH sequence is SEQ ID NO: 102 and the selected VL sequence is SEQ ID NO: 103. In some embodiments, the selected VH sequence is SEQ ID NO: 104, and the selected VL sequence is SEQ ID NO: 105. In some embodiments, the selected VH sequence is SEQ ID NOL 106 and the selected VL sequence is SEQ ID NO: 107. In some embodiments, the selected VH sequence is SEQ ID NO: 108 and the selected VL sequence is SEQ ID NO: 109.

The disclosure also provides antibodies or antigen-binding fragments thereof that can compete with the antibodies described herein. In some aspects, the antibodies or antigen-binding fragments can bind to the same epitope as the antibodies described herein.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) . The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of illustration, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The disclosure also provides nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises CDRs as shown in FIG. 23 or FIG. 24, or have sequences as shown in FIG. 26 and FIG. 27. When the polypeptides are paired with corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region) , the paired polypeptides bind to CD47 (e.g., human CD47) .

The anti-CD47 antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multi-specific (e.g., bi-specific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multi-specific (multimeric, e.g., bi-specific) , human antibodies, chimeric antibodies (e.g., human-mouse chimera) , single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies) , and antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) , or subclass. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.

Fragments of antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity of the full-length antibody. Thus, a fragment of an antibody that binds to CD47 will retain an ability to bind to CD47. An Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site.

Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. In some embodiments, the linker connecting scFv VH and VL domains is GGGGSGGGGSGGGGS (SEQ ID NO: 111) .

The Fab fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CH1) of the heavy chain. F (ab′) 2 antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.

Diabodies are small antibody fragments with two antigen-binding sites, which fragments comprise a VH connected to a VL in the same polypeptide chain (VH and VL) . By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

Linear antibodies comprise a pair of tandem Fd segments (VH-CH 1-VH-CH 1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

Antibodies and antibody fragments of the present disclosure can be modified in the Fc region to provide desired effector functions or serum half-life.

Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.

Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents including, but not limited to SMCC (succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acethylthio-acetate) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Sci. U.S.A. 94: 7509-7514, 1997) . Antibody homodimers can be converted to Fab’ ₂ homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao et al. (J. Immunol. 25: 396-404, 2002) .

In some embodiments, the multi-specific antibody is a bi-specific antibody. Bi-specific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan) . Compensatory “cavities” of identical or similar size to the large side chain (s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) . This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96/27011, which is incorporated by reference in its entirety.

Bi-specific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can also be made using any convenient cross-linking methods. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

Methods for generating bi-specific antibodies from antibody fragments are also known in the art. For example, bi-specific antibodies can be prepared using chemical linkage. Brennan et al. (Science 229: 81, 1985) describes a procedure where intact antibodies are proteolytically cleaved to generate F (ab’) ₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab’ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab’ TNB derivatives is then reconverted to the Fab’ thiol by reduction with mercaptoethylamine, and is mixed with an equimolar amount of another Fab’ TNB derivative to form the bi-specific antibody.

Any of the antibodies or antigen-binding fragments described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution) . Non-limiting examples of stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin) . The conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of an antibody or an antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human) .

In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent. The antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can covalently or non-covalently bind to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4, dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs) .

In some embodiments, the antibody or antigen-binding fragment thereof described herein recognizes a recombinant CD47, e.g., on the cell surface of MC38 cells. In some embodiments, the antibody or antigen-binding fragment thereof described herein recognizes an endogenous CD47, e.g., on the cell surface of Raji cells.

In some embodiments, binding affinity of the antibody or antigen-binding fragment thereof described herein to CD47 (e.g., human CD47) is determined (e.g., by surface plasma resonance (SPR) ) . In some embodiments, the determined KD is less than or about 5 × 10 ^-8 M, less than or about 2 × 10 ^-8 M, less than or about 1 × 10 ^-8 M, less than or about 5 × 10 ^-9 M, less than or about 2 × 10 ^-9 M, less than or about 1 × 10 ^-9 M, less than or about 5 × 10 ^-10 M, less than or about 2 × 10 ^-10 M, or less than or about 1 × 10 ^-10 M.

In some embodiments, the antibody or antigen-binding fragment thereof described herein exhibits immune-stimulating effects (e.g., promoting phagocytosis) . In some embodiments, the antibody or antigen-binding fragment thereof described herein exhibits immune-suppressing effects. In some embodiments, the antibody or antigen-binding fragment thereof described herein stimulates one or more immune functions (e.g., phagocytosis) to at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, at least 1000-fold, or higher, as compared to the same immune function (e.g., phagocytosis) when the antibody or antigen-binding fragment thereof is not administered. In some embodiments, the one or more immune functions include phagocytosis (e.g., by macrophage) .

In some embodiments, the antibody or antigen-binding fragment thereof described herein decreases CD47 binding to SIRPα to less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%as compared to the CD47/SIRPα binding when the antibody or antigen-binding fragment thereof is not administered.

Antibody Characteristics

In some embodiments, the antibodies or antigen-binding fragments thereof described herein can block the binding between CD47 and the CD47 ligand (e.g., SIRPα) .

The antibodies or antigen-binding fragments thereof as described herein can be CD47 agonist or antagonist. In some embodiments, by binding to CD47, the antibody can reduce the “don’t eat me” signal and/or promote phagocytosis. In some embodiments, the antibody can upregulate immune response or downregulate immune response.

In some embodiments, the antibodies or antigen-binding fragments thereof as described herein can increase immune response, activity or number of immune cells (e.g., T cells, CD8+ T cells, CD4+ T cells, macrophages, antigen presenting cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds. In some embodiments, the antibodies or antigen-binding fragments thereof as described herein can decrease the activity or number of immune cells (e.g., T cells, CD8+ T cells, CD4+ T cells, macrophages, antigen presenting cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds.

In some implementations, the antibody (or antigen-binding fragments thereof) specifically binds to CD47 (e.g., human CD47, monkey (e.g., rhesus macaques, Macaca fascicularis) CD47, mouse CD47, and/or chimeric CD47) with a dissociation rate (koff) of less than 0.1 s ^-1, less than 0.01 s ^-1, less than 0.001 s ^-1, less than 0.0001 s ^-1, or less than 0.00001 s ^-1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s ^-1, greater than 0.001 s ^-1, greater than 0.0001 s ^-1, greater than 0.00001 s ^-1, or greater than0.000001 s ^-1.

In some embodiments, kinetic association rates (kon) is greater than 1 x 10 ²/Ms, greater than 1 x 10 ³/Ms, greater than 1 x 10 ⁴/Ms, greater than 1 x 10 ⁵/Ms, or greater than 1 x 10 ⁶/Ms. In some embodiments, kinetic association rates (kon) is less than 1 x 10 ⁵/Ms, less than 1 x 10 ⁶/Ms, or less than 1 x 10 ⁷/Ms.

Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff/kon) . In some embodiments, KD is less than 1 x 10 ^-6 M, less than 1 x 10 ^-7M, less than 1 x 10 ^-8 M, less than 1 x 10 ^-9 M, or less than 1 x 10 ^-10 M. In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1 x 10 ^- ⁷ M, greater than 1 x 10 ^-8 M, greater than 1 x 10 ^-9 M, greater than 1 x 10 ^-10 M, greater than 1 x 10 ^-11 M, or greater than 1 x 10 ^-12 M. In some embodiments, the antibody binds to CD47 (e.g., human CD47) with KD less than or equal to about 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. In some embodiments, the antibody binds to CD47 (e.g., human CD47) with KD less than or equal to about 0.5 nM.

General techniques for measuring the affinity of an antibody for an antigen include, e.g., ELISA, RIA, and surface plasmon resonance (SPR) . In some embodiments, the antibody binds to human CD47 (SEQ ID NO: 85) , monkey CD47 (e.g., rhesus macaque CD47, SEQ ID NO: 87) , chimeric CD47 (SEQ ID NO: 88) , and/or mouse CD47 (SEQ ID NO: 86) . In some embodiments, the antibody does not bind to human CD47 (SEQ ID NO: 85) , monkey CD47 (e.g., rhesus macaque CD47, SEQ ID NO: 87; or cynomolgus CD47) , chimeric CD47 (SEQ ID NO: 88) , and/or mouse CD47 (SEQ ID NO: 86) .

In some embodiments, thermal stabilities are determined. The antibodies or antigen binding fragments as described herein can have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 ℃.

In some embodiments, the antibody has a tumor growth inhibition percentage (TGI%) that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the antibody has a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI%can be determined, e.g., at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the treatment starts, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the treatment starts. As used herein, the tumor growth inhibition percentage (TGI%) is calculated using the following formula:

TGI (%) = [1- (Ti-T0) / (Vi-V0) ] × 100

Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day zero.

In some embodiments, the antibodies or antigen-binding fragments thereof as described herein are CD47 agonist. In some embodiments, the antibodies or antigen-binding fragments thereof as described herein are CD47 antagonist. In some embodiments, the antibodies or antigen binding fragments can increase or decrease CD47-SIRPa signal transduction (e.g., immune and/or angiogenic responses) .

In some embodiments, the antibodies or antigen-binding fragments thereof blocks CD47 binding to SIRPα. In some embodiments, the decreased CD47 binding to SIRPα causes decreased phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs) of the SIRPα; decreased activation or recruitment of SHP-1 and SHP-2 phosphatases; and/or increased accumulation of myosin (e.g., myosin-IIA) at cell surface (e.g., synapses) ; thereby promoting phagocytosis.

In some embodiments, the antibodies or antigen binding fragments have a functional Fc region. In some embodiments, effector function of a functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC) . In some embodiments, effector function of a functional Fc region is phagocytosis. In some embodiments, effector function of a functional Fc region is ADCC and phagocytosis. In some embodiments, the antibodies or antigen binding fragments can induce complement mediated cytotoxicity (CMC) .

In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.

In some embodiments, the antibodies or antigen binding fragments do not have a functional Fc region. For example, the antibodies or antigen binding fragments are Fab, Fab’, F (ab’) 2, and Fy fragments. In some embodiments, the Fc region has LALA mutations (L234A and L235A mutations in EU numbering) , or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering) . In some embodiments, the antibodies or antigen binding fragments have a FLAA mutation (F234A and L235A mutations in EU numbering) .

In some embodiments, the antibodies or antigen-binding fragments thereof described herein have IgG4 (e.g., human IgG4) constant domains. IgG4 is known to have poor ability to trigger effector functions (e.g., ADCC) . Details are described in, e.g., Crescioli, Silvia, et al., "IgG4 characteristics and functions in cancer immunity. " Current Allergy and Asthma Reports 16.1 (2016) : 7; which is incorporated herein by reference in its entirety. Because CD47 is widely expressed in many different tumor cells as well as normal cells, antibody-induced effector functions (e.g., ADCC) may kill normal cells expressing CD47 thereby causing side effects. Thus, in some embodiments, the antibodies or antigen-binding fragments thereof comprise IgG4 constant domains to have low effector functions (e.g., ADCC) .

In some embodiments, the antibodies or antigen binding fragments thereof described herein induce red blood cell agglutination. In some embodiments, the antibodies or antigen binding fragments thereof described herein do not induce red blood cell agglutination. In some embodiments, the antibodies or antigen binding fragments thereof described herein induce red blood cell (e.g., 2%red blood cell suspension) agglutination at a concentration of at least or about 0.5 ng/ml. 1 ng/ml, 10 ng/ml, 100 ng/ml, 500 ng/ml, 1000 ng/ml, 5000 ng/ml, 10000 ng/ml, 20000 ng/ml, 30000 ng/ml, or higher. In some embodiments, the red blood cell agglutination occurs after mixing the anti-CD47 antibodies with the red blood cell suspension for at least or about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 1 hour.

Methods of Making Anti-CD47 Antibodies

An isolated fragment of human CD47 can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species to be immunized. Animals can be injected with the antigenic peptide or protein more than one time (e.g., twice, three times, or four times) .

The full-length polypeptide or protein can be used or, alternatively, antigenic peptide fragments thereof can be used as immunogens. The antigenic peptide of a protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of CD47 and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein. As described above, the full length sequence of human CD47 is known in the art (SEQ ID NO: 85) .

An immunogen typically is used to prepare antibodies by immunizing a suitable subject (e.g., human or transgenic animal expressing at least one human immunoglobulin locus) . An appropriate immunogenic preparation can contain, for example, a recombinantly-expressed or a chemically-synthesized polypeptide (e.g., a fragment of human CD47) . The preparation can further include an adjuvant, such as Freund’s complete or incomplete adjuvant, or a similar immunostimulatory agent.

Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a CD47 polypeptide, or an antigenic peptide thereof (e.g., part of CD47) as an immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme-linked immunosorbent assay (ELISA) using the immobilized CD47 polypeptide or peptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A of protein G chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 256: 495-497, 1975) , the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72, 1983) , the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985) , or trioma techniques. The technology for producing hybridomas is well known (see, generally, Current Protocols in Immunology, 1994, Coligan et al. (Eds. ) , John Wiley & Sons, Inc., New York, NY) . Hybridoma cells producing a monoclonal antibody are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest, e.g., using a standard ELISA assay.

Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human, humanized, or chimeric antibody, or antigen-binding fragment thereof described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acids sequences that make-up the antigen-binding site of the antibody or an antigen-binding domain. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for the target protein, e.g., CD47. Any combination of deletions, insertions, and/or combinations can be made to arrive at an antibody or antigen-binding fragment thereof that has increased binding affinity for the target. The amino acid changes introduced into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications into the antibody or antigen-binding fragment, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence such that a different sugar is attached by enzymes present in a cell) , or introducing new glycosylation sites.

Antibodies disclosed herein can be derived from any species of animal, including mammals. Non-limiting examples of native antibodies include antibodies derived from humans, primates, e.g., monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas) , chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits) , including transgenic rodents genetically engineered to produce human antibodies.

Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as those derived from) human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) , for example in the CDRs.

A humanized antibody, typically has a human framework (FR) grafted with non-human CDRs. Thus, a humanized antibody has one or more amino acid sequence introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed by e.g., substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. These methods are described in e.g., Jones et al., Nature, 321: 522-525 (1986) ; Riechmann et al., Nature, 332: 323-327 (1988) ; Verhoeyen et al., Science, 239: 1534-1536 (1988) ; each of which is incorporated by reference herein in its entirety. Accordingly, “humanized” antibodies are chimeric antibodies wherein substantially less than an intact human V domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically mouse antibodies in which some CDR residues and some FR residues are substituted by residues from analogous sites in human antibodies.

The choice of human VH and VL domains to be used in making the humanized antibodies is very important for reducing immunogenicity. According to the so-called “best-fit” method, the sequence of the V domain of a mouse antibody is screened against the entire library of known human-domain sequences. The human sequence which is closest to that of the mouse is then accepted as the human FR for the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993) ; Chothia et al., J. Mol. Biol., 196: 901 (1987) ) .

It is further important that antibodies be humanized with retention of high specificity and affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen (s) , is achieved.

Ordinarily, amino acid sequence variants of the human, humanized, or chimeric anti-CD47 antibody will contain an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%percent identity with a sequence present in the light or heavy chain of the original antibody.

Identity with respect to an original sequence is usually the percentage of amino acid residues present within the candidate sequence that are identical with a sequence present within the human, humanized, or chimeric anti-CD47 antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

Additional modifications to the anti-CD47 antibodies or antigen-binding fragments can be made. For example, a cysteine residue (s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have any increased half-life in vitro and/or in vivo. Homodimeric antibodies with increased half-life in vitro and/or in vivo can also be prepared using heterobifunctional cross-linkers as described, for example, in Wolff et al. (Cancer Res. 53: 2560-2565, 1993) . Alternatively, an antibody can be engineered which has dual Fc regions (see, for example, Stevenson et al., Anti-Cancer Drug Design 3: 219-230, 1989) .

In some embodiments, a covalent modification can be made to the anti-CD47 antibody or antigen-binding fragment thereof. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatization agent that is capable of reacting with selected side chains or the N-or C-terminal residues.

In some embodiments, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1%to 80%, from 1%to 65%, from 5%to 65%or from 20%to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering) ; however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can be further engineered to replace the Asparagine at position 297 with Alanine (N297A) .

In some embodiments, to facilitate production efficiency by avoiding Fab-arm exchange, the Fc region of the antibodies was further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P) . A detailed description regarding S228 mutation is described, e.g., in Silva et al. "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation. " Journal of Biological Chemistry 290.9 (2015) : 5462-5469, which is incorporated by reference in its entirety.

In some aspects, the disclosure also provides the use of the antibodies or antigen fragments thereof described herein for manufacture of a medicament for cancer treatment.

Recombinant Vectors

The present disclosure also provides recombinant vectors (e.g., an expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) , host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide) , and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.

As used herein, a “vector” is any construct capable of delivering one or more polynucleotide (s) of interest to a host cell when the vector is introduced to the host cell. An “expression vector” is capable of delivering and expressing the one or more polynucleotide (s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.

A vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran) , transformation, transfection, and infection and/or transduction (e.g., with recombinant virus) . Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant virus) , naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

In some implementations, a polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus) , which may involve the use of a non-pathogenic (defective) , replication competent virus, or may use a replication defective virus. In the latter case, viral propagation generally will occur only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86: 317-321; Flexner et al., 1989, Ann. N.Y. Acad Sci. 569: 86-103; Flexner et al., 1990, Vaccine, 8: 17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner-Biotechniques, 6: 616-627, 1988; Rosenfeld et al., 1991, Science, 252: 431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 11498-11502; Guzman et al., 1993, Circulation, 88: 2838-2848; and Guzman et al., 1993, Cir. Res., 73: 1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be “naked, ” as described, for example, in Ulmer et al., 1993, Science, 259: 1745-1749, and Cohen, 1993, Science, 259: 1691-1692. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cells.

For expression, the DNA insert comprising an antibody-encoding or polypeptide-encoding polynucleotide disclosed herein can be operatively linked to an appropriate promoter (e.g., a heterologous promoter) , such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan. The expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Appropriate culture mediums and conditions for the host cells described herein are known in the art.

Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.

Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters ofretroviral LTRs, such as those of the Rous sarcoma virus (RSV) , and metallothionein promoters, such as the mouse metallothionein-I promoter.

In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y, and Grant et al., Methods Enzymol., 153: 516-544 (1997) .

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986) , which is incorporated herein by reference in its entirety.

Transcription of DNA encoding an antibody of the present disclosure by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals.

The polypeptide (e.g., antibody) can be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion) or with a histidine-tag, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition ofpeptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.

The disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any nucleotide sequence as described herein, and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any amino acid sequence as described herein.

The disclosure also provides a nucleic acid sequence that has a homology of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%to any nucleotide sequence as described herein, and an amino acid sequence that has a homology of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%to any amino acid sequence as described herein.

In some embodiments, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.

In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.

In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.

The percentage of residues conserved with similar physicochemical properties (percent homology) , e.g. leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues having similar physicochemical properties have been defined in the art. These families include e.g., amino acids with 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) . The homology percentage, in many cases, is higher than the identity percentage.

Methods of Treatment

The antibodies or antibody or antigen-binding fragments thereof of the present disclosure can be used for various therapeutic purposes.

In one aspect, the disclosure provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject. In some embodiments, the treatment can halt, slow, retard, or inhibit progression of a cancer. In some embodiments, the treatment can result in the reduction of in the number, severity, and/or duration of one or more symptoms of the cancer in a subject.

In one aspect, the disclosure features methods that include administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, a cancer) , e.g., breast cancer (e.g., triple-negative breast cancer) , carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematologic malignancy. In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung carcinoma (NSCLC) , small cell lung cancer (SCLC) , bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN) , renal cell carcinoma (RCC) , triple-negative breast cancer (TNBC) , or colorectal carcinoma. In some embodiments, the subject has Hodgkin′s lymphoma. In some embodiments, the subject has triple-negative breast cancer (TNBC) , gastric cancer, urothelial cancer, Merkel-cell carcinoma, or head and neck cancer. In some embodiments, the cancer described herein is selected from solid tumors, hematologic malignancies (e.g., relapsed or refractory hematologic malignancies) , acute myeloid leukemia, non-Hodgkin’s lymphoma, breast cancer, bladder cancer, ovarian cancer, and small cell lung cancer tumors. In some embodiments, the antibody or antigen-binding fragment thereof is an anti-CD47 antibody or antigen-binding fragment thereof. In some embodiments, the antibody is an IgG4 anti-CD47 antibody (e.g., human IgG4 anti-CD47 antibody) .

In some embodiments, the compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer. Patients with cancer can be identified with various methods known in the art.

Furthermore, as the anti-CD47 antibodies can promote immune response, the disclosure provides methods for treating infection in a subject. Types of infection include e.g., bacterial, fungal, viral, protozoan, and parasitic diseases. In some embodiments, the treatment can halt, slow, retard, or inhibit progression of the disease. In addition, CD47 antibody treatment (e.g., agonistic antibody or antagonistic antibody) can also be used to treat autoimmune disease, asthma, and additionally as a means to improve vaccination. These methods generally involve administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject in need thereof.

As used herein, by an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a cancer, or an autoimmune disease. An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the antibody, antigen binding fragment, antibody-encoding polynucleotide, vector comprising the polynucleotide, and/or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.

An effective amount can be administered in one or more administrations. By way of example, an effective amount of an antibody or an antigen binding fragment is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and/or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line) ) in vitro. As is understood in the art, an effective amount of an antibody or antigen binding fragment may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of antibody used.

Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein, the route of administration, the particular type of antibodies, antibody-encoding polynucleotides, antigen binding fragments, and/or compositions disclosed herein used and other drugs being administered to the mammal. Guidance in selecting appropriate doses for antibody or antigen binding fragment can be found in the literature on therapeutic uses of antibodies and antigen binding fragments, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., 1985, ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp. 365-389.

A typical daily dosage of an effective amount of an antibody is 0.01 mg/kg to 100 mg/kg. In some embodiments, the dosage can be less than 100 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.1 mg/kg. In some embodiments, the dosage can be greater than 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, 0.1 mg/kg, 0.05 mg/kg, or 0.01 mg/kg. In some embodiments, the dosage is about 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.9 mg/kg, 0.8 mg/kg, 0.7 mg/kg, 0.6 mg/kg, 0.5 mg/kg, 0.4 mg/kg, 0.3 mg/kg, 0.2 mg/kg, or 0.1 mg/kg.

In any of the methods described herein, the at least one antibody, antigen-binding fragment thereof, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein) and, optionally, at least one additional therapeutic agent can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day) . In some embodiments, at least two different antibodies and/or antigen-binding fragments are administered in the same composition (e.g., a liquid composition) . In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition) . In some embodiments, the at least one antibody or antigen-binding fragment and the at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent) . In some embodiments, the at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained-release oral formulation.

In some embodiments, the one or more additional therapeutic agents can be administered to the subject prior to, or after administering the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) . In some embodiments, the one or more additional therapeutic agents and the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to the subject such that there is an overlap in the bioactive period of the one or more additional therapeutic agents and the at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) in the subject.

In some embodiments, the subject can be administered the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years) . A skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of cancer) . As described herein, a skilled medical professional can also change the identity and number (e.g., increase or decrease) of antibodies or antigen-binding antibody fragments (and/or one or more additional therapeutic agents) administered to the subject and can also adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one antibody or antigen-binding antibody fragment (and/or one or more additional therapeutic agents) to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art) .

In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of B-Raf, an EGFR inhibitor, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, an inhibitor of anaplastic lymphoma kinase (ALK) , an inhibitor of a phosphatidylinositol 3-kinase (PI3K) , an inhibitor of an Akt, an inhibitor of mTOR, a dual PI3K/mTOR inhibitor, an inhibitor of Bruton′s tyrosine kinase (BTK) , and an inhibitor of Isocitrate dehydrogenase 1 (IDH 1) and/or Isocitrate dehydrogenase 2 (IDH2) . In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2, 3-dioxygenase-1) (IDO1) (e.g., epacadostat) .

In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of HER3, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.

In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB 1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a Selectin agonist.

In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject.

In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody.

In some embodiments, the additional therapy is chemotherapy or chemoradiation. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab) , an anti-CD20 antibody (e.g., rituximab) , an anti-EGFR antibody (e.g., cetuximab) , an anti-CD319 antibody (e.g., elotuzumab) , or an anti-PD 1 antibody (e.g., nivolumab) .

In some embodiments, the additional therapeutic agent is an antibody that specifically binds to PD-1, CTLA-4, BTLA, PD-L1, CD20, CD27, CD28, CD40, CD137, CD154, TIGIT, TIM-3, GITR, or OX40. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to CD20, e.g., Rituximab.

In some embodiments, the additional therapeutic agent is an antibody that specifically binds to a cancer specific antigen. Some exemplary cancer specific antigens include, e.g., CD20, PSA, PSCA, PD-L1, Her2, Her3, Her1, β-Catenin, CD19, CEACAM3, EGFR, c-Met, EPCAM, PSMA, CD40, MUC1, and IGF 1R.

Pharmaceutical Compositions and Routes of Administration

Also provided herein are pharmaceutical compositions that contain at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described herein. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein can be present in a pharmaceutical composition in any combination. The pharmaceutical compositions may be formulated in any manner known in the art.

Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) . The compositions can include a sterile diluent (e.g., sterile water or saline) , a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose) , polyalcohols (e.g., mannitol or sorbitol) , or salts (e.g., sodium chloride) , or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811) . Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations) , proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin) . Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc. ) .

Compositions containing one or more of any of the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage) .

Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys) . One can, for example, determine the LD50 (the dose lethal to 50%of the population) and the ED50 (the dose therapeutically effective in 50%of the population) : the therapeutic index being the ratio of LD50: ED50. Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects) . Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dosage of any given agent for use in a subject (e.g., a human) . A therapeutically effective amount of the one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) will be an amount that treats the disease in a subject (e.g., kills cancer cells ) in a subject (e.g., a human subject identified as having cancer) , or a subject identified as being at risk of developing the disease (e.g., a subject who has previously developed cancer but now has been cured) , decreases the severity, frequency, and/or duration of one or more symptoms of a disease in a subject (e.g., a human) . The effectiveness and dosing of any of the antibodies or antigen-binding fragments described herein can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more symptoms of disease in a subject (e.g., a human) . Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and the presence of other diseases) .

Exemplary doses include milligram or microgram amounts of any of the antibodies or antigen-binding fragments described herein per kilogram of the subject's weight (e.g., about 1 μg/kg to about 500 mg/kg; about 100 μg/kg to about 500 mg/kg; about 100 μg/kg to about 50 mg/kg; about 10 μg/kg to about 5 mg/kg; about 10 μg/kg to about 0.5 mg/kg; about 1 μg/kg to about 50 μg/kg; about 500 μg/kg to about 5 mg/kg; or about 500 μg/kg to about 2 mg/kg) . While these doses cover a broad range, one of ordinary skill in the art will understand that therapeutic agents, including antibodies and antigen-binding fragments thereof, vary in their potency, and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the antibody or antibody fragment in vivo.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The disclosure also provides methods of manufacturing the antibodies or antigen binding fragments thereof for various uses as described herein.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1. Generating Mouse Anti-hCD47 Antibodies

To generate mouse antibodies against human CD47 (hCD47; SEQ ID NO: 85) , mice were immunized with human CD47. Anti-hCD47 antibodies were made by the methods as described below and shown in FIG. 1 and FIG. 2.

Immunization of mice

Mice were immunized with His-tagged human CD47 proteins (Absin Bioscience Inc.; Cat#abs04007) . The His-tagged human CD47 proteins were emulsified with adjuvant and injected at four positions on the back of the mice. For the first subcutaneous (s.c. ) injection, the diluted antigen was emulsified with Complete Freund's Adjuvant (CFA) in equal volume. In the following subcutaneous injections, the protein was emulsified with Incomplete Freund's Adjuvant (IFA) in equal volume. Three days after the third injection or the booster immunization, blood (serum) was collected and analyzed for antibody titer using ELISA.

In another experiment, several mice are immunized by injecting the expression plasmid encoding human CD47 into the mice. The plasmids encoding the antigen are injected into the tibialis anterior muscle (intramuscular injection; i.m. injection) . At least four injections are performed with at least 14 days between two injections. Blood (serum) is collected seven days after the last immunization and the serum is tested for antibody titer by ELISA.

Procedures to enhance immunization were also performed at least fourteen days after the previous immunization (either by injecting the plasmid or by injecting the proteins) . CHO cells that express CD47 antigen on the surface were intravenously injected into the mice through tail veins. Spleen was then collected four days after the injection.

Fusion of SP2/0 cells and spleen cells

Spleen tissues were grinded. Spleen cells were first selected by CD3ε Microbeads and Anti-Mouse IgM Microbeads, and then fused with SP2/0 cells. The cells were then plated in 96-well plates with hypoxanthine-aminopterin-thymidine (HAT) medium.

Primary screening of hybridorna

Primary screening of the hybridoma supematant in the 96-well plates was performed using Fluorescence-Activated Cell Sorting (FACS) pursuant to standard procedures. Chinese hamster ovary (CHO) cells were added to 96-well plates (2 × 10 ⁴ cells per well) before the screening. 50 μl of supernatant was used. The antibodies that were used in experiments were

(1) Fluorescein (FITC) -conjugated AffiniPure F (ab) 2 Fragment Goat Anti-Mouse IgG, Fcγ Fragment Specific, and

(2) Alexa

647-conjugated AffiniPure F (ab) 2 Fragment Goat Anti-Human lgG, Fcγ Fragment Specific.

Sub-cloning

Sub-cloning was performed using ClonePix2. In short, the positive wells identified during the primary screening were transferred to semisolid medium, and IgG positive clones were identified and tested. FITC anti-mouse IgG Fc antibody was used.

Ascites fluid antibodies

1 × 10 ⁶ positive hybridoma cells were injected intraperitoneally (i.p. ) to B-NDG ^TM mice (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., Beijing, China; Cat#B-CM-002) . Monoclonal antibodies were produced by growing hybridoma cells within the peritoneal cavity of the mouse. The hybridoma cells multiplied and produced ascites fluid in the abdomens of the mice. The fluid contained a high concentration of antibody which can be harvested for later use.

Purification of antibodies

Antibodies in ascites fluid were purified using GE AKTA protein chromatography (GE Healthcare, Chicago, Illinois, United States) . 20-1A5 ( “1A5” ) , 23-1D7 ( “1D7” ) , 23-2B5 ( “2B5” ) , 24-3G9 ( “3G9” ) , 28-3H9 ( “3H9” ) , 29-4E11 ( “4E11” ) , and 18-10C10 ( “10C10” ) were among the mouse antibodies produced by the methods described above.

The VH, VL and CDR regions of the antibodies were determined. The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acid sequences of 1A5 are shown in SEQ ID NOs: 1-6 (Kabat numbering) or SEQ ID NOs: 43-48 (Chothia numbering) .

The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acid sequences of 1D7 are shown in SEQ ID NOs: 7-12 (Kabat numbering) or SEQ ID NOs: 49-54 (Chothia numbering) .

The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acid sequences of 2B5 are shown in SEQ ID NOs: 13-18 (Kabat numbering) or SEQ ID NOs: 55-60 (Chothia numbering) .

The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acid sequences of 3G9 are shown in SEQ ID NOs: 19-24 (Kabat numbering) or SEQ ID NOs: 61-66 (Chothia numbering) .

The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acid sequences of 3H9 are shown in SEQ ID NOs: 25-30 (Kabat numbering) or SEQ ID NOs: 67-72 (Chothia numbering) .

The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acid sequences of 4E11 are shown in SEQ ID NOs: 31-36 (Kabat numbering) or SEQ ID NOs: 73-78 (Chothia numbering) .

The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acid sequences of 10C10 are shown in SEQ ID NOs: 37-42 (Kabat numbering) or SEQ ID NOs: 79-84 (Chothia numbering) .

Example 2. Humanization of mouse antibodies

The starting point for humanization was the mouse antibody. The amino acid sequences for the heavy chain variable region and the light chain variable region of these mouse antibodies were determined.

Three humanized heavy chain variable region variants (SEQ ID NOs: 89-91) and four humanized light chain variable region variants (SEQ ID NOs: 92-95) for 1A5 were constructed, containing different modifications or substitutions. One amino acid in VH CDR2 (Kabat) has been modified during humanization. It is SEQ ID NO: 110.

These humanized heavy chain variable region variants can be combined with any of the light chain variable region variants derived from the same mouse antibody. For example, 1A5-H1 (SEQ ID NO: 89) can be combined with any humanized light chain variable region variant based on the same mouse antibody 1A5 (e.g., SEQ ID NO: 92-95) , and the antibody will be labeled accordingly. For example, if 1A5-H1 is combined with 1A5-K3 (SEQ ID NO: 94) , the antibody is labeled as 1A5-H1K3.

Example 3. In vitro testing of anti-hCD47 antibodies: blocking the binding of human CD47 (hCD47) and human SIRPα (hSIRPα)

In vitro blocking assays were performed to determine whether anti-hCD47 antibodies can block the binding between hCD47 and hSIRPα. CHO cells expressing hCD47 were added to each well (25 μl per well) of a 96-well plate. Purified anti-hCD47 antibodies 1A5-mHvKv-IgG4, 1D7-mHvKv-IgG4, 2B5-mHvKv-IgG4, 3G9-mHvKv- IgG4, 3H9-mHvKv-IgG4, 4E11-mHvKv-IgG4, 10C10-mHvKv-IgG4, and a positive control (PC) antibody Hu5F9-IgG4, were titrated to final concentrations of 50, 5, 0.5, 0.05, and 0.005 μg/ml. The titrated antibodies were added to corresponding wells at 25 μl per well and incubated at 4 ℃ for 30 minutes. Alternatively, 25 μl phosphate-buffered saline (PBS) was added to negative control (NC) and ligand control (contained no anti-hCD47 antibodies) wells. Next, Biotin-hSIRPα (mFc) (ACROBiosystems Inc.; Cat#SIA-H52A8) was diluted to 0.5 μg/ml and added to each well at 50 μl per well, followed by an incubation at 4 ℃ for 15 minutes. After being washed with PBS twice (1200 rpm, 5 min) , 50 ul of anti-human-IgG Fc-FITC (Jackson ImmunoResearch Inc., Cat#109-096-170) at 1∶500 dilution and anti-mouse-IgG Fc-647 (Jackson ImmunoResearch Inc., Cat#115-606-071) at 1∶500 (or 1∶2000) dilution were mixed and added into each well, followed by an incubation of 30 minutes at 4 ℃. After being washed with PBS (1200 rpm, 5 min) , each well was added with 20 μl PBS. Signals for FITC and Alexa

647 were determined by flow cytometry.

As shown in FIGS. 3A-3C, when the concentration of the anti-hCD47 antibodies increased, the signal for FITC increased, suggesting that the binding between human CD47 and SIRPA was blocked by the anti-hCD47 antibodies.

Example 4. In vitro testing of anti-hCD47 antibodies: binding of anti-hCD47 antibodies with human CD47

In vitro binding assays were performed to determine whether anti-hCD47 antibodies can bind to human CD47. CHO cells expressing hCD47 were added to each well (25 μl per well) of a 96-well plate. Purified anti-hCD47 antibodies 2B5-mHvKv-IgG4, 4E11-mHvKv-IgG4, 1A5-mHvKv-IgG4, 3H9-mHvKv-IgG4, 3G9-mHvKv-IgG4, and a positive control (PC) antibody Hu5F9-IgG4, were titrated to final concentrations of 10, 1, 0.1, 0.01, and 0.001 μg/ml. The titrated antibodies were added to corresponding wells at 25 μl per well and incubated at 4 ℃ for 30 minutes. Alternatively, 25 μl PBS was added to wells used as negative control (NC) . After being washed with phosphate-buffered saline (PBS) twice (1200 rpm, 5 min) , 50 μl of anti-human IgG Fc-647 (Jackson ImmunoResearch Inc., Cat#109-606-170) at 1∶500 dilution was added into each well, and incubated at 4 ℃ for 30 minutes, followed by PBS wash (1200 rpm, 5 min) . 20 μl PBS was added to each well and signals for Alexa

647 was determined by flow cytometry.

As shown in FIGS. 4A-4B, when the concentration of the anti-hCD47 antibodies increased, the signal for cells binding to the anti-hCD47 antibodies increased, suggesting that the anti-hCD47 antibodies can bind to cells expressing human CD47.

Example 5. Cross-reactivity of anti-hCD47 antibodies against monkey, mouse, and human-mouse chimeric CD47

Cross-reactivity of the anti-hCD47 antibodies were determined as follows. CHO-K1 cells were transfected to express mouse CD47 (mCD47, SEQ ID NO: 86) , monkey (rhesus macaque) CD47 (rmCD47 or rCD47, SEQ ID NO: 87) , or chimeric (human-mouse) CD47 (chiCD47 or cCD47, SEQ ID NO: 88) . Specifically, 25 μl transfected CHO-K1 cells were added to each well of a 96-well plate. Purified anti-hCD47 antibodies 1A5-mHvKv-IgG4, 1D7-mHvKv-IgG4, 2B5-mHvKv-IgG4, 3G9-mHvKv-IgG4, 3H9-mHvKv-IgG4, 4E11-mHvKv-IgG4, 10C10-mHvKv-IgG4 and a positive control (PC) antibody Hu5F9-IgG4, were diluted to 5 μg/ml. The diluted antibodies were added to corresponding wells at 25 μl per well and incubated at 4 ℃ for 30 minutes. Alternatively, 25 μl PBS was added to wells used as negative control (NC) . After being washed with PBS (1200 rpm, 5 min) twice, 50 μl of anti-human IgG Fc-647 (Jackson ImmunoResearch Inc., Cat#109-606-170) was added into each well at 1: 500 dilution, followed by incubating at 4 ℃ for 30 minutes, and then PBS wash (1200 rpm, 5 min) . Signals for Alexa

647 were detected by flow cytometry (Sartorius-IntelliCyt

Screener PLUS) .

As shown in FIGS. 5A-5C, the anti-hCD47 antibodies did not cross react with mouse CD47, but had strong cross reactivity with rmCD47 and chimeric CD47. The tables below summarize the results from Examples 3-5.

Table 2

Example 6. Binding affinity of anti-hCD47 antibodies to human CD47

The binding affinity of the anti-hCD47 antibodies were measured using surface plasmon resonance (SPR) using Biacore (Biacore, INC, Piscataway N.J. ) 8K biosensor equipped with pre-immobilized Protein A sensor chips.

Purified anti-hCD47 antibodies were diluted 10-50 folds and then injected into the Biacore 8K biosensor at 10 μL/min for about 30 seconds to achieve a desired protein density (e.g., about 50 response units (RU) ) . His-tagged human CD47 at concentrations of 100, 25, 6.25, 1.56, or 0.39 nM were then injected at 30 μL/min for 180 seconds. Dissociation was monitored for 400 seconds. The chip was regenerated after the last injection of each titration with Glycine (pH 2.0, 30 μL/min for 12 seconds) .

Kinetic association rates (kon) and dissociation rates (koff) were obtained simultaneously by fitting the data globally to a 1∶1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) using Biacore 8K Evaluation Software 3.0. Affinities were deduced from the quotient of the kinetic rate constants (KD=koff/kon) .

As a person of ordinary skill in the art would understand, the same method with appropriate adjustments for parameters (e.g., antibody concentration) was performed for each tested antibody. For example, the result of 1A5-mHvKv-IgG4 is shown in FIG. 6. The results for the tested antibodies are summarized in the table below.

Table 3

The tested antibodies, including 1A5-mHvKv-IgG4, 1D7-mHvKv-IgG4, 2B5-mHvKv-IgG4, 3G9-mHvKv-IgG4, 3H9-mHvKv-IgG4, 4E11-mHvKv-IgG4, and 10C10-mHvKv-IgG4, are chimeric anti-hCD47 antibodies. The names and the corresponding SEQ ID NOs of the chimeric anti-CD47 antibodies are summarized in Table 1. Hu5F9-IgG4 is a humanized monoclonal antibody against CD47. It was included in the experiment form comparison purpose. The results show that these chimeric antibodies have very high binding affinity with human CD47.

Example 7. In vitro red blood cell agglutination of anti-hCD47 antibodies

Human CD47 is expressed on the surface of red blood cells (RBCs) . Therefore, anti-hCD47 antibodies may bind to red blood cells, causing red blood cell agglutination. The red blood cells were isolated from human peripheral blood. The experiments were performed to detect whether the anti-hCD47 antibodies are toxic, which can cause red blood cell to form aggregation. Red blood cells (2%) were mixed with different concentrations of the anti-hCD47 antibodies in a 96-well plate, followed by an incubation for 30 minutes. Without antibodies, red blood cells would settle on the bottom of the wells, as dark red dots. In contrast, agglutinated red blood cells would sink to the bottom of the wells evenly, with a pink color.

The experiment was performed as follows. First, red blood cells were harvested using Ficoll-Paque density gradient media and then transferred to a 15 ml centrifuge tube, followed by PBS wash twice. Supematant was discarded and the washed cells were diluted in PBS to obtain the 2%suspension solution. Next, 50 μl of the anti-hCD47 antibodies 1A5-mHvKv-IgG4, 1D7-mHvKv-IgG4, 2B5-mHvKv-IgG4, 3G9-mHvKv-IgG4, 3H9-mHvKv-IgG4, 4E11-mHvKv-IgG4, Hu5F9-IgG4, or 2H3-mHvKv-IgG4 (3-fold serially diluted from 30 μg/ml) were gently mixed with the 2%cell suspension by pipetting and then incubated at room temperature for 30 minutes. The results are shown in FIG. 7. The agglutination effects of the anti-hCD47 antibodies at different concentrations are summarized in the table below.

Table 4

According to the results, 3G9-mHvKv-IgG4 exhibited the highest safe concentration, indicating low toxicity to red blood cells. Chimeric anti-hCD47 antibodies 1A5-mHvKv-IgG4, 1D7-mHvKv-IgG4, 2B5-mHvKv-IgG4, 3G9-mHvKv-IgG4, 3H9-mHvKv-IgG4, and 4E11-mHvKv-IgG4 exhibited higher safe concentrations than Hu5F9-IgG and 2H3-mHvKv-IgG4. Hu5F9-IgG4 is a humanized monoclonal antibody against CD47. It was included in the experiment form comparison purpose. 2H3-mHvKv-IgG4 was used as a positive control (PC) , showing a high toxicity by inducing red blood cell agglutination at 0.51 ng/ml.

Example 8. In vitro testing of humanized anti-hCD47 antibodies: blocking the binding of human CD47 (hCD47) and human SIRPα (hSIRPα)

In vitro blocking assays were performed to determine whether humanized anti-hCD47 antibodies can block the binding between hCD47 and hSIRPα. CHO cells expressing hCD47 were added to each well (25 μl per well) of a 96-well plate. Purified chimeric anti-hCD47 antibodies 1A5-mHvKv-IgG4; and humanized anti-hCD47 antibodies 1A5-H1K1-IgG4, 1A5-H2K1-IgG4, 1A5-H3K1-IgG4, 1A5-H1K2-IgG4, 1A5-H2K2-IgG4, 1A5-H3K2-IgG4, 1A5-H1K3-IgG4, 1A5-H2K3-IgG4, 1A5-H3K3-IgG4, 1A5-H1K4-IgG4, 1A5-H2K4-IgG4, and 1A5-H3K4-IgG4, were titrated to final concentrations of 5, 0.5, 0.05, and 0.005 μg/ml. The titrated antibodies were added to corresponding wells at 25 μl per well and incubated at 4 ℃ for 30 minutes. Alternatively, 25 μl PBS was added to wells used as negative control (NC) and ligand control (contained no anti-hCD47 antibodies) wells. Next, Biotin-hSIRPα (mFc) was diluted to 0.5 μg/ml and added to each well at 50 μl per well, followed by an incubation at 4 ℃ for 15 minutes. A ligand control well was set up which contained no anti-hCD47 antibodies. After being washed with PBS twice (1200 rpm, 5 min) , 50 ul of anti-human-IgG Fc-FITC (Jackson ImmunoResearch Inc., Cat#109-096-170) at 1∶500 dilution and anti-mouse-IgG Fc-647 (Jackson ImmunoResearch Inc., Cat#115-606-071) at 1∶500 dilution were mixed and added into each well, followed by an incubation of 30 minutes at 4 ℃. After being washed with PBS (1200 rpm, 5 min) , each well was added with 20 μl PBS. Signals for FITC and Alexa

647 were determined by flow cytometry.

As shown in FIGS. 8A-8B, when the concentration of the anti-hCD47 antibodies increased, the signal for FITC increased, suggesting that the binding between human CD47 and SIRPA was blocked by the humanized anti-hCD47 antibodies.

Example 9. Cross-reactivity of humanized anti-hCD47 antibodies against monkey, mouse, and human-mouse chimeric CD47

Cross-reactivity of the humanized anti-hCD47 antibodies were determined as follows. CHO-K1 cells were transfected to express mouse CD47 (mCD47, SEQ ID NO: 86) , monkey (rhesus macaque) CD47 (rmCD47 or rCD47, SEQ ID NO: 87) , or chimeric (human-mouse) CD47 (chiCD47 or cCD47, SEQ ID NO: 88) . Specifically, 25 μl transfected CHO-K1 cells were added to each well of a 96-well plate. Purified chimeric anti-hCD47 antibodies 1A5-mHvKv-IgG4; and humanized anti-hCD47 antibodies 1A5-H1K1-IgG4, 1A5-H2K1-IgG4, 1A5-H3K1-IgG4, 1A5-H1K2-IgG4, 1A5-H2K2-IgG4, 1A5-H3K2-IgG4, 1A5-H1K3-IgG4, 1A5-H2K3-IgG4, 1A5-H3K3-IgG4, 1A5-H1K4-IgG4, 1A5-H2K4-IgG4, and 1A5-H3K4-IgG4, were diluted to 5 μg/ml. The diluted antibodies were added to corresponding wells at 25 μl per well and incubated at 4 ℃ for 30 minutes. Alternatively, 25 μl PBS was added to wells used as negative control (NC) . After being washed with PBS (1200 rpm, 5 min) twice, 50 μl of anti-human IgG Fc-647 (Jackson ImmunoResearch Inc., Cat#109-606-170) was added into each well at 1∶500 dilution, followed by incubating at 4 ℃ for 30 minutes, and then PBS wash (1200 rpm, 5 min) . Signals for Alexa

647 were detected by flow cytometry (Sartorius-IntelliCyt

Screener PLUS) .

As shown in FIGS. 9A-9B, most of the humanized anti-hCD47 antibodies did not cross react with mouse CD47, but all had strong cross reactivity with rmCD47 and chimeric CD47.

Example 10. Binding affinity of humanized anti-hCD47 antibodies to human CD47

The binding affinity of the humanized anti-hCD47 antibodies were measured using surface plasmon resonance (SPR) using Biacore (Biacore, INC, Piscataway N.J. ) 8K biosensor equipped with pre-immobilized Protein A sensor chips.

The methods were discussed the early example. The result of 1A5-H1K1-IgG4 is shown in FIG. 10 as an example The results for the tested antibodies are summarized in the table below.

Table 5

The tested antibodies included 1A5-mHvKv-IgG4 (a chimeric anti-hCD47 antibody) . 1A5-H1K1-IgG4, 1A5-H1K2-IgG4, 1A5-H1K3-IgG4, 1A5-H1K4-IgG4, 1A5-H2K1-IgG4, 1A5-H2K2-IgG4, 1A5-H2K3-IgG4, 1A5-H2K4-IgG4, 1A5-H3K1-IgG4, 1A5-H3K2-IgG4, 1A5-H3K3-IgG4, and 1A5-H3K4-IgG4, are humanized anti-hCD47 antibodies. The name and the sequences of variable regions of the chimeric and humanized anti-CD47 antibodies are summarized in Table 1, and FIGS. 26-27. 1A5-mHvKv-IgG4 was included in the experiment form comparison purpose. The results show that these chimeric and humanized antibodies have very high binding affinity with human CD47. Further, the results indicate that humanization of the anti-hCD47 antibodies did not affect their binding affinities to human CD47.

Example 11: Thermal stability measurement of anti-hCD47 antibodies

Thermal stability of humanized anti-hCD47 antibodies 1A5-mHvKv-IgG4, 1A5-H1K1-IgG4, 1A5-H2K2-IgG4, 1A5-H3K3-IgG4, 1A5-H3K4-IgG4, and Hu5F9-IgG4, were measured by a Protein Thermal Shift ^TM Dye Kit using QuantStudio ^TM 5 Real Time PCR Systems.

The experiments were performed according to the manufacturer’s protocol. Reactions were performed continuously in two steps. Specifically, the first step was carried out at 1.6℃ per second at 25℃ for 2 minutes and the second step was carried out at 0.05℃ per second at 99℃ for 2 minutes. Melting temperature (Tm) of each anti-hCD47 antibody was determined, as shown in the table below.

Table 6

Protein	Tm (℃)
1A5-mHvKv-IgG4	71.26
1A5-H1K1-IgG4	71.86
1A5-H2K2-IgG4	73.18
1A5-H3K3-IgG4	73.13
1A5-H3K4-IgG4	72.85
Hu5F9-IgG4	73.90

The results show that Tm of the chimeric and humanized anti-hCD47 antibodies are comparable to Hu5F9-IgG4.

Example 12. In vivo testing of anti-hCD47 antibodies

In vivo results for anti-hCD47 antibodies 20-1A5 ( “1A5” ) against colon cancer

In order to test the anti-hCD47 antibodies in vivo and to predict the effects of these antibodies in human, a humanized CD47 mouse model was generated. The humanized CD47 mouse model was engineered to express a chimeric CD47 protein (SEQ ID NO: 88) wherein a part of the extracellular region of the mouse CD47 protein was replaced with the corresponding human CD47 extracellular region. The amino acid residues 23-124 of mouse CD47 (SEQ ID NO: 86) were replaced by amino acid residues 23-126 of human CD47 (SEQ ID NO: 85) . A double humanized CD47/SIRPα mouse model was also generated by crossing the CD47 humanized mice with SIRPα humanized mice. The humanized mouse models (e.g., B-hCD47 mice, or double humanized CD47/SIRPα mice (B-hSIRPα/hCD47 mice) ) provide a new tool for testing new therapeutic treatments in a clinical setting by significantly decreasing the difference between clinical outcome in human and in ordinary mice expressing mouse CD47 or SIRPα. A detailed description regarding humanized CD47, humanized SIRPα, or double humanized CD47/SIRPα mouse models can be found in PCT/CN2018/081628 and PCT/CN2018/081629; each of which is incorporated herein by reference in its entirety.

The anti-hCD47 antibodies were tested for their effect on tumor growth in vivo in a model of colon carcinoma. About 1 × 10 ⁶ MC-38 cancer tumor cells (colon adenocarcinoma cell) expressing human CD47 were injected subcutaneously in double humanized B-hSIRPα/hCD47 mice. When the tumors in the mice reached a volume of 100-150 mm ³, the mice were randomly placed into different groups based on the volume of the tumor.

The mice were then injected with physiological saline (PS) or anti-hCD47 antibodies by intraperitoneal (i.p. ) administration.

The injected volume was calculated based on the weight of the mouse at 3 mg/kg or 10 mg/kg. The length of the long axis and the short axis of the tumor were measured and the volume of the tumor was calculated as 0.5 × (long axis) × (short axis) ². The weight of the mice was also measured twice a week.

The tumor growth inhibition percentage (TGI%) was calculated using the following formula: TGI (%) = [1- (Ti-T0) / (Vi-V0) ] × 100. Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day zero.

T-test was performed for statistical analysis. A TGI%higher than 60%indicates clear suppression of tumor growth. P ＜ 0.05 is a threshold to indicate significant difference.

In each group, B-hSIRPα/hCD47 mice were injected with physiological saline (PS) (G1) , 3 mg/kg 1A5-mHvKv-IgG4 (G2) , 3 mg/kg Hu5F9-IgG4 (G3) , 20-1A5-mHvKv-IgG4 3 mg/kg twice and then10 mg/kg for 4 times (G4) , Hu5F9-IgG4 3 mg/kg twice and then 10 mg/kg for 4 times (G5) , or 10 mg/kg Hu5F9-IgG4 (G6) .

Table 7

The weight of the mice was monitored during the entire treatment period. The average weight of mice in different groups all increased to different extents (FIG. 11, and FIG. 12) . No obvious difference in weight was observed among different groups at the end of the treatment periods. The results showed that 20-1A5-mHvKv-IgG4 was well tolerated and were not obviously toxic to the mice.

The tumor size in groups treated with 1A5-mHvKv-IgG4 and Hu5F9-IgG4 is shown in FIG. 13. The TGI%at day 21 (21 days after grouping) was also calculated as shown in the table below.

Table 8

The results show that anti-hCD47 antibody 1A5-mHvKv-IgG4 inhibited tumor growth. Particularly, -1A5-mHvKv-IgG4 had higher TGI%than Hu5F9-IgG4.

In vivo results for anti-hCD47 antibodies 20-1A5 ( “1A5” ) against lymphoma

In order to test the anti-hCD47 antibodies in vivo and to predict the effects of these antibodies in human, an immunodeficient animal model (B-NDG) having a disruption (knockout) at the endogenous CD132 gene was generated. A detailed description regarding the B-NDG mouse models can be found in PCT/CN2018/079365; which is incorporated herein by reference in its entirety.

The anti-hCD47 antibodies were tested for their effect on tumor growth in vivo in a model of human B cell lymphoma. About 5 × 10 ⁵ Raji cells were injected subcutaneously in B-NDG mice. When the tumors in the mice reached a volume of 100-150 mm ³, the mice were randomly placed into different groups based on the volume of the tumor.

The mice were then injected with physiological saline (PS) or antibodies by intraperitoneal (i.p. ) administration.

The injected volume was calculated based on the weight of the mouse at 10 mg/kg. The length of the long axis and the short axis of the tumor were measured and the volume of the tumor was calculated as 0.5 × (long axis) × (short axis) ². The weight of the mice was also measured twice a week.

The tumor growth inhibition percentage (TGI%) was calculated as described herein. T-test was performed for statistical analysis. A TGI%higher than 60%indicates clear suppression of tumor growth. P ＜ 0.05 is a threshold to indicate significant difference.

In each group, B-NDG mice were injected with physiological saline (PS) (G1) , 10 mg/kg 1A5-mHvKv-IgG4 (G2) , 10 mg/kg Hu5F9-IgG4 (G3) , or 10 mg/kg Rituximab-IgG1 (G4) .

Table 9

The weight of the mice was monitored during the entire treatment period. The average weight of mice in different groups all increased to different extents (FIG. 14, and FIG. 15) . No obvious difference in weight was observed among different groups at the end of the treatment periods. The results showed that 1A5-mHvKv-IgG4 were well tolerated and were not obviously toxic to the mice.

The tumor size in groups treated with 1A5-mHvKv-IgG4, Hu5F9-IgG4, and Rituximab-IgG1 is shown in FIG. 16. The TGI%at day 24 (24 days after grouping) was also calculated as shown in the table below.

Table 10

The results show that both anti-hCD47 antibody 1A5-mHvKv-IgG4 (G2) and Hu5F9-IgG4 (G3) significantly inhibited tumor growth because of phagocytosis induced by anti-CD47 antibodies. In contrast, Rituximab-IgG1 (G4) did not exhibit tumor inhibitory effects in B-NDG mice, which is likely due to lack of NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) in immuno-deficient B-NDG mice.

In vivo results for anti-hCD47 antibodies 20-1A5 ( “1A5” ) , 11-2G4 ( “2G4” ) , 17-2C12 ( “2C12” ) , 23-1D7 ( “1D7” ) , 24-3G9 ( “3G9” ) , and 28-3H9 ( “3H9” ) against lymphoma

In order to test the anti-hCD47 antibodies in vivo and to predict the effects of these antibodies in human, the anti-hCD47 antibodies were tested for their effect on tumor growth in vivo in a model of human B cell lymphoma. About 5 × 10 ⁵ Raji cells were injected subcutaneously in B-NDG mice. When the tumors in the mice reached a volume of 100-150 mm ³, the mice were randomly placed into different groups based on the volume of the tumor.

The mice were then injected with physiological saline (PS) and anti-hCD47 antibodies by intraperitoneal (i.p. ) administration.

The injected volume was calculated based on the weight of the mouse at 3 mg/kg. The length of the long axis and the short axis of the tumor were measured and the volume of the tumor was calculated as 0.5 × (long axis) × (short axis) ². The weight of the mice was also measured before the injection, when the mice were placed into different groups (before the first antibody injection) , twice a week during the antibody injection period, and before euthanization.

In each group, B-NDG mice were injected with physiological saline (PS) (G1) , 3 mg/kg1A5-mHvKv-IgG4 (G2) , 3 mg/kg 1G4-mHvKv-IgG4 (G3) , 3 mg/kg 2C12-mHvKv-IgG4 (G4) , 3 mg/kg 1D7-mHvKv-IgG4 (G5) , 3 mg/kg 3G9-mHvKv-IgG4 (G6) , 3 mg/kg 3H9-mHvKv-IgG4 (G7) , or 3 mg/kg Hu5F9-IgG4 (G8) .

Table 11

The weight of the mice was monitored during the entire treatment period. The average weight of mice in different groups all increased to different extents (FIG. 17, and FIG. 18) . The results showed that the anti-hCD47 antibodies were well tolerated.

The tumor size in groups treated with anti-hCD47 antibodies is shown in FIG. 19. The TGI%at day 23 (23 days after grouping) was also calculated as shown in the table below.

Table 12

The results show that anti-hCD47 antibody 1A5-mHvKv-IgG4 (G2) , 2C 12-mHvKv-IgG4 (G4) , 1D7-mHvKv-IgG4 (G5) , 3G9-mHvKv-IgG4 (G6) , 3H9-mHvKv-IgG4 (G7) , and Hu5F9-IgG4 (G8) significantly inhibited tumor growth.

In vivo results for anti-hCD47 antibodies 15-6F2 ( “6F2” ) , 23-2B5 ( “2B5” ) , 23-1D7 ( “1D7” ) , 24-3G9 ( “3G9 ” ) , 28-3H9 ( “3H9” ) , and 29-4E11 ( “4E11” ) against lymphoma

In order to test the anti-hCD47 antibodies in vivo and to predict the effects of these antibodies in human, a B-NDG/hSIRPα mouse model was also generated by crossing the B-NDG mice with the SIRPα humanized mice. The anti-hCD47 antibodies were tested for their effect on tumor growth in vivo in a model of human B cell lymphoma. About 5 × 10 ⁵ Raji cells were injected subcutaneously in B-NDG/hSIRPαmice. When the tumors in the mice reached a volume of 100-150 mm ³, the mice were randomly placed into different groups based on the volume of the tumor.

In each group, B-NDG mice were injected with physiological saline (PS) (G1) , 3 mg/kg 6F2-mHvKv-IgG4 (G2) , 3 mg/kg 2B5-mHvKv-IgG4 (G3) , 3 mg/kg 1D7-mHvKv-IgG4 (G4) , 3 mg/kg 3G9-mHvKv-IgG4 (G5) , 3 mg/kg 3H9-mHvKv-IgG4 (G6) , 3 mg/kg 4E11-mHvKv-IgG4 (G7) , or 3 mg/kg Hu5F9-IgG4 (G8) .

Table 13

The weight of the mice was monitored during the entire treatment period. The average weight of mice in different groups all increased to different extents (FIG. 20, and FIG. 21) . No obvious difference in weight was observed among different groups at the end of the treatment periods. The results showed that the anti-hCD47 antibodies were well tolerated and were not obviously toxic to the mice.

The tumor size in groups treated with anti-hCD47 antibodies is shown in FIG. 22. The TGI%at day 23 (23 days after grouping) was also calculated as shown in the table below.

Table 14

The results show that anti-hCD47 antibody 2B5-mHvKv-IgG4 (G3) , 23-1D7-mHvKv-IgG4 (G4) , 24-3G9-mHvKv-IgG4 (G5) , 28-3H9-mHvKv-IgG4 (G6) , 29-4E11-mHvKv-IgG4 (G7) , or Hu5F9-IgG4 (G8) significantly inhibited tumor growth.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

An antibody or antigen-binding fragment thereof that binds to CD47 comprising:

a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR3 amino acid sequence; and

a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence,

wherein the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:

(1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;

(2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7, 8, 9, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively;

(3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13, 14, 15, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, 18, respectively;

(4) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19, 20, 21, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 23, 24, respectively;

(5) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 25, 26, 27, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 28, 29, 30, respectively;

(6) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 32, 33, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 34, 35, 36, respectively;

(7) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 37, 38, 39, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 40, 41, 42, respectively; and

(8) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 110, 3, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3 respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 110, and 3, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.
The antibody or antigen-binding fragment thereof of any one of claims 1-9, wherein the antibody or antigen-binding fragment specifically binds to human CD47.
The antibody or antigen-binding fragment thereof of any one of claims 1-10, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.
The antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) .
A nucleic acid comprising a polynucleotide encoding a polypeptide comprising:

(1) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 92, 93, 94, 95, or 97 binds to CD47;

(2) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 89, 90, 91, or 96 binds to CD47;

(3) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 99 binds to CD47;

(4) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 98 binds to CD47;

(5) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 101 binds to CD47;

(6) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 100 binds to CD47;

(7) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20, 21, respectively, and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 103 binds to CD47;

(8) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23, 24, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 102 binds to CD47;

(9) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 25, 26, 27, respectively, and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 105 binds to CD47;

(10) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 29, 30, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 104 binds to CD47;

(11) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 31, 32, 33, respectively, and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 107 binds to CD47;

(12) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 34, 35, 36, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 106 binds to CD47;

(13) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 37, 38, 39, respectively, and wherein the VH, when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 109 binds to CD47; or

(14) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 40, 41, 42, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 108 binds to CD47.
The nucleic acid of claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively.
The nucleic acid of claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.
The nucleic acid of claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively.
The nucleic acid of claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.
The nucleic acid of claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively.
The nucleic acid of claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively.
The nucleic acid of claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively.
The nucleic acid of claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively.
The nucleic acid of claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively.
The nucleic acid of claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively.
The nucleic acid of claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively.
The nucleic acid of claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively.
The nucleic acid of claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively.
The nucleic acid of claim 13, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively.
The nucleic acid of any one of claims 13-27, wherein the VH when paired with a VL specifically binds to human CD47, or the VL when paired with a VH specifically binds to human CD47.
The nucleic acid of any one of claims 13-28, wherein the immunoglobulin heavy chain or the fragment thereof is a humanized immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or the fragment thereof is a humanized immunoglobulin light chain or a fragment thereof.
The nucleic acid of any one of claims 13-29, wherein the nucleic acid encodes a single-chain variable fragment (scFv) .
The nucleic acid of any one of claims 13-30, wherein the nucleic acid is cDNA.
A vector comprising one or more of the nucleic acids of any one of claims 13-31.
A vector comprising two of the nucleic acids of any one of claims 13-31, wherein the vector encodes the VL region and the VH region that together bind to CD47.
A pair of vectors, wherein each vector comprises one of the nucleic acids of any one of claims 13-31, wherein together the pair of vectors encodes the VL region and the VH region that together bind to CD47.
A cell comprising the vector of claim 32 or 33, or the pair of vectors of claim 34.
The cell of claim 35, wherein the cell is a CHO cell.
A cell comprising one or more of the nucleic acids of any one of claims 13-31.
A cell comprising two of the nucleic acids of any one of claims 13-31.
The cell of claim 38, wherein the two nucleic acids together encode the VL region and the VH region that together bind to CD47.
A method of producing an antibody or an antigen-binding fragment thereof, the method comprising

(a) culturing the cell of any one of claims 35-39 under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment; and

(b) collecting the antibody or the antigen-binding fragment produced by the cell.
An antibody or antigen-binding fragment thereof that binds to CD47 comprising

a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

(1) the selected VH sequence is SEQ ID NO: 89, 90, 91, or 96, and the selected VL sequence is SEQ ID NO: 92, 93, 94, 95, or 97;

(2) the selected VH sequence is SEQ ID NO: 98, and the selected VL sequence is SEQ ID NO: 99;

(3) the selected VH sequence is SEQ ID NO: 100, and the selected VL sequence is SEQ ID NO: 101;

(4) the selected VH sequence is SEQ ID NO: 102, and the selected VL sequence is SEQ ID NO: 103;

(5) the selected VH sequence is SEQ ID NO: 104, and the selected VL sequence is SEQ ID NO: 105;

(6) the selected VH sequence is SEQ ID NO: 106, and the selected VL sequence is SEQ ID NO: 107; and

(7) the selected VH sequence is SEQ ID NO: 108, and the selected VL sequence is SEQ ID NO: 109.
The antibody or antigen-binding fragment thereof of claim 41, wherein the VH comprises the sequence of SEQ ID NO: 89 and the VL comprises the sequence of SEQ ID NO: 92.
The antibody or antigen-binding fragment thereof of claim 41, wherein the VH comprises the sequence of SEQ ID NO: 90 and the VL comprises the sequence of SEQ ID NO: 93.
The antibody or antigen-binding fragment thereof of claim 41, wherein the VH comprises the sequence of SEQ ID NO: 91 and the VL comprises the sequence of SEQ ID NO: 94.
The antibody or antigen-binding fragment thereof of claim 41, wherein the VH comprises the sequence of SEQ ID NO: 91 and the VL comprises the sequence of SEQ ID NO: 95.
The antibody or antigen-binding fragment thereof of any one of claims 41-45, wherein the antibody or antigen-binding fragment specifically binds to human CD47.
The antibody or antigen-binding fragment thereof of any one of claims 41-46, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.
The antibody or antigen-binding fragment thereof of any one of claims 41-47, wherein the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) .
An antibody or antigen-binding fragment thereof comprising the VH CDRs 1, 2, 3, and the VL CDRs 1, 2, 3 of the antibody or antigen-binding fragment thereof of any one of claims 1-12 and 41-48.
An antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof of any one of claims 1-12 and 41-49.
An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-12 and 41-50 covalently bound to a therapeutic agent.
The antibody drug conjugate of claim 51, wherein the therapeutic agent is a cytotoxic or cytostatic agent.
A method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-12 and 41-50, or the antibody-drug conjugate of claims 51 or 52, to the subject.
The method of claim 53, wherein the subject has a solid tumor.
The method of claim 53, wherein the cancer is hematologic malignancy, and/or relapsed or refractory hematologic malignancy.
The method of claim 53, wherein the cancer is acute myeloid leukemia, non-Hodgkin’s lymphoma, breast cancer, bladder cancer, ovarian cancer, and/or small cell lung cancer tumor.
A method of decreasing the rate of tumor growth, the method comprising

contacting a tumor cell with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof of any one of claims 1-12 and 41-50, or the antibody-drug conjugate of claims 51 or 52.
A method of killing a tumor cell, the method comprising

contacting a tumor cell with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-12 and 41-50, or the antibody-drug conjugate of claims 51 or 52.
A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-12 and 41-50, and a pharmaceutically acceptable carrier.
A pharmaceutical composition comprising the antibody drug conjugate of claim 51 or 52, and a pharmaceutically acceptable carrier.
The antibody or antigen-binding fragment thereof of any one of claims 1-12 and 41-50, or the antibody-drug conjugate of claims 51 or 52, wherein the antibody is a IgG4 antibody.
The antibody or antigen-binding fragment thereof of any one of claims 1-12 and 41-50, or the antibody-drug conjugate of claims 51 or 52, wherein the antibody is a human IgG4 antibody.
An IgG4 antibody or antigen-binding fragment thereof that binds to CD47 comprising:

a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR3 amino acid sequence; and

a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence,

wherein the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are selected from one of the antibodies as set forth in FIG. 23 and FIG. 24.
The IgG4 antibody or antigen-binding fragment thereof of claim 63, wherein the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively.
The IgG4 antibody or antigen-binding fragment thereof of claim 63, wherein the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 110, and 3, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively.
The IgG4 antibody or antigen-binding fragment thereof of any one of claims 63-65, wherein the antibody is a human IgG4 antibody.
An IgG4 antibody or antigen-binding fragment thereof that binds to CD47 comprising:

a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are selected from one of the antibodies as set forth in FIG. 26 and FIG. 27.
The IgG4 antibody or antigen-binding fragment thereof of claim 67, wherein the selected VH sequence is SEQ ID NO: 89, and the selected VL sequence is SEQ ID NO: 92.
The IgG4 antibody or antigen-binding fragment thereof of claim 67 or 68, wherein the antibody is a human IgG4 antibody.