Combination therapy for treating cancer

Abstract

Methods for treating cancer are provided, including administering a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant and an Fc domain variant in combination with at least one chemotherapeutic agent and / or at least one therapeutic antibody. Related kits are also provided.

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Application No. 62 / 941,390, filed on November 27, 2019; U.S. Provisional Application No. 63 / 022,998, filed on May 11, 2020; U.S. Provisional Application No. 63 / 030,686, filed on May 27, 2020; U.S. Provisional Application No. 63 / 106,225, filed on October 27, 2020; and U.S. Provisional Application No. 63 / 109,044, filed on November 3, 2020, the entire contents of each of which are incorporated herein by reference.

[0002] Submission of Sequence Listing in ASCII text file The contents of the following submissions in ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in computer - readable form (CRF) (file name: 757972001140SEQLIST.TXT, recording date: November 25, 2020, size: 333KB).

[0003] The present invention relates to a method for treating cancer, the method comprising administering an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) in combination with a chemotherapeutic agent and at least one additional anti - cancer agent and / or at least one additional modality of cancer treatment.

Background Art

[0004] Many cancers have a poor prognosis even when treated with available therapeutic agents. There is a need in the art for new treatments that provide additional treatment options and improve patient outcomes.

[0005] Tumor cells manipulate the bone marrow compartment to evade the anti-tumor host immune response (Gabrilovich et al., Nat Rev Immunol (2012) 12(4):253-68). For example, CD47, which is expressed on the surface of normal cells, binds to SIRPα of macrophages to issue a "don't eat me" signal, but it has also been found that tumor cells overexpress CD47 to evade the macrophage component of immune surveillance (Oldenborg, ISRN Hematol (2013) 614619).

[0006] The destruction of cancer cells via macrophages requires both the disruption of the "don't eat me" signal (such as CD47-SIRPα, etc.) and the activation of the "eat me" signal. Only either component is not sufficient to elicit the maximum phagocytic response against tumor cells. As described above, CD47 provides a basic "don't eat me" signal through its interaction with SIRPα on macrophages. A phagocytosis-promoting "eat me" signal can be provided to the same macrophages by binding to their activated Fc gamma receptors. For example, a phagocytosis-promoting "eat me" signal can be provided by the binding of an anti-tumor antibody to the Fc receptor on macrophages.

[0007] All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are incorporated herein by reference in their entirety as if each individual reference were specifically and individually indicated to be incorporated by reference. SUMMARY OF THE INVENTION

[0008] A method of treating cancer in an individual is provided, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) a Bcl-2 inhibitor, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index). In some embodiments, the cancer is leukemia, multiple myeloma or non-Hodgkin lymphoma. In some embodiments, the non-Hodgkin lymphoma is diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), or follicular lymphoma (FL). In some embodiments, the leukemia is acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), or myelodysplastic syndrome (MDS). In some embodiments, the leukemia is acute lymphoblastic leukemia. In some embodiments, the Bcl-2 inhibitor is venetoclax, ABT-737, navitoclax, BCL201, or AZD-0466. In some embodiments, the Bcl-2 inhibitor is venetoclax.

[0009] Also provided is a method of treating cancer in an individual, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) a platinum-based chemotherapeutic agent, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index). In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is colon cancer, lung cancer, head and neck cancer, esophageal cancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer, testicular cancer, endometrial cancer, liver cancer, gastric cancer, gastroesophageal junction cancer, brain tumor, mesothelioma, or neuroblastoma. In some embodiments, the colon cancer is colon carcinoma. In some embodiments, the platinum-based chemotherapeutic agent is carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin. In some embodiments, the platinum-based chemotherapeutic agent is cisplatin or carboplatin.

[0010] Also provided is a method of treating cancer in an individual, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, (b) a PD-1 inhibitor, (c) an antimetabolite, and (d) a platinum-based chemotherapeutic agent, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; and the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index). Here, the cancer is head and neck squamous cell carcinoma (HNSCC), and the individual has not received prior treatment for HNSCC. In some embodiments, the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant is administered at a dose of 10 mg / kg once a week (qw). In some embodiments, the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant is administered at a dose of 15 mg / kg once a week (qw).

[0011] In some embodiments, the HNSCC is progressive and / or metastatic HNSCC. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody, such as pembrolizumab, nivolumab, pidilizumab, semipilimab, or BMS-936559. In some embodiments, the anti-PD-1 antibody is pembrolizumab. In some embodiments, the antimetabolite is 5-fluorouracil, 6-mercaptopurine, capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxycarbamide, methotrexate, pemetrexed, fotemustine. In some embodiments, the antimetabolite is 5-fluorouracil. In some embodiments, the platinum-based chemotherapeutic agent is carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin. In some embodiments, the platinum-based chemotherapeutic agent is cisplatin or carboplatin.

[0012] In another aspect, a method of treating cancer in an individual is provided, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, (b) an anti-HER2 antibody, and (c) an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody), wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index). In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is a colon cancer, lung cancer, head and neck cancer, esophageal cancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer, testicular cancer, endometrial cancer, liver cancer, gastric cancer, gastroesophageal junction cancer, brain tumor, mesothelioma, or neuroblastoma. In some embodiments, the solid tumor is HER2 + a solid tumor. In some embodiments, the solid tumor is a colon cancer (e.g., HER2 + colon cancer). In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab.

[0013] In some embodiments, a method of treating cancer in an individual is provided, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, (b) an anti-HER2 antibody, (c) an anti-VEGF2 antibody, and (d) paclitaxel, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index). The cancer is gastric cancer or gastroesophageal junction (GEJ) cancer, and the individual has received at least one prior treatment for gastric cancer or GEJ cancer. In some embodiments, the gastric cancer or GEJ cancer is HER2 overexpressing (e.g., HER2 + ) gastric cancer or HER2 overexpressing GEJ cancer. In some embodiments, the individual has received prior treatment with an anti-HER2 antibody, with an anti-HER2 antibody and a fluoropyrimidine, or with an anti-HER2 antibody and a platinum-based chemotherapeutic agent. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the anti-VEGF antibody is ramucirumab. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 10 mg / kg once a week (qw). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 15 mg / kg once a week (qw).

[0014] Also provided is a method of treating cancer in an individual, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) an anti-TROP2 antibody, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; and the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index). In some embodiments, the cancer is a solid tumor, gastric cancer, nasopharyngeal cancer, gallbladder cancer, cervical cancer, extranodal NK / T cell lymphoma, lung cancer, laryngeal squamous cell carcinoma, colon cancer, hilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrioid endometrial cancer, or ovarian cancer.

[0015] In some embodiments of any of the methods described herein, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, the Fc domain variant is a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, numbered according to the Kabat EU index. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 136. In some embodiments, the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant forms a homodimer. In some embodiments, the subject is human.

[0016] In another aspect, provided is a kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier for use in combination with a Bcl-2 inhibitor in an individual in need of treatment for cancer, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), and the kit comprises instructions for administering the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant in combination with the Bcl-2 inhibitor to an individual in need thereof. In some embodiments, the cancer is leukemia, multiple myeloma or non-Hodgkin lymphoma. In some embodiments, the Bcl-2 inhibitor is venetoclax.

[0017] Also provided is a kit comprising a polypeptide comprising an SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier for use in combination with a platinum-based chemotherapeutic agent for treating cancer in an individual in need thereof, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), and the kit comprises instructions for administering the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant in combination with the chemotherapeutic agent to an individual in need thereof. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is colon cancer, colon carcinoma, lung cancer, head and neck cancer, esophageal cancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer, testicular cancer, endometrial cancer, liver cancer, gastric cancer, brain tumor, mesothelioma, or neuroblastoma. In some embodiments, the platinum-based chemotherapeutic agent is cisplatin or carboplatin.

[0018] In some embodiments, provided is a kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier for use in combination with a PD-1 inhibitor, an antimetabolite, and a platinum-based chemotherapeutic agent in an individual in need of treatment of cancer, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), and the kit comprises instructions for administering the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant in combination with a PD-1 inhibitor, an antimetabolite, and a platinum-based chemotherapeutic agent to an individual having HNSCC who has not previously been treated for HNSCC. In some embodiments, the PD-1 inhibitor is pembrolizumab. In some embodiments, the antimetabolite is 5-fluorouracil. In some embodiments, the platinum-based chemotherapeutic agent is cisplatin or carboplatin.

[0019] In some embodiments, a kit is provided that includes a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, the polypeptide for use in combination with an anti-HER2 antibody, an anti-VEGFR2 antibody, and paclitaxel for treating cancer in an individual in need thereof, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), and the kit includes instructions for administering the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant in combination with the anti-HER2 antibody, the anti-VEGFR2 antibody, and paclitaxel to an individual having gastric cancer or gastroesophageal junction (GEJ) cancer and who has received at least one prior treatment for gastric cancer or GEJ cancer. In some embodiments, the gastric cancer or GEJ cancer is HER2 + Gastric cancer or HER2 +It is GEJ cancer. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the anti-VEGFR2 antibody is ramucirumab. In some embodiments, the individual has received prior treatment (or multiple prior treatments) with an anti-HER2 antibody (e.g., trastuzumab) and / or a fluoropyrimidine, and / or a platinum-based chemotherapeutic agent. In some embodiments, the individual's gastric cancer or GEJ cancer has progressed during or after prior treatment (or multiple prior treatments) comprising an anti-HER2 antibody (e.g., trastuzumab), and / or a fluoropyrimidine, and / or a platinum-based chemotherapeutic agent. In some embodiments, the individual did not respond (e.g., relapsed after prior treatment or did not respond to prior treatment) to prior treatment (or multiple prior treatments) comprising an anti-HER2 antibody (e.g., trastuzumab) and / or a fluoropyrimidine, and / or a platinum-based chemotherapeutic agent. In some embodiments, the prior treatment (or multiple prior treatments) included an anti-HER2 antibody and a fluoropyrimidine (e.g., administered during the same or different treatment regimens). In some embodiments, the prior treatment (or multiple prior treatments) included an anti-HER2 antibody and a platinum-based chemotherapeutic agent (e.g., administered during the same or different treatment regimens).

[0020] Also provided is a kit comprising a polypeptide comprising an SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier for use in combination with an anti-TROP2 antibody for use in an individual in need of treatment of cancer, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), and the kit comprises instructions for administering the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant in combination with the anti-TROP2 antibody to an individual in need thereof. In some embodiments, the cancer is solid tumor, gastric cancer, nasopharyngeal cancer, gallbladder cancer, cervical cancer, extranodal NK / T cell lymphoma, lung cancer, laryngeal squamous cell carcinoma, colon cancer, hilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrioid endometrial cancer, or ovarian cancer.

[0021] Also provided is a kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, for use in combination with an anti-HER2 antibody and an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody) in an individual in need of cancer treatment, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), and the kit comprises instructions for administering the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant in combination with an anti-HER2 antibody and an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody) to an individual in need thereof. In some embodiments, the cancer is colon cancer. In some embodiments, the colon cancer is HER2 + colon cancer. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab.

[0022] In some embodiments of the kit, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, the Fc domain variant is a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, numbered according to the Kabat EU index. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 136. In some embodiments, the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant forms a homodimer. In some embodiments, the subject is human.

Brief Description of the Drawings

[0023]

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Mode for Carrying Out the Invention

[0024] In the following description, exemplary methods, parameters, and the like are set forth. However, such description is not intended to limit the scope of the present disclosure. Instead, it should be recognized that it is provided as an illustration of exemplary embodiments.

[0025] Definitions The terms "about" or "approximately" mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, and depend in part on the method of measuring or determining the value, i.e., the limitations of the measuring system. For example, "about" can mean within one or more standard deviations in accordance with the conventions in the art. Alternatively, "about" can mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of the value, preferably within fivefold, more preferably within twofold. Unless otherwise specified, when a particular value is recited in the present application and claims, the term "about" is considered to mean within an acceptable error range for the particular value.

[0026] The terms used herein are for the purpose of describing particular instances only and are not intended to be limiting. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Further, to the extent that the terms "including", "includes", "having", "has", "with", or variations thereof are used in either the detailed description or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising".

[0027] As used herein, terms such as "treatment" and "treating" refer to administering an agent or performing a procedure to obtain an effect. In some embodiments, the effect is prophylactic in that it completely or partially prevents a disease or its symptoms. In some embodiments, the effect is therapeutic in that it affects the partial or complete cure of a disease or a symptom of the disease.

[0028] As used herein, the term "antibody" refers to intact antibodies, antibody fragments, monoclonal antibodies; polyclonal antibodies; monospecific antibodies; multispecific antibodies (e.g., bispecific antibodies); and antibody-like proteins when they exhibit the desired biological activity (e.g., epitope binding).

[0029] As used herein, the term "antibody variable domain" refers to portions of the light and heavy chains of an antibody that include the amino acid sequences of the complementarity determining regions (CDRs, e.g., CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3) and the framework regions (FR).

[0030] As used herein, the term "linker" refers to a connection between two elements, e.g., protein domains. In some embodiments, the linker can be a covalent bond or a spacer. The term "spacer" refers to a moiety (e.g., a polyethylene glycol (PEG) polymer) or amino acid sequence (e.g., a 1-200 amino acid sequence) that occurs between two polypeptides or polypeptide domains to provide space or flexibility (or both space and flexibility) between the two polypeptides or polypeptide domains. In some embodiments, the amino acid spacer is part of the primary sequence of a polypeptide (e.g., attached to a polypeptide or polypeptide domain spaced via the polypeptide backbone).

[0031] As used herein, the term "effective amount" refers to an amount of a polypeptide or a pharmaceutical composition comprising a polypeptide described herein, such as a polypeptide having a SIRPα D1 domain or a variant thereof, that is sufficient and effective to achieve a desired therapeutic effect when treating a patient having a disease such as cancer, e.g., solid tumor or hematological cancer. In some embodiments, an effective amount of the polypeptide will avoid adverse side effects.

[0032] As used herein, the term "pharmaceutical composition" refers to a pharmaceutical or a pharmaceutical preparation that contains an active ingredient as well as an excipient or a diluent (or both an excipient and a diluent), and the active ingredient can be administered by a suitable method of administration. In some embodiments, the pharmaceutical compositions disclosed herein contain pharmaceutically acceptable ingredients that are compatible with the polypeptide. In some embodiments, the pharmaceutical composition is in the form of a tablet or capsule for oral administration, or in an aqueous form for intravenous or subcutaneous administration, e.g., by injection.

[0033] As used herein, the terms "subject", "individual", and "patient" are used interchangeably to refer to a vertebrate, e.g., a mammal. Mammals include, but are not limited to, mice, monkeys, humans, livestock, sport animals, and pets. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro. None of the terms require the supervision of a medical professional.

[0034] As used herein, the term "affinity" or "binding affinity" refers to the strength of the binding interaction between two molecules. Generally, binding affinity refers to the total strength of non-covalent interactions between a molecule and its binding partner (such as SIRPα D1 domain variant and CD47). Unless otherwise specified, binding affinity refers to the native binding affinity that reflects the 1:1 interaction between members of the binding pair. The binding affinity between two molecules is generally the dissociation constant (K D ) or the association constant (K A) are described. Two molecules having a low binding affinity for each other generally bind slowly and tend to dissociate readily, exhibiting a large K D . Two molecules having a high affinity for each other generally bind readily and tend to remain bound for a long time, exhibiting a small K D . In some embodiments, the K D of two interacting molecules is determined using known methods and techniques, such as surface plasmon resonance (SPR). K D can be calculated as the ratio of koff / kon.

[0035] As used herein, the term "less K D " refers to a numerically small K D value and an increasing binding affinity compared to the recited K D value. As used herein, the term "greater K D " refers to a numerically greater K D value and a decreasing binding affinity compared to the recited K D value.

[0036] As used herein, "in combination with" refers to the administration of another therapy in addition to one therapy. Thus, "in combination with" refers to the administration of another therapy before, during, or after the administration of one therapy to an individual.

[0037] Overview Provided herein is a method of treating cancer in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) and (b) a chemotherapeutic agent (at least one chemotherapeutic agent, e.g., at least two, at least three, or at least four chemotherapeutic agents, etc.). In some embodiments, the method further comprises administering to the individual an effective amount of a therapeutic antibody (at least one therapeutic antibody, e.g., at least two, at least three, or at least four therapeutic antibodies). Additionally or alternatively, in some embodiments, the method further comprises administering to the individual an effective amount of an immunotherapeutic agent (at least one immunotherapeutic agent, e.g., at least two, at least three, or at least four immunotherapeutic agents). Additionally or alternatively, in some embodiments, the method comprises administering the polypeptide and the chemotherapeutic agent in combination with one or more additional treatment modalities, including but not limited to, for example, radiation therapy, surgery, cryoablation, and bone marrow transplantation, etc.

[0038] In some embodiments, the agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is a small molecule inhibitor of the CD47-SIRPα pathway (e.g., RRX-001, etc.). See, for example, Miller et al. (2019) “Quantitative high-throughput screening assays for the discovery and development of SIRPα-CD47 interaction inhibitors.” PLoS ONE 14(7):e0218897 and Sasikumar et al. ACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; October 26 - 30, 2017; Philadelphia, PA; Abstract B007.

[0039] In some embodiments, an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) binds to CD47 (e.g., hCD47). In some embodiments, the agent has a K of about 10 nM D or better K D(For example, at least any one of about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM, or less than 10 pM) binds to CD47 (e.g., hCD47). In some embodiments, the agent that binds to CD47 (e.g., hCD47) exhibits a CD47 receptor occupancy of at least about 50% (e.g., at least any one of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%) in a human subject. In some embodiments, the agent that binds to CD47 (e.g., hCD47) has an EC50 of about 80 ng / ml or less, for example, any one of about 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 ng / ml. In some embodiments, the agent that binds to CD47 (e.g., hCD47) is an anti-CD47 antibody (e.g., a therapeutic anti-CD47 antibody) or an antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment is a Fab, Fab’, Fab’-SH, F(ab’)2, Fv, scFv, single-chain antibody, or diabody. In some embodiments, the anti-CD47 antibody is a monospecific antibody. In some embodiments, the anti-CD47 antibody is a multispecific (e.g., bispecific) antibody. In some embodiments, the term “anti-CD47 antibody” includes antibody-based constructs (such as multispecific constructs), for example, but not limited to, triomab, DART (i.e., dual affinity retargeting antibody), TandAb (i.e., tandem diabody), tandem scFv, CrossMab, DNL (i.e., dock and lock antibody), DVD-Ig (i.e., dual variable domain immunoglobulin), tetravalent bispecific IgG, nanobody, dual targeting domain, and ART-Ig (i.e., asymmetric re-engineering technology-immunoglobulin).Additional details regarding exemplary antibody constructs (both monospecific and multispecific) are provided in Husain et al. (2018) Biodrugs 32(5):441-464 and Spiess et al. (2015) Molecular Immunology 67(2):95-106. In some embodiments, the anti-CD47 antibody is Hu5F9-G4, B6H12.2, BRIC126, CC-90002, SRF231, or IBI188 (Innovent Biologics) (for additional information regarding these anti-CD47 antibodies, see, for example, Zhao et al. (2011), PNAS USA 108:18342-18347; Chao et al. (2010) Cell 142:699-713, Kim et al. (2012) Leukemia 26:2538-2545; Chao et al. (2011) Blood 118:4890-4891; Goto et al. (2014) Eur J. Cancer 50:1836-1846; and Edris et al. (2012) PNAS USA 109:6656-61).

[0040] In some embodiments, an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) binds to SIRPα (e.g., hSIRPα). In some embodiments, the agent has a K of about 10 nM D or better K D(For example, at least any one of about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM or less than 10 pM) binds to SIRPα (for example, hSIRPα). In some embodiments, the agent that binds to SIRPα (for example, hSIRPα) exhibits an SIRPα receptor occupancy of at least about 50% (for example, at least any one of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%) in a human subject. In some embodiments, the agent that binds to SIRPα (for example, hSIRPα) has an EC50 of about 80 ng / ml or less, for example, any one of about 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 ng / ml. In some embodiments, the agent that binds to SIRPα (for example, hSIRPα) is an anti-SIRPα antibody (for example, a therapeutic anti-SIRPα antibody) or an antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment is a Fab, Fab’, Fab’-SH, F(ab’)2, Fv, scFv, single-chain antibody, or diabody. In some embodiments, the anti-SIRPα antibody is a monospecific antibody or a monospecific antibody construct (including, but not limited to, those described above). In some embodiments, the anti-SIRPα antibody is a multispecific (for example, bispecific) antibody or a multispecific antibody construct (including, but not limited to, those described above). In some embodiments, the anti-SIRPα antibody is KWAR23, SE12C3, 040, or MY-1 (for additional information regarding these anti-SIRPα antibodies, see, for example, Ring et al. (2017) PNAS USA 114(49):E10578-E10585); Murata et al. (2018) Cancer Sci 109(5):1300-1308; and Yanigata et al. (2017) JCI Insight 2:e89140).In some embodiments, the anti-SIRPα antibody is an antibody described in WO2018 / 057669, US-2018-0105600-A1; US20180312587; WO2018107058; WO2019023347; US20180037652; WO2018210795; WO2017178653; WO2018149938; WO2017068164; and WO2016063233, the content of which is hereby incorporated by reference in its entirety.

[0041] In some embodiments, an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is an anti-SIRPβ antibody or an anti-SIRPγ antibody (e.g., an anti-SIRPβ antibody or an anti-SIRPγ antibody that can bind to SIRPα), or an antigen-binding fragment thereof. In some embodiments, the agent is an antibody (or an antigen-binding fragment thereof) that can bind to two or more of SIRPα, SIRPβ, and SIRPγ. In some embodiments, such antibodies have a K of about 10 nM D or better K DBind to SIRPα (e.g., hSIRPα) at (such as at least any one of about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM or less than 10 pM). In some embodiments, the antibody exhibits an SIRPα receptor occupancy of at least about 50% (e.g., at least any one of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%) in a human subject. In some embodiments, the antibody has an EC50 of about 80 ng / ml or less, such as any one of about 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 ng / ml. In some embodiments, the antigen-binding fragment is a Fab, Fab’, Fab’-SH, F(ab’)2, Fv, scFv, single-chain antibody, or diabody. In some embodiments, the antibody is a monospecific antibody or monospecific antibody construct (including but not limited to those described above). In some embodiments, the antibody is a multispecific (e.g., bispecific) antibody or multispecific antibody construct (including but not limited to those described above).

[0042] In some embodiments, the agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is a fusion polypeptide comprising a portion that binds to CD47. In some embodiments, the fusion polypeptide comprises an antibody Fc region and a portion that binds to CD47. In some embodiments, the portion of the fusion polypeptide that binds to CD47 (e.g., hCD47) has a K D or better K D(such as at least any one of about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM, or less than 10 pM) binds to CD47 (e.g., hCD47). In some embodiments, the fusion polypeptide exhibits a CD47 receptor occupancy of at least about 50% (e.g., at least any one of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%) in a human subject. In some embodiments, the fusion polypeptide has an EC50 of about 80 ng / ml or less, e.g., any one of about 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 ng / ml. In some embodiments, the fusion polypeptide comprises a WT human antibody Fc region. In some embodiments, the fusion polypeptide comprises an Fc variant (e.g., a variant of the WT human antibody Fc region) that exhibits reduced (e.g., removed, etc.) effector function compared to the WT Fc region. Exemplary Fc variants are described in WO2017 / 027422 and US2017 / 0107270, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the portion that binds to CD47 (e.g., hCD47) is WT SIRPα (e.g., hSIRPα), or WT SIRPγ (e.g., hSIRPγ). In some embodiments, the portion that binds to CD47 (e.g., hCD47) is a CD47-binding fragment (e.g., the d1 domain) of WT SIRPα (e.g., hSIRPα) or WT SIRPγ (e.g., hSIRPγ). In some embodiments, the portion that binds to CD47 (e.g., hCD47) is a SIRPα variant, a SIRPγ variant, a SIRPβ variant, or a CD47-binding fragment thereof (e.g., the d1 domain).Exemplary SIRPγ variants, SIRPβ1 variants, and SIRPβ2 variants are described, for example, in WO2013 / 109752; US2015 / 0071905; USP9,944,911; WO2016 / 023040; WO2017 / 027422; US2017 / 0107270; USP10,259,859; US9845345; WO2016187226; US20180155405; WO2017177333; WO2014094122; US2015329616; US20180312563; WO2018176132; WO2018081898; WO2018081897; PCT / US2019 / 048921; US20180141986A1; and EP3287470A1, the contents of which are hereby incorporated by reference in their entirety.

[0043] In some embodiments, the agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is a fusion polypeptide comprising an antibody Fc region and a SIRPα variant. In some embodiments, the SIRPα variant has a K of about 10 nM D or better K D(For example, at least any one of about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM, or less than 10 pM) binds to CD47 (e.g., hCD47). In some embodiments, the fusion polypeptide exhibits at least about 50% (e.g., at least any one of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%) CD47 receptor occupancy in a human subject. In some embodiments, the fusion polypeptide has an EC50 of about 80 ng / ml or less, for example, any one of about 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 ng / ml. In some embodiments, the fusion polypeptide comprises a WT human antibody Fc region. In some embodiments, the fusion polypeptide comprises an Fc variant (e.g., a variant of the WT human antibody Fc region) that exhibits reduced (e.g., removed, etc.) effector function compared to the WT Fc region, including, for example, those described in the references cited herein. In some embodiments, the fusion polypeptide comprises a SIRPα variant described in WO2013 / 109752; US2015 / 0071905; WO2016 / 023040; WO2017 / 027422; US2017 / 0107270; USP10,259,859; US9845345; WO2016187226; US20180155405; WO2017177333; WO2014094122; US2015329616; US20180312563; WO2018176132; WO2018081898; WO2018081897; US20180141986A1; and EP3287470A1, the content of which is hereby incorporated by reference in its entirety.In some embodiments, the fusion polypeptide comprising the antibody Fc region and the SIRPα variant is TTI-621, TTI-622, or IMM01 (see, e.g., Petrova et al. (2017) Clin Cancer Res 23:1086-1079; Russ et al. (2018) Blood Rev S0268-960X(17)30093-0; Zhang, X, Chen, W, Fan, J et al. Disrupting CD47-SIRPα axis alone or combined with autophagy depletion for the therapy of glioblastoma. Carcinogenesis 2018;39:689-99).

[0044] In some embodiments, the agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is a fusion polypeptide comprising an SIRPα D1 domain variant (e.g., an SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein).

[0045] In some embodiments, provided is a method of treating cancer (e.g., leukemia such as acute lymphoblastic leukemia) in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), and (b) a BCL2 inhibitor (e.g., a selective BCL2 inhibitor such as venetoclax). In some embodiments, the agent is a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85, and the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index).

[0046] In some embodiments, provided is a method of treating cancer (e.g., colon cancer) in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), and (b) a platinum-based chemotherapeutic agent (e.g., cisplatin). In some embodiments, the agent is a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85, and the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index).

[0047] In some embodiments, provided is a method of treating cancer (e.g., head and neck cancer, head and neck squamous cell carcinoma, etc.) in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) a PD-1 inhibitor, (c) an antimetabolite, and (d) a platinum-based chemotherapeutic agent. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85, and the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index).

[0048] In some embodiments, provided is a method of treating cancer (e.g., gastric cancer or gastroesophageal cancer) in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-HER2 antibody, (c) an anti-VEGFR2 antibody, and (d) paclitaxel. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85, and the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index).

[0049] Further details regarding the method of treatment with a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant are described below. See also WO2017 / 027422 and U.S. Patent No. 10,259,859. The entire contents of each of these are hereby incorporated by reference in their entirety.

[0050] Signal regulatory protein alpha (SIRPα) D1 domain and variants thereof In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein alpha (SIRP-α) D1 variant, wherein the polypeptide has an amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain (e.g., the wild-type SIRPα D1 domain shown in SEQ ID NO: 1 or 2); and at least one additional amino acid mutation at a residue selected from the group consisting of residues 6, 27, 31, 47, 53, 54, 56, 66, and 92 relative to the wild-type SIRPα D1 domain (the wild-type SIRPα D1 domain shown in SEQ ID NO: 1 or 2), and comprises an SIRPα D1 domain or a fragment thereof.

[0051] Also disclosed herein is, in some embodiments, a polypeptide comprising an Fc domain variant, wherein the Fc domain variant dimer comprises two Fc domain variants, and each Fc domain variant is independently selected from (i) a human IgG1 Fc region consisting of the mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of the mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A.

[0052] Signal regulatory protein alpha (referred to herein as "SIRP-α" or "SIRP-alpha") is a transmembrane glycoprotein belonging to the immunoglobulin superfamily that is widely expressed on the membranes of myeloid cells. SIRPα interacts with CD47, a protein that is widely expressed in many cell types in the body. The interaction between SIRPα and CD47 prevents the engulfment of "self" cells that can be recognized by the immune system in other ways. High expression of CD47 in tumor cells has been observed to act as a negative prognostic factor for survival in acute myeloid leukemia and some solid tumor cancers.

[0053] Native SIRPα is composed of three highly homologous immunoglobulin (Ig)-like extracellular domains (D1, D2, and D3). The SIRPα D1 domain (the "D1 domain") refers to the membrane-distal extracellular domain of SIRPα and mediates the binding of SIRPα to CD47. As used herein, the term "SIRPα polypeptide" refers to any SIRPα polypeptide or fragment thereof that can bind to CD47. There are at least 10 variants of wild-type human SIRPα. Table 1 shows the amino acid sequences of the D1 domains of naturally occurring wild-type human SIRPα D1 domain variants (SEQ ID NOs: 1 and 2). In some embodiments, the SIRPα polypeptide comprises the SIRPα D1 domain. In some embodiments, the SIRPα polypeptide comprises a wild-type D1 domain such as those provided in SEQ ID NOs: 1 and 2. In some embodiments, the SIRPα polypeptide comprises the D2 or D3 domain (or both the D2 and D3 domains) of wild-type human SIRPα (see Table 3). [Table 1]

[0054] As used herein, the term "SIRPα D1 domain variant" refers to a polypeptide comprising the SIRPα D1 domain or the CD47-binding portion of an SIRPα polypeptide that has a higher affinity for CD47 than wild-type SIRPα. SIRPα D1 domain variants contain at least one amino acid substitution, deletion, or insertion (or combinations thereof) relative to wild-type SIRPα.

[0055] In some embodiments, the SIRPα D1 domain variants disclosed herein include the SIRPα D1 domain or variants thereof. In some embodiments, the SIRPα D1 domain variants include one or more amino acid substitutions, insertions, additions, or deletions relative to the wild-type D1 domain shown in SEQ ID NOs: 1 and 2. Table 2 lists exemplary amino acid substitutions in each SIRPα D1 domain variant (SEQ ID NOs: 13-14). In some embodiments, the SIRPα D1 domain polypeptide or SIRPα D1 domain variant includes a fragment of the D1 domain. In some embodiments, the SIRPα polypeptide fragment or SIRPα D1 domain variant fragment includes an amino acid sequence less than 10 amino acids in length, about 10 amino acids in length, about 20 amino acids in length, about 30 amino acids in length, about 40 amino acids in length, about 50 amino acids in length, about 60 amino acids in length, about 70 amino acids in length, about 80 amino acids in length, about 90 amino acids in length, about 100 amino acids in length, or about 100 amino acids or more in length. In some embodiments, the SIRPα D1 domain fragment retains the ability to bind to CD47.

[0056] In some embodiments, the polypeptides of the present disclosure that include the SIRPα D1 domain variant bind to CD47 with a higher binding affinity than the wild-type human SIRPα D1 domain. In some embodiments, the SIRPα D1 domain variant binds to human CD47 with an affinity that is at least 1-fold (e.g., at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 5-fold or more) higher than the affinity of the naturally occurring D1 domain. In some embodiments, the SIRPα D1 domain variant binds to human CD47 with an affinity that is at least 1-fold (e.g., at least 10-fold, 100-fold, 1000-fold, or 1000-fold or more) higher than the affinity of the naturally occurring D1 domain.

[0057] As used herein, the terms "optimized affinity" or "optimized binding affinity" refer to the optimized strength of the binding interaction between a polypeptide disclosed herein, such as a SIRPα D1 domain variant, and CD47. For example, in some embodiments, the polypeptide binds primarily or with higher affinity to CD47 on cancer cells and does not substantially bind or binds with lower affinity to CD47 on non-cancer cells. In some embodiments, the binding affinity between the polypeptide and CD47 is optimized such that the interaction does not cause clinically relevant toxicity or reduces toxicity as compared to variants that bind with maximal affinity. In some embodiments, to achieve the optimized binding affinity between a polypeptide provided herein and CD47, a polypeptide comprising a SIRPα D1 domain variant is developed to have a binding affinity to CD47 that is lower than that which can be maximally achieved. In some embodiments, the SIRPα D1 domain variants disclosed herein cross-react with murine, non-human primate (NHP), and human CD47.

[0058] As used herein, the term "immunogenicity" refers to the property of a protein (e.g., a therapeutic protein) that elicits an immune response in a host as if it were a foreign antigen. The immunogenicity of a protein can be assayed in vitro by various methods, such as in vitro T cell proliferation assays.

[0059] As used herein, the term "minimal immunogenicity" refers to the immunogenicity of a protein (e.g., a therapeutic protein) that has been modified, e.g., by amino acid substitution, to be lower (e.g., at least 10%, 25%, 50%, or 100% lower) than the immunogenicity before the amino acid substitution was introduced (e.g., the unmodified protein). In some embodiments, the protein (e.g., a therapeutic protein) is modified to have minimal immunogenicity and elicits little or no host immune response even if it is considered a foreign antigen.

[0060] In some embodiments, the SIRPα D1 domain variant exhibits minimal immunogenicity. In some embodiments, the SIRPα polypeptide of the present disclosure administered to a subject has the same amino acid sequence as the SIRPα polypeptide in the subject's biological sample, except for amino acid changes that increase the affinity of the SIRPα D1 domain variant. In some embodiments, the polypeptide variants disclosed herein have a reduced risk of side effects compared to anti-CD47 antibodies or wild-type SIRPα. In some embodiments, the polypeptide variants disclosed herein have a reduced risk of anemia compared to anti-CD47 antibodies or wild-type SIRPα. In some embodiments, the polypeptide variants disclosed herein do not cause acute anemia in rodent or non-human primate (NHP) studies.

[0061] Table 2 shows the specific amino acid substitutions of the SIRPα D1 domain variant for each D1 domain sequence. In some embodiments, the SIRPα D1 domain variant comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more) of the substitutions listed in Table 2. In some embodiments, the SIRPα D1 domain variant comprises up to 14 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant comprises up to 10 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant comprises up to 7 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant of the present disclosure has at least 90% (e.g., at least 92%, 95%, 97% or greater than 97%) amino acid sequence identity to the sequence of the wild-type D1 domain.

[0062] In some embodiments, the SIRPα D1 domain variant is a chimeric SIRPα D1 domain variant that includes two or more wild-type D1 domains or portions thereof (e.g., one wild-type D1 domain or a portion thereof and another wild-type D1 domain or a portion thereof). In some embodiments, the chimeric SIRPα D1 domain variant includes at least two portions of a wild-type D1 domain or a variant thereof (e.g., three, four, five, or more portions), where each of the portions is derived from a different wild-type D1 domain. In some embodiments, the chimeric SIRPα D1 domain variant further includes one or more amino acid substitutions listed in Table 2. [Table 2]

[0063] In some embodiments, the polypeptide includes a SIRPα D1 domain variant having the following sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGNITPADAGTYYCX 12 KX 13 RKGSPDDVEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 13), X1 is L, I, or V, X2 is V, L, or I, X3 is A or V, X4 is A, I, or L, X5 is I, T, S, or F, X6 is E, V, or L, X7 is K or R, X8 is E or Q, X9 is H, P, or R, X 10 is L, T, or G, X 11 is K or R, X 12 is V or I, X 13 is F, L, or V, X 14is F or V, and the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO: 1.

[0064] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant comprising the sequence of SEQ ID NO: 13, wherein X1 is L, I, or V. In any of the foregoing embodiments, X2 is V, L, or I. In some embodiments, X3 is A or V. In some embodiments, X4 is A, I, or L. In some embodiments, X5 is I, T, S, or F. In some embodiments, X6 is E, V, or L. In some embodiments, X7 is K or R. In some embodiments, X8 is E or Q. In some embodiments, X9 is H, P, or R. In some embodiments, X 10 is L, T, or G. In some embodiments, X 11 is K or R. In some embodiments, X 12 is V or I. In some embodiments, X 13 is F, L, V. In some embodiments, X 14 is F or V. In some embodiments, the polypeptide of this aspect of the disclosure comprises six or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO: 1.

[0065] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof is at 1×10 -8 M or less, 5x10-9 less than M, 1 x 10 -9 less than M, 5 x 10 -10 less than M, 1 x 10 -10 less than M or 1 x 10 -11 K less than M D and binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM D and binds to CD47.

[0066] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant comprising the following sequence: EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 14), X1 is L, I, or V, X2 is V, L, or I, X3 is A or V, X4 is V, I, or L, X5 is I, T, S, or F, X6 is E, V, or L, X7 is K or R, X8 is E or Q, X9 is H, P, or R, X 10 is S, T, or G, X 11 is K or R, X 12 is V or I, X 13 is F, L, or V, X 14 is F or V, and the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO: 2.

[0067] In some embodiments of this aspect of the disclosure, the polypeptide comprises the sequence of SEQ ID NO: 14, where X1 is L, I, or V. In some embodiments, X2 is V, L, or I. In some embodiments, X3 is A or V. In some embodiments, X4 is V, I, or L. In some embodiments, X5 is I, T, S, or F. In some embodiments, X6 is E, V, or L. In some embodiments, X7 is K or R. In some embodiments, X8 is E or Q. In some embodiments, X9 is H, P, or R. In some embodiments, X 10 is S, T, or G. In some embodiments, X 11 is K or R. In some embodiments, X 12 is V or I. In some embodiments, X 13 is F, L, or V. In some embodiments, X 14 is F or V. In some embodiments, the polypeptide of this aspect of the disclosure comprises six or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO: 2.

[0068] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K of less than 1×10 -8 M, less than 5x10 -9 M, less than 1x10 -9 M, less than 5x10 -10 M, less than 1x10 -10 M, less than 1x10 -11 M or less.D It binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM for binding to CD47. D It binds to CD47.

[0069] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSLX 11 PVGPIQWFRGAGPX 12 RX 13 LIYNQX 14 X 15 GX 16 FPRVTTVSX 17 X 18 TX 19 RX 20 NMDFX 21 IX 22 IX 23 NITPADAGTYYCX 24 KX 25 RKGSPDX 26 X 27 EX 28 KSGAGTELSVRX 29 KPS (SEQ ID NO: 23), X1 is E or G; X2 is L, I, or V; X3 is V, L, or I; X4 is S or F; X5 is L or S; X6 is S or T; X7 is A or V; X8 is I or T; X9 is H or R; X 10 is A, V, I, or L; X 11 is I, T, S, or F; X 12 is A or G; X 13 is E, V, or L; X 14 is K or R; X 15 is E or Q; X 16is H, P, or R; X 17 is D or E; X 18 is S, L, T, or G; X 19 is K or R; X 20 is E or D; X 21 is S or P; X 22 is S or R; X 23 is S or G; X 24 is V or I; X 25 is F, L, V; X 26 is D or absent; X 27 is T or V; X 28 is F or V; X 29 is A or G; wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2.

[0070] In any of the foregoing embodiments of this aspect of the present disclosure, X2 is L, I, or V. In any of the foregoing embodiments, X3 is V, L, or I. In some embodiments, X4 is S or F. In some embodiments, X5 is L or S. In some embodiments, X6 is S or T. In some embodiments, X7 is A or V. In some embodiments, X8 is I or T. In some embodiments, X9 is H or R. In some embodiments, X 10 is A, V, I, or L. In some embodiments, X 11 is I, T, S, or F. In some embodiments, X 12 is A or G. In some embodiments, X 13 is E, V, or L. In some embodiments, X 14 is K or R. In some embodiments, X 15 is E or Q. In some embodiments, X 16 is H, P, or R. In some embodiments, X 17 is D or E. In some embodiments, X18 is S, L, T, or G. In some embodiments, X 19 is K or R. In some embodiments, X 20 is E or D. In some embodiments, X 21 is S or P. In some embodiments, X 22 is S or R. In some embodiments, X 23 is S or G. In some embodiments, X 24 is V or I. In some embodiments, X 25 is F, L, V. In some embodiments, X 26 is D or absent. In some embodiments, X 27 is T or V. In some embodiments, X 28 is F or V. In some embodiments, X 29 is A or G. In some embodiments, the polypeptide of this aspect of the present disclosure comprises six or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2.

[0071] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K of less than 1×10 -8 M, less than 5x10 -9 M, less than 1x10 -9 M, less than 5x10 -10 M, less than 1x10 -10 M or less than 1x10 -11 M of K DIt binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM D It binds to CD47.

[0072] In some embodiments, the polypeptide of the present disclosure comprising the SIRPα D1 domain variant further comprises the D2 domain having the sequence of SEQ ID NO: 24, the D3 domain having the sequence of SEQ ID NO: 25, or the D2 domain having the sequence of SEQ ID NO: 24 and the D3 domain having the sequence of SEQ ID NO: 25 of the wild-type human SIRPα shown in Table 3. In some embodiments, the SIRPα D1 domain variant further comprises a fragment or variant of the D2 domain, or a fragment or variant of the D3 domain. In some embodiments, the SIRPα D1 domain variant further comprises a fragment or variant of the D2 domain and a fragment or variant of the D3 domain. In some embodiments, the SIRPα D1 domain variant is linked to the D2 or D3 domain via a linker. In some embodiments, the SIRPα D1 domain variant is linked to the D2 and D3 domains via a linker.

Table 3

[0073] In some embodiments, the polypeptides of the present disclosure comprising an SIRPα D1 domain variant are attached to an Fc domain variant to improve the pharmacokinetic properties of the polypeptide, for example, to increase the serum half-life. In some embodiments, the SIRPα D1 domain variant is attached to an Fc domain variant that cannot dimerize. In some embodiments, the Fc domain variant serves to increase the serum half-life of the polypeptides described herein. In some embodiments, the polypeptides of the present disclosure comprising an SIRPα D1 domain variant do not include any of the sequences of SEQ ID NOs: 26-36 shown in Table 4. [Table 4]

[0074] In some embodiments, the polypeptides and polypeptide constructs described herein are utilized in vitro for binding assays such as immunoassays. For example, in some embodiments, the polypeptides and polypeptide constructs described herein are utilized in solution or are bound to a solid support. In some embodiments, the polypeptides utilized in immunoassays are detectably labeled in various ways.

[0075] In some embodiments, the polypeptides and polypeptide constructs described herein are bound to various carriers and used to detect the presence of specific antigen-expressing cells. Examples of carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamide, agarose, and magnetite. The nature of the carrier can be either soluble or insoluble.

[0076] A variety of different labels and labeling methods are known. Examples of labels include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds. A variety of techniques are available for attaching labels to the polypeptides disclosed herein.

[0077] In some embodiments, the polypeptide is conjugated to a low molecular weight hapten. These haptens are then specifically detected by a second reaction. For example, in some embodiments, hapten biotin is used with avidin, or hapten dinitrophenol, pyridoxal, or fluorescein is detected with a specific anti-hapten antibody (e.g., anti-dinitrophenol antibody, anti-pyridoxal antibody, and anti-fluorescein antibody, respectively).

[0078] SIRPα D1 domain variant having an altered glycosylation pattern In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein alpha (SIRP-α) D1 variant, wherein the polypeptide has an amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain (e.g., the wild-type SIRPα D1 domain shown in SEQ ID NO: 1 or 2); and at least one additional amino acid mutation at a residue selected from the group consisting of residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92 relative to the wild-type SIRPα D1 domain (the wild-type SIRPα D1 domain shown in SEQ ID NO: 1 or 2), and comprises the SIRPα D1 domain or a fragment thereof.

[0079] Also disclosed herein is, in some embodiments, a polypeptide comprising an Fc domain variant, wherein the Fc domain variant dimer comprises two Fc domain variants, and each Fc domain variant is independently selected from (i) a human IgG1 Fc region consisting of the mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of the mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A.

[0080] In some embodiments, the polypeptide in the compositions disclosed herein comprises a SIRPα D1 domain variant with reduced or minimal glycosylation. The D1 domains of SEQ ID NOs: 1 and 2 in Table 1 each contain a single potential N-linked glycosylation site at amino acid N80 in the sequence N80ITP. Expression of the SIRPα D1 domain in Chinese hamster ovary (CHO) cells results in a major band at 16 kDa (non-glycosylated) and minor high molecular weight bands that are removed by Endo Hf. Endo Hf is a recombinant protein fusion of endoglycosidase H and maltose binding protein. Endo Hf cleaves within the high mannose chitobiose core and some hybrid oligosaccharides from N-linked glycoproteins. This means that the proline at amino acid position 83 can reduce the efficiency of glycosylation, resulting in proteins with different degrees of glycosylation and thus heterogeneity. In the case of drug development, heterogeneity can pose challenges in process development. Thus, in some embodiments, amino acid N80 of the SIRPα D1 variant is mutated to Ala to investigate the possibility of generating a homogeneous non-glycosylated form of the SIRPα D1 domain variant. In some embodiments, for generating a non-glycosylated, SIRPα D1 domain variant, amino acid N80 in the SIRPα D1 domain variant is replaced by any natural and non-natural amino acid, such as any amino acid like N80A and N80Q. In some embodiments, the SIRPα D1 domain variant comprises the N80A mutation and at least one additional mutation (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more additional mutations). In some embodiments, the additional mutation is in the CD47 binding site. In some embodiments, the additional mutation is within the hydrophobic core of the D1 domain.

[0081] In some embodiments, the polypeptide in the compositions disclosed herein comprises a SIRPα D1 domain variant with increased glycosylation compared to the wild-type SIRPα D1 domain. In another option to enhance the homogeneity of the final product, the efficiency of glycosylation at amino acid N80 is enhanced to produce a SIRPα D1 domain variant with increased glycosylation compared to the wild-type. In some embodiments, the amino acid P83 in the sequence NITP83 affects the degree of glycosylation at amino acid N80. In some embodiments, changing P83 to any amino acid increases the efficiency of glycosylation at N80. In some embodiments, the amino acid P83 in the SIRPα D1 domain variant is replaced by any amino acid, such as natural and non-natural amino acids, for example, P83V, P83A, P83I, and P83L. In some embodiments, the polypeptides of the present disclosure are expressed in cells optimized to not glycosylate proteins expressed, for example, by genetic engineering of cell lines (e.g., genetically engineered yeast or mammalian hosts), or by changing cell culture conditions such as the addition of kifunensine, or using natural non-glycosylating hosts such as prokaryotes (e.g., E. coli).

[0082] Table 5 shows specific amino acid substitutions of the SIRPα D1 domain variant for each D1 domain variant sequence. In some embodiments, the SIRPα D1 domain variant comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more) of the substitutions listed in Table 5. In some embodiments, the SIRPα D1 domain variant is not glycosylated or minimally glycosylated. In some embodiments, the SIRPα D1 domain variant is fully glycosylated or nearly fully glycosylated. In some embodiments, the SIRPα D1 domain variant comprises up to 14 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant comprises up to 10 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant comprises up to 7 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant of the present disclosure has at least 90% (e.g., at least 92%, 95%, 97% or more than 97%) amino acid sequence identity to the sequence of the wild-type D1 domain.

[0083] In some embodiments, the SIRPα D1 domain variant is a chimeric SIRPα D1 domain variant comprising two or more wild-type D1 domains or portions thereof (e.g., one wild-type D1 domain or a portion thereof and another wild-type D1 domain or a portion thereof). In some embodiments, the chimeric SIRPα D1 domain variant comprises at least two portions (e.g., 3, 4, 5, or more portions) of the wild-type D1 domain or its variant, where each of the portions is derived from a different wild-type D1 domain. In some embodiments, the chimeric SIRPα D1 domain variant further comprises one or more amino acid substitutions listed in Table 5.

Table 5-1

Table 5-2

Table 5-3

[0084] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSPDDVEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 37), X1 is L, I, or V; X2 is V, L, or I; X3 is A, or V; X4 is A, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K, or R; X8 is E, or Q; X9 is H, P, or R; X 10 is L, T, or G; X 11 is K, or R; X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; X 14 is V, or I; X 15 is F, L, or V; X 16 is F or V; the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1.

[0085] In some embodiments of this aspect of the present disclosure, the polypeptide comprises a SIRPα D1 domain variant having the sequence of SEQ ID NO: 37, and X1 is L, I, or V. In some embodiments, X2 is V, L, or I. In some embodiments, X3 is A or V. In some embodiments, X4 is A, I, or L. In some embodiments, X5 is I, T, S, or F. In some embodiments, X6 is E, V, or L. In some embodiments, X7 is K or R. In some embodiments, X8 is E or Q. In some embodiments, X9 is H, P, or R. In some embodiments, X 10 is L, T, or G. In some embodiments, X 11 is K or R. In some embodiments, X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. In some embodiments, X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. In some embodiments, X 14 is V or I. In some embodiments, X 15 is F, L, V. In some embodiments, X 16 is F or V.

[0086] In some embodiments, the polypeptides provided herein comprise 10 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptides provided herein comprise 7 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1.

[0087] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M or a K D less than 1×10 D M. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K

[0088] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSPDTEX 16KSGAGTELSVRAKPS (SEQ ID NO: 38), X1 is L, I, or V; X2 is V, L, or I; X3 is A or V; X4 is V, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K or R; X8 is E or Q; X9 is H, P, or R; X 10 is S, T, or G; X 11 is K or R; X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; X 14 is V or I; X 15 is F, L, or V; and X 16 is F or V; the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2.

[0089] In some embodiments of this aspect of the present disclosure, the polypeptide comprises a SIRPα D1 domain variant having the sequence of SEQ ID NO: 38, and X1 is L, I, or V. In some embodiments, X2 is V, L, or I. In some embodiments, X3 is A or V. In some embodiments, X4 is V, I, or L. In some embodiments, X5 is I, T, S, or F. In some embodiments, X6 is E, V, or L. In some embodiments, X7 is K or R. In some embodiments, X8 is E or Q. In some embodiments, X9 is H, P, or R. In some embodiments, X 10 is S, T, or G. In some embodiments, X 11 is K or R. In some embodiments, X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. In some embodiments, X 13is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. In some embodiments, X 14 is V or I. In some embodiments, X 15 is F, L, or V. In some embodiments, X 16 is F or V.

[0090] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having 10 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having 7 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2.

[0091] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M or less than 1×10 D M. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pMD and binds to CD47.

[0092] In another aspect, the disclosure features a polypeptide comprising an SIRPα D1 domain variant having the following sequence: EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSLX 11 PVGPIQWFRGAGPX 12 RX 13 LIYNQX 14 X 15 GX 16 FPRVTTVSX 17 X 18 TX 19 RX 20 NMDFX 21 IX 22 IX 23 X 24 ITX 25 ADAGTYYCX 26 KX 27 RKGSPDX 28 X 29 EX 30 KSGAGTELSVRX 31 KPS (SEQ ID NO: 47), X1 is E or G; X2 is L, I, or V; X3 is V, L, or I; X4 is S or F; X5 is L or S; X6 is S or T; X7 is A or V; X8 is I or T; X9 is H, R, or L; X 10 is A, V, I, or L; X 11 is I, T, S, or F; X 12 is A or G; X 13 is E, V, or L; X 14 is K or R; X 15 is E or Q; X 16 is H, P, or R; X 17 is D or E; X 18 is S, L, T, or G; X 19 is K or R; X 20 is E or N; X 21 is S or P; X22 is S or R; X 23 is S or G; X 24 is any amino acid; X 25 is any amino acid; X 26 is V or I; X 27 is F, L, V; X 28 is D or absent; X 29 is T or V; X 30 is F or V; X 31 is A or G; The variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2.

[0093] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 47 and X1 is E or G. In any of the foregoing embodiments of this aspect of the present disclosure, X2 is L, I, or V. In any of the foregoing embodiments, X3 is V, L, or I. In any of the foregoing embodiments, X4 is S or F. In any of the foregoing embodiments, X5 is L or S. In any of the foregoing embodiments, X6 is S or T. In any of the foregoing embodiments, X7 is A or V. In any of the foregoing embodiments, X8 is I or T. In any of the foregoing embodiments, X9 is H or R. In any of the foregoing embodiments, X 10 is A, V, I, or L. In any of the foregoing embodiments, X 11 is I, T, S, or F. In any of the foregoing embodiments, X 12 is A or G. In any of the foregoing embodiments, X 13 is E, V, or L. In any of the foregoing embodiments, X 14 is K or R. In any of the foregoing embodiments, X 15 is E or Q. In any of the foregoing embodiments, X 16 is H, P, or R. In any of the foregoing embodiments, X 17is D or E. In any of the foregoing embodiments, X 18 is S, L, T, or G. In any of the foregoing embodiments, X 19 is K or R. In any of the foregoing embodiments, X 20 is E or N. In any of the foregoing embodiments, X 21 is S or P. In any of the foregoing embodiments, X 22 is S or R. In any of the foregoing embodiments, X 23 is S or G. In any of the foregoing embodiments, X 24 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. In any of the foregoing embodiments, X 25 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. In any of the foregoing embodiments, X 26 is V or I. In any of the foregoing embodiments, X 27 is F, L, V. In any of the foregoing embodiments, X 28 is D or absent. In any of the foregoing embodiments, X 29 is T or V. In any of the foregoing embodiments, X 30 is F or V. In any of the foregoing embodiments, X 31 is A or G.

[0094] In some embodiments, the polypeptide of this aspect of the disclosure comprises 10 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptide of this aspect of the disclosure comprises 7 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2.

[0095] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M or less than 1×10 D M. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K D of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM.

[0096] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVX2VAAGEX3AX4LX5CTX6TSLX7PVGPIQWFRGAGPX8RX9LIYNQX 10 X 11 GX 12 FPRVTTVSX 13 X 14 TKRX 15 NMDFSIX 16 IX 17 X 18 ITPADAGTYYCX 19 KFRKGX 20 X 21 X 22 DX23 EFKSGAGTELSVRAKPS (SEQ ID NO: 48), X1 is V or I; X2 is L or S; X3 is T or S; X4 is T or I; X5 is R or H; X6 is A, V, or I; X7 is I, R, Y, K or F; X8 is G or A; X9 is E or V; X 10 is K or R; X 11 is E, D or Q; X 12 is H or P; X 13 is D or E; X 14 is S, L or T; X 15 is N or E; X 16 is R or S; X 17 is G or S; X 18 is N or A; X 19 is V or I; X 20 is S, I or M; X 21 is P or absent; X 22 is D or P; X 23 is V or T, or a fragment thereof.

[0097] In another aspect, the present disclosure features a polypeptide comprising a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9ITPADAGTYYCX 10 KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 49), X1 is V, L, or I; X2 is A, I, V, or L; X3 is I, F, S, or T; X4 is E, V, or L; X5 is K or R; X6 is E or Q; X7 is H, P, or R; X8 is L, T, S, or G; X9 is A; X 10is V or I; wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1.

[0098] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 49, and X1 is V, L, or I. In any of the foregoing embodiments of this aspect of the disclosure, X2 is A, I, V, or L. In any of the foregoing embodiments, X3 is I, F, S, or T. In any of the foregoing embodiments, X4 is E, V, or L. In any of the foregoing embodiments, X5 is K or R. In any of the foregoing embodiments, X6 is E or Q. In any of the foregoing embodiments, X7 is H, P, or R. In any of the foregoing embodiments, X8 is L, T, S, or G. In any of the foregoing embodiments, X9 is A. In any of the foregoing embodiments, X 10 is V or I.

[0099] In some embodiments, the polypeptide comprises a SIRPα D1 domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the sequence of SEQ ID NO: 49, and each of X1, X2, X3, X4, X5, X6, X7, X8, X9, and X 10 is not a wild-type amino acid.

[0100] In some embodiments, the polypeptide of this aspect of the disclosure comprises 10 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1. In some embodiments, the polypeptide of this aspect of the disclosure comprises 7 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1.

[0101] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M or less than 1×10 D M. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K D of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM.

[0102] In another aspect, the disclosure features a polypeptide comprising a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVSVAAGESAILHCTX2TSLX3PVGPIQWFRGAGPARX4LIYNQX5X6GX7FPRVTTVSEX8TKRENMDFSISISX9ITPADAGTYYCX 10 KFRKGSPDTEFKSGAGTELSVRAKPS (SEQ ID NO: 50), where X1 is V or I; X2 is V or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is E or Q; X7 is H or P; X8 is S or T; X9 is N or A; X 10is V or I; wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2.

[0103] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 50, and X1 is V or I. In any of the foregoing embodiments of this aspect of the present disclosure, X2 is V or I. In any of the foregoing embodiments, X3 is I or F. In any of the foregoing embodiments, X4 is E or V. In any of the foregoing embodiments, X5 is K or R. In any of the foregoing embodiments, X6 is E or Q. In any of the foregoing embodiments, X7 is H or P. In any of the foregoing embodiments, X8 is S or R. In any of the foregoing embodiments, X9 is N or A. In any of the foregoing embodiments, X 10 is V or I.

[0104] In some embodiments, the polypeptide comprises a SIRPα D1 domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the sequence of SEQ ID NO: 50, and each of X1, X2, X3, X4, X5, X6, X7, X8, X9, and X 10 is not a wild-type amino acid.

[0105] In some embodiments, the polypeptide of this aspect of the present disclosure comprises no more than 10 amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide of this aspect of the present disclosure comprises no more than 7 amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2.

[0106] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M or a K D of less than 1×10 D M. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K

[0107] In another aspect, the disclosure features a polypeptide comprising a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMDFSIRIGX8ITPADAGTYYCX9KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 51), where X1 is V or I; X2 is A or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is H or P; X7 is L or T; X8 is N or A; X9 is V or I; provided that the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1.

[0108] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 51 and X1 is V or I. In any of the foregoing embodiments of this aspect of the disclosure, X2 is A or I. In any of the foregoing embodiments, X3 is I or F. In any of the foregoing embodiments, X4 is E or V. In any of the foregoing embodiments, X5 is K or R. In any of the foregoing embodiments, X6 is H or P. In any of the foregoing embodiments, X7 is L or T. In any of the foregoing embodiments, X8 is N or A. In any of the foregoing embodiments, X9 is V or I. In some embodiments, X4 is not V.

[0109] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 51, and X8 is A. In any of the foregoing embodiments in this aspect of the present disclosure, X8 is A and X1 is V or I. In any of the foregoing embodiments in this aspect of the present disclosure, X8 is A and X2 is A or I. In any of the foregoing embodiments, X8 is A and X3 is I or F. In any of the foregoing embodiments, X8 is A and X4 is E or V. In some embodiments, X4 is not V. In any of the foregoing embodiments, X8 is A and X5 is K or R. In any of the foregoing embodiments, X8 is A and X6 is H or P. In any of the foregoing embodiments, X8 is A and X7 is A or V. In any of the foregoing embodiments, X8 is A and X9 is V or I.

[0110] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 51, and X8 is A. In any of the foregoing embodiments in this aspect of the present disclosure, X8 is A and X1 is I. In any of the foregoing embodiments in this aspect of the present disclosure, X8 is A and X2 is I. In any of the foregoing embodiments, X8 is A and X3 is F. In any of the foregoing embodiments, X8 is A and X4 is V. In any of the foregoing embodiments, X8 is A and X5 is R. In any of the foregoing embodiments, X8 is A and X6 is P. In any of the foregoing embodiments, X8 is A and X7 is T. In any of the foregoing embodiments, X8 is A and X9 is I.

[0111] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 51, and each of X1, X2, X3, X4, X5, X6, X7, X8, and X9 is not a wild-type amino acid.

[0112] In some embodiments, the polypeptide of this aspect of the disclosure comprises 10 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide of this aspect of the disclosure comprises 7 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1.

[0113] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M or less than 1×10 D M. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K of about 500 nM to about 100 nM, about 100 nM to about 50 nM, about 50 nM to about 10 nM, about 10 nM to about 5 nM, about 5 nM to about 1 nM, about 1 nM to about 500 pM, about 500 pM to about 100 pM, about 100 pM to about 50 pM, or about 50 pM to about 10 pMD binds to CD47 in D .

[0114] In another aspect, the present disclosure features a polypeptide comprising a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRELIYNQX4EGX5FPRVTTVSDX6TKRNNMDFSIRIGX7ITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 222), where X1 is V, L, or I; X2 is A, I, or L; X3 is I, T, S, or F; X4 is K or R; X5 is H or P; X6 is L, T, or G; X7 is N or A; wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence set forth in SEQ ID NO: 1.

[0115] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 222, where X1 is V, L, or I. In any of the foregoing embodiments of this aspect of the present disclosure, X2 is A, I, or L. In any of the foregoing embodiments, X3 is I, T, S, or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In the foregoing embodiments, X6 is L, T, or G. In any of the foregoing embodiments, X7 is N or A.

[0116] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 222, where X1 is V or I. In any of the foregoing embodiments of this aspect of the present disclosure, X2 is A or I. In any of the foregoing embodiments, X3 is I or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In the foregoing embodiments, X6 is L or T. In any of the foregoing embodiments, X7 is N or A.

[0117] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 222, where X7 is A. In any of the foregoing embodiments of this aspect of the present disclosure, X7 is A and X1 is V or I. In any of the foregoing embodiments of this aspect of the present disclosure, X7 is A and X2 is A or I. In any of the foregoing embodiments, X7 is A and X3 is I or F. In any of the foregoing embodiments, X7 is A and X4 is K or R. In any of the foregoing embodiments, X7 is A and X5 is H or P. In any of the foregoing embodiments, X7 is A and X6 is L or T.

[0118] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 222, where X7 is A. In any of the foregoing embodiments of this aspect of the present disclosure, X7 is A and X1 is I. In any of the foregoing embodiments of this aspect of the present disclosure, X7 is A and X2 is I. In any of the foregoing embodiments, X7 is A and X3 is F. In any of the foregoing embodiments, X7 is A and X4 is R. In any of the foregoing embodiments, X7 is A and X5 is P. In any of the foregoing embodiments, X7 is A and X6 is T.

[0119] In some embodiments, the polypeptide comprises an SIRPα D1 domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 222, and each of X1, X2, X3, X4, X5, X6, and X7 is not a wild-type amino acid.

[0120] In some embodiments, the polypeptide of this aspect of the disclosure comprises 10 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide of this aspect of the disclosure comprises 7 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1.

[0121] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the fragment comprises a polypeptide that is less than 10 amino acids in length, about 10 amino acids in length, about 20 amino acids in length, about 30 amino acids in length, about 40 amino acids in length, about 50 amino acids in length, about 60 amino acids in length, about 70 amino acids in length, about 80 amino acids in length, about 90 amino acids in length, about 100 amino acids in length, or about 100 amino acids or more in length. The fragment retains the ability to bind to CD47. Preferably, the SIRPα D1 domain variant polypeptide and fragments thereof bind to CD47 with a higher affinity than the SIRPα polypeptide binds to CD47. For example, in some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M or less than 1×10 DIt binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM D It binds to CD47.

[0122] In another aspect, the present disclosure features a polypeptide comprising a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVSVAAGESAILHCTX2TSLX3PVGPIQWFRGAGPARELIYNQX4EGX5FPRVTTVSEX6TKRENMDFSISISX7ITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS (SEQ ID NO: 212), where X1 is V, L, or I; X2 is V, I, or L; X3 is I, T, S, or F; X4 is K or R; X5 is H, P, or R; X6 is S, T, or G; X7 is N or A; where the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2.

[0123] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, where X1 is V, L, or I. In any of the foregoing embodiments in this aspect of the present disclosure, X2 is V, I, or L. In any of the foregoing embodiments, X3 is I, T, S, or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In the foregoing embodiments, X6 is S, T, or G. In any of the foregoing embodiments, X7 is N or A.

[0124] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, where X1 is V or I. In any of the foregoing embodiments of this aspect of the present disclosure, X2 is V or I. In any of the foregoing embodiments, X3 is I or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In the foregoing embodiments, X6 is S or T. In any of the foregoing embodiments, X7 is N or A.

[0125] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, where X7 is A. In any of the foregoing embodiments of this aspect of the present disclosure, X7 is A and X1 is V or I. In any of the foregoing embodiments of this aspect of the present disclosure, X7 is A and X2 is V or I. In any of the foregoing embodiments, X7 is A and X3 is I or F. In any of the foregoing embodiments, X7 is A and X4 is K or R. In any of the foregoing embodiments, X7 is A and X5 is H or P. In any of the foregoing embodiments, X7 is A and X6 is S or T.

[0126] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, where X7 is A. In any of the foregoing embodiments of this aspect of the present disclosure, X7 is A and X1 is I. In any of the foregoing embodiments of this aspect of the present disclosure, X7 is A and X2 is I. In any of the foregoing embodiments, X7 is A and X3 is F. In any of the foregoing embodiments, X7 is A and X4 is R. In any of the foregoing embodiments, X7 is A and X5 is P. In any of the foregoing embodiments, X7 is A and X6 is T.

[0127] In some embodiments, the polypeptide comprises an SIRPα D1 domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 212, and each of X1, X2, X3, X4, X5, X6, and X7 is not a wild-type amino acid.

[0128] In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than 10 amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than 7 amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2.

[0129] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the fragment comprises a polypeptide less than 10 amino acids in length, about 10 amino acids in length, about 20 amino acids in length, about 30 amino acids in length, about 40 amino acids in length, about 50 amino acids in length, about 60 amino acids in length, about 70 amino acids in length, about 80 amino acids in length, about 90 amino acids in length, about 100 amino acids in length, or about 100 amino acids or more in length. The fragment retains the ability to bind to CD47. Preferably, the SIRPα D1 domain variant polypeptide and fragments thereof bind to CD47 with a higher affinity than the SIRPα polypeptide binds to CD47. For example, in some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof is less than 1 × 10 -8 M, less than 5 × 10 -9 M, less than 1 × 10-9 less than M, 5 x 10 -10 less than M, 1 x 10 -10 less than M or 1 x 10 -11 K less than M D and binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof has a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM D and binds to CD47.

[0130] Described herein are, in some embodiments, polypeptides comprising SIRPα D1 domain variants having the following sequences: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9X 10 X 11 X 12 ADAGTYYCX 13 KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 218), X1 is V, L, or I; X2 is A, V, L, or I; X3 is I, S, T, or F; X4 is E, L, or V; X5 is K or R; X6 is E or Q; X7 is H, R, or P; X8 is S, G, L, or T; X9 is any amino acid; X 10 is any amino acid; X 11 is any amino acid; X 12 is any amino acid; X 13 is V or I; wherein the SIRPα D1 domain variant comprises at least two amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence set forth in SEQ ID NO: 1.

[0131] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, where X1 is such that X9 is A. In any of the foregoing embodiments of this aspect of the present disclosure, X9 is N. In any of the foregoing embodiments of this aspect of the present disclosure, X 10 is I. In any of the foregoing embodiments of this aspect of the present disclosure, X9 is N and X10 is P. In any of the foregoing embodiments of this aspect of the present disclosure, X9 is N and X 11 is any amino acid other than S, T, or C. In any of the foregoing embodiments of this aspect of the present disclosure, X 11 is T. In any of the foregoing embodiments of this aspect of the present disclosure, X 11 is any amino acid other than T. In any of the foregoing embodiments of this aspect of the present disclosure, X 12 is P. In any of the foregoing embodiments of this aspect of the present disclosure, X9 is N and X 12 is any amino acid other than P.

[0132] Described herein are, in some embodiments, polypeptides comprising an SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9ITX 10 ADAGTYYCX 11 KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 219), where X1 is V, L, or I; X2 is A, V, L, or I; X3 is I, S, T, or F; X4 is E, L, or V; X5 is K or R; X6 is E or Q; X7 is H, R, or P; X8 is S, G, L, or T; X9 is N; X 10 is any の amino acid other than P; X 11is V or I; wherein the SIRPα D1 domain variant comprises at least two amino acid substitutions compared to the wild-type SIRPα D1 domain variant having the sequence according to SEQ ID NO: 1.

[0133] In another aspect of the present disclosure, a composition comprising a SIRPα D1 domain variant polypeptide having the amino acid sequence of SEQ ID NO: 48, or a fragment thereof, is disclosed herein. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a higher affinity compared to the affinity of the SIRPα polypeptide for binding to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide binds to CD47 with a K -8 less than 1×10 -9 M, or less than 1×10 -10 M, less than 1×10 -11 M, or less than 1×10 D M. In some embodiments, the above-mentioned SIRPα D1 domain variant polypeptide is attached to or fused with a second polypeptide. In some embodiments, the second polypeptide includes, but is not limited to, an Fc polypeptide, an Fc variant, or a fragment as described above.

[0134] Without limiting the foregoing, in some embodiments, the SIRPα D1 domain variant polypeptide is selected from any one of SEQ ID NOs: 53 to 87 and 213 shown in Table 6.

Table 6-1

Table 6-2

Table 6-3

Table 6-4

Table 6-5

[0135] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to any of the variants set forth in Table 6.

[0136] In some embodiments, the polypeptide comprises a SIRPα D1 domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 80, 81, or 85 set forth in Table 6.

[0137] Fc domain variants and fusion polypeptides comprising Fc domain variants In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein alpha (SIRP-α) D1 variant, wherein the polypeptide has an amino acid mutation at residue 80 relative to the wild-type SIRP-α D1 domain (e.g., the wild-type SIRPα D1 domain shown in SEQ ID NO: 1 or 2); and at least one additional amino acid mutation at a residue selected from the group consisting of residues 6, 27, 31, 47, 53, 54, 56, 66, and 92 relative to the wild-type SIRPα D1 domain (the wild-type SIRPα D1 domain shown in SEQ ID NO: 1 or 2), and comprises a SIRPα D1 domain or a fragment thereof.

[0138] Also disclosed herein, in some embodiments, are Fc domain variant dimers, where the Fc domain variant dimer comprises two Fc domain variants, and each Fc domain variant is independently selected from (i) a human IgG1 Fc region consisting of the mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of the mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A.

[0139] Antibodies that target cell surface antigens can elicit immune stimulation and effector functions associated with the binding of Fc receptors (FcRs) on immune cells. There are multiple Fc receptors specific for particular classes of antibodies, such as IgG (gamma receptor), IgE (epsilon receptor), IgA (alpha receptor), and IgM (mu receptor). Binding of the antibody's Fc region to Fc receptors on the cell surface can trigger multiple biological responses, such as phagocytosis of antibody-coated particles (antibody-dependent cell-mediated phagocytosis, or ADCP), clearance of immune complexes, lysis of antibody-coated cells by killer cells (antibody-dependent cell-mediated cytotoxicity, or ADCC), and release of inflammatory mediators, placental transfer, and regulation of immunoglobulin production. Additionally, binding of the C1 component of complement to the antibody can activate the complement system. Complement activation can be important for the lysis of pathogenic cells, but it can also stimulate an inflammatory response and be involved in autoimmune hypersensitivity or other immune disorders. Variant Fc regions with reduced or eliminated ability to bind to specific Fc receptors are useful for the development of therapeutic antibodies and Fc fusion polypeptide constructs that act by targeting, activating, or neutralizing ligand functions without damaging or destroying local cells or tissues.

[0140] In some embodiments, the SIRPα D1 polypeptide construct comprises a non-native SIRPα D1 domain variant linked to an Fc domain variant that forms an Fc domain having reduced or eliminated effector function.

[0141] In some embodiments, the Fc domain variant refers to a polypeptide chain comprising the second and third antibody constant domains (e.g., CH2 and CH3). In some embodiments, the Fc domain variant also comprises a hinge domain. In some embodiments, the Fc domain variant is of any immunoglobulin antibody isotype such as IgG, IgE, IgM, IgA, and IgD. Further, in some embodiments, the Fc domain variant is of any IgG subtype (e.g., IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, and IgG4). In some embodiments, the Fc domain variant has 10 amino acid modifications (e.g., insertions, deletions, and / or substitutions) that alter the interaction between the Fc domain and the Fc receptor relative to the wild-type Fc domain monomer sequence (e.g., 1-10, 1-8, 1-6, 1-4 amino acid substitutions, additions or insertions, deletions, or combinations thereof).

[0142] As used herein, the term "Fc domain dimer" refers to a dimer of two Fc domains. In a wild-type Fc domain dimer, the two wild-type Fc domains dimerize by the interaction between the two CH3 antibody constant domains and one or more disulfide bonds formed between the hinge domains of the two dimerized Fc domains.

[0143] As used herein, the term "Fc domain dimer variant" includes at least one Fc domain variant. In some embodiments, the Fc domain dimer variant includes an Fc domain variant that has been mutated to lack effector function, e.g., a "dead" Fc domain dimer variant. In some embodiments, each of the Fc domains in the Fc domain dimer variant includes an amino acid substitution in the CH2 antibody constant domain to reduce the interaction or binding between the Fc domain dimer variant and an Fc receptor such as an Fc gamma receptor (FcγR), an Fc alpha receptor (FcαR), or an Fc epsilon receptor (FcεR).

[0144] In some embodiments, a SIRPα D1 domain variant (e.g., any of the variants described in Tables 2, 5, and 6) is fused to an Fc domain variant of an immunoglobulin or a fragment of an Fc domain variant. In some embodiments, an Fc domain variant of an immunoglobulin or a fragment of an Fc domain variant can form an Fc domain dimer with another Fc domain variant. In some embodiments, an Fc domain variant of an immunoglobulin or a fragment of an Fc domain variant cannot form an Fc domain dimer with another Fc domain variant. In some embodiments, an Fc domain variant or a fragment of an Fc domain variant is fused to a polypeptide of the present disclosure to increase the serum half-life of the polypeptide. In some embodiments, an Fc domain variant or a fragment of an Fc domain variant fused to a polypeptide of the present disclosure dimerizes with a second Fc domain variant to form an Fc domain dimer variant that binds to an Fc receptor, or the Fc domain variant binds to an Fc receptor. In some embodiments, an Fc domain variant or a fragment of an Fc domain variant fused to a polypeptide to increase the serum half-life of the polypeptide does not induce any immune system-related response.

[0145] In some embodiments, the SIRPα polypeptide or construct provided herein comprises a SIRPα D1 domain or variant thereof bound to a first Fc domain variant, and an antibody variable domain bound to a second Fc domain variant, wherein the first and second Fc domain variants combine to form an Fc domain dimer variant (e.g., a heterodimeric Fc domain dimer variant). The Fc domain dimer is a protein structure found at the C-terminus of immunoglobulins. The Fc domain dimer includes two Fc domains dimerized by interactions between the CH3 antibody constant domains. The wild-type Fc domain dimer forms the minimal structure that binds to Fc receptors, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and FcγRIV.

[0146] The Fc domain dimer is not directly involved in the binding of the antibody to its target, but can be involved in various effector functions such as the involvement of the antibody in antibody-dependent cell cytotoxicity. In some embodiments, the Fc domain in the SIRPα polypeptide or construct of the present disclosure results in a decrease in effector function such as a decrease in antibody-dependent cell-mediated cytotoxicity (ADCC), a decrease in complement-dependent cytotoxicity (CDC), a decrease in antibody-dependent cell-mediated phagocytosis (ADCP), or any combination thereof, including amino acid substitutions, additions or insertions, deletions, or any combination thereof. In some embodiments, the SIRPα polypeptide or construct of the present disclosure is characterized by a decrease in binding to human Fc receptors (e.g., minimal or no binding) and a decrease in binding to complement protein C1q (e.g., minimal or no binding). In some embodiments, the SIRPα construct of the present disclosure is characterized by a decrease in binding to human FcγRI, FcγRIIA, FcγRIIB, FcγRIIIB, or any combination thereof, and C1q (e.g., minimal or no binding). In some embodiments, in order to alter or reduce antibody-dependent effector functions such as ADCC, CDC, ADCP, or any combination thereof, in some embodiments, the Fc domain in the SIRPα construct of the present disclosure is of the IgG class and contains one or more amino acid substitutions at E233, L234, L235, G236, G237, D265, D270, N297, E318, K320, K322, A327, A330, P331, or P329 (numbering according to the Kabat EU index) (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991))).

[0147] In some embodiments, the polypeptide construct comprising the non-natural Fc region described herein exhibits a decrease or elimination of binding to at least one of the Fcγ receptors CD16a, CD32a, CD32b, CD32c, and CD64 as compared to a polypeptide construct comprising a natural Fc region. In some cases, the polypeptide construct described herein exhibits a decrease or elimination of binding to the CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors.

[0148] CDC refers to a form of cytotoxicity in which the complement cascade is activated by the complement component C1q that binds to the antibody Fc domain. In some embodiments, the polypeptide construct comprising the non-natural Fc region described herein shows a decrease in C1q binding of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more as compared to a polypeptide construct comprising a wild-type Fc region. In some cases, the polypeptide construct comprising the non-natural Fc region described herein exhibits a decrease in CDC as compared to a polypeptide construct comprising a wild-type Fc region. In some embodiments, the polypeptide construct comprising the non-natural Fc region described herein shows a decrease in CDC of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more as compared to a polypeptide construct comprising a wild-type Fc region. In some cases, the polypeptide construct comprising the non-natural Fc domain variant or Fc domain dimer variant described herein exhibits negligible CDC as compared to a polypeptide construct comprising a wild-type Fc region.

[0149] In some embodiments, the Fc domain variant or Fc domain dimer variant described herein is minimally glycosylated or has reduced glycosylation relative to the wild-type sequence. In some embodiments, deglycosylation is achieved by a mutation of N297A or by mutating N297 to any amino acid other than N. In some embodiments, deglycosylation is achieved by disrupting the motif N-Xaa1-Xaa2-Xaa3. Wherein, N = asparagine; Xaa1 = any amino acid other than P (proline); Xaa2 = T (threonine), S (serine), or C (cysteine); and Xaa3 = any amino acid other than P (proline). In one embodiment, the N-Xaa1-Xaa2-Xaa3 motif refers to residues 297-300 designated according to Kabat et al., (1991). In some embodiments, mutations to any one or more of N, Xaa1, Xaa2, or Xaa3 result in deglycosylation of the Fc domain variant or Fc domain dimer variant.

[0150] In some embodiments, variants of the antibody IgG constant region (e.g., Fc domain variants or Fc domain dimer variants) have a reduced ability to specifically bind to Fcγ receptors or a reduced ability to induce phagocytosis. In some embodiments, variants of the antibody IgG constant region (e.g., Fc domain variants or Fc domain dimer variants) have a reduced ability to specifically bind to Fcγ receptors and a reduced ability to induce phagocytosis. For example, in some embodiments, the Fc domain variant is mutated to lack effector functions typical of a "dead" Fc domain variant. For example, in some embodiments, the Fc domain variant includes specific amino acid substitutions known to minimize the interaction between the Fc domain dimer and the Fcγ receptor. In some embodiments, the Fc domain variant is derived from an IgG1 antibody and includes one or more of the amino acid substitutions L234A, L235A, G237A, and N297A (designated according to the EU numbering system by Kabat et al., (1991)). In some embodiments, one or more additional mutations are included in such IgG1 Fc domain variants. Non-limiting examples of such additional mutations to the human IgG1 Fc domain variant include E318A and K322A. In some cases, the human IgG1 Fc domain variant has a total of up to 12, 11, 10, 9, 8, 7, 6, 5, or 4 or fewer mutations compared to the wild-type human IgG1 sequence. In some embodiments, one or more additional deletions are included in such IgG1 Fc domain variants. For example, in some embodiments, the C-terminal lysine of the Fc domain IgG1 heavy chain constant region provided in SEQ ID NO: 88 in Table 7 is deleted, for example, to enhance the homogeneity of the polypeptide when the polypeptide is produced in bacterial or mammalian cells. In some cases, the human IgG1 Fc domain variant has a total of up to 12, 11, 10, 9, 8, 7, 6, 5, or 4 or fewer deletions compared to the wild-type human IgG1 sequence (see, for example, SEQ ID NO: 161 below).In some embodiments, the IgG1 Fc domain variant has the sequence set forth in any one of SEQ ID NO: 135, SEQ ID NO: 136, or SEQ ID NO: 137. SEQ ID NO: 161: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0151] In some embodiments, the Fc domain variant is derived from an IgG2 or IgG4 antibody and comprises the amino acid substitutions A330S, P331S, or both A330S and P331S. The aforementioned amino acid positions are defined according to Kabat et al., (1991). The Kabat numbering of amino acid residues can be determined for a given antibody by alignment in the homology region of the antibody's sequence with the "standard" Kabat numbering sequence. In some embodiments, the Fc domain variant comprises a human IgG2 Fc domain sequence comprising one or more of the A330S, P331S, and N297A amino acid substitutions (designated according to the EU numbering system by Kabat et al., (1991)). In some embodiments, one or more additional mutations are included in such IgG2 Fc domain variants. Non-limiting examples of such additional mutations to the human IgG2 Fc domain variant include V234A, G237A, P238S, V309L, and H268A (designated according to the EU numbering system by Kabat et al., (1991)). In some cases, the human IgG2 Fc domain variant has a total of up to 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 or fewer mutations compared to the wild-type human IgG2 sequence. In some embodiments, one or more additional deletions are included in such IgG2 Fc domain variants. For example, in some embodiments, the C-terminal lysine of the Fc domain IgG2 heavy chain constant region provided in SEQ ID NO: 89 in Table 7 is deleted to enhance the homogeneity of the polypeptide, for example, when the polypeptide is produced in bacterial or mammalian cells. In some cases, the human IgG2 Fc domain variant has a total of up to 12, 11, 10, 9, 8, 7, 6, 5, or 4, or fewer deletions compared to the wild-type human IgG2 sequence (see, for example, SEQ ID NO: 162 below). SEQ ID NO: 162: ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0152] When the Fc domain variant is an IgG4 Fc domain variant, in some embodiments, such an Fc domain variant comprises the S228P mutation (designated according to Kabat et al., (1991)). Optionally, the human IgG4 Fc domain variant has a total of up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations compared to the wild-type human IgG4 sequence. In some embodiments, the Fc domain variant comprises a human IgG4 Fc sequence comprising one or more of the S228P, E233P, F234V, L235A, and delG236 amino acid substitutions (designated according to the EU numbering system by Kabat et al., (1991)). In some embodiments, the Fc domain variant comprises a human IgG4 Fc sequence comprising one or more of the S228P, E233P, F234V, L235A, delG236, and N297A amino acid substitutions (designated according to the EU numbering system by Kabat et al., (1991)).

[0153] In some embodiments, the Fc domain variant comprises at least one of the mutations L234A, L235A, G237A, or N297A in the IgG1 Fc region, or at least one of the mutations A330S, P331S, or N297A in the IgG2 Fc region. In some embodiments, the Fc domain variant comprises at least two of the mutations L234A, L235A, G237A, or N297A in the IgG1 Fc region, or at least two of the mutations A330S, P331S, or N297A in the IgG2 Fc region. In some embodiments, the Fc domain variant comprises at least three of the mutations L234A, L235A, G237A, or N297A in the IgG1 Fc region, or consists of the mutations A330S, P331S, and N297A in the IgG2 Fc region. In some embodiments, the Fc domain variant consists of the mutations L234A, L235A, G237A, and N297A.

[0154] In some embodiments, the Fc domain variant exhibits a decrease in binding to the target Fc receptor as compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain variant exhibits a removal of binding to the target Fc receptor as compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain variant exhibits a decrease in phagocytosis as compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain variant exhibits a removal of phagocytosis as compared to the wild-type human IgG Fc region.

[0155] SEQ ID NO: 88 and SEQ ID NO: 89 provide the amino acid sequences of the Fc domain IgG1 and IgG2 heavy chain constant regions. In some embodiments, the Fc domain variant is any of the variants of SEQ ID NOs: 90-95 as shown in Table 7.

Table 7-1

Table 7-2

[0156] As used herein, antibody-dependent cell-mediated cytotoxicity, also referred to as ADCC, refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells and neutrophils), thereby enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells. As used herein, antibody-dependent cell-mediated phagocytosis, also referred to as ADCP, refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain phagocytic cells (e.g., macrophages), thereby enabling these phagocytic effector cells to specifically bind to antigen-bearing target cells and subsequently engulf and digest the target cells. Ligand-specific high-affinity IgG antibodies directed against the surface of target cells can stimulate cytotoxic or phagocytic cells and can be used for such killing. In some embodiments, polypeptide constructs comprising the Fc domain variants or Fc domain dimer variants described herein exhibit reduced ADCC or ADCP as compared to polypeptide constructs comprising a wild-type Fc region. In some embodiments, polypeptide constructs comprising the Fc domain variants or Fc domain dimer variants described herein exhibit at least a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more reduction in ADCC or ADCP as compared to polypeptide constructs comprising a wild-type Fc region. In some embodiments, polypeptide constructs comprising the Fc domain variants or Fc domain dimer variants described herein exhibit ablation of ADCC or ADCP as compared to polypeptide constructs comprising a wild-type Fc region.

[0157] As used herein, complement-directed cytotoxicity, also referred to as CDC, refers to a form of cytotoxicity in which the complement cascade is activated by complement component C1q that binds to the antibody Fc domain. In some embodiments, a polypeptide construct comprising an Fc domain variant or an Fc domain dimer variant described herein exhibits a decrease in C1q binding of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more as compared to a polypeptide construct comprising a wild-type Fc region. Optionally, a polypeptide construct comprising an Fc domain variant or an Fc domain dimer variant described herein exhibits decreased CDC as compared to a polypeptide construct comprising a wild-type Fc region. In some embodiments, a polypeptide construct comprising an Fc domain variant or an Fc domain dimer variant described herein exhibits a decrease in CDC of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more as compared to a polypeptide construct comprising a wild-type Fc region. In some embodiments, a polypeptide construct comprising an Fc domain variant or an Fc domain dimer variant described herein exhibits negligible CDC as compared to a polypeptide construct comprising a wild-type Fc region.

[0158] Fc domain variants or Fc domain dimer variants as described herein include those that exhibit a reduced binding to Fcγ receptors as compared to the wild-type human IgG Fc region. For example, in some embodiments, the Fc domain variant or Fc domain dimer variant exhibits a reduced binding to Fcγ receptors as compared to the binding to Fcγ receptors exhibited by the wild-type human IgG Fc region as described in the examples. In some cases, the Fc domain variant or Fc domain dimer variant has a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (where effector function is completely removed) reduction in binding to Fcγ receptors. In some embodiments, the reduction in binding is for any one or more Fcγ receptors, such as CD16a, CD32a, CD32b, CD32c, or CD64.

[0159] In some cases, the Fc domain variants or Fc domain dimer variants disclosed herein exhibit a reduced phagocytosis as compared to their wild-type human IgG Fc region. Such Fc domain variants or Fc domain dimer variants exhibit a reduced phagocytosis as compared to their wild-type human IgG Fc region, where the reduction in phagocytic activity is, for example, by a factor of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some cases, the Fc domain variant or Fc domain dimer variant exhibits an ablation of phagocytosis as compared to its wild-type human IgG Fc region.

[0160] In some embodiments, the Fc domain variant or Fc domain dimer variant disclosed herein is conjugated to one or more fusion partners. Optionally, the fusion partner is a therapeutic moiety. Optionally, the fusion partner is selected to enable targeting, purification, screening, presentation, etc. of the expressed protein. In some embodiments, the fusion partner also affects the degree of binding to Fc receptors or the degree of reduction of phagocytosis. As described herein, in some embodiments, when an Fc domain variant or Fc domain dimer variant is conjugated to a fusion partner, a polypeptide construct as described below is formed.

[0161] In some embodiments, the fusion partner is linked to the Fc domain variant or Fc domain dimer variant sequence via a linker sequence. In some embodiments, the linker sequence generally comprises a small number of amino acids such as less than 10 amino acids, although longer linkers are also utilized. Optionally, the linker has a length of 10, 9, 8, 7, 6, or 5 amino acids or less. Optionally, the linker has a length of at least 10, 11, 12, 13, 14, 15, 20, 25, 30, or 35, or more amino acids. Optionally, in some embodiments, a cleavable linker is used.

[0162] In some embodiments, the fusion partner is a targeting or signal sequence that directs the Fc domain variant or Fc domain dimer variant protein and any associated fusion partner to a desired cellular location or the extracellular culture medium. In some embodiments, certain signaling sequences target proteins that are secreted either into the growth medium or into the periplasmic space located between the inner and outer membranes of the cell. In some embodiments, the fusion partner is a sequence encoding a peptide or protein that enables purification or screening. Such fusion partners include, but are not limited to, polyhistidine tags (His tags) (e.g., His6 (SEQ ID NO: 223) and His10 (SEQ ID NO: 224)), or tags for use with immobilized metal affinity chromatography (IMAC) systems (e.g., Ni+2 affinity columns), GST fusions, MBP fusions, Strep tags, the BSP biotinylation target sequence of the bacterial enzyme BirA, and epitope tags that are targets for antibodies (e.g., c-myc tag, flag tag, etc.).

[0163] In some embodiments, such tags are useful for purification, screening, or both. For example, in some embodiments, the Fc domain variant or Fc domain dimer variant is purified by immobilizing it on a Ni+2 affinity column using a His tag, and then, after purification, the same His tag is used to immobilize the antibody on a Ni+2-coated plate and an ELISA or other binding assay as described elsewhere herein is performed. In some embodiments, the fusion partner enables the use of a selection method for screening Fc domain variants or Fc domain dimer variants as described herein.

[0164] A variety of fusion partners are available that enable various selection methods. For example, phage display can be used by fusing members of an Fc domain variant or Fc domain dimer variant library to the gene III protein. In some embodiments, the fusion partner is an Fc domain variant or Fc domain dimer variant that is labeled. Alternatively, in some embodiments, the fusion partner binds to a specific sequence on an expression vector, thereby enabling the fusion partner and the associated Fc domain variant or Fc domain dimer variant to be linked to the nucleic acid encoding them by covalent or non-covalent bonds.

[0165] In some embodiments, when the fusion partner is a therapeutic moiety, the therapeutic moiety is, for example, a peptide, protein, antibody, siRNA, or small molecule. Non-limiting examples of therapeutic antibodies that bind to the Fc domain variants or Fc domain dimer variants of the present disclosure include, but are not limited to, antibodies that recognize CD47. Non-limiting examples of therapeutic polypeptides that bind to the Fc domain variants or Fc domain dimer variants of the present disclosure include, but are not limited to, CD47-binding polypeptides such as SIRPα polypeptides. In such cases, the CD47-binding polypeptide is attached or fused to the Fc domain variant or Fc domain dimer variant of the present disclosure. Examples of CD47-binding polypeptides include, but are not limited to, anti-CD47 antibodies or fragments thereof, and ligands of CD47 such as SIRPα or fragments thereof. Additional examples of CD47-binding polypeptides include, but are not limited to, naturally occurring forms of SIRPα and variants thereof.

[0166] In some embodiments, disclosed herein is a polypeptide comprising an Fc domain dimer variant, wherein the Fc domain dimer variant comprises two Fc domain variants, and each Fc domain variant is independently selected from (i) a human IgG1 Fc region consisting of the mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of the mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A. In some embodiments, the Fc domain variants are identical (i.e., homodimer). In some embodiments, the Fc domain variants are different (i.e., heterodimer). In some embodiments, at least one of the Fc domain variants in the Fc domain dimer is a human IgG1 Fc region consisting of the mutations L234A, L235A, G237A, and N297A. In some embodiments, at least one of the Fc domain variants in the Fc domain dimer is a human IgG2 Fc region consisting of the mutations A330S, P331S, and N297A. In some embodiments, the Fc domain dimer variant exhibits removal or reduction of binding to Fcγ receptors as compared to the wild-type version of the human IgG Fc region. In some embodiments, the Fc domain dimer variant exhibits removal or reduction of binding to the CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors as compared to the wild-type version of the human IgG Fc region. In some embodiments, the Fc domain dimer variant exhibits removal or reduction of binding to C1q as compared to the wild-type version of the human IgG Fc fusion. In some embodiments, at least one of the Fc domain variants in the Fc domain dimer variant is a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A. In some embodiments, the Fc domain dimer variant exhibits removal or reduction of binding to Fcγ receptors as compared to the wild-type human IgG4 Fc region.In some embodiments, the Fc domain dimer variant exhibits removal or reduction of binding to CD16a and CD32b Fcγ receptors as compared to the wild-type version of its human IgG4 Fc region. In some embodiments, the Fc domain dimer variant has a K -6 greater than about 5×10 D M and binds to the Fcγ receptor.

[0167] In some embodiments, the Fc domain dimer variant further comprises a CD47-binding polypeptide. In some embodiments, the Fc domain dimer variant exhibits removal or reduction of binding to the Fcγ receptor as compared to the wild-type version of the human IgG Fc region. In some embodiments, the CD47-binding polypeptide does not cause acute anemia in rodents and non-human primates. In some embodiments, the CD47-binding polypeptide does not cause acute anemia in humans.

[0168] In some embodiments, the CD47-binding polypeptide is a signal regulatory protein alpha (SIRP-α) polypeptide or a fragment thereof. In some embodiments, the SIRPα polypeptide comprises a SIRPα D1 domain variant comprising the following amino acid sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMDFSIRIGX8ITPADAGTYYCX9KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 221), where X1 is V or I; X2 is A or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is H or P; X7 is L or T; X8 is any amino acid other than N; X9 is V or I. In some embodiments, the SIRPα polypeptide comprises a SIRPα D1 domain variant, where X1 is V or I; X2 is A or I; X3 is I or F; X4 is E; X5 is K or R; X6 is H or P; X7 is L or T; X8 is not N; and X9 is V.

[0169] In some embodiments disclosed herein, it is a polypeptide comprising a SIRPα D1 domain variant, where the SIRPα D1 domain variant is a non-naturally occurring high-affinity SIRPα D1 domain, and the SIRPα D1 domain variant binds to human CD47 with an affinity that is at least 10-fold higher than the affinity of the naturally occurring D1 domain and the Fc domain variant, and the Fc domain variant is linked to a second polypeptide comprising a second Fc domain variant to form an Fc domain dimer variant, and the Fc domain dimer variant has its effector function removed or reduced. In some embodiments, the non-naturally occurring high-affinity SIRPα D1 domain comprises an amino acid mutation at residue 80.

[0170] In some embodiments, disclosed herein is a SIRPα D1 domain variant, and the SIRPα D1 domain variant binds to a first type of CD47 with a K D of less than 250 nM, and the SIRPα D1 domain variant binds to a first type of CD47 with a K Dbinds to the second type of CD47 and the K of the first type of CD47 D and the K of the second type of CD47 D are within 100-fold of each other, and the first and second types are selected from the group consisting of humans, rodents, and non-human primates. In some embodiments, the SIRPα D1 domain variant binds to CD47 of at least three different species. In some embodiments, the non-human primate is a cynomolgus monkey.

[0171] In some embodiments, disclosed herein is a polypeptide comprising (a) an SIRPα D1 domain that binds human CD47 with a K D less than 250 nM, and (b) an Fc domain or a variant thereof linked to the N-terminus or C-terminus of the SIRPα D1 domain, wherein the polypeptide does not cause acute anemia in rodents and non-human primates. In some embodiments, the polypeptide is a non-naturally occurring variant of human SIRP-α. In some embodiments, administration of the polypeptide in vivo results in less than a 50% decrease in hemoglobin during the first week after administration. In some embodiments, administration of the polypeptide in humans results in less than a 50% decrease in hemoglobin during the first week after administration. In some embodiments, the polypeptide further comprises at least one Fc domain dimer variant, wherein the Fc domain dimer variant comprises an Fc domain variant selected from (i) a human IgG1 Fc region consisting of the mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of the mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A. In some embodiments, the Fc domain variant is a human IgG1 Fc region consisting of the mutations L234A, L235A, G237A, and N297A. In some embodiments, the Fc domain variant is a human IgG2 Fc region consisting of the mutations A330S, P331S, and N297A.

[0172] The SIRPα constructs of the present disclosure include an SIRPα domain or a variant thereof having a C-terminus that is linked to the N-terminus of an Fc domain or a variant thereof via a linker using conventional genetic or chemical means, such as chemical conjugation. In some embodiments, a linker (e.g., a spacer) is inserted between the polypeptide and the Fc domain or a variant thereof. In some embodiments, the polypeptides of the present disclosure that include an SIRPα D1 domain variant are fused to an Fc domain variant that cannot form a dimer. In some embodiments, the polypeptides of the present disclosure are fused to an Fc domain or a variant thereof that can form a dimer, such as a heterodimer, with another Fc domain or a variant thereof. In some embodiments, the polypeptides of the present invention are fused to an Fc domain or a variant thereof, and this fusion protein forms a homodimer. In some embodiments, the polypeptides of the present disclosure are fused to a first Fc domain or a variant thereof, and a different protein or peptide (e.g., an antibody variable region) is fused to a second Fc domain or a variant thereof. In some embodiments, the SIRPα D1 domain or a variant thereof is linked to a first Fc domain or a variant thereof, and a therapeutic protein (e.g., a cytokine, an interleukin, an antigen, a steroid, an anti-inflammatory agent, or an immunomodulatory agent) is linked to a second Fc domain or a variant thereof. In some embodiments, the first and second Fc domains or variants thereof form a heterodimer.

[0173] Without the foregoing limitations, in some embodiments, an SIRPα D1 domain variant polypeptide (e.g., any of the variants described in Tables 2, 5, and 6) is fused to an Fc polypeptide, or an Fc variant polypeptide such as an Fc domain or a variant thereof. Examples of polypeptides that include an SIRPα D1 domain variant polypeptide and a fused Fc domain variant polypeptide include, but are not limited to, SEQ ID NOs: 96-137, 214, and 216 shown in Table 8.

Table 8-1

Table 8-2

Table 8-3

Table 8-4

Table 8-5

Table 8-6

Table 8-7

Table 8-8

Table 8-9

Table 8-10

Table 8-11

[0174] In some embodiments, the polypeptide comprises an SIRPα D1 variant domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to any of the variants described in Table 8.

[0175] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the sequences of SEQ ID NOs: 98 - 104, 107 - 113, 116 - 122, or 135 - 137 in Table 8.

[0176] In some embodiments, the polypeptide comprises (a) a signal regulatory protein alpha (SIRP-α) D1 variant and (b) an fc domain dimer variant, wherein the SIRPα D1 domain variant comprises the following amino acid sequence: EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSLX 11 PVGPIQWFRGAGPX 12 RX 13 LIYNQX 14 X 15 GX 16 FPRVTTVSX 17 X 18 TX 19 RX 20 NMDFX 21 IX 22 IX 23 X 24 ITX 25 ADAGTYYCX 26 KX 27 RKGSPDX 28 X 29 EX 30 KSGAGTELSVRX 31 KPS (SEQ ID NO: 47), X1 is E or G; X2 is L, I, or V; X3 is V, L, or I; X4 is S or F; X5 is L or S; X6 is S or T; X7 is A or V; X8 is I or T; X9 is H, R, or L; X 10 is A, V, I, or L; X 11 is I, T, S, or F; X 12 is A or G; X 13is E, V, or L; X 14 is K, or R; X 15 is E, or Q; X 16 is H, P, or R; X 17 is D, or E; X 18 is S, L, T, or G; X 19 is K, or R; X 20 is E, or N; X 21 is S, or P; X 22 is S, or R; X 23 is S, or G; X 24 is any amino acid; X 25 is any amino acid; X 26 is V, or I; X 27 is F, L, or V; X 28 is D or absent; X 29 is T, or V; X 30 is F, or V; X 31 is A, or G; wherein the SIRPα D1 domain variant contains at least two amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence set forth in any one of SEQ ID NOs: 1-10. Each Fc domain variant is independently (i) a human IgG1 Fc region containing the N297A mutation; (ii) a human IgG1 Fc region containing the L234A, L235A, and G237A mutations; (iii) a human IgG1 Fc region containing the L234A, L235A, G237A, and N297A mutations; (iv) a human IgG2 Fc region containing the N297A mutation; (v) a human IgG2 Fc region containing the A330S and P331S mutations; (vi) a human IgG2 Fc region containing the A330S, P331S, and N297A mutations; (vii) a human IgG4 Fc region containing the S228P, E233P, F234V, L235A, and delG236 mutations; or (viii) a human IgG4 Fc region containing the S228P, E233P, F234V, L235A, delG236, and N297A mutations.

[0177] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant and an Fc domain dimer having two Fc domains, the SIRPα D1 domain variant comprises the amino acid sequence according to SEQ ID NO: 47, and one of the Fc domains is an Fc domain variant comprising a human IgG1 Fc region containing the L234A, L235A, G237A, and N297A mutations.

[0178] Dimerization of the Fc domain In some embodiments, a SIRPα D1 domain variant polypeptide (e.g., any of the variants described in Tables 2, 5, and 6) is fused to a first Fc domain (e.g., an Fc domain variant) at either the N-terminus or the C-terminus. In some embodiments, the first Fc domain is a variant that cannot form a dimer. In some embodiments, the first Fc domain forms a dimer with a second Fc domain. In some embodiments, the first Fc domain and the second Fc domain comprise amino acid substitutions that promote heterodimerization between the first domain Fc domain and the second domain Fc domain.

[0179] In some embodiments, each of the two Fc domains in the Fc domain dimer comprises amino acid substitutions that promote heterodimerization of the two monomers. In some embodiments, the SIRPα construct is formed from a first subunit such as a SIRPα D1 domain variant polypeptide fused to a first Fc domain and a second subunit such as a second Fc domain (e.g., not including a SIRPα D1 domain variant polypeptide or any other polypeptide). In some embodiments, the construct has a single SIRPα D1 domain variant polypeptide linked to an Fc domain dimer (e.g., a single arm). In some embodiments, the construct has two SIRPα D1 domain variant polypeptides linked to an Fc domain dimer (e.g., a double arm). In some embodiments, a K of about 500 nM DThe SIRPα D1 domain variant having [it] is particularly useful in a double-arm construct. In some embodiments, a K of about 50 nM D The SIRPα D1 domain variant having [it] is particularly useful in a double-arm construct. In some embodiments, a K of about 5 nM D The SIRPα D1 domain variant having [it] is useful in both double-arm constructs and single-arm constructs. In some embodiments, a K of about 500 pM D The SIRPα D1 domain variant having [it] is useful in both double-arm constructs and single-arm constructs. In some embodiments, a K of about 100 pM D The SIRPα D1 domain variant having [it] is useful in both double-arm constructs and single-arm constructs. In some embodiments, a K of about 50 pM D The SIRPα D1 domain variant having [it] is useful in both double-arm constructs and single-arm constructs. In some embodiments, a K of about 10 pM D The SIRPα D1 domain variant having [it] is useful in both double-arm constructs and single-arm constructs.

[0180] In some embodiments, the heterodimerization of the Fc domain is facilitated by introducing different but compatible substitutions into the two Fc domains, such as "knob-into-hole" residue pairs and charged residue pairs. The knob and hole interactions are favorable for the formation of the heterodimer, but the knob-knob and hole-hole interactions prevent the formation of the homodimer due to steric clashes and the lack of favorable interactions. A hole refers to a cavity created when the original amino acid in a protein is replaced with a different amino acid having a small side-chain volume. A knob refers to a protrusion created when the original amino acid in a protein is replaced with a different amino acid having a large side-chain volume. For example, in some embodiments, the amino acids being substituted are in the CH3 antibody constant domain of the Fc domain and are involved in the dimerization of the two Fc domains. In some embodiments, the hole in one CH3 antibody constant domain is created to accommodate the knob of another CH3 antibody constant domain such that the knob and hole amino acids act to promote or favor the heterodimerization of the two Fc domains. In some embodiments, the hole in one CH3 antibody constant domain is created to better accommodate the original amino acid in another CH3 antibody constant domain. In some embodiments, the knob in one CH3 antibody constant domain is created to form additional interactions with the original amino acid in another CH3 antibody constant domain.

[0181] In some embodiments, the hole is constructed by replacing an amino acid having a larger side chain, such as tyrosine or tryptophan, with an amino acid having a smaller side chain, such as alanine, valine, or threonine, for example, the Y407V mutation in the CH3 antibody constant domain. Similarly, in some embodiments, the knob is constructed by replacing an amino acid having a smaller side chain with an amino acid having a larger side chain, such as the T366W mutation in the CH3 antibody constant domain. In some embodiments, one Fc domain contains the knob mutation T366W and the other Fc domain contains the hole mutations T366S, L358A, and Y407V. In some embodiments, the polypeptides of the present disclosure comprising the SIRPα D1 domain variant are fused to an Fc domain containing the knob mutation T366W to limit unwanted homodimer formation between knobs. Examples of knob-into-hole amino acid pairs are listed in Table 9, but are not limited thereto. Examples of knob-into-hole Fc domain variants and SIRPα-Fc fusions are shown in Table 10.

Table 9

Table 10-1

Table 10-2

Table 10-3

[0182] In addition to the knob-into-hole strategy, in some embodiments, electrostatic steering is also used to control dimerization of the Fc domain. Electrostatic steering refers to controlling the formation of higher-order protein molecules by taking advantage of favorable electrostatic interactions between peptides, protein domains, and oppositely charged amino acids within a protein. In particular, to control dimerization of the Fc domain using electrostatic steering, one or more amino acid residues that make up the CH3-CH3 interface are replaced with positively or negatively charged amino acid residues such that the interaction is either electrostatically favorable or unfavorable, depending on the particular charged amino acids introduced. In some embodiments, positively charged amino acids at the interface such as lysine, arginine, or histidine are replaced with negatively charged amino acids such as aspartic acid or glutamic acid. In some embodiments, negatively charged amino acids at the interface are replaced with positively charged amino acids. In some embodiments, the charged amino acids are introduced into one or both of the interacting CH3 antibody constant domains. In some embodiments, by introducing charged amino acids into the interacting CH3 antibody constant domains of two Fc domains, selective formation of heterodimers of the Fc domain is promoted such that it is controlled by the electrostatic steering effect resulting from the interaction between the charged amino acids. Examples of electrostatic steering amino acid pairs are shown in Table 11, but are not limited thereto. [Table 11]

[0183] In particular, in the context of constructing bispecific antibodies, other methods are available for controlling heterodimerization of the Fc domain.

[0184] In some embodiments, the first Fc domain and the second Fc domain each comprise one or more of the amino acid substitutions of T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, and K409I with respect to the sequence of human IgG1.

[0185] In some embodiments, the Fc domain comprises: (a) with respect to wild-type human IgG1, one of the following amino acid substitutions: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, or K409I; or (b) (i) the N297A mutation with respect to the human IgG1 Fc region; (ii) the L234A, L235A, and G237A mutations with respect to the human IgG1 Fc region; (iii) the L234A, L235A, G237A, and N297A mutations with respect to the human IgG1 Fc region; (iv) the N297A mutation with respect to the human IgG2 Fc region; (v) the A330S and P331S mutations with respect to the human IgG2 Fc region; (vi) the A330S, P331S, and N297A mutations with respect to the human IgG2 Fc region; (vii) the S228P, E233P, F234V, L235A, and delG236 mutations with respect to the human IgG4 Fc region; or (viii) the S228P, E233P, F234V, L235A, delG236, and N297A mutations with respect to the human IgG4 Fc region.In some embodiments, the Fc domain variant comprises: (a) with respect to wild-type human IgG1, one of the following amino acid substitutions: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, or K409I; and further (b) (i) the N297A mutation with respect to the human IgG1 Fc region; (ii) the L234A, L235A, and G237A mutations with respect to the human IgG1 Fc region; (iii) the L234A, L235A, G237A, and N297A mutations with respect to the human IgG1 Fc region; (iv) the N297A mutation with respect to the human IgG2 Fc region; (v) the A330S and P331S mutations with respect to the human IgG2 Fc region; (vi) the A330S, P331S, and N297A mutations with respect to the human IgG2 Fc region; (vii) the S228P, E233P, F234V, L235A, and delG236 mutations with respect to the human IgG4 Fc region; or (viii) the S228P, E233P, F234V, L235A, delG236, and N297A mutations with respect to the human IgG4 Fc region.

[0186] In some embodiments, the first Fc domain and the second Fc domain comprise different amino acid substitutions. In some embodiments, the first Fc domain comprises T366W. In some embodiments, the second Fc domain comprises T366S, L368A, and Y407V. In some embodiments, the first Fc domain comprises D399K. In some embodiments, the second Fc domain comprises K409D.

[0187] Linker In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein alpha (SIRP-α) D1 variant comprising a SIRPα D1 domain or a fragment thereof having an amino acid mutation at residue 80 relative to the wild-type SIRPα D1 domain; and at least one additional amino acid mutation relative to the wild-type SIRPα D1 domain at a residue selected from the group consisting of residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92.

[0188] Also disclosed herein is, in some embodiments, a polypeptide comprising an Fc variant, wherein the Fc variant comprises an Fc domain dimer comprising two Fc domain variants, and each Fc domain variant is independently selected from (i) a human IgG1 Fc region comprising the mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region comprising the mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A.

[0189] In the present disclosure, a linker is used to describe a linkage or connection between a polypeptide or protein domain or an associated non-protein moiety. In some embodiments, the linker is a linkage or connection between an Fc domain (or a variant thereof) and a SIRPα D1 domain variant. In some embodiments, the linker connects the C-terminus of the SIRPα D1 domain variant and the N-terminus of the Fc domain variant such that two polypeptides are joined to each other in a tandem series.

[0190] In some embodiments, the linker is any type of bond formed from a simple covalent bond, such as a peptide bond, a synthetic polymer, or a chemical reaction, such as a chemical conjugate. When the linker is a peptide bond, in some embodiments, the carboxylic acid group at the C-terminus of a protein domain reacts with the amino group at the N-terminus of another protein domain in a condensation reaction to form a peptide bond. In some embodiments, the peptide bond is formed from synthetic means by conventional organic chemical reactions or by natural production from host cells, and the nucleic acid molecule encoding the DNA sequences of both proteins in a tandem series (e.g., the Fc domain variant and the SIRPα D1 domain variant) can be directly transcribed and translated in a host cell by the necessary molecular machinery (e.g., DNA polymerase and ribosome) into a continuous polypeptide encoding both proteins.

[0191] When the linker is a synthetic polymer, in some embodiments, the polymer is functionalized with reactive chemical functional groups at each end to react with the terminal amino acids at the connecting ends of the two proteins.

[0192] When the linker (excluding the above peptide bond) is made from a chemical reaction, in some embodiments, chemical functional groups (e.g., amine, carboxylic acid, ester, azide, or other functional groups) are synthetically attached to the C-terminus of one protein and the N-terminus of another protein, respectively. Next, in some embodiments, the two functional groups react via synthetic chemical means to form a chemical bond, thus connecting the two proteins together.

[0193] Spacer In some embodiments of the present disclosure, the linker between the Fc domain monomer and the SIRPα D1 variant polypeptide of the present disclosure is an amino acid spacer such as about 1 to 200 amino acids. Suitable peptide spacers include peptide linkers containing flexible amino acid residues such as glycine and serine. Examples of linker sequences are shown in Table 12. In some embodiments, the spacer comprises a motif of GS, GG, GGS, GGG, GGGGS (SEQ ID NO: 163), GGSG (SEQ ID NO: 164), or SGGG (SEQ ID NO: 165), for example, a plurality or repetitive motifs. In some embodiments, the spacer comprises a motif of GS, for example, 2 to 12 amino acids such as GS, GSGS (SEQ ID NO: 166), GSGSGS (SEQ ID NO: 167), GSGSGSGS (SEQ ID NO: 168), GSGSGSGSGS (SEQ ID NO: 169), or GSGSGSGSGSGS (SEQ ID NO: 170). In some embodiments, the spacer comprises a motif of GGS, for example, 3 to 12 amino acids including GGS, GGSGGS (SEQ ID NO: 171), GGSGGSGGS (SEQ ID NO: 172), and GGSGGSGGSGGS (SEQ ID NO: 173). In some embodiments, the spacer comprises a motif of GGSG (SEQ ID NO: 164), for example, 4 to 12 amino acids such as GGSG (SEQ ID NO: 164), GGSGGGSG (SEQ ID NO: 174), or GGSGGGSGGGSG (SEQ ID NO: 175). In some embodiments, the spacer comprises a motif of GGGGS (SEQ ID NO: 163), for example, GGGGSGGGGSGGGGS (SEQ ID NO: 176).In some embodiments, the spacer comprises an amino acid other than glycine and serine, such as AAS (SEQ ID NO: 177), AAAL (SEQ ID NO: 178), AAAK (SEQ ID NO: 179), AAAR (SEQ ID NO: 180), EGKSSGSGSESKST (SEQ ID NO: 181), GSAGSAAGSGEF (SEQ ID NO: 182), AEAAAKEAAAKA (SEQ ID NO: 183), KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), GGGGAGGGG (SEQ ID NO: 185), GENLYFQSGG (SEQ ID NO: 186), SACYCELS (SEQ ID NO: 187), RSIAT (SEQ ID NO: 188), RPACKIPNDLKQKVMNH (SEQ ID NO: 189), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 190), AAANSSIDLISVPVDSR (SEQ ID NO: 191), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 192).

[0194] In some embodiments, the spacer comprises a motif of EAAAK (SEQ ID NO: 193), such as a plurality or repeating motif. In some embodiments, the spacer comprises a motif of a proline-rich sequence, such as (XP)n, where X is any amino acid (e.g., A, K, or E), n is from 1 to 5, and PAPAP (SEQ ID NO: 194), a plurality or repeating motif. [Table 12]

[0195] In some embodiments, the length of the peptide spacer and the amino acids used are adjusted according to the degree of flexibility desired for the two proteins involved and the final protein fusion polypeptide. In some embodiments, the length of the spacer is adjusted to ensure proper protein folding and avoid aggregate formation. In some embodiments, the spacer is A or AAAL (SEQ ID NO: 178).

[0196] Vectors, host cells, and protein production In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein alpha (SIRP-α) D1 variant comprising a SIRPα D1 domain or a fragment thereof having an amino acid mutation at residue 80 relative to the wild-type SIRPα D1 domain; and at least one additional amino acid mutation relative to the wild-type SIRPα D1 domain at a residue selected from the group consisting of residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92.

[0197] Also disclosed herein is, in some embodiments, a polypeptide comprising an Fc variant, wherein the Fc variant comprises an Fc domain dimer having two Fc domain monomers, and each Fc domain monomer is independently selected from (i) a human IgG1 Fc region comprising the mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region comprising the mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A.

[0198] In some embodiments, the polypeptides of the present disclosure are produced from host cells. Host cells refer to vehicles containing the necessary cellular components, such as organelles, required to express the polypeptides and fusion polypeptides described herein from their corresponding nucleic acids. In some embodiments, the nucleic acid is contained in a nucleic acid vector that is introduced into the host cell by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, or the like. In some embodiments, the selection of the nucleic acid vector depends on the host cell to be used. In some embodiments, the host cell is of either prokaryotic (e.g., bacterial) or eukaryotic (e.g., mammalian) origin.

[0199] In some embodiments, a polypeptide construct comprising a polypeptide, such as a SIRPα D1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and a fusion partner such as an Fc variant, is produced by culturing a host cell transformed with a nucleic acid, preferably an expression vector, comprising a nucleic acid encoding the polypeptide construct (e.g., an Fc variant, a linker, and a fusion partner) under appropriate conditions to induce or cause expression of the polypeptide construct. In some embodiments, the appropriate conditions for expression vary depending on the selected expression vector and host cell. In some embodiments, a variety of suitable host cells are used, including but not limited to mammalian cells, bacteria, insect cells, and yeast. For example, various cell lines found to be useful in the present disclosure are described in the ATCC® cell line catalog available from the American Type Culture Collection. In some embodiments, the Fc domain variants of the present disclosure are expressed in cells optimized such that the proteins expressed by such cells do not glycosylate the proteins expressed by such cells, either by genetic engineering of the cell line or by changing cell culture conditions such as the addition of kifunensine, or by using a natural non-glycosylated host such as a prokaryote (e.g., E. coli), and in some cases, modification of the glycosylation sequences within the Fc is not necessary.

[0200] Construction of Nucleic Acid Vectors and Host Cells The nucleic acid sequences encoding the amino acid sequences of the polypeptides of the present disclosure can be prepared by various methods. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. In some embodiments, the nucleic acid molecules encoding the polypeptides of the present disclosure are obtained using standard techniques, such as gene synthesis. Alternatively, nucleic acid molecules encoding wild-type SIRPα D1 domain are mutated to include specific amino acid substitutions using standard techniques, such as QuikChange™ mutagenesis. In some cases, the nucleic acid molecules are synthesized using nucleotide synthesizers or PCR techniques.

[0201] In some embodiments, a nucleic acid encoding a polypeptide construct, such as a fusion partner including a SIRPα D1 domain variant (e.g., any variant shown in Tables 2, 5, and 6) and an Fc variant, is incorporated into an expression vector to express a protein. Various expression vectors can be utilized for protein expression. The expression vector can include an extrachromosomal vector that self-replicates or a vector that is incorporated into the host genome. The vector can also include various components or elements. For example, in some embodiments, vector components include, but are not limited to, transcriptional and translational regulatory sequences, such as promoter sequences, ribosome binding sites, signal sequences, transcription start and stop sequences, translation start and stop sequences, 3' and 5' untranslated regions (UTRs), and enhancer or activator sequences; an origin of replication; a selectable marker gene; as well as a nucleic acid sequence encoding the polypeptide of interest and a transcription termination sequence. In some embodiments, the expression vector includes a protein operably linked to a control or regulatory sequence, a selectable marker, any fusion partner, additional elements, or any combination thereof. The term "operably linked" means that a nucleic acid is placed in a functional relationship with another nucleic acid sequence. Generally, these expression vectors include transcriptional and translational regulatory nucleic acids operably linked to a nucleic acid encoding an Fc variant and are typically appropriate for the host cell used to express the protein. A selectable gene or marker, such as an antibiotic resistance gene or a fluorescent protein gene, can be used to select host cells containing the expression vector, for example, by antibiotic or fluorescence expression. Various selectable genes are available.

[0202] In some embodiments, the components or elements of the vector are optimized such that the expression vector is compatible with the host cell type. Expression vectors found to be useful in the present disclosure include, but are not limited to, those enabling protein expression in mammalian cells, bacteria, insect cells, yeast, and in vitro systems.

[0203] In some embodiments, mammalian cells are used as host cells for producing the polypeptides of the present disclosure. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NS0, Sp2 / 0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells. In some embodiments, E. coli cells are used as host cells for producing the polypeptides of the present disclosure. Examples of E. coli strains include, but are not limited to, E. coli 294 (ATCC® 31,446), E. coli λ1776 (ATCC® 31,537, E. coli BL21(DE3) (ATCC® BAA-1025)), and E. coli RV308 (ATCC® 31,608).

[0204] Different host cells have characteristic and specific mechanisms for post-translational processing and modification (e.g., glycosylation) of protein products. In some embodiments, an appropriate cell line or host system is selected to ensure proper modification and processing of the expressed polypeptide. When a vector is introduced into a host cell for protein production, the host cell is cultured in a conventional nutrient medium that has been appropriately modified for induction of the promoter, selection of transformants, or amplification of the gene encoding the desired sequence.

[0205] In some embodiments, a polypeptide construct, such as a polypeptide construct comprising a fusion partner such as an SIRPα D1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and an Fc variant, is expressed in a mammalian expression system, such as a system in which an expression construct is introduced into mammalian cells using a virus such as a retrovirus or an adenovirus. In some embodiments, cells of humans, mice, rats, hamsters, or primates are utilized. Suitable cells include, but are not limited to, known research cells such as Jurkat T cells, NIH3T3, CHO, COS, and 293 cells. Alternatively, in some embodiments, the protein is expressed in bacterial cells. Bacterial expression systems are well known in the art and include Escherichia coli (E. coli), Bacillus subtilis, Streptococcus cremoris, and Streptococcus lividans. In some cases, a polypeptide construct comprising an Fc domain variant is produced in insect cells such as Sf9 and Sf21 cells, or in yeast cells such as organisms of the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, and Yarrowia. In some cases, a polypeptide construct comprising an Fc domain variant is expressed in vitro using a cell-free translation system. In vitro translation systems derived from both prokaryotic (e.g., E. coli) and eukaryotic (e.g., wheat germ, rabbit reticulocyte) cells are available and, in some embodiments, are selected based on the expression level and functional properties of the protein of interest. For example, as understood by those skilled in the art, in vitro translation is required for some display technologies, such as ribosome display. Further, in some embodiments, the Fc domain variant is produced by chemical synthesis methods such as liquid-phase peptide synthesis and solid-phase peptide synthesis.In the case of in vitro transcription using a non-glycosylated system such as a bacterial extract, even if a natural glycosylation site exists, the Fc is not glycosylated, so inactivation of the Fc can be obtained equivalently.

[0206] In some embodiments, the polypeptide construct includes unnatural amino acids, amino acid analogs, amino acid mimetics, or any combination thereof that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids generally refer to the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), and pyrrolysine and selenocysteine. An amino acid analog is a compound having the same basic chemical structure as a naturally occurring amino acid, for example, hydrogen, a carboxyl group, an amino group, and a carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. In some embodiments, such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but generally retain the same basic chemical structure as natural amino acids.

[0207] Protein production, recovery, and purification In some embodiments, the host cells used to produce the polypeptides of the present disclosure are grown in a medium suitable for culturing the selected host cells. Examples of suitable media for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Expi293™ Expression Medium, DMEM supplemented with fetal bovine serum (FBS), and RPMI-1640. Examples of suitable media for bacterial host cells include Luria broth (LB) supplemented with necessary supplements such as a selective agent, e.g., ampicillin. In some embodiments, the host cells are cultured at a suitable temperature, such as about 20°C to about 39°C, for example, about 25°C to about 37°C, preferably 37°C, and at a CO2 level of 5% to 10%. In some embodiments, the pH of the medium is about pH 6.8 to pH 7.4, for example, pH 7.0, mainly depending on the host organism. When an inducible promoter is used in the expression vector, protein expression can be induced under conditions suitable for activation of the promoter.

[0208] In some embodiments, the recovery of the protein involves disruption of the host cell, for example, by osmotic shock, sonication, or lysis. Once the cells are disrupted, the cell debris is removed by centrifugation or filtration. Thereafter, the protein can be further purified. In some embodiments, the polypeptides of the present disclosure are purified by various methods of protein purification, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard method for protein purification. For example, in some embodiments, the protein is isolated and purified by appropriately selecting and combining an affinity column, such as a Protein A column (e.g., POROS Protein A chromatography), a chromatography column (e.g., POROS HS-50 cation exchange chromatography), filtration, ultrafiltration, desalting, and dialysis procedures. In some embodiments, the polypeptide is conjugated to a marker sequence, such as a peptide, to facilitate purification. An example of a marker amino acid sequence is a hexahistidine peptide (His6-tag (SEQ ID NO: 223)) that can bind to a nickel-functionalized agarose affinity column with micromolar affinity. Alternatively, a hemagglutinin "HA" tag corresponding to an epitope derived from the influenza hemagglutinin protein can be used.

[0209] In some embodiments, polypeptide constructs of the disclosure, such as polypeptides comprising a fusion partner such as an SIRPα D1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and an Fc variant, are produced by a cell of a subject (e.g., a human) by administering a vector (e.g., a viral vector such as a retroviral vector, an adenoviral vector, a poxviral vector (e.g., a vaccinia virus vector, e.g., modified vaccinia Ankara (MVA)), an adeno-associated viral vector, and an alphavirus vector) comprising a nucleic acid molecule encoding a polypeptide of the disclosure. The vector can be used for expression of the polypeptides disclosed herein when it enters the cells of the subject (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.). Optionally, the polypeptide is secreted from the cell. In some embodiments, no further action is required if treatment of a disease or disorder is the desired outcome. In some embodiments, if protein collection is desired, blood is collected from the subject and the protein is purified from the blood by various methods.

[0210] Method for treating cancer Provided herein is a method of treating cancer in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) and (b) a chemotherapeutic agent (at least one chemotherapeutic agent, e.g., at least two, at least three, or at least four chemotherapeutic agents, etc.). Provided herein is a method of treating cancer in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein) and (b) a chemotherapeutic agent (at least one chemotherapeutic agent, e.g., at least two, at least three, or at least four chemotherapeutic agents, etc.). In some embodiments, the method further comprises administering to the individual an effective amount of a therapeutic antibody (at least one therapeutic antibody, e.g., at least two, at least three, or at least four therapeutic antibodies). Additionally or alternatively, in some embodiments, the method further comprises administering to the individual an effective amount of an immunotherapeutic agent (at least one immunotherapeutic agent, e.g., at least two, at least three, or at least four immunotherapeutic agents). Additionally or alternatively, in some embodiments, the method comprises administering the polypeptide and the chemotherapeutic agent in combination with one or more additional treatment modalities, including but not limited to, for example, radiation therapy, surgery, cryoablation, and bone marrow transplantation, etc.

[0211] Combination therapies comprising chemotherapeutic agents, and exemplary chemotherapeutic agents Exemplary chemotherapeutic agent(s) that can be used in the method of treating cancer described herein include, but are not limited to, methotrexate (RHEUMATREX®, amethopterin), cyclophosphamide (CYTOXAN®), abiraterone, abemaciclib, altretamine, thalidomide (THALIDOMID®), acridine carboxamide, actimid®, actinomycin, actinomycin-D, afatinib, 17-N-allylamino-17-demethoxygeldanamycin, alectinib, alpelisib, aminopterin, amsacrine, anlotinib, anthracycline, antineoplastic agent, antineoplaston, apartinib, 5-azacitidine, 6-mercaptopurine, 6-thioguanine, arabinosylcytosine, axitinib, azacitidine, azathioprine, BL22, bendamustine, binimetinib, bilicodal, bleomycin, bortezomib, bosutinib, brigatinib, bryostatin, busulfan, cabozantinib, calyculin, camptothecin, capecitabine, carboplatin, carmustine, ceritinib, chlorambucil, cisplatin, cladribine, clofarabine, cobimetinib, crizotinib, cytarabine, dabrafenib, dacarbazine, dacomitinib, dasatinib, daunorubicin, dexamethasone, dichloroacetic acid, discodermolide, docetaxel, doxorubicin, encorafenib, epirubicin, enzastaurin, enzalutamide, epothilone, erdafitinib, eribulin, erlotinib, estramustine, etoposide, everolimus, exatecan, exisulind, feruginol, floxuridine, fludarabine, fluorouracil (such as 5-fluorouracil), folic acid, phosphoestrol, fotemustine, fruquintinib, ganciclovir, gefitinib, gemcitabine, gilteritinib, goserelin, hexamethylmelamine, hydroxycarbamide, hydroxyurea, IT-101, ibrutinib, icotinib, idarubicin, idelalisib, ifosfamide, imatinib, irinoimiquimod, irinotecan, irinotecan, irinotecan, irinotecan, laniquidar,Lapatinib, larotrectinib, lenalidomide, lenvatinib, lorlatinib, romidepsin, larotrectinib, mafosfamide, masoprocol, mechlorethamine, melphalan, mercaptopurine, methotrexate, methylprednisolone, mitomycin, mitotane, mitoxantrone, nelarabine, neratinib, niraparib, nilotinib, nintedanib, oblimersen, olaparib, osimertinib, oxaliplatin, nedaplatin, phenanthriplatin, picoplatin, PAC-1, paclitaxel, palbociclib, pazopanib, pemetrexed, pegfilgrastim, pentostatin, pipobroman, pixantrone, plicamycin, prednisone, ponatinib, procarbazine, proteasome inhibitor (e.g., bortezomib), pyrotinib, raltitrexed, rebeccamycin, leflamide (registered trademark), regorafenib, ribociclib, rubitecan, rucaparib, luxolitinib, SN-38, salinosporamide A, satraplatin, sirolimus, sonidegib, sorafenib, streptozocin, streptozotocin, sunitinib, swainsonine, talazoparib, talidomide, taxane, tegafur-uracil, temsirolimus, teniposide, temozolomide, testolactone, thiotepa, thioguanine, topotecan, trabectedin, trametinib, tretinoin, trifluridine, triplatin tetranitrate, tris(2-chloroethyl)amine, troxacitabine, uracil mustard, valrubicin, vandetanib, vemurafenib, venetoclax (ABT-199), navitoclax (ABT-263), vinblastine, vincristine, vinorelbine, visimodegib, vorinostat, ziv-aflibercept (ZALTRAP (registered trademark)), zoledronic acid, etc.

[0212] In some embodiments, a method of treating cancer comprises administering an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), in combination with a particular class of chemotherapeutic agent(s). In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is a polypeptide described herein (e.g., a fusion polypeptide comprising a SIRPα d1 domain variant and an Fc variant; a fusion polypeptide comprising a SIRPγ variant, a SIRPβ1 variant, or a SIRPβ2 variant and an Fc variant). For example, in some embodiments, a method of treating cancer comprises administering a polypeptide described herein (e.g., a fusion polypeptide) in combination with an adrenocortical inhibitor (including but not limited to the adrenocortical inhibitors described herein). For example, in some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with an anthracycline (including but not limited to the anthracyclines described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with an alkylating agent (including but not limited to the alkylating agents described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with an androgen inhibitor (including but not limited to the androgen inhibitors described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with an antimetabolite, such as a purine analog (including but not limited to the antimetabolites, such as purine analogs, described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with an antitumor antibiotic (including but not limited to the antitumor antibiotics described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with a BCL-2 inhibitor (including but not limited to the BCL-2 inhibitors described herein).In some embodiments, a method of treating cancer comprises administering a polypeptide as described herein in combination with a BTK inhibitor (including but not limited to the BTK inhibitors described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide as described herein in combination with a CDK4 / 6 inhibitor (including but not limited to the CDK4 / 6 inhibitors described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide as described herein in combination with a colony stimulating factor (including but not limited to the colony stimulating factors described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide as described herein in combination with a corticosteroid (including but not limited to the corticosteroids described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide as described herein in combination with an EGFR inhibitor (including but not limited to the EGFR inhibitors described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide as described herein in combination with a gonadotropin-releasing hormone (GnRH) agonist (including but not limited to the GnRH agonists described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide as described herein in combination with a mitotic inhibitor / microtubule inhibitor (including but not limited to the mitotic inhibitor / microtubule inhibitors described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide as described herein in combination with an mTOR kinase inhibitor (including but not limited to the mTOR kinase inhibitors described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide as described herein in combination with a proteasome inhibitor (including but not limited to the proteasome inhibitors described herein).In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with a signaling inhibitor, such as a protein tyrosine kinase inhibitor, a PAK4 inhibitor, a PI3K inhibitor (including but not limited to the signaling inhibitors described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with a topoisomerase inhibitor (including but not limited to the topoisomerase inhibitors described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with a tyrosine kinase inhibitor (including but not limited to the tyrosine kinase inhibitors described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with a VEGF inhibitor, such as a VEGF1 inhibitor, a VEGF2 inhibitor, and / or a VEGF3 inhibitor (including but not limited to the VEGF inhibitors described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with an agent that regulates apoptosis, for example, by regulating the activity of Bcl-2, Mcl1, Bcl-1x, etc. (including but not limited to agents that regulate apoptosis by regulating the activity of Bcl-2, Mcl1, Bcl-1x, etc. described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with a platinum-based agent (including but not limited to the platinum-based agents described herein).In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with an inhibitor of NTRK1, NTRK2, and / or NTRK3, an ALK inhibitor, a ROS inhibitor, an FLT3 inhibitor, a BRAF inhibitor, an inhibitor of MEK1 and / or MEK2, an inhibitor of HER2, HER3, and / or HER4, an inhibitor of RET / PTC, an inhibitor of BCR-ABL, a c-KIT inhibitor, an inhibitor of PDGFR-alpha and / or PDGFR-beta, an inhibitor of FGFR1, FGFR2, FGFR3, and / or FGFR4, a smoothened inhibitor and / or an inhibitor of PARP1, PARP2, and / or PARP3 (including, but not limited to, the inhibitors described herein). In some embodiments, the inhibitor is an antisense polynucleotide (such as siRNA or RNAi). In some embodiments, the inhibitor is a small molecule inhibitor as further described in detail below.

[0213] In some embodiments, the chemotherapeutic agent is a small molecule anti-cancer agent (such as a small molecule inhibitor). In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with a small molecule inhibitor of VEGFR and / or PDGFR, a small molecule EGFR inhibitor, a small molecule ALK inhibitor, a small molecule CDK4 / 6 inhibitor, a small molecule PARP inhibitor, a small molecule PAK4 inhibitor, a small molecule mTOR inhibitor, a small molecule KRAS inhibitor, a small molecule TRK inhibitor, a small molecule BCL2 inhibitor, a small molecule B-raf inhibitor, a small molecule IDH inhibitor, a small molecule PI3K inhibitor, a small molecule DDR (DNA damage response) inhibitor, or a small molecule hypomethylating agent. In other cases, the targeted small molecule modulates a cell signaling pathway of cells expressing CD47, such as an IDO / TDO inhibitor, an AhR inhibitor, an arginase inhibitor, an A2a R inhibitor, a TLR agonist, a STING agonist, or a Rig-1 agonist.

[0214] In some embodiments, a method of treating cancer comprises administering a polypeptide described herein (e.g., a fusion polypeptide comprising an SIRPα d1 domain variant and an Fc variant) in combination with at least one, at least two, at least three, or at least four chemotherapeutic agents. In some embodiments where two or more chemotherapeutic agents are administered, the two or more chemotherapeutic agents are from different classes (as described above) and / or exert their anti-cancer effects via different mechanisms of action.

[0215] Further details regarding exemplary pharmaceutical compositions and preparations, exemplary dosages, and exemplary routes of administration of the fusion polypeptides described herein are provided in WO2017 / 027422 and U.S. Patent No. 10,259,859, the entire contents of each of which are incorporated by reference.

[0216] Combination therapies comprising therapeutic antibodies, and exemplary therapeutic antibodies. In some embodiments, a method of treating cancer provided herein comprises administering to a subject an effective amount of a therapeutic antibody (e.g., at least one therapeutic antibody, e.g., at least two, at least three, or at least four therapeutic antibodies), i.e., an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., a fusion polypeptide described herein) in combination with a chemotherapeutic agent described herein (e.g., at least one chemotherapeutic agent, e.g., at least two, at least three, or at least four chemotherapeutic agents). In some embodiments, the therapeutic antibody is conjugated to a drug (i.e., an antibody-drug conjugate or "ADC").

[0217] Exemplary therapeutic antibodies (e.g., therapeutic monoclonal antibodies) for use in the methods of this specification include, but are not limited to, 3F8, 8H9, abagovomab, abciximab, abituzumab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afelimomab, afutuzumab, alacizumab pegol, ALD518, alemtuzumab, alirocumab, Altumomab pentetate, amatuximab, Anatumomab mafenatox, Anetumab ravtansine, anifrolumab, Anrukinzumab (IMA-638), apolizumab, Arcitumomab, Ascrinvacumab, Aselizumab, atezolizumab, Atinumab, Atorolizumab (tositumomab), avelumab, bapineuzumab, basiliximab, babiximab, bectumomab, begelomab, belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, bezlotoxumab, bictiromab, bimagrumab, Bimekizumab, Bivatuzumab mertansine, blinatumomab, brodalumab, brodalumab, bronchiolizumab, cabiralizumab (FPA008), camrelizumab, canakinumab, Cantuzumab mertansine, Cantuzumab ravtansine, caplacizumab, Capromab pendetidependetide), carlumab, catumaxomab, cBR96-doxorubicin immunoconjugate, CC49, cedelizumab, certolizumab pegol, cetuximab, Ch.14.18, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, codrituzumab, coltuximab ravtansine, conatumumab, concizumab, crenezumab, CR6261, dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumumab, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, derlotuximab biotin, detumomab, dinutuximab, diridavumab, dorlimomab aritoxaritox), Drozitumab, Duligotumab, Dupilumab, Durvalumab, Dusigitumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Eldelumab, Elgemtumab, Elotuzumab, Elsirimomab, Emactuzumab (RG7155), Emibetuzumab, Enavatuzumab, Enfortumab vedotin, Enlimomab pegol, Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epitumomabcituxetan), Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, Ficlatuzumab, Figitumumab, Firivumab, Flanvotumab, Fletikumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab, Ganitumumab, Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Guselkumab, Ibalizumab, Ibritumomab tiuxetantiuxetan), Icrucumab, Idarucizumab, Igovomab, IMAB362, Imalumab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Indusatumab vedotin, Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Isatuximab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lambrolizumab, Lampalizumab, Lebrikizumab, Lemalesomab, Lenzilumab, Lerdelimumab, Lexatumumab, Libivirumab, Lifastuzumab vedotin, Ligelizumab, Lilotomab satetraxetan, Lintuzumab, Lirilumab, Lodelcizumab, Lokibetumab, Lorvotuzumab mertansine, Lucatumumab, Lulizumabpegol), Lumiliximab, Lumretuzumab, MSB0010718C (avelumab), Mapatumumab, Margetuximab, Maslimomab, Mavrilimumab, Matuzumab, MEDI6469, MEDI0680, MEDI6383, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Mogamulizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox, Muromonab - CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox, Narnatumab, Natalizumab, Nebacumab, Necitumumab, Nemolizumab, Nere limomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentan, Obiltoxaximab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Ontuxizumab, Opisimumab (Opicinumab), Oportuzumab monatox, Oregovomab, Orticumab, Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab, Pankomab, Panobacumab, Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Polatuzumab vedotinvedotin), Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO140, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab, Refanezumab, Regavirumab, Reslizumab, Rilotumumab, Rinucumab, Rituximab, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Sacituzumab govitecan, Samalizumab, SAR650984 (Isatuximab), Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, Sibrotuzumab, SGN-CD19A, SGN-CD33A, Sifalimumab, Siltuximab, Simtuzumab, Sintilimab, Siplizumab, Sirukumab, Sofituzumab vedotinvedotin), Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanespumab, Taplitumomab paptox, Tarextumab, Tefibazumab, Telimomab aritox, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, Tesidolumab, TGN1412, Ticilimumab (tremelimumab), Tildrakizumab, Tigatuzumab, TNX-650, Tocilizumab (atlizumab), Toralizumab, Toripalimab, Tosatoxumab, Tositumomab, Tovetumab, Tralokinumab, Trastuzumab, Trastuzumab emtansine, TRBS07, Tregalizumab, tremelimumab, Tucotuzumab celmoleukin, Tuvirumab, Ublituximab, Ulocuplumab, Urelumab, Urtoxazumab, Ustekinumab, Utomilumab (PF-05082566), Vandortuzumabvedotin), Vantictumab, Vanucizumab, Vapaliximab, Varlilumab, Vatelizumab, Vedolizumab, Bertuzumab, Vepalimomab, Vesencumab, Visilizumab, Volociximab, Vonlerolizumab (RG7888), Vorsetuzumab mafodotin, Votumumab, Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab, or Zolimomab aritox, and biosimilars of any of the aforementioned therapeutic antibodies.

[0218] Other exemplary therapeutic antibodies (e.g., therapeutic monoclonal antibodies) that can be used in the methods of this specification are antibodies and include, but are not limited to, for example, anti-CD20 antibodies, anti-EGFR antibodies, anti-Her2 / Neu (ERBB2) antibodies, anti-EPCAM antibodies, anti-GL2 antibodies, anti-GD2 antibodies, anti-GD3 antibodies, anti-CD2 antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD8 antibodies, anti-CD19 antibodies, anti-CD22 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD39 antibodies, anti-CD45 antibodies, anti-CD47 antibodies, anti-CD52 antibodies, anti-CD56 antibodies, anti-CD70 antibodies, anti-CD73 antibodies, anti-CD117 antibodies, anti-SIRPα antibodies, anti-LILRB1 antibodies, anti-LILRB2 antibodies, anti-LILRB4 antibodies, anti-PD1 antibodies (e.g., anti-PD-1 antagonist antibodies), anti-PD-L1 antibodies (e.g., anti-PD-L1 antagonist antibodies), anti-PD-L2 antibodies, antibodies designed to bind to tumor cells, virus- or bacteria-infected cells, immune cells, or healthy normal cells, or cytokines, chemokines, or any type of hormone.

[0219] In some embodiments, the therapeutic antibodies used in the methods herein are, for example, CS1 / SLAMF7, Trop-2, VWF, Vimentin, VEGFR2, VEGFR-1, VEGF, VEGF-A, TYRP1 (glycoprotein 75), TWEAK receptor, tumor-specific glycosylation of MUC1, tumor antigen CTAA16.88, TRAIL-R2, TRAIL-R1, TNF-α, TGF-beta, TGF beta 2, TGF beta 1, TFPI, Tenascin C, TEM1, TAG-72, T cell receptor, STEAP1, sphingosine-1-phosphate, SOST, SLAMF7, BCL-2, selectin P, SDC1, sclerostin, RTN4, RON, Rh factor, RHD, RS virus, RANKL, rabies virus glycoprotein, platelet-derived growth factor receptor beta, phosphatidylserine, sodium phosphate cotransporter, PDGF-R alpha, PDCD1, PD-1, PD-L1, PCSK9, oxLDL, OX-40, NRP1, Notch receptor 4, Notch receptor 3, Notch receptor 2, Notch receptor 1, NOGO-A, NGF, neural apoptosis-regulated proteinase 1, NCA-90 (granulocyte antigen), NARP-1, N-glycolylneuraminic acid, myostatin, myelin-associated glycoprotein, mucin CanAg, MUC1, MSLN, MS4A1, MIF, mesothelin, MCP-1, LTA, LOXL2, lipoteichoic acid, LINGO-1, LFA-1 (CD11a), Lewis-Y antigen, L-selectin (CD62L), KIR2D, ITGB2 (CD18), ITGA2, interferon alpha / beta receptor, interferon receptor, interferon gamma-induced protein, integrin alpha v beta 3, integrin alpha II beta 3, integrin alpha 7 beta 7, integrin alpha 5 beta 1, integrin alpha 4 beta 7, integrin alpha 4, insulin-like growth factor I receptor, influenza A hemagglutinin, ILGF2, IL9, IL6, IL4, IL3 IRA, IL23, ILI 7A, IL-6 receptor, IL-6, IL-S, IL-4, IL-23, IL-22, IL-I, IL-I 7A, IL-I 7, IL-13, IL-I 2, IL-I, IL20, IGHE, IgG4, IGF-I, IGF-I receptor, IgE Fc region, IFN-gamma, IFN-alpha, ICAM-1 (CD54), human TNF, human scatter factor receptor kinase, Hsp90, HNGF, HLA-DR, HIV-1, histone complex, HHGFR, HGF, HER3, HER2, HER2 / neu, HER1, hepatitis B surface antigen, hemagglutinin, GUCY2C, GPNMB, GMCSF receptor alpha chain, glypican 3, GD3 ganglioside, GD2, ganglioside GD2, Frizzled receptor, folate receptor 1, folate hydrolase, fibronectin extra domain-B, fibrin II, beta chain, FAP, respiratory syncytial virus F protein, ERBB3, epicyanin, EpCAM, endotoxin, EGFR, EGFL7, Shiga toxin-producing Escherichia coli type 2 (E. coli shiga toxin type-2), Shiga toxin-producing Escherichia coli type I (E. coli shiga toxin type-I), DRS, DPP4, DLL4, dabigatran, cytomegalovirus glycoprotein B, CTLA-4, CSF2, CSF1R, clamping factor A, CLDN18.2, ch4DS, CFD, CEA-related antigen, CEA, CD80, CD79B, CD74, CD73, CD70, CD6, CD56, CD52, CD51, CD5, CD44 v6, CD41, CD40 ligand, CD40, CD4, CD39, CD38, CD37, CD33, CD30 (TNFRSF8), CD123, CD138, CD3 epsilon, CD3, CD28, CD274, CD27, CD2S (chain of IL-2 receptor), CD23 (IgE receptor), CD221, CD22, CD200, CD20, CD2, CD19, CD137, CD154, CD152, CD15, CD147 (basigin), CD140a, CD125, CD11, CD-18, CCR5, CCR4, CCL11 (eotaxin-I), cardiac myosin, carbonic anhydrase 9 (CA-IX), Canis lupus familiarisIt is an antibody that binds to IL31, CA-125, C5, C242 antigen, CXC chemokine receptor type 4, beta amyloid, BAFF, B7-H3, B lymphoma cells, AOC3 (VAP-I), anthrax toxin, protective antigen, angiopoietin 3, angiopoietin 2, alpha-fetoprotein, AGS-22M6, adenocarcinoma antigen, ACVR2B, activin receptor-like kinase I, 5T4, 5AC, 4-IBB or 1-40-beta-amyloid.

[0220] In some embodiments, the therapeutic antibodies used in the methods herein bind to antigens expressed by cancer cells (e.g., expressed on the surface of cancer cells). Exemplary antigens expressed by cancer are known in the art and include, but are not limited to, for example, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD56, CD70, CD74, CD79b, CD123, CD138, CS1 / SLAMF7, Trop-2, 5T4, BCMA, mucin 1, mucin 16, PTK7, PD-L1, STEAP1, endothelin B receptor, mesothelin, EGFRvIII, ENPP3, SLC44A4, GNMB, nectin 4, NaPi2b, LIV-1A, guanylyl cyclase C, DLL3, EGFR, HER2, VEGF, VEGFR, integrin αVβ3, integrin α5β1, MET, IGF1R, TRAILR1, TRAILR2, RANKL, FAP, tenascin, Le y, EpCAM, CEA, gpA33, PSMA, TAG72, mucin, CAIX, EPHA3, folate receptor alpha, GD2, GD3, MHC / peptide complexes containing peptides from NY-ESO-1 / LAGE, SSX-2, MAGE family proteins, MAGE-A3, gp100 / pmel17, Melan-A / MART1, gp75 / TRP1, tyrosinase, TRP2, CEA, PSA, TAG-72, immature laminin receptor, MOK / RAGE-1, WT-1, SAP-1, BING-4, EpCAM, MUC1, PRAME, survivin, BRCA1, BRCA2, CDK4, CML66, MART-2, p53, Ras, beta-catenin, TGF-betaRII, HPV E6, or HPV E7. For example, in some embodiments, the polypeptides described herein are administered in combination with a monoclonal antibody that binds to a chemotherapeutic agent (e.g., at least one chemotherapeutic agent) and CD123 (also known as interleukin-3 receptor alpha), such as talacotuzumab (also known as CSL362 and JNJ-56022473).

[0221] In some embodiments, the therapeutic antibodies (e.g., therapeutic monoclonal antibodies) used in the methods herein are antibodies that bind to antigens expressed by NK cells. Exemplary antigens expressed by NK cells include, but are not limited to, NKR-P1A (KLRB1), CD94 (NKG2A), KLRG1, KIR2DL5A, KIR2DL5B, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, KIR2DS1, CD94 (NKG2C / E), NKG2D, CD160 (BY55), CD16 (FcγRIIIA), NKp46 (NCR1), NKp30 (NCR3), NKp44 (NCR2), DNAM1 (CD226), CRTAM, CD27, NTB-A (SLAMF6), PSGL1, CD96 (Tactile), CD100 (SEMA4D), NKp80 (KLRF1, CLEC5C), SLAMF7 (CRACC, CS1, CD319), and CD244 (2B4, SLAMF4).

[0222] Combination therapies comprising an immunotherapeutic agent, and exemplary immunotherapeutic agents In some embodiments, the methods of treating cancer provided herein comprise administering to a subject an effective amount of an immunotherapeutic agent (e.g., at least one immunotherapeutic agent, e.g., at least two, at least three, or at least four immunotherapeutic agents), i.e., an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., the polypeptides described herein) in combination with a chemotherapeutic agent described herein (e.g., at least one chemotherapeutic agent, e.g., at least two, at least three, or at least four chemotherapeutic agents).

[0223] In some embodiments, an immunotherapeutic agent targets the immune system and refers to any therapeutic agent that promotes the therapeutic reorientation of the immune system, such as a co-stimulatory pathway regulator, a cancer vaccine, a recombinant modified immune cell, etc. Exemplary and non-limiting immunotherapeutic agents are described below. In some embodiments, the immunotherapeutic agent is an antibody or comprises an antibody. Exemplary targets of immunotherapeutic antibodies are known in the art and include, but are not limited to, BDCA2, BDCA4, ILT7, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, Siglec-3, Siglec-7, Siglec-9, Siglec-10, Siglec-15, FGL-1, CD200, CD200R, CSF-1R, CD24, CD40, CD40L, CD163, CD206, DEC205, CD47, CD123, arginase, IDO, TDO, AhR, EP2, COX-2, CCR2, CCR-7, CXCR1, CX3CR1, CXCR2, CXCR3, CXCR4, CXCR7, TGF-β RI, TGF-β RII, c-Kit, CD244, L-selectin / CD62L, CD11b, CD11c, CD68, 41BB, CTLA4, PD1, PD-L1, PD-L2, TIM-3, BTLA, VISTA, LAG-3, CD28, OX40, GITR, CD137, CD27, HVEM, CCR4, CD25, CD103, KIrg1, Nrp1, CD278, Gpr83, TIGIT, CD154, CD160, TNFR2, PVRIG, DNAM, and ICOS.

[0224] Approved or investigational immunotherapeutic agents include, but are not limited to, ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, and the like. In certain embodiments, an agent that blocks the interaction between CD47 and SIRPα (such as the polypeptides described herein) is administered in combination with an inhibitor of the PD-L1 / PD-1 pathway, such as an antibody, small molecule, or polypeptide that blocks the interaction between PD-L1 and PD-1 (e.g., by binding to PD-1 or PD-L1). In some embodiments, the inhibitor of the PD-L1 / PD-1 pathway is an antisense polynucleotide. In some embodiments, the inhibitor of the PD-L1 / PD-1 pathway is an anti-PD-1 or anti-PD-L1 antagonist antibody (e.g., the anti-PD-1 or anti-PD-L1 antagonist antibodies described elsewhere herein). As shown herein, combination administration of an agent that blocks the interaction between CD47 and SIRPα (such as the polypeptides described herein) and an inhibitor of the PD-L1 / PD-1 pathway can result in synergistic antitumor activity. In some embodiments, the immunotherapeutic agent is, or comprises, a vaccine, oncolytic virus, adoptive cell therapy, cytokine, or small molecule immunotherapeutic agent. Examples of such immunotherapeutic agents are known in the art. For example, adoptive cell therapies and therapeutic agents include, but are not limited to, chimeric antigen receptor T cell therapy (CAR-T), tumor infiltrating lymphocytes (TIL), TCR-engineered T cells, TCR-engineered NK cells, and macrophage cell products. Vaccines include, but are not limited to, polynucleotide vaccines, polypeptide vaccines, or cell-based (e.g., tumor or dendritic cell-based) vaccines. A variety of cytokines useful in the treatment of cancer are known, including, but not limited to, IL-2, IL-15, IL-7, IL-10, IL-12, IL21, TNFa, IFN, GM-CSF, and engineered cytokine mutants.Examples of small molecule immunotherapeutic agents include, but are not limited to, IDO / TDO inhibitors, AhR inhibitors, arginase inhibitors, A2a R inhibitors, TLR agonists, STING agonists, and Rig-1 agonists.

[0225] In some embodiments, an agent that blocks the interaction between CD47 and SIRPα (such as a polypeptide described herein) and a chemotherapeutic agent (e.g., at least one chemotherapeutic agent) are administered in combination with one or more additional agents (e.g., therapeutic antibodies, small molecule inhibitors, immunotherapeutic agents, etc.) described herein. In some such embodiments, the additional agent(s) are of different classes and / or exert their anti-cancer effects via different mechanisms of action. For example, in some embodiments, a method of treating cancer comprises administering an agent that blocks the interaction between CD47 and SIRPα (such as a polypeptide described herein) in combination with a chemotherapeutic agent (including but not limited to those described herein) and a therapeutic antibody (including but not limited to those described herein, such as an anti-HER2 antibody). In some embodiments, an agent that blocks the interaction between CD47 and SIRPα (such as a polypeptide described herein) is administered in combination with a chemotherapeutic agent (including but not limited to those described herein) and a small molecule inhibitor (including but not limited to those described herein). Other combinations are contemplated.

[0226] In some embodiments, an agent that blocks the interaction between CD47 and SIRPα (such as a polypeptide described herein) is administered in combination with one or more agents, including but not limited to, antidiarrheal agents, antiemetic agents, analgesics, opioids, and / or non-steroidal anti-inflammatory agents.

[0227] Combination therapies including additional treatment modalities (s) In some embodiments, an agent that blocks the interaction between CD47 and SIRPα (such as a polypeptide described herein) is administered in combination with at least one chemotherapeutic agent and one or more additional treatment modalities. In some embodiments, the one or more additional treatment modalities include radiation therapy (e.g., gamma rays, X-rays, and / or direct delivery of a radioisotope to tumor cells, microwaves, UV radiation, or gene therapy. For example, therapeutic genes for gene therapy include, but are not limited to, antisense versions of cell growth inducers (oncogenes), cell growth inhibitors (tumor suppressors), or inducers of programmed cell death (apoptosis-promoting genes). In some embodiments, any one or more of the combination therapies described herein are administered in conjunction with surgery (e.g., resection).

[0228] Exemplary therapeutic combinations In some embodiments, a method of treating cancer comprises administering an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) in combination with nivolumab and one or more agents selected from the following: lenalidomide, ibrutinib, palbociclib, enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin, cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine, gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin, docetaxel, estramustine, paclitaxel, vinblastine, etoposide, irinotecan, teniposide, topotecan, prednisone, methylprednisolone, and dexamethasone. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is a polypeptide described herein (e.g., a fusion polypeptide comprising a SIRPα d1 domain variant and an Fc variant).

[0229] In some embodiments, a method of treating cancer comprises administering an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) in combination with pembrolizumab and one or more agents selected from the following: lenalidomide, ibrutinib, palbociclib, enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin, cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine, gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin, docetaxel, estramustine, paclitaxel, vinblastine, etoposide, irinotecan, teniposide, topotecan, prednisone, methylprednisolone, and dexamethasone. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is a polypeptide described herein (e.g., a fusion polypeptide comprising an SIRPα d1 domain variant and an Fc variant).

[0230] In some embodiments, a method of treating cancer comprises administering an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) in combination with trastuzumab and one or more agents selected from the following: lenalidomide, ibrutinib, palbociclib, enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin, cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine, gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin, docetaxel, estramustine, paclitaxel, vinblastine, etoposide, irinotecan, teniposide, topotecan, prednisone, methylprednisolone, and dexamethasone. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is a polypeptide described herein (e.g., a fusion polypeptide comprising an SIRPα d1 domain variant and an Fc variant).

[0231] In some embodiments, a method of treating cancer comprises administering an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) in combination with bevacizumab and one or more agents selected from the following: lenalidomide, ibrutinib, palbociclib, enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin, cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine, gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin, docetaxel, estramustine, paclitaxel, vinblastine, etoposide, irinotecan, teniposide, topotecan, prednisone, methylprednisolone, and dexamethasone. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is a polypeptide described herein (e.g., a fusion polypeptide comprising an SIRPα d1 domain variant and an Fc variant).

[0232] In some embodiments, a method of treating cancer comprises administering an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) in combination with rituximab and one or more agents selected from the following: lenalidomide, ibrutinib, palbociclib, enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin, cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine, gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin, docetaxel, estramustine, paclitaxel, vinblastine, etoposide, irinotecan, teniposide, topotecan, prednisone, methylprednisolone, and dexamethasone. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is a polypeptide described herein (e.g., a fusion polypeptide comprising SIRPα d1 domain variants and Fc variants).

[0233] In some embodiments, a method of treating cancer comprises administering an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) in combination with pertuzumab and one or more agents selected from the following: lenalidomide, ibrutinib, palbociclib, enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin, cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine, gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin, docetaxel, estramustine, paclitaxel, vinblastine, etoposide, irinotecan, teniposide, topotecan, prednisone, methylprednisolone, and dexamethasone. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is a polypeptide described herein (e.g., a fusion polypeptide comprising an SIRPα d1 domain variant and an Fc variant).

[0234] In some embodiments, a method of treating cancer comprises administering an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) in combination with denosumab and one or more agents selected from the following: lenalidomide, ibrutinib, palbociclib, enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin, cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine, gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin, docetaxel, estramustine, paclitaxel, vinblastine, etoposide, irinotecan, teniposide, topotecan, prednisone, methylprednisolone, and dexamethasone. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is a polypeptide described herein (e.g., a fusion polypeptide comprising a SIRPα d1 domain variant and an Fc variant).

[0235] Exemplary cancers In some embodiments, the cancer treated by the methods provided herein is breast cancer, lung cancer, lung adenocarcinoma, squamous cell lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), head and neck cancer, mesothelioma, brain cancer, brain tumor, abdominal cancer, colon cancer, colorectal cancer, esophageal cancer, paraganglioma, gastrointestinal cancer, glioma, liver cancer, gastric cancer, oral cancer, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, kidney cancer, bladder cancer, urinary tract cancer, pancreatic cancer, retinoblastoma, cervical cancer, uterine cancer, Wilms tumor, multiple myeloma, skin cancer, lymphoma, leukemia, blood cancer, thyroid cancer, bone cancer, adenoid cystic tumor, chondrosarcoma, islet cell tumor, neuroendocrine tumor, prostate cancer, glioblastoma, endometrial carcinoma, endometrial cancer, leiomyosarcoma, gallbladder cancer, hepatocellular carcinoma, melanoma, or solid tumor.

[0236] In some embodiments, the cancer treated by the methods provided herein is a blood cancer. In some embodiments, the blood cancer is multiple myeloma, or leukemia, including but not limited to acute or chronic myeloid leukemia, acute or chronic lymphoblastic leukemia, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), hairy cell leukemia, chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), large granular lymphocyte (LGL) leukemia, plasmacytoma, blastic plasmacytoid dendritic cell neoplasm (BPDCN), B-cell prolymphocytic leukemia (B-PLL), T-cell prolymphocytic leukemia (T-PLL), multiple myeloma (MM), and non-Hodgkin lymphoma (such as diffuse large B-cell lymphoma (DLBCL)), Burkitt lymphoma, mantle cell lymphoma (MCL), peripheral T-cell lymphoma (PTCL), lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma (MZL), and follicular lymphoma (FL).

[0237] Method for treating leukemia In some embodiments, a method of treating leukemia (e.g., acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), hairy cell leukemia, chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), large granular lymphocytic (LGL) leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN), B-cell prolymphocytic leukemia (B-PLL), T-cell prolymphocytic leukemia (T-PLL), multiple myeloma (MM), and non-Hodgkin lymphoma (such as diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, mantle cell lymphoma (MCL), peripheral T-cell lymphoma (PTCL), lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma (MZL), and follicular lymphoma (FL)) in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), and (b) a Bcl2 inhibitor. In some embodiments, the Bcl2 inhibitor is venetoclax (also known as ABT-199), ABT-737, navitoclax (also known as ABT-263), BCL201, or AZD-0466. In some embodiments, the agent is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein). In some embodiments, the polypeptide (e.g., a fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85.In some embodiments, the Fc domain variant is: (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (where numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows the Kabat EU index). In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to the individual comprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., fusion polypeptide) forms a homodimer. In some embodiments, the polypeptide (e.g., fusion polypeptide) and a Bcl2 inhibitor (e.g., venetoclax) are administered simultaneously, in parallel, or sequentially.

[0238] Bcl2 inhibitors are a class of anti-cancer drugs thought to exert a cytotoxic effect by competing with pro-apoptotic Bcl2 and occupying the BH3 docking groove on the surface of anti-apoptotic family members. By binding to one or more Bcl2 family members, these inhibitors induce apoptosis and restore apoptosis in tumor cells by mimicking the activity of natural antagonists of BCL-2 and other related proteins.

[0239] Venetoclax (also known as GDC-0199, ABT-199, and RG7601) is an exemplary selective Bcl2 inhibitor used in the methods described herein. Venetoclax is experimental C 45 H 50Venetoclax is a pale to dark yellow solid with the chemical formula ClN7O7S and a molecular weight of 868.44 g / mol. Venetoclax has very low water solubility. Venetoclax is described chemically as 4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide and has the following chemical structure: [Chemical formula]

[0240] The CAS registration number of Venetoclax is 1257044-40-8. Venetoclax is administered orally and is sold under the trade names Venclexta and Venclyxto. Complete information regarding the preparation, formulation, dosage, and administration schedule of Venetoclax may be described in the package insert for each country (in the United States, see, for example, www.accessdata.fda.gov / drugsatfda_docs / label / 2016 / 208573s000lbl.pdf; in Europe, see, for example, www.ema.europa.eu / en / medicines / human / EPAR / venclyxto#product-information-section). In some embodiments, Venetoclax is administered according to the usage and frequency recommended in the package insert for each country.

[0241] ABT-737 is another exemplary selective Bcl2 inhibitor used in the methods described herein. ABT-737 inhibits both Bcl2 and Bcl-xL and experimental C 42 H 45It has ClN6O5S2 and a molecular weight of 813.43 g / mol. The CAS registration number of ABT-737 is 852-808-04-9. ABT-737 is described chemically as 4-{4-[(4’-chloro-2-biphenylyl)methyl]-1-piperazinyl}-N-[(4-{[(2R)-4-(dimethylamino)-1-(phenylsulfanyl)-2-butanoyl]amino}-3-nitrophenyl)sulfonyl]benzamide and has the following chemical structure: [Chemical formula]

[0242] Another exemplary selective Bcl2 inhibitor used in the methods described herein is navitoclax (also known as ABT-263). Navitoclax inhibits both Bcl2 and Bcl-xL, and has the empirical formula C 47 H 55 It has ClF3N5O6S3 and a molecular weight of 974.6 g / mol. The CAS registration number of navitoclax is 923564-51-6. ABT-737 is described chemically as 4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-morpholin-4-yl-1-phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide and has the following chemical structure. Additional details regarding navitoclax are described, for example, in Tse et al. (2008) Cancer Res. 68(9):3421-3429. [Chemical formula]

[0243] Another exemplary selective Bcl2 inhibitor used in the methods described herein is S55746 (also known as BCL201 and Servier-1). S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile shows no significant binding to MCL-1, and S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile shows no significant binding to MCL-1, BFL-1 (BCL2A1 / A1), and insufficient affinity for BCL-XL. S55746 is not cytotoxic to BCL-XL-dependent cells such as platelets (see, for example, Casara et al. (2008) Oncotarget. 9(28):29975-20088). S55746 is 43 H 42 has N4O6 and a molecular weight of 710.82 g / mol. The CAS registration number of S55746 is 1448584-12-0. S55746 is chemically described as (S)-N-(4-hydroxyphenyl)-3-(6-(3-(morpholinomethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)benzo[d][1,3]dioxol-5-yl)-N-phenyl-5,6,7,8-tetrahydroindolizine-1-carboxamide and has the following chemical structure:

Chemical formula

[0244] Therapeutic methods for solid tumors In some embodiments, provided is a method of treating a solid tumor in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), and (b) a platinum-based chemotherapeutic agent. In some embodiments, the solid tumor is colon cancer (e.g., colorectal cancer), lung cancer, head and neck cancer, esophageal cancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer, testicular cancer, brain tumor, mesothelioma, or neuroblastoma. In some embodiments, the platinum-based chemotherapeutic agent is carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and / or satraplatin. In some embodiments, the platinum-based chemotherapeutic agent is cisplatin. In some embodiments, the agent is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein). In some embodiments, the polypeptide (e.g., a fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index). In some embodiments, the polypeptide (e.g., a fusion polypeptide) administered to the individual comprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135.In some embodiments, the polypeptide (e.g., a fusion polypeptide) forms a homodimer. In some embodiments, the polypeptide (e.g., a fusion polypeptide) and a platinum-based chemotherapeutic agent (e.g., cisplatin) are administered simultaneously, in parallel, or sequentially.

[0245] Platinum agents (such as carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin, etc.) are widely used antitumor drugs that cause cross-linking of DNA as monoadducts, interstrand cross-links, intrastrand cross-links, or DNA-protein cross-links. Platinum agents typically act on the adjacent N-7 position of guanine to form 1,2 intrastrand cross-links (Poklar et al. (1996). Proc. Natl. Acad. Sci. U.S.A. 93(15):7606-11; Rudd et al. (1995). Cancer Chemother. Pharmacol. 35(4):323-6). The resulting cross-links inhibit DNA repair and / or DNA synthesis in cancer cells.

[0246] Cisplatin is an exemplary platinum coordination compound used in the methods described herein. The chemical name of cisplatin is dichloroplatinum diammoniate, and cisplatin has the following structural formula:

Chemical formula

[0247] Cisplatin is an inorganic water-soluble platinum complex with a molecular formula of Pt(NH3)2Cl2 and a molecular weight of 300.046. After undergoing hydrolysis, cisplatin reacts with DNA to form both intrastrand and interstrand cross-links. These cross-links are thought to impair DNA replication and transcription. The cytotoxicity of cisplatin correlates with cell arrest in the G2 phase of the cell cycle. Cisplatin, assigned the CAS Registry Number 15663-27-1, is commercially available under the names PLATINOL®, PLATINOL®-AQ, CDDP, CISPLAN, CISPLAT, PLATIKEM, PLATIONCO, PRACTICIS, PLATICIS, BLASTOLEM, CISMAX, CISPLAN, CISPLATINUM, CISTEEN, DUPLAT, KEMOPLAT, ONCOPLATIN-AQ, PLATINEX, PLATIN, TEVAPLATIN, etc. Complete information regarding the preparation, formulation, dosage, and administration schedule of cisplatin can be described in the package insert of each country (in the United States, for example, see www.accessdata.fda.gov / drugsatfda_docs / label / 2011 / 018057s080lbl.pdf and www.accessdata.fda.gov / drugsatfda_docs / label / 2015 / 018057s083lbl.pdf). In some embodiments, cisplatin is administered according to the usage and frequency recommended in the package insert of each country.

[0248] Carboplatin is another exemplary platinum coordination compound used in the methods described herein. The chemical name of carboplatin is platinum, diammin[1,1-cyclobutanedicarboxylato(2-)-O,O]-, (SP-4-2), and carboplatin has the following structural formula: [Chemical formula]

[0249] Carboplatin has a molecular formula of C6H 12It is a water-soluble platinum complex that is N2O4Pt and has a molecular weight of 373.26. Carboplatin is assigned the CAS registration number 41575-94-4, and its mechanism of action is the same as that of cisplatin. Carboplatin is typically more commonly prescribed than cisplatin. Carboplatin is commercially available under the names PARAPLATIN (registered trademark), BLASTOCARB (registered trademark), BLASTOPLATIN (registered trademark), CARBOKEM (registered trademark), CARBOMAX (registered trademark), PARAPLATIN (registered trademark), CARBOPA (registered trademark), KARPLAT (registered trademark), etc. Complete information regarding the preparation, formulation, dosage, and dosing schedule of carboplatin can be described in the package inserts of each country (in the United States, for example, see www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2010 / 020452s005lbl(dot)pdf and www(dot)accessdata.fda(dot)gov / drugsatfda_docs / label / 2012 / 077139Orig1s016lbl(dot)pdf). In some embodiments, carboplatin is administered according to the usage and frequency recommended in the package inserts of each country.

[0250] In some embodiments, provided is a method of treating a solid tumor in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-HER2 antibody, and (c) an anti-PDL1 antibody. In some embodiments, the anti-HER2 antibody is trastuzumab (CAS Registry Number: 180288-69-1). In some embodiments, the anti-PDL1 antibody is atezolizumab (CAS Registry Number 1380723-44-3), avelumab (CAS Registry Number 1537032-82-8), or durvalumab (CAS Registry Number 1428935-60-7). In some embodiments, the agent is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein). In some embodiments, the polypeptide (e.g., the fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index). In some embodiments, the polypeptide (e.g., the fusion polypeptide) administered to the individual comprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., the fusion polypeptide) forms a homodimer.In some embodiments, the polypeptide (e.g., a fusion polypeptide), anti-HER2 antibody, anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody) are administered simultaneously, concurrently, or sequentially. In some embodiments, the solid tumor is a colon cancer, lung cancer, head and neck cancer, esophageal cancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer, testicular cancer, endometrial cancer, liver cancer, gastric cancer, gastroesophageal junction cancer, brain tumor, mesothelioma, or neuroblastoma. In some embodiments, the solid tumor is HER2. + is a solid tumor. In some embodiments, the solid tumor is a colon cancer (e.g., HER2 + colon cancer).

[0251] Methods for treating gastric cancer or gastroesophageal junction (GEJ) cancer In some embodiments, provided is a method of treating gastric cancer or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-HER2 antibody, (c) an anti-VEGFR2 antibody, and (d) paclitaxel. In some embodiments, the anti-HER2 antibody is trastuzumab (CAS Registry Number: 180288-69-1). In some embodiments, the anti-VEGFR2 antibody is ramucirumab (CAS Registry Number: 947687-13-0). In some embodiments, the agent is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein). In some embodiments, the polypeptide (e.g., the fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index). In some embodiments, the polypeptide (e.g., the fusion polypeptide) administered to the individual comprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., the fusion polypeptide) forms a homodimer.In some embodiments, the polypeptide (e.g., a fusion polypeptide), anti-HER2 antibody, anti-VEGFR2 antibody, and paclitaxel are administered simultaneously, concurrently, or sequentially. In some embodiments, the polypeptide (e.g., a fusion polypeptide) is administered to an individual at a dose of 10 mg / kg once a week or 15 mg / kg once a week. In some embodiments, the individual being treated has gastric or GEJ adenocarcinoma. In some embodiments, the individual being treated is HER2. + Gastric cancer or HER2 + Has GEJ cancer (e.g., HER2-overexpressing gastric or GEJ cancer). In some embodiments, HER2 + Gastric cancer or HER2 + The GEJ cancer is progressive and / or metastatic. In some embodiments, the individual being treated has gastric or GEJ cancer that progressed during or after previous treatment(s) including an anti-HER2 antibody (e.g., trastuzumab). In some embodiments, the individual being treated has gastric or GEJ cancer that progressed during or after previous treatment(s) including an anti-HER2 antibody (e.g., trastuzumab) and a fluoropyrimidine. In some embodiments, the individual being treated has gastric or GEJ cancer that progressed during or after previous treatment(s) including an anti-HER2 antibody (e.g., trastuzumab) and a platinum-based chemotherapeutic agent. In some embodiments, the individual being treated has gastric or GEJ cancer (e.g., HER2 that progressed during or after previous treatment(s) including an anti-HER2 antibody (e.g., trastuzumab) and / or a fluoropyrimidine, and / or a platinum-based chemotherapeutic agent +has gastric cancer or GEJ cancer. In some embodiments, the individual has not responded (e.g., has relapsed or not responded) to prior treatment with an anti-HER2 antibody, an anti-HER2 antibody and a fluoropyrimidine, or an anti-HER2 antibody and a platinum-based chemotherapeutic agent. In some embodiments, the fluoropyrimidine was fluorouracil (also known as 5-fluorouracil). In some embodiments, treatment with the polypeptide, anti-HER2 antibody, anti-VEGFR2 antibody, and paclitaxel does not result in adverse effects. In some embodiments, treatment with the polypeptide, anti-HER2 antibody, anti-VEGFR2 antibody, and paclitaxel results in only mild adverse effects.

[0252] In some embodiments, a method of treating gastric cancer or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-PD-1 antibody (e.g., an anti-PD-1 antagonist antibody), (c) an anti-HER2 antibody, (d) 5-fluorouracil, and (e) a platinum-based chemotherapeutic agent. In some embodiments, a method of treating gastric cancer or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-PD-1 antibody (e.g., an anti-PD-1 antagonist antibody), (c) an anti-HER2 antibody, (d) capecitabine, and (e) a platinum-based chemotherapeutic agent. In some embodiments, the anti-PD-1 antibody is pembrolizumab (CAS Registry Number: 1374853-91-4). In some embodiments, the anti-HER2 antibody is trastuzumab (CAS Registry Number: 180288-69-1). In some embodiments, the platinum-based chemotherapeutic agent is cisplatin. In some embodiments, the agent is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein). In some embodiments, the polypeptide (e.g., a fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85.In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (where numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows the Kabat EU index). In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to the individual comprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., fusion polypeptide) forms a homodimer. In some embodiments, the polypeptide (e.g., fusion polypeptide), anti-PD-1 antibody, anti-HER2 antibody, 5-fluorouracil, and platinum-based chemotherapeutic agent are administered simultaneously, concurrently, or sequentially. In some embodiments, the polypeptide (e.g., fusion polypeptide), anti-PD-1 antibody, anti-HER2 antibody, capecitabine, and platinum-based chemotherapeutic agent are administered simultaneously, concurrently, or sequentially. In some embodiments, the individual being treated has HER2-overexpressing gastric cancer or HER2-overexpressing GEJ cancer. In some embodiments, the gastric cancer or GEJ cancer is progressive and / or metastatic. In some embodiments, the individual has not received prior treatment for gastric cancer or GEJ cancer.

[0253] Method for treating head and neck cancer In some embodiments, a method of treating head and neck cancer (e.g., head and neck squamous cell carcinoma or HNSCC) in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) a PD-1 inhibitor, (c) an antimetabolite, and (d) a platinum-based agent. In some embodiments, the PD-1 inhibitor is a small molecule inhibitor, an antisense nucleotide, or a peptide. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is pembrolizumab, nivolumab, pidilizumab, semipramab, or BMS-936559. In some embodiments, the anti-PD-1 antibody is pembrolizumab (CAS Registry Number: 1374853-91-4). In some embodiments, the antimetabolite is 5-fluorouracil, 6-mercaptopurine, capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxycarbamide, methotrexate, pemetrexed, fotemustine. In some embodiments, the antimetabolite is 5-fluorouracil. In some embodiments, the platinum-based chemotherapeutic agent is carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin. In some embodiments, the platinum-based chemotherapeutic agent is cisplatin or carboplatin. In some embodiments, the agent is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein). In some embodiments, the polypeptide (e.g., a fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85.In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (where numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows the Kabat EU index). In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to an individual comprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., fusion polypeptide) forms a homodimer. In some embodiments, the polypeptide (e.g., fusion polypeptide), a PD-1 inhibitor (e.g., anti-PD-1 antibody, e.g., pembrolizumab), an antimetabolite (e.g., 5-fluorouracil), and a platinum-based chemotherapeutic agent (e.g., cisplatin or carboplatin) are administered simultaneously, concurrently, or sequentially. In some embodiments, the polypeptide (e.g., fusion polypeptide) is administered to an individual at a dose of 10 mg / kg once a week or 15 mg / kg once a week. In some embodiments, the individual being treated has HNSCC. In some embodiments, the HNSCC is progressive and / or metastatic HNSCC. In some embodiments, the HNSCC is inoperable and / or recurrent. In some embodiments, the individual has not previously been treated for head and neck cancer (e.g., HNSCC). In some embodiments, treatment with the polypeptide, a PD-1 inhibitor (e.g., pembrolizumab), an antimetabolite (e.g., 5-fluorouracil), and a platinum-based chemotherapeutic agent (e.g., cisplatin or carboplatin) does not result in adverse effects.In some embodiments, treatment with a polypeptide, a PD-1 inhibitor (e.g., pembrolizumab), an antimetabolite (e.g., 5-fluorouracil), and a platinum-based chemotherapeutic agent (e.g., cisplatin or carboplatin) results in only mild adverse effects.

[0254] Combination cancer therapy comprising an anti-TROP2 antibody In some embodiments, provided is a method of treating cancer in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), and (b) an anti-TROP2 antibody. In some embodiments, the anti-TROP2 antibody is RS7 as described in U.S. Patent No. 10,179,171, the content of which is incorporated herein by reference in its entirety. In some embodiments, the anti-TROP2 antibody is conjugated to a drug (i.e., an antibody-drug conjugate or “ADC”). In some embodiments, the anti-TROP2 ADC is sacituzumab govitecan (also known as hRS7-SN38 or IMMU-132), which is described in US2017 / 0281791, the content of which is incorporated herein by reference in its entirety. In some embodiments, the agent is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein). In some embodiments, the polypeptide (e.g., a fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index).In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to the individual comprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., fusion polypeptide) forms a homodimer. In some embodiments, the polypeptide (e.g., fusion polypeptide) and the anti-TROP2 antibody are administered simultaneously, in parallel, or sequentially. In some embodiments, the cancer is a solid tumor, gastric cancer, nasopharyngeal cancer, gallbladder cancer, cervical cancer, extranodal NK / T cell lymphoma, lung cancer, laryngeal squamous cell carcinoma, colon cancer, hilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrioid endometrial cancer, or ovarian cancer. In some embodiments, the cancer is characterized by overexpression of TROP2. In some embodiments, the cancer is not characterized by overexpression of TROP2.

[0255] Method for increasing phagocytosis of target cells In some embodiments, provided is a method of increasing the phagocytosis of target cells (e.g., cancer cells), the method comprising contacting the target cells with (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), and (b) an anti-TROP2 antibody. In some embodiments, the anti-TROP2 antibody is RS7 as described in U.S. Patent No. 10,179,171, the content of which is incorporated herein by reference in its entirety. In some embodiments, the anti-TROP2 antibody is conjugated to a drug (i.e., an antibody-drug conjugate or “ADC”). In some embodiments, the anti-TROP2 ADC is sacituzumab govitecan (also known as hRS7-SN38 or IMMU-132), which is described in US2017 / 0281791, the content of which is incorporated herein by reference in its entirety. In some embodiments, the agent is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein). In some embodiments, the polypeptide (e.g., a fusion polypeptide) comprises a SIRPα D1 domain variant having the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index).In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to the individual comprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., fusion polypeptide) forms a homodimer. In some embodiments, the target cell is a cancer cell. In some embodiments, the cancer cell is a solid tumor cell, gastric cancer cell, nasopharyngeal cancer cell, gallbladder cancer cell, cervical cancer cell, extranodal NK / T cell lymphoma cell, lung cancer cell, laryngeal squamous cell carcinoma cell, colon cancer cell, hilar cholangiocarcinoma cell, pancreatic cancer cell, oral squamous cell carcinoma cell, endometrioid endometrial cancer cell, or ovarian cancer cell.

[0256] In some embodiments, provided is a method of increasing the phagocytosis of target cells, the method comprising contacting the target cells with (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), and (b) a second agent that can enhance phagocytosis. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein). In some embodiments, the polypeptide (e.g., a fusion polypeptide) comprises a SIRPα D1 domain variant having the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index). In some embodiments, the polypeptide (e.g., a fusion polypeptide) administered to an individual comprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., a fusion polypeptide) forms a homodimer. In some embodiments, the second agent enhances phagocytosis, for example, by blocking the "don't eat me" signal.Exemplary agents include, but are not limited to, for example, anti-LILRB2 antibody, anti-LILRB1 antibody, anti-SIGLEC-10 antibody, anti-CD24 antibody, anti-SIRPα antibody, anti-PD1 antibody (e.g., anti-PD1 antagonist antibody), and anti-PD-L1 antibody (e.g., anti-PD-L1 antagonist antibody). In some embodiments, the second agent enhances the phagocytosis, for example, by enhancing the "eat me" signal. Exemplary agents include, but are not limited to, for example, BTK activator, TLR agonist, an agent that promotes the interaction between Mac-1 and SLAMF7, and an agent that promotes the interaction between calreticulin and LRP1. Additional exemplary agents that enhance phagocytosis include, but are not limited to, for example, an agent that regulates podosome adhesion, an agent that regulates the expression level of lamin A, an activator of SHP-1 phosphatase activity, and an activator of myosin IIa assembly. In some embodiments, the method comprises contacting the target cell with (a) a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein) and (b) an anti-LILBR2 antibody, an anti-CD24 antibody, or an anti-SIGLEC-10 antibody. In some embodiments, the method comprises contacting the target cell with (a) a fusion polypeptide and (b) a BTK activator. In some embodiments, the method comprises contacting the target cell with (a) a fusion polypeptide and (b) a TLR agonist.

[0257] In some embodiments, the method comprises contacting a target cell with (a) a polypeptide (e.g., a fusion polypeptide) comprising an SIRPα D1 domain variant (e.g., an SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein), and (b) two or more agents capable of enhancing phagocytosis (e.g., but not limited to, two or more agents described herein). In some embodiments, the method comprises contacting a target cell with (a) a polypeptide (e.g., a fusion polypeptide) comprising an SIRPα D1 domain variant (e.g., an SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein), (b) an anti-LILBR2 antibody, an anti-CD24 antibody, or an anti-SIGLEC-10 antibody, and (c) an anti-PD1 antibody (e.g., an anti-PD-1 antagonist antibody) or an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody). In some embodiments, the method comprises contacting a target cell with (a) a fusion polypeptide, (b) an anti-LILBR2 antibody, and (c) an anti-PD1 antibody (e.g., an anti-PD-1 antagonist antibody). In some embodiments, the method comprises contacting a target cell with (a) a fusion polypeptide, (b) an anti-LILBR2 antibody, and (c) an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody).

[0258] In some embodiments, the contacting is performed in vitro. In some embodiments, the contacting is performed in vivo. In some embodiments, the target cells are cancer cells. In some embodiments, the target cells are contacted with (a) a polypeptide comprising an SIRPα D1 domain variant (e.g., an SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein), and (b) one or more agents capable of enhancing phagocytosis, such that the phagocytosis of the target cells is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99% or more as compared to (i.e., in the absence of) the polypeptide comprising an SIRPα D1 domain variant (e.g., an SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein).

[0259] Kits and Manufactured Articles In another embodiment of the present invention, there is provided a manufactured product or kit comprising a polypeptide (e.g., a fusion polypeptide described herein) comprising an SIRPα D1 domain variant and an Fc domain variant. In some embodiments, the SIRPα D1 domain variant comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 81 and SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index). In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 135 or SEQ ID NO: 136. In some embodiments, the kit or manufactured product is for use in accordance with the therapeutic methods provided herein.

[0260] In some embodiments, the kit or article of manufacture further comprises a BCL2 inhibitor. In some embodiments, the BCL2 inhibitor is venetoclax. In some embodiments, the kit includes instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with a BCL2 inhibitor (e.g., venetoclax) to treat or delay the progression of cancer (e.g., leukemia, including but not limited to acute or chronic lymphoblastic leukemia, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), hairy cell leukemia, chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), large granular lymphocytic (LGL) leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN), B-cell prolymphocytic leukemia (B-PLL), T-cell prolymphocytic leukemia (T-PLL), multiple myeloma (MM), and non-Hodgkin lymphoma (such as diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, mantle cell lymphoma (MCL), peripheral T-cell lymphoma (PTCL), lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma (MZL), and follicular lymphoma (FL), etc.) in an individual (a human individual).

[0261] In some embodiments, the kit or product further comprises a platinum-based chemotherapeutic agent. In some embodiments, the platinum-based chemotherapeutic agent is carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin. In some embodiments, the kit comprises a package insert or label including instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with a platinum-based chemotherapeutic agent (e.g., cisplatin) to treat or delay the progression of solid tumors (e.g., colon cancer, colon carcinoma, lung cancer, head and neck cancer, esophageal cancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer, testicular cancer, endometrial cancer, liver cancer, gastric cancer, brain tumor, mesothelioma, or neuroblastoma) in an individual (such as a human individual).

[0262] In some embodiments, the kit or product further comprises an anti-HER2 antibody (e.g., trastuzumab), and a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody such as atezolizumab, avelumab, or durvalumab). In some embodiments, the kit includes a package insert or label including instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-HER2 antibody (e.g., trastuzumab) and a PD-L1 inhibitor (e.g., atezolizumab, avelumab, or durvalumab) to treat or delay the progression of cancer (e.g., solid tumors) in an individual (such as a human individual). In some embodiments, the cancer (e.g., solid tumors) is colon cancer, lung cancer, head and neck cancer, esophageal cancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer, testicular cancer, endometrial cancer, liver cancer, gastric cancer, gastroesophageal junction cancer, brain tumor, mesothelioma, or neuroblastoma. In some embodiments, the cancer (e.g., solid tumors) is HER2 + cancer. In some embodiments, the cancer is colon cancer (e.g., HER2 + colon cancer).

[0263] In some embodiments, the kit or product further comprises an anti-HER2 antibody (e.g., trastuzumab), an anti-VEGFR2 antibody (e.g., ramucirumab), and paclitaxel. In some embodiments, the kit includes a package insert or label containing instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-HER2 antibody (e.g., trastuzumab), an anti-VEGFR2 antibody (e.g., ramucirumab), and paclitaxel for treating or delaying the progression of gastric cancer or gastroesophageal junction (GEJ) cancer in an individual (such as a human individual), for example, by the methods described herein.

[0264] In some embodiments, the kit or product further comprises an anti-HER2 antibody (e.g., trastuzumab), a PD-1 inhibitor (e.g., an anti-PD-1 antibody such as pembrolizumab), 5-fluorouracil, and a platinum-based agent (e.g., cisplatin or carboplatin). In some embodiments, the kit includes a package insert or label containing instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-HER2 antibody (e.g., trastuzumab), a PD-1 inhibitor (e.g., pembrolizumab), 5-fluorouracil, and a platinum-based agent (e.g., cisplatin or carboplatin) for treating or delaying the progression of gastric cancer or gastroesophageal junction (GEJ) cancer in an individual (such as a human individual). In some embodiments, the kit or product further comprises an anti-HER2 antibody (e.g., trastuzumab), a PD-1 inhibitor (e.g., an anti-PD-1 antibody such as pembrolizumab), capecitabine, and a platinum-based agent (e.g., cisplatin or carboplatin). In some embodiments, the kit includes a package insert or label containing instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-HER2 antibody (e.g., trastuzumab), a PD-1 inhibitor (e.g., pembrolizumab), capecitabine, and a platinum-based agent (e.g., cisplatin or carboplatin) for treating or delaying the progression of gastric cancer or gastroesophageal junction (GEJ) cancer in an individual (such as a human individual).

[0265] In some embodiments, the kit or product further comprises a PD-1 inhibitor (e.g., an anti-PD-1 antibody such as pembrolizumab, nivolumab, pidilizumab, semaprilumab, BMS936559), an antimetabolite (e.g., 5-fluorouracil, 6-mercaptopurine, capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxycarbamide, methotrexate, pemetrexed, fotemustine), and a platinum-based agent (e.g., cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin). In some embodiments, the kit includes, for example, a PD-1 inhibitor (e.g., pembrolizumab, nivolumab, pidilizumab, semaprilumab, or BMS936559), an antimetabolite (e.g., 5-fluorouracil, 6-mercaptopurine, capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxycarbamide, methotrexate, pemetrexed, fotemustine), and a platinum-based agent (e.g., cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin) in combination for use in treating or delaying the progression of head and neck cancer (e.g., head and neck squamous cell carcinoma) in an individual (e.g., a human individual) according to the methods provided herein. The kit includes a package insert or label containing instructions for using a polypeptide (e.g., a fusion polypeptide).

[0266] In some embodiments, the kit or product further comprises a therapeutic anti-TROP2 antibody. In some embodiments, the anti-TROP2 antibody is RS7 (see, e.g., U.S. Patent No. 10,179,171) or sacituzumab govitecan. In some embodiments, the kit includes instructions for using a polypeptide (e.g., a fusion polypeptide) in an individual (such as a human individual) with respect to TROP2 +Instructions for use, including package inserts or labels, are included for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-TROP2 antibody (e.g., cisplatin) to treat cancer (e.g., solid tumors, gastric cancer, nasopharyngeal cancer, gallbladder cancer, cervical cancer, extranodal NK / T cell lymphoma, lung cancer, laryngeal squamous cell carcinoma, colon cancer, hilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrioid endometrial cancer, or ovarian cancer) or delay its progression.

[0267] In some embodiments, the polypeptide (e.g., a fusion polypeptide) and one or more additional anti-cancer agents (e.g., as outlined in the above embodiments) are provided together in a kit. In some embodiments, the polypeptide (e.g., a fusion polypeptide) and one or more additional anti-cancer agents are provided in the same container or separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The container can be formed from various materials, such as glass, plastic (such as polyvinyl chloride or polyolefin), or alloys (such as stainless steel or Hastelloy). In some embodiments, the container holds the formulation, and the label on the container or a label associated with the container can indicate instructions for use. The manufactured product or kit can further include other buffers, diluents, filters, needles, syringes, and other materials desirable from a commercial and user perspective, including package inserts with instructions for use. In some embodiments, the manufactured product further includes one or more of another agent (e.g., a chemotherapeutic agent and an anti-tumor agent, a therapeutic antibody, etc.). Suitable containers for the one or more agents include, for example, bottles, vials, bags, and syringes.

[0268] This specification is considered to be sufficient to enable one of ordinary skill in the art to practice the invention. In addition to the modifications shown and described herein, various modifications of the invention will become apparent to those of ordinary skill in the art from the foregoing description and are within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Examples

[0269] The present disclosure will be more fully understood by reference to the following examples. However, the examples should not be construed as limiting the scope of the present disclosure. The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes suggested to those skilled in the art will be understood to be within the spirit and scope of this application, as well as within the scope of the appended claims.

[0270] Example 1A: Antitumor activity of Drug A combined with Venetoclax in an acute leukemia model. In this example, in an RS4;11 xenograft model, the antitumor activity of Drug A, an exemplary polypeptide containing the SIRPα d1 domain variant and the Fc variant, was evaluated in combination with Venetoclax.

[0271] Materials and Methods RS4;11 Xenograft Model RS4;11 cells (described in Stong et al. (1985) Blood. 65(1):21-31) were injected into the right flank of NOD-SCID female mice at a concentration of 5×10 6 cells / mouse using a 1:1 ratio of Matrigel (Corning) to RPMI 1640. The average size of all tumors was 190 mm 3The tumors were monitored until they reached . Mice were randomized into PBS control, Venetoclax (Selleckchem), Drug A, and Venetoclax / Drug A combination cohorts, with 10 mice per cohort. The formulation of Venetoclax was in the ratio of DMSO:ethanol:Cremophor EL:dextrose-containing water 5% (D5W) at a volume ratio of 2.5:5:10:20:67.5. Venetoclax-treated mice were force-fed a total of 2 doses of 250 μg of Venetoclax at 3-day intervals. Drug A-treated mice were administered IP at 10 mg / kg, a total of 4 times at 3 - 4-day intervals. Venetoclax / Drug A-treated mice were force-fed a total of 2 doses of 250 μg of Venetoclax at 3-day intervals, and 1 day after the Venetoclax administration, Drug A at 10 mg / kg was administered a total of 4 times at 3 - 4-day intervals. Tumors were measured two-dimensionally using calipers, and tumor volume was calculated as length × width × width × 0.5: the length was the larger of the two measurements.

[0272] Results Single-agent Venetoclax inhibited tumor growth (see Figure 1A), while single-agent Drug A had no perceptible effect on tumor growth. The combination of Venetoclax and Drug A significantly inhibited tumor growth more than Venetoclax alone (see Figure 1A). On day 41, 1 out of 10 mice treated with Venetoclax alone had no tumor ("TF"), while 6 out of 10 mice treated with the Venetoclax / Drug A combination were TF (Figure 1A).

[0273] Next, Venetoclax-treated mice (n = 10) were divided into two groups (n = 5 / group) and treated with (a) re-treatment with single-agent Venetoclax on day 45 or (b) a combination of Venetoclax and Drug A (administered on day 45) and Drug A (administered on day 46). As shown in Figure 1B, in mice previously administered Venetoclax, treatment with Venetoclax in combination with Drug A significantly suppressed tumor growth more than re-treatment with Venetoclax alone. The average tumor volume on day 65 in mice re-treated with Venetoclax was approximately 1685 mm 3However, the average tumor volume on day 65 of the mice treated with the combination was about 970 mm 3 Mice that showed tumor regression when treated with single-agent venetoclax received a single dose of venetoclax on day 45. In particular, tumor regrowth was observed in this mouse.

[0274] Example 1B: Effect of Drug A combined with venetoclax on phagocytosis by macrophages in an in vitro model In this example, the effects of Drug A alone, venetoclax alone, and Drug A combined with venetoclax on the phagocytosis of HL60 and OCIAML3 human acute myeloid leukemia cells by macrophages were evaluated in an in vitro assay.

[0275] Materials and methods Induction and culture of human monocyte-derived macrophages for phagocytosis CD14 + Monocytes were purified by negative selection using the Classical Monocyte Isolation Kit, human (Miltenyi Biotec) and LS column (Miltenyi Biotec) according to the manufacturer's protocol. CD14 + Monocytes were seeded at 6 million cells per dish in 25 mL of medium composed of RPMI complete medium supplemented with 50 ng / mL M-CSF (Miltenyi Biotec), 10% human FBS serum (Thermo Fisher Scientific), 1% penicillin / streptomycin, and 1% GlutaMAX in 150 mm tissue culture dishes (Corning). The cells were cultured for 7 - 11 days.

[0276] In vitro phagocytosis assay HL60 and OCI-AML3 cells were washed once with PBS and labeled with the Celltrace CFSE Cell Proliferation Kit (Thermo Fisher Scientific) in suspension containing 300 nM CFSE (carboxyfluorescein succinimidyl ester) according to the manufacturer's instructions, and then resuspended in RPMI complete medium. The target cells were incubated overnight with two-fold serial dilutions of venetoclax at 39 nM to 2.5 μM in RPMI complete medium. Prior to incubation with macrophages, the cells were resuspended in RPMI. Macrophages were detached from the culture plates by washing once with PBS and incubating at 37 °C for 20 min with TrypLE Select. The cells were removed with a cell scraper (Corning), washed with PBS, and resuspended in RPMI.

[0277] CFSE-labeled target cells treated with benetoclax for 48 hours were rotated and added to an ultra-low attachment U-bottom 96-well plate (Corning) at 100,000 cells per well. Next, drug A was added. The plate was incubated at 37°C for 30 minutes in a humidified incubator containing 5% carbon dioxide, and then 50,000 macrophages were added. The plate was incubated at 37°C for 2 hours in a humidified incubator containing 5% carbon dioxide. The cells were pelleted by centrifugation at 400×g for 5 minutes and stained with Fixable Viability Dye eFluor 780 (ebioscience) diluted 1:4000 in PBS at 4°C for 30 minutes. The cells were washed with FACS buffer (PBS containing 0.5% BSA) and stained with FACS buffer containing human FcR blocking reagent (Miltenyi Biotec), BV421 anti-CD33 (Biolegend), APC anti-CD14 (Biolegend), and PE-Cyanine7 anti-CD11b (Invitrogen) at 4°C for 45 minutes. The cells were washed twice with FACS buffer and fixed overnight at 4°C with 0.5% paraformaldehyde diluted in PBS. The cells were analyzed on a FACS Canto II (BD Biosciences), and then data analysis was performed using Flowjo 10.6.1 (Becton Dickinson & Company). Dead cells were excluded by gating the e780-negative population. Macrophages were identified as cells positive for the lineage markers CD33, CD11b, and CD14. Among this population, macrophages that had phagocytosed tumor cells were identified as CFSE-positive cells.

[0278] Results Briefly described, HL60 cells and OCI-AML3 cells (i.e., "target cells") were labeled with CFSE (carboxyfluorescein succinimidyl ester) and treated with venetoclax for 48 hours. Next, the target cells were rotated and added to the wells of a 96-well plate at 100,000 cells per well. Next, drug A was added. Untreated control target cells, as well as control target cells treated with venetoclax alone or drug A alone, were prepared in parallel. Macrophages were added to the wells and the plate was incubated at 37°C for 2 hours. Macrophage cells were pelleted, stained, and analyzed by flow cytometry. Dead cells were excluded by gating the e780-negative population. Macrophages were identified as cells positive for the lineage markers CD33, CD11b, and CD14. Among this population, macrophages that phagocytosed tumor cells were identified as CFSE-positive cells.

[0279] As shown in FIG. 5A, venetoclax as a single agent stimulated macrophage-mediated phagocytosis of HL60 cells, while drug A as a single agent had little effect on phagocytosis. (Compare cells treated with drug A to untreated cells). The combination of 20 nM drug A and 125 nM venetoclax stimulated phagocytosis of HL60 cells by macrophages to a greater extent than either drug A alone or venetoclax alone. Similar results were observed in OCI-AML3 cells using 20 nM drug A and 1 μM venetoclax. See FIG. 5B.

[0280] Example 2: Antitumor activity of drug A in combination with cisplatin in a colon cancer model. In this example, the antitumor activity of drug A in combination with cisplatin was evaluated in a CT26 syngeneic mouse colon cancer model. See, for example, Mosely et al. (2016) “Rational Selection of Syngeneic Preclinical Tumor Models for Immunotherapeutic Drug Discovery” Cancer Immunol Res. 5(1):29-41.

[0281] Materials and Methods CT26 Syngeneic Model CT26 cells (see Wang et al. (1995) J. Immunol. 154:4685-4692) were injected into the right flank of BALB / c female mice at a concentration of 5×10 5 cells per mouse in RPMI 1640. Tumors were monitored until the average size of all tumors reached 65-70 mm 3 . Mice were randomized into PBS control, cisplatin (Selleckchem), drug A, and cisplatin / drug A combination cohorts, and 5-10 mice per cohort were used. Drug A was administered intraperitoneally (IP) twice at a dose of 30 mg / kg (“mpk”). Two 30 mpk doses were given 10 days apart. Cisplatin was administered IP according to either of two regimens: once at a dose of 10 mpk or twice at a dose of 5 mpk. The cisplatin 5 mpk dose was given twice 10 days apart. Mice receiving both cisplatin and the drug were administered cisplatin (IP) according to one of the above regimens and drug A as described above. Mice receiving combination therapy were administered drug A 1 day after cisplatin. Tumors were measured two-dimensionally using calipers, and tumor volume was calculated as length × width × width × 0.5: the length being the larger of the two measurements.

[0282] Results As shown in Figure 2A, on the 20th day, tumor growth in mice treated with single-agent cisplatin (two 5 mpk doses given at 10-day intervals each) was somewhat inhibited, but drug A had no perceivable effect on tumor growth. Treatment with the combination of cisplatin and drug A delayed the growth of CT26 tumors in mice to a greater extent than either drug alone. Furthermore, as shown in Figure 2B, mice treated with cisplatin in combination with drug A gained more weight during the course of treatment than mice administered cisplatin alone. Additionally, in each of the PBS control, cisplatin, and drug A treatment groups, only 10% of the mice were found to have a tumor volume < 500 mm 3 However, in the cisplatin + drug A treatment group, 33% of the mice had a tumor volume < 500 mm 3 .

[0283] Similar results were observed in groups treated with a single 10 mpk dose of cisplatin alone or in combination with drug A. Treatment with the combination of cisplatin and drug A delayed the growth of CT26 tumors in mice to a greater extent than either drug A alone. See Figure 2C. Mice treated with cisplatin alone or in combination with drug A showed similar weight changes (*p < 0.0106 and **p < 0.0021, with two-sided t-tests performed on days 24 and 27 respectively between the cisplatin and drug A + cisplatin treatment groups). See Figure 2D.

[0284] Example 3: Antitumor activity of drug A in combination with an anti-TROP2 antibody The DLD-1 cells were washed twice with 20 ml of PBS and incubated with 10 ml of TRYPLE (trademark) Select (Gibco) cell dissociation enzyme at 37 °C for 10 minutes to detach the cells from the culture plate. Next, the detached cells were centrifuged, washed with PBS, and resuspended in the medium. The cells were labeled with the fluorescent label attached to the CELLTRACE (trademark) CFSE Cell Proliferation kit (Thermo Fisher) according to the manufacturer's instructions and resuspended in IMDM (Iscove's Modified Dulbecco's Medium). The macrophages were washed twice with 20 ml of PBS and detached from the culture plate by incubating with 10 ml of TRYPLE (trademark) Select (Gibco) cell dissociation enzyme at 37 °C for 20 minutes. The cells were removed with a cell scraper (Corning), washed with PBS, and resuspended in IMDM.

[0285] The phagocytosis assay was assembled in an ultra-low attachment U-bottom 96-well plate (Corning) containing 100,000 DLD-1, 50,000 macrophages, 5-fold serial dilutions of drug A at 100 nM to 6.4 pM or a negative control antibody, and 0.01 μg / ml of anti-TROP2 antibody. The plate was incubated at 37 °C for 2 hours in a humidified incubator containing 5% carbon dioxide. Next, the cells were pelleted by centrifugation at 400 × g for 5 minutes and washed with 250 μl of FACS buffer. The macrophages were stained on ice for 15 minutes with 50 μl of FACS buffer containing 10 μl of human FcR blocking reagent (Miltenyi Biotec), 0.5 μl of anti-CD33 Ab conjugated to BV421 (Biolegend), and 0.5 μl of anti-CD206 conjugated to allophycocyanin-Cy7 (Biolegend). Next, the cells were washed with 200 μl of FACS buffer, washed with 250 μl of PBS, and stained with 50 μl of Fixable Viability Dye EFLUOR™ 506 (ebioscience) viability dye diluted 1:1000 in PBS for 30 minutes on ice. Next, the cells were washed twice with 250 μl of FACS buffer and fixed overnight with 0.5% paraformaldehyde. The fixed cells were analyzed using a FACS CANTO II™ (BD Biosciences) fluorescence-activated cell sorting analyzer, and then data analysis was performed using FlowJo 10.7 (Treestar) flow cytometry software. Dead cells were excluded by gating the e506-negative population. Macrophages that had phagocytosed tumor cells were identified as cells positive for CD33, CD206, and CFSE (i.e., carboxyfluorescein succinimidyl ester).

[0286] Enhanced phagocytosis of CFSE-labeled DLD-1 tumor cells by human monocyte-derived macrophages in the presence of Drug A combined with anti-Trop2. In Figure 3, the percentage of macrophages that phagocytosed CFSE-labeled tumor cells is shown on the y-axis. Macrophages were incubated with Drug A at the indicated concentrations and 10 ng / mL of anti-TROP2 antibody. Cells were also incubated with 10 ng / mL of anti-TROP2 antibody alone, a combination of a negative control human IgG antibody and anti-TROP2 antibody, and medium alone. Phagocytosis of CFSE-labeled DLD-1 tumor cells by human monocyte-derived macrophages was enhanced in the presence of Drug A combined with anti-TROP2 antibody. See Figure 3.

[0287] Example 4: Antitumor activity of Drug A combined with trastuzumab and anti-PD1 antibody in a colon cancer model. MC38m / h HER2 cells were generated by infecting MC38 mouse colon cancer cells with a lentiviral vector encoding a chimeric mouse and human HER2 transmembrane and extracellular domains. MC38m / h HER2 cells were maintained in DMEM (Thermo Fisher Scientific 11965092) supplemented with 10% FBS, 1% penicillin-streptomycin, 1% GlutaMAX, and 1 mM sodium pyruvate (Thermo Fisher Scientific 11360070) in a 37 °C, 5% CO2 incubator. All tissue culture was performed under sterile conditions.

[0288] Prior to transplantation, a master cell bank of each cell line was generated to confirm that the cells used in subsequent experiments were of the same passage number. Cells were harvested and washed twice with 50 mL of cold PBS (Life Technologies 10010072). After the final wash, in the case of the MC38m / h HER2 cell line, the cells were resuspended in PBS or RPMI at 5 × 10 6 cells / mL. In the case of MC38m / h HER2, 100 μL of the cell suspension was subcutaneously injected into the right flank of C57BL / 6 mice. The tumor size of MC38m / h HER2 tumors averaged 65 - 69 mm 3When that was reached, the animals were randomized into eight groups consisting of 10 mice each. Each group was assigned to a treatment group outlined in Table A:

Table 13

[0289] Tumor volume (mm 3 ) and body weight were recorded twice or three times a week using a Mitutoyo Digital Caliper (Mitutoyo America, Aurora, Illinois). Mice with a tumor volume exceeding 2000 mm 3 or a 20% decrease in body weight were euthanized according to IACUC guidelines. Tumor volume was calculated as ([length × {width × width}] × 0.5 = volume (mm 3 ). Statistical analysis and p-values were calculated using GraphPad Prism software.

[0290] A chimeric m / h HER2 having the extracellular domain of human HER2 and the intracellular domain of mouse HER2 was expressed on MC38 colon cells so as to enable evaluation of the activity of trastuzumab against MC38 mouse tumors. As shown in Figure 5, monotherapy with trastuzumab had no effect on tumor growth, whereas monotherapy with drug A and monotherapy with the anti-PD-L1 antibody each had a moderate effect on tumor growth. Treatment with the drug A + anti-PD-L1 antibody doublet or the trastuzumab + anti-PD-L1 antibody doublet showed improved tumor growth inhibition compared to monotherapy alone. Treatment with the drug A + anti-PD-L1 + trastuzumab triple combination showed improved tumor inhibition when compared to each doublet. The effect of the triple combination on reducing tumor growth was most evident on days 19 and 22 (3 - 6 days after the last dose) compared to the drug A + anti-PD-L1 antibody doublet or the trastuzumab + anti-PD-L1 antibody doublet. By day 26, the triple combination was minimally better in reducing tumor growth compared to the drug A + anti-PD-L1 antibody doublet or the trastuzumab + anti-PD-L1 antibody doublet. No adverse effects were observed in any of the treatment cohorts within the MC38 m / hHER2 colon tumor model.

[0291] Example 5A: Exemplary clinical trial for evaluating the anti-tumor activity of drug A combination therapy in human patients Gastric or gastroesophageal junction (GEJ) adenocarcinoma HER2 that progressed during or after previous treatment with trastuzumab and fluoropyrimidine-containing chemotherapy (e.g., fluorouracil); during or after previous treatment with trastuzumab and platinum-containing chemotherapy; or during or after previous treatment with trastuzumab, fluoropyrimidine-containing chemotherapy (e.g., fluorouracil), and a platinum-containing chemotherapeutic agent +A clinical trial is conducted to evaluate the safety, tolerability, and efficacy of the combination of Drug A, trastuzumab, ramucirumab, and paclitaxel in patients with overexpressing progressive or metastatic gastric cancer or GEJ adenocarcinoma. Patients enrolled in the trial are suitable for treatment with trastuzumab. The patients have not received prior treatment with an anti-CD47 agent or an anti-SIRPα agent.

[0292] The clinical trial is conducted in patients with gastric or GEJ adenocarcinoma (e.g., HER2 + overexpressing gastric or GEJ adenocarcinoma) to evaluate the safety, tolerability, and efficacy of the combination of Drug A, pembrolizumab, cisplatin, and 5-fluorouracil or capecitabine. Patients enrolled in this trial have not received prior treatment with an anti-CD47 agent or an anti-SIRPα agent. The patients have appropriate organ function and hemoglobin is 9 g / dL or more.

[0293] Head and neck squamous cell carcinoma (HNSCC) The clinical trial is conducted to evaluate the safety, tolerability, and efficacy of the combination of Drug A, pembrolizumab, 5-fluorouracil, and either carboplatin or cisplatin in patients with metastatic or unresectable recurrent HNSCC who have not received treatment for advanced disease.

[0294] Example 5B: Preliminary safety results from the exemplary clinical trial described in Example 5A One patient with untreated progressive head and neck squamous cell carcinoma received treatment with Drug A (10 mg / kg IV QW), pembrolizumab (200 mg IV Q3W), 5-fluorouracil (1,000 mg / m 2 / day, days 1, 2, 3, 4, Q3W×6), and carboplatin (AUC = 5 mg / ml / min, day 1, Q3W×6). (In the expansion trial, cisplatin (100 mg / m 2, carboplatin (AUC = 5 mg / ml / min, day 1, Q3W×6) or cisplatin (AUC = 5 mg / ml / min, day 1, Q3W×6) is administered in combination with drug A, pembrolizumab, and fluorouracil. Patients receiving carboplatin will continue to receive carboplatin throughout the expansion study. Patients receiving cisplatin will continue to receive cisplatin throughout the expansion study. Three patients with HER2-positive gastric / gastroesophageal cancer who had progressed on prior treatment (if any) with trastuzumab, fluorouracil, and a platinum agent received treatment with drug A (10 mg / kg IV QW), trastuzumab (loading dose 8 mg / kg IV, then 6 mg / kg Q3W), ramucirumab (8 mg / kg, days 1 and 15, Q4W), and paclitaxel (80 mg / m2, days 1, 8, and 15, Q4W). Another three patients with HER2-positive gastric / gastroesophageal cancer who had progressed on prior treatment (if any) with trastuzumab, fluorouracil, and a platinum agent received treatment with drug A (15 mg / kg IV QW), trastuzumab (loading dose 8 mg / kg IV, then 6 mg / kg Q3W), ramucirumab (8 mg / kg, days 1 and 15, Q4W), and paclitaxel (80 mg / m2, days 1, 8, and 15, Q4W).

[0295] Initial results suggest that drug A is well tolerated without dose-limiting toxicity when administered at doses of 10 mg / kg or 15 mg / kg QW in the above combination regimens. Three patients (50%) receiving drug A + trastuzumab + ramucirumab + paclitaxel experienced treatment-related adverse events (TRAEs), while patients receiving drug A + pembrolizumab + fluorouracil + carboplatin did not (0%). There was no dose-limiting toxicity in patients receiving drug A + pembrolizumab + fluorouracil + carboplatin or drug A + trastuzumab + ramucirumab + paclitaxel. Also, there were no treatment-related adverse events (TRAEs) occurring in two or more patients in the following three cohorts. Drug A (10 mg / kg QW) + pembrolizumab + fluorouracil + carboplatin (N = 1) Drug A (10 mg / kg QW) + Trastuzumab + Ramucirumab + Paclitaxel (N = 3) Drug A (15 mg / kg QW) + Trastuzumab + Ramucirumab + Paclitaxel (N = 3)

[0296] Finally, there were no treatment-related adverse events of grade 3 or higher (TRAE ≥ grade 3) reported in patients treated with Drug A + Pembrolizumab + Fluorouracil + Carboplatin or Drug A + Trastuzumab + Ramucirumab + Paclitaxel.

[0297] Example 5C: Preliminary Efficacy Results from the Exemplary Clinical Trial Described in Example 5A Patients with untreated advanced head and neck squamous cell carcinoma who were treated with Drug A, Pembrolizumab, 5-Fluorouracil, and a platinum agent at the dosages and administration schedules described in Example 5B achieved a partial response (PR) based on the response evaluated by the investigator using the RECIST v1.1 criteria.

[0298] Of the three patients with HER2-positive gastric / gastroesophageal cancer who were treated with Drug A (10 mg / kg QW), Trastuzumab, Ramucirumab, and Paclitaxel (see Example 5B), two were still not evaluable. One patient achieved a PR based on the response evaluated by the investigator using the RECIST v1.1 criteria.

[0299] Of the three patients with HER2-positive gastric / gastroesophageal cancer who were treated with Drug A (15 mg / kg QW), Trastuzumab, Ramucirumab, and Paclitaxel (see Example 5B), two were still not evalua...

Claims

**Claim 1** A medicament for treating gastric cancer or gastroesophageal junction (GEJ) cancer in an individual in combination with an anti-HER2 antibody, an anti-VEGFR2 antibody, and paclitaxel, wherein the medicament comprises a polypeptide comprising an effective amount of a SIRPα D1 domain variant and an Fc domain variant, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85, wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, numbered according to the Kabat EU index, said region; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, numbered according to the Kabat EU index, said region; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to the Kabat EU index, said region, or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, numbered according to the Kabat EU index, said region, wherein the individual has received at least one previous treatment for the gastric cancer or the GEJ cancer, said medicament. **Claim 2** The medicament according to claim 1, wherein the anti-HER2 antibody is trastuzumab. **Claim 3** The medicament according to claim 1 or 2, wherein the individual has received previous treatment with an anti-HER2 antibody, with an anti-HER2 antibody and a fluoropyrimidine, or with an anti-HER2 antibody and a platinum-based chemotherapeutic agent. **Claim 4** The medicament according to claim 3, wherein the anti-HER2 antibody in the previous treatment is trastuzumab. **Claim 5** The medicament according to any one of claims 1 to 4, wherein the anti-VEGFR2 antibody is ramucirumab. **Claim 6** The gastric cancer or the GEJ cancer is HER2 ＋ gastric cancer or HER2 ＋ The pharmaceutical according to any one of claims 1 to 5, wherein the cancer is a gastric cancer or a GEJ cancer **Claim 7** The medicament according to any one of claims 1 to 6, wherein the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant is administered at a dose of 10 mg / kg once a week. **Claim 8** The pharmaceutical according to any one of claims 1 to 6, wherein the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant is administered at a dose of 15 mg / kg once a week.

9. The pharmaceutical according to any one of claims 1 to 8, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO:

85.

10. The pharmaceutical according to any one of claims 1 to 8, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO:

81.

11. The pharmaceutical according to any one of claims 1 to 10, wherein the Fc domain variant is a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, and the numbering follows the Kabat EU index.

12. The pharmaceutical according to claim 11, wherein the Fc domain variant comprises the amino acid sequence of SEQ ID NO:

91.

13. The pharmaceutical according to any one of claims 1 to 9, 11, and 12, wherein the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO:

136.

14. The pharmaceutical according to any one of claims 1 to 8 and 10 to 12, wherein the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO:

135.

15. The pharmaceutical according to any one of claims 1 to 14, wherein the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant forms a homodimer.

16. The pharmaceutical according to any one of claims 1 to 15, wherein the individual is human.

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