Combination therapies for treating cancer

Abstract

To provide a method for treating cancer in an individual.SOLUTION: Provided are methods of treating cancer that comprise administering a polypeptide (e.g. a fusion polypeptide) that comprises a SIRPα D1 domain variant and an Fc domain variant in combination with at least one chemotherapy agent and / or at least one therapeutic antibody. Also provided are related kits.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 941,390, filed November 27, 2019; U.S. Provisional Application No. 63 / 022,998, filed May 11, 2020; U.S. Provisional Application No. 63 / 030,686, filed May 27, 2020; U.S. Provisional Application No. 63 / 106,225, filed October 27, 2020; and U.S. Provisional Application No. 63 / 109,044, filed November 3, 2020, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 757972001140SEQLIST.TXT, Recorded: November 25, 2020, Size: 333KB).

[0003] The present invention relates to a method of 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 mode of cancer therapy. [Background technology]

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

[0005] Tumor cells manipulate the myeloid compartment to evade anti-tumor host immune responses (Gabrilovich et al., Nat Rev Immunol (2012) 12(4):253-68). For example, CD47, expressed on the surface of normal cells, binds to SIRPα on macrophages, signaling "don't eat me," but tumor cells have also been shown to overexpress CD47 to evade the macrophage component of immune surveillance (Oldenborg, ISRN Hematol (2013) 614619).

[0006] Macrophage-mediated cancer cell destruction requires both disruption of a "don't eat me" signal (e.g., CD47-SIRPα) and activation of an "eat me" signal. Neither component alone is sufficient to trigger a maximal phagocytic response against tumor cells. As noted above, CD47 provides the basal "don't eat me" signal through its interaction with SIRPα on macrophages. A pro-phagocytic "eat me" signal can be provided to the same macrophages by binding to their activating Fc gamma receptor. For example, a pro-phagocytic "eat me" signal can be provided by binding of an anti-tumor antibody to an Fc receptor on macrophages.

[0007] All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are incorporated by reference in their entirety, as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0008] Methods of treating cancer in an individual are provided, the methods comprising administering to the individual effective amounts 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; and the Fc domain variant is selected from the group consisting of (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); and (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to the EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein numbering is according to the EU index of Kabat). In some embodiments, the cancer is leukemia, multiple myeloma, or non-Hodgkin's lymphoma. In some embodiments, the non-Hodgkin's 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 myeloid 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 are methods of treating cancer in an individual, the method comprising administering to the individual effective amounts 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; and the Fc domain variant comprises (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); or (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein numbering is according to EU index of Kabat). 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 are methods of treating cancer in an individual, the methods comprising administering to the individual effective amounts 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 comprises (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); or (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A, wherein the cancer is head and neck squamous cell carcinoma (HNSCC) and the individual has not received previous treatment for HNSCC. 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 weekly (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 weekly (qw).

[0011] In some embodiments, the HNSCC is advanced and / or metastatic HNSCC. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody, e.g., pembrolizumab, nivolumab, pidilizumab, cemiplimab, 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, or phototrexate. 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 effective amounts 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; and the Fc domain variant is selected from the group consisting of: (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); or (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to the EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein numbering is according to the EU index of Kabat). 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 solid tumor is HER2 + In some embodiments, the solid tumor is a colon cancer (e.g., HER2 + 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 effective amounts 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; and the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); or (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein numbering is according to EU index of Kabat). The cancer is gastric cancer or gastroesophageal junction (GEJ) cancer, and the individual has received at least one prior treatment for the 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, an anti-HER2 antibody and a fluoropyrimidine, or an anti-HER2 antibody and a platinum-based chemotherapy 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 weekly (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 weekly (qw).

[0014] Also provided are methods of treating cancer in an individual, the methods comprising administering to the individual effective amounts 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 comprises (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); or (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to the EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein numbering is according to the EU index of Kabat). In some embodiments, the cancer is a solid tumor, gastric cancer, nasopharyngeal carcinoma, gallbladder cancer, cervical cancer, extranodal NK / T-cell lymphoma, lung cancer, laryngeal squamous cell carcinoma, colon cancer, perihilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrioid endometrial carcinoma, 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 L234A, L235A, G237A, and N297A mutations, where numbering is according to the EU index of Kabat. 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 individual is human.

[0016] In another aspect, a kit is also provided comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, the polypeptide being for use in combination with a Bcl-2 inhibitor to treat 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; and the Fc domain variant is selected from the group consisting of: (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); or (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the kit comprises instructions for administering the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant in combination with a Bcl-2 inhibitor to an individual in need thereof. In some embodiments, the cancer is leukemia, multiple myeloma, or non-Hodgkin's lymphoma. In some embodiments, the Bcl-2 inhibitor is venetoclax.

[0017] Also provided is a kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, the polypeptide being for use in combination with a platinum-based chemotherapy agent to treat 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; and the Fc domain variant comprises (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); or (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein numbering is according to the EU index of Kabat), and the kit comprises instructions for administering the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant in combination with a 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] Also provided in some embodiments are kits comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, the polypeptide being for use in combination with a PD-1 inhibitor, an antimetabolite, and a platinum-based chemotherapy agent to treat 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; and the Fc domain variant comprises: (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); or (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein 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 with head and neck squamous cell carcinoma (HNSCC) who has not received a previous treatment 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] Also provided in some embodiments is a kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, the polypeptide being 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; and the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); or (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, and the kit comprises instructions for administering a polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant in combination with an anti-HER2 antibody, an anti-VEGFR2 antibody, and paclitaxel to an individual with gastric cancer or gastroesophageal junction (GEJ) cancer who has received at least one prior treatment for the gastric cancer or GEJ cancer. In some embodiments, the gastric cancer or GEJ cancer is an HER2 antibody. + Gastric cancer or HER2 +The cancer 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 a previous treatment (or multiple previous treatments) with an anti-HER2 antibody (e.g., trastuzumab) and / or a fluoropyrimidine and / or platinum-based chemotherapy agent. In some embodiments, the individual's gastric cancer or GEJ cancer has progressed during or after a previous treatment (or multiple previous treatments) including an anti-HER2 antibody (e.g., trastuzumab) and / or a fluoropyrimidine and / or platinum-based chemotherapy agent. In some embodiments, the individual has failed a previous treatment (or multiple previous treatments) including an anti-HER2 antibody (e.g., trastuzumab) and / or a fluoropyrimidine and / or platinum-based chemotherapy agent (e.g., relapsed after the previous treatment or did not respond to the previous treatment). In some embodiments, the prior treatment(s) included an anti-HER2 antibody and a fluoropyrimidine (e.g., administered during the same or different treatments). In some embodiments, the prior treatment(s) included an anti-HER2 antibody and a platinum-based chemotherapeutic agent (e.g., administered during the same or different treatments).

[0020] Also provided is a kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, the polypeptide being for use in combination with an anti-TROP2 antibody to treat 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; and the Fc domain variant comprises (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); or (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein numbering is according to the EU index of Kabat), 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-TROP2 antibody to an individual in need thereof. In some embodiments, the cancer is a solid tumor, gastric cancer, nasopharyngeal carcinoma, gallbladder cancer, cervical cancer, extranodal NK / T-cell lymphoma, lung cancer, laryngeal squamous cell carcinoma, colon cancer, perihilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrioid endometrial carcinoma, 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, the polypeptide being for use in combination with an anti-HER2 antibody and an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody) to treat 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; and the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); or (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein 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 + 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 L234A, L235A, G237A, and N297A mutations, numbering according to the EU index of Kabat. 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 individual is human. [Brief explanation of the drawings]

[0023] [Figure 1A] Tumor volumes (mm3) are shown at the indicated times after implantation in NOD-SCID female mice injected with RS4;11 leukemia cells and subsequently treated with drug A, venetoclax, the venetoclax / drug A combination, or vehicle (PBS). Dashed arrows indicate administration of venetoclax (250 μg) by oral gavage, a total of two times, 3 days apart. Dotted arrows indicate administration of drug A (10 mg / kg), a total of four times, 3-4 days apart. SEM = standard error of mean (SEM). TF = no tumor. [Figure 1B] Tumor volume (mm3) at the indicated times post-implantation in NOD-SCID female mice injected with RS4;11 leukemia cells, treated with venetoclax, and then retreated with single-agent venetoclax or the venetoclax / drug A combination. [Figure 2A]Tumor volumes (mm3) and body weights of BALB / c female mice injected with CT26 tumor cells and subsequently treated with Drug A, cisplatin, a cisplatin / Drug A combination, or vehicle (PBS) are shown at the indicated times after implantation. Mean tumor volumes (+ / - SEM) are shown for the indicated treatments. Dashed arrows indicate administration of cisplatin (two doses of 5 mg / kg, 10 days apart). Dotted arrows indicate administration of Drug A (two doses of 30 mg / kg, 10 days apart). Both drugs were administered intraperitoneally. Mice treated with both agents received Drug A one day after treatment with cisplatin. [Figure 2B] Tumor volume (mm) and body weight of BALB / c female mice injected with CT26 tumor cells and subsequently treated with Drug A, cisplatin, a cisplatin / Drug A combination, or vehicle (PBS) are shown at the indicated times after implantation. The mean percent change in body weight from day 7 (D7) is shown for mice treated according to the regimen shown in Figure 2A. [Figure 2C] Tumor volumes (mm3) and body weights of BALB / c female mice injected with CT26 tumor cells and subsequently treated with Drug A, cisplatin, a cisplatin / Drug A combination, or vehicle (PBS) are shown at the indicated times after implantation. Mean tumor volumes (+ / - SEM) are shown for the indicated treatments. Dashed arrows indicate administration of cisplatin (10 mg / kg, given once). Dotted arrows indicate administration of Drug A (30 mg / kg, given twice, 10 days apart). Both drugs were administered intraperitoneally. Mice treated with both agents received Drug A one day after treatment with cisplatin. [Figure 2D] Tumor volume (mm) and body weight of BALB / c female mice injected with CT26 tumor cells and subsequently treated with Drug A, cisplatin, a cisplatin / Drug A combination, or vehicle (PBS) are shown at the indicated times after implantation. The mean percent change in body weight from day 7 (D7) is shown for mice treated according to the regimens shown in Figure 2C. [Figure 3]1 shows the results of an experiment performed to determine the effect of Drug A in combination with an anti-TROP2 antibody on the phagocytosis of CFSE-labeled DLD-1 tumor cells by human monocyte-derived macrophages. [Figure 4] 1 shows the results of experiments conducted to determine the effect of Drug A in combination with (a) an anti-HER2 antibody, (b) an anti-PD-L1 antibody, or (c) an anti-HER2 antibody and anti-PD-L1 on tumor growth in an MC38m / h colon cancer model. [Figure 5A] 1 shows the results of experiments conducted to evaluate the effect of adding drug A, venetoclax, or both drug A and venetoclax on the phagocytosis of HL60 cells by macrophages in an in vitro assay. [Figure 5B] 1 shows the results of experiments performed to evaluate the effect of adding drug A, venetoclax, or both drug A and venetoclax on the phagocytosis of OCI-AML3 cells by macrophages in an in vitro assay. [Figure 6A] 1 shows the results of experiments performed to evaluate the effect of Drug A or Drug C on the activation of CD8+ dendritic cells. [Figure 6B] 1 shows the results of experiments performed to evaluate the effect of Drug A or Drug C on the activation of CD8-dendritic cells. [Figure 7A] 1 shows the results of experiments performed to evaluate the effect of Drug A or Drug B on the activation of CD8+ dendritic cells. [Figure 7B] 1 shows the results of experiments performed to evaluate the effect of Drug A or Drug B on the activation of CD8-dendritic cells. [Figure 8A] 1 shows the results of experiments performed to evaluate the binding of Drug A, F59 / magrolimab, TTI-621, and TTI-622 to hCD47. [Figure 8B] 1 shows the results of quantitative experiments performed to evaluate the effects of Drug A, F59 / magrolimab, TTI-621, and TTI-622 on SIRPα signaling. DETAILED DESCRIPTION OF THE INVENTION

[0024] The following description sets forth example methods, parameters, etc. However, it should be recognized that such description is not intended to limit the scope of the present disclosure, but is instead provided as a description of example embodiments.

[0025] definition The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on the method for measuring or determining the value, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more standard deviations, as is customary in the art. Alternatively, "about" can mean 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, preferably within 5-fold, and more preferably within 2-fold, of a value. Unless otherwise stated, when a particular value is described in this application and claims, the term "about" is deemed to mean within an acceptable error range for the particular value.

[0026] The terminology used herein is for the purpose of describing particular instances only and is 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 indicates otherwise. Furthermore, to the extent that "including," "includes," "having," "has," "involving," or variations thereof are used in either the detailed description or the claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0027] As used herein, the terms "treatment," "treating," and the like 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 a partial or complete cure of a disease or a symptom of a disease.

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

[0029] As used herein, the term "antibody variable domain" refers to the portions of the light and heavy chains of an antibody comprising 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 framework regions (FRs).

[0030] As used herein, the term "linker" refers to a link between two elements, e.g., protein domains. In some embodiments, a 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 the polypeptide (e.g., attached to spaced polypeptides or polypeptide domains via the polypeptide backbone).

[0031] As used herein, the term "effective amount" refers to an amount of a polypeptide or pharmaceutical composition comprising a polypeptide described herein, e.g., a polypeptide having a SIRPα D1 domain or a variant thereof, that is sufficient and effective to achieve a desired therapeutic effect in treating a patient with a disease such as cancer, e.g., a solid tumor or a blood 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 product or formulation that includes an active ingredient and an excipient or diluent (or both an excipient and a diluent), such that the active ingredient may be administered by a suitable method of administration. In some embodiments, the pharmaceutical compositions disclosed herein include pharmaceutically acceptable ingredients that are compatible with the polypeptide. In some embodiments, the pharmaceutical composition is in tablet or capsule form for oral administration, or in 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 vertebrates, e.g., mammals. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Also encompassed are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro. Neither term requires 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 strength of the sum of non-covalent interactions between a molecule and its binding partner (such as a SIRPα D1 domain variant and CD47). Unless otherwise specified, binding affinity refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair. Binding affinity between two molecules is generally measured by a dissociation constant (K D ) or binding constant (K A) Two molecules that have low binding affinity for each other generally tend to bind slowly and dissociate easily, with a large K D Two molecules that have a high affinity for each other generally bind easily, tend to remain long-lasting, and exhibit a small K D In some embodiments, the K of two interacting molecules D is determined using known methods and techniques, for example, surface plasmon resonance (SPR). D can be calculated as the ratio koff / kon.

[0035] As used herein, "less than K" D The term "" refers to the numerically small K D Value and stated K D As used herein, "greater K" refers to an increased binding affinity compared to the K value. D The term "K" refers to the numerically larger D Value and stated K D It refers to a decreased binding affinity compared to the original value.

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

[0037] Overview Provided herein are methods of treating cancer in an individual (e.g., a human individual), the methods comprising administering to the individual effective amounts 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, such as at least two, at least three, or at least four chemotherapeutic agents). In some embodiments, the method further comprises administering to the individual an effective amount of a therapeutic antibody (at least one therapeutic antibody, such as 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 therapeutic modalities, such as, but not limited to, radiation therapy, surgery, cryoablation, and bone marrow transplantation.

[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, e.g., 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, the 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 D or better K D(e.g., at least 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 agent that binds CD47 (e.g., hCD47) exhibits a CD47 receptor occupancy of at least about 50% (e.g., at least 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 CD47 (e.g., hCD47) has an EC50 of about 80 ng / ml or less, e.g., about any one of 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 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" encompasses antibody-based constructs (such as multispecific constructs) including, but not limited to, triomabs, DARTs (i.e., dual affinity retargeting antibodies), TandAbs (i.e., tandem diabodies), tandem scFvs, CrossMabs, DNLs (i.e., dock-and-lock antibodies), DVD-Igs (i.e., dual variable domain immunoglobulins), tetravalent bispecific IgGs, nanobodies, dual targeting domains, and ART-Igs (i.e., asymmetric reengineering technology-immunoglobulins).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, e.g., 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, the 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 D or better K D(e.g., at least 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, an agent that binds to SIRPα (e.g., hSIRPα) exhibits a SIRPα receptor occupancy of at least about 50% (e.g., at least 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 (e.g., hSIRPα) has an EC50 of about 80 ng / ml or less, e.g., about any one of 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 (e.g., hSIRPα) is an anti-SIRPα antibody (e.g., 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 (e.g., 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, e.g., 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 contents of which are incorporated by reference herein in their entireties.

[0041] In some embodiments, the 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 capable of binding to SIRPα), or an antigen-binding fragment thereof. In some embodiments, the agent is an antibody (or antigen-binding fragment thereof) capable of binding to two or more of SIRPα, SIRPβ, and SIRPγ. In some embodiments, such an antibody has a K D or better K DIn some embodiments, the antibody binds to SIRPα (e.g., hSIRPα) with an occupancy of at least about 50% (e.g., at least 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 antibody 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 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 a monospecific antibody construct (including but not limited to those described above). In some embodiments, the antibody is a multispecific (e.g., bispecific) antibody or a 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 DIn some embodiments, the fusion polypeptide binds to CD47 (e.g., hCD47) at a binding affinity of at least about 50% (e.g., at least 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., about any one of 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 a WT human antibody Fc region) that exhibits reduced (e.g., eliminated) effector function compared to the WT Fc region. Exemplary Fc variants are described in WO2017 / 027422 and US2017 / 0107270, the contents of which are incorporated herein by reference in their entireties. In some embodiments, the moiety that binds to CD47 (e.g., hCD47) is WT SIRPα (e.g., hSIRPα) or WT SIRPγ (e.g., hSIRPγ). In some embodiments, the moiety 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 moiety 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 in, e.g., WO2013 / 109752; US2015 / 0071905; USP9,944,911; WO2016 / 023040; WO2017 / 027422; US2017 / 0107270; USP10,259,859; US9845345; WO2016187226; US201801554 05; WO2017177333; WO2014094122; US2015329616; US20180312563; WO2018176132; WO2018081898; WO2018081897; PCT / US2019 / 048921; US20180141986A1; and EP3287470A1, the contents of which are incorporated herein by reference in their entireties.

[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 D or better K D(e.g., 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) bind to CD47 (e.g., hCD47). In some embodiments, the fusion polypeptide exhibits a CD47 receptor occupancy of at least about 50% (e.g., 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., about any one of 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 a WT human antibody Fc region) that exhibits reduced (e.g., eliminated) effector function compared to the WT Fc region, e.g., including those described in the references cited herein. In some embodiments, the fusion polypeptide is selected from the group consisting of WO2013 / 109752; US2015 / 0071905; WO2016 / 023040; WO2017 / 027422; US2017 / 0107270; USP10,259,859; US9845345; WO2016187226; US20180155405; WO201717733 3; WO2014094122; US2015329616; US20180312563; WO2018176132; WO2018081898; WO2018081897; US20180141986A1; and EP3287470A1, the contents of which are incorporated herein by reference in their entireties.In some embodiments, the fusion polypeptide comprising an antibody Fc region and a 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 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).

[0045] In some embodiments, provided are methods 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 effective amounts 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 selected from the group consisting of: (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations (wherein numbering is according to EU index of Kabat); or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations. Fc region (where the numbering is according to the EU index of Kabat).

[0046] In some embodiments, provided are methods of treating cancer (e.g., colon cancer) in an individual (e.g., a human individual), the method comprising administering to the individual effective amounts 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 selected from the group consisting of: (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations (wherein numbering is according to EU index of Kabat); or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations. Fc region (where the numbering is according to the EU index of Kabat).

[0047] In some embodiments, methods are provided for treating cancer (e.g., head and neck cancer, head and neck squamous cell carcinoma, etc.) in an individual (e.g., a human individual), the methods comprising administering to the individual effective amounts 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 selected from the group consisting of: (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); and (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. Fc region (wherein numbering is according to EU index of Kabat); or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) containing the S228P, E233P, F234V, L235A, delG236, and N297A mutations.

[0048] In some embodiments, provided are methods 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 effective amounts 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 selected from the group consisting of: (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); and (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. Fc region (wherein numbering is according to EU index of Kabat); or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) containing the S228P, E233P, F234V, L235A, delG236, and N297A mutations.

[0049] Further details regarding methods of treatment with polypeptides comprising SIRPα D1 domain variants and Fc domain variants are provided below. See also WO2017 / 027422 and U.S. Patent No. 10,259,859, the contents of each of which are incorporated herein by reference in their entireties.

[0050] Signal regulatory protein alpha (SIRPα) D1 domain and its variants In some embodiments, disclosed herein are polypeptides comprising a signal-regulatory protein alpha (SIRP-α) D1 variant, wherein the polypeptide comprises a SIRPα D1 domain or a fragment thereof comprising an amino acid mutation at residue 80 relative to a wild-type SIRP-α D1 domain (e.g., a wild-type SIRPα D1 domain set forth 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 (e.g., a wild-type SIRPα D1 domain set forth in SEQ ID NO: 1 or 2).

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

[0052] Signal regulatory protein α ("SIRP-α" or "SIRP-alpha") is a transmembrane glycoprotein belonging to the Ig superfamily that is widely expressed on the membranes of myeloid cells. SIRPα interacts with CD47, a protein that is widely expressed on many cell types in the body. The interaction of SIRPα with CD47 prevents the engulfment of "self" cells that might otherwise be recognized by the immune system. It has been observed that high expression of CD47 on tumor cells can 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 ("D1 domain") refers to the membrane-distal extracellular domain of SIRPα and mediates binding of SIRPα to CD47. As used herein, the term "SIRPα polypeptide" refers to any SIRPα polypeptide or fragment thereof capable of binding to CD47. At least 10 wild-type human SIRPα variants exist. Table 1 shows the amino acid sequences of the D1 domain of naturally occurring wild-type human SIRPα D1 domain variants (SEQ ID NOs: 1 and 2). In some embodiments, a SIRPα polypeptide comprises a SIRPα D1 domain. In some embodiments, a SIRPα polypeptide comprises a wild-type D1 domain, such as that provided in SEQ ID NOs: 1 and 2. In some embodiments, a 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 a SIRPα D1 domain or a CD47-binding portion of a SIRPα polypeptide that has a higher affinity for CD47 than wild-type SIRPα. A SIRPα D1 domain variant comprises at least one amino acid substitution, deletion, or insertion (or a combination thereof) relative to wild-type SIRPα.

[0055] In some embodiments, the SIRPα D1 domain variants disclosed herein comprise a SIRPα D1 domain or variants thereof. In some embodiments, the SIRPα D1 domain variants comprise one or more amino acid substitutions, insertions, additions, or deletions relative to the wild-type D1 domain set forth 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 comprises a fragment of the D1 domain. In some embodiments, the SIRPα polypeptide fragment or SIRPα D1 domain variant fragment comprises 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 more than about 100 amino acids in length. In some embodiments, the SIRPα D1 domain fragment retains the ability to bind to CD47.

[0056] In some embodiments, polypeptides of the present disclosure comprising a SIRPα D1 domain variant bind to CD47 with higher binding affinity than 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, or more) greater than that of a 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 more) greater than that of a naturally occurring D1 domain.

[0057] As used herein, the term "optimized affinity" or "optimized binding affinity" refers 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 to CD47 on non-cancer cells or binds with lower affinity. In some embodiments, the binding affinity between the polypeptide and CD47 is optimized so that the interaction does not cause clinically relevant toxicity or reduces toxicity compared to a variant that binds with maximum affinity. In some embodiments, to achieve optimized binding affinity between a polypeptide provided herein and CD47, a polypeptide comprising a SIRPα D1 domain variant is developed to have a lower binding affinity to CD47 than is maximally achievable. In some embodiments, the SIRPα D1 domain variants disclosed herein cross-react with rodent, 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) to provoke an immune response in a host as if it were a foreign antigen. The immunogenicity of a protein can be assayed in vitro in a variety of ways, such as in vitro T cell proliferation assays.

[0059] As used herein, the term "minimally immunogenic" refers to the immunogenicity of a protein (e.g., a therapeutic protein) that has been modified, e.g., by amino acid substitution, to be less immunogenic (e.g., at least 10%, 25%, 50%, or 100% less) than the immunogenicity before the amino acid substitutions were introduced (e.g., the unmodified protein). In some embodiments, the protein (e.g., a therapeutic protein) is modified to have minimal immunogenicity, eliciting no or little host immune response even though it is 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 affinity of 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 reduce the risk of side effects compared to anti-CD47 antibodies or wild-type SIRPα. In some embodiments, the polypeptide variants disclosed herein reduce the 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 SIRPα D1 domain variants relative to each D1 domain sequence. In some embodiments, the SIRPα D1 domain variant contains 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 contains up to 14 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant contains up to 10 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant contains up to 7 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variants of the present disclosure have 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.

[0062] In some embodiments, the SIRPα D1 domain variant is a chimeric SIRPα D1 domain variant comprising portions of two or more wild-type D1 domains or variants thereof (e.g., a portion of one wild-type D1 domain or variant thereof and a portion of another wild-type D1 domain or variant thereof). In some embodiments, the chimeric SIRPα D1 domain variant comprises at least two portions (e.g., three, four, five, or more portions) of the wild-type D1 domain or variant thereof, where each portion 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 2. [Table 2]

[0063] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant comprising the following sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGNITPADAGTYYCX 12 KX 13 RKGSPDDVEX 14 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, and X 10 is L, T, or G, and X 11 is K or R, and X 12 is V or I, and X 13 is F, L, or V, and 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, or V. In some embodiments, X 14 is F or V. In some embodiments, the polypeptides of this aspect of the disclosure comprise 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 a 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 a 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 a wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1 x 10 -8 Under M, 5x10-9 Under M, 1x10 -9 Under M, 5x10 -10 Under M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with 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.

[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 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, and X 10 is S, T, or G, and X 11 is K or R, and X 12 is V or I, and X 13 is F, L, or V, and 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, wherein 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 a 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 a 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 a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1 x 10 -8 Under M, 5x10 -9 Under M, 1x10 -9 Under M, 5x10 -10 Under M, 1x10 -10 Less than M or 1x10 -11 K less than MD In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with 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.

[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 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 are 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 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, or V. In some embodiments, X 26 is D or absent. 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 polypeptides of this aspect of the disclosure comprise 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 a 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 a 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 a 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 fragment thereof is at least 1 x 10 -8 Under M, 5x10 -9 Under M, 1x10 -9 Under M, 5x10 -10 Under M, 1x10 -10 Less than M or 1x10 -11 K less than M DIn some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with 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, a polypeptide of the present disclosure comprising a SIRPα D1 domain variant further comprises a D2 domain having the sequence of SEQ ID NO: 24, a D3 domain having the sequence of SEQ ID NO: 25, or a D2 domain having the sequence of SEQ ID NO: 24 and a D3 domain having the sequence of SEQ ID NO: 25 of wild-type human SIRPα as 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 linkers. [Table 3]

[0073] In some embodiments, a polypeptide of the present disclosure comprising a SIRPα D1 domain variant is attached to an Fc domain variant to improve the pharmacokinetic properties of the polypeptide, e.g., to increase serum half-life. In some embodiments, the SIRPα D1 domain variant is attached to an Fc domain variant that is unable to dimerize. In some embodiments, the Fc domain variant serves to increase serum half-life of a polypeptide described herein. In some embodiments, a polypeptide of the present disclosure comprising a SIRPα D1 domain variant does not comprise any 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 liquid phase or bound to a solid support. In some embodiments, 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, amylase, natural and modified cellulose, 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, polypeptides are conjugated to low molecular weight haptens. These haptens are then specifically detected by a second reaction. For example, in some embodiments, the hapten biotin is used with avidin, or the haptens dinitrophenol, pyridoxal, or fluorescein are detected with specific anti-hapten antibodies (e.g., anti-dinitrophenol, anti-pyridoxal, and anti-fluorescein antibodies, respectively).

[0078] SIRPα D1 domain variants with altered glycosylation patterns In some embodiments, disclosed herein are polypeptides comprising a signal-regulatory protein alpha (SIRP-α) D1 variant, wherein the polypeptide comprises a SIRPα D1 domain or a fragment thereof having an amino acid mutation at residue 80 relative to a wild-type SIRP-α D1 domain (e.g., a wild-type SIRPα D1 domain set forth 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 (e.g., a wild-type SIRPα D1 domain set forth in SEQ ID NO: 1 or 2).

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

[0080] In some embodiments, the polypeptides in the compositions disclosed herein comprise SIRPα D1 domain variants 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 of 16 kDa (unglycosylated) and a minor band of higher molecular weight that is 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 glycosylation efficiency, resulting in proteins with different degrees of glycosylation and therefore heterogeneity. In the context of drug development, heterogeneity can pose challenges in process development. Therefore, to explore the possibility of generating a homogeneously aglycosylated form of a SIRPα D1 domain variant, in some embodiments, amino acid N80 of the SIRPα D1 variant is mutated to Ala. In some embodiments, to create an aglycosylated SIRPα D1 domain variant, amino acid N80 in the SIRPα D1 domain variant is replaced with any natural or unnatural amino acid, such as N80A or 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. Another option for increasing the homogeneity of the final product is to increase the efficiency of glycosylation at amino acid N80, generating a SIRPα D1 domain variant with increased glycosylation compared to the wild-type. In some embodiments, 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, amino acid P83 in a SIRPα D1 domain variant is substituted with any amino acid, including natural and unnatural amino acids, for example, P83V, P83A, P83I, and P83L. In some embodiments, polypeptides of the disclosure are expressed in cells that have been optimized not to glycosylate the expressed protein, for example, by genetically engineering the cell line (e.g., a genetically engineered yeast or mammalian host), or by altering cell culture conditions, such as adding kifunensine, or by using a naturally non-glycosylating host, such as a prokaryote (e.g., E. coli).

[0082] Table 5 lists the specific amino acid substitutions of SIRPα D1 domain variants for each D1 domain variant sequence. In some embodiments, the SIRPα D1 domain variant contains 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 unglycosylated 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 contains up to 14 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant contains up to 10 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant contains up to 7 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, a SIRPα D1 domain variant of the present disclosure has at least 90% (eg, at least 92%, 95%, 97%, or greater 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 portions of two or more wild-type D1 domains or variants thereof (e.g., a portion of one wild-type D1 domain or variant thereof and a portion of another wild-type D1 domain or variant thereof). In some embodiments, the chimeric SIRPα D1 domain variant comprises at least two portions (e.g., three, four, five, or more portions) of the wild-type D1 domain or variant thereof, where each portion 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 X is L, I, or V; X is V, L, or I; X is A or V; X is A, I, or L; X is I, T, S, or F; X is E, V, or L; X is K or R; X is E or Q; X is H, P, or R; 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 disclosure, the polypeptide comprises a SIRPα D1 domain variant having the sequence of SEQ ID NO: 37, wherein 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, or V. In some embodiments, X 16 is F or V.

[0086] In some embodiments, the polypeptides provided herein comprise no more than 10 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptides provided herein comprise no more than 7 amino acid substitutions relative to a 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 a 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 a 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 a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1 x 10 -8 Under M, 5x10 -9 Under M, 1x10 -9 Under M, 5x10 -10 Under M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with 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.

[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 16X is L, I, or V; X is V, L, or I; X is A or V; X is V, I, or L; X is I, T, S, or F; X is E, V, or L; X is K or R; X is E or Q; X is H, P, or R; 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 disclosure, the polypeptide comprises a SIRPα D1 domain variant having the sequence of SEQ ID NO: 38, wherein 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 no more than 10 amino acid substitutions relative to a 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 no more than 7 amino acid substitutions relative to a 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 a 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 a 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 a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1 x 10 -8 Under M, 5x10 -9 Under M, 1x10 -9 Under M, 5x10 -10 Under M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with 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.

[0092] In another aspect, the disclosure features a polypeptide including 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 X 24 ITX 25 ADAGTYYCX 26 KX 27 RKGSPDX 28 X 29 EX 30 KSGAGTELSVRX 31 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 are 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 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 above embodiments, X 28 In any of the foregoing embodiments, X is D or absent. 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 polypeptides of this aspect of the disclosure comprise no more than 10 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 7 amino acid substitutions relative to a 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 a 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 a 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 a 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 fragment thereof is at least 1 x 10 -8 Under M, 5x10 -9 Under M, 1x10 -9 Under M, 5x10 -10 Under M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with 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.

[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 X is V or I; X is L or S; X is T or S; X is T or I; X is R or H; X is A, V, or I; X is I, R, Y, K, or F; X is G or A; X is E or V; 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 disclosure features a polypeptide including a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9ITPADAGTYYCX 10 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; 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 comprising 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: 49, and comprises X1, X2, X3, X4, X5, X6, X7, X8, X9, and X 10 Each of the amino acids is not a wild-type amino acid.

[0100] In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 10 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NO: 1. In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 7 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of any one 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 a 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 a 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 a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1 x 10 -8 Under M, 5x10 -9 Under M, 1x10 -9 Under M, 5x10 -10 Under M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with 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.

[0102] In another aspect, the disclosure features a polypeptide including a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVSVAAGESAILHCTX2TSLX3PVGPIQWFRGAGPARX4LIYNQX5X6GX7FPRVTTVSEX8TKRENMDFSISISX9ITPADAGTYYCX 10 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; 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 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 comprising 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: 50, and comprises X1, X2, X3, X4, X5, X6, X7, X8, X9, and X10. 10 Each of the amino acids is not a wild-type amino acid.

[0105] In some embodiments, the polypeptides of this aspect of the disclosure comprise 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 polypeptides of this aspect of the disclosure comprise 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 a 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 a 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 a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1 x 10 -8 Under M, 5x10 -9 Under M, 1x10 -9 Under M, 5x10 -10 Under M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with 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.

[0107] In another aspect, the disclosure features a polypeptide including a SIRPα D1 domain variant having the following sequence: X6 is H or P; X7 is L or T; X8 is N or A; and X9 is 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.

[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 of this aspect of the disclosure, X8 is A and X1 is V or I. In any of the foregoing embodiments of this aspect of the 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 of this aspect of the disclosure, X8 is A and X1 is I. In any of the foregoing embodiments of this aspect of the 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 comprising 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, wherein each of X1, X2, X3, X4, X5, X6, X7, X8, and X9 is not a wild-type amino acid.

[0112] In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 10 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 7 amino acid substitutions relative to a 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 a 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 a 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 a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1 x 10 -8 Under M, 5x10 -9 Under M, 1x10 -9 Under M, 5x10 -10 Under M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with 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.

[0114] In another aspect, the disclosure features a polypeptide including a SIRPα D1 domain variant having the following sequence: X4 is K or R; X5 is H or P; X6 is L, T, or G; and 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, wherein X1 is V, L, or I. In any of the foregoing embodiments of this aspect of the 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 any of 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, wherein 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 K or R. In any of the foregoing embodiments, X5 is H or P. In any of 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, wherein X7 is A. In any of the foregoing embodiments of this aspect of the disclosure, X7 is A and X1 is V or I. In any of the foregoing embodiments of this aspect of the 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, wherein X7 is A. In any of the foregoing embodiments of this aspect of the disclosure, X7 is A and X1 is I. In any of the foregoing embodiments of this aspect of the 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 a SIRPα D1 domain comprising 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 polypeptides of this aspect of the disclosure comprise no more than 10 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 7 amino acid substitutions relative to a 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 greater than that of a 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 greater than that of a 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 greater than that of a 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 greater than about 100 amino acids in length. Fragments retain the ability to bind to CD47. Preferably, the SIRPα D1 domain variant polypeptides 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 fragment thereof is administered in an amount of 1×10 -8 Under M, 5x10 -9 Under M, 1x10 -9 Under M, 5x10 -10 Under M, 1x10 -10 Less than M or 1x10 -11 K less than M DIn some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with 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 disclosure features a polypeptide including a SIRPα D1 domain variant having the following sequence: X4 is K or R; X5 is H, P, or R; X6 is S, T, or G; and 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 of SEQ ID NO: 2.

[0123] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, wherein X1 is V, L, or I. In any of the foregoing embodiments of this aspect of the 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 any of 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, wherein X1 is V or I. In any of the foregoing embodiments of this aspect of the 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 any of 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, wherein X7 is A. In any of the foregoing embodiments of this aspect of the disclosure, X7 is A and X1 is V or I. In any of the foregoing embodiments of this aspect of the 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, wherein X7 is A. In any of the foregoing embodiments of this aspect of the disclosure, X7 is A and X1 is I. In any of the foregoing embodiments of this aspect of the 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 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: 212, and each of X1, X2, X3, X4, X5, X6, and X7 is not a wild-type amino acid.

[0128] In some embodiments, the polypeptides of this aspect of the disclosure comprise 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 polypeptides of this aspect of the disclosure comprise 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 greater than that of a 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 greater than that of a 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 greater than that of a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. 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 greater than about 100 amino acids in length. Fragments retain the ability to bind to CD47. Preferably, the SIRPα D1 domain variant polypeptides 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 fragment thereof is administered in an amount of 1×10 -8 Under M, 5x10 -9 Under M, 1x10-9 Under M, 5x10 -10 Under M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with 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.

[0130] Described herein, in some embodiments, is a polypeptide comprising a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9X 10 X 11 X 12 ADAGTYYCX 13 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; 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, wherein X is A and X is A. In any of the foregoing embodiments of this aspect of the disclosure, X is N. In any of the foregoing embodiments of this aspect of the disclosure, X is N. 10 In any of the foregoing embodiments of this aspect of the disclosure, X is N and X is P. In any of the foregoing embodiments of this aspect of the disclosure, X is N and X is P. 11 is any amino acid other than S, T, or C. In any of the foregoing embodiments of this aspect of the disclosure, X 11 is T. In any of the foregoing embodiments of this aspect of the disclosure, X 11 is any amino acid other than T. In any of the foregoing embodiments of this aspect of the disclosure, X 12 In any of the foregoing embodiments of this aspect of the disclosure, X is N and X 12 is any amino acid other than P.

[0132] Described herein, in some embodiments, is a polypeptide comprising a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9ITX 10 ADAGTYYCX 11 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; 10 is any other than P の It is an amino acid;X 11is V or I; wherein the SIRPα D1 domain variant comprises at least two amino acid substitutions compared to a wild-type SIRPα D1 domain variant having a sequence according to SEQ ID NO:1.

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

[0134] Without limiting the foregoing, in some embodiments, the SIRPα D1 domain variant polypeptide is selected from any one of SEQ ID NOs: 53-87 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 variant listed 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 containing Fc domain variants In some embodiments, disclosed herein are polypeptides comprising a signal-regulatory protein alpha (SIRP-α) D1 variant, wherein the polypeptide comprises a SIRPα D1 domain or a fragment thereof having an amino acid mutation at residue 80 relative to a wild-type SIRP-α D1 domain (e.g., a wild-type SIRPα D1 domain set forth 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 (e.g., a wild-type SIRPα D1 domain set forth in SEQ ID NO: 1 or 2).

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

[0139] Antibodies targeting cell surface antigens can trigger immune stimulation and effector functions associated with binding of Fc receptors (FcRs) on immune cells. There are multiple Fc receptors specific to particular classes of antibodies, such as IgG (gamma receptors), IgE (eta receptors), IgA (alpha receptors), and IgM (mu receptors). Binding of the Fc region of an antibody to an Fc receptor on the cell surface can trigger multiple biological responses, including 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), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. Furthermore, binding of the C1 component of complement to antibodies can activate the complement system. Complement activation may be important for the lysis of cellular pathogens. However, complement activation can also stimulate inflammatory responses and may 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 antibody and Fc fusion polypeptide constructs that act by targeting, activating, or neutralizing ligand function 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 to form an Fc domain with eliminated or reduced effector function.

[0141] In some embodiments, an Fc domain variant refers to a polypeptide chain comprising a second and third antibody constant domain (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. Furthermore, 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 comprises as many as 10 amino acid modifications (e.g., insertions, deletions, and / or substitutions) 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) that alter the interaction between the Fc domain and an Fc receptor.

[0142] As used herein, the term "Fc domain dimer" refers to a dimer of two Fc domains. In a wild-type Fc domain dimer, two wild-type Fc domains dimerize via interactions between 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 not contain effector function, e.g., a "dead Fc domain dimer variant." In some embodiments, each of the Fc domains in the Fc domain dimer variant comprises an amino acid substitution in the CH2 antibody constant domain to reduce interaction or binding between the Fc domain dimer variant and an Fc receptor, such as an Fcγ receptor (FcγR), an Fcα receptor (FcαR), or an Fcε (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 immunoglobulin Fc domain variant or a fragment of an Fc domain variant. In some embodiments, an immunoglobulin Fc domain variant or a fragment of an Fc domain variant can form an Fc domain dimer with another Fc domain variant. In some embodiments, an immunoglobulin Fc domain variant 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, the Fc domain variant or 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, a SIRPα polypeptide or construct provided herein comprises a SIRPα D1 domain or variant thereof linked to a first Fc domain variant and an antibody variable domain linked 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). An Fc domain dimer is a protein structure found at the C-terminus of immunoglobulins. An Fc domain dimer contains two Fc domains that are dimerized by interaction between CH3 antibody constant domains. Wild-type Fc domain dimers form the minimal structures that bind to Fc receptors, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and FcγRIV.

[0146] The Fc domain dimer is not directly involved in binding of the antibody to its target, but may be involved in various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity. In some embodiments, the Fc domain in a SIRPα polypeptide or construct of the present disclosure comprises an amino acid substitution, addition or insertion, deletion, or any combination thereof that results in a reduced effector function, such as reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated phagocytosis (ADCP), or any combination thereof. In some embodiments, the SIRPα polypeptide or construct of the present disclosure is characterized by reduced binding to human Fc receptors (e.g., minimal or no binding) and reduced binding to complement protein C1q (e.g., minimal or no binding). In some embodiments, the SIRPα constructs of the present disclosure are characterized by reduced binding (e.g., minimal or no binding) to human FcγRI, FcγRIIA, FcγRIIB, FcγRIIIB, or any combination thereof, and C1q. In some embodiments, to alter or reduce antibody-dependent effector function such as ADCC, CDC, ADCP, or any combination thereof, in some embodiments, the Fc domain in the SIRPα constructs of the 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 EU index of Kabat) (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0147] In some embodiments, polypeptide constructs comprising a non-native Fc region described herein exhibit reduced or ablated binding to at least one of the Fcγ receptors CD16a, CD32a, CD32b, CD32c, and CD64 compared to polypeptide constructs comprising a native Fc region. In some cases, the polypeptide constructs described herein exhibit reduced or ablated 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 complement component C1q binding to an antibody Fc domain. In some embodiments, polypeptide constructs comprising a non-native Fc region described herein exhibit at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more reduced C1q binding compared to a polypeptide construct comprising a wild-type Fc region. In some cases, polypeptide constructs comprising a non-native Fc region described herein exhibit reduced CDC compared to a polypeptide construct comprising a wild-type Fc region. In some embodiments, polypeptide constructs comprising a non-native Fc region described herein exhibit at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more reduced CDC compared to a polypeptide construct comprising a wild-type Fc region. In some cases, polypeptide constructs comprising a non-naturally occurring Fc domain variant or Fc domain dimer variant described herein exhibit negligible CDC compared to polypeptide constructs comprising a wild-type Fc region.

[0149] In some embodiments, the Fc domain variants or Fc domain dimer variants described herein are minimally glycosylated or have reduced glycosylation relative to the wild-type sequence. In some embodiments, deglycosylation is achieved by mutating 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, where 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, antibody IgG constant region variants (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, antibody IgG constant region variants (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 variants are mutated so that they do not contain effector functions typical of "dead" Fc domain variants. For example, in some embodiments, the Fc domain variants contain specific amino acid substitutions known to minimize interactions between Fc domain dimers and Fcγ receptors. In some embodiments, the Fc domain variants are derived from an IgG1 antibody and contain 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 in human IgG1 Fc domain variants include E318A and K322A. In some cases, the human IgG1 Fc domain variants have 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 to increase the homogeneity of the polypeptide, for example, when the polypeptide is produced in bacteria or mammalian cells. In some cases, the human IgG1 Fc domain variants have 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, e.g., 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: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[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 of the antibody's sequence at regions of homology with the "standard" Kabat numbering sequence. In some embodiments, the Fc domain variant comprises a human IgG2 Fc domain sequence (designated according to the EU numbering system according to Kabat et al. (1991)) comprising one or more of the A330S, P331S, and N297A amino acid substitutions. In some embodiments, one or more additional mutations are included in such an IgG2 Fc domain variant. Non-limiting examples of such additional mutations for human IgG2 Fc domain variants 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 of Table 7 is deleted to increase the homogeneity of the polypeptide, for example, when the polypeptide is produced in bacteria 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: ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPI EKTISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0152] Where the Fc domain variant is an IgG4 Fc domain variant, in some embodiments, such an Fc domain variant comprises an S228P mutation (designated according to Kabat et al., (1991)). In some cases, 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 following amino acid substitutions (designated according to the EU numbering system according to Kabat et al., (1991)). In some embodiments, the Fc domain variant comprises a human IgG4 Fc sequence containing one or more of the following amino acid substitutions (designated according to the EU numbering system according to Kabat et al., (1991)): S228P, E233P, F234V, L235A, delG236, and N297A.

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

[0154] In some embodiments, the Fc domain variants exhibit reduced binding to an Fc receptor of interest compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain variants exhibit abolished binding to an Fc receptor of interest compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain variants exhibit reduced phagocytosis compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain variants exhibit abolished phagocytosis 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 variant of SEQ ID NOs: 90-95, as shown in Table 7. [Table 7-1] [Table 7-2]

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

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

[0158] Fc domain variants or Fc domain dimer variants herein include those that exhibit reduced binding to Fcγ receptors compared to wild-type human IgG Fc regions. For example, in some embodiments, the Fc domain variants or Fc domain dimer variants exhibit less binding to Fcγ receptors than that exhibited by wild-type human IgG Fc regions, as described in the Examples. In some cases, the Fc domain variants or Fc domain dimer variants exhibit reduced binding to Fcγ receptors by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (effector function completely eliminated). In some embodiments, the reduced binding is to 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 reduced phagocytosis compared to the wild-type human IgG Fc region. Such Fc domain variants or Fc domain dimer variants exhibit reduced phagocytosis compared to the 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 variants or Fc domain dimer variants exhibit ablated phagocytosis compared to the wild-type human IgG Fc region.

[0160] In some embodiments, the Fc domain variants or Fc domain dimer variants disclosed herein are conjugated to one or more fusion partners. In some cases, the fusion partner is a therapeutic moiety. In some cases, the fusion partner is selected to allow for targeting, purification, screening, display, 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 in phagocytosis. As described herein, in some embodiments, when the Fc domain variants or Fc domain dimer variants are conjugated to a fusion partner, they form the polypeptide constructs described below.

[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 contains a small number of amino acids, such as fewer than 10 amino acids, although longer linkers are also utilized. In some cases, the linker has a length of 10, 9, 8, 7, 6, or 5 amino acids or less. In some cases, 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 partners to a desired cellular location or extracellular medium. In some embodiments, specific signaling sequences target proteins for secretion either to the growth medium or to the periplasmic space located between the inner and outer membranes of a cell. In some embodiments, the fusion partner is a sequence encoding a peptide or protein that allows for 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 other 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 targeted by antibodies (e.g., c-myc tags, Flag tags, etc.).

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

[0164] A variety of fusion partners are available that allow for a variety of selection methods. For example, phage display can be used by fusing members of an Fc domain variant or Fc domain dimer variant library to gene III protein. In some embodiments, the fusion partner is a labeled Fc domain variant or Fc domain dimer variant. Alternatively, in some embodiments, the fusion partner binds to a specific sequence on an expression vector, allowing the fusion partner and associated Fc domain variant or Fc domain dimer variant to be covalently or noncovalently linked to the nucleic acid that encodes them.

[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 conjugated 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 conjugated 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 to or fused to the Fc domain variants or Fc domain dimer variants of the present disclosure. Examples of CD47-binding polypeptides include, but are not limited to, anti-CD47 antibodies or fragments thereof, and CD47 ligands, 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 are polypeptides comprising an Fc domain dimer variant, wherein the Fc domain dimer variant comprises two Fc domain variants, each Fc domain variant independently selected from: (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236, and N297A. In some embodiments, the Fc domain variants are identical (i.e., homodimers). In some embodiments, the Fc domain variants are different (i.e., heterodimers). 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 eliminated or reduced binding to Fcγ receptors compared to a wild-type version of the human IgG Fc region. In some embodiments, the Fc domain dimer variant exhibits eliminated or reduced binding to CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors compared to a wild-type version of the human IgG Fc region. In some embodiments, the Fc domain dimer variant exhibits eliminated or reduced binding to C1q compared to a 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 ablated or reduced binding to Fcγ receptors compared to a wild-type human IgG4 Fc region.In some embodiments, the Fc domain dimer variant exhibits ablated or reduced binding to CD16a and CD32b Fcγ receptors compared to a wild-type version of the human IgG4 Fc region. ... -6 K exceeds M D It binds to Fcγ receptors.

[0167] In some embodiments, the Fc domain dimer variant further comprises a CD47-binding polypeptide. In some embodiments, the Fc domain dimer variant exhibits ablated or reduced binding to Fcγ receptors compared to a wild-type version of a 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 (sequence number 221), wherein 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; and X9 is V or I. In some embodiments, the SIRPα polypeptide comprises a SIRPα D1 domain variant, wherein 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, the polypeptide comprises a SIRPα D1 domain variant, wherein the SIRPα D1 domain variant is a non-naturally occurring high-affinity SIRPα D1 domain that binds to human CD47 with an affinity that is at least 10-fold higher than the affinity of a naturally occurring D1 domain and 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, wherein the Fc domain dimer variant has eliminated or reduced effector function. 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 are SIRPα D1 domain variants, wherein the SIRPα D1 domain variants have a K D The SIRPα D1 domain variant binds to the first class of CD47 with a K of less than 250 nM. Dbinds to the second type of CD47 and the K D and type 2 CD47 K D are within 100-fold of each other, and the first and second species are selected from the group consisting of human, rodent, and non-human primate. 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 are compounds that (a) bind to human CD47 at a K of less than 250 nM. D 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 mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236, and N297A. In some embodiments, the Fc domain variant is a human IgG1 Fc region consisting of mutations L234A, L235A, G237A, and N297A. In some embodiments, the Fc domain variant is a human IgG2 Fc region consisting of mutations A330S, P331S, and N297A.

[0172] The SIRPα constructs of the present disclosure comprise a SIRPα domain or variant thereof having its C-terminus linked to the N-terminus of the Fc domain or variant thereof via a linker using conventional genetic or chemical means, e.g., chemical conjugation. In some embodiments, a linker (e.g., a spacer) is inserted between the polypeptide and the Fc domain or variant thereof. In some embodiments, a polypeptide of the present disclosure comprising a SIRPα D1 domain variant is fused to an Fc domain variant that is unable to form a dimer. In some embodiments, a polypeptide of the present disclosure is fused to an Fc domain or variant thereof that can form a dimer, e.g., a heterodimer, with another Fc domain or variant thereof. In some embodiments, a polypeptide of the present disclosure is fused to an Fc domain or variant thereof, and the fusion protein forms a homodimer. In some embodiments, a polypeptide of the present disclosure is fused to a first Fc domain or variant thereof, and a different protein or peptide (e.g., an antibody variable region) is fused to a second Fc domain or variant thereof. In some embodiments, the SIRPα D1 domain or variant thereof is linked to a first Fc domain or variant thereof, and a therapeutic protein (e.g., a cytokine, interleukin, antigen, steroid, anti-inflammatory agent, or immunomodulatory agent) is linked to a second Fc domain or variant thereof. In some embodiments, the first and second Fc domains or variants thereof form a heterodimer.

[0173] Without limiting the foregoing, 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 an Fc polypeptide, or an Fc variant polypeptide, such as an Fc domain or variant thereof. Examples of polypeptides comprising SIRPα D1 domain variant polypeptides and fused Fc domain variant polypeptides 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 a 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 variant listed 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 SEQ ID NOs: 98-104, 107-113, 116-122, or 135-137 of 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 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 comprises at least two amino acid substitutions relative to a wild-type SIRPα D1 domain having a sequence set forth in any one of SEQ ID NOs: 1-10. Each Fc domain variant is independently: (i) a human IgG1 Fc region comprising an N297A mutation; (ii) a human IgG1 Fc region comprising L234A, L235A, and G237A mutations; (iii) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations; (iv) a human IgG2 Fc region comprising an N297A mutation; (v) a human IgG2 Fc region comprising A330S and P331S mutations; (vi) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations; and (vii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. Fc region; or (viii) a human IgG4 Fc region containing 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, wherein the SIRPα D1 domain variant comprises an 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 comprising L234A, L235A, G237A, and N297A mutations.

[0178] Fc domain dimerization In some embodiments, a SIRPα D1 domain variant polypeptide (e.g., any of the variants described in Tables 2, 5, and 6) is fused at either the N- or C-terminus to a first Fc domain (e.g., an Fc domain variant). In some embodiments, the first Fc domain is a variant that is unable to form a dimer. In some embodiments, the first Fc domain dimerizes 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 Fc domain and the second Fc domain.

[0179] In some embodiments, each of the two Fc domains in the Fc domain dimer contains an amino acid substitution that promotes heterodimerization of the two monomers. In some embodiments, a SIRPα construct is formed from a first subunit, such as, for example, 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 the Fc domain dimer (e.g., a single arm). In some embodiments, the construct has two SIRPα D1 domain variant polypeptides linked to the Fc domain dimer (e.g., a double arm). In some embodiments, the construct has a K of about 500 nM. DSIRPα D1 domain variants having a K of about 50 nM are particularly useful in dual-arm constructs. D SIRPα D1 domain variants having a K of about 5 nM are particularly useful in dual-arm constructs. D SIRPα D1 domain variants having a K of about 500 pM are useful in dual-arm and single-arm constructs. D SIRPα D1 domain variants having a K of about 100 pM are useful in dual-arm and single-arm constructs. D SIRPα D1 domain variants having a K of about 50 pM are useful in dual-arm and single-arm constructs. D SIRPα D1 domain variants having a K of about 10 pM are useful in dual-arm and single-arm constructs. D SIRPα D1 domain variants having the following are useful in double-arm and single-arm constructs.

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

[0181] In some embodiments, holes are constructed by replacing amino acids with larger side chains, such as tyrosine or tryptophan, with amino acids with smaller side chains, such as alanine, valine, or threonine, e.g., the Y407V mutation in the CH3 antibody constant domain. Similarly, in some embodiments, knobs are constructed by replacing amino acids with smaller side chains with amino acids with larger side chains, e.g., 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, a polypeptide of the present disclosure containing a SIRPα D1 domain variant is fused to an Fc domain containing the knob mutation T366W to limit undesired homodimer formation between the knobs. Examples of knob-into-hole amino acid pairs are listed, but are not limited to, in Table 9. Examples of knob-into-hole Fc domain variants and SIRPα-Fc fusions are listed in Table 10. [Table 9] [Table 10-1] [Table 10-2] [Table 10-3]

[0182] In addition to the knobs-into-holes strategy, in some embodiments, electrostatic steering is also used to control the dimerization of Fc domains. Electrostatic steering refers to the use of favorable electrostatic interactions between oppositely charged amino acids within peptides, protein domains, and proteins to control the formation of higher-order protein molecules. Specifically, to control the dimerization of Fc domains using electrostatic steering, one or more amino acid residues constituting the CH3-CH3 interface are replaced with positively or negatively charged amino acid residues so that the interaction is electrostatically favorable or unfavorable, depending on the specific charged amino acid introduced. In some embodiments, a positively charged amino acid at the interface, such as lysine, arginine, or histidine, is replaced with a negatively charged amino acid, such as aspartic acid or glutamic acid. In some embodiments, a negatively charged amino acid at the interface is replaced with a positively charged amino acid. In some embodiments, a charged amino acid is introduced into one or both of the interacting CH3 antibody constant domains. In some embodiments, the introduction of charged amino acids into the interacting CH3 antibody constant domains of two Fc domains promotes the selective formation of Fc domain heterodimers as 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] Other methods are available that can be used to control heterodimerization of Fc domains, particularly in the context of constructing bispecific antibodies.

[0184] In some embodiments, the first Fc domain and the second Fc domain each have the following amino acids with respect to the sequence of human IgG1: 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 amino acid substitutions.

[0185] In some embodiments, the Fc domain has: (a) one of the following amino acid substitutions relative to wild-type human IgG1: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S36 4A, 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) human IgG1 (ii) an N297A mutation for the human IgG1 Fc region; (iii) an L234A, L235A, G237A, and N297A mutation for the human IgG1 Fc region; (iv) an N297A mutation for the human IgG2 Fc region; (v) an A330S and P331S mutation for the human IgG2 Fc region; (vi) an A330S, P331S, and N297A mutation for the human IgG2 Fc region; (vii) an S228P, E233P, F234V, L235A, and delG236 mutation for the human IgG4 Fc region; or (viii) an S228P, E233P, F234V, L235A, delG236, and N297A mutation for the human IgG4 Fc region.In some embodiments, the Fc domain variant comprises: (a) one of the following amino acid substitutions relative to wild-type human IgG1: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S353S, S354D, S354E, S354F, S354H ... 64A, 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 (b)(i) human IgG1 (ii) an N297A mutation for the human IgG1 Fc region; (iii) an L234A, L235A, G237A, and N297A mutation for the human IgG1 Fc region; (iv) an N297A mutation for the human IgG2 Fc region; (v) an A330S and P331S mutation for the human IgG2 Fc region; (vi) an A330S, P331S, and N297A mutation for the human IgG2 Fc region; (vii) an S228P, E233P, F234V, L235A, and delG236 mutation for the human IgG4 Fc region; or (viii) an S228P, E233P, F234V, L235A, delG236, and N297A mutation for 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, in some embodiments, is a polypeptide comprising an Fc variant, wherein the Fc variant comprises an Fc domain dimer comprising two Fc domain variants, each Fc domain variant independently selected from: (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236, and N297A.

[0189] In this disclosure, linker is used to describe a link or connection between a polypeptide or protein domain or related non-protein moiety. In some embodiments, the linker is a link or connection between an Fc domain (or 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 the two polypeptides are linked to each other in tandem series.

[0190] In some embodiments, the linker is a simple covalent bond, e.g., a peptide bond, a synthetic polymer, or any type of bond formed by a chemical reaction, e.g., chemical conjugation. When the linker is a peptide bond, in some embodiments, a carboxylic acid group at the C-terminus of one protein domain reacts with an 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 by synthetic means using conventional organic chemistry reactions or by natural generation from host cells, and a nucleic acid molecule encoding the DNA sequences of both proteins in a tandem series (e.g., an Fc domain variant and a SIRPα D1 domain variant) can be directly transcribed and translated into consecutive polypeptides encoding both proteins in a host cell by the necessary molecular machinery (e.g., DNA polymerase and ribosomes).

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

[0192] When a linker (other than a peptide bond as described above) is created from a chemical reaction, in some embodiments, a chemical functional group (e.g., an amine, carboxylic acid, ester, azide, or other functional group) is synthetically attached to the C-terminus of one protein and the N-terminus of another protein, respectively. Then, in some embodiments, the two functional groups react via synthetic chemistry 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 includes a GS, GG, GGS, GGG, GGGGS (SEQ ID NO: 163), GGSG (SEQ ID NO: 164), or SGGG (SEQ ID NO: 165) motif, e.g., multiple or repeated motifs. In some embodiments, the spacer includes a GS motif, e.g., 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 3 to 12 amino acids including a GGS motif, e.g., GGS, GGSGGS (SEQ ID NO: 171), GGSGGSGGS (SEQ ID NO: 172), and GGSGGSGGSGGS (SEQ ID NO: 173). In some embodiments, the spacer comprises 4 to 12 amino acids including a GGSG motif (SEQ ID NO: 164), e.g., GGSG (SEQ ID NO: 164), GGSGGGSG (SEQ ID NO: 174), or GGSGGGSGGGSG (SEQ ID NO: 175). In some embodiments, the spacer comprises a GGGGS (SEQ ID NO: 163) motif, e.g., GGGGSGGGGSGGGGGS (SEQ ID NO: 176).In some embodiments, the spacer includes amino acids other than glycine and serine, for example, 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 includes a motif, e.g., multiple or repeated motifs, of EAAAK (SEQ ID NO: 193). In some embodiments, the spacer includes a motif, e.g., multiple or repeated motifs of proline-rich sequences, such as (XP)n, where X is any amino acid (e.g., A, K, or E) and n is from 1 to 5, and PAPAP (SEQ ID NO: 194). [Table 12]

[0195] In some embodiments, the length of the peptide spacer and the amino acids used are adjusted depending on the two proteins involved and the degree of flexibility desired in 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, in some embodiments, is a polypeptide comprising an Fc variant, wherein the Fc variant comprises an Fc domain dimer having two Fc domain monomers, each Fc domain monomer independently selected from: (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region comprising 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, e.g., organelles, required to express the polypeptides and fusion polypeptides described herein from their corresponding nucleic acids. In some embodiments, the nucleic acids are contained in nucleic acid vectors that are introduced into host cells by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc. In some embodiments, the choice of nucleic acid vector depends on the host cell 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, e.g., a SIRPα D1 domain variant (e.g., any of the variants 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., the Fc variant, a linker, and a fusion partner), under conditions appropriate to induce or cause expression of the polypeptide construct. In some embodiments, conditions appropriate for expression vary depending on the expression vector and host cell selected. In some embodiments, a wide variety of suitable host cells are used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. For example, various cell lines that find use 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 that have been optimized to not glycosylate proteins expressed by such cells, either by genetic engineering of the cell line or by altering cell culture conditions such as the addition of kifunensine, or by using naturally non-glycosylating hosts such as prokaryotes (e.g., E. coli), and in some cases, modification of glycosylation sequences within the Fc is not required.

[0200] Nucleic acid vector construction and host cells Nucleic acid sequences encoding the amino acid sequences of the polypeptides of the present disclosure can be prepared by a variety of methods. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. In some embodiments, 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 domains are mutated to contain specific amino acid substitutions using standard techniques, such as QuikChange™ mutagenesis. In some cases, nucleic acid molecules are synthesized using a nucleotide synthesizer or PCR techniques.

[0201] In some embodiments, nucleic acids encoding polypeptide constructs, e.g., polypeptide constructs comprising a fusion partner such as a SIRPα D1 domain variant (e.g., any of the variants listed in Tables 2, 5, and 6) and an Fc variant, are incorporated into an expression vector for protein expression. A variety of expression vectors are available for protein expression. Expression vectors can include self-replicating extrachromosomal vectors or vectors that integrate into a host genome. Vectors 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, ribosomal binding sites, signal sequences, transcriptional start and stop sequences, translational start and stop sequences, 3' and 5' untranslated regions (UTRs), and enhancer or activator sequences; an origin of replication; a selectable marker gene; and a nucleic acid sequence encoding a polypeptide of interest, and a transcription termination sequence. In some embodiments, an expression vector comprises a protein operably linked to control or regulatory sequences, a selectable marker, any fusion partner, additional elements, or any combination thereof. The term "operably linked" means that a nucleic acid is placed into a functional relationship with another nucleic acid sequence. Generally, these expression vectors contain transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the Fc variant and are typically appropriate for the host cell used to express the protein. A selection gene or marker, such as, but not limited to, an antibiotic resistance gene or a fluorescent protein gene, can be used to select host cells containing the expression vector, e.g., by antibiotic or fluorescent expression. A variety of selection genes are available.

[0202] In some embodiments, vector components or elements are optimized so that the expression vector is compatible with the host cell type. Expression vectors that find use in the present disclosure include, but are not limited to, those that allow for 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. Exemplary 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 the 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 the correct modification and processing of the expressed polypeptide. Once the vector is introduced into the host cells for protein production, the host cells are cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0205] In some embodiments, a polypeptide construct, e.g., a polypeptide construct comprising a fusion partner such as a SIRPα D1 domain variant (e.g., any of the variants provided in Tables 2, 5, and 6) and an Fc variant, is expressed in a mammalian expression system, such as a system in which the expression construct is introduced into mammalian cells using a virus, such as a retrovirus or adenovirus. In some embodiments, human, mouse, rat, hamster, or primate cells are utilized. Suitable cells also include known laboratory cells, such as, but not limited to, 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, polypeptide constructs comprising Fc domain variants are produced in insect cells, such as, but not limited to, Sf9 and Sf21 cells, or yeast cells, such as organisms from the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, and Yarrowia. In some cases, polypeptide constructs comprising Fc domain variants are expressed in vitro using cell-free translation systems. 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 will be appreciated by those skilled in the art, in vitro translation is required for some display technologies, such as ribosome display. Furthermore, in some embodiments, Fc domain variants are produced by chemical synthesis methods, such as, but not limited to, solution-phase peptide synthesis and solid-phase peptide synthesis.In vitro transcription using a non-glycosylated system such as a bacterial extract results in equivalent inactivation of the Fc, since the Fc is not glycosylated even when natural glycosylation sites are present.

[0206] In some embodiments, polypeptide constructs include unnatural amino acids, amino acid analogs, amino acid mimetics, or any combination thereof that function similarly 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), as well as pyrrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, e.g., carbons attached to hydrogen, carboxyl groups, amino groups, and R groups, e.g., homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. In some embodiments, such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but generally retain the same basic chemical structure as naturally occurring amino acids.

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

[0208] In some embodiments, protein recovery involves disruption of host cells, for example, by osmotic shock, sonication, or lysis. Once the cells are disrupted, cellular debris is removed by centrifugation or filtration. The protein can then be further purified. In some embodiments, 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, proteins are isolated and purified by appropriately selecting and combining affinity columns, such as Protein A columns (e.g., POROS Protein A chromatography), chromatography columns (e.g., POROS HS-50 cation exchange chromatography), filtration, ultrafiltration, desalting, and dialysis procedures. In some embodiments, polypeptides are 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 can be used, which corresponds to an epitope derived from the influenza hemagglutinin protein.

[0209] In some embodiments, a polypeptide of the present disclosure, e.g., a polypeptide construct comprising a fusion partner such as a SIRPα D1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and an Fc variant, is produced by cells of a subject (e.g., a human) by administering a vector, such as a viral vector (e.g., a retroviral vector, an adenoviral vector, a poxvirus vector (e.g., a vaccinia virus vector, e.g., Modified Vaccinia Ankara (MVA)), an adeno-associated virus vector, and an alphavirus vector), comprising a nucleic acid molecule encoding a polypeptide of the present disclosure, e.g., in the context of gene therapy. The vector can be used to express a polypeptide disclosed herein upon entry into the subject's cells (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.). In some embodiments, the polypeptide is secreted from the cells. In some embodiments, if treatment of a disease or disorder is the desired outcome, no further action is required. In some embodiments, if collection of protein is desired, blood is collected from the subject and the protein is purified from the blood by various methods.

[0210] Cancer treatment methods Provided herein are methods of treating cancer in an individual (e.g., a human individual), the methods comprising administering to the individual effective amounts 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, such as at least two, at least three, or at least four chemotherapeutic agents). Provided herein are methods of treating cancer in an individual (e.g., a human individual), the methods comprising administering to the individual effective amounts 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, such as at least two, at least three, or at least four chemotherapeutic agents). 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 chemotherapeutic agent in combination with one or more additional therapeutic modalities, such as, but not limited to, radiation therapy, surgery, cryoablation, and bone marrow transplantation.

[0211] Combination Therapies Including Chemotherapeutic Agents and Exemplary Chemotherapeutic Agents Exemplary chemotherapeutic agent(s) that can be used in the methods 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, ...cyclophosphamide (CYTOXAN®), abiraterone, abemaciclib, altretamine, thalidomide (THALIDOMID®), acridine carboxamide, actimid®, actinomycin, actinomycin-D, afatinib, 17-N-allylamino-17-demethoxygeldanamycin, alectinib, cyclophosphamide, cyclophosphamide, cyclophosphamide, cyclophosphamide, cyclophosphamide, cyclophosphamide, cyclophosphamide, cyclophosphamide, cyclophosphamide, cyclophosphamide, cyclophosphamide, cyclophosphamide, cyclophosphamide, cyclophosphamide, cyclophosphamide, cyclophosphamide, cyclophosphamide, cyclophosphamide, cyclophosphamide, cyclophosphamide, cyclophosphamide, cyclophos Nib, alpelisib, aminopterin, amsacrine, anlotinib, anthracycline, antitumor agent, antineoplaston, apartinib, 5-azacytidine, 6-mercaptopurine, 6-thioguanine, arabinosylcytosine, axitinib, azacitidine, azathioprine, BL22, bendamustine, binimetinib, biricodar, bleomycin, bortezomib, bosutinib, brigutinib, bryostatin, busulfan, cabozantinib, calyculin, camptothecin, capecitabine, carboplatin, carmus tin, ceritinib, chlorambucil, cisplatin, cladribine, clofarabine, cobimetinib, crizotinib, cytarabine, dabrafenib, dacarbazine, dacomitinib, dasatinib, daunorubicin, dexamethasone, dichloroacetic acid, discodermolide, docetaxel, doxorubicin, encorafenib, epirubicin, entrectinib, enzalutamide, epothilone, erdafitinib, eribulin, erlotinib, estramustine, etoposide, everolimus, exatecan, exisulind, ferruginol, floxuridine, Fludarabine, fluorouracil (such as 5-fluorouracil), folinic acid, fosfestrol, fotemustine, fruquintinib, ganciclovir, gefitinib, gemcitabine, gilteritinib, goserelin, hexamethylmelamine, hydroxycarbamide, hydroxyurea, IT-101, ibrutinib, icotinib, idarubicin, idelalisib, ifosfamide, imatinib, irinoimiquimod, irinotecan, irofulven, ivosidenib, ixabepilone, laniquidar,Lapatinib, larotrectinib, lenalidomide, lenvatinib, lorlatinib, lomustine, raltotecan, mafosfamide, masoprocol, mechlorethamine, melphalan, mercaptopurine, methotrexate, methylprednisolone, mitomycin, mitotane, mitoxantrone, nelarabine, neratinib, niraparib, nilotinib, nintedanib, oblimersen, olaparib, osimertinib, oxaliplatin, nedap platin, phenanthriplatin, picoplatin, PAC-1, paclitaxel, palbociclib, pazopanib, pemetrexed, pegfilgrastim, pentostatin, pipobroman, pixantrone, plicamycin, prednisone, ponatinib, procarbazine, proteasome inhibitors (e.g., bortezomib), pyrotinib, raltitrexed, rebeccamycin, Revlimid®, regorafenib, ribociclib, rubite Can, rucaparib, ruxolitinib, SN-38, salinosporamide A, satraplatin, sirolimus, sonidegib, sorafenib, streptozocin, streptozotocin, sunitinib, swainsonine, talazoparib, tariquidar, taxane, tegafur-uracil, temsirolimus, teniposide, temozolomide, testolactone, thiotepa, thioguanine, topotecan, trabectedin, trametinib, Examples of such anti-inflammatory drugs include tretinoin, trifluridine, triplatin tetranitrate, tris(2-chloroethyl)amine, troxacitabine, uracil mustard, valrubicin, vandetanib, vemurafenib, venetoclax (ABT-199), navitoclax (ABT-263), vinblastine, vincristine, vinorelbine, vismodegib, vorinostat, ziv-aflibercept (ZALTRAP®), and zosuquidar.

[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 adrenal inhibitor (including but not limited to, an adrenal inhibitor 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, an anthracycline 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, an alkylating agent 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 an androgen inhibitor described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with an antimetabolite, e.g., a purine analog (including but not limited to an antimetabolite described herein, e.g., a purine analog). 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 an antitumor antibiotic described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with a BLC-2 inhibitor (including but not limited to a BLC-2 inhibitor described herein).In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with a BTK inhibitor (including but not limited to, a BTK inhibitor described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with a CDK4 / 6 inhibitor (including but not limited to, a CDK4 / 6 inhibitor described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with a colony-stimulating factor (including but not limited to, a colony-stimulating factor described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with a corticosteroid (including but not limited to, a corticosteroid described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with an EGFR inhibitor (including but not limited to, an EGFR inhibitor described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with a gonadotropin-releasing hormone (GnRH) agonist (including but not limited to, a GnRH agonist described herein). In some embodiments, methods of treating cancer include administering a polypeptide described herein in combination with an antimitotic / antimicrotubule agent (including but not limited to, an antimitotic / antimicrotubule agent described herein). In some embodiments, methods of treating cancer include administering a polypeptide described herein in combination with an mTOR kinase inhibitor (including but not limited to, an mTOR kinase inhibitor described herein). In some embodiments, methods of treating cancer include administering a polypeptide described herein in combination with a proteasome inhibitor (including but not limited to, a proteasome inhibitor described herein).In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with a signal transduction inhibitor, e.g., a protein tyrosine kinase inhibitor, a PAK4 inhibitor, a PI3K inhibitor (including but not limited to, the signal transduction 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, e.g., a VEGF1 inhibitor, a VEGF2 inhibitor, and / or a VEGF3 inhibitor (including but not limited to, the VEGF inhibitors described herein). In some embodiments, methods of treating cancer include administering a polypeptide described herein in combination with an agent that modulates apoptosis, e.g., by modulating the activity of Bcl-2, Mcl1, Bcl-1x, etc. (including but not limited to, an agent that modulates apoptosis by modulating the activity of Bcl-2, Mcl1, Bcl-1x, etc. described herein). In some embodiments, methods of treating cancer include administering a polypeptide described herein in combination with a platinum-based agent (including but not limited to, a platinum-based agent 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, a 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 an siRNA or RNAi). In some embodiments, the inhibitor is a small molecule inhibitor, as described in more detail below.

[0213] In some embodiments, the chemotherapeutic agent is a small molecule anticancer agent (e.g., 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 in cells expressing CD47, e.g., 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, methods of treating cancer include administering a polypeptide described herein (e.g., a fusion polypeptide comprising a 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 in which 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 through 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. Pat. No. 10,259,859, the contents of each of which are incorporated by reference in their entirety.

[0216] Combination Therapies Including Therapeutic Antibodies, and Exemplary Therapeutic Antibodies. In some embodiments, the methods of treating cancer provided herein comprise administering to an individual 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., in combination with an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., a fusion polypeptide described herein) and 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 herein 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, Tocilizumab, Atorolimumab, Avelumab, Pinezumab, Basiliximab, Bavituximab, Bectumomab, Begelomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Bivatuzumab-mertansine mertansine, blinatumomab, brosozumab, bococizumab, brentuximab vedotin, briakinumab, brodalumab, brolucizumab, brontuximab, cabilalizumab (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, Crivatuzumab tetraxetan, Codrituzumab, Coltuximab Ravtansine, Conatumumab, Concizumab, Crenezumab, CR6261, Dacetuzumab, Daclizumab, Dalotuzumab, Dapirolizumab pegol, Daratumumab, Dectrekumab, Demcizumab, Denintuzumab mafodotin, Denosumab, Derlotuximab biotin Biotin, Detumomab, Dinutuximab, Diridavumab, Dorlimomabaritox, drozitumab, durigotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab izumab, Efungumab, Eldelumab, Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab (RG7155), Emibetuzumab, Enavatuzumab, Enfortumab vedotin, Enlimomab pegol, Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epitumomab situxetanCituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, Ficlatuzumab Ficlatuzumab, Figitumumab, Filibuumab, Flanvotumab, Fletikumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab-ozogamicin 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 ozogamicin, ipilimumab, iratumumab, isatuximab, itolizumab, ixekizumab, keliximab, labetuzumab, lambrolizumab, lampalizumab, lebrikizumab, lemaresomab, lenzilumab, lerdelimumab, lexatumumab, ribivirumab, rifastuzumab vedotin vedotin, Ligelizumab, Lilotomab satetlaxetan, Lintuzumab, Lirilumab, Lodelcizumab, Lokivetmab, Lorvotuzumab mertansine, Lucatumumab, Lulizumab pegolpegol), Lumiliximab, Lumretuzumab, MSB0010718C (Avelumab), Mapatumumab, Margetuximab, Maslimomab, Mavrilimumab, Matuzumab, MEDI6469, MEDI0680, MEDI 6383, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Mogamulizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentane merpentan, obiltoxaximab, obinutuzumab, ocralizumab, ocrelizumab, odulimomab, ofatumumab, olaritumab, olokizumab, omalizumab, onartuzumab, ontuxizumab, opicinumab Opicinumab, Oportuzumab monatox, Oregovomab, Orticumab, Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab, Pancomab, Panobacumab, Pulsatilla Operandi Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, PolatuzumabVedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO140, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab , Refanezumab, Regavirumab, Reslizumab, Rilotumumab, Linucumab, Rituximab, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Sacituzumab govitecan govitecan), samalizumab, SAR650984 (isatuximab), sarilumab, satumomab pendetide 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, tanezumab, taplitumomab paptox, tarextumab, tefibazumab, telimomab aritox, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, TGN1412, ticilimumab (tremelimumab), tildrakizumab, tigatuzumab, TNX-650, tocilizumab ab) (atlizumab), toralizumab, toripalimab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, trastuzumab-emtansine, TRBS07, tregalizumab, tremelimumab, tucotuzumab-celmoleukin Celmoleukin, Tuvirumab, Ublituximab, Ulocuplumab, Urelumab, Urtoxazumab, Ustekinumab, Utomilumab (PF-05082566), Vandortuzumab vedotinvedotin, vantictumab, vanucizumab, vapaliximab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vonlerolizumab (RG7888), borsetuzumab-mafodotin These include rituximab, zalutumumab, zanolimumab, zatuximab, ziralimumab, or zolimomab aritox, as well as 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 herein are antibodies, including, but not limited to, 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, and anti-CD19 antibodies. Examples of antibodies include D56 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, and antibodies designed to bind to tumor cells, virus- or bacteria-infected cells, immune cells, or healthy normal cells, or cytokines, chemokines, or hormones of any kind.

[0219] In some embodiments, the therapeutic antibody used in the methods herein is selected from the group consisting of, 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, TGFbeta2, TGFbeta1, TFPI, tenascin-C, TEM1, TAG-72, T cell receptor, STEAP1, sphingosine-1-phosphate, S OST, SLAMF7, BCL-2, selectin P, SDC1, sclerostin, RTN4, RON, Rh factor, RHD, respiratory syncytial 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, neuronal apoptosis-regulating protease 1 (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-inducible protein, integrin αvβ3, integrin αIIβ3, integrin α7β7, integrin α5β1, integrin α4β7, integrin α4, 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, IGF-1, IgG4, IGF-1, IGF-1 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, episialin, EpCAM, endotoxin, EGFR, EGFL7, Shiga toxin-producing Escherichia coli type 2 (E. coli shiga toxin) type-2), Shiga toxin-producing E. coli type I (E. coli shiga toxin type-I), DRS, DPP4, DLL4, dabigatran, cytomegalovirus glycoprotein B, CTLA-4, CSF2, CSF1R, clumping 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 (IL-2 receptor chain), 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 familiarisAn 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 antibody used in the methods herein binds to an antigen expressed by (eg, expressed on the surface of) a cancer cell. 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, β-catenin, TGF-βRII, HPV E6, or HPV E7. For example, in some embodiments, a polypeptide described herein is administered in combination with a chemotherapeutic agent (e.g., at least one chemotherapeutic agent) and a monoclonal antibody that binds to CD123 (e.g., also known as IL-3 receptor alpha), such as talacuzumab (also known as CSL362 and JNJ-56022473).

[0221] In some embodiments, the therapeutic antibody (e.g., therapeutic monoclonal antibody) used in the methods herein is an antibody that binds to an antigen 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), C D16 (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 Including Immunotherapeutic Agents and Exemplary Immunotherapeutic Agents In some embodiments, the methods of treating cancer provided herein include administering to an individual 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., in combination with an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., a polypeptide described herein) and 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, immunotherapeutic agents refer to any therapeutic agent that targets the immune system and promotes therapeutic redirection of the immune system, such as modulators of costimulatory pathways, cancer vaccines, recombinant modified immune cells, etc. Exemplary, non-limiting immunotherapeutic agents are described below. In some embodiments, the immunotherapeutic agent is 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] Immunotherapeutic agents that are approved or in late-stage clinical trials 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 a polypeptide described herein) is administered in combination with a PD-L1 / PD-1 pathway inhibitor, e.g., 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 PD-L1 / PD-1 pathway inhibitor is an antisense polynucleotide. In some embodiments, the PD-L1 / PD-1 pathway inhibitor is an anti-PD-1 or anti-PD-L1 antagonist antibody (e.g., an anti-PD-1 or anti-PD-L1 antagonist antibody described elsewhere herein). As demonstrated herein, the combined administration of an agent that blocks the interaction between CD47 and SIRPα (such as a polypeptide described herein) with an inhibitor of the PD-L1 / PD-1 pathway can result in synergistic anti-tumor activity. In some embodiments, the immunotherapeutic agent is or includes a vaccine, an oncolytic virus, an adoptive cell therapy, a cytokine, or a small molecule immunotherapeutic agent. Examples of such immunotherapeutics are known in the art. For example, adoptive cell therapies and therapeutic agents can 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 can include, but are not limited to, polynucleotide vaccines, polypeptide vaccines, or cell-based (e.g., tumor- or dendritic cell-based) vaccines. Various cytokines are known to be useful in the treatment of cancer, including, but not limited to, IL-2, IL-15, IL-7, IL-10, IL-12, IL21, TNFα, IFN, GM-CSF, and engineered cytokine mutants.Small molecule immunotherapeutics can 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 additional agent(s) described herein (e.g., a therapeutic antibody, a small molecule inhibitor, an immunotherapeutic agent, etc.), where the additional agent(s) are of a different class and / or exert their anti-cancer effect through a different mechanism 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, e.g., 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 (such as, but not limited to, those described herein) and a small molecule inhibitor (such as, but not limited to, those described herein). Other combinations are also 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, an antidiarrheal, an antiemetic, an analgesic, an opioid, and / or a nonsteroidal anti-inflammatory agent.

[0227] Combination therapy including additional treatment modality(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 therapeutic modalities. In some embodiments, the one or more additional therapeutic modalities include radiation therapy (e.g., gamma rays, X-rays, and / or direct delivery of radioisotopes 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 inducers of cell proliferation (oncogenes), inhibitors of cell proliferation (tumor suppressors), or inducers of programmed cell death (pro-apoptotic genes). In some embodiments, any one or more combination therapies described herein are administered in conjunction with surgery (e.g., resection).

[0228] Exemplary Therapeutic Combinations In some embodiments, the method of treating cancer includes 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, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, pemetrexed, nilotinib, abiraterone, pemetrexed, nilotinib, abiraterone, pemetrexed, nilotinib, nivolumab ... Zalutamide, 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, the method of treating cancer includes 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 a SIRPα d1 domain variant and an Fc variant).

[0230] In some embodiments, the 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, entrectinib, cefotaxime ... 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).

[0231] In some embodiments, the 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, entrectinib, cefotaxime ... 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).

[0232] In some embodiments, the method of treating cancer includes 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, entrectinib, cefotaxime ... 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).

[0233] In some embodiments, the 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, entrectinib, cefotaxime ... 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).

[0234] In some embodiments, the method of treating cancer includes 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, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, pemetrexed, nilotinib, abiraterone, pemetrexed, nilotinib ... Zalutamide, 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, lung squamous cell carcinoma, 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, parapharyngeal cancer, gastrointestinal cancer, glioma, liver cancer, gastric cancer, oral cancer, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, renal 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, pancreatic islet cell tumor, neuroendocrine tumor, prostate cancer, glioblastoma, endometrial carcinoma, endometrial cancer, leiomyosarcoma, gallbladder cancer, hepatocellular carcinoma, melanoma, or a 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 myelogenous leukemia, acute or chronic lymphoblastic leukemia, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), chronic myeloid leukemia (CML), hairy cell leukemia, chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), large granular lymphocytic (LGL) leukemia, These include 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's lymphoma (such as diffuse large B-cell lymphoma (DLBCL)), Burkitt lymphoma, mantle cell lymphoma (MCL), peripheral T-cell lymphoma (PTCL), lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, marginal zone lymphoma (MZL), and follicular lymphoma (FL).

[0237] Treatment methods for leukemia In some embodiments, the individual (e.g., a human individual) is diagnosed with leukemia (e.g., acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), myelodysplastic syndromes (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), Methods of treating lymphomas including multiple myeloma (MM), non-Hodgkin's lymphoma (such as diffuse large B-cell lymphoma (DLBCL)), Burkitt's lymphoma, mantle cell lymphoma (MCL), peripheral T-cell lymphoma (PTCL), lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, marginal zone lymphoma (MZL), and follicular lymphoma (FL) are provided, the methods comprising administering to an individual effective amounts 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 L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat. 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 Bcl2 inhibitor (e.g., venetoclax) are administered simultaneously, concurrently, or sequentially.

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

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

[0240] The CAS Registry Number for 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 can be found in the local package insert (in the United States, see, e.g., www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2016 / 208573s000lbl(dot)pdf; in Europe, see, e.g., www(dot)ema(dot)europa(dot)eu / en / medicines / human / EPAR / venclyxto#product-information-section). In some embodiments, venetoclax is administered according to the dosing and frequency recommended in the local package insert.

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

[0242] Another exemplary selective Bcl2 inhibitor for use in the methods described herein is navitoclax (also known as ABT-263). Navitoclax inhibits both Bcl2, Bcl-xL, and Bcl-w and has the empirical formula C 47 H 55 It has a molecular weight of 974.6 g / mol. The CAS registry number for navitoclax is 923564-51-6. ABT-737 is chemically described 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. [ka]

[0243] Another exemplary selective Bcl2 inhibitor for use 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 indicates no significant binding to MCL-1 and BFL-1 (BCL2A1 / A1), and poor affinity for BCL-XL. S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile indicates no significant binding to MCL-1, BFL-1 (BCL2A1 / A1), and poor affinity for BCL-XL. S55746 is not cytotoxic to BCL-XL-dependent cells, such as platelets (see, e.g., Casara et al. (2008) Oncotarget. 9(28):29975-20088). S55746 has the empirical formula C 43 H 42 NO and has a molecular weight of 710.82 g / mol. The CAS Registry Number for 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: [ka]

[0244] Treatment of solid tumors In some embodiments, provided are methods of treating a solid tumor in an individual (e.g., a human individual), the method comprising administering to the individual effective amounts 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., colon cancer), lung cancer, head and neck cancer, esophageal cancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer, testicular cancer, brain cancer, 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 L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat. In some embodiments, the polypeptide (eg, 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., fusion polypeptide) forms a homodimer. In some embodiments, the polypeptide (e.g., fusion polypeptide) and the platinum-based chemotherapeutic agent (e.g., cisplatin) are administered simultaneously, concurrently, or sequentially.

[0245] Platinum drugs (e.g., carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin) are widely used antitumor drugs that induce DNA crosslinking as monoadducts, interstrand crosslinks, intrastrand crosslinks, or DNA-protein crosslinks. Platinum drugs typically act at the adjacent N-7 position of guanine, forming 1,2 intrastrand crosslinks (Poklar et al. (1996). Proc. Natl. Acad. Sci. USA 93(15):7606-11; Rudd et al. (1995). Cancer Chemother. Pharmacol. 35(4):323-6). The resulting crosslinks inhibit DNA repair and / or DNA synthesis in cancer cells.

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

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

[0248] Carboplatin is another exemplary platinum coordination compound for use in the methods described herein. The chemical name of carboplatin is platinum, diamine[1,1 cyclobutane-dicarboxylato(2-)-0,0]-, (SP-4-2), and carboplatin has the following structural formula: [ka]

[0249] Carboplatin has the molecular formula CH 12Carboplatin is a water-soluble platinum complex with a molecular weight of 373.26 and a molecular weight of N2O4Pt. Carboplatin has been assigned the CAS Registry Number 41575-94-4 and its mechanism of action is similar to that of cisplatin. Carboplatin is typically more commonly prescribed than cisplatin. Carboplatin is commercially available as PARAPLATIN®, BLASTOCARB®, BLASTOPLATIN®, CARBOKEM®, CARBOMAX®, PARAPLATIN®, CARBOPA®, KARPLAT®, and others. Complete information regarding the preparation, dispensing, dosage, and administration schedule of carboplatin can be found in the national package insert (in the United States, see, e.g., 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 dosage and frequency recommended in the national package insert.

[0250] In some embodiments, provided are methods of treating a solid tumor in an individual (e.g., a human individual), the method comprising administering to the individual effective amounts 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., 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 L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat. 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-HER2 antibody, and anti-PD-L1 antibody (e.g., anti-PD-L1 antagonist antibody) are administered simultaneously, concurrently, or sequentially. 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 solid tumor is HER2. + In some embodiments, the solid tumor is a colon cancer (e.g., HER2 + colon cancer).

[0251] Treatment for gastric or gastroesophageal junction (GEJ) cancer In some embodiments, methods of treating gastric cancer or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual) are provided, the methods comprising administering to the individual effective amounts 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, a polypeptide (eg, 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 L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat. 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-HER2 antibody, anti-VEGFR2 antibody, and paclitaxel are administered simultaneously, concurrently, or sequentially. In some embodiments, the polypeptide (e.g., fusion polypeptide) is administered to the individual at a dose of 10 mg / kg once weekly or 15 mg / kg once weekly. In some embodiments, the individual being treated has gastric or GEJ adenocarcinoma. In some embodiments, the individual being treated has HER2. + Gastric cancer or HER2 + GEJ cancer (e.g., HER2-overexpressing gastric or GEJ cancer). In some embodiments, HER2 + Gastric cancer or HER2 + The GEJ cancer is advanced and / or metastatic. In some embodiments, the individual being treated has gastric or GEJ cancer that has progressed during or after previous treatment(s) comprising an anti-HER2 antibody (e.g., trastuzumab). In some embodiments, the individual being treated has gastric or GEJ cancer that has progressed during or after previous treatment(s) comprising an anti-HER2 antibody (e.g., trastuzumab) and a fluoropyrimidine. In some embodiments, the individual being treated has gastric or GEJ cancer that has progressed during or after previous treatment(s) comprising an anti-HER2 antibody (e.g., trastuzumab) and a platinum-based chemotherapy agent. In some embodiments, the individual being treated has gastric or GEJ cancer (e.g., HER2) that has progressed during or after previous treatment(s) comprising an anti-HER2 antibody (e.g., trastuzumab) and / or a fluoropyrimidine, and / or a platinum-based chemotherapy agent. +In some embodiments, the individual has cancer of the esophagus (e.g., gastric or GEJ cancer). In some embodiments, the individual has failed (e.g., relapsed or failed to respond to) previous treatment with an anti-HER2 antibody, an anti-HER2 antibody and a fluoropyrimidine, or an anti-HER2 antibody and a platinum-based chemotherapy agent. In some embodiments, the fluoropyrimidine is fluorouracil (also known as 5-fluorouracil). In some embodiments, treatment with the polypeptide, anti-HER2 antibody, anti-VEGFR2 antibody, and paclitaxel results in no 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, methods are provided for treating gastric cancer or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual), the methods comprising administering to the individual effective amounts 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, methods of treating gastric cancer or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual) are provided, the methods comprising administering to the individual effective amounts 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 L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat. 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 advanced and / or metastatic. In some embodiments, the individual has not received previous treatment for gastric cancer or GEJ cancer.

[0253] Treatment methods for head and neck cancer In some embodiments, methods of treating head and neck cancer (e.g., head and neck squamous cell carcinoma or HNSCC) in an individual (e.g., a human individual) are provided, the methods comprising administering to the individual effective amounts 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, cemiplimab, 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, or phototrexate. 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, a polypeptide (eg, 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 L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat. 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), PD-1 inhibitor (e.g., an anti-PD-1 antibody, e.g., pembrolizumab), an antimetabolite (e.g., 5-fluorouracil), and platinum-based chemotherapy (e.g., cisplatin or carboplatin) are administered simultaneously, concurrently, or sequentially. In some embodiments, the polypeptide (e.g., fusion polypeptide) is administered to the individual at a dose of 10 mg / kg once weekly or 15 mg / kg once weekly. In some embodiments, the individual receiving treatment has HNSCC. In some embodiments, the HNSCC is advanced and / or metastatic HNSCC. In some embodiments, the HNSCC is unresectable and / or recurrent. In some embodiments, the individual has not received previous treatment for head and neck cancer (e.g., HNSCC). 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) does not result in adverse effects.In some embodiments, treatment with polypeptides, PD-1 inhibitors (e.g., pembrolizumab), antimetabolites (e.g., 5-fluorouracil), and platinum-based chemotherapeutic agents (e.g., cisplatin or carboplatin) results in only mild adverse effects.

[0254] Combination cancer therapy including anti-TROP2 antibodies In some embodiments, provided are methods of treating cancer in an individual (e.g., a human individual), the method comprising administering to the individual effective amounts 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, described in U.S. Patent No. 10,179,171, the contents of which are incorporated herein by reference in their 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 US 2017 / 0281791, the contents of which are incorporated herein by reference in their 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 L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat.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, concurrently, or sequentially. In some embodiments, the cancer is a solid tumor, gastric cancer, nasopharyngeal carcinoma, gallbladder cancer, cervical cancer, extranodal NK / T-cell lymphoma, lung cancer, laryngeal squamous cell carcinoma, colon cancer, perihilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrioid endometrial carcinoma, 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] Methods for increasing phagocytosis of target cells In some embodiments, provided are methods of increasing phagocytosis of target cells (e.g., cancer cells), the methods 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, described in U.S. Patent No. 10,179,171, the contents of which are incorporated herein by reference in their 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 US 2017 / 0281791, the contents of which are incorporated herein by reference in their 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 L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat.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, a gastric cancer cell, a nasopharyngeal carcinoma cell, a gallbladder cancer cell, a cervical cancer cell, an extranodal NK / T-cell lymphoma cell, a lung cancer cell, a laryngeal squamous cell carcinoma cell, a colon cancer cell, a perihilar cholangiocarcinoma cell, a pancreatic cancer cell, an oral squamous cell carcinoma cell, an endometrioid endometrial cancer cell, or an ovarian cancer cell.

[0256] In some embodiments, methods of increasing phagocytosis of target cells are provided, the methods 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 capable of enhancing 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 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 L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat. 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 second agent enhances phagocytosis, for example, by blocking a "don't eat me" signal.Exemplary agents include, but are not limited to, for example, an anti-LILRB2 antibody, an anti-LILRB1 antibody, an anti-SIGLEC-10 antibody, an anti-CD24 antibody, an anti-SIRPα antibody, an anti-PD1 antibody (e.g., an anti-PD1 antagonist antibody), and an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody). In some embodiments, the second agent enhances phagocytosis, for example, by enhancing the "eat me" signal. Exemplary agents include, but are not limited to, for example, a BTK activator, a TLR agonist, an agent that promotes the interaction between Mac-1 and SLAMF7, or 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 modulates podosome adhesion, an agent that modulates 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 a 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) the fusion polypeptide and (b) a BTK activator. In some embodiments, the method comprises contacting the target cell with (a) the 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 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) two or more agents capable of enhancing phagocytosis (such as, but not limited to, two or more agents described herein). 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), (b) and 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 the target cell with (a) the 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 the target cell with (a) the 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 occurs in vitro. In some embodiments, the contacting occurs in vivo. In some embodiments, the target cell is a cancer cell. In some embodiments, contacting target cells with (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) one or more agents capable of enhancing phagocytosis increases phagocytosis of the target cells 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 compared to contacting the target cells with one or more agents capable of enhancing phagocytosis (i.e., in the absence of 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)).

[0259] Kits and manufactured products In another embodiment of the present invention, an article of manufacture or kit is provided that includes a polypeptide (e.g., a fusion polypeptide described herein) comprising a 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 numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations (wherein numbering is according to EU index of Kabat); or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein numbering is according to EU index of Kabat). 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 article of manufacture is for use in accordance with the methods of treatment 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 is used to treat a 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 syndromes (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 lymphoma (T-cell lymphoma), or leukemia (e.g., leukemia ... The package insert or labeling contains instructions for using the polypeptide (e.g., fusion polypeptide) in combination with a BCL2 inhibitor (e.g., venetoclax) to treat or delay the progression of prolymphocytic leukemia (T-PLL), multiple myeloma (MM), and non-Hodgkin's lymphoma (such as diffuse large B-cell lymphoma (DLBCL)), Burkitt's lymphoma, mantle cell lymphoma (MCL), peripheral T-cell lymphoma (PTCL), lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, marginal zone lymphoma (MZL), and follicular lymphoma (FL), etc.

[0261] In some embodiments, the kit or article of manufacture 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 containing instructions for using the polypeptide (e.g., the fusion polypeptide) in combination with the platinum-based chemotherapeutic agent (e.g., cisplatin) to treat or delay the progression of a solid tumor (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 article of manufacture 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 containing instructions for using the polypeptide (e.g., a fusion polypeptide) in combination with an anti-HER2 antibody (e.g., trastuzumab), a PD-L1 inhibitor (e.g., atezolizumab, avelumab, or durvalumab) to treat or delay the progression of cancer (e.g., a solid tumor) in an individual (e.g., a human individual). In some embodiments, the cancer (e.g., a 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 cancer (e.g., solid tumor) is HER2 + In some embodiments, the cancer is colon cancer (e.g., HER2 + colon cancer).

[0263] In some embodiments, the kit or article of manufacture 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 the polypeptide (e.g., the fusion polypeptide) in combination with the anti-HER2 antibody (e.g., trastuzumab), an anti-VEGFR2 antibody (e.g., ramucirumab), and paclitaxel to treat or delay the progression of gastric cancer or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual), e.g., by a method described herein.

[0264] In some embodiments, the kit or article of manufacture 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 the polypeptide (e.g., the fusion polypeptide) in combination with the anti-HER2 antibody (e.g., trastuzumab), the PD-1 inhibitor (e.g., pembrolizumab), 5-fluorouracil, and a platinum-based agent (e.g., cisplatin or carboplatin) to treat or delay the progression of gastric cancer or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual). In some embodiments, the kit or article of manufacture 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 the polypeptide (e.g., the fusion polypeptide) in combination with the anti-HER2 antibody (e.g., trastuzumab), the PD-1 inhibitor (e.g., pembrolizumab), capecitabine, and a platinum-based agent (e.g., cisplatin or carboplatin) to treat or delay the progression of gastric cancer or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual).

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

[0266] In some embodiments, the kit or article of manufacture further comprises a therapeutic anti-TROP2 antibody. In some embodiments, the anti-TROP2 antibody is RS7 (see, e.g., U.S. Pat. No. 10,179,171) or sacituzumab govitecan. In some embodiments, the kit also includes an antibody for the treatment of TROP2 in an individual (such as a human individual). +The package insert or label includes instructions for using the polypeptide (e.g., the fusion polypeptide) in combination with an anti-TROP2 antibody (e.g., cisplatin) to treat or delay the progression of cancer (e.g., solid tumors, gastric cancer, nasopharyngeal carcinoma, gallbladder cancer, cervical cancer, extranodal NK / T-cell lymphoma, lung cancer, laryngeal squamous cell carcinoma, colon cancer, perihilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrioid endometrial carcinoma, or ovarian cancer).

[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 embodiments above) 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 in separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers can be formed from a variety of 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 a label on or associated with the container can indicate instructions for use. The article of manufacture or kit can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further includes one or more additional agents (e.g., chemotherapeutic and anti-tumor agents, therapeutic antibodies, etc.). Suitable containers for one or more agents include, for example, bottles, vials, bags, and syringes.

[0268] The specification is considered to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall 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. [Example]

[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. It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are within the spirit and scope of the present application and the scope of the appended claims.

[0270] Example 1A: Antitumor activity of Drug A in combination with venetoclax in an acute leukemia model. In this example, the anti-tumor activity of Drug A, an exemplary polypeptide comprising a SIRPα d1 domain variant and an Fc variant, was evaluated in combination with venetoclax in an RS4;11 xenograft model.

[0271] Materials and Methods RS4;11 xenograft model RS4;11 cells (described in Stong et al. (1985) Blood. 65(1):21-31) were cultured at 5 × 10 6 The tumors were injected into the right flank of female NOD-SCID mice at a concentration of 100 cells / mouse. The average size of all tumors was 190 mm. 3Tumors were monitored until tumor size reached 100%. Mice were randomized into PBS control, venetoclax (Selleckchem), drug A, and venetoclax / drug A combination cohorts, with 10 mice per cohort. Venetoclax was formulated in a 2.5:5:10:20:67.5 by volume ratio of DMSO:ethanol:Cremophor EL:dextrose in water 5% (D5W). Venetoclax-treated mice were administered 250 μg of venetoclax by oral gavage twice, 3 days apart. Drug A-treated mice were administered 10 mg / kg of venetoclax IP four times, 3–4 days apart. Venetoclax / drug A-treated mice were administered 250 μg of venetoclax by oral gavage twice, 3 days apart, and then 1 day after venetoclax administration, 10 mg / kg of drug A was administered 4 times, 3-4 days apart. Tumors were measured in two dimensions using calipers, and tumor volume was calculated as length × width × width × 0.5: length was the larger of the two measurements.

[0272] result Single-agent venetoclax inhibited tumor growth (see Figure 1A), while single-agent drug A had no appreciable effect on tumor growth. The combination of venetoclax and drug A inhibited tumor growth to a greater extent than venetoclax alone (see Figure 1A). At day 41, 1 in 10 mice treated with venetoclax alone was tumor-free ("TF"), while 6 in 10 mice treated with the venetoclax / drug A combination were TF (Figure 1A).

[0273] Venetoclax-treated mice (n = 10) were then divided into two groups (n = 5 / group) and either (a) retreated with single-agent venetoclax on day 45 or (b) treated with a combination of venetoclax (administered on day 45) and drug A (administered on day 46). As shown in Figure 1B, in mice previously treated with venetoclax, treatment with venetoclax in combination with drug A suppressed tumor growth to a greater extent than retreated treatment with venetoclax alone. The mean tumor volume at day 65 in venetoclax-retreated mice was approximately 1685 mm. 3whereas the mean tumor volume at day 65 in the combination-treated mice was approximately 970 mm 3 Mice that showed tumor regression when treated with single-agent venetoclax received single-agent venetoclax on day 45. Notably, tumor regrowth was observed in these mice.

[0274] Example 1B: Effect of Drug A in Combination 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 in combination 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 columns (Miltenyi Biotec) according to the manufacturer's protocol. + Monocytes were seeded at 6 million cells per dish in 25 mL 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®. Cells were cultured for 7–11 days.

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

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

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

[0279] As shown in Figure 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 drug A-treated cells with 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 or venetoclax alone. Similar results were observed in OCI-AML3 cells using 20 nM drug A and 1 μM venetoclax. See Figure 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 the CT26 syngeneic mouse colon cancer model. See, e.g., 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 similar model CT26 cells (see Wang et al. (1995) J. Immunol. 154:4685-4692) were cultured in RPMI 1640 at 5 × 10 cells per mouse. 5 The cells were injected into the right flank of BALB / c female mice at a concentration of 1000 x 1000 cells / mL. The average size of all tumors was 65-70 mm. 3 Tumors were monitored until tumor size reached 100%. Mice were randomized into PBS control, cisplatin (Selleckchem), drug A, and cisplatin / drug A combination cohorts, with 5–10 mice per cohort. Drug A was administered intraperitoneally (IP) at a dose of 30 mg / kg ("mpk") twice. Two 30 mpk doses were given 10 days apart. Cisplatin was administered IP according to one of two regimens: one 10 mpk dose or two 5 mpk doses. Two 5 mpk doses of cisplatin were given 10 days apart. Mice receiving both cisplatin and drug received cisplatin (IP) according to one of the regimens listed above, followed by drug A as described above. Mice receiving combination treatment received drug A one day after cisplatin. Tumors were measured in two dimensions using calipers, and tumor volume was calculated as length × width × width × 0.5: length is the larger of the two measurements.

[0282] result As shown in Figure 2A, on day 20, tumor growth was somewhat inhibited in mice treated with single-agent cisplatin (two 5 mpk doses each given 10 days apart), whereas drug A had no appreciable effect on tumor growth. Treatment with cisplatin in combination with drug A delayed CT26 tumor growth 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 over the course of treatment than mice administered cisplatin alone. Furthermore, in each of the PBS control, cisplatin, and drug A treatment groups, only 10% of mice developed tumor volumes <500 mm 3 In the cisplatin + drug A treatment group, 33% of mice had tumor volumes <500 mm 3 had the following characteristics:

[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 a combination of cisplatin and drug A delayed CT26 tumor growth 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 body weight changes (*p<0.0106 and **p<0.0021, two-tailed t-test performed between cisplatin and drug A + cisplatin treatment groups on days 24 and 27, respectively). See Figure 2D.

[0284] Example 3: Phagocytic activity of Drug A in combination with anti-TROP2 antibody DLD-1 cells were washed twice with 20 ml of PBS and detached from the culture plate by incubation with 10 ml of TRYPLE™ Select (Gibco) cell dissociation enzyme at 37°C for 10 minutes. The detached cells were then centrifuged, washed with PBS, and resuspended in culture medium. The cells were labeled with the fluorescent label provided in the CELLTRACE™ CFSE Cell Proliferation kit (Thermo Fisher) according to the manufacturer's instructions and resuspended in IMDM (Iscove's Modified Dulbecco's Medium). Macrophages were detached from the culture plate by washing twice with 20 ml of PBS and incubating with 10 ml of TRYPLE™ 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] Phagocytosis assays were assembled in ultra-low attachment U-bottom 96-well plates (Corning) containing 100,000 DLD-1, 50,000 macrophages, 5-fold serial dilutions of drug A or negative control antibody from 100 nM to 6.4 pM, and 0.01 μg / ml anti-TROP2 antibody. The plates were incubated for 2 hours at 37°C in a humidified incubator containing 5% carbon dioxide. Cells were then pelleted by centrifugation at 400 x g for 5 minutes and washed with 250 μl of FACS buffer. Macrophages were stained for 15 minutes on ice 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 label (Biolegend), and 0.5 μl of anti-CD206 conjugated to allophycocyanin-Cy7 label (Biolegend). Cells were then washed with 200 μl of FACS buffer, 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. Cells were then washed twice with 250 μl of FACS buffer and fixed overnight with 0.5% paraformaldehyde. Fixed cells were analyzed using a FACS CANTO II™ (BD Biosciences) fluorescence-activated cell sorting analyzer, followed by data analysis using FlowJo 10.7 (Treestar) flow cytometry software. Dead cells were excluded by gating on 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 in combination with anti-TROP2. Figure 3 shows the percentage of macrophages that phagocytosed CFSE-labeled tumor cells on the y-axis. Macrophages were incubated with the indicated concentrations of Drug A and 10 ng / mL of anti-TROP2 antibody. Cells were also incubated with 10 ng / mL of anti-TROP2 antibody alone, a negative control combination of 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 in combination with anti-TROP2 antibody. See Figure 3.

[0287] Example 4: Antitumor activity of Drug A in combination with trastuzumab and anti-PD1 antibody in a colon cancer model. MC38m / h HER2 cells were generated by infecting MC38 mouse colon carcinoma cells with a lentiviral vector encoding a chimera of 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) at 37°C in a 5% CO2 incubator. All tissue culture was performed under sterile conditions.

[0288] Prior to implantation, a master cell bank was generated for each cell line to ensure that cells used in subsequent experiments were at the same passage number. Cells were harvested and washed twice with 50 mL of cold PBS (Life Technologies 10010072). After the final wash, for the MC38m / h HER2 cell line, cells were diluted to 5 × 10 6 The cells were resuspended in PBS or RPMI at 100 cells / mL. For MC38m / h HER2, 100 μL of the cell suspension was injected subcutaneously into the right flank of C57BL / 6 mice. MC38m / h HER2 tumors had an average tumor size of 65–69 mm. 3When the NIH score reached 0.01, the animals were randomized into eight groups of 10 mice. Each group was assigned to a treatment group as outlined in Table A: [Table 13]

[0289] Tumor volume (mm ) was measured using a Mitutoyo Digital Caliper (Mitutoyo America, Aurora, Illinois). 3 ) and body weight were recorded two or three times a week. 3 Mice that exceeded 100 mg / kg or lost 20% of their body weight were euthanized according to IACUC guidelines. Tumor volume was calculated ([length × {width × width}] × 0.5 = volume (mm 3 )) Statistical analyses and p-values were calculated using GraphPad Prism software.

[0290] A chimeric m / h HER2 construct containing the extracellular domain of human HER2 and the intracellular domain of mouse HER2 was expressed in MC38 colon cells, allowing the activity of trastuzumab against MC38 mouse tumors to be evaluated. As shown in Figure 5, monotherapy with trastuzumab had no effect on tumor growth, whereas monotherapy with drug A and anti-PD-L1 antibody each had a moderate effect on tumor growth. Treatment with drug A plus an anti-PD-L1 antibody doublet or trastuzumab plus an anti-PD-L1 antibody doublet demonstrated improved tumor growth inhibition compared with monotherapy alone. Treatment with the triple combination of drug A plus anti-PD-L1 plus trastuzumab demonstrated improved tumor inhibition compared with each doublet. The effect of the triple combination in reducing tumor growth compared to drug A plus anti-PD-L1 antibody doublet or trastuzumab plus anti-PD-L1 antibody doublet was most evident on days 19 and 22 (3 to 6 days after the last dose). By day 26, the triple combination was minimally better at reducing tumor growth compared to drug A plus anti-PD-L1 antibody doublet or trastuzumab plus anti-PD-L1 antibody doublet. No adverse effects were observed in any of the treatment cohorts within the MC38m / hHER2 colon tumor model.

[0291] Example 5A: Exemplary Clinical Trial to Evaluate the Antitumor Activity of Drug A Combination Therapy in Human Patients Gastric or gastroesophageal junction (GEJ) adenocarcinoma HER2 tumors that have progressed during or after prior treatment with trastuzumab and fluoropyrimidine-containing chemotherapy (e.g., fluorouracil); during or after prior treatment with trastuzumab and platinum-containing chemotherapy; or during or after prior treatment with trastuzumab, fluoropyrimidine-containing chemotherapy (e.g., fluorouracil), and a platinum-containing chemotherapy agent +A clinical trial will be conducted to evaluate the safety, tolerability, and efficacy of the combination of Drug A, trastuzumab, ramucirumab, and paclitaxel in patients with overexpressing advanced or metastatic gastric cancer or GEJ adenocarcinoma. Patients enrolled in the trial will be eligible for treatment with trastuzumab. Patients will not have received prior treatment with anti-CD47 or anti-SIRPα agents.

[0292] Clinical trials are being conducted in patients with gastric or GEJ adenocarcinoma (e.g., HER2 + This study is being conducted to evaluate the safety, tolerability, and efficacy of the combination of Drug A, pembrolizumab, cisplatin, and 5-fluorouracil or capecitabine, in patients with CD47-overexpressing gastric or GEJ adenocarcinoma. Patients enrolled in this study have not received prior treatment with anti-CD47 or anti-SIRPα agents. Patients have adequate organ function and a hemoglobin level of 9 g / dL or higher.

[0293] Head and neck squamous cell carcinoma (HNSCC) The clinical trial will be 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 previously untreated advanced head and neck squamous cell carcinoma was treated with drug A (10 mg / kg IV QW), pembrolizumab (200 mg IV Q3W), and 5-fluorouracil (1,000 mg / m 2 / day, days 1, 2, 3, and 4, Q3W × 6), and carboplatin (AUC = 5 mg / ml / min, day 1, Q3W × 6). (In the expansion study, cisplatin (100 mg / m 2Drug A (10 mg / kg IV QW), trastuzumab (8 mg / kg IV on day 1, followed by 6 mg / kg Q3W on day 1), ramucirumab (8 mg / kg on day 1 and 15, Q4W on day 1), and paclitaxel (80 mg / m2 on day 1, 8, and 15, Q4W on day 1) were 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(s) with trastuzumab, fluorouracil, and a platinum agent were treated with drug A (10 mg / kg IV QW), trastuzumab (8 mg / kg IV on day 1 and 15, Q4W on day 1), and paclitaxel (80 mg / m2 on day 1, 8, and 15, Q4W on day 1). Three additional patients with HER2-positive gastric / gastroesophageal cancer who had progressed on prior treatment(s) with trastuzumab, fluorouracil, and a platinum agent were treated with drug A (15 mg / kg IV QW), trastuzumab (initial 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 any dose-limiting toxicities 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 none (0%) receiving drug A + pembrolizumab + fluorouracil + carboplatin experienced them. There were no dose-limiting toxicities in patients receiving drug A + pembrolizumab + fluorouracil + carboplatin or drug A + trastuzumab + ramucirumab + paclitaxel. Additionally, no TRAEs occurred in more than two 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 (TRAEs ≥ 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 previously untreated advanced squamous cell carcinoma of the head and neck treated with Drug A, pembrolizumab, 5-fluorouracil, and a platinum agent at the doses and schedules described in Example 5B achieved partial responses (PRs) based on investigator-assessed response using RECIST v1.1 criteria.

[0298] Of three patients with HER2-positive gastric / gastroesophageal cancer treated with Drug A (10 mg / kg QW), trastuzumab, ramucirumab, and paclitaxel (see Example 5B), two remained unevaluable. One patient achieved a PR based on investigator-assessed response using RECIST v1.1 criteria.

[0299] Of three patients with HER2-positive gastric / gastroesophageal cancer treated with Drug A (15 mg / kg QW), trastuzumab, ramucirumab, and paclitaxel (see Example 5B), two remained unevaluable. One patient achieved a PR based on investigator-assessed response using RECIST v1.1 criteria. Low rates of cytopenias were observed.

[0300] Drug A in combination with pembrolizumab, 5-fluorouracil, and a platinum agent (i.e., as initial treatment for patients with advanced HNSCC who have not received prior treatment for HNSCC) has demonstrated clinical activity in the treatment of advanced 1L HNSCC. Drug A in combination with trastuzumab, ramucirumab, and paclitaxel has demonstrated clinical activity in the treatment of advanced >2L gastric / gastroesophageal cancer (i.e., as treatment for patients who have received at least one prior therapy for gastric or GEJ cancer).

[0301] Pharmacodynamic analysis results showed that when combined with chemotherapy-containing regimens, nearly complete CD47 target occupancy (also known as receptor occupancy) was maintained throughout the entire Drug A administration interval.

[0302] Example 5D: Additional Results from the Exemplary Clinical Trial Described in Example 5A CD47 is a myeloid checkpoint receptor upregulated by tumors to evade anti-cancer immune responses. Drug A is an exemplary high-affinity CD47-blocking fusion protein with an inactive Fc region designed to safely potentiate anti-cancer therapeutics (Kauder et al. (2018) PLoS ONE. 13(8): e0201832; Chow et al. (2020) Journal of Clinical Oncology. 38: 15_suppl, 3056-3056). Drug A was evaluated in combination with standard chemotherapy and antibody regimens in patients with advanced HER2-positive gastric cancer (GC) or head and neck squamous cell carcinoma (HNSCC).

[0303] method Previously treated patients with advanced HER2-positive GC received drug A (A) 10 mg / kg QW or 15 mg / kg QW in combination with trastuzumab (T) + ramucirumab (ram) + paclitaxel (pac) as second-line or subsequent therapy. GC patients had progressed during or after prior fluoropyrimidine therapy (or fluoropyrimidine-containing therapy). GC patients who had progressed during or after prior treatment with trastuzumab and / or platinum-based chemotherapy were included. Patients with previously untreated advanced HNSCC received drug A (A) 10 mg / kg QW or 15 mg / kg QW in combination with pembrolizumab (P) + 5FU + platinum (cisplatin or carboplatin) as first-line therapy. The primary endpoint was dose-limiting toxicity (DLT). Tumor response, pharmacokinetic (PK), and pharmacodynamic (PD) markers were assessed in all patients.

[0304] result Fifty-five patients were enrolled in the study. Their baseline characteristics are shown in Table B. [Table 14]

[0305] One patient with GC ≥2L received A+T+ramucirumab and was evaluated for safety. No dose-limiting toxicities (DLTs) were reported, and the maximum dose of drug A was 15 mg / kg QW. Of the nine patients who experienced any adverse events, eight reported treatment-related adverse events (TRAEs). The most common TRAEs were mild diarrhea, fatigue, pruritus / urticaria, and rash (n=21% each). TRAEs of grade 3 or greater severity were infrequent. No treatment-related SAEs were reported in GC patients treated with A+T+ramucirumab. Of 11 GC patients who received 15 mg / kg qw drug A + trastuzumab + ramucirumab + paclitaxel, seven had partial responses, three had stable disease, and one had progressive disease. Of the three patients who received 10 mg / kg qw of drug A + trastuzumab + ramucirumab + paclitaxel, two had a partial response and one had stable disease.

[0306] As described above, three patients with previously untreated HNSCC received A + P + 5-fluorouracil + platinum. No DLTs were reported. Three patients experienced any adverse events (AEs), none of which were treatment-related. The HNSCC patient who received drug A + pembrolizumab at 15 mg / kg qw + 5-fluorouracil + platinum-based chemotherapy was CPI-naive and demon...

Claims

1. 1. A method of treating cancer in an individual, the method comprising administering to the individual effective amounts of: (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) a Bcl-2 inhibitor; the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; The Fc domain variant comprises: (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat.

2. 10. The method of claim 1, wherein the cancer is leukemia, multiple myeloma, or non-Hodgkin's lymphoma.

3. 3. The method of claim 2, wherein the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), or follicular lymphoma (FL).

4. 3. The method of claim 2, wherein the leukemia is acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), or myelodysplastic syndrome (MDS).

5. 5. The method of claim 4, wherein the leukemia is acute lymphoblastic leukemia.

6. 6. The method of any one of claims 1 to 5, wherein the Bcl-2 inhibitor is venetoclax, ABT-737, navitoclax, BCL201, or AZD-0466.

7. 7. The method of claim 6, wherein the Bcl-2 inhibitor is venetoclax.

8. 1. A method of treating cancer in an individual, the method comprising administering to the individual effective amounts of: (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) a platinum-based chemotherapeutic agent; the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat.

9. 9. The method of claim 8, wherein the cancer is a solid tumor.

10. 10. The method of claim 9, wherein 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, stomach cancer, gastroesophageal junction cancer, brain tumor, mesothelioma, or neuroblastoma.

11. 10. The method of claim 9, wherein the colon cancer is colon cancer.

12. 1. A method of treating cancer in an individual, the method comprising administering to the individual effective amounts 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; the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat; The method, wherein the cancer is head and neck squamous cell carcinoma (HNSCC) and the individual has not received previous treatment for HNSCC.

13. 13. The method of claim 12, wherein the HNSCC is advanced and / or metastatic HNSCC.

14. The method of claim 12 or 13, wherein the PD-1 inhibitor is an anti-PD-1 antibody.

15. 15. The method of claim 14, wherein the anti-PD-1 antibody is pembrolizumab, nivolumab, pidilizumab, cemiplimab, or BMS-936559.

16. 16. The method of claim 15, wherein the anti-PD-1 antibody is pembrolizumab.

17. The method according to any one of claims 12 to 16, wherein the antimetabolite is 5-fluorouracil, 6-mercaptopurine, capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxycarbamide, methotrexate, pemetrexed, or phototrexate.

18. 18. The method of claim 17, wherein the antimetabolite is 5-fluorouracil.

19. The method of any one of claims 8 to 18, wherein the platinum-based chemotherapeutic agent is carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin.

20. 20. The method of claim 19, wherein the platinum-based chemotherapeutic agent is cisplatin.

21. 20. The method of claim 19, wherein the platinum-based chemotherapeutic agent is carboplatin.

22. 1. A method of treating cancer in an individual, the method comprising administering to the individual effective amounts 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; the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat.

23. 23. The method of claim 22, wherein the cancer is a solid tumor.

24. 24. The method of claim 23, wherein 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, stomach cancer, gastroesophageal junction cancer, brain tumor, mesothelioma, or neuroblastoma.

25. The solid tumor is HER2 ＋ 25. The method of claim 23 or 24, wherein the tumor is a solid tumor.

26. The method of any one of claims 23 to 25, wherein the solid tumor is colon cancer.

27. The method of any one of claims 22 to 26, wherein the anti-HER2 antibody is trastuzumab.

28. The method of any one of claims 22 to 27, wherein the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab.

29. 1. A method of treating cancer in an individual, the method comprising administering to the individual effective amounts 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; the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat; The method, wherein the cancer is gastric cancer or gastroesophageal junction (GEJ) cancer, and the individual has received at least one prior treatment for the gastric cancer or the GEJ cancer.

30. 30. The method of claim 29, wherein the individual has undergone 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 chemotherapy agent.

31. 31. The method of claim 29 or 30, wherein the anti-HER2 antibody is trastuzumab.

32. The method of any one of claims 29 to 31, wherein the anti-VEGF antibody is ramucirumab.

33. the gastric cancer or the GEJ cancer is HER2 ＋ Gastric cancer or HER2 ＋ The method of any one of claims 29 to 32, wherein the cancer is GEJ cancer.

34. 34. The method of any one of claims 12-21 and 29-33, wherein 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.

35. 34. The method of any one of claims 12-21 and 29-33, wherein 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.

36. 1. A method of treating cancer in an individual, the method comprising administering to the individual effective amounts of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) an anti-TROP2 antibody; the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat.

37. 37. The method of claim 36, wherein 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, perihilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrioid endometrial carcinoma, or ovarian cancer.

38. The method of any one of claims 1 to 37, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO:

85.

39. The method of any one of claims 1 to 37, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO:

81.

40. 40. The method of any one of claims 1 to 39, wherein the Fc domain variant is a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, numbering according to the EU index of Kabat.

41. 41. The method of claim 40, wherein the Fc domain variant comprises the amino acid sequence of SEQ ID NO:

91.

42. 42. The method of any one of claims 1 to 38 or 40 to 41, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant comprises the amino acid sequence of SEQ ID NO:

136.

43. 42. The method of any one of claims 1 to 37 or 39 to 41, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant comprises the amino acid sequence of SEQ ID NO:

135.

44. 44. The method of any one of claims 1 to 43, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant forms a homodimer.

45. The method of any one of claims 1 to 44, wherein the individual is a human.

46. 1. A kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, said polypeptide for use in combination with a Bcl-2 inhibitor to treat cancer in an individual in need thereof, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat; The kit further comprises instructions for administering the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in combination with the Bcl-2 inhibitor to the individual in need thereof.

47. 47. The kit of claim 46, wherein the cancer is leukemia, multiple myeloma, or non-Hodgkin's lymphoma.

48. The kit of claim 46 or 47, wherein the Bcl-2 inhibitor is venetoclax.

49. 1. A kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, said polypeptide for use in combination with a platinum-based chemotherapeutic agent to treat cancer in an individual in need thereof, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat; The kit further comprises instructions for administering the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in combination with the platinum-based chemotherapeutic agent to the individual in need thereof.

50. 50. The kit of claim 49, wherein the cancer is a solid tumor.

51. 51. The kit of claim 50, wherein 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.

52. 1. A kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, said polypeptide for use in combination with a PD-1 inhibitor, an antimetabolite, and a platinum-based chemotherapeutic agent to treat cancer in an individual in need thereof, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat; the kit comprising instructions for administering the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in combination with the PD-1 inhibitor, the antimetabolite, and the platinum-based chemotherapeutic agent to an individual with head and neck squamous cell carcinoma (HNSCC), who has not received previous treatment for HNSCC.

53. The kit of any one of claims 49 to 52, wherein the PD-1 inhibitor is pembrolizumab.

54. 54. The kit of any one of claims 49 to 53, wherein the antimetabolite is 5-fluorouracil.

55. The kit according to any one of claims 49 to 54, wherein the platinum-based chemotherapeutic agent is cisplatin or carboplatin.

56. 1. A kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, said polypeptide for use in combination with an anti-HER2 antibody, an anti-VEGFR2 antibody, and paclitaxel, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat; the kit comprising instructions for administering the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in combination with the anti-HER2 antibody, the anti-VEGFR2 antibody, and the paclitaxel to an individual with gastric cancer or gastroesophageal (GEJ) cancer, wherein the individual has received at least one previous treatment for gastric cancer or GEJ cancer.

57. the gastric cancer or the GEJ cancer is HER2 ＋ Gastric cancer or HER2 ＋ 57. The kit of claim 56, wherein the cancer is GEJ cancer.

58. 58. The kit of claim 56 or 57, wherein the anti-HER2 antibody is trastuzumab.

59. The kit of any one of claims 56 to 58, wherein the anti-VEGFR2 antibody is ramucirumab.

60. 60. The kit of any one of claims 56 to 59, wherein the individual has undergone 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 chemotherapy agent.

61. 1. A kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, said polypeptide for use in combination with an anti-TROP2 antibody to treat cancer in an individual in need thereof, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat; The kit further comprises instructions for administering the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in combination with the anti-TROP2 antibody to the individual in need thereof.

62. 62. The kit of claim 61, wherein 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, perihilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrioid endometrial carcinoma, or ovarian cancer.

63. 1. A kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, said polypeptide for use in combination with an anti-HER2 antibody and an anti-PD-L1 antibody to treat cancer in an individual in need thereof, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat; The kit further comprises instructions for administering the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in combination with the anti-HER2 antibody and the anti-PD-L1 antibody to the individual in need thereof.

64. 64. The kit of claim 63, wherein the cancer is colon cancer.

65. the colon cancer is HER2 ＋ 65. The kit of claim 64, wherein the treatment is colon cancer.

66. The kit of any one of claims 63 to 65, wherein the anti-HER2 antibody is trastuzumab.

67. The kit of any one of claims 63 to 66, wherein the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab.

68. The kit of any one of claims 46 to 67, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO:

85.

69. The kit of any one of claims 46 to 67, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO:

81.

70. 70. The kit of any one of claims 46 to 69, wherein the Fc domain variant is a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, numbering according to the EU index of Kabat.

71. 71. The kit of claim 70, wherein the Fc domain variant comprises the amino acid sequence of SEQ ID NO:

91.

72. The kit of any one of claims 46 to 68 and 70 to 71, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant comprises the amino acid sequence of SEQ ID NO:

136.

73. The kit of any one of claims 46 to 67 and 69 to 71, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant comprises the amino acid sequence of SEQ ID NO:

135.

74. The kit of any one of claims 46 to 73, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant forms a homodimer.

75. The kit of any one of claims 46 to 74, wherein the individual is a human.

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