Combination Therapies for Treating Cancer

Abstract

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

Description

[Technical Field]

[0001] Cross-reference of 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 which are incorporated herein by reference in their entirety.

[0002] Submission of sequence listings in ASCII text files. The contents of the following submission in ASCII text file are incorporated herein by reference in their entirety: a computer-readable format (CRF) sequence listing (filename: 757972001140SEQLIST.TXT, date: November 25, 2020, size: 333KB).

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

[0004] Many cancers have a poor prognosis, even when treated with available medications. Novel therapies are needed in this field to provide additional treatment options and improve patient outcomes.

[0005] Tumor cells can evade the anti-tumor host immune response by manipulating the bone marrow compartment (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 and sends a "don't eat me" signal, but it has been found that tumor cells can overexpress CD47 to evade the macrophage component of immune surveillance (Oldenborg, ISRN Hematol (2013) 614619).

[0006] Destroying cancer cells via macrophages requires both the disruption of "don't eat me" signals (e.g., CD47-SIRPα) and the activation of "eat me" signals. Neither component alone is sufficient to elicit a maximum phagocytic response against tumor cells. As mentioned above, CD47 provides the basic "don't eat me" signal through its interaction with SIRPα on macrophages. Phagocytic "eat me" signals can be provided to the same macrophages by binding to their activated Fc gamma receptors. For example, phagocytic "eat me" signals can be provided by the binding of antitumor antibodies to Fc receptors on macrophages.

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

[0008] A method is provided for treating cancer in an individual, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) a Bcl-2 inhibitor, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant comprises (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); and (iii) a human IgG4 region comprising S228P, E233P, F234V, L235A, and delG236 mutations. Fc regions (wherein numbering follows Kabat's EU index); or (iv) human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein numbering follows Kabat's EU index). In some embodiments, cancer is leukemia, multiple myeloma, or non-Hodgkin lymphoma. In some embodiments, non-Hodgkin lymphoma is diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), or follicular lymphoma (FL). In some embodiments, leukemia is acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), chronic myeloid 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] Furthermore, a method is provided for treating cancer in an individual, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) a platinum-based chemotherapeutic agent, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant comprises (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); and (iii) a human IgG4 region comprising S228P, E233P, F234V, L235A, and delG236 mutations. Fc regions (where numbering follows Kabat's EU index); or (iv) human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows Kabat's EU index). In some embodiments, cancer is a solid tumor. In some embodiments, solid tumors are 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, colon cancer is colon carcinoma. In some embodiments, platinum-based chemotherapeutic agents are carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenantriplatin, picoplatin, or satraplatin. In some embodiments, the platinum-based chemotherapeutic agent is cisplatin or carboplatin.

[0010] Furthermore, a method for treating cancer in an individual is provided, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, (b) 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; the Fc domain variant comprises (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); and (iii) a human IgG4 region comprising S228P, E233P, F234V, L235A, and delG236 mutations. Fc regions (where numbering follows Kabat's EU index); or (iv) human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows Kabat's EU index). Here, the cancer is head and neck squamous cell carcinoma (HNSCC), and the individual has not received prior treatment for HNSCC. In some embodiments, the polypeptide containing the SIRPα D1 domain variant and the Fc domain variant is administered once weekly (qw) at a dose of 10 mg / kg. In some embodiments, the polypeptide containing the SIRPα D1 domain variant and the Fc domain variant is administered once weekly (qw) at a dose of 15 mg / kg.

[0011] In some embodiments, HNSCC is progressive and / or metastatic HNSCC. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody, e.g., pembrolizumab, nivolumab, pizilizumab, semiprimab, or BMS-936559. In some embodiments, the anti-PD-1 antibody is pembrolizumab. In some embodiments, the antimetabolites are 5-fluorouracil, 6-mercaptopurine, capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxycarbamide, methotrexate, pemetrexed, or phototrexate. In some embodiments, the antimetabolites are 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 embodiment, a method is provided for treating cancer in an individual, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, (b) an anti-HER2 antibody, and (c) an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody), wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant comprises (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); and (iii) a human IgG4 region comprising S228P, E233P, F234V, L235A, and delG236 mutations Fc regions (where numbering follows Kabat's EU index); or (iv) human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows Kabat's EU index). In some embodiments, cancer is a solid tumor. In some embodiments, solid tumors are 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, solid tumors are HER2 + It is a solid tumor. In some embodiments, the solid tumor is colon cancer (e.g., HER2 + (Colon cancer). In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab.

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

[0014] Furthermore, a method for treating cancer in an individual is provided, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) 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; the Fc domain variant comprises (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); and (iii) a human IgG4 region comprising S228P, E233P, F234V, L235A, and delG236 mutations. Fc regions (where numbering follows Kabat's EU index); or (iv) human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows Kabat's EU index). In some embodiments, cancer is a solid tumor, gastric cancer, nasopharyngeal cancer, gallbladder cancer, cervical cancer, extranodal NK / T cell lymphoma, lung cancer, laryngeal squamous cell carcinoma, colon cancer, hilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrial cancer, or ovarian cancer.

[0015] In some embodiments 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, numbered according to the Kabat EU index. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 136. In some embodiments, the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant forms a homodimer. In some embodiments, the individual is human.

[0016] In another embodiment, a kit is also provided comprising a polypeptide containing a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, for use in combination with a Bcl-2 inhibitor to treat cancer in an individual requiring treatment, 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 containing L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); and (iii) a human IgG4 region containing S228P, E233P, F234V, L235A, and delG236 mutations. The Fc region (where numbering follows the Kabat EU index); or (iv) the human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows the Kabat EU index), and the kit includes instructions for administering the polypeptide containing the SIRPα D1 domain variant and the Fc domain variant in combination with a Bcl-2 inhibitor to an individual requiring it. In some embodiments, the cancer is leukemia, multiple myeloma, or non-Hodgkin lymphoma. In some embodiments, the Bcl-2 inhibitor is venetoclax.

[0017] Also provided are kits containing polypeptides comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, for use in combination with platinum-based chemotherapeutic agents for treating cancer in individuals requiring such treatment, 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) human IgG1 Fc regions containing L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) human IgG2 Fc regions containing A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); and (iii) human IgG4 regions containing S228P, E233P, F234V, L235A, and delG236 mutations. The Fc region (wherein numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein numbering follows the Kabat EU index), and the kit includes instructions for administering a polypeptide containing the SIRPα D1 domain variant and the Fc domain variant in combination with a chemotherapeutic agent to an individual requiring it. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is colon cancer, colon carcinoma, lung cancer, head and neck cancer, esophageal cancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer, testicular cancer, endometrial cancer, liver cancer, gastric cancer, brain tumor, mesothelioma, or neuroblastoma. In some embodiments, the platinum-based chemotherapeutic agent is cisplatin or carboplatin.

[0018] In some embodiments, kits are also provided that contain polypeptides comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, for use in combination with PD-1 inhibitors, antimetabolites, and platinum-based chemotherapeutic agents to treat cancer in individuals requiring such treatment, 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) human IgG1 Fc regions containing L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) human IgG2 Fc regions containing A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); and (iii) human IgG4 regions containing S228P, E233P, F234V, L235A, and delG236 mutations. The Fc region (wherein numbering follows the Kabat EU index); or (iv) the human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein numbering follows the Kabat EU index), and the kit includes instructions for administering a polypeptide containing the SIRPα D1 domain variant and the Fc domain variant in combination with a PD-1 inhibitor, an antimetabolites, and a platinum-based chemotherapeutic agent to an individual with head and neck squamous cell carcinoma (HNSCC) that has not received prior treatment. In some embodiments, the PD-1 inhibitor is pembrolizumab. In some embodiments, the antimetabolites is 5-fluorouracil. In some embodiments, the platinum-based chemotherapeutic agent is cisplatin or carboplatin.

[0019] In some embodiments, kits are also provided that include a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, for use in combination with an anti-HER2 antibody, an anti-VEGFR2 antibody, and paclitaxel to treat cancer in an individual requiring such treatment, 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 containing L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); and (iii) a human IgG4 region containing S228P, E233P, F234V, L235A, and delG236 mutations. The Fc region (where numbering follows the EU index of Kabat); or (iv) the human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows the EU index of Kabat), and the kit includes 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 having gastric cancer or gastroesophageal junction (GEJ) cancer and having received at least one prior treatment for gastric cancer or GEJ cancer. In some embodiments, gastric cancer or GEJ cancer is HER2 + 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 prior treatment (or multiple prior treatments) with an anti-HER2 antibody (e.g., trastuzumab) and / or fluoropyrimidine and / or platinum-based chemotherapeutic agents. In some embodiments, the individual's gastric cancer or GEJ cancer progressed during or after prior treatment (or multiple prior treatments) including an anti-HER2 antibody (e.g., trastuzumab) and / or fluoropyrimidine and / or platinum-based chemotherapeutic agents. In some embodiments, the individual did not respond to prior treatment (or multiple prior treatments) including an anti-HER2 antibody (e.g., trastuzumab) and / or fluoropyrimidine and / or platinum-based chemotherapeutic agents (e.g., relapsed after prior treatment or did not respond to prior treatment). In some embodiments, the prior treatment (or multiple prior treatments) included an anti-HER2 antibody and a fluoropyrimidine (e.g., administered during the same treatment or a different treatment). In some embodiments, the prior treatment (or multiple prior treatments) included an anti-HER2 antibody and a platinum-based chemotherapeutic agent (e.g., administered during the same treatment or a different treatment).

[0020] Also provided are kits containing polypeptides comprising SIRPα D1 domain variants and Fc domain variants in a pharmaceutically acceptable carrier, for use in combination with an anti-TROP2 antibody to treat cancer in individuals requiring such treatment, 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) human IgG1 Fc regions containing L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) human IgG2 Fc regions containing A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); and (iii) human IgG4 regions containing S228P, E233P, F234V, L235A, and delG236 mutations. The Fc region (where numbering follows the EU index of Kabat); or (iv) human IgG4 Fc region (where numbering follows the EU index of Kabat) containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, and the kit includes instructions for administering a polypeptide containing the SIRPα D1 domain variant and the Fc domain variant in combination with an anti-TROP2 antibody to an individual requiring it. In some embodiments, the cancer is a solid tumor, gastric cancer, nasopharyngeal cancer, gallbladder cancer, cervical cancer, extranodal NK / T cell lymphoma, lung cancer, laryngeal squamous cell carcinoma, colon cancer, hilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrial cancer, or ovarian cancer.

[0021] Also provided are kits containing polypeptides comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, for use in combination with anti-HER2 antibodies and anti-PD-L1 antibodies (e.g., anti-PD-L1 antagonist antibodies) to treat cancer in individuals requiring such treatment, 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) human IgG1 Fc regions containing L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) human IgG2 Fc regions containing A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); and (iii) human IgG4 regions containing S228P, E233P, F234V, L235A, and delG236 mutations. Fc regions (where numbering follows Kabat's EU index); or (iv) human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows Kabat's EU index), and the kit includes instructions for administering a polypeptide containing 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 requiring it. In some embodiments, the cancer is colon cancer. In some embodiments, the colon cancer is HER2 + The cancer is colon cancer. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab.

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

[0023] [Figure 1A] This image shows tumor volume (mm3) in NOD-SCID female mice at specified post-transplant time after injection of RS4;11 leukemia cells, followed by treatment with drug A, venetoclax, a combination of venetoclax and drug A, or vehicle (PBS). Dashed arrows indicate administration of venetoclax (250 μg) by forced oral administration a total of two times at 3-day intervals. Dotted arrows indicate administration of drug A (10 mg / kg) a total of four times at 3-4 day intervals. SEM = standard error. TF = no tumor. [Figure 1B] This shows the tumor volume (mm3) of NOD-SCID female mice at specified time post-transplantation after being injected with RS4;11 leukemia cells, treated with venetoclax, and then retreated with venetoclax monotherapy or a venetoclax / drug A combination. [Figure 2A]This shows tumor volume (mm3) and body weight of BALB / c female mice at specified post-transplant time after injection of CT26 tumor cells and subsequent treatment with drug A, cisplatin, a combination of cisplatin and drug A, or a vehicle (PBS). Mean tumor volume (+ / -SEM) for the indicated treatment is shown. Dashed arrows indicate cisplatin administration (5 mg / kg twice, 10 days apart). Dotted arrows indicate drug A administration (30 mg / kg twice, 10 days apart). Both drugs were administered intraperitoneally. Mice treated with both drugs received drug A one day after cisplatin treatment. [Figure 2B] Figure 2A shows the tumor volume (mm3) and body weight of BALB / c female mice at specified post-transplant time after injection of CT26 tumor cells and subsequent treatment with drug A, cisplatin, a cisplatin / drug A combination, or vehicle (PBS). The mean percentage change in body weight from day 7 (D7) in mice treated according to the regimen shown is shown. [Figure 2C] This shows tumor volume (mm3) and body weight of BALB / c female mice at specified post-transplant time after injection of CT26 tumor cells and subsequent treatment with drug A, cisplatin, a combination of cisplatin and drug A, or a vehicle (PBS). Mean tumor volume (+ / -SEM) for the indicated treatment is shown. Dashed arrows indicate cisplatin administration (10 mg / kg, single dose). Dotted arrows indicate drug A administration (30 mg / kg, two doses, 10 days apart). Both drugs were administered intraperitoneally. Mice treated with both drugs received drug A one day after cisplatin treatment. [Figure 2D] Figure 2C shows the tumor volume (mm3) and body weight of BALB / c female mice at specified post-transplant time after injection of CT26 tumor cells and subsequent treatment with drug A, cisplatin, a cisplatin / drug A combination, or vehicle (PBS). It also shows the mean percentage change in body weight from day 7 (D7) in mice treated according to the regimen shown. [Figure 3]The results of an experiment conducted 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 are shown. [Figure 4] The results of experiments conducted to determine the effects of (a) an anti-HER2 antibody, (b) an anti-PD-L1 antibody, or (c) drug A in combination with an anti-HER2 antibody and anti-PD-L1 on tumor growth in an MC38m / h colon cancer model are shown. [Figure 5A] The results of experiments conducted to evaluate the effects of drug A, venetoclax, or both drugs A and venetoclax on macrophage phagocytosis of HL60 cells in in vitro assays are shown. [Figure 5B] The results of experiments conducted to evaluate the effects of drug A, venetoclax, or both drugs A and venetoclax on macrophage phagocytosis of OCI-AML3 cells in in vitro assays are shown. [Figure 6A] The results of experiments conducted to evaluate the effects of drug A or drug C on the activation of CD8+ dendritic cells are shown. [Figure 6B] The results of experiments conducted to evaluate the effects of drug A or drug C on the activation of CD8-dendritic cells are shown. [Figure 7A] The results of experiments conducted to evaluate the effects of drug A or drug B on the activation of CD8+ dendritic cells are shown. [Figure 7B] The results of experiments conducted to evaluate the effects of drug A or drug B on the activation of CD8-dendritic cells are shown. [Figure 8A] The results of experiments conducted to evaluate the binding of drug A, F59 / maglorimab, TTI-621, and TTI-622 to hCD47 are shown. [Figure 8B] The results of quantitative experiments conducted to evaluate the effects of drugs A, F59 / maglorimab, TTI-621, and TTI-622 on SIRPα signaling are shown. [Modes for carrying out the invention]

[0024] The following description includes exemplary methods, parameters, etc. However, it should be noted that such descriptions are not intended to limit the scope of this disclosure, but rather are provided as descriptions of exemplary embodiments.

[0025] definition The terms “approximately” or “about” mean that a particular value is within an acceptable margin of error, as determined by those skilled in the art, and depend in part on the limits of the method of measuring or determining the value, i.e., the measuring system. For example, “approximately” can mean within one standard deviation or more than one, according to convention in the art. Alternatively, “approximately” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term can mean within five orders of magnitude of the value, preferably more than two orders of magnitude. Unless otherwise stated, where a particular value is described in this application and claims, the term “approximately” is considered to mean that the particular value is within an acceptable margin of error.

[0026] The terms used herein are for illustrative purposes only and are not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context otherwise explicitly indicates. Furthermore, terms such as “including,” “includes,” “having,” “has,” “accompany,” or variations thereof, are intended to be comprehensive in the same manner as the term “comprising.”

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

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

[0029] As used herein, the term “antibody variable domain” refers to portions of the light and heavy chains of an antibody that include the amino acid sequences of complementary determination 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, for example, protein domains. In some embodiments, the linker may be a covalent bond or a spacer. The term “spacer” refers to a portion (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 them. 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 polypeptide or a pharmaceutical composition comprising the polypeptide described herein, for example, a polypeptide having the SIRPα D1 domain or a variant thereof, that is sufficient and effective to achieve the desired therapeutic effect when treating a patient with a disease such as cancer, for example, a solid tumor or a hematological cancer. In some embodiments, an effective amount of polypeptide would avoid adverse side effects.

[0032] As used herein, the term “pharmaceutical composition” refers to a pharmaceutical or pharmaceutical preparation comprising an active ingredient and excipients or diluents (or both), wherein the active ingredient can be administered by a preferred method of administration. In some embodiments, the pharmaceutical compositions disclosed herein include a polypeptide and a pharmaceutically acceptable component compatible with it. In some embodiments, the pharmaceutical composition is in the form of a tablet or capsule for oral administration, or in an aqueous form for intravenous or subcutaneous administration, for example, by injection.

[0033] As used herein, the terms “subject,” “individual,” and “patient” are used synonymously to refer to vertebrates, such as mammals. Mammals include, but are not limited to, mice, monkeys, humans, livestock, sports animals, and pets. Tissues, cells, and their offspring of biological entities obtained in vivo or cultured in vitro are also included. None of the terms require medical professional oversight.

[0034] As used herein, the terms “affinity” or “binding affinity” refer 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 the SIRPα D1 domain variant and CD47). Unless otherwise specified, binding affinity refers to the intrinsic binding affinity, which reflects the 1:1 interaction between members of a binding pair. The binding affinity between two molecules is generally expressed in terms of the dissociation constant (K). D ) or coupling constant (K A) are described. Two molecules having low binding affinity for each other generally bind slowly and tend to dissociate easily, exhibiting a large K D . Two molecules having high affinity for each other generally bind easily and tend to have a long binding, exhibiting a small K D . In some embodiments, the K D of two interacting molecules is determined using known methods and techniques, such as surface plasmon resonance (SPR). K D can be calculated as the ratio of koff / kon.

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

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

[0037] Overview Provided herein are methods for treating cancer in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) and (b) a chemotherapeutic agent (at least one chemotherapeutic agent, e.g., at least two, at least three, or at least four chemotherapeutic agents). 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 immunotherapy agent (at least one immunotherapy agent, e.g., at least two, at least three, or at least four immunotherapy agents). Additionally or alternatively, in some embodiments, the method comprises administering the polypeptide and the chemotherapeutic agent in combination with one or more additional modes of treatment, not limited to these, e.g., radiotherapy, surgery, cryoablation, and bone marrow transplantation.

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

[0039] In some embodiments, 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 is about 10 nM K D Or a better K DThe agents bind to CD47 (e.g., hCD47) at concentrations of approximately 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 at least one of these concentrations less than 10 pM. In some embodiments, the agents that bind to CD47 (e.g., hCD47) exhibit a CD47 receptor occupancy of at least approximately 50% (e.g., at least one of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or approximately 100%) in human subjects. In some embodiments, the agent that binds to CD47 (e.g., hCD47) has an EC50 of approximately 80 ng / ml or less, for example, one of approximately 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 ng / ml. In some embodiments, the agent that binds to CD47 (e.g., hCD47) is an anti-CD47 antibody (e.g., therapeutic anti-CD47 antibody) or its antigen-binding fragment. In some embodiments, the antigen-binding fragment is 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, triomab, DART (i.e., dual affinity retargeting antibody), TandAb (i.e., tandem diabody), tandem scFv, CrossMab, DNL (i.e., dock-and-lock antibody), DVD-Ig (i.e., dual variable domain immunoglobulin), tetravalent bispecific IgG, nanobody, dual targeting domain, and ART-Ig (i.e., asymmetric reengineered technology-immunoglobulin), among others.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 on these anti-CD47 antibodies, see, for example, Zhao et al. (2011), PNAS USA 108:18342-18347; Chao et al. (2010) Cell 142:699-713; Kim et al. (2012) Leukemia 26:2538-2545; Chao et al. (2011) Blood 118:4890-4891; Goto et al. (2014) Eur J. Cancer 50:1836-1846; and Edris et al. (2012) PNAS USA.) See 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 is about 10 nM K D Or a better K DThe agents bind to SIRPα (e.g., hSIRPα) at ​​concentrations of approximately 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 at least one of these concentrations less than 10 pM. In some embodiments, the agents that bind to SIRPα (e.g., hSIRPα) exhibit a SIRPα receptor occupancy of at least approximately 50% (e.g., at least one of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or approximately 100%) in human subjects. In some embodiments, the agent that binds to SIRPα (e.g., hSIRPα) has an EC50 of approximately 80 ng / ml or less, for example, one of approximately 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., therapeutic anti-SIRPα antibody) or its antigen-binding fragment. In some embodiments, the antigen-binding fragment is 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 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 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 on these anti-SIRPα antibodies, see, for example, Ring et al. (2017) PNAS USA 114(49):E10578-E10585; Murata et al. (2018) Cancer Sci 109(5):1300-1308; and Yanigata et al. (2017) JCI Insight 2:e89140).In some embodiments, the anti-SIRPα antibody is the 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 herein by reference in their entirety.

[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 anti-SIRPγ antibody that can bind to SIRPα), or an antigen-binding fragment thereof. In some embodiments, the agent is an antibody (or an antigen-binding fragment thereof) that can bind to two or more of SIRPα, SIRPβ, and SIRPγ. In some embodiments, such an antibody is approximately 10 nM K D Or a better K DThe antibody binds to SIRPα (e.g., hSIRPα) at ​​concentrations of approximately 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 at least one of these less than 10 pM. In some embodiments, the antibody exhibits a SIRPα receptor occupancy of at least approximately 50% in a human subject (e.g., at least one of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or approximately 100%). In some embodiments, the antibody has an EC50 of approximately 80 ng / ml or less, for example, one of approximately 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 Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv, single-chain antibody, or diabody. In some embodiments, the antibody is a monospecific antibody or monospecific antibody construct (including, but not limited to, those described above). In some embodiments, the antibody is a multispecific (e.g., bispecific) antibody or multispecific antibody construct (including, but not limited to, those described above).

[0042] In some embodiments, the agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is a fusion polypeptide comprising a CD47-binding portion. In some embodiments, the fusion polypeptide comprises an antibody Fc region and a CD47-binding portion. In some embodiments, the CD47-binding portion of the fusion polypeptide is approximately 10 nM K D Or a better K DThe fusion polypeptide binds to CD47 (e.g., hCD47) at concentrations of approximately 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 at least one of these less than 10 pM. In some embodiments, the fusion polypeptide exhibits a CD47 receptor occupancy of at least approximately 50% (e.g., at least one of approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or approximately 100%) in human subjects. In some embodiments, the fusion polypeptide has an EC50 of approximately 80 ng / ml or less, for example, one of approximately 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 ng / ml. In some embodiments, the fusion polypeptide includes a WT human antibody Fc region. In some embodiments, the fusion polypeptide includes an Fc variant (e.g., a variant of the WT human antibody Fc region) that exhibits reduced effector function (e.g., removed) 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 entirety. In some embodiments, the CD47-binding portion (e.g., hCD47) is WT SIRPα (e.g., hSIRPα) or WT SIRPγ (e.g., hSIRPγ). In some embodiments, the CD47-binding portion (e.g., d1 domain) is a CD47-binding fragment (e.g., d1 domain) of WT SIRPα (e.g., hSIRPα) or WT SIRPγ (e.g., hSIRPγ). In some embodiments, the CD47-binding portion (e.g., hCD47) is a SIRPα variant, a SIRPγ variant, a SIRPβ variant, or their CD47-binding fragment (e.g., d1 domain).Exemplary SIRPγ variants, SIRPβ1 variants, and SIRPβ2 variants are, for example, WO2013 / 109752;US2015 / 0071905;USP9,944,911;WO2016 / 023040;WO2017 / 027422;US2017 / 0107270;USP10,259,859;US9845345;WO2016187226;US201801554 The contents of the following documents are described in 05;WO2017177333;WO2014094122;US2015329616;US20180312563;WO2018176132;WO2018081898;WO2018081897;PCT / US2019 / 048921;US20180141986A1; and EP3287470A1, and are incorporated herein by reference in their entirety.

[0043] In some embodiments, the agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is a fusion polypeptide comprising an antibody Fc region and a SIRPα variant. In some embodiments, the SIRPα variant is approximately 10 nM K D Or a better K DThe fusion polypeptide binds to CD47 (e.g., hCD47) at concentrations of at least one of approximately 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 fusion polypeptide exhibits a CD47 receptor occupancy of at least approximately 50% (e.g., at least one of approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or approximately 100%) in human subjects. In some embodiments, the fusion polypeptide has an EC50 of approximately 80 ng / ml or less, for example, one of approximately 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 ng / ml. In some embodiments, the fusion polypeptide includes a WT human antibody Fc region. In some embodiments, the fusion polypeptide includes an Fc variant (e.g., a variant of the WT human antibody Fc region) that exhibits reduced effector function (e.g., removed) compared to the WT Fc region, including, for example, those described in the references cited herein. In some embodiments, the fusion polypeptide is 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 thereof are incorporated herein by reference in their entirety) including the SIRPα variant.In some embodiments, the fusion polypeptide comprising the antibody Fc region and the SIRPα variant is TTI-621, TTI-622, or IMM01 (see, for example, 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 for blocking the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is a fusion polypeptide comprising a SIRPα D1 domain variant (e.g., the SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., the Fc domain variant described herein).

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

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

[0047] In some embodiments, a method is 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 method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) a PD-1 inhibitor, (c) an antimetabolite, and (d) a platinum-based chemotherapeutic agent. In some embodiments, an agent for blocking 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 comprises (i) a human IgG1 Fc region containing L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); and (iii) a human IgG4 region containing S228P, E233P, F234V, L235A, and delG236 mutations. Fc regions (where numbering follows Kabat's EU index); or (iv) human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows Kabat's EU index).

[0048] In some embodiments, the provided method is for treating cancer (e.g., gastric cancer or gastroesophageal cancer) in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-HER2 antibody, (c) an anti-VEGFR2 antibody, and (d) paclitaxel. In some embodiments, an agent for blocking 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 comprises (i) a human IgG1 Fc region containing L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); and (iii) a human IgG4 region containing S228P, E233P, F234V, L235A, and delG236 mutations. Fc regions (where numbering follows Kabat's EU index); or (iv) human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows Kabat's EU index).

[0049] Further details regarding therapeutic methods using polypeptides containing 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 these are incorporated herein by reference in their entirety.

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

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

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

[0053] Native SIRPα consists 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, which mediates the binding of SIRPα to CD47. As used herein, the term "SIRPα polypeptide" refers to any SIRPα polypeptide or fragment thereof that can bind to CD47. At least 10 variants of wild-type human SIRPα 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, the SIRPα polypeptide contains the SIRPα D1 domain. In some embodiments, the SIRPα polypeptide contains the wild-type D1 domain, such as those provided in SEQ ID NOs: 1 and 2. In some embodiments, the SIRPα polypeptide contains the D2 or D3 domain (or both 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 containing 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 combination thereof) compared to wild-type SIRPα.

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

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

[0057] As used herein, the terms “optimized affinity” or “optimized binding affinity” refer to the optimized strength of the binding interaction between the polypeptides disclosed herein, such as SIRPα D1 domain variants, and CD47. For example, in some embodiments, a polypeptide binds primarily or with higher affinity to CD47 on cancer cells and substantially not to CD47 on non-cancer cells, or 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, in order to achieve the optimized binding affinity between the polypeptide and CD47 provided herein, polypeptides containing SIRPα D1 domain variants are developed to have a binding affinity to CD47 lower than the maximum achievable. In some embodiments, the SIRPα D1 domain variants disclosed herein cross-react with rodents, non-human primates (NHPs), and human CD47.

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

[0059] As used herein, the term “minimal immunogenicity” refers to the immunogenicity of a protein modified by an amino acid substitution (e.g., a therapeutic protein) to be lower than the immunogenicity of the protein before the introduction of the amino acid substitution (e.g., an unmodified protein) (e.g., by at least 10%, 25%, 50%, or 100%). In some embodiments, a protein (e.g., a therapeutic protein) is modified to have minimal immunogenicity and, even if it is an exogenous antigen, does not evoke a host immune response of any or little.

[0060] In some embodiments, the SIRPα D1 domain variant exhibits minimal immunogenicity. In some embodiments, the SIRPα polypeptide of the Disclosure administered to a subject has the same amino acid sequence as the SIRPα polypeptide in the biological sample of the subject, except for amino acid changes that increase the affinity of the SIRPα D1 domain variant. In some embodiments, the polypeptide variant disclosed herein reduces the risk of adverse events compared to anti-CD47 antibody or wild-type SIRPα. In some embodiments, the polypeptide variant disclosed herein reduces the risk of anemia compared to anti-CD47 antibody or wild-type SIRPα. In some embodiments, the polypeptide variant disclosed herein does not cause acute anemia in rodent or non-human primate (NHP) studies.

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

[0062] In some embodiments, a SIRPα D1 domain variant is a chimeric SIRPα D1 domain variant comprising two or more wild-type D1 domains or parts of their variants (e.g., one wild-type D1 domain or part of its variant and another wild-type D1 domain or part of its variant). In some embodiments, a chimeric SIRPα D1 domain variant comprises at least two parts (e.g., three, four, five, or more parts) of a wild-type D1 domain or its variant, where each part originates from a different wild-type D1 domain. In some embodiments, a 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 containing the following sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGNITPADAGTYYCX 12 KX 13 RKGSPDDVEX 14 KSGAGTELSVRAKPS (Sequence ID 13), X1 is L, I, or V, X2 is V, L, or I, X3 is A or V, X4 is A, I, or L, X5 is I, T, S, or F, X6 is E, V, or L, X7 is K or R, X8 is E or Q, X9 is H, P, or R, X 10 is L, T, or G, and X 11 is K or R, and X 12 is V or I, and X 13 is F, L, or V, and X 14The variant is either F or V, and the variant contains at least one amino acid substitution relative to the wild-type SIRPα D1 domain containing the sequence of SEQ ID NO: 1.

[0064] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant containing the sequence of sequence number 13, where X1 is L, I, or V. In any of the embodiments described above, 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 These are F, L, and V. In some embodiments, X 14 is F or V. In some embodiments, the polypeptide of this embodiment of the present disclosure comprises six or fewer amino acid substitutions to the wild-type SIRPα D1 domain containing the sequence of SEQ ID NO: 1.

[0065] In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 10 times higher than that of the wild-type SIRPα D1 domain containing the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 100 times higher than that of the wild-type SIRPα D1 domain containing the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 1000 times higher than that of the wild-type SIRPα D1 domain containing the sequence of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a binding affinity of 1 × 10⁻¹⁶. -8 Less than M, 5x10-9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 Less than M K D It binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K content 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 Then combine it with CD47.

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

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

[0068] In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 10 times higher 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 at least 100 times higher 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 at least 1000 times higher than that of a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a binding affinity of 1 × 10⁻¹⁶ -8 Less than M, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 Less than M KD It binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM for binding to CD47. D It binds to CD47.

[0069] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSLX 11 PVGPIQWFRGAGPX <000​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​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 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 does not exist; X 27 is T or V; X 28 is F or V; X 29 is A or G; where the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2.

[0070] In any of the embodiments described herein, X2 is L, I, or V. In any of the embodiments described herein, 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 These are F, L, and V. In some embodiments, X 26 is either D or absent. In some embodiments, X 27 is T or V. In some embodiments, X 28 is F or V. In some embodiments, X 29 is A or G. In some embodiments, the polypeptides of this embodiment of the present disclosure include six or fewer amino acid substitutions 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 at least 10 times higher than that of 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 at least 100 times higher than that of 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 at least 1000 times higher than that of 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 a fragment thereof binds to CD47 with a binding affinity of 1 × 10⁻¹⁶ -8 Less than M, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 Less than M K DIt binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K content 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 Then combine it with CD47.

[0072] In some embodiments, the polypeptides of the Disclosure comprising a SIRPα D1 domain variant further comprise 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, 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 a D2 or D3 domain via a linker. In some embodiments, the SIRPα D1 domain variant is linked to both the D2 and D3 domains via a linker. [Table 3]

[0073] In some embodiments, the polypeptides of the Disclosure comprising a SIRPα D1 domain variant are attached to an Fc domain variant to improve the pharmacokinetic properties of the polypeptide, for example, to extend the serum half-life. In some embodiments, the SIRPα D1 domain variant is attached to a non-dimerizable Fc domain variant. In some embodiments, the Fc domain variant helps to extend the serum half-life of the polypeptides described herein. In some embodiments, the polypeptides of the Disclosure comprising a SIRPα D1 domain variant do not contain any of the sequences of SEQ ID NOs. 26-36 shown in Table 4. [Table 4]

[0074] In some embodiments, the polypeptides and polypeptide constructs described herein are used in vitro for binding assays such as immunoassays. For example, in some embodiments, the polypeptides and polypeptide constructs described herein are used in a liquid phase or are bound to a solid support. In some embodiments, the polypeptides used in immunoassays are labeled in a detectable manner 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 properties of the carriers may be either soluble or insoluble.

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

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

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

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

[0080] In some embodiments, the polypeptides in the compositions disclosed herein include SIRPα D1 domain variants in which glycosylation is reduced or minimized. 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 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 high molecular weight removed by Endo Hf. Endo Hf is a recombinant protein fusion of endoglycosidase H and a maltose-binding protein. Endo Hf cleaves within several hybrid oligosaccharides from a high-mannose chitobiose core and N-linked glycoprotein. This means that proline at amino acid position 83 can reduce the efficiency of glycosylation, resulting in proteins with different degrees of glycosylation, and thus heterogeneity. In drug development, heterogeneity can present challenges in process development. Therefore, in some embodiments, to investigate the possibility of generating a homogeneous nonglycosylated form of the SIRPα D1 domain variant, the amino acid N80 of the SIRPα D1 variant is mutated to Ala. In some embodiments, to produce a nonglycosylated, SIRPα D1 domain variant, the amino acid N80 in the SIRPα D1 domain variant is replaced with any native or non-native amino acid, e.g., any amino acid such as N80A and N80Q. In some embodiments, the SIRPα D1 domain variant includes an 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 located at the CD47 binding site. In some embodiments, the additional mutation is located within the hydrophobic core of the D1 domain.

[0081] In some embodiments, the polypeptides in the compositions disclosed herein include a SIRPα D1 domain variant with increased glycosylation compared to the wild-type SIRPα D1 domain. Another option to enhance the homogeneity of the final product is to increase the efficiency of glycosylation at amino acid N80, producing a SIRPα D1 domain variant with increased glycosylation compared to the wild type. In some embodiments, amino acid P83 in the sequence NITP83 influences 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 the SIRPα D1 domain variant is substituted with any amino acid, such as native and non-native amino acids, e.g., P83V, P83A, P83I, and P83L. In some embodiments, the polypeptides of the Disclosure are expressed in cells optimized to prevent glycosylation of the expressed proteins, for example, by genetically engineering cell lines (e.g., genetically modified yeast or mammalian hosts), or by modifying cell culture conditions, such as by adding kifunensin, or by using a natural non-glycosylated host, such as a prokaryote (e.g., E. coli).

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

[0083] In some embodiments, a SIRPα D1 domain variant is a chimeric SIRPα D1 domain variant comprising two or more wild-type D1 domains or parts of their variants (e.g., one wild-type D1 domain or part of its variant and another wild-type D1 domain or part of its variant). In some embodiments, a chimeric SIRPα D1 domain variant comprises at least two parts (e.g., three, four, five, or more parts) of a wild-type D1 domain or its variant, where each part originates from a different wild-type D1 domain. In some embodiments, a chimeric SIRPα D1 domain variant further comprises one or more amino acid substitutions listed in Table 5. [Table 5-1] [Table 5-2] [Table 5-3]

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

[0085] In some embodiments of this disclosure, the polypeptide comprises a SIRPα D1 domain variant having the sequence of SEQ ID NO: 37, where X1 is L, I, or V. In some embodiments, X2 is V, L, or I. In some embodiments, X3 is A or V. In some embodiments, X4 is 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 These are F, L, and V. In some embodiments, X 16 It is either F or V.

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

[0087] In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 10 times higher 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 at least 100 times higher 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 at least 1000 times higher than that of a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a binding affinity of 1 × 10⁻¹⁶ -8 Less than M, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 Less than M K D It binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K content 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 Then combine it with 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 16KSGAGTELSVRAKPS (Sequence ID 38), X1 is L, I, or V; X2 is V, L, or I; X3 is A or V; X4 is V, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K or R; X8 is E or Q; X9 is H, P, or R; X 10 is S, T, or G; X 11 is K or R; X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; X 14 is V or I; X 15 is F, L, or V; and X 16 is F or V; the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2.

[0089] In some embodiments of this disclosure, the polypeptide comprises a SIRPα D1 domain variant having the sequence of SEQ ID NO: 38, where X1 is L, I, or V. In some embodiments, X2 is V, L, or I. In some embodiments, X3 is A or V. In some embodiments, X4 is V, I, or L. In some embodiments, X5 is I, T, S, or F. In some embodiments, X6 is E, V, or L. In some embodiments, X7 is K or R. In some embodiments, X8 is E or Q. In some embodiments, X9 is H, P, or R. In some embodiments, X 10 is S, T, or G. In some embodiments, X 11 is K or R. In some embodiments, X 12 is 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 It is either F or V.

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

[0091] In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 10 times higher 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 at least 100 times higher 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 at least 1000 times higher than that of a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a binding affinity of 1 × 10⁻¹⁶ -8 Less than M, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 Less than M K D It binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K content 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 Then combine it with CD47.

[0092] In another embodiment, the disclosure features a polypeptide comprising 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 KPS (Sequence ID 47), X1 is E or G; X2 is L, I, or V; X3 is V, L, or I; X4 is S or F; X5 is L or S; X6 is S or T; X7 is A or V; X8 is I or T; X9 is H, R, or L; X 10 is A, V, I, or L; X 11 is I, T, S, or F; X 12 is A or G; X 13 is E, V, or L; X 14 is K or R; X 15 is E or Q; X 16 is H, P, or R; X 17 is D or E; X 18 is S, L, T, or G; X 19 is K or R; X 20 is E or N; X 21 is S or P; X22 is S or R; X 23 is S or G; X 24 is any amino acid; X 25 is any amino acid; X 26 is V or I; X 27 are F, L, V; X 28 is D or does not exist; 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 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 Sequence ID No. 47, where X1 is E or G. In any of the embodiments described above in this aspect of the Disclosure, X2 is L, I, or V. In any of the embodiments described above, X3 is V, L, or I. In any of the embodiments described above, X4 is S or F. In any of the embodiments described above, X5 is L or S. In any of the embodiments described above, X6 is S or T. In any of the embodiments described above, X7 is A or V. In any of the embodiments described above, X8 is I or T. In any of the embodiments described above, X9 is H or R. In any of the embodiments described above, X 10 is A, V, I, or L. In any of the embodiments described above, X 11 is I, T, S, or F. In any of the embodiments described above, X 12 is A or G. In any of the embodiments described above, X 13 is E, V, or L. In any of the embodiments described above, X 14 is K or R. In any of the embodiments described above, X 15 is E or Q. In any of the embodiments described above, X 16 is H, P, or R. In any of the embodiments described above, X 17is D or E. In any of the embodiments described above, X 18 is S, L, T, or G. In any of the embodiments described above, X 19 is K or R. In any of the embodiments described above, X 20 is E or N. In any of the embodiments described above, X 21 is S or P. In any of the embodiments described above, X 22 is S or R. In any of the embodiments described above, X 23 is S or G. In any of the embodiments described above, 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 embodiments described above, 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 embodiments described above, X 26 is V or I. In any of the embodiments described above, X 27 These are F, L, and V. In any of the embodiments described above, X 28 X is either D or does not exist. In any of the embodiments described above, X 29 is T or V. In any of the embodiments described above, X 30 is F or V. In any of the embodiments described above, X 31 It is either A or G.

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

[0095] In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 10 times higher than that of 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 at least 100 times higher than that of 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 at least 1000 times higher than that of 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 a fragment thereof binds to CD47 with a binding affinity of 1 × 10⁻¹⁶ -8 Less than M, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 Less than M K D It binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K content 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 Then combine it with 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 EFKSGAGTELSVRAKPS (Sequence ID 48), X1 is V or I; X2 is L or S; X3 is T or S; X4 is T or I; X5 is R or H; X6 is A, V, or I; X7 is I, R, Y, K, or F; X8 is G or A; X9 is E or V; X 10 is K or R; X 11 is E, D, or Q; X 12 is H or P; X 13 is D or E; X 14 is S, L, or T; X 15 is N or E; X 16 is R or S; X 17 is G or S; X 18 is N or A; X 19 is V or I; X 20 is S, I, or M; X 21 P or not present; X 22 is D or P; X 23 is V or T, or a fragment thereof.

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

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

[0099] In some embodiments, the polypeptide comprises a SIRPα D1 domain with at least 85% sequence identity to SEQ ID NO: 49 (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity), and includes X1, X2, X3, X4, X5, X6, X7, X8, X9, and X 10 None of these are wild-type amino acids.

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

[0101] In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 10 times higher 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 at least 100 times higher 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 at least 1000 times higher than that of a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a binding affinity of 1 × 10⁻¹⁶ -8 Less than M, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 Less than M K D It binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K content 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 Then combine it with CD47.

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

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

[0104] In some embodiments, the polypeptide comprises a SIRPα D1 domain with at least 85% sequence identity to SEQ ID NO: 50 (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity), and includes X1, X2, X3, X4, X5, X6, X7, X8, X9, and X 10 None of these are wild-type amino acids.

[0105] In some embodiments, the polypeptide of this embodiment of the disclosure comprises 10 or fewer amino acid substitutions to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide of this embodiment of the disclosure comprises 7 or fewer amino acid substitutions to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2.

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

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

[0108] In some embodiments, the polypeptide comprises the sequence of Sequence ID No. 51, where X1 is V or I. In any of the embodiments described herein, X2 is A or I. In any of the embodiments described herein, X3 is I or F. In any of the embodiments described herein, X4 is E or V. In any of the embodiments described herein, X5 is K or R. In any of the embodiments described herein, X6 is H or P. In any of the embodiments described herein, X7 is L or T. In any of the embodiments described herein, X8 is N or A. In any of the embodiments described herein, X9 is V or I. In some embodiments, X4 is not V.

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

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

[0111] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant with respect to SEQ ID NO: 51 containing 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), where each of X1, X2, X3, X4, X5, X6, X7, X8, and X9 is not a wild-type amino acid.

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

[0113] In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 10 times higher 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 at least 100 times higher 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 at least 1000 times higher than that of a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a binding affinity of 1 × 10⁻¹⁶ -8 Less than M, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 Less than M K D It binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K content 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 Then combine it with CD47.

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

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

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

[0117] In some embodiments, the polypeptide comprises the sequence of Sequence ID No. 222, where X7 is A. In any of the embodiments described above in this embodiment of the present disclosure, X7 is A and X1 is V or I. In any of the embodiments described above in this embodiment of the present disclosure, X7 is A and X2 is A or I. In any of the embodiments described above, X7 is A and X3 is I or F. In any of the embodiments described above, X7 is A and X4 is K or R. In any of the embodiments described above, X7 is A and X5 is H or P. In any of the embodiments described above, X7 is A and X6 is L or T.

[0118] In some embodiments, the polypeptide comprises the sequence of Sequence ID No. 222, where X7 is A. In any of the embodiments described above in this embodiment of the present disclosure, X7 is A and X1 is I. In any of the embodiments described above in this embodiment of the present disclosure, X7 is A and X2 is I. In any of the embodiments described above, X7 is A and X3 is F. In any of the embodiments described above, X7 is A and X4 is R. In any of the embodiments described above, X7 is A and X5 is P. In any of the embodiments described above, X7 is A and X6 is T.

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

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

[0121] In some embodiments, the polypeptide binds to CD47 with at least 10 times higher binding affinity than the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 100 times higher binding affinity than the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 1000 times higher binding affinity than the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the fragments include polypeptides with a length of less than 10 amino acids, about 10 amino acids, about 20 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 60 amino acids, about 70 amino acids, about 80 amino acids, about 90 amino acids, about 100 amino acids, or about 100 amino acids or more. The fragments retain the ability to bind to CD47. Preferably, the SIRPα D1 domain variant polypeptide and its fragments bind to CD47 with a higher affinity than the SIRPα polypeptide binds to CD47. For example, in some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof is 1 × 10 -8 Less than M, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 Less than M K DIt binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K content 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 Then combine it with CD47.

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

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

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

[0125] In some embodiments, the polypeptide comprises the sequence of Sequence ID No. 212, where X7 is A. In any of the embodiments described herein, X7 is A and X1 is V or I. In any of the embodiments described herein, X7 is A and X2 is V or I. In any of the embodiments described herein, X7 is A and X3 is I or F. In any of the embodiments described herein, X7 is A and X4 is K or R. In any of the embodiments described herein, X7 is A and X5 is H or P. In any of the embodiments described herein, X7 is A and X6 is S or T.

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

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

[0128] In some embodiments, the polypeptide of this embodiment of the disclosure comprises 10 or fewer amino acid substitutions to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide of this embodiment of the disclosure comprises 7 or fewer amino acid substitutions 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 at least 10 times higher binding affinity than the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with at least 100 times higher binding affinity than the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with at least 1000 times higher binding affinity than the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the fragments include polypeptides with a length of less than 10 amino acids, about 10 amino acids, about 20 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 60 amino acids, about 70 amino acids, about 80 amino acids, about 90 amino acids, about 100 amino acids, or about 100 amino acids or more. The fragments retain the ability to bind to CD47. Preferably, the SIRPα D1 domain variant polypeptide and its fragments bind to CD47 with a higher affinity than the SIRPα polypeptide binds to CD47. For example, in some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof is 1 × 10 -8 Less than M, 5x10 -9 Less than M, 1x10-9 less than M, 5 x 10 -10 less than M, 1 x 10 -10 less than M or 1 x 10 -11 K less than M D and binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM D and binds to CD47.

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

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

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

[0133] In another aspect of this disclosure, compositions comprising a SIRPα D1 domain variant polypeptide having the amino acid sequence of SEQ ID NO: 48, or a fragment thereof, are disclosed herein. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a higher affinity compared to the affinity with which the SIRPα polypeptide binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide binds to 1 × 10⁻¹⁶ -8 Less than M, or 1 × 10 -9 Less than M, 1 x 10 -10 Less than M, or 1 × 10 -11 Less than M K D It binds to CD47. In some embodiments, the above SIRPα D1 domain variant polypeptide is attached to or fused to a second polypeptide. In some embodiments, the second polypeptide includes, but is not limited to, an Fc polypeptide, an Fc variant, or the aforementioned fragment.

[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 of the variants listed in Table 6.

[0136] In some embodiments, the polypeptide includes a SIRPα D1 domain having at least 85% sequence identity with respect to sequence numbers 80, 81, or 85 listed in Table 6 (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity).

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

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

[0139] Antibodies targeting cell surface antigens can trigger immune stimulation and effector functions related to the binding of Fc receptors (FcRs) on immune cells. There are multiple Fc receptors specific to certain classes of antibodies, including IgG (gamma receptor), IgE (eta receptor), IgA (alpha receptor), and IgM (mu receptor). Binding of antibodies to Fc receptors in the Fc region 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 regulation of immunoglobulin production. Furthermore, the complement system can be activated by the binding of the C1 component of complement to antibodies. 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 antibodies 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-natural SIRPα D1 domain variant linked to an Fc domain variant that forms an Fc domain having removed or reduced effector function.

[0141] In some embodiments, the Fc domain variant refers to a polypeptide chain containing second and third antibody constant domains (e.g., CH2 and CH3). In some embodiments, the Fc domain variant also includes 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 includes as many as 10 amino acid modifications (e.g., insertions, deletions, and / or substitutions) that alter the interaction between the Fc domain and the Fc receptor, compared to the wild-type Fc domain monomer sequence (e.g., amino acid substitutions, additions, or insertions, deletions, or combinations thereof of 1-10, 1-8, 1-6, and 1-4).

[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 through 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 contains an amino acid substitution in the CH2 antibody constant domain to reduce the interaction or binding between the Fc domain dimer variant and an Fc receptor such as the Fcγ receptor (FcγR), Fcα receptor (FcαR), or Fcε (FcεR).

[0144] In some embodiments, a SIRPα D1 domain variant (e.g., any of the variants listed in Tables 2, 5, and 6) is fused to an Fc domain variant or fragment of an Fc domain variant of an immunoglobulin. In some embodiments, the Fc domain variant or fragment of an immunoglobulin can form an Fc domain dimer with another Fc domain variant. In some embodiments, the Fc domain variant or fragment of an immunoglobulin cannot form an Fc domain dimer with another Fc domain variant. In some embodiments, the Fc domain variant or fragment of an Fc domain variant is fused to the polypeptide of the disclosure to extend the serum half-life of the polypeptide. In some embodiments, the Fc domain variant or fragment of an Fc domain variant fused to the polypeptide of the 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, Fc domain variants or fragments of Fc domain variants fused to a polypeptide to extend its serum half-life do not induce any immune system-related response.

[0145] In some embodiments, the SIRPα polypeptide or construct provided herein comprises a SIRPα D1 domain or a variant thereof conjugated to a first Fc domain variant, and an antibody variable domain conjugated to a second Fc domain variant, where the first and second Fc domain variants combine to form an Fc domain dimer variant (e.g., a heterodimer Fc domain dimer variant). An Fc domain dimer is a protein structure found at the C-terminus of an immunoglobulin. An Fc domain dimer contains two Fc domains that are dimerized by an interaction between CH3 antibody constant domains. The wild-type Fc domain dimer forms minimal structures that bind to Fc receptors, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and FcγRIV.

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

[0147] In some embodiments, polypeptide constructs containing a non-natural Fc region as described herein exhibit reduced or eliminated binding to at least one of the Fcγ receptors CD16a, CD32a, CD32b, CD32c, and CD64 compared to polypeptide constructs containing a natural Fc region. In some cases, polypeptide constructs as described herein exhibit reduced or eliminated binding to the CD16a, CD32a, CD32b, CD32c, and CD64Fcγ receptors.

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

[0149] In some embodiments, the Fc domain variants or Fc domain dimer variants described herein are minimally glycosylated or reducedly glycosylated compared to the wild-type sequence. In some embodiments, deglycosylation is achieved by a mutation at 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 as specified according to Kabat et al., (1991). In some embodiments, mutations to one or more of N, Xaa1, Xaa2, or Xaa3 result in deglycosylation of the Fc domain variant or Fc domain dimer variant.

[0150] In some embodiments, variants in the constant region of the antibody IgG (e.g., Fc domain variants or Fc domain dimer variants) have reduced ability to specifically bind to the Fcγ receptor or to induce phagocytosis. In some embodiments, variants in the constant region of the antibody IgG (e.g., Fc domain variants or Fc domain dimer variants) have reduced ability to specifically bind to the Fcγ receptor and reduced ability to induce phagocytosis. For example, in some embodiments, the Fc domain variant is mutated so as not to contain the effector function typical of a “dead” Fc domain variant. For example, in some embodiments, the Fc domain variant includes certain amino acid substitutions known to minimize interaction between the Fc domain dimer and the Fcγ receptor. In some embodiments, the Fc domain variant is derived from an IgG1 antibody and includes one or more of the amino acid substitutions L234A, L235A, G237A, and N297A (designated according to the EU numbering system by Kabat et al., (1991)). In some embodiments, one or more additional mutations are included in such IgG1 Fc domain variants. Non-limiting examples of such additional mutations to human IgG1 Fc domain variants include E318A and K322A. In some cases, 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 when the polypeptide is produced in bacterial or mammalian cells. In some cases, 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, for example, SEQ ID NO: 161 below).In some embodiments, the IgG1 Fc domain variant has the sequence described in any one of SEQ ID NO: 135, SEQ ID NO: 136, or SEQ ID NO: 137. Sequence ID 161: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0151] In some embodiments, the Fc domain variant is derived from an IgG2 or IgG4 antibody and contains 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 sequence homology region with a “standard” Kabat numbered sequence. In some embodiments, the Fc domain variant contains a human IgG2 Fc domain sequence containing one or more of the A330S, P331S, and N297A amino acid substitutions (specified according to the EU numbering system by Kabat et al., (1991)). In some embodiments, one or more additional mutations are included in such IgG2 Fc domain variants. Non-limiting examples of such additional mutations to 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, human IgG2 Fc domain variants have a total of up to 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 or fewer mutations compared to the wild-type human IgG2 sequence. In some embodiments, one or more additional deletions are included in such IgG2 Fc domain variants. For example, in some embodiments, the C-terminal lysine of the Fc domain IgG2 heavy chain constant region, provided in SEQ ID NO: 89 in Table 7, is deleted to increase the uniformity of the polypeptide when the polypeptide is produced in bacterial or mammalian cells. In some cases, human IgG2 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 IgG2 sequence (see, for example, SEQ ID NO: 162 below). Sequence ID 162: ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPI EKTISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

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

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

[0154] In some embodiments, the Fc domain variant exhibits reduced binding to the target Fc receptor compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain variant exhibits elimination of binding to the target Fc receptor compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain variant exhibits reduced phagocytosis compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain variant exhibits elimination of phagocytosis compared to the wild-type human IgG Fc region.

[0155] Sequence IDs 88 and 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 Sequence IDs 90-95, as shown in Table 7. [Table 7-1] [Table 7-2]

[0156] Antibody-dependent cell-mediated cytotoxicity (CMC), also referred to herein as ADCC, refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on specific cytotoxic cells (e.g., natural killer (NK) cells and neutrophils), thereby enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill them. Antibody-dependent cell-mediated phagocytosis (CCP), also referred to herein as ADCP, refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on specific phagocytic cells (e.g., macrophages), thereby enabling these phagocytic effector cells to specifically bind to antigen-bearing target cells and subsequently engulf and digest them. Ligand-specific high-affinity IgG antibodies directed at the surface of target cells can stimulate cytotoxic or phagocytic cells and can be used for such killing. In some embodiments, polypeptide constructs containing the Fc domain variant or Fc domain dimer variant described herein exhibit reduced ADCC or ADCP compared to polypeptide constructs containing the wild-type Fc region. In some embodiments, polypeptide constructs comprising the 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 reductions in ADCC or ADCP compared to polypeptide constructs comprising the wild-type Fc region. In some embodiments, polypeptide constructs comprising the Fc domain variant or Fc domain dimer variant described herein exhibit removal of ADCC or ADCP compared to polypeptide constructs comprising the 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 the antibody Fc domain. In some embodiments, polypeptide constructs containing the Fc domain variants or Fc domain dimer variants described herein exhibit at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more C1q binding compared to polypeptide constructs containing the wild-type Fc region. In some cases, polypeptide constructs containing the Fc domain variants or Fc domain dimer variants described herein exhibit reduced CDC compared to polypeptide constructs containing the wild-type Fc region. In some embodiments, polypeptide constructs comprising the Fc domain variant or Fc domain dimer variant described herein exhibit a reduction in CDC of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to polypeptide constructs comprising the wild-type Fc region. In some embodiments, polypeptide constructs comprising the Fc domain variant or Fc domain dimer variant described herein exhibit negligible CDC compared to polypeptide constructs comprising the wild-type Fc region.

[0158] Fc domain variants or Fc domain dimer variants as described herein include those exhibiting reduced binding to the Fcγ receptor compared to the wild-type human IgG Fc region. For example, in some embodiments, the Fc domain variant or Fc domain dimer variant exhibits less binding to the Fcγ receptor than that exhibited by the wild-type human IgG Fc region, as described in the examples. In some cases, the Fc domain variant or Fc domain dimer variant exhibits reduced binding to the Fcγ receptor by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (effector function completely removed). In some embodiments, the reduced binding is for any one or more Fcγ receptors, e.g., 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 their wild-type human IgG Fc region. Such Fc domain variants or Fc domain dimer variants exhibit reduced phagocytosis compared to their wild-type human IgG Fc region, where the reduction in phagocytic activity is, for example, a coefficient 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 phagocytic cleavage compared to their wild-type human IgG Fc region.

[0160] In some embodiments, an Fc domain variant or Fc domain dimer variant disclosed herein is bound to one or more fusion partners. In some cases, the fusion partners are therapeutic moieties. In some cases, the fusion partners are selected to enable targeting, purification, screening, presentation, etc., of the expressed protein. In some embodiments, the fusion partners also influence the degree of binding to the Fc receptor or the degree of phagocytosis reduction. In some embodiments, as described herein, when an Fc domain variant or Fc domain dimer variant is bound to a fusion partner, it forms the polypeptide construct described below.

[0161] In some embodiments, the fusion partner is linked to an 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, but 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 amino acids, or more. Optionally, in some embodiments, a cleavable linker is used.

[0162] In some embodiments, the fusion partner is a targeting or signaling sequence that directs an Fc domain variant or Fc domain dimeric variant protein and any associated fusion partner to a desired cellular location or extracellular medium. In some embodiments, a specific signaling sequence targets a protein secreted into either growth medium or the peripheral 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 enables purification or screening. Examples of 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, BSP biotinylation target sequences 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 is performed as described elsewhere herein. In some embodiments, the fusion partner enables the use of a selection method for screening Fc domain variants or Fc domain dimer variants as described herein.

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

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

[0166] In some embodiments, disclosed herein is a polypeptide comprising an Fc domain dimer variant, wherein the Fc domain dimer variant comprises two Fc domain variants, each Fc domain variant independently selected from (i) a human IgG1 Fc region consisting of mutants L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of mutants A330S, P331S, and N297A; or (iii) a human IgG4 Fc region consisting of mutants 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 mutants 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 mutants A330S, P331S, and N297A. In some embodiments, the Fc domain dimer variant exhibits elimination or reduction of binding to the Fcγ receptor compared to the wild-type version of the human IgG Fc region. In some embodiments, the Fc domain dimer variant exhibits elimination or reduction of binding to the CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors compared to the wild-type version of the human IgG Fc region. In some embodiments, the Fc domain dimer variant exhibits elimination or reduction of binding to C1q compared to the wild-type version of the human IgG Fc fusion. In some embodiments, at least one of the Fc domain variants in the Fc domain dimer variant is a human IgG4 Fc region containing mutants S228P, E233P, F234V, L235A, delG236, and N297A. In some embodiments, the Fc domain dimer variant exhibits deprivation or reduction of binding to the Fcγ receptor compared to the wild-type human IgG4 Fc region.In some embodiments, the Fc domain dimer variant exhibits desorption or reduction in binding to CD16a and CD32b Fcγ receptors compared to the wild-type version of its human IgG4 Fc region. In some embodiments, the Fc domain dimer variant exhibits approximately 5 × 10⁻¹⁴. -6 K that exceeds M D It then binds to the Fcγ receptor.

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

[0168] In some embodiments, the CD47-binding polypeptide is a signal-regulating protein α (SIRP-α) polypeptide or a fragment thereof. In some embodiments, the SIRPα polypeptide includes a SIRPα D1 domain variant having the following amino acid sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMDFSIRIGX8ITPADAGTYYCX9KFRKGSPDDVEFKSGAGTELSVRAKPS (Sequence ID 221), where X1 is V or I; X2 is A or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is H or P; X7 is L or T; X8 is any amino acid other than N; X9 is V or I. In some embodiments, the SIRPα polypeptide comprises a SIRPα D1 domain variant, where X1 is V or I; X2 is A or I; X3 is I or F; X4 is E; X5 is K or R; X6 is H or P; X7 is L or T; X8 is not N; and X9 is V.

[0169] In some embodiments disclosed herein, the polypeptide comprises a SIRPα D1 domain variant, wherein the SIRPα D1 domain variant is a naturally occurring high-affinity SIRPα D1 domain that binds to human CD47 with an affinity at least 10 times higher than that of naturally occurring D1 domains and Fc domain variants, and the Fc domain variant is ligated to a second polypeptide comprising a second Fc domain variant to form an Fc domain dimer variant, the Fc domain dimer variant having either de-effector function or reduced effector function. In some embodiments, the naturally occurring high-affinity SIRPα D1 domain comprises an amino acid mutation at residue 80.

[0170] In some embodiments, disclosed herein is a SIRPα D1 domain variant, wherein the SIRPα D1 domain variant has a K content of less than 250 nM. D It binds to type 1 CD47, and the SIRPα D1 domain variant has a K content of less than 250 nM. DIt binds to the second type of CD47, and the K of the first type of CD47 D and the second type CD47 K D The relative sizes are within 100 times each, and the first and second species are selected from the group consisting of humans, rodents, and non-human primates. In some embodiments, the SIRPα D1 domain variant binds to CD47 of at least three different species. In some embodiments, the non-human primate is a cynomolgus macaque.

[0171] In some embodiments, disclosed herein is (a) human CD47 at a concentration of less than 250 nM. D A polypeptide comprising (b) a SIRPα D1 domain bound by (b) an Fc domain or a variant thereof ligated 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 variant of human SIRP-α that does not exist in nature. In some embodiments, administration of the polypeptide in vivo results in a hemoglobin reduction of less than 50% during the first week after administration. In some embodiments, administration of the polypeptide in humans results in a hemoglobin reduction of less than 50% 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 mutants L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of mutants A330S, P331S, and N297A; or (iii) a human IgG4 Fc region including mutants S228P, E233P, F234V, L235A, delG236, and N297A. In some embodiments, the Fc domain variant is a human IgG1 Fc region consisting of mutants L234A, L235A, G237A, and N297A. In some embodiments, the Fc domain variant is a human IgG2 Fc region consisting of mutants A330S, P331S, and N297A.

[0172] The SIRPα construct of this disclosure comprises a SIRPα domain or a variant thereof having its C-terminus linked to the N-terminus of an Fc domain or a 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 a variant thereof. In some embodiments, a polypeptide of this disclosure comprising a SIRPα D1 domain variant is fused to an Fc domain variant that cannot form a dimer. In some embodiments, a polypeptide of this disclosure is fused to an Fc domain or a variant thereof that can form a dimer, e.g., a heterodimer, with another Fc domain or a variant thereof. In some embodiments, a polypeptide of the present invention is fused to an Fc domain or a variant thereof, and this fusion protein forms a homodimer. In some embodiments, a polypeptide of this disclosure is fused to a first Fc domain or a variant thereof, and a different protein or peptide (e.g., an antibody variable region) is fused to a second Fc domain or a variant thereof. In some embodiments, the SIRPα D1 domain or a variant thereof is ligated to a first Fc domain or a variant thereof, and a therapeutic protein (e.g., cytokine, interleukin, antigen, steroid, anti-inflammatory agent, or immunomodulator) is ligated to a second Fc domain or a variant thereof. In some embodiments, the first and second Fc domains or variant thereof form a heterodimer.

[0173] Without limitation as stated above, in some embodiments, a SIRPα D1 domain variant polypeptide (e.g., any of the variants listed in Tables 2, 5, and 6) is fused to an Fc polypeptide, or an Fc variant polypeptide such as an Fc domain or a variant thereof. Examples of polypeptides containing a SIRPα D1 domain variant polypeptide and a fused Fc domain variant polypeptide include, but are not limited to, SEQ ID NOs: 96-137, 214, and 216 shown in Table 8. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8] [Table 8-9] [Table 8-10] [Table 8-11]

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

[0175] In some embodiments, the polypeptide includes 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 sequence numbers 98-104, 107-113, 116-122, or 135-137 in Table 8.

[0176] In some embodiments, the polypeptide comprises (a) a signal regulatory protein α(SIRP-α)D1 variant and (b) an fc domain dimer variant, where the SIRPα D1 domain variant comprises the following amino acid sequence: EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSLX 11 PVGPIQWFRGAGPX 12 RX 13 LIYNQX 14 X 15 GX 16 FPRVTTVSX 17 X 18 TX 19 RX 20 NMDFX 21 IX 22 IX 23 X 24 ITX 25 ADAGTYYCX 26 KX 27 RKGSPDX 28 X 29 EX 30 KSGAGTELSVRX 31 KPS (Sequence ID 47), X1 is E or G; X2 is L, I, or V; X3 is V, L, or I; X4 is S or F; X5 is L or S; X6 is S or T; X7 is A or V; X8 is I or T; X9 is H, R, or L; X 10 is A, V, I, or L; X 11 is I, T, S, or F; X 12 is A or G; X 13is E, V, or L; X 14 is K, or R; X 15 is E, or Q; X 16 is H, P, or R; X 17 is D or E; X 18 is S, L, T, or G; X 19 is K, or R; X 20 is E or N; X 21 is S, or P; X 22 is S, or R; X 23 is S, or G; X 24 is any amino acid; X 25 is any amino acid; X 26 is V, or I; X 27 is F, L, or V; X 28 is D or does not exist; X 29 is T, or V; X 30 is F, or V; X 31 is A or G; where the SIRPα D1 domain variant comprises at least two amino acid substitutions to the wild-type SIRPα D1 domain having the sequence described in any one of SEQ ID NOs: 1 to 10. Each Fc domain variant independently includes: (i) a human IgG1 Fc region containing the N297A mutation; (ii) a human IgG1 Fc region containing the L234A, L235A, and G237A mutations; (iii) a human IgG1 Fc region containing the L234A, L235A, G237A, and N297A mutations; (iv) a human IgG2 Fc region containing the N297A mutation; (v) a human IgG2 Fc region containing the A330S and P331S mutations; (vi) a human IgG2 Fc region containing the A330S, P331S, and N297A mutations; and (vii) a human IgG4 Fc region containing the S228P, E233P, F234V, L235A, and delG236 mutations. Fc region; or (viii) 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 the amino acid sequence of SEQ ID NO: 47, and one of the Fc domains is an Fc domain variant containing a human IgG1 Fc region including L234A, L235A, G237A, and N297A mutations.

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

[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, the SIRPα construct is formed from a first subunit, such as a SIRPα D1 domain variant polypeptide fused to a first Fc domain, and a second subunit, such as a second Fc domain (e.g., not containing a SIRPα D1 domain variant polypeptide or any other polypeptide). In some embodiments, the construct has a single SIRPα D1 domain variant polypeptide linked to an Fc domain dimer (e.g., a single arm). In some embodiments, the construct has two SIRPα D1 domain variant polypeptides linked to an Fc domain dimer (e.g., a double arm). In some embodiments, about 500 nM of K DSIRPα D1 domain variants having about 50 nM K are particularly useful in double-arm constructs. In some embodiments, D SIRPα D1 domain variants having about 5 nM K are particularly useful in double-arm constructs. In some embodiments, D SIRPα D1 domain variants having this property are useful in dual-arm and single-arm constructions. In some embodiments, a K of about 500 pM is used. D SIRPα D1 domain variants having are useful in dual-arm and single-arm constructions. In some embodiments, K is present at approximately 100 pM. D SIRPα D1 domain variants having are useful in dual-arm and single-arm constructions. In some embodiments, about 50 pM K D SIRPα D1 domain variants having are useful in dual-arm and single-arm constructions. In some embodiments, about 10 pM K D SIRPα D1 domain variants possessing this feature are useful in both dual-arm and single-arm constructions.

[0180] In some embodiments, heterodimerization of Fc domains is facilitated by introducing different but compatible substitutions into two Fc domains, such as "knob-into-hole" residue pairs and charge residue pairs. While knob-and-hole interactions are favorable for heterodimer formation, interactions between knobs and holes hinder homodimer formation due to steric collisions and the loss of favorable interactions. A hole refers to the void created when an original amino acid in a protein is replaced by a different amino acid with a smaller side-chain volume. A knob refers to the bulge created when an original amino acid in a protein is replaced by a different amino acid with a larger side-chain volume. For example, in some embodiments, the substituted amino acid is located in the CH3 antibody constant domain of an Fc domain and is involved in the dimerization of two Fc domains. In some embodiments, a hole in one CH3 antibody constant domain is created to accommodate a knob in another CH3 antibody constant domain so that the amino acids in the knob and hole act to facilitate or favor the heterodimerization of two Fc domains. In some embodiments, a hole in one CH3 antibody constant domain is created to better accommodate the original amino acid in another CH3 antibody constant domain. In some embodiments, a knob in one CH3 antibody constant domain is created to form an additional interaction with the original amino acid in another CH3 antibody constant domain.

[0181] In some embodiments, holes are constructed by replacing an amino acid with a larger side chain, such as tyrosine or tryptophan, with an amino acid with a smaller side chain, such as alanine, valine, or threonine, for example, the Y407V mutation in the CH3 antibody constant domain. Similarly, in some embodiments, knobs are constructed by replacing an amino acid with a smaller side chain with an amino acid with a larger side chain, for example, the T366W mutation in the CH3 antibody constant domain. In some embodiments, one Fc domain contains the knob mutation T366W, and the other Fc domains contain the hole mutations T366S, L358A, and Y407V. In some embodiments, polypeptides of the present disclosure containing a SIRPα D1 domain variant are fused to an Fc domain containing the knob mutation T366W to restrict homodimerization between undesirable knobs. Examples of knob-into-hole amino acid pairs are listed in Table 9, but are not limited to these. Examples of knob-into-hole Fc domain variants and SIRPα-Fc fusions are shown in Table 10. [Table 9] [Table 10-1] [Table 10-2] [Table 10-3]

[0182] In addition to the knob-into-hole strategy, in some embodiments, electrostatic steering is also used to control the dimerization of Fc domains. Electrostatic steering refers to the control of higher-order protein molecule formation by utilizing favorable electrostatic interactions between peptides, protein domains, and oppositely charged amino acids within a protein. In particular, 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 becomes 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, the 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 domain of two Fc domains promotes the selective formation of heterodimers of the Fc domains, controlled by the electrostatic steering effect resulting from the interaction between the charged amino acids. Examples of electrostatic steering amino acid pairs, but not limited to these, are shown in Table 11. [Table 11]

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

[0184] In some embodiments, the first Fc domain and the second Fc domain are, respectively, T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A relative to the human IgG1 sequence. It contains one or more amino acid substitutions from 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.

[0185] In some embodiments, the Fc domain is: (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) N297A mutation in the Fc region; (ii) L234A, L235A, and G237A mutations in the human IgG1 Fc region; (iii) L234A, L235A, G237A, and N297A mutations in the human IgG1 Fc region; (iv) N297A mutation in the human IgG2 Fc region; (v) A330S and P331S mutations in the human IgG2 Fc region; (vi) A330S, P331S, and N297A mutations in the human IgG2 Fc region; (vii) S228P, E233P, F234V, L235A, and delG236 mutations in the human IgG4 Fc region; or (viii) S228P, E233P, F234V, L235A, delG236, and N297A mutations in the human IgG4 Fc region.In some embodiments, the Fc domain variant is: (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, S3 (b)(i) human IgG1 (ii) N297A mutation in the Fc region; (ii) L234A, L235A, and G237A mutations in the human IgG1 Fc region; (iii) L234A, L235A, G237A, and N297A mutations in the human IgG1 Fc region; (iv) N297A mutation in the human IgG2 Fc region; (v) A330S and P331S mutations in the human IgG2 Fc region; (vi) A330S, P331S, and N297A mutations in the human IgG2 Fc region; (vii) S228P, E233P, F234V, L235A, and delG236 mutations in the human IgG4 Fc region; or (viii) S228P, E233P, F234V, L235A, delG236, and N297A mutations in the human IgG4 Fc region.

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

[0187] Linker In some embodiments, disclosed herein are polypeptides comprising a signal-regulating protein α(SIRP-α)D1 variant 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 residues selected from the group consisting of residues 6, 27, 31, 47, 53, 54, 56, 66, and 92.

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

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

[0190] In some embodiments, the linker is any type of bond produced from a simple covalent bond, e.g., a peptide bond, a synthetic polymer, or a chemical reaction, e.g., a chemical conjugate. When the linker is a peptide bond, in some embodiments, the carboxylic acid group at the C-terminus of one protein domain reacts with the amino group at the N-terminus of another protein domain in a condensation reaction to form a peptide bond. In some embodiments, the peptide bond is formed from synthetic means by conventional organic chemical reactions or by spontaneous generation from a host cell, and the nucleic acid molecules 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 a continuous polypeptide encoding both proteins in the host cell by the necessary molecular mechanisms (e.g., DNA polymerase and ribosomes).

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

[0192] When linkers (excluding the peptide bonds mentioned above) are created by chemical reactions, in some embodiments, chemical functional groups (e.g., amines, carboxylic acids, esters, azides, or other functional groups) are synthetically attached to the C-terminus of one protein and the N-terminus of another, respectively. Then, in some embodiments, the two functional groups react via synthetic chemical means to form a chemical bond, thus linking the two proteins together.

[0193] Spacer In some embodiments of this disclosure, the linker between the Fc domain monomer and the SIRPα D1 variant polypeptide of this disclosure is an amino acid spacer, such as about 1 to 200 amino acids. Preferred 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 motif of GS, GG, GGS, GGG, GGGGS (SEQ ID NO: 163), GGSG (SEQ ID NO: 164), or SGGG (SEQ ID NO: 165), for example, multiple or repeating motifs. In some embodiments, the spacer includes a motif of GS, for example, 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), consisting of 2 to 12 amino acids. In some embodiments, the spacer comprises 3 to 12 amino acids including the 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 the 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 the GGGGS motif (SEQ ID NO: 163), e.g., GGGGSGGGGSGGGGGS (SEQ ID NO: 176).In some embodiments, the spacer includes amino acids other than glycine and serine, such as AAS (SEQ ID NO: 177), AAAL (SEQ ID NO: 178), AAAK (SEQ ID NO: 179), AAAR (SEQ ID NO: 180), EGKSSGSGSESKST (SEQ ID NO: 181), GSAGSAAGSGEF (SEQ ID NO: 182), AEAAAKEAAAKA (SEQ ID NO: 183), KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), GGGGAGGGG (SEQ ID NO: 185), GENLYFQSGG (SEQ ID NO: 186), SACYCELS (SEQ ID NO: 187), RSIAT (SEQ ID NO: 188), RPACKIPNDLKQKVMNH (SEQ ID NO: 189), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 190), AAANSSIDLISVPVDSR (SEQ ID NO: 191), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 192).

[0194] In some embodiments, the spacer includes the motif EAAAK (SEQ ID NO: 193), for example, multiple or repeating motifs. In some embodiments, the spacer includes the motif of a proline-rich sequence, for example, (XP)n, multiple or repeating motifs, where X is any amino acid (e.g., A, K, or E), 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 according to the desired degree of flexibility of the two proteins involved and the final protein fusion polypeptide. In some embodiments, the length of the spacer is adjusted to ensure proper protein folding and avoid aggregate formation. In some embodiments, the spacer is A or AAAL (SEQ ID NO: 178).

[0196] Vectors, host cells, and protein production In some embodiments, disclosed herein are polypeptides comprising a signal-regulating protein α(SIRP-α)D1 variant 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 residues selected from the group consisting of residues 6, 27, 31, 47, 53, 54, 56, 66, and 92.

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

[0198] In some embodiments, the polypeptides of this disclosure are produced from host cells. The host cells refer to a vehicle 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 introduced into host cells by means of transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc. In some embodiments, the choice of nucleic acid vector depends on the host cells used. In some embodiments, the host cells are either of prokaryotic (e.g., bacteria) or eukaryotic (e.g., mammals) origin.

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

[0200] Nucleic acid vector construction and host cells The nucleic acid sequences encoding the amino acid sequences of the polypeptides of this 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, the nucleic acid molecules encoding the polypeptides of this disclosure are obtained using standard techniques, e.g., gene synthesis. Alternatively, a nucleic acid molecule encoding the wild-type SIRPα D1 domain may be mutated to include specific amino acid substitutions using standard techniques, e.g., QuikChange® mutagenesis. In some cases, the nucleic acid molecules are synthesized using nucleotide synthesizers or PCR techniques.

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

[0202] In some embodiments, the components or elements of the vector are optimized so that the expression vector is compatible with the host cell type. Expression vectors found to be used in this disclosure include, but are not limited to, those that enable 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 Disclosure. Examples of mammalian cell types, but not limited to, include human fetal 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 Disclosure. Examples of E. coli strains include, but are not limited to, E. coli294 (ATCC® 31,446), E. coliλ1776 (ATCC® 31,537, E. coli BL21(DE3) (ATCC® BAA-1025)), and E. coliRV308 (ATCC® 31,608).

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

[0205] In some embodiments, the polypeptide construct, including a polypeptide construct such as a SIRPα D1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and a fusion partner such as an Fc variant, is 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 used. Preferred cells, but not limited to these, include known research cells such as Jarcutt 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 containing Fc domain variants are produced in insect cells such as Sf9 and Sf21 cells, or in yeast cells such as organisms of the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, and Yarrowia. In some cases, polypeptide constructs containing 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 reticulocytes) cells are available and, in some embodiments, are selected based on the expression level and functional characteristics of the protein of interest. For example, as will be understood by those skilled in the art, in vitro translation is required for some display techniques, e.g., ribosome display. Furthermore, in some embodiments, Fc domain variants are produced by chemical synthesis methods such as liquid-phase peptide synthesis and solid-phase peptide synthesis, among others.In in vitro transcription using non-glycosylated systems such as bacterial extracts, Fc is not glycosylated even if a natural glycosylation site is present, thus achieving equivalent inactivation of Fc.

[0206] In some embodiments, the polypeptide construct includes unnatural amino acids, amino acid analogs, amino acid mimics, or any combination thereof that function similarly to naturally occurring amino acids. Naturally encoded amino acids generally refer to 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 pyrrolicine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, e.g., hydrogen, carboxyl group, amino group, and carbon atom bonded to an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. In some embodiments, such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but generally retain the same basic chemical structure as natural amino acids.

[0207] Protein production, recovery, and purification In some embodiments, the host cells used to produce the polypeptides of this disclosure are grown in a medium suitable for culturing selected host cells. Examples of suitable media for mammalian host cells include minimal essential medium (MEM), Dulbecco's modified Eagle medium (DMEM), Expi293® expression medium, DMEM supplemented with fetal bovine serum (FBS), and RPMI-1640. Examples of suitable media for bacterial host cells include Luria broth (LB) with the addition of a selective agent, such as ampicillin, or any other necessary supplement. In some embodiments, the host cells are cultured at a suitable temperature, such as about 20°C to about 39°C, for example, about 25°C to about 37°C, preferably 37°C, and at a CO2 level of 5% to 10%. In some embodiments, the pH of the medium is about pH 6.8 to pH 7.4, for example, pH 7.0, depending mainly 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, the cell debris is removed by centrifugation or filtration. The protein can then be further purified. In some embodiments, the polypeptides of the present disclosure are purified by various methods of protein purification, e.g., chromatography (e.g., ion exchange chromatography, affinity chromatography, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard method for protein purification. For example, in some embodiments, the protein is isolated and purified by appropriately selecting and combining 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, the polypeptide is conjugated to a marker sequence, such as a peptide, to facilitate purification. An example of a marker amino acid sequence is a hexahistidine peptide (His6-tag (SEQ ID NO: 223)) that can be conjugated to a nickel-functionalized agarose affinity column with micromolar affinity. Alternatively, a hemagglutinin "HA" tag corresponding to an epitope derived from influenza hemagglutinin protein can be used.

[0209] In some embodiments, a polypeptide construct comprising the polypeptide of the Disclosure, e.g., a SIRPα D1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and a fusion partner such as an Fc variant, is produced by cells of a subject (e.g., human) by administering a vector (e.g., retroviral vector, adenovirus vector, poxvirus vector (e.g., vaccinia virus vector, e.g., modified vaccinia ankara (MVA)), adeno-associated virus vector, and alphavirus vector) containing a nucleic acid molecule encoding the polypeptide of the Disclosure, for example, in the context of gene therapy. The vector can be used to express the polypeptide disclosed herein once it has entered the cells of the subject (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.). In some cases, the polypeptide is secreted from the cells. In some embodiments, if the treatment of a disease or disorder is the desired outcome, no further action is required. In some embodiments, if protein collection is desired, blood is collected from the subject, and the protein is purified from the blood by various means.

[0210] Cancer treatment methods Provided herein is a method for treating cancer in an organism (e.g., a human organism), comprising administering to the organism an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) and (b) a chemotherapeutic agent (at least one chemotherapeutic agent, e.g., at least two, at least three, or at least four chemotherapeutic agents). Provided herein is a method for treating cancer in an organism (e.g., a human organism), comprising administering to the organism an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant (e.g., the SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., the Fc domain variant described herein) and (b) a chemotherapeutic agent (at least one chemotherapeutic agent, e.g., at least two, at least three, or at least four chemotherapeutic agents). In some embodiments, the method further includes administering an effective amount of therapeutic antibodies (at least one therapeutic antibody, e.g., at least two, at least three, or at least four therapeutic antibodies) to the individual. Additionally or alternatively, in some embodiments, the method further includes administering an effective amount of immunotherapy agents (at least one immunotherapy agent, e.g., at least two, at least three, or at least four immunotherapy agents) to the individual. Additionally or alternatively, in some embodiments, the method includes administering polypeptides and chemotherapeutic agents in combination with one or more additional modes of treatment, but not limited to these, such as radiotherapy, surgery, cryoablation, and bone marrow transplantation.

[0211] Combination therapies including chemotherapeutic agents, and exemplary chemotherapeutic agents Examples of chemotherapeutic agents that may be used in the methods of treating cancer described herein include, but are not limited to, methotrexate (RHEUMATREX®, ametopterin), cyclophosphamide (CYTOXAN®), abiraterone, abemaciclib, altretamine, thalidomide (THALIDOMID®), acridine carboxamide, actimid®, actinomycin, actinomycin-D, afatinib, 17-N-allylamino-17-demethoxygeldanamycin, and alectinib. Nib, alpelisib, aminopterin, amsacrin, anlotinib, anthracycline, antitumor agent, antineoplaston, apartinib, 5-azacitidine, 6-mercaptopurine, 6-thioguanine, arabinosylcytosine, axitinib, azacitidine, azathioprine, BL22, bendamustine, binimetinib, bilicodal, bleomycin, bortezomib, bosutinib, brigutinib, bryostatin, busulfan, cabozantinib, kallikrin, camptothecin, capecitabine, carboplatin, calmus Chlorambucil, cisplatin, cladribine, clofarabine, cobimetinib, crizotinib, cytarabine, dabrafenib, dacarbazine, dacomitinib, dasatinib, daunorubicin, dexamethasone, dichloroacetate, discodermorid, docetaxel, doxorubicin, encorafenib, epirubicin, entrectinib, enzalutamide, epotilon, erdafitinib, eribulin, erlotinib, estramustine, etoposide, everolimus, exatecan, exislind, ferginol, floxuridine, Fludarabine, fluorouracil (5-fluorouracil, etc.), folinic acid, phosfestrol, fotemustine, fluquintinib, ganciclovir, gefitinib, gemcitabine, gilteritinib, goserelin, hexamethylmelamine, hydroxycarbamide, hydroxyurea, IT-101, ibrutinib, icotinib, idarubicin, idelalisib, ifosfamide, imatinib, irinoimiquimod, irinotecan, ilofluben, ivosidenib, ixabepyrone, laniquidar,Lapatinib, lalotrectinib, lenalidomide, lenvatinib, lorlatinib, lomustine, raltotecan, maphosfamide, masopropyl, mechloretamine, melphalan, mercaptopurine, methotrexate, methylprednisolone, mitomycin, mitotane, mitoxantrone, nelarabine, neratinib, niraparib, nilotinib, nintedanib, oblimersen, olaparib, osimertinib, oxaliplatin, nedap Latin, phenanthiplatin, picoplatin, PAC-1, paclitaxel, palbociclib, pazopanib, pemetrexed, pegfilgrastim, pentostatin, pipobromane, pixantrone, plicamycin, prednisone, ponatinib, procarbazine, proteasome inhibitors (e.g., bortezomib), pirotinib, larcitrexed, rebeccamycin, Revlimid (registered trademark), regorafenib, ribociclib, rubite Can, lucaparib, ruxolitinib, SN-38, salinosporamide A, satoraplatin, sirolimus, sonidegib, sorafenib, streptozosin, streptozotocin, sunitinib, swinesonin, thalazoparib, talikidal, taxane, tegafur-uracil, temsirolimus, teniposide, temozolomide, testactone, thiotepa, thioguanine, topotecan, trabectedin, trametinib, Examples include tretinoin, trifluridine, triplatin tetranitrate, tris(2-chloroethyl)amine, troxacitabine, uracil mustard, barrubicin, vandetanib, vemurafenib, venetoclax (ABT-199), navitoclax (ABT-263), vinblastine, vincristine, vinorelbine, bismodegib, vorinostat, ziv-aflibercept (ZALTRAP®), and zoskidal.

[0212] In some embodiments, a method for treating cancer includes administering an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) in combination with a specific class of chemotherapeutic agents. 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 for treating cancer includes 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 for treating cancer includes administering a polypeptide described herein in combination with an anthracycline (including, but not limited to, anthracyclines described herein). In some embodiments, a method for treating cancer includes 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 for treating cancer includes administering the polypeptide described herein in combination with an androgen inhibitor (including, but not limited to, the androgen inhibitors described herein). In some embodiments, a method for treating cancer includes administering the polypeptide described herein in combination with an antimetabolite, such as a purine analog (including, but not limited to, the antimetabolite described herein, such as a purine analog). In some embodiments, a method for treating cancer includes administering the polypeptide described herein in combination with an antitumor antibiotic (including, but not limited to, the antitumor antibiotics described herein). In some embodiments, a method for treating cancer includes administering the polypeptide described herein in combination with a BLC-2 inhibitor (including, but not limited to, the BLC-2 inhibitors described herein).In some embodiments, a method for treating cancer includes administering the polypeptide described herein in combination with a BTK inhibitor (including, but not limited to, the BTK inhibitors described herein). In some embodiments, a method for treating cancer includes administering the polypeptide described herein in combination with a CDK4 / 6 inhibitor (including, but not limited to, the CDK4 / 6 inhibitors described herein). In some embodiments, a method for treating cancer includes administering the polypeptide described herein in combination with a colony-stimulating factor (including, but not limited to, the colony-stimulating factor described herein). In some embodiments, a method for treating cancer includes administering the polypeptide described herein in combination with a corticosteroid (including, but not limited to, the corticosteroids described herein). In some embodiments, a method for treating cancer includes administering the polypeptide described herein in combination with an EGFR inhibitor (including, but not limited to, the EGFR inhibitors described herein). In some embodiments, a method for treating cancer includes administering the polypeptide described herein in combination with a gonadotropin-releasing hormone (GnRH) agonist (including, but not limited to, the GnRH agonists described herein). In some embodiments, a method for treating cancer includes administering the polypeptide described herein in combination with a mitotic inhibitor / microtubule inhibitor (including, but not limited to, the mitotic inhibitor / microtubule inhibitor described herein). In some embodiments, a method for treating cancer includes administering the polypeptide described herein in combination with an mTOR kinase inhibitor (including, but not limited to, the mTOR kinase inhibitor described herein). In some embodiments, a method for treating cancer includes administering the polypeptide described herein in combination with a proteasome inhibitor (including, but not limited to, the proteasome inhibitor described herein).In some embodiments, a method for treating cancer includes administering the polypeptide described herein in combination with a signaling inhibitor, such as a protein tyrosine kinase inhibitor, a PAK4 inhibitor, or a PI3K inhibitor (including, but not limited to, the signaling inhibitors described herein). In some embodiments, a method for treating cancer includes administering the polypeptide described herein in combination with a topoisomerase inhibitor (including, but not limited to, the topoisomerase inhibitors described herein). In some embodiments, a method for treating cancer includes administering the 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 for treating cancer includes administering the polypeptide described herein in combination with a VEGF inhibitor, such as a VEGF1 inhibitor, a VEGF2 inhibitor, and / or a VEGF3 inhibitor (including, but not limited to, the VEGF inhibitors described herein). In some embodiments, a method for treating cancer includes administering the polypeptide described herein in combination with an agent that modulates apoptosis (including, but not limited to, agents that modulate apoptosis by modulating the activity of Bcl-2, Mcl1, Bcl-1x, etc., as described herein). In some embodiments, a method for treating cancer includes administering the polypeptide described herein in combination with a platinum-based agent (including, but not limited to, the platinum-based agents described herein).In some embodiments, a method of treating cancer involves administering polypeptides described herein in combination with inhibitors of NTRK1, NTRK2, and / or NTRK3, ALK inhibitors, ROS inhibitors, FLT3 inhibitors, BRAF inhibitors, MEK1 and / or MEK2 inhibitors, HER2, HER3, and / or HER4 inhibitors, RET / PTC inhibitors, BCR-ABL inhibitors, c-KIT inhibitors, PDGFR-alpha and / or PDGFR-beta inhibitors, FGFR1, FGFR2, FGFR3, and / or FGFR4 inhibitors, Smoothund inhibitors, and / or PARP1, PARP2, and / or PARP3 inhibitors (including, but not limited to, the inhibitors described herein). In some embodiments, the inhibitor is an antisense polynucleotide (such as siRNA or RNAi). In some embodiments, the inhibitor is a small molecule inhibitor, as will be described in more detail below.

[0213] In some embodiments, the chemotherapeutic agent is a small molecule anticancer agent (such as a small molecule inhibitor). In some embodiments, a method of treating cancer involves administering the polypeptides 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 the cellular signaling pathway of cells expressing CD47, for example, an IDO / TDO inhibitor, an AhR inhibitor, an arginase inhibitor, an A2a R inhibitor, a TLR agonist, a STING agonist, or a Rig-1 agonist.

[0214] In some embodiments, a method of treating cancer involves 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 derived from different classes (as described above) and / or exert their anticancer effects through different mechanisms of action.

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

[0216] Combination therapies including therapeutic antibodies, and exemplary therapeutic antibodies. In some embodiments, methods for treating cancer provided herein include administering to an individual an effective amount of therapeutic antibodies (e.g., at least one therapeutic antibody, e.g., at least two, at least three, or at least four therapeutic antibodies) in combination with an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., the 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 antibodies are conjugated with a drug (i.e., an antibody-drug conjugate or "ADC").

[0217] Examples of therapeutic antibodies (e.g., therapeutic monoclonal antibodies) for use in the methods described herein include, but are not limited to, 3F8, 8H9, avagovomab, absiximab, abituzumab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, aferimomab, aftuzumab, aracizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatsuximab, anatumomab mafenatox, and anetumab labtansine. ravtansine), aniflorumab, anrukinzumab (IMA-638), apolizumab, arcitumomab, ascrinvacumab, aselizumab, atezolizumab, atinumab, tocilizumab, atorolimumab, avelumab, ba Pineozumab, basiliximab, bavituximab, vectumomab, begeromab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, bisilomab, bimaglumab, bimekizumab, bibatuzumab meltansine mertansine), blinatumomab, brosozumab, vococizumab, brentuximab vedotin, briakinumab, brodalumab, brolucizumab, brontuzumab, kabilizumab (FPA008), camrelizumab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide (Capromabpendetide), carlumab, catumakisomab, cBR96-doxorubicin immunoconjugate, CC49, cedelizumab, certolizumab pegol, cetuximab, Ch.14.18, citatuzumab bogatox, cixutumumab, crazakizumab, clenoliximab, clivatuzumab tetraxetan, codrituzumab, coltuximab labtansine ravtansine), conatumumab, concizumab, crenezumab, CR6261, dasetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumumab, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, derlotuximab biotin biotin), detumomab, dinutuximab, diridavumab, dorlimomabaritox), drozitumab, duligotumab, 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, Epitumomabcituxetan), epratuzumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, facinumab, FBTA05, felvizumab, fezakinumab, ficratuzumab Ficlatuzumab, Figitumumab, Firivumab, Flambotumab, 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, Lemalesomab, Lenzilumab, Lerdelimumab, Lexatumumab, Libivirumab, Lifastuzumab vedotin Vedotin, Ligelizumab, Lilotomab satetraxetan, Lintuzumab, Lirilumab, Lodelcizumab, Lokivetomab, Lorvotuzumab mertansine, Lucatumumab, Lulizumab pegolpegol), Lumiliximab, Lumretuzumab, MSB0010718C (Avelumab), Mapatumumab, Margetuximab, Maslimomab, Mavrilimumab, Matuzumab, MEDI6469, MEDI0680, MEDI 6383, Mepolizumab, Metelimumab, Miratuzumab, Minretumomab, Mitumomab, Mogamulizumab, Morolimmumab, Motavizumab, Moxetumomab Pasdotox pasudotox), Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox, Narnatumab, Natalizumab, Nebacumab, Necitumumab, Nemolizumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentan Merpentan, Obiltoxaximab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Ontuxizumab, Opicinumab (Opicinumab), Oportuzumab monatox, Oregovomab, Orticumab, Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab, Pankomab, Panobacumab, Pulsat Zumab (Parsatuzumab), Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Polatuzumab vedotinVedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO140, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab Refanezumab, Regavirumab, Reslizumab, Rilotumumab, Rinucumab, Rituximab, Robatumumab, Rolledumab, Romosozumab, Rontalizumab, Robelizumab, Ruplizumab, Sacituzumab (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, tripalimab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, trastuzumab emtansine, TRBS07, tregalizumab, tremelimumab, tucotuzumab sermoloukin celmoleukin), tuvirumab, ublituximab, urocuplumab, urerumab, urtoxazumab, ustekinumab, utomirumab (PF-05082566), vandortuzumab vedotinVedotin, Vantictumab, Vanucizumab, Vapaliximab, Varlilumab, Vatelizumab, Vedolizumab, Beltuzumab, Vepalimomab, Vesencumab, Visilizumab, Volociximab, Vonlerolizumab (RG7888), Vorsetuzumab mafodotin Examples include mafodotin, botumumab, zalutumumab, zanolimmab, zatuximab, diralimumab, or zolimomab aritox, or 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, but are not limited to these, including, for example, anti-CD20 antibody, anti-EGFR antibody, anti-Her2 / Neu(ERBB2) antibody, anti-EPCAM antibody, anti-GL2 antibody, anti-GD2 antibody, anti-GD3 antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CDI9 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD39 antibody, anti-CD45 antibody, anti-CD47 antibody, anti-CD52 antibody, anti-C Examples 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 bacterial-infected cells, immune cells, or healthy normal cells, or to cytokines, chemokines, or any type of hormone.

[0219] In some embodiments, the therapeutic antibodies used in the methods herein include, for example, CS1 / SLAMF7, Trop-2, VWF, vimentin, VEGFR2, VEGFR-1, VEGF, VEGF-A, TYRP1 (glycoprotein 75), TWEAK receptor, tumor-specific glycosylation of MUC1, tumor antigen CTAA16.88, TRAIL-R2, TRAIL-R1, TNF-α, TGF-beta, TGF-beta2, TGF-beta1, 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, RS virus, RANKL, Rabies virus glycoprotein, Platelet-derived growth factor receptor beta, Phosphatidylserine, Sodium phosphate cotransporter, PDGF-R alpha, PDCD1, PD-1, PD-L1, PCSK9, oxLDL, OX-40, NRP1, Notch receptor 4, Notch receptor 3, Notch receptor 2, Notch receptor 1, NOGO-A, NGF, Neuronal apoptosis-regulating protease 1 (neural apoptosis-regulated proteinase 1), NCA-90 (granulocyte antigen), NARP-1, N-glycolylneuraminic acid, myostatin, myelin-related 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, IGHE, IgG4, IGF-I, IGF-I receptor, IgE Fc region, IFN-gamma, IFN-alpha, ICAM-1 (CD54), human TNF, human scattering 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 extradomain-B, fibrin II, beta chain, FAP, respiratory syncytial virus F protein, ERBB3, epicyalin, EpCAM, endotoxin, EGFR, EGFL7, Shiga toxin-producing Escherichia coli type 2 (E. coli shiga toxin Type 2), Shiga toxin-producing Escherichia coli type I (E. coli shiga toxin type-I), DRS, DPP4, DLL4, dabigatran, cytomegalovirus glycoprotein B, CTLA-4, CSF2, CSF1R, clamping factor A, CLDN18.2, ch4DS, CFD, CEA-related antigen, CEA, CD80, CD79B, CD74, CD73, CD70, CD6, CD56, CD52, CD51, CD5, CD44 v6, CD41, CD40 ligand, CD40, CD4, CD39, CD38, CD37, CD33, CD30 (TNFRSF8), CD123, CD138, CD3 epsilon, CD3, CD28, CD274, CD27, CD2S (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 familiarisThis antibody 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 cancer cells (for example, expressed on the surface of cancer cells). Exemplary antigens expressed by cancer are known in the art, but are not limited to these, including, 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, Guanyle cyclase C, DLL3, EGFR, HER2, VEGF, VEGFR, Integrin αVβ3, Integrin α5β1, MET, IGF1R, TRAILR1, TRAILR2, RANKL, FAP, Tenascin, Le yExamples include EpCAM, CEA, gpA33, PSMA, TAG72, mucin, CAIX, EPHA3, folate receptor α, 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, the polypeptides described herein are 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 talakotzumab (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 Examples include 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, methods for treating cancer provided herein include administering to an individual an effective amount of immunotherapy agents (e.g., at least one immunotherapy agent, e.g., at least two, at least three, or at least four immunotherapy agents) in combination with an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., the 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 the therapeutic reorientation of the immune system, such as modifiers of co-stimulatory pathways, cancer vaccines, and recombinant modified immune cells. Exemplary and non-limiting immunotherapeutic agents are described below. In some embodiments, immunotherapeutic agents are antibodies or contain antibodies. Exemplary targets for immunotherapy antibodies are known in the art, 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 Examples include 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, and durvalumab. In certain embodiments, an agent that blocks the interaction between CD47 and SIRPα (such as the polypeptides described herein) is administered in combination with a PD-L1 / PD-1 pathway inhibitor, e.g., an antibody, a small molecule, or a 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., the anti-PD-1 or anti-PD-L1 antagonist antibodies described elsewhere herein). As described herein, the combined administration of an agent that blocks the interaction between CD47 and SIRPα (such as a polypeptide described herein) and an inhibitor of the PD-L1 / PD-1 pathway may result in synergistic antitumor activity. In some embodiments, the immunotherapeutic agent is or comprises a vaccine, an oncolytic virus, adoptive cell therapy, a cytokine, or a small molecule immunotherapeutic agent. Examples of such immunotherapeutic agents are known in the art. For example, adoptive cell therapies and therapeutic agents include, but are not limited to, chimeric antigen receptor T cell therapy (CAR-T), tumor-infiltrating lymphocytes (TILs), TCR-modified T cells, TCR-modified NK cells, and macrophage cell products. Vaccines include, but are not limited to, polynucleotide vaccines, polypeptide vaccines, or cell-based vaccines (e.g., tumor or dendritic cell-based). Various cytokines useful for cancer treatment are known, but are not limited to, IL-2, IL-15, IL-7, IL-10, IL-12, IL-21, TNFα, IFN, GM-CSF, and engineered cytokine mutants.Examples of small molecule immunotherapies, though not limited to these, include 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 further agents (may include therapeutic antibodies, small molecule inhibitors, immunotherapeutic agents, etc.) described herein, the further agents (may include) of different classes and / or exert their anticancer effects through different mechanisms of action. For example, in some embodiments, a method of treating cancer involves administering an agent that blocks the interaction between CD47 and SIRPα (such as a polypeptide described herein) in combination with a chemotherapeutic agent (including, but not limited to, those described herein) and a therapeutic antibody (including, but not limited to, those described herein, such as an anti-HER2 antibody). In some embodiments, an agent that blocks the interaction between CD47 and SIRPα (such as a polypeptide described herein) is administered in combination with a chemotherapeutic agent (including, but not limited to, those described herein) and a small molecule inhibitor (including, but not limited to, those described herein). Other combinations are also conceivable.

[0226] In some embodiments, agents that block the interaction between CD47 and SIRPα (such as polypeptides described herein) are administered in combination with one or more agents, including but not limited to antidiarrheal agents, antiemetics, analgesics, opioids and / or nonsteroidal anti-inflammatory drugs.

[0227] Combination therapy including additional treatment(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 modes. In some embodiments, the one or more additional therapeutic modes include radiotherapy (e.g., gamma rays, X-rays, and / or direct delivery of radioisotopes to tumor cells, microwaves, UV radiation, or gene therapy). Examples of therapeutic genes for gene therapy include, but are not limited to, antisense versions of cell proliferation inducers (oncogenes), cell proliferation inhibitors (tumor suppressors), or programmed cell death inducers (pro-apoptotic genes). In some embodiments, any one or more combination therapies described herein are administered in conjunction with surgery (e.g., resection).

[0228] Example therapeutic combinations In some embodiments, a 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, leerlotinib, bortezomib, enzalutamide, lemetrexed, nilotinib, abiraterone, iris, iris, iris, iris, 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 blocking the interaction between CD47 and SIRPα is a polypeptide described herein (e.g., a fusion polypeptide comprising a SIRPα d1 domain variant and an Fc variant).

[0229] In some embodiments, a method of treating cancer 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 blocking 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, a 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 trastuzumab and one or more agents selected from the following: lenalidomide, ibrutinib, palbociclib, enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, etc. Nzalutamide, 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 blocking 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, a 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 bevacizumab and one or more agents selected from the following: lenalidomide, ibrutinib, palbociclib, enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, etc. Nzalutamide, 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 blocking 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, a 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, etc. Nzalutamide, 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 blocking 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, a 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 pertuzumab and one or more agents selected from the following: lenalidomide, ibrutinib, palbociclib, enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, etc. Nzalutamide, 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 blocking 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, a 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, lemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, lemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, lemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, erlotinib, bortezomib, enzalutamide, erlotinib, bortezomib, enzalutamide, erlotinib, erlotinib, bortezomib, erlotinib 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 blocking 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 cancer In some embodiments, cancers treated by the methods provided herein include 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, stomach cancer, oral cancer, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, kidney cancer, bladder cancer, urinary tract cancer, pancreatic cancer, retinoblastoma, cervical cancer, uterine cancer, Wilms' tumor, multiple myeloma, skin cancer, lymphoma, leukemia, hematological cancer, thyroid cancer, bone cancer, adenoid cystic tumor, chondrosarcoma, islet cell tumor, neuroendocrine tumor, prostate cancer, glioblastoma, endometrial carcinoma, endometrial cancer, leiomyosarcoma, gallbladder cancer, hepatocellular carcinoma, melanoma, or solid tumors.

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

[0237] Treatment methods for leukemia In some embodiments, in an individual (e.g., a human individual), leukemia (e.g., acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), hairy cell leukemia, chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), large granular lymphocytic (LGL) leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN), B-cell prelymphocytic leukemia (B-PLL), T-cell prelymphocytic leukemia (T-PLL) A method is provided for treating multiple myeloma (MM), and non-Hodgkin lymphomas (such as diffuse large B-cell lymphoma (DLBCL)), Burkitt lymphoma, mantle cell lymphoma (MCL), peripheral T-cell lymphoma (PTCL), lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma (MZL), and follicular lymphoma (FL), the method comprising administering to an individual an effective dose 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., the SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., the 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 containing L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations (where numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows the Kabat EU index). In some embodiments, the polypeptide administered to the individual (e.g., a fusion polypeptide) contains the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., a fusion polypeptide) forms a homodimer. In some embodiments, the polypeptide (e.g., a fusion polypeptide) and the Bcl2 inhibitor (e.g., venetoclax) are administered simultaneously, in parallel, or sequentially.

[0238] Bcl2 inhibitors are a class of anticancer drugs that are thought to exert cytotoxic effects by competing with pro-apoptotic Bcl2 and occupying the BH3 docking groove on the surface of anti-apoptotic family members. By binding to one or more Bcl2 family members, these inhibitors induce apoptosis by mimicking the activity of native 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 used in the methods described herein. Venetoclax is empirically formulated C 45 H 50ClN7O7S is a pale yellow to dark yellow solid 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)-4,4dimethylcyclohexa-1-en-1-yl]methyl}piperazine-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridine-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 marketed under the trade names Venclexta and Venclyxto. Complete information regarding the preparation, dispensing, dosage, and administration schedule of venetoclax may be found in the national prescribing information (in the United States, see, for example, www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2016 / 208573s000lbl(dot)pdf; in Europe, see, for example, www(dot)ema(dot)europa(dot)eu / en / medicines / human / EPAR / venclyxto#product-information-section). In some embodiments, venetoclax is administered according to the dosage and frequency recommended in the national prescribing information.

[0241] ABT-737 is another exemplary selective Bcl2 inhibitor used in the method described herein. ABT-737 inhibits both Bcl2 and Bcl-xL, and empirically formula C 42 H 45It contains ClN6O5S2 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 used in the method described herein is Navitocrax (also known as ABT-263). Navitocrax inhibits both Bcl2, Bcl-xL, and Bcl-w, and empirically formulated C 47 H 55 It has ClF3N5O6S3 and a molecular weight of 974.6 g / mol. The CAS registry number for Navitocrax is 923564-51-6. ABT-737 is chemically described as 4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazine-1-yl]-N-[4-[[(2R)-4-morpholine-4-yl-1-phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide and has the following chemical structure. Additional details regarding Navitocrax are described, for example, in Tse et al. (2008) Cancer Res. 68(9):3421-3429. [ka]

[0243] Another exemplary selective Bcl2 inhibitor used in the methods described herein is S55746 (also known as BCL201 and Servier-1). S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile shows no significant binding to MCL-1 and BFL-1 (BCL2A1 / A1), indicating insufficient affinity for BCL-XL. S55746 does not exhibit cytotoxicity to BCL-XL-dependent cells such as platelets (see, e.g., Casara et al. (2008) Oncotarget. 9(28):29975-20088). S55746 is empirically formulated C 43 H 42 It contains N4O6 and has a molecular weight of 710.82 g / mol. The CAS registry number for S55746 is 1448584-12-0. Chemically, S55746 is 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-tetrahydroindridine-1-carboxamide and has the following chemical structure: [ka]

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

[0245] Platinum-based drugs (such as carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenantriplatin, picoplatin, and satraplatin) are widely used antitumor drugs that cause DNA crosslinking as monoadducts, interstrand crosslinks, intrastrand crosslinks, or DNA protein crosslinks. Platinum-based drugs typically act on the N-7 position adjacent to 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-coordinate compound used in the methods described herein. The chemical name of cisplatin is dichloroplatinate 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 hydrolysis, cisplatin reacts with DNA to form both intra- and inter-strand crosslinks. These crosslinks are thought to impair DNA replication and transcription. The cytotoxicity of cisplatin correlates with cell arrest in the G2 phase of the cell cycle. It is commercially available under various names including CAS registry number 15663-27-1, PLATINOL (trademark), PLATINOL (trademark)-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 may be found in the package inserts of each country (see, for example, 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 the United States). In some embodiments, cisplatin is administered according to the dosage and frequency recommended in the package inserts of each country.

[0248] Carboplatin is another exemplary platinum-coordinate compound used in the method described herein. The chemical name of carboplatin is platinum, diammine[1,1-cyclobutane-dicarboxylat(2-)-0,0]-, (SP-4-2), and carboplatin has the following structural formula: [ka]

[0249] Carboplatin has the molecular formula C6H 12Carboplatin is a water-soluble platinum complex with the molecular weight 373.26, represented as N2O4Pt. CAS registry number 41575-94-4 is assigned to carboplatin, and its mechanism of action is similar to that of cisplatin. Carboplatin is typically prescribed more commonly than cisplatin. Carboplatin is marketed under various names, including PARAPLATIN®, BLASTOCARB®, BLASTOPLATIN®, CARBOKEM®, CARBOMAX®, PARAPLATIN®, CARBOPA®, and KARPLAT®. Complete information regarding the preparation, dispensing, dosage, and administration schedule of carboplatin may be found in the package inserts of each country (see, for example, www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2010 / 020452s005lbl(dot)pdf and www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2012 / 077139Orig1s016lbl(dot)pdf in the United States). In some embodiments, carboplatin is administered according to the dosage and frequency recommended in the package inserts of each country.

[0250] In some embodiments, the provided method is for treating a solid tumor in an organism (e.g., a human organism), the method comprising administering to the organism an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-HER2 antibody, and (c) an anti-PDL1 antibody. In some embodiments, the anti-HER2 antibody is trastuzumab (CAS registry number: 180288-69-1). In some embodiments, the anti-PDL1 antibody is atezolizumab (CAS registry number: 1380723-44-3), avelumab (CAS registry number: 1537032-82-8), or durvalumab (CAS registry number: 1428935-60-7). In some embodiments, the agent is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., the SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., the 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 containing L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations (where numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows the Kabat EU index). In some embodiments, the polypeptide administered to the individual (e.g., a fusion polypeptide) contains the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., a fusion polypeptide) forms a homodimer.In some embodiments, polypeptides (e.g., fusion polypeptides), anti-HER2 antibodies, and anti-PD-L1 antibodies (e.g., anti-PD-L1 antagonist antibodies) are administered simultaneously, in parallel, 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. + It is a solid tumor. In some embodiments, the solid tumor is colon cancer (e.g., HER2 + It is colon cancer.

[0251] Treatment methods for gastric cancer or gastroesophageal junction (GEJ) cancer In some embodiments, a method is provided for treating gastric cancer or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-HER2 antibody, (c) an anti-VEGFR2 antibody, and (d) paclitaxel. In some embodiments, the anti-HER2 antibody is trastuzumab (CAS registry number: 180288-69-1). In some embodiments, the anti-VEGFR2 antibody is ramucirumab (CAS registry number: 947687-13-0). In some embodiments, the agent is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., the SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., the Fc domain variant described herein). In some embodiments, the polypeptide (e.g., fusion polypeptide) comprises a SIRPα D1 domain variant containing 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 containing L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations (where numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows the Kabat EU index). In some embodiments, the polypeptide administered to the individual (e.g., a fusion polypeptide) contains the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., a fusion polypeptide) forms a homodimer.In some embodiments, polypeptides (e.g., fusion polypeptides), anti-HER2 antibodies, anti-VEGFR2 antibodies, and paclitaxel are administered simultaneously, in parallel, or sequentially. In some embodiments, polypeptides (e.g., fusion polypeptides) are administered to the individual at a dose of 10 mg / kg 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 + The patient has GEJ cancer (e.g., HER2-overexpressing gastric or GEJ cancer). In some embodiments, HER2 + Gastric cancer or HER2 + GEJ cancer is progressive and / or metastatic. In some embodiments, the treated individual has gastric cancer or GEJ cancer that has progressed during or after a prior treatment (multiple) including an anti-HER2 antibody (e.g., trastuzumab). In some embodiments, the treated individual has gastric cancer or GEJ cancer that has progressed during or after a prior treatment (multiple) including an anti-HER2 antibody (e.g., trastuzumab) and fluoropyrimidine. In some embodiments, the treated individual has gastric cancer or GEJ cancer that has progressed during or after a prior treatment (multiple) including an anti-HER2 antibody (e.g., trastuzumab) and platinum-based chemotherapy agents. In some embodiments, the treated individual has gastric cancer or GEJ cancer (e.g., HER2) that has progressed during or after a prior treatment (multiple) including an anti-HER2 antibody (e.g., trastuzumab) and / or fluoropyrimidine and / or platinum-based chemotherapy agents. +The patient has gastric cancer or GEJ cancer. In some embodiments, the patient had not responded to previous treatment with an anti-HER2 antibody, an anti-HER2 antibody and a fluoropyrimidine, or an anti-HER2 antibody and a platinum-based chemotherapeutic agent (e.g., relapsed or did not respond). In some embodiments, the fluoropyrimidine was fluorouracil (also known as 5-fluorouracil). In some embodiments, treatment with a polypeptide, an anti-HER2 antibody, an anti-VEGFR2 antibody, and paclitaxel does not result in adverse effects. In some embodiments, treatment with a polypeptide, an anti-HER2 antibody, an 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 method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-PD-1 antibody (e.g., an anti-PD-1 antagonist antibody), (c) an anti-HER2 antibody, (d) 5-fluorouracil, and (e) a platinum-based chemotherapeutic agent. In some embodiments, a method is provided for treating gastric cancer or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-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., the SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., the 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 containing L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations (where numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows the Kabat EU index). In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to the individual contains the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., fusion polypeptide) forms homodimers. In some embodiments, the polypeptide (e.g., fusion polypeptide), anti-PD-1 antibody, anti-HER2 antibody, 5-fluorouracil, and platinum-based chemotherapeutic agents are administered simultaneously, in parallel, or sequentially. In some embodiments, the polypeptide (e.g., fusion polypeptide), anti-PD-1 antibody, anti-HER2 antibody, capecitabine, and platinum-based chemotherapeutic agents are administered simultaneously, in parallel, or sequentially. In some embodiments, the individual being treated has HER2-overexpressing gastric cancer or HER2-overexpressing GEJ cancer. In some embodiments, the gastric cancer or GEJ cancer is progressive and / or metastatic. In some embodiments, the individual has not received prior treatment for gastric cancer or GEJ cancer.

[0253] Treatment methods for head and neck cancer In some embodiments, a method is provided for treating head and neck cancer (e.g., squamous cell carcinoma or HNSCC) in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) a PD-1 inhibitor, (c) an antimetabolite, and (d) a platinum-based 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, pizilizumab, semiprimab, 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, phloxuridine, 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, phenantriplatin, 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., the SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., the Fc domain variant described herein). In some embodiments, the polypeptide (e.g., fusion polypeptide) comprises a SIRPα D1 domain variant containing 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 containing L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations (where numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows the Kabat EU index). In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to the individual contains the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., fusion polypeptide) forms homodimers. In some embodiments, the polypeptide (e.g., fusion polypeptide), PD-1 inhibitor (e.g., anti-PD-1 antibody, e.g., pembrolizumab), antimetabolites (e.g., 5-fluorouracil), and platinum-based chemotherapeutic agents (e.g., cisplatin or carboplatin) are administered simultaneously, in parallel, 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 HNSCC. In some embodiments, the HNSCC is progressive and / or metastatic HNSCC. In some embodiments, the HNSCC is unresectable and / or recurrent. In some embodiments, the individual has not received prior treatment for head and neck cancer (e.g., HNSCC). 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) 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, the provided method is for treating cancer in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), and (b) an anti-TROP2 antibody. In some embodiments, the anti-TROP2 antibody is RS7 as described in U.S. Patent No. 10,179,171, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the anti-TROP2 antibody is conjugated with 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), as described in U.S.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., the SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., the 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 containing L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations (where numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows the Kabat EU index).In some embodiments, the polypeptide administered to the individual (e.g., a fusion polypeptide) contains the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., a fusion polypeptide) forms homodimers. In some embodiments, the polypeptide (e.g., a fusion polypeptide) and the anti-TROP2 antibody are administered simultaneously, in parallel, or sequentially. In some embodiments, the cancer is a solid tumor, gastric cancer, nasopharyngeal cancer, gallbladder cancer, cervical cancer, extranodal NK / T cell lymphoma, lung cancer, laryngeal squamous cell carcinoma, colon cancer, hilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrial cancer, or ovarian cancer. In some embodiments, the cancer is characterized by overexpression of TROP2. In some embodiments, the cancer is not characterized by overexpression of TROP2.

[0255] Methods to increase the phagocytic activity of target cells In some embodiments, a method is provided for increasing the phagocytosis of target cells (e.g., cancer cells), the method comprising contacting the target cells with (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), and (b) an anti-TROP2 antibody. In some embodiments, the anti-TROP2 antibody is RS7 as described in U.S. Patent No. 10,179,171, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the anti-TROP2 antibody is conjugated with 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 U.S.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., the SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., the 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 containing L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations (where numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows the Kabat EU index).In some embodiments, the polypeptide administered to the individual (e.g., a fusion polypeptide) comprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., a fusion polypeptide) forms homodimers. In some embodiments, the target cells are cancer cells. In some embodiments, the cancer cells are solid tumor cells, gastric cancer cells, nasopharyngeal cancer cells, gallbladder cancer cells, cervical cancer cells, extranodal NK / T cell lymphoma cells, lung cancer cells, laryngeal squamous cell carcinoma cells, colon cancer cells, hilar cholangiocarcinoma cells, pancreatic cancer cells, oral squamous cell carcinoma cells, endometrial cancer cells, or ovarian cancer cells.

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

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

[0258] In some embodiments, the contact is performed in vitro. In some embodiments, the contact is performed in vivo. In some embodiments, the target cells are cancer cells. In some embodiments, target cells are brought into contact with (a) a polypeptide comprising a SIRPα D1 domain variant (e.g., the SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., the Fc domain variant described herein), and (b) one or more agents capable of enhancing phagocytosis, thereby increasing the phagocytosis of 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 target cells with one or more agents capable of enhancing phagocytosis (i.e., in the absence of polypeptides comprising a SIRPα D1 domain variant (e.g., the SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., the Fc domain variant described herein).

[0259] Kits and manufactured products In another embodiment of the present invention, a product or kit is provided comprising a polypeptide (e.g., a fusion polypeptide as 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 NOs: 81 and 85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region containing L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations (where numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows the Kabat EU index). In some embodiments, the Fc domain variant contains the amino acid sequence of Sequence 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 product is intended for use in accordance with the therapeutic methods provided herein.

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

[0261] In some embodiments, the kit or product further comprises a platinum-based chemotherapeutic agent. In some embodiments, the platinum-based chemotherapeutic agent is carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenantriplatin, picoplatin, or satraplatin. In some embodiments, the kit includes a package insert or label containing instructions for using a polypeptide (e.g., fusion polypeptide) in combination with a 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 (e.g., a human individual).

[0262] In some embodiments, the kit or product further comprises an anti-HER2 antibody (e.g., trastuzumab) and a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody such as atezolizumab, avelumab, or durvalumab). In some embodiments, the kit includes a package insert or label containing instructions for using a 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 slow 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, cancer (e.g., solid tumors) is HER2 + It is cancer. In some embodiments, the cancer is colon cancer (e.g., HER2 + It is colon cancer.

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

[0264] In some embodiments, the kit or product further comprises an anti-HER2 antibody (e.g., trastuzumab), a PD-1 inhibitor (e.g., an anti-PD-1 antibody such as pembrolizumab), 5-fluorouracil, and a platinum-based agent (e.g., cisplatin or carboplatin). In some embodiments, the kit includes a package insert or label containing instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-HER2 antibody (e.g., trastuzumab), a PD-1 inhibitor (e.g., pembrolizumab), 5-fluorouracil, and a platinum-based agent (e.g., cisplatin or carboplatin) 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 product further comprises an anti-HER2 antibody (e.g., trastuzumab), a PD-1 inhibitor (e.g., an anti-PD-1 antibody such as pembrolizumab), capecitabine, and a platinum-based agent (e.g., cisplatin or carboplatin). In some embodiments, the kit includes a package insert or label containing instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-HER2 antibody (e.g., trastuzumab), a PD-1 inhibitor (e.g., pembrolizumab), capecitabine, and a platinum-based agent (e.g., cisplatin or carboplatin) 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 product further comprises a PD-1 inhibitor (e.g., an anti-PD-1 antibody such as pembrolizumab, nivolumab, pizilizumab, semiprimab, or BMS936559), antimetabolites (e.g., 5-fluorouracil, 6-mercaptopurine, capecitabine, cytarabine, phloxuridine, fludarabine, gemcitabine, hydroxycarbamide, methotrexate, pemetrexed, or phototrexate), and a platinum-based agent (e.g., cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenantriplatin, picoplatin, or satraplatin). In some embodiments, the kit includes, for example, a package insert or label containing instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with a PD-1 inhibitor (e.g., pembrolizumab, nivolumab, pizilizumab, semiprimab, or BMS936559), antimetabolites (e.g., 5-fluorouracil, 6-mercaptopurine, capecitabine, cytarabine, phloxuridine, fludarabine, gemcitabine, hydroxycarbamide, methotrexate, pemetrexed, phototrexate) and a platinum-based agent (e.g., cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenantriplatin, 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) according to the methods provided herein.

[0266] In some embodiments, the kit or product further comprises a therapeutic anti-TROP2 antibody. In some embodiments, the anti-TROP2 antibody is RS7 (see, for example, U.S. Patent No. 10,179,171) or sacituzumab govitecan. In some embodiments, the kit contains a substance that treats TROP2 in an organism (such as a human organism). +The package insert or label includes instructions for using a polypeptide (e.g., 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 cancer, gallbladder cancer, cervical cancer, extranodal NK / T cell lymphoma, lung cancer, laryngeal squamous cell carcinoma, colon cancer, hilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrial cancer, or ovarian cancer).

[0267] In some embodiments, a polypeptide (e.g., a fusion polypeptide) and one or more additional anticancer 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 anticancer agents are provided in the same container or in separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. Containers may be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or alloy (e.g., stainless steel or Hastelloy). In some embodiments, the container holds the formulation, and a label on the container or a label associated with the container may indicate instructions for use. The product or kit may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and accompanying documentation with instructions for use. In some embodiments, the product further includes one or more other agents (e.g., chemotherapeutic agents and antitumor agents, therapeutic antibodies, etc.). Suitable containers for one or more agents include, for example, bottles, vials, bags, and syringes.

[0268] This specification is deemed sufficient to enable those skilled in the art to practice the invention. In addition to the modifications shown and described herein, various modifications of the invention are evident to those skilled in the art from the foregoing description and are included in the appended claims. All publications, patents, and patent applications referenced herein are incorporated herein by reference in their entirety for all purposes. [Examples]

[0269] This disclosure will be better understood by reference to the following examples. However, the examples should not be construed as limiting the scope of this disclosure. The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes taking them into account will be suggested to those skilled in the art, and will be understood to be included within the spirit and scope of this application and within 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, in an RS4;11 xenograft model, the antitumor activity of drug A, an exemplary polypeptide containing SIRPα d1 domain variants and Fc variants, was evaluated in combination with venetoclax.

[0271] Materials and methods RS4;11 heterogeneous port model RS4;11 cells (described in Strong et al. (1985) Blood. 65(1):21-31) were sampled using a Matrigel (Corning) vs. RPMI 1640 ratio of 1:1 for 5 × 10⁶ cells. 6 The drug was injected into the right flank of NOD-SCID female mice at a cell / mouse concentration. The average size of all tumors was 190 mm. 3Tumors were monitored until a certain level was reached. Mice were randomized into three cohorts: PBS control, venetoclax (Selleckchem), drug A, and a combination of venetoclax and drug A, with 10 mice per cohort. The venetoclax formulation was DMSO:ethanol:cremohol EL:5% dextrose-containing water (D5W) in a volume ratio of 2.5:5:10:20:67.5. Venetoclax-treated mice were orally administered 250 μg of venetoclax twice at 3-day intervals. Drug A-treated mice were intravenously administered 10 mg / kg four times at 3-4 day intervals. Mice treated with venetoclax / drug A were orally administered 250 μg of venetoclax twice at 3-day intervals, and drug A 10 mg / kg was administered four times at 3-4 day intervals, one day after venetoclax administration. Tumors were measured two-dimensionally using calipers, and tumor volume was calculated as length × width × width × 0.5; the length was the larger of the two measured values.

[0272] result Venetoclax alone inhibited tumor growth (see Figure 1A), but drug A alone had no noticeable effect on tumor growth. The combination of venetoclax and drug A significantly inhibited tumor growth more effectively than venetoclax alone (see Figure 1A). On day 41, one out of ten mice treated with venetoclax alone was tumor-free ("TF"), while six out of ten mice treated with the venetoclax / drug A combination were TF (Figure 1A).

[0273] Next, venetoclax-treated mice (n=10) were divided into two groups (n=5 / group): (a) re-treated with venetoclax monotherapy on day 45, or (b) treated with a combination of venetoclax and drug A (administered on day 46) (administered on day 45). As shown in Figure 1B, in mice previously treated with venetoclax, treatment with venetoclax in combination with drug A significantly suppressed tumor growth compared to re-treatment with venetoclax monotherapy. The average tumor volume at day 65 in mice re-treated with venetoclax was approximately 1685 mm². 3However, the average tumor volume at day 65 of mice treated with the combination therapy was approximately 970 mm². 3 In mice that showed tumor regression when treated with venetoclax monotherapy, venetoclax monotherapy was administered again on day 45. In particular, tumor regrowth was observed in these mice.

[0274] Example 1B: Effect of drug A in combination with venetoclax on macrophage phagocytosis in an in vitro model. In this example, the effects of drug A alone, venetoclax alone, and drug A in combination with venetoclax on macrophage phagocytosis of HL60 and OCIAML3 human acute myeloid leukemia cells were evaluated in vitro using assays.

[0275] Materials and methods Induction and culture of human monocyte-derived macrophages for phagocytosis CD14 + Monocytes were purified by negative selection using the Classical Monocyte Isolation Kit, human (Miltenyi Biotec), and LS column (Miltenyi Biotec) according to the manufacturer's protocol. CD14 + Monocytes were seeded at a rate of 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 in 150 mm tissue culture dishes (Corning). The cells were cultured for 7–11 days.

[0276] In vitro phagocytic assay HL60 and OCI-AML3 cells were washed once with PBS and labeled with the Celltrace CFSE Cell Proliferation Kit (Thermo Fisher Scientific) in a suspension containing 300 nM CFSE (carboxyfluorescein succinimimidyl ester) according to the manufacturer's instructions, and then resuspended in RPMI complete medium. Target cells were incubated overnight in RPMI complete medium with 2-fold serial dilutions of 39 nM to 2.5 μM venetoclax. Cells were resuspended in RPMI before incubation with macrophages. Macrophages were separated from the culture plate by washing once with PBS and incubating at 37°C for 20 minutes using TrypLE Select. 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 rotated and added to a 96-well ultra-low adhesion U-bottom plate (Corning) at a rate of 100,000 cells per well. Drug A was then added. The plate was incubated in a humidified incubator containing 5% carbon dioxide at 37°C for 30 minutes, and then 50,000 macrophages were added. The plate was incubated in a humidified incubator containing 5% carbon dioxide at 37°C for 2 hours. Cells were pelleted by centrifugation at 400×g for 5 minutes and stained with Fixable Viability Dye eFluor 780 (ebioscience) diluted 1:4000 in PBS at 4°C for 30 minutes. Cells were washed with FACS buffer (PBS containing 0.5% BSA) and stained in FACS buffer containing human FcR blocking reagent (Miltenyi Biotec), BV421 anti-CD33 (Biolegend), APC anti-CD14 (Biolegend), and PE-Cyanine7 anti-CD11b (Invitrogen) for 45 minutes at 4°C. Cells were washed twice with FACS buffer and fixed overnight at 4°C in 0.5% paraformaldehyde diluted in PBS. Cells were analyzed using FACS Canto II (BD Biosciences), followed by data analysis using Flowjo 10.6.1 (Becton Dickinson & Company). Dead cells were excluded by gating the e780-negative population. Macrophages were identified as cells positive for the lineage markers CD33, CD11b, and CD14. Of this population, macrophages that phagocytosed tumor cells were identified as CFSE-positive cells.

[0278] result In short, HL60 cells and OCI-AML3 cells (i.e., "target cells") were labeled with CFSE (carboxyfluorescein succinimimidyl ester) and treated with venetoclax for 48 hours. The target cells were then rotated and added to the wells of a 96-well plate at a rate of 100,000 cells per well. Drug A was then added. Untreated control target cells, as well as control target cells treated with venetoclax alone or drug A alone, were prepared in parallel. Macrophages were added to the wells, and the 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 the e780-negative population. Macrophages were identified as cells positive for the lineage markers CD33, CD11b, and CD14. Of this population, macrophages that phagocytosed tumor cells were identified as CFSE-positive cells.

[0279] As shown in Figure 5A, venetoclax alone stimulated macrophage-mediated phagocytosis in HL60 cells, while drug A alone had little effect on phagocytosis (cells treated with drug A were compared to untreated cells). The combination of 20 nM drug A and 125 nM venetoclax stimulated macrophage-mediated phagocytosis of HL60 cells to a higher degree than either drug A alone or venetoclax alone. Similar results were observed in OCI-AML3 cells using 20 nM drug A and 1 μM venetoclax. See 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 a CT26 syngeneic mouse colon cancer model. See, for example, Mosely et al. (2016) “Rational Selection of Syngeneic Preclinical Tumor Models for Immunotherapeutic Drug Discovery” Cancer Immunol Res. 5(1):29-41.

[0281] Materials and methods CT26 equivalent model CT26 cells (see Wang et al. (1995) J. Immunol. 154:4685-4692) were incubated with RPMI 1640 at a rate of 5 × 10⁶ cells per mouse. 5 The cells were injected into the right flank of BALB / c female mice at a concentration. The average size of all tumors was 65-70 mm. 3 Tumors were monitored until they reached a certain level. 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) twice at a dose of 30 mg / kg ("mpk"). The two 30 mpk doses were given 10 days apart. Cisplatin was administered via IP according to one of two regimens: one dose of 10 mpk or two doses of 5 mpk. The cisplatin 5 mpk dose was given twice 10 days apart. Mice treated with both cisplatin and drug A were administered cisplatin (IP) according to one of the regimens above, followed by drug A as described above. Mice receiving combination therapy were administered drug A 1 day after cisplatin. The tumor was measured in two dimensions using a caliper, and the tumor volume was calculated as length × width × width × 0.5: the length is the larger of the two measured values.

[0282] result As shown in Figure 2A, tumor growth was somewhat inhibited on day 20 in mice treated with monotherapy cisplatin (two doses of 5 mpk each administered 10 days apart), but drug A had no noticeable effect on tumor growth. Combination therapy with cisplatin and 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 combined with drug A gained more body weight during the course of treatment than mice treated with cisplatin alone. In addition, in each of the PBS control, cisplatin, and drug A treatment groups, only 10% of mice had a tumor volume <500 mm². 3 It was found that the mice had a tumor volume of <500 mm² in the cisplatin + drug A treatment group. 3 He possessed it.

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

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

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

[0286] Enhanced phagocytosis of CFSE-labeled DLD-1 tumor cells by human monocyte-derived macrophages in the presence of drug A combined with anti-Trop2. 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, in combination with a negative control human IgG antibody and anti-TROP2 antibody, and in medium alone. Phagocytosis of CFSE-labeled DLD-1 tumor cells by human monocyte-derived macrophages was enhanced in the presence of drug A combined with anti-TROP2 antibody. See Figure 3.

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

[0288] Before transplantation, a master cell bank was created for each cell line to ensure that the cells used in subsequent experiments were of the same passage number. The 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, the cells were divided into 5 × 10⁶ cells. 6 Cells were resuspended in PBS or RPMI at a concentration of cells / mL. For MC38m / h HER2, 100 μL of cell suspension was subcutaneously injected into the right flank of C57BL / 6 mice. The average tumor size of MC38m / h HER2 tumors was 65–69 mm. 3When this was reached, the animals were randomized into eight groups of 10 mice each. Each group was assigned to one of the treatment groups outlined in Table A: [Table 13]

[0289] Tumor volume (mm) measured using Mitutoyo Digital Caliper (Mitutoyo America, Aurora, Illinois) 3 ) and weight were recorded two or three times a week. Tumor volume was 2000 mm 3 Mice exceeding a certain size or those with a 20% weight loss were euthanized according to IACUC guidelines. Tumor volume is calculated as follows: ([length × {width × width}] × 0.5 = volume (mm) 3 Statistical analysis and p-values ​​were calculated using GraphPad Prism software.

[0290] A chimeric m / h HER2, possessing the extracellular domain of human HER2 and the intracellular domain of mouse HER2, was expressed on MC38 colon cells to evaluate the activity of trastuzumab against MC38 mouse tumors. As shown in Figure 5, trastuzumab monotherapy had no effect on tumor growth, while drug A monotherapy and anti-PD-L1 antibody monotherapy each had a moderate effect on tumor growth. Treatment with drug A + anti-PD-L1 antibody doublet or trastuzumab + anti-PD-L1 antibody doublet showed improved tumor growth inhibition compared to monotherapy alone. Treatment with the drug A + anti-PD-L1 + trastuzumab triple combination showed improved tumor inhibition compared to each doublet. Compared to drug A + anti-PD-L1 antibody doublet or trastuzumab + anti-PD-L1 antibody doublet, the triple combination's effect on reducing tumor growth was most evident on days 19 and 22 (3–6 days after the last dose). By day 26, the triple combination showed minimally superior reduction in tumor growth compared to drug A + anti-PD-L1 antibody doublet or trastuzumab + 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 progression 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 platinum-containing chemotherapy agents. +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 are suitable for treatment with trastuzumab. Patients have not received prior treatment with anti-CD47 or anti-SIRPα agents.

[0292] Clinical trials are conducted for gastric or GEJ adenocarcinoma (e.g., HER2). + This study will evaluate the safety, tolerability, and efficacy of the combination of drug A, pembrolizumab, cisplatin, and 5-fluorouracil or capecitabine in patients with 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 hemoglobin levels of 9 g / dL or higher.

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

[0294] Example 5B: Preliminary safety results from the exemplary clinical trial described in Example 5A. One patient with untreated advanced head and neck squamous cell carcinoma received drug A (10 mg / kg IV QW), pembrolizumab (200 mg IV Q3W), and 5-fluorouracil (1,000 mg / m²). 2 (Q3W×6 on days 1, 2, 3, and 4) and carboplatin (AUC=5mg / ml / min, day 1, Q3W×6) were administered. (In the expanded study, cisplatin (100mg / m²) was used.) 2Carboplatin (AUC=5 mg / ml / min, day 1, Q3W x 6) or cisplatin (AUC=5 mg / ml / min, day 1, Q3W x 6) is administered in combination with drug A, pembrolizumab, and fluorouracil. Patients who received carboplatin will continue to receive carboplatin throughout the expansion study. Patients who received cisplatin will continue to receive cisplatin throughout the expansion study. Three patients with HER2-positive gastric / esophageal cancer that had progressed to previous treatment(s) with trastuzumab, fluorouracil, and platinum-based agents were treated with drug A (10 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). Three additional patients with HER2-positive gastric / esophageal cancer that had progressed after prior treatment (multiple) with trastuzumab, fluorouracil, and platinum-based agents 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 dose-limiting toxicities when administered at doses of 10 mg / kg or 15 mg / kg QW in the above combination regimens. Three patients (50%) who received drug A + trastuzumab + ramucirumab + paclitaxel experienced treatment-related adverse events (TRAEs), while none (0%) of patients who received drug A + pembrolizumab + fluorouracil + carboplatin experienced any. There were no dose-limiting toxicities in patients who received drug A + pembrolizumab + fluorouracil + carboplatin or drug A + trastuzumab + ramucirumab + paclitaxel. Furthermore, no treatment-related adverse events (TRAEs) occurred in two or more patients in the following three cohorts. Drug A (10 mg / kg QW) + Pembrolizumab + Fluorouracil + Carboplatin (N=1) Drug A (10 mg / kg QW) + trastuzumab + ramucirumab + paclitaxel (N=3) Drug A (15 mg / kg QW) + trastuzumab + ramucirumab + paclitaxel (N=3)

[0296] Finally, no treatment-related adverse events (TRAEs ≥ Grade 3) of grade 3 or higher were 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 head and neck squamous cell carcinoma treated with drug A, pembrolizumab, 5-fluorouracil, and platinum at the dosage and administration schedule described in Example 5B achieved partial responses (PRs) based on investigator-assessed responses using RECIST v1.1 criteria.

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

[0299] Of the three patients with HER2-positive gastric / esophageal cancer treated with drug A (15 mg / kg QW), trastuzumab, ramucirumab, and paclitaxel (see Example 5B), two remained unevaluable. One patient achieved a partial response (PR) based on investigator-assessed responses using RECIST v1.1 criteria. A low incidence of cytopenia was observed.

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

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

[0302] Example 5D: Additional results from the exemplary clinical trial described in Example 5A CD47 is a myeloid checkpoint that is 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 enhance anticancer drugs (Kauder et al. (2018) PLoS ONE. 13(8): e0201832; Chow et al. (2020) Journal of Clinical Oncology. 38:15_suppl, 3056-3056). Drug A, in combination with standard chemotherapy and antibody regimens, was evaluated in patients with advanced HER2-positive gastric cancer (GC) or head and neck squamous cell carcinoma (HNSCC).

[0303] method Patients with previously treated 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 treatment. GC patients had progressed during or after prior fluoropyrimidine therapy (or fluoropyrimidine-containing therapy). This included GC patients who progressed during or after prior treatment with trastuzumab and / or platinum-based chemotherapy. Patients with 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 treatment. The primary endpoint was dose-limiting toxicity (DLT). Tumor response, pharmacokinetics (PK), and pharmacodynamic (PD) markers were evaluated in all patients.

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

[0305] One patient with GC of 2L or more received A+T+ramucirumab+paclitaxel, and safety was evaluated. 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, ...

Claims

1. 1. A medicament for treating gastric cancer or gastroesophageal junction (GEJ) cancer in an individual in combination with an anti-HER2 antibody, an anti-VEGFR2 antibody, and paclitaxel, comprising: The medicament comprises an effective amount of a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, 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 the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat; The individual has undergone at least one prior treatment for the gastric cancer or the GEJ cancer.

2. The pharmaceutical described in claim 1, wherein the anti-HER2 antibody is trastuzumab.

3. The method of claim 1 or 2, 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.

4. The pharmaceutical composition described in claim 3, wherein the anti-HER2 antibody in the previous treatment is trastuzumab.

5. The pharmaceutical agent according to any one of claims 1 to 4, wherein the anti-VEGFR2 antibody is ramucirumab.

6. The gastric cancer or the GEJ cancer is HER2 ＋ Gastric cancer or HER2 ＋ The pharmaceutical agent according to any one of claims 1 to 5, which is for GEJ cancer.

7. The pharmaceutical according to any one of claims 1 to 6, 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.

8. The pharmaceutical according to any one of claims 1 to 6, 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.

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

85.

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

81.

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

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

91.

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

136.

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

135.

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

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

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