Combination therapy for treating cancer

Abstract

The present invention relates to combination therapies for the treatment of cancer, and specifically provides methods of treating cancer comprising administering a polypeptide (e.g., a fusion polypeptide) comprising a SIRP [alpha] 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

[0001] This invention application is a divisional application of the invention patent application filed on November 25, 2020, with application number 202080090447.6 (international application number PCT / US2020 / 062402) entitled "Combination Therapy for Treating Cancer".

[0002] Cross-references to related applications

[0003] 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.

[0004] Submitting sequence lists on ASCII text files

[0005] The following submission regarding an ASCII text file is incorporated herein by reference in its entirety: Computer-readable form of a sequence list (CRF) (filename: 757972001140SEQLIST.TXT, record date: November 25, 2020, size: 333KB). Technical Field

[0006] This invention relates to a method of treating cancer, 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 cancer treatment modality. Background Technology

[0007] Many cancers have poor prognoses, even when treated with available therapies. There is a need in the field for new treatment approaches to provide additional treatment options and improve patient outcomes.

[0008] Tumor cells manipulate myeloid compartments to evade anti-tumor host immune responses (Gabrilovich et al., Nat RevImmunol (2012) 12(4): 253-68). For example, while CD47 expressed on the surface of normal cells binds to SIRPα on macrophages and provides a “don’t eat me” signal, tumor cells have also been found to overexpress CD47 to evade the macrophage component of immune surveillance (Oldenborg, ISRN Hematol (2013) 614619).

[0009] Macrophage-mediated destruction of cancer cells requires 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 trigger a maximal phagocytic response against tumor cells. As mentioned above, CD47 provides the basic "don't eat me" signal through its interaction with SIRPα on macrophages. Prophagocytic "eat me" signals can be provided to the same macrophages by binding to their activating Fcγ receptors. For example, prophagocytic "eat me" signals can be provided by the binding of antitumor antibodies to Fc receptors on macrophages.

[0010] All references cited in this article, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are incorporated herein by reference in their entirety, as if each individual reference were specifically and individually indicated as incorporated by reference. Summary of the Invention

[0011] A method of treating cancer in an individual is provided, comprising administering to the individual an effective amount of: (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) a Bcl-2 inhibitor; wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is based on the Kabat EU index; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is based on the Kabat EU index; and (iii) a human IgG4 region comprising S228P, E233P, F234V, L235A, and delG236 mutations. The Fc region, numbered according to the Kabat EU index; or (iv) the human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, numbered according to the Kabat EU index. In some embodiments, the cancer is leukemia, multiple myeloma, or non-Hodgkin lymphoma. In some embodiments, the non-Hodgkin lymphoma is diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), or follicular lymphoma (FL). In some embodiments, the leukemia is acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), small lymphoblastic 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.

[0012] A method of treating cancer in an individual is also provided, comprising administering to the individual an effective amount of: (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) a platinum-based chemotherapeutic agent; wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is based on the Kabat EU index; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is based on the Kabat EU index; and (iii) a human IgG4 region comprising S228P, E233P, F234V, L235A, and delG236 mutations. The Fc region, numbered according to the Kabat EU index; or (iv) the human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, numbered according to the Kabat EU index. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is colon cancer, lung cancer, head and neck cancer, esophageal cancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer, testicular cancer, endometrial cancer, liver cancer, gastric cancer, gastroesophageal junction cancer, brain tumor, mesothelioma, or neuroblastoma. In some embodiments, the colon cancer is carcinoma. In some embodiments, the platinum-based chemotherapy agent is carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthreneplatin, pyridine, or saxaplatin. In some embodiments, the platinum-based chemotherapy agent is cisplatin or carboplatin.

[0013] A method of treating cancer in an individual is also provided, comprising administering to the individual an effective amount of: (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, (b) a PD-1 inhibitor, (c) an antimetabolite, and (d) a platinum-based chemotherapeutic agent; wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is based on the Kabat EU index; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is based on the Kabat EU index; and (iii) a human IgG4 region comprising S228P, E233P, F234V, L235A, and delG236 mutations. The Fc region, numbered according to the Kabat EU index; or (iv) the human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, numbered according to the Kabat EU index, wherein the cancer is head and neck squamous cell carcinoma (HNSCC), and wherein the individual has not received prior treatment for HNSCC. In some embodiments, the peptide containing the SIRPαD1 domain variant and the Fc domain variant is administered once weekly (qw) at a dose of 10 mg / kg. In some embodiments, the peptide containing the SIRPαD1 domain variant and the Fc domain variant is administered once weekly (qw) at a dose of 15 mg / kg.

[0014] In some embodiments, HNSCC is advanced and / or metastatic HNSCC. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody, such as pembrolizumab, nivolumab, pildizumab, cimipril, or BMS-936559. In some embodiments, the anti-PD-1 antibody is pembrolizumab. In some embodiments, the antimetabolite is 5-fluorouracil, 6-mercaptopurine, capecitabine, cytarabine, fluorouridine, fludarabine, gemcitabine, hydroxyurea, 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, triplatinum tetranitrate, phenanthreneplatin, pyrplatin, or saplatin. In some embodiments, the platinum-based chemotherapeutic agent is cisplatin or carboplatin.

[0015] In another aspect, a method of treating cancer in an individual is provided, comprising administering to the individual an effective amount of: (a) a polypeptide comprising a SIRPαD1 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 SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is based on the Kabat EU index; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is based on the Kabat EU index; and (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. The Fc region, numbered according to the Kabat EU index; or (iv) the human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, numbered according to the Kabat EU index. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is colon cancer, lung cancer, head and neck cancer, esophageal cancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer, testicular cancer, endometrial cancer, liver cancer, gastric cancer, gastroesophageal junction cancer, brain tumor, mesothelioma, or neuroblastoma. In some embodiments, the solid tumor is HER2. + Solid tumor. In some embodiments, the solid tumor is colon cancer (e.g., HER2). + (Colorectal cancer). In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the anti-PD-L1 antibody is atezolizumab, avelumab, or duruvazumab.

[0016] In some embodiments, a method of treating an individual's cancer is provided, comprising administering to the individual an effective amount of: (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, (b) an anti-HER2 antibody, (c) an anti-VEGF2 antibody, and (d) paclitaxel; wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is based on the Kabat EU index; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is based on the Kabat EU index; and (iii) a human IgG4 region comprising S228P, E233P, F234V, L235A, and delG236 mutations. Fc region, numbered according to the Kabat EU index; or (iv) containing human IgG4 Fc region with mutations of S228P, E233P, F234V, L235A, delG236, and N297A, numbered according to the Kabat EU index, wherein the cancer is gastric cancer or gastroesophageal junction (GEJ) cancer, and wherein the individual has received at least one prior treatment for gastric cancer or GEJ cancer. In some embodiments, the gastric cancer or GEJ cancer is HER2 overexpressing (e.g., HER2...). + The individual is diagnosed with 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 chemotherapy agent. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the anti-VEGF antibody is ramucirumab. In some embodiments, the peptide comprising a SIRPαD1 domain variant and an Fc domain variant is administered once weekly (qw) at a dose of 10 mg / kg. In some embodiments, the peptide comprising a SIRPαD1 domain variant and an Fc domain variant is administered once weekly (qw) at a dose of 15 mg / kg.

[0017] A method of treating cancer in an individual is also provided, comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) an anti-TROP2 antibody; wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is based on the Kabat EU index; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is based on the Kabat EU index; and (iii) a human IgG4 region comprising S228P, E233P, F234V, L235A, and delG236 mutations. The Fc region, numbered according to the Kabat EU index; or (iv) the human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, numbered according to the Kabat EU index. In some embodiments, the cancer is a solid tumor, gastric cancer, nasopharyngeal carcinoma, gallbladder cancer, cervical cancer, extranodal NK / T-cell lymphoma, lung cancer, laryngeal squamous cell carcinoma, colon cancer, hilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrioid endometrial cancer, or ovarian cancer.

[0018] In some embodiments of any of the methods described herein, the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:85. In some embodiments, the SIRPαD1 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 mutations of L234A, L235A, G237A, and N297A, wherein the numbering is based on 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 SIRPαD1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO:136. In some embodiments, the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO:135. In some embodiments, the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant forms a homodimer. In some embodiments, the individual is a human.

[0019] In another aspect, a kit is provided comprising a polypeptide containing a SIRPαD1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, said polypeptide for use in combination with a Bcl-2 inhibitor for the treatment of cancer in individuals of need, wherein said SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein said Fc domain variant is (i) a human IgG1 Fc region containing L234A, L235A, G237A, and N297A mutations, wherein the numbering is based on the Kabat EU index; (ii) a human IgG2 region containing A330S, P331S, and N297A mutations. The kit contains: (iii) an Fc region, numbered according to the Kabat EU index; or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to the Kabat EU index; or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, numbered according to the Kabat EU index; and wherein the kit contains instructions for administering the peptide containing the SIRPαD1 domain variant and the Fc domain variant in combination with the Bcl-2 inhibitor to the individual in need. In some embodiments, the cancer is leukemia, multiple myeloma, or non-Hodgkin's lymphoma. In some embodiments, the Bcl-2 inhibitor is venetoclax.

[0020] A kit is also provided comprising a polypeptide containing a SIRPαD1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, the polypeptide being used in combination with a platinum-based chemotherapeutic agent for the treatment of cancer in individuals of need, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region containing L234A, L235A, G237A, and N297A mutations, numbered according to the Kabat EU index; (ii) a human IgG2 region containing A330S, P331S, and N297A mutations. The kit contains: (iii) an Fc region, numbered according to the Kabat EU index; or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to the Kabat EU index; or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, numbered according to the Kabat EU index; and wherein the kit contains instructions for administering the combination of the peptide containing the SIRPαD1 domain variant and the Fc domain variant with the chemotherapeutic agent to the individual in need. 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 implementations, the platinum-based chemotherapeutic agent is cisplatin or carboplatin.

[0021] In some embodiments, a kit is provided comprising a polypeptide containing a SIRPαD1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, the polypeptide being used in combination with a PD-1 inhibitor, an antimetabolite, and a platinum-based chemotherapeutic agent for the treatment of cancer in an individual of need, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region containing L234A, L235A, G237A, and N297A mutations, numbered according to the Kabat EU index; (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations, numbered according to the Kabat EU index; and (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations. The kit contains an Fc region, numbered according to the Kabat EU index; or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, numbered according to the Kabat EU index, and wherein the kit contains instructions for administering the peptide containing the SIRPαD1 domain variant and the Fc domain variant in combination with the anti-PD-1 antibody, the antimetabolite, and the platinum-based chemotherapeutic agent to an individual with head and neck squamous cell carcinoma (HNSCC) who has not received prior treatment for HNSCC. In some embodiments, the PD-1 inhibitor is pembrolizumab. In some embodiments, the antimetabolite is 5-fluorouracil. In some embodiments, the platinum-based chemotherapeutic agent is cisplatin or carboplatin.

[0022] In some embodiments, a kit is provided comprising a polypeptide containing a SIRPαD1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, said polypeptide for use in combination with an anti-HER2 antibody, an anti-VEGFR2 antibody, and paclitaxel; wherein said SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein said Fc domain variant is (i) a human IgG1 Fc region containing L234A, L235A, G237A, and N297A mutations, wherein the numbering is based on the Kabat EU index; (ii) a human IgG1 Fc region containing A330S, P331S, and N297A mutations. (iii) Fc regions, numbered according to the Kabat EU index; or (iv) human IgG4 Fc regions containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to the Kabat EU index; and (iv) human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, numbered according to the Kabat EU index, wherein the kit contains instructions for administering the peptide containing the SIRPαD1 domain variant and the Fc domain variant in combination with the anti-HER2 antibody, the anti-VEGFR2 antibody, and the paclitaxel to an individual with gastric cancer or gastroesophageal junction (GEJ) cancer who has received at least one prior treatment for gastric cancer or GEJ cancer. In some embodiments, the gastric cancer or GEJ cancer is HER2-positive. + Stomach cancer or HER2 + 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 received prior treatment (or multiple treatments) using an anti-HER2 antibody (e.g., trastuzumab) and / or fluoropyrimidine and / or platinum-based chemotherapy. In some embodiments, the individual's gastric cancer or GEJ cancer progressed during or after prior treatment (or multiple treatments) containing an anti-HER2 antibody (e.g., trastuzumab) and / or fluoropyrimidine and / or platinum-based chemotherapy. In some embodiments, the individual's prior treatment (or multiple treatments) containing an anti-HER2 antibody (e.g., trastuzumab) and / or fluoropyrimidine and / or platinum-based chemotherapy failed (e.g., relapsed or did not respond). In some embodiments, the prior treatment (or multiple treatments) included an anti-HER2 antibody and fluoropyrimidine (e.g., administered during the same treatment line or during different treatment lines). In some implementations, the prior treatment (or multiple treatments) comprises an anti-HER2 antibody and a platinum-based chemotherapy agent (e.g., administered during the same treatment line or during different treatment lines).

[0023] A kit is also provided comprising a peptide containing a SIRPαD1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, the peptide being used in combination with an anti-TROP2 antibody for the treatment of cancer in individuals of need, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region containing L234A, L235A, G237A, and N297A mutations, numbered according to the Kabat EU index; (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations, numbered according to the Kabat EU index; and (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations. The Fc region, numbered according to the Kabat EU index; or (iv) containing the human IgG4 Fc region containing mutations of S228P, E233P, F234V, L235A, delG236, and N297A, numbered according to the Kabat EU index; and wherein the kit contains instructions for administering to the individual in need the combination of the peptide containing the SIRPαD1 domain variant and the Fc domain variant with the anti-TROP2 antibody. In some embodiments, the cancer is a solid tumor, gastric cancer, nasopharyngeal carcinoma, gallbladder cancer, cervical cancer, extranodal NK / T-cell lymphoma, lung cancer, laryngeal squamous cell carcinoma, colon cancer, hilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrioid endometrial cancer, or ovarian cancer.

[0024] A kit is also provided comprising a peptide containing a SIRPαD1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, the peptide being used in combination with an anti-HER2 antibody and an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody) for the treatment of cancer in individuals of need, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region containing L234A, L235A, G237A, and N297A mutations, numbered according to the Kabat EU index; (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations, numbered according to the Kabat EU index; and (iii) a human IgG4 region containing S228P, E233P, F234V, L235A, and delG236 mutations. The Fc region, numbered according to the Kabat EU index; or (iv) containing the human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, numbered according to the Kabat EU index; and wherein the kit contains instructions for administering to the individual in need the combination of the peptide containing the SIRPαD1 domain variant and the Fc domain variant with the anti-HER2 antibody and the anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody). In some embodiments, the cancer is colon cancer. In some embodiments, the colon cancer is HER2. + Colorectal cancer. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the anti-PD-L1 antibody is atezolizumab, avelumab, or duruvazumab.

[0025] In some embodiments of the kit, the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:85. In some embodiments, the SIRPαD1 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 containing mutations of L234A, L235A, G237A, and N297A, where the numbering is based on 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 SIRPαD1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO:136. In some embodiments, the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO:135. In some embodiments, the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant forms a homodimer. In some embodiments, the individual is a human.

[0026] Specifically, the present invention includes, but is not limited to, the following:

[0027] 1. A method of treating cancer in an individual, comprising administering to the individual an effective amount of: (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) a Bcl-2 inhibitor;

[0028] The SIRPαD1 domain variant therein comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85;

[0029] The Fc domain variants mentioned above are

[0030] (i) Human IgG1 Fc regions containing L234A, L235A, G237A and N297A mutations, where the numbering is based on the Kabat EU index;

[0031] (ii) Human IgG2 Fc regions containing A330S, P331S and N297A mutations, where the numbering is based on the EU index of Kabat;

[0032] (iii) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to Kabat's EU index; or

[0033] (iv) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236 and N297A mutations, where the numbering is based on the Kabat EU index.

[0034] 2. The method as described in item 1, wherein the cancer is leukemia, multiple myeloma, or non-Hodgkin's lymphoma.

[0035] 3. The method of claim 2, wherein the non-Hodgkin lymphoma is diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), or follicular lymphoma (FL).

[0036] 4. The method as described in item 2, wherein the leukemia is acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), small lymphoblastic lymphoma (SLL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), or myelodysplastic syndrome (MDS).

[0037] 5. The method as described in item 4, wherein the leukemia is acute lymphoblastic leukemia.

[0038] 6. The method of any one of items 1 to 5, wherein the Bcl-2 inhibitor is venetoclax, ABT-737, navittoclax, BCL201 or AZD-0466.

[0039] 7. The method of claim 6, wherein the Bcl-2 inhibitor is venetoclax.

[0040] 8. A method of treating cancer in an individual, comprising administering to the individual an effective amount of: (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) a platinum-based chemotherapeutic agent;

[0041] The SIRPαD1 domain variant therein comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85;

[0042] The Fc domain variants mentioned above are

[0043] (i) Human IgG1 Fc regions containing L234A, L235A, G237A and N297A mutations, where the numbering is based on the Kabat EU index;

[0044] (ii) Human IgG2 Fc regions containing A330S, P331S and N297A mutations, where the numbering is based on the EU index of Kabat;

[0045] (iii) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to Kabat's EU index; or

[0046] (iv) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236 and N297A mutations, where the numbering is based on the Kabat EU index.

[0047] 9. The method as described in item 8, wherein the cancer is a solid tumor.

[0048] 10. The method of claim 9, wherein the solid tumor is colon cancer, lung cancer, head and neck cancer, esophageal cancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer, testicular cancer, endometrial cancer, liver cancer, stomach cancer, gastroesophageal junction cancer, brain tumor, mesothelioma, or neuroblastoma.

[0049] 11. The method as described in item 9, wherein the colon cancer is colon carcinoma.

[0050] 12. A method of treating cancer in an individual, comprising administering to the individual an effective amount of: (a) a polypeptide comprising a variant of the SIRPαD1 domain and a variant of the Fc domain, (b) a PD-1 inhibitor, (c) an antimetabolite, and (d) a platinum-based chemotherapeutic agent;

[0051] The SIRPαD1 domain variant therein comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85;

[0052] The Fc domain variants mentioned above are

[0053] (i) Human IgG1 Fc regions containing L234A, L235A, G237A and N297A mutations, where the numbering is based on the Kabat EU index;

[0054] (ii) Human IgG2 Fc regions containing A330S, P331S and N297A mutations, where the numbering is based on the EU index of Kabat;

[0055] (iii) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to Kabat's EU index; or

[0056] (iv) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, where the numbers are based on the Kabat EU index.

[0057] The cancer described is head and neck squamous cell carcinoma (HNSCC), and the individual described has not received prior treatment for HNSCC.

[0058] 13. The method of claim 12, wherein the HNSCC is late and / or metastatic HNSCC.

[0059] 14. The method of claim 12 or 13, wherein the PD-1 inhibitor is an anti-PD-1 antibody.

[0060] 15. The method of claim 14, wherein the anti-PD-1 antibody is pembrolizumab, nivolumab, pildizumab, cimipril, or BMS-936559.

[0061] 16. The method of claim 15, wherein the anti-PD-1 antibody is pembrolizumab.

[0062] 17. The method of any one of claims 12 to 16, wherein the antimetabolite is 5-fluorouracil, 6-mercaptopurine, capecitabine, cytarabine, fluorouracil, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, or chloroquine.

[0063] 18. The method of claim 17, wherein the antimetabolite is 5-fluorouracil.

[0064] 19. The method of any one of items 8 to 18, wherein the platinum-based chemotherapeutic agent is carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatinum tetranitrate, phenanthreneplatin, pyridineplatin, or saxaplatin.

[0065] 20. The method of claim 19, wherein the platinum-based chemotherapeutic agent is cisplatin.

[0066] 21. The method of claim 19, wherein the platinum-based chemotherapeutic agent is carboplatin.

[0067] 22. A method of treating cancer in an individual, comprising administering to the individual an effective amount of: (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, (b) an anti-HER2 antibody, and (c) an anti-PD-L1 antibody;

[0068] The SIRPαD1 domain variant therein comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85;

[0069] The Fc domain variants mentioned above are

[0070] (i) Human IgG1 Fc regions containing L234A, L235A, G237A and N297A mutations, where the numbering is based on the Kabat EU index;

[0071] (ii) Human IgG2 Fc regions containing A330S, P331S and N297A mutations, where the numbering is based on the EU index of Kabat;

[0072] (iii) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to Kabat's EU index; or

[0073] (iv) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236 and N297A mutations, where the numbering is based on the Kabat EU index.

[0074] 23. The method as described in item 22, wherein the cancer is a solid tumor.

[0075] 24. The method of claim 23, wherein the solid tumor is colon cancer, lung cancer, head and neck cancer, esophageal cancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer, testicular cancer, endometrial cancer, liver cancer, stomach cancer, gastroesophageal junction cancer, brain tumor, mesothelioma, or neuroblastoma.

[0076] 25. The method as described in item 23 or 24, wherein the solid tumor is HER2. + Solid tumor.

[0077] 26. The method of any one of items 23 to 25, wherein the solid tumor is colon cancer.

[0078] 27. The method of any one of items 22 to 26, wherein the anti-HER2 antibody is trastuzumab.

[0079] 28. The method of any one of items 22 to 27, wherein the anti-PD-L1 antibody is atezolizumab, avelumab, or duruvazumab.

[0080] 29. A method of treating cancer in an individual, comprising administering to the individual an effective amount of: (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, (b) an anti-HER2 antibody, (c) an anti-VEGF2 antibody, and (d) paclitaxel;

[0081] The SIRPαD1 domain variant therein comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85;

[0082] The Fc domain variants mentioned above are

[0083] (i) Human IgG1 Fc regions containing L234A, L235A, G237A and N297A mutations, where the numbering is based on the Kabat EU index;

[0084] (ii) Human IgG2 Fc regions containing A330S, P331S and N297A mutations, where the numbering is based on the EU index of Kabat;

[0085] (iii) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to Kabat's EU index; or

[0086] (iv) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, where the numbers are based on the Kabat EU index.

[0087] 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.

[0088] 30. The method of claim 29, wherein 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 chemotherapy agent.

[0089] 31. The method of claim 29 or 30, wherein the anti-HER2 antibody is trastuzumab.

[0090] 32. The method of any one of items 29 to 31, wherein the anti-VEGF antibody is ramucirumab.

[0091] 33. The method of any one of items 29 to 32, wherein the gastric cancer or GEJ cancer is HER2 + Stomach cancer or HER2 + GEJ cancer.

[0092] 34. The method of any one of items 12 to 21 and 29 to 33, wherein the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant is administered once weekly at a dose of 10 mg / kg.

[0093] 35. The method of any one of items 12 to 21 and 29 to 33, wherein the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant is administered once weekly at a dose of 15 mg / kg.

[0094] 36. A method of treating cancer in an individual, comprising administering to the individual an effective amount of (a) a polypeptide comprising a variant of the SIRPαD1 domain and a variant of the Fc domain, and (b) an anti-TROP2 antibody;

[0095] The SIRPαD1 domain variant therein comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85;

[0096] The Fc domain variants mentioned above are

[0097] (i) Human IgG1 Fc regions containing L234A, L235A, G237A and N297A mutations, where the numbering is based on the Kabat EU index;

[0098] (ii) Human IgG2 Fc regions containing A330S, P331S and N297A mutations, where the numbering is based on the EU index of Kabat;

[0099] (iii) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to Kabat's EU index; or

[0100] (iv) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236 and N297A mutations, where the numbering is based on the Kabat EU index.

[0101] 37. The method as described in item 36, wherein the cancer is a solid tumor, gastric cancer, nasopharyngeal carcinoma, gallbladder cancer, cervical cancer, extranodal NK / T-cell lymphoma, lung cancer, laryngeal squamous cell carcinoma, colon cancer, hilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrioid endometrial cancer, or ovarian cancer.

[0102] 38. The method of any one of claims 1 to 37, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 85.

[0103] 39. The method of any one of claims 1 to 37, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81.

[0104] 40. The method of any one of items 1 to 39, wherein the Fc domain variant is a human IgG1 Fc region containing mutations of L234A, L235A, G237A, and N297A, wherein the numbering is based on the EU index of Kabat.

[0105] 41. The method of claim 40, wherein the Fc domain variant comprises the amino acid sequence of SEQ ID NO:91.

[0106] 42. The method of any one of claims 1 to 38 and 40 to 41, wherein the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO:136.

[0107] 43. The method of any one of items 1 to 37 and 39 to 41, wherein the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO:135.

[0108] 44. The method of any one of claims 1 to 43, wherein the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant forms a homodimer.

[0109] 45. The method of any one of items 1 to 44, wherein the individual is a person.

[0110] 46. ​​A kit comprising a peptide containing a SIRPαD1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, said peptide for use in combination with a Bcl-2 inhibitor for the treatment of cancer in an individual of need.

[0111] The SIRPαD1 domain variant therein comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85;

[0112] The Fc domain variants mentioned above are

[0113] (i) Human IgG1 Fc regions containing L234A, L235A, G237A and N297A mutations, where the numbering is based on the Kabat EU index;

[0114] (ii) Human IgG2 Fc regions containing A330S, P331S and N297A mutations, where the numbering is based on the EU index of Kabat;

[0115] (iii) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to Kabat's EU index; or

[0116] (iv) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236 and N297A mutations, where the numbering is based on the Kabat EU index;

[0117] The kit contains instructions for administering the combination of the peptide containing the SIRPαD1 domain variant and the Fc domain variant with the Bcl-2 inhibitor to the individual in need.

[0118] 47. The kit as described in item 46, wherein the cancer is leukemia, multiple myeloma, or non-Hodgkin's lymphoma.

[0119] 48. The kit as described in item 46 or 47, wherein the Bcl-2 inhibitor is venetoclax.

[0120] 49. A kit comprising a polypeptide containing a SIRPαD1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, said polypeptide for use in combination with a platinum-based chemotherapeutic agent for the treatment of cancer in an individual of need.

[0121] The SIRPαD1 domain variant therein comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85;

[0122] The Fc domain variants mentioned above are

[0123] (i) Human IgG1 Fc regions containing L234A, L235A, G237A and N297A mutations, where the numbering is based on the Kabat EU index;

[0124] (ii) Human IgG2 Fc regions containing A330S, P331S and N297A mutations, where the numbering is based on the EU index of Kabat;

[0125] (iii) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to Kabat's EU index; or

[0126] (iv) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236 and N297A mutations, where the numbering is based on the Kabat EU index;

[0127] The kit contains instructions for administering the combination of the peptide containing the SIRPαD1 domain variant and the Fc domain variant with the platinum-based chemotherapeutic agent to the individual in need.

[0128] 50. The kit as described in item 49, wherein the cancer is a solid tumor.

[0129] 51. The kit as described in item 50, wherein the solid tumor is colon cancer, colon carcinoma, lung cancer, head and neck cancer, esophageal cancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer, testicular cancer, endometrial cancer, liver cancer, gastric cancer, brain tumor, mesothelioma, or neuroblastoma.

[0130] 52. A kit comprising a peptide containing a SIRPαD1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, said peptide for use in combination with a PD-1 inhibitor, an antimetabolite, and a platinum-based chemotherapeutic agent for the treatment of cancer in an individual of need.

[0131] The SIRPαD1 domain variant therein comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85;

[0132] The Fc domain variants mentioned above are

[0133] (i) Human IgG1 Fc regions containing L234A, L235A, G237A and N297A mutations, where the numbering is based on the Kabat EU index;

[0134] (ii) Human IgG2 Fc regions containing A330S, P331S and N297A mutations, where the numbering is based on the EU index of Kabat;

[0135] (iii) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to Kabat's EU index; or

[0136] (iv) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, where the numbers are based on the Kabat EU index.

[0137] The kit contains instructions for administering the peptide comprising a SIRPαD1 domain variant and an Fc domain variant in combination with the PD-1 inhibitor, the antimetabolite, and the platinum-based chemotherapeutic agent to an individual with head and neck squamous cell carcinoma (HNSCC) who has not received prior treatment for HNSCC.

[0138] 53. The kit according to any one of items 49 to 52, wherein the PD-1 inhibitor is pembrolizumab.

[0139] 54. The kit according to any one of items 49 to 53, wherein the antimetabolite is 5-fluorouracil.

[0140] 55. The kit according to any one of items 49 to 54, wherein the platinum-based chemotherapeutic agent is cisplatin or carboplatin.

[0141] 56. A kit comprising a polypeptide containing a SIRPαD1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, said polypeptide being used in combination with an anti-HER2 antibody, an anti-VEGFR2 antibody, and paclitaxel;

[0142] The SIRPαD1 domain variant therein comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85;

[0143] The Fc domain variants mentioned above are

[0144] (i) Human IgG1 Fc regions containing L234A, L235A, G237A and N297A mutations, where the numbering is based on the Kabat EU index;

[0145] (ii) Human IgG2 Fc regions containing A330S, P331S and N297A mutations, where the numbering is based on the EU index of Kabat;

[0146] (iii) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to Kabat's EU index; or

[0147] (iv) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, where the numbers are based on the Kabat EU index.

[0148] The kit contains instructions for administering the peptide comprising a SIRPαD1 domain variant and an Fc domain variant in combination with the anti-HER2 antibody, the anti-VEGFR2 antibody, and the paclitaxel to an individual with gastric cancer or gastroesophageal (GEJ) cancer who has received at least one prior treatment for gastric cancer or GEJ cancer.

[0149] 57. The kit as described in item 56, wherein the gastric cancer or GEJ cancer is HER2-positive. + Stomach cancer or HER2 + GEJ cancer.

[0150] 58. The kit as described in item 56 or 57, wherein the anti-HER2 antibody is trastuzumab.

[0151] 59. The kit as described in any one of items 56 to 58, wherein the anti-VEGFR2 antibody is ramucirumab.

[0152] 60. The kit of any one of claims 56 to 59, wherein 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 chemotherapy agent.

[0153] 61. A kit comprising a polypeptide containing a SIRPαD1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, said polypeptide for use in combination with an anti-TROP2 antibody for the treatment of cancer in an individual of need.

[0154] The SIRPαD1 domain variant therein comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85;

[0155] The Fc domain variants mentioned above are

[0156] (i) Human IgG1 Fc regions containing L234A, L235A, G237A and N297A mutations, where the numbering is based on the Kabat EU index;

[0157] (ii) Human IgG2 Fc regions containing A330S, P331S and N297A mutations, where the numbering is based on the EU index of Kabat;

[0158] (iii) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to Kabat's EU index; or

[0159] (iv) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236 and N297A mutations, where the numbering is based on the Kabat EU index;

[0160] The kit contains instructions for administering the combination of the peptide containing the SIRPαD1 domain variant and the Fc domain variant with the anti-TROP2 antibody to the individual in need.

[0161] 62. The kit as described in item 61, wherein the cancer is a solid tumor, gastric cancer, nasopharyngeal carcinoma, gallbladder cancer, cervical cancer, extranodal NK / T-cell lymphoma, lung cancer, laryngeal squamous cell carcinoma, colon cancer, hilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrioid endometrial cancer, or ovarian cancer.

[0162] 63. A kit comprising a peptide containing a SIRPαD1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, said peptide for use in combination with an anti-HER2 antibody and an anti-PD-L1 antibody for the treatment of cancer in an individual of need.

[0163] The SIRPαD1 domain variant therein comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85;

[0164] The Fc domain variants mentioned above are

[0165] (i) Human IgG1 Fc regions containing L234A, L235A, G237A and N297A mutations, where the numbering is based on the Kabat EU index;

[0166] (ii) Human IgG2 Fc regions containing A330S, P331S and N297A mutations, where the numbering is based on the EU index of Kabat;

[0167] (iii) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A and delG236 mutations, where the numbering is based on the Kabat EU index;

[0168] (iv) Human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236 and N297A mutations, where the numbering is based on the Kabat EU index;

[0169] The kit contains instructions for administering the peptide containing the SIRPαD1 domain variant and the Fc domain variant in combination with the anti-HER2 antibody and the anti-PD-L1 antibody to the individual in need.

[0170] 64. The kit as described in item 63, wherein the cancer is colon cancer.

[0171] 65. The kit as described in item 64, wherein the colon cancer is HER2-positive. + Colon cancer.

[0172] 66. The kit as described in any one of items 63 to 65, wherein the anti-HER2 antibody is trastuzumab.

[0173] 67. The kit according to any one of items 63 to 66, wherein the anti-PD-L1 antibody is atezolizumab, avelumab, or duruvazumab.

[0174] 68. The kit of any one of claims 46 to 67, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 85.

[0175] 69. The kit of any one of claims 46 to 67, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81.

[0176] 70. The kit as described in any one of items 46 to 69, wherein the Fc domain variant is a human IgG1 Fc region containing L234A, L235A, G237A, and N297A mutations, wherein the numbering is based on the Kabat EU index.

[0177] 71. The kit as described in item 70, wherein the Fc domain variant comprises the amino acid sequence of SEQ ID NO:91.

[0178] 72. The kit of any one of items 46 to 68 and 70 to 71, wherein the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO:136.

[0179] 73. The kit of any one of items 46 to 67 and 69 to 71, wherein the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO:135.

[0180] 74. The kit of any one of claims 46 to 73, wherein the peptide comprising the SIRPαD1 domain variant and the Fc domain variant forms a homodimer.

[0181] 75. The kit as described in any one of items 46 to 74, wherein the individual is a human being. Attached Figure Description

[0182] Figure 1A Tumor volume (mm²) at a specified time post-implantation was provided in NOD-SCID female mice injected with RS4;11 leukemia cells after treatment with drug A, venetoclax, a combination of venetoclax and drug A, or a carrier (PBS). 3 The dashed arrows indicate that venetoclax (250 μg) was administered orally via tube feeding twice, with an interval of 3 days. The dotted arrows indicate that drug A (10 mg / kg) was administered four times, with an interval of 3-4 days. SEM = standard error of the mean; TF = no tumor.

[0183] Figure 1B Tumor volume (mm²) was provided in NOD-SCID female mice injected with RS4;11 leukemia cells at a specified time post-implantation. 3 These mice had been treated with venetoclax and then retreated with either venetoclax alone or with a combination of venetoclax and drug A.

[0184] Figures 2A-2DTumor volume (mm²) at a specified time post-implantation was provided in BALB / c female mice injected with CT26 tumor cells after treatment with drug A, cisplatin, cisplatin / drug A combination, or a carrier (PBS). 3 ) and weight. Figure 2A The mean tumor volume (+ / - SEM) for the specified treatment is provided. The dashed arrow indicates cisplatin administration (two 5 mg / kg doses, 10 days apart). The dotted arrow indicates drug A administration (two 30 mg / kg doses, 10 days apart). Both drugs were administered intraperitoneally. Mice treated with both agents received drug A one day after cisplatin treatment. Figure 2B Provided according to Figure 2A The mice treated with the protocol shown in the diagram show the average percentage change in body weight starting from day 7 (D7). Figure 2C The mean tumor volume (+ / - SEM) for the specified treatment is provided. The dashed arrow indicates cisplatin administration (a single dose of 10 mg / kg). The dotted arrow indicates drug A administration (two doses of 30 mg / kg, 10 days apart). Both drugs are administered intraperitoneally. Mice treated with both agents received drug A one day after cisplatin treatment. Figure 2D Provided according to Figure 2C The average percentage change in body weight of mice treated with the protocol shown begins on day 7 (D7).

[0185] Figure 3 Results of experiments conducted to determine the effect of the combination of drug A and anti-TROP2 antibody on the phagocytosis of CFSE-labeled DLD-1 tumor cells by human monocyte-derived macrophages are provided.

[0186] Figure 4 Results are provided for experiments conducted to determine the effects of drug A on tumor growth in an MC38 m / h colon cancer model, in combination with (a) anti-HER2 antibody, (b) anti-PD-L1 antibody, or (c) anti-HER2 antibody and anti-PD-L1.

[0187] Figure 5A Results of experiments conducted to evaluate the effects of adding drug A, venetoclax, or both drug A and venetoclax in in vitro assays on the phagocytosis of macrophages on HL60 cells are provided. Figure 5B Results of experiments conducted to evaluate the effects of adding drug A, venetoclax, or both drug A and venetoclax in in vitro assays on the phagocytosis of OCI-AML3 cells by macrophages are provided.

[0188] Figure 6A Provided for evaluating the effect of drug A or drug C on CD8 + The results of experiments conducted to investigate the effects of dendritic cell activation. Figure 6BProvided for evaluating the effect of drug A or drug C on CD8 - The results of experiments conducted to investigate the effects of dendritic cell activation.

[0189] Figure 7A Provides a method for evaluating the effect of drug A or drug B on CD8. + The results of experiments conducted to investigate the effects of dendritic cell activation. Figure 7B Provides a method for evaluating the effect of drug A or drug B on CD8. - The results of experiments conducted to investigate the effects of dendritic cell activation.

[0190] Figure 8A Results of experiments conducted to evaluate the binding of drugs A, F59 / magrolimab, TTI-621, and TTI-622 to hCD47 are provided. Figure 8B Results of quantitative experiments conducted to evaluate the effects of drugs A, F59 / molololimab, TTI-621, and TTI-622 on SIRPα signaling are provided. Detailed Implementation

[0191] The following description illustrates exemplary methods, parameters, etc. However, it should be understood that such description is not intended to limit the scope of this disclosure, but is provided as a description of exemplary embodiments.

[0192] definition

[0193] The terms "about" or "approximately" refer to an acceptable margin of error for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" may mean within 1 or greater than 1 standard deviation. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly for biological systems or methods, the term may refer to orders of magnitude of the numerical value, preferably within 5 times, more preferably within 2 times. In cases where a particular value is described in this application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable margin of error for the particular value.

[0194] The terminology used herein is for the purpose of describing a particular situation only and not for limitation. As used herein, the singular forms “a,” “an,” and “the” are also intended to include the plural forms unless the context clearly indicates otherwise. Furthermore, with respect to the use of the terms “including,” “includes,” “having,” “has,” “with,” or variations thereof in the detailed description or claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0195] As used herein, the terms "treatment," "treating," etc., refer to the administration of an agent or procedure for the purpose of achieving an effect. In some embodiments, the effect is preventative in relation to the complete or partial prevention of a disease or its symptoms. In some embodiments, the effect is therapeutic in relation to the partial or complete cure of a disease or its symptoms.

[0196] As used herein, the term “antibody” refers to an intact antibody; an antibody fragment provided that it exhibits the desired biological activity (e.g., epitope binding); a monoclonal antibody; a polyclonal antibody; a monospecific antibody; a multispecific antibody (e.g., a bispecific antibody); and an antibody-like protein.

[0197] As used herein, the term “antibody variable domain” refers to portions of the light and heavy chains of an antibody, including the amino acid sequences of complementarity-determining regions (CDRs, such as CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3) and framework regions (FRs).

[0198] As used herein, the term "connector" refers to a bond between two elements (e.g., a protein domain). In some embodiments, the connector can be a covalent bond or a spacer. The term "spacer" refers to a portion (e.g., a polyethylene glycol (PEG) polymer) or an amino acid sequence (e.g., a sequence of 1-200 amino acids) present between two polypeptides or polypeptide domains to provide space or flexibility (or both) between the two polypeptides or polypeptide domains. In some embodiments, the amino acid spacer is part of the primary sequence of the polypeptide (e.g., a polypeptide or polypeptide domain linked to the spacer via the polypeptide backbone).

[0199] As used herein, the term "effective amount" refers to the amount of a polypeptide or a pharmaceutical composition containing the polypeptide described herein (e.g., a polypeptide having a SIRPαD1 domain or a variant thereof) sufficient and effective to achieve the desired therapeutic effect in a patient with a disease (such as cancer, e.g., a solid tumor or hematologic malignancy). In some embodiments, the effective amount of the polypeptide will avoid adverse side effects.

[0200] As used herein, the term "pharmaceutical composition" refers to a pharmaceutical or pharmaceutical formulation that comprises an active ingredient and excipients or diluents (or both) and enables the active ingredient to be administered by a suitable method of application. In some embodiments, the pharmaceutical compositions disclosed herein comprise pharmaceutically acceptable components compatible with peptides. In some embodiments, the pharmaceutical compositions are in tablet or capsule form for oral administration or in an aqueous form for intravenous or subcutaneous administration (e.g., by injection).

[0201] As used herein, the terms “subject,” “individual,” and “patient” are used interchangeably to refer to vertebrates, such as mammals. Mammals include, but are not limited to, rodents, apes, humans, farm animals, sporting animals, and pets. This also includes tissues, cells, and their progeny from biological entities obtained in vivo or cultured in vitro. These terms do not require the supervision of a medical professional.

[0202] As used herein, the term "affinity" or "binding affinity" refers to the strength of the binding interaction between two molecules. Generally, binding affinity refers to the strength of the sum of non-covalent interactions between a molecule and its binding partner (such as SIRPαD1 domain variants and CD47). Unless otherwise specified, binding affinity refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of the binding pair. The binding affinity between two molecules is typically determined by the dissociation constant (K0). D ) or association constant (K) A Description. Two molecules with low binding affinity to each other typically bind slowly, tend to dissociate easily, and exhibit a large K-value. D Two molecules with high affinity for each other usually bind easily, tend to maintain the bond for longer, and exhibit small K-values. D In some implementations, known methods and techniques, such as surface plasmon resonance (SPR), are used to determine the Kk of the two interacting molecules. D K D It can be calculated as the ratio of koff / kon.

[0203] As used in this article, the term "less than K" D "Refers to K, which is numerically smaller" D Value and relative to K D The value increases the binding affinity. As used in this paper, the term "greater than K" refers to the binding affinity. D "" refers to a numerically larger K D Value and relative to K D The binding affinity decreases.

[0204] As used in this article, “combination” means administering a treatment in addition to another treatment. Therefore, “combination” means administering a treatment before, during, or after administering another treatment to an individual.

[0205] Overview

[0206] This document provides a method for treating cancer in an individual (e.g., a human individual), 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 (e.g., at least one chemotherapeutic agent, such as at least two, at least three, or at least four chemotherapeutic agents). In some embodiments, the method further comprises administering to the individual an effective amount of a therapeutic antibody (e.g., at least one therapeutic antibody, such as at least two, at least three, or at least four therapeutic antibodies). Alternatively or additionally, in some embodiments, the method further comprises administering to the individual an effective amount of an immunotherapeutic agent (e.g., at least one immunotherapeutic agent, such as at least two, at least three, or at least four immunotherapeutic agents). Alternatively or additionally, in some embodiments, the method comprises administering the peptide and the chemotherapeutic agent in combination with one or more other treatment modalities (including, but not limited to, radiotherapy, surgery, cryoablation, and bone marrow transplantation).

[0207] In some implementations, the 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, etc.). See, for example, Miller et al. (2019) “Quantitative high-throughput screening assays for the discovery and development of SIRPα-CD47 interaction inhibitors.” PLoS ONE 14(7):e0218897 and Sasikumar et al. ACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; October 26-30, 2017; Philadelphia, PA; Abstract B007.

[0208] In some embodiments, an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) binds to CD47 (e.g., hCD47). In some embodiments, the agent is present at about 10 nM or better K. D(e.g., at least about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM, or less than 10 pM) binds to CD47 (e.g., hCD47). In some embodiments, in human subjects, the agent that binds to CD47 (e.g., hCD47) exhibits at least about 50% CD47 receptor occupancy (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%). In some embodiments, the CD47-binding agent (e.g., hCD47) has an EC50 of about 80 ng / ml or less, such as any one of about 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 ng / ml. In some embodiments, the CD47-binding agent (e.g., hCD47) is an anti-CD47 antibody (e.g., a therapeutic anti-CD47 antibody) or an antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment is Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv, a single-arm antibody, or a bispecific antibody. 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 implementations, the term "anti-CD47 antibody" encompasses antibody-based constructs (such as multispecific constructs) including, but not limited to, triomabs, DART (i.e., biparental and heavily targeted antibodies), TandAb (i.e., tandem biantibodies), tandem scFv, CrossMab, DNL (i.e., dock and lock antibodies), DVD-Ig (i.e., dual variable domain immunoglobulin), tetravalent bispecific IgG, nanobodies, dual targeting domains, and ART-Ig (i.e., asymmetric recombination technology-immunoglobulin). Further 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 (from Innovent Biologics) (see, for example, Zhao et al. (2011), PNAS USA 108:18342-18347; Chao et al. (2010) Cell 142:699-713; Kim et al. (2012) Leukemia 26:2538-2545; Chao et al. (2011) Blood 118:4890-4891; Goto et al. (2014) Eur J. Cancer 50:1836-1846; and Edris et al. (2012) PNAS USA 109:6656-61 for further information on these anti-CD47 antibodies).

[0209] In some embodiments, an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) binds to SIRPα (e.g., hSIRPα). In some embodiments, the agent is in a K+ concentration of about 10 nM or better. D(e.g., at least about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM, or less than 10 pM) binds to SIRPα (e.g., hSIRPα). In some embodiments, in human subjects, the agent that binds to SIRPα (e.g., hSIRPα) exhibits at least about 50% SIRPα receptor occupancy (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%). In some embodiments, the SIRPα-binding agent (e.g., hSIRPα) has an EC50 of about 80 ng / ml or less, such as any one of about 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 ng / ml. In some embodiments, the SIRPα-binding agent (e.g., hSIRPα) is an anti-SIRPα antibody (e.g., a therapeutic anti-SIRPα antibody) or an antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment is Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv, a single-arm antibody, or a bispecific antibody. In some embodiments, the anti-SIRPα antibody is a monospecific antibody or a monospecific antibody construct (including, but not limited to, those described above). In some embodiments, the anti-SIRPα antibody is a multispecific (e.g., bispecific) antibody or a multispecific antibody construct (including, but not limited to, those described above). In some embodiments, the anti-SIRPα antibody is KWAR23, SE12C3, O40, or MY-1 (see, for example, Ring et al. (2017) PNASUSA 114(49):E10578-E10585); Murata et al. (2018) Cancer Sci 109(5):1300-1308; and Yanigata et al. (2017) JCI Insight 2:e89140 for further information on these anti-SIRPα antibodies). In some embodiments, the anti-SIRPα antibody is the antibody described in WO 2018 / 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.

[0210] In some embodiments, the agent blocking 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 capable of binding SIRPα) or an antigen-binding fragment thereof. In some embodiments, the agent is an antibody (or an antigen-binding fragment thereof) capable of binding two or more of SIRPα, SIRPβ, and SIRPγ. In some embodiments, such antibodies are expressed at about 10 nM or better KJ. D (e.g., at least about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM, or less than 10 pM) binds to SIRPα (e.g., hSIRPα). In some embodiments, in human subjects, the antibody exhibits at least about 50% SIRPα receptor occupancy (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%). In some embodiments, the antibody has an EC50 of about 80 ng / ml or less, such as any one of about 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 ng / ml. In some embodiments, the antigen-binding fragment is Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv, a single-arm antibody, or a bispecific antibody. In some embodiments, the antibody is a monospecific antibody or a monospecific antibody construct (including, but not limited to, those described above). In some embodiments, the antibody is a multispecific (e.g., bispecific) antibody or a multispecific antibody construct (including, but not limited to, those described above).

[0211] 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 moiety. In some embodiments, the fusion polypeptide comprising an antibody Fc region and a CD47-binding moiety. In some embodiments, the CD47-binding (e.g., hCD47)-binding fusion polypeptide moiety is at about 10 nM or better K. D(e.g., at least about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM, or less than 10 pM) binds to CD47 (e.g., hCD47). In some embodiments, in human subjects, the fusion peptide exhibits at least about 50% CD47 receptor occupancy (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%). In some embodiments, the fusion peptide has an EC50 of about 80 ng / ml or less, such as any one of about 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 ng / ml. In some embodiments, the fusion peptide comprises a WT human antibody Fc region. In some embodiments, the fusion peptide comprises an Fc variant (e.g., a variant of the WT human antibody Fc region) exhibiting reduced (e.g., ablation-like) effector function compared to the WT Fc region. Exemplary Fc variants are described in WO 2017 / 027422 and US2017 / 0107270, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the portion binding CD47 (e.g., hCD47) is WT SIRPα (e.g., hSIRPα) or WT SIRPγ (e.g., hSIRPγ). In some embodiments, the portion binding CD47 (e.g., hCD47) is a CD47-binding fragment (e.g., a d1 domain) of WT SIRPα (e.g., hSIRPα) or WT SIRPγ (e.g., hSIRPγ). In some embodiments, the portion binding CD47 (e.g., hCD47) is a SIRPα variant, a SIRPγ variant, a SIRPβ variant, or a CD47-binding fragment (e.g., a d1 domain) thereof.Exemplary SIRPγ variants, SIRPβ1 variants, and SIRPβ2 variants are described in, for example, WO 2013 / 109752; US2015 / 0071905; USP 9,944,911; WO 2016 / 023040; WO 2017 / 027422; US2017 / 0107270; USP The contents of the patents mentioned in 10,259,859; US9845345; WO2016187226; US20180155405; WO2017177333; WO2014094122; US2015329616; US20180312563; WO2018176132; WO2018081898; WO2018081897; PCT / US2019 / 048921; US20180141986A1 and EP3287470A1 are incorporated herein by reference in their entirety.

[0212] In some embodiments, the agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is a fusion peptide comprising an antibody Fc region and a SIRPα variant. In some embodiments, the SIRPα variant is at about 10 nM or better K D(e.g., at least about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM, or less than 10 pM) binds to CD47 (e.g., hCD47). In some embodiments, in human subjects, the fusion peptide exhibits at least about 50% CD47 receptor occupancy (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%). In some embodiments, the fusion peptide has an EC50 of about 80 ng / ml or less, such as any one of about 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 ng / ml. In some embodiments, the fusion peptide comprises the Fc region of a WT human antibody. In some embodiments, the fusion peptide comprises an Fc variant (e.g., a variant of the WT human antibody Fc region) that exhibits reduced (e.g., ablation-like) effector function compared to the WT Fc region (such as those described in the references cited herein). In some embodiments, the fusion peptide is contained in WO 2013 / 109752; US 2015 / 0071905; WO 2016 / 023040; WO 2017 / 027422; US2017 / 0107270; USP The SIRPα variants described in 10,259,859; US9845345; WO2016187226; US20180155405; WO2017177333; WO2014094122; US2015329616; US20180312563; WO2018176132; WO2018081898; WO2018081897; US20180141986A1 and EP3287470A1, the contents of which are incorporated herein by reference in their entirety.In some embodiments, the fusion peptide 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 RevS0268-960X(17)30093-0; Zhang, X, Chen, W, Fan, J et al. Disrupting CD47-SIRPαaxis alone or combined with autophagy depletion for the therapy of glioblastoma. Carcinogenesis 2018; 39:689–99).

[0213] In some embodiments, the agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is a fusion peptide comprising a SIRPαD1 domain variant (e.g., the SIRPαD1 domain variant described herein) and an Fc domain variant (e.g., the Fc domain variant described herein).

[0214] In some embodiments, a method of treating cancer (e.g., leukemia, such as acute lymphoblastic leukemia) in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) and (b) a BCL2 inhibitor (e.g., a selective BCL2 inhibitor, such as venetoclax). In some embodiments, the agent is a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is based on the Kabat EU index; and (ii) a human IgG1 Fc region comprising A330S, P331S, and N297A mutations. (iii) Fc regions containing S228P, E233P, F234V, L235A, and delG236 mutations, with numbers based on the Kabat EU index; or (iv) Fc regions containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, with numbers based on the Kabat EU index.

[0215] In some embodiments, a method of treating cancer (e.g., colon cancer) in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), and (b) a platinum-based chemotherapeutic agent (e.g., cisplatin). In some embodiments, the agent is a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is based on the Kabat EU index; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is based on the Kabat EU index; (iii) a human IgG4 region comprising S228P, E233P, F234V, L235A, and delG236 mutations. Fc regions, numbered according to the Kabat EU index; or (iv) human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236 and N297A mutations, numbered according to the Kabat EU index.

[0216] In some embodiments, a method of treating cancer (e.g., head and neck cancer, such as squamous cell carcinoma of the head and neck) in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) a PD-1 inhibitor, (c) an antimetabolite, and (d) a platinum-based chemotherapeutic agent. In some embodiments, the agent blocking the interaction between CD47 and SIRPα is a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is based on the Kabat EU index; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is based on the Kabat EU index; (iii) a human IgG4 region comprising S228P, E233P, F234V, L235A, and delG236 mutations. Fc regions, numbered according to the Kabat EU index; or (iv) human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236 and N297A mutations, numbered according to the Kabat EU index.

[0217] In some embodiments, a method is provided 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, the agent blocking the interaction between CD47 and SIRPα is a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is based on the Kabat EU index; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is based on the Kabat EU index; (iii) a human IgG4 region comprising S228P, E233P, F234V, L235A, and delG236 mutations. Fc regions, numbered according to the Kabat EU index; or (iv) human IgG4 Fc regions containing S228P, E233P, F234V, L235A, delG236 and N297A mutations, numbered according to the Kabat EU index.

[0218] Further details of the method of treatment with peptides containing SIRPαD1 domain variants and Fc domain variants are described below. See also WO 2017 / 027422 and U.S. Patent No. 10,259,859, the contents of which are each incorporated herein by reference in their entirety.

[0219] Signal regulatory protein α (SIRP-α) D1 domain and its variants

[0220] In some embodiments, this document discloses polypeptides comprising a variant of the signal regulatory protein α (SIRP-α)D1, the variant comprising a SIRPαD1 domain or a fragment thereof, the SIRPαD1 domain comprising an amino acid mutation at residue 80 relative to the wild-type SIRPαD1 domain (e.g., the wild-type SIRPαD1 domain described in SEQ ID NO: 1 or 2); and comprising at least one additional amino acid mutation at residues selected from the group consisting of the wild-type SIRPαD1 domain (e.g., the wild-type SIRPαD1 domain described in SEQ ID NO: 1 or 2): residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92.

[0221] In some embodiments, this document also discloses peptides comprising Fc domain variants, wherein the Fc domain variant dimer comprises two Fc domain variants, wherein each Fc domain variant is independently selected from (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236, and N297A.

[0222] Signal regulatory protein α (“SIRP-α” or “SIRP-alpha”) is a transmembrane glycoprotein belonging to the Ig superfamily, widely expressed on the membranes of bone marrow cells. SIRPα interacts with CD47, a protein widely expressed in many cell types in vivo. This interaction between SIRPα and CD47 prevents phagocytosis of “self” cells, which would otherwise be recognized by the immune system. High CD47 expression on tumor cells has been observed to act as a negative prognostic factor for survival in acute myeloid leukemia and several solid tumor cancers.

[0223] Natural SIRPα contains three highly homologous immunoglobulin (Ig)-like extracellular domains—D1, D2, and D3. The SIRPα D1 domain (“D1 domain”) refers to the distal membrane extracellular domain of SIRPα and mediates the binding of SIRPα to CD47. As used herein, the term “SIRPα polypeptide” refers to any SIRPα polypeptide or fragment thereof capable of binding CD47. At least ten 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 NO: 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 NO: 1 and 2. In some embodiments, the SIRPα polypeptide contains the D2 or D3 domain of wild-type human SIRPα (or both the D2 and D3 domains) (see Table 3).

[0224] Table 1. Sequence of wild-type SIRPαD1 domains

[0225]

[0226]

[0227] As used herein, the term "SIRPαD1 domain variant" refers to a polypeptide containing the SIRPαD1 domain or the CD47-binding portion of the SIRPα polypeptide, which has a higher affinity for CD47 than wild-type SIRPα. SIRPαD1 domain variants contain at least one amino acid substitution, deletion, or insertion (or a combination thereof) relative to wild-type SIRPα.

[0228] In some embodiments, the SIRPαD1 domain variants disclosed herein comprise the SIRPαD1 domain or variants thereof. In some embodiments, the SIRPαD1 domain variants comprise one or more amino acid substitutions, insertions, additions, or deletions relative to the wild-type D1 domain shown in SEQ ID NO:1 and 2. Table 2 lists exemplary amino acid substitutions (SEQ ID NO:13-14) in each SIRPαD1 domain variant. In some embodiments, the SIRPαD1 domain peptide or SIRPαD1 domain variant comprises a fragment of the D1 domain. In some embodiments, the SIRPα peptide fragment or SIRPαD1 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 more than about 100 amino acids. In some implementations, the SIRPαD1 domain segment retains the ability to bind CD47.

[0229] In some embodiments, the disclosed polypeptide comprising a variant of the SIRPαD1 domain binds CD47 with a higher binding affinity than the wild-type human SIRPαD1 domain. In some embodiments, the SIRPαD1 domain variant binds human CD47 with an affinity at least 1 times (e.g., at least 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 5 times, or greater than 5 times) that of the naturally occurring D1 domain. In some embodiments, the SIRPαD1 domain variant binds human CD47 with an affinity at least 1 times (e.g., at least 10 times, 100 times, 1000 times, or greater than 1000 times) that of the naturally occurring D1 domain.

[0230] As used herein, the terms "optimized affinity" or "optimized binding affinity" refer to the optimized binding interaction strength between the disclosed peptides (including SIRPαD1 domain variants) and CD47. For example, in some embodiments, the peptide binds CD47 primarily or with a high affinity to cancer cells, while substantially not binding or binding CD47 on non-cancer cells with a low affinity. In some embodiments, the binding affinity between the peptide and CD47 is optimized such that the interaction does not cause clinically relevant toxicity or reduces toxicity compared to variants that bind with maximum affinity. In some embodiments, to achieve the optimized binding affinity between the peptides provided herein and CD47, peptides containing SIRPαD1 domain variants are developed to have a lower binding affinity for CD47 than the maximum achievable binding affinity. In some embodiments, the SIRPαD1 domain variants disclosed herein cross-react with rodent, non-human primate (NHP), and human CD47.

[0231] As used herein, the term "immunogenicity" refers to the property of a protein (e.g., a therapeutic protein) that elicits an immune response in the host, just as if it were a foreign antigen. The immunogenicity of a protein can be determined in vitro in a variety of different ways, such as by in vitro T-cell proliferation assays.

[0232] As used herein, the term "minimal immunogenicity" refers to the immunogenicity of a modified protein (e.g., a therapeutic protein) that is lower (e.g., at least 10%, 25%, 50%, or 100% lower) than that of the protein before the introduction of the amino acid substitution (e.g., an unmodified protein). In some embodiments, the protein (e.g., a therapeutic protein) is modified to have minimal immunogenicity and to elicit little or no host immune response, even if it is a foreign antigen.

[0233] In some embodiments, the SIRPαD1 domain variant exhibits minimal immunogenicity. In some embodiments, the SIRPα peptide of this disclosure administered to a subject has the same amino acid sequence as the SIRPα peptide in the subject's biological sample, except for the amino acid alterations that increase the affinity of the SIRPαD1 domain variant. In some embodiments, the peptide variants disclosed herein reduce the risk of side effects compared to anti-CD47 antibodies or wild-type SIRPα. In some embodiments, the peptide variants disclosed herein reduce the risk of anemia compared to anti-CD47 antibodies or wild-type SIRPα. In some embodiments, the peptide variants disclosed herein do not induce acute anemia in rodent or non-human primate (NHP) studies.

[0234] Table 2 lists specific amino acid substitutions relative to each D1 domain sequence in SIRPαD1 domain variants. In some embodiments, the SIRPαD1 domain variants include one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more) substitutions listed in Table 2. In some embodiments, the SIRPαD1 domain variants contain up to fourteen amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPαD1 domain variants contain up to ten amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPαD1 domain variants contain up to seven amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPαD1 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 the wild-type D1 domain.

[0235] In some embodiments, the SIRPαD1 domain variant is a chimeric SIRPαD1 domain variant comprising portions of two or more wild-type D1 domains or variants thereof (e.g., a portion of one wild-type D1 domain or variant thereof and a portion of another wild-type D1 domain or variant thereof). In some embodiments, the chimeric SIRPαD1 domain variant comprises at least two portions (e.g., three, four, five or more portions) of a wild-type D1 domain or variant thereof, wherein each portion originates from a different wild-type D1 domain. In some embodiments, the chimeric SIRPαD1 domain variant further comprises one or more amino acid substitutions listed in Table 2.

[0236] Table 2. Amino acid substitutions in SIRPαD1 domain variants

[0237]

[0238] In some embodiments, the peptide comprises a variant of the SIRPαD1 domain containing the following sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGNITPADAGTYYCX 12 KX 13 RKGSPDDVEX 14KSGAGTELSVRAKPS (SEQ ID NO:13), wherein 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 For L, T, or G; X 11 For K or R; X 12 For V or I; X 13 For F, L, or V; and X 14 It is F or V; and said variant contains at least one amino acid substitution relative to the wild-type SIRPαD1 domain containing the sequence of SEQ ID NO:1.

[0239] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant containing the sequence of SEQ ID NO:13, wherein X1 is L, I, or V. In any of the above 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 For L, T, or G. In some implementations, X 11 For K or R. In some implementations, X 12 For V or I. In some implementations, X 13 For F, L, and V. In some implementations, X 14 It is F or V. In some embodiments, the polypeptide of this aspect of the present disclosure contains no more than six amino acid substitutions relative to the wild-type SIRPαD1 domain containing the sequence of SEQ ID NO:1.

[0240] In some embodiments, the peptide binds CD47 with a binding affinity of at least 10 times that of the wild-type SIRPαD1 domain comprising the sequence of SEQ ID NO:1. In some embodiments, the peptide binds CD47 with a binding affinity of at least 100 times that of the wild-type SIRPαD1 domain comprising the sequence of SEQ ID NO:1. In some embodiments, the peptide binds CD47 with a binding affinity of at least 1000 times that of the wild-type SIRPαD1 domain comprising the sequence of SEQ ID NO:1. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof binds with a binding affinity of less than 1 x 10^6. -8 M, less than 5 x 10 -9 M, less than 1 x 10 -9M, less than 5x10 -10 M, less than 1 x 10 -10 M or less than 1 x 10 -11 M of K D Binding to CD47. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof is in a Kc ratio between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Combined with CD47.

[0241] In some embodiments, the peptide comprises a variant containing a SIRPαD1 domain with the following sequence: EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14 KSGAGT ELSVRAKPS (SEQ ID NO:14), wherein 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 For S, T, or G; X 11 For K or R; X 12 For V or I; X 13 For F, L, or V; and X 14 It is F or V; and said variant contains at least one amino acid substitution relative to the wild-type SIRPαD1 domain containing the sequence of SEQ ID NO:2.

[0242] In some embodiments of this aspect of the disclosure, the polypeptide comprises the sequence of SEQ ID NO: 14, wherein X1 is L, I, or V. In some embodiments, X2 is V, L, or I. In some embodiments, X3 is A or V. In some embodiments, X4 is V, I, or L. In some embodiments, X5 is I, T, S, or F. In some embodiments, X6 is E, V, or L. In some embodiments, X7 is K or R. In some embodiments, X8 is E or Q. In some embodiments, X9 is H, P, or R. In some embodiments, X...10 For S, T, or G. In some implementations, X 11 For K or R. In some implementations, X 12 For V or I. In some implementations, X 13 For F, L, or V. In some implementations, X 14 It is F or V. In some embodiments, the polypeptide of this aspect of the present disclosure contains no more than six amino acid substitutions relative to the wild-type SIRPαD1 domain containing the sequence of SEQ ID NO:2.

[0243] In some embodiments, the peptide binds CD47 with a binding affinity of at least 10 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2. In some embodiments, the peptide binds CD47 with a binding affinity of at least 100 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2. In some embodiments, the peptide binds CD47 with a binding affinity of at least 1000 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof binds CD47 with a binding affinity of less than 1 x 10^6. -8 M, less than 5 x 10 -9 M, less than 1 x 10 -9 M, less than 5x10 -10 M, less than 1 x 10 -10 M or less than 1 x 10 -11 M of K D Binding to CD47. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof is in a Kc ratio between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Combined with CD47.

[0244] In some embodiments, the peptide comprises a SIRPαD1 domain variant having the following sequence: EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSLX 11 PVGPIQWFRGAGPX 12 RX 13 LI YNQX 14 X 15 GX 16 FPRVTTVSX17 X 18 TX19RX 20 NMDFX 21 IX 22 IX 23 NITPADAGTYYCX 24 KX 25 RKGSPDX 26 X 27 EX 28 KSGAGTELSVRX 29 KPS (SEQ ID NO:23), wherein X1 is E or G; X2 is L, I or V; X3 is V, L or I; X4 is S or F; X5 is L or S; X6 is S or T; X7 is A or V; X8 is I or T; X9 is H or R; X 10 For A, V, I, or L; X 11 For I, T, S, or F; X 12 For A or G; X 13 For E, V, or L; X 14 For K or R; X 15 For E or Q; X 16 For H, P, or R; X 17 For D or E; X 18 For S, L, T or G; X 19 For K or R; X 20 For E or D; X 21 For S or P; X 22 For S or R; X 23 For S or G; X 24 For V or I; X 25 For F, L, V; X 26 D or does not exist; X 27 For T or V; X 28 For F or V; and X 29 The variant is A or G; and the variant contains at least one amino acid substitution relative to the wild-type SIRPαD1 domain containing the sequence of SEQ ID NO:1 or 2.

[0245] In any of the above embodiments of this aspect of the disclosure, X2 is L, I, or V. In any of the above embodiments, X3 is V, L, or I. In 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 It can be A, V, I, or L. In some implementations, X 11 It can be I, T, S, or F. In some implementations, X12 It is either A or G. In some implementations, X 13 For E, V, or L. In some implementations, X 14 For K or R. In some implementations, X 15 For E or Q. In some implementations, X 16 For H, P, or R. In some implementations, X 17 It is D or E. In some implementations, X 18 For S, L, T, or G. In some implementations, X 19 For K or R. In some implementations, X 20 For E or D. In some implementations, X 21 For S or P. In some implementations, X 22 For S or R. In some implementations, X 23 For S or G. In some implementations, X 24 For V or I. In some implementations, X 25 For F, L, and V. In some implementations, X 26 It is either D or does not exist. In some implementations, X 27 For T or V. In some implementations, X 28 For F or V. In some implementations, X 29 It is A or G. In some embodiments, the polypeptide of this aspect of the present disclosure contains no more than six amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1 or 2.

[0246] In some embodiments, the peptide binds CD47 with a binding affinity of at least 10-fold to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the peptide binds CD47 with a binding affinity of at least 100-fold to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the peptide binds CD47 with a binding affinity of at least 1000-fold to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof binds with a binding affinity of less than 1 x 10^6 ppm. -8 M, less than 5 x 10 -9 M, less than 1x10 -9 M, less than 5 x 10 -10 M, less than 1 x 10 -10 M or less than 1 x 10 -11 M of K DBinding to CD47. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof is in a Kc ratio between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Combined with CD47.

[0247] In some embodiments, the polypeptide of this disclosure comprising a variant of the SIRPαD1 domain further comprises the D2 domain of wild-type human SIRPα having the sequence of SEQ ID NO:24, the D3 domain having the sequence of SEQ ID NO:25, or the D2 domain having the sequence of SEQ ID NO:24 and the D3 domain having the sequence of SEQ ID NO:25. In some embodiments, the SIRPαD1 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 SIRPαD1 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 SIRPαD1 domain variant is linked to the D2 or D3 domain via a linker. In some embodiments, the SIRPαD1 domain variant is linked to both the D2 and D3 domains via a linker.

[0248] Table 3. Amino acid sequences of SIRPα D2 and D3 domains

[0249]

[0250]

[0251] In some embodiments, the peptide of this disclosure comprising a SIRPαD1 domain variant is attached to an Fc domain variant to improve the pharmacokinetic properties of the peptide, such as increasing serum half-life. In some embodiments, the SIRPαD1 domain variant is attached to a non-dimerizable Fc domain variant. In some embodiments, the Fc domain variant is used to increase the serum half-life of the peptide described herein. In some embodiments, the peptide of this disclosure comprising a SIRPαD1 domain variant does not include the sequence of any one of SEQ ID NO:26-36 shown in Table 4.

[0252] Table 4.

[0253]

[0254] In some embodiments, the peptides and peptide constructs described herein are used in vitro for binding assays, such as immunoassays. For example, in some embodiments, the peptides and peptide constructs described herein are used in a liquid phase or bound to a solid-phase carrier. In some embodiments, the peptides used for immunoassays are detectably labeled in various ways.

[0255] In some embodiments, the peptides and peptide constructs described herein are bound to various carriers and used to detect the presence of cells expressing a specific antigen. Examples of carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The carriers may be soluble or insoluble.

[0256] Various labeling methods are known. Examples of labeling include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds. Labels can be attached to the peptides disclosed herein using a variety of techniques.

[0257] In some embodiments, the peptide is conjugated to a low molecular weight hapten. These haptens are then specifically detected by a second reaction. For example, in some embodiments, the hapten biotin is used in conjunction with avidin, or the hapten dinitrophenol, pyridoxal, or fluorescein is detected using specific anti-hapten antibodies (e.g., anti-dinitrophenol antibody, anti-pyridoxal antibody, and anti-fluorescein antibody, respectively).

[0258] SIRPαD1 domain variant with altered glycosylation patterns

[0259] In some embodiments, this document discloses polypeptides comprising a variant of the signal regulatory protein α (SIRP-α)D1, the variant comprising a SIRPαD1 domain or a fragment thereof, the SIRPαD1 domain having an amino acid mutation at residue 80 relative to the wild-type SIRPαD1 domain (e.g., the wild-type SIRPαD1 domain described in SEQ ID NO:1 or 2); and having at least one additional amino acid mutation at residues selected from the group consisting of the wild-type SIRPαD1 domain (e.g., the wild-type SIRPαD1 domain described in SEQ ID NO:1 or 2): residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92.

[0260] In some embodiments, this document also discloses peptides comprising Fc domain variants, wherein the Fc domain variant dimer comprises two Fc domain variants, each Fc domain variant being independently selected from (i) the human IgG1 Fc region composed of L234A, L235A, G237A, and N297A mutations; (ii) the human IgG2 Fc region composed of A330S, P331S, and N297A mutations; or (iii) the human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations.

[0261] In some embodiments, the peptides in the compositions disclosed herein comprise variants of the SIRPαD1 domain with reduced or minimal glycosylation. The D1 domains of SEQ ID NO:1 and 2 in Table 1 contain a single potential N-linked glycosylation site at amino acid N80 in sequence N80ITP. Expression of the SIRPαD1 domain in Chinese hamster ovary (CHO) cells produces a major band of 16 kDa (non-glycosylated) and a higher molecular weight minor band removed by Endo Hf. Endo Hf is a recombinant protein fusion of endoglucosidase H and a maltose-binding protein. Endo Hf is cleaved within a high-mannose chitobiose core and some heterozygous oligosaccharides from N-linked glycoproteins. This means that the proline at amino acid position 83 reduces the efficiency of glycosylation, resulting in proteins with varying degrees of glycosylation and thus heterogeneity. For drug development, heterogeneity can pose challenges in process development. Therefore, to investigate the possibility of generating homogeneous, non-glycosylated variants of the SIRPαD1 domain, in some embodiments, the amino acid N80 of the SIRPαD1 variant is mutated to Ala. In some embodiments, to prepare non-glycosylated SIRPαD1 domain variants, the amino acid N80 in the SIRPαD1 domain variant is substituted with any amino acid, including any naturally occurring and non-naturally occurring amino acids, such as N80A and N80Q. In some embodiments, the SIRPαD1 domain variant contains the N80A mutation and at least one additional mutation (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional mutations or more). In some embodiments, the additional mutation is in the CD47 binding site. In some embodiments, the additional mutation is in the hydrophobic core of the D1 domain.

[0262] In some embodiments, the peptides in the compositions disclosed herein comprise SIRPαD1 domain variants with increased glycosylation relative to the wild-type SIRPαD1 domain. Another option to increase the homogeneity of the final product is to enhance the glycosylation efficiency at amino acid N80 and produce SIRPαD1 domain variants with increased glycosylation relative 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 glycosylation efficiency at N80. In some embodiments, amino acid P83 in the SIRPαD1 domain variant is replaced by any amino acid, including native and non-native amino acids such as P83V, P83A, P83I, and P83L. In some embodiments, the polypeptides of this disclosure are expressed in cells optimized to prevent the glycosylation of proteins expressed by such cells, for example by genetic engineering of the cell line (e.g., genetically engineered yeast or mammalian hosts) or modification of cell culture conditions such as the addition of chitosan or by using a naturally non-glycosylated host such as a prokaryote (Escherichia coli, etc.).

[0263] Table 5 lists the specific amino acid substitutions in the SIRPαD1 domain variants relative to the sequence of each D1 domain variant. In some embodiments, the SIRPαD1 domain variants include one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more) substitutions listed in Table 5. In some embodiments, the SIRPαD1 domain variants are unglycosylated or minimally glycosylated. In some embodiments, the SIRPαD1 domain variants are fully or almost fully glycosylated. In some embodiments, the SIRPαD1 domain variants contain up to fourteen amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPαD1 domain variants contain up to ten amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPαD1 domain variants contain up to seven amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPαD1 domain variant of this disclosure has at least 90% (e.g., at least 92%, 95%, 97% or greater than 97%) amino acid sequence identity with the wild-type D1 domain.

[0264] In some embodiments, the SIRPαD1 domain variant is a chimeric SIRPαD1 domain variant comprising portions of two or more wild-type D1 domains or variants thereof (e.g., a portion of one wild-type D1 domain or variant thereof and a portion of another wild-type D1 domain or variant thereof). In some embodiments, the chimeric SIRPαD1 domain variant comprises at least two portions (e.g., three, four, five or more portions) of a wild-type D1 domain or variant thereof, wherein each portion originates from a different wild-type D1 domain. In some embodiments, the chimeric SIRPαD1 domain variant further includes one or more amino acid substitutions listed in Table 5.

[0265] Table 5. Amino acid substitutions in SIRPαD1 domain variants

[0266]

[0267]

[0268]

[0269] In some embodiments, the peptide comprises a variant of the SIRPαD1 domain having the following sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSPDDVEX 16 KSGAGTELSVRAKPS (SEQ ID NO:37), wherein 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 For L, T, or G; X 11 For K or R; X 12 For N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y; X 13 For P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W or Y; X 14 For V or I; X 15 For F, L, or V; and X 16It is F or V; and said variant contains at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1.

[0270] In some embodiments of this aspect of the disclosure, the polypeptide comprises a variant of the SIRPαD1 domain having the sequence of SEQ ID NO:37, wherein X1 is L, I, or V. In some embodiments, X2 is V, L, or I. In some embodiments, X3 is A or V. In some embodiments, X4 is A, I, or L. In some embodiments, X5 is I, T, S, or F. In some embodiments, X6 is E, V, or L. In some embodiments, X7 is K or R. In some embodiments, X8 is E or Q. In some embodiments, X9 is H, P, or R. In some embodiments, X... 10 For L, T, or G. In some implementations, X 11 For K or R. In some implementations, X 12 It can be N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. In some implementations, X 13 It can be P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. In some implementations, X 14 For V or I. In some implementations, X 15 For F, L, or V. In some implementations, X 16 It can be F or V.

[0271] In some embodiments, the polypeptide provided herein contains no more than ten amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1. In some embodiments, the polypeptide provided herein contains no more than seven amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1.

[0272] In some embodiments, the peptide binds CD47 with a binding affinity of at least 10 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1. In some embodiments, the peptide binds CD47 with a binding affinity of at least 100 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1. In some embodiments, the peptide binds CD47 with a binding affinity of at least 1000 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof binds CD47 with a binding affinity of less than 1 x 10^6. -8 M, less than 5 x 10 -9 M, less than 1 x 10-9 M, less than 5x10 -10 M, less than 1 x 10 -10 M or less than 1 x 10 -11 M of K D Binding to CD47. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof is in a Kc ratio between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Combined with CD47.

[0273] In some embodiments, the peptide comprises a SIRPαD1 domain variant having the following sequence: EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSPDTEX 16 KS GAGTELSVRAKPS (SEQ ID NO:38), wherein 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 For S, T, or G; X 11 For K or R; X 12 For N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y; X 13 For P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W or Y; X 14 For V or I; X 15 For F, L, or V; and X 16 It is F or V; and said variant contains at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2.

[0274] In some embodiments of this aspect of the disclosure, the polypeptide comprises a variant of the SIRPαD1 domain having the sequence of SEQ ID NO:38, wherein X1 is L, I, or V. In some embodiments, X2 is V, L, or I. In some embodiments, X3 is A or V. In some embodiments, X4 is V, I, or L. In some embodiments, X5 is I, T, S, or F. In some embodiments, X6 is E, V, or L. In some embodiments, X7 is K or R. In some embodiments, X8 is E or Q. In some embodiments, X9 is H, P, or R. In some embodiments, X... 10 For S, T, or G. In some implementations, X 11 For K or R. In some implementations, X 12 It can be N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. In some implementations, X 13 It can be P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. In some implementations, X 14 For V or I. In some implementations, X 15 For F, L, or V. In some implementations, X 16 It can be F or V.

[0275] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having no more than ten amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2. In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having no more than seven amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2.

[0276] In some embodiments, the peptide binds CD47 with a binding affinity of at least 10 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2. In some embodiments, the peptide binds CD47 with a binding affinity of at least 100 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2. In some embodiments, the peptide binds CD47 with a binding affinity of at least 1000 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof binds CD47 with a binding affinity of less than 1 x 10^6. -8 M, less than 5 x 10 -9 M, less than 1 x 10 -9 M, less than 5x10 -10 M, less than 1 x 10 -10 M or less than 1x10-11 M of K D Binding to CD47. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof is in a Kc ratio between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Combined with CD47.

[0277] On the other hand, this disclosure is characterized by a polypeptide comprising a variant of the SIRPαD1 domain having the following sequence: EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSLX 11 PVGPIQWFRGAGPX 12 RX 13 LI YNQX 14 X 15 GX 16 FPRVTTVSX 17 X 18 TX 19 RX 20 NMDFX 21 IX 22 IX 23 X 24 ITX 25 ADAGTYYCX 26 KX 27 RKGSPDX 28 X 29 EX 30 KSGAGTELSVRX 31 KPS (SEQ ID NO:47), wherein 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 For A, V, I, or L; X 11 For I, T, S, or F; X 12 For A or G; X 13 For E, V, or L; X 14 For K or R; X 15 For E or Q; X 16 For H, P, or R; X 17 For D or E; X 18 For S, L, T or G; X 19 For K or R; X20 For E or N; X 21 For S or P; X 22 For S or R; X 23 For S or G; X 24 For any amino acid; X 25 For any amino acid; X 26 For V or I; X 27 For F, L, V; X 28 D or does not exist; X 29 For T or V; X 30 For F or V; and X 31 The variant is A or G; and the variant contains at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence SEQ ID NO: 1 or 2.

[0278] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:47, wherein X1 is E or G. In any of the above embodiments of this aspect of the present disclosure, X2 is L, I, or V. In any of the above embodiments, X3 is V, L, or I. In any of the above embodiments, X4 is S or F. In any of the above embodiments, X5 is L or S. In any of the above embodiments, X6 is S or T. In any of the above embodiments, X7 is A or V. In any of the above embodiments, X8 is I or T. In any of the above embodiments, X9 is H or R. In any of the above embodiments, X 10 It can be A, V, I, or L. In any of the above embodiments, X 11 It is I, T, S, or F. In any of the above embodiments, X 12 It is either A or G. In any of the above embodiments, X 13 For E, V, or L. In any of the above embodiments, X 14 For K or R. In any of the above embodiments, X 15 For E or Q. In any of the above embodiments, X 16 For H, P, or R. In any of the above embodiments, X 17 It is D or E. In any of the above embodiments, X 18 For S, L, T, or G. In any of the above embodiments, X 19 For K or R. In any of the above embodiments, X 20 It is E or N. In any of the above embodiments, X 21 For S or P. In any of the above embodiments, X 22 For S or R. In any of the above embodiments, X 23 For S or G. In any of the above embodiments, X 24It can be 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 above embodiments, X 25 It can be 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 above embodiments, X 26 V or I. In any of the above embodiments, X 27 For F, L, and V. In any of the above embodiments, X 28 It is either D or does not exist. In any of the above embodiments, X 29 For T or V. In any of the above embodiments, X 30 For F or V. In any of the above embodiments, X 31 It can be either A or G.

[0279] In some embodiments, the polypeptide of this aspect of the present disclosure contains no more than ten amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1 or 2. In some embodiments, the polypeptide of this aspect of the present disclosure contains no more than seven amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1 or 2.

[0280] In some embodiments, the peptide binds CD47 with a binding affinity of at least 10-fold to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the peptide binds CD47 with a binding affinity of at least 100-fold to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the peptide binds CD47 with a binding affinity of at least 1000-fold to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof binds with a binding affinity of less than 1 x 10^6 ppm. -8 M, less than 5 x 10 -9 M, less than 1x10 -9 M, less than 5 x 10 -10 M, less than 1 x 10 -10 M or less than 1 x 10 -11 M of K DBinding to CD47. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof is in a Kc ratio between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Combined with CD47.

[0281] In some embodiments, the peptide comprises a SIRPαD1 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 DX 23 EFKSGAGTELSVRAKPS (SEQ ID NO:48) or a fragment thereof, wherein 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 For K or R; X 11 For E, D, or Q; X 12 For H or P; X 13 For D or E; X 14 For S, L, or T; X 15 For N or E; X 16 For R or S; X 17 For G or S; X 18 For N or A; X 19 For V or I; X 20 For S, I, or M; X 21 P or does not exist; X 22 For D or P; and X 23 It can be V or T.

[0282] On the other hand, this disclosure is characterized by a polypeptide comprising a variant of the SIRPαD1 domain having the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9ITPADAGTYYCX 10 KFRKGSPDDVEFKSGAGTE LSVRAKPS (SEQ ID NO:49), wherein 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; and X 10 It is V or I; and the variant contains at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1.

[0283] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:49, wherein X1 is V, L, or I. In any of the above embodiments of this aspect of the present disclosure, X2 is A, I, V, or L. In any of the above embodiments, X3 is I, F, S, or T. In any of the above embodiments, X4 is E, V, or L. In any of the above embodiments, X5 is K or R. In any of the above embodiments, X6 is E or Q. In any of the above embodiments, X7 is H, P, or R. In any of the above embodiments, X8 is L, T, S, or G. In any of the above embodiments, X9 is A. In any of the above embodiments, X 10 It can be V or I.

[0284] In some embodiments, the polypeptide comprises a SIRPαD1 domain having at least 85% sequence identity with 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), wherein X1, X2, X3, X4, X5, X6, X7, X8, X9, and X 10 None of them are wild-type amino acids.

[0285] In some embodiments, the polypeptide of this aspect of the present disclosure contains no more than ten amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1. In some embodiments, the polypeptide of this aspect of the present disclosure contains no more than seven amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1.

[0286] In some embodiments, the peptide binds CD47 with a binding affinity of at least 10 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1. In some embodiments, the peptide binds CD47 with a binding affinity of at least 100 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1. In some embodiments, the peptide binds CD47 with a binding affinity of at least 1000 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof binds CD47 with a binding affinity of less than 1 x 10^6. -8 M, less than 5 x 10 -9 M, less than 1 x 10 -9 M, less than 5x10 -10 M, less than 1 x 10 -10 M or less than 1 x 10 -11 M of K D Binding to CD47. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof is in a Kc ratio between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Combined with CD47.

[0287] On the other hand, this disclosure is characterized by a polypeptide comprising a variant of the SIRPαD1 domain having the following sequence: EEELQX1IQPDKSVSVAAGESAILHCTX2TSLX3PVGPIQWFRGAGPARX4LIYNQX5X6GX7FPRVTTVSEX8TKRENMDFSISISX9ITPADAGTYYCX 10 KFRKGSPDTEFKSGAGTELS VRAKPS, (SEQ ID NO:50), wherein 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; and X 10 It is V or I; and the variant contains at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence in SEQ ID NO:2.

[0288] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:50, wherein X1 is V or I. In any of the above embodiments of this aspect of the present disclosure, X2 is V or I. In any of the above embodiments, X3 is I or F. In any of the above embodiments, X4 is E or V. In any of the above embodiments, X5 is K or R. In any of the above embodiments, X6 is E or Q. In any of the above embodiments, X7 is H or P. In any of the above embodiments, X8 is S or R. In any of the above embodiments, X9 is N or A. In any of the above embodiments, X 10 It can be V or I.

[0289] In some embodiments, the polypeptide comprises a SIRPαD1 domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with respect to SEQ ID NO:50, wherein X1, X2, X3, X4, X5, X6, X7, X8, X9, and X 10 None of them are wild-type amino acids.

[0290] In some embodiments, the polypeptide of this aspect of the present disclosure contains no more than ten amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2. In some embodiments, the polypeptide of this aspect of the present disclosure contains no more than seven amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2.

[0291] In some embodiments, the peptide binds CD47 with a binding affinity of at least 10 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2. In some embodiments, the peptide binds CD47 with a binding affinity of at least 100 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2. In some embodiments, the peptide binds CD47 with a binding affinity of at least 1000 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof binds CD47 with a binding affinity of less than 1 x 10^6. -8 M, less than 5 x 10 -9 M, less than 1 x 10 -9 M, less than 5x10 -10 M, less than 1 x 10 -10 M or less than 1 x 10 -11 M of K DBinding to CD47. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof is in a Kc ratio between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Combined with CD47.

[0292] In another aspect, this disclosure is characterized by a polypeptide comprising a variant of the SIRPαD1 domain having the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMDFSIRIGX8ITPADAGTYYCX9KFRKGSPDDVEFKSGAGTELS VRAKPS (SEQ ID NO:51), wherein X1 is V or I; X2 is A or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is H or P; X7 is L or T; X8 is N or A; and X9 is V or I; and wherein said variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1.

[0293] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:51, wherein X1 is V or I. In any of the above embodiments of this aspect of the present disclosure, X2 is A or I. In any of the above embodiments, X3 is I or F. In any of the above embodiments, X4 is E or V. In any of the above embodiments, X5 is K or R. In any of the above embodiments, X6 is H or P. In any of the above embodiments, X7 is L or T. In any of the above embodiments, X8 is N or A. In any of the above embodiments, X9 is V or I. In some embodiments, X4 is not V.

[0294] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:51, wherein X8 is A. In any of the above embodiments of this aspect of the present disclosure, X8 is A and X1 is V or I. In any of the above embodiments of this aspect of the present disclosure, X8 is A and X2 is A or I. In any of the above embodiments, X8 is A and X3 is I or F. In any of the above embodiments, X8 is A and X4 is E or V. In some embodiments, X4 is not V. In any of the above embodiments, X8 is A and X5 is K or R. In any of the above embodiments, X8 is A and X6 is H or P. In any of the above embodiments, X8 is A and X7 is A or V. In any of the above embodiments, X8 is A and X9 is V or I.

[0295] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:51, wherein X8 is A. In any of the above embodiments of this aspect of the present disclosure, X8 is A and X1 is I. In any of the above embodiments of this aspect of the present disclosure, X8 is A and X2 is I. In any of the above embodiments, X8 is A and X3 is F. In any of the above embodiments, X8 is A and X4 is V. In any of the above embodiments, X8 is A and X5 is R. In any of the above embodiments, X8 is A and X6 is P. In any of the above embodiments, X8 is A and X7 is T. In any of the above embodiments, X8 is A and X9 is I.

[0296] In some embodiments, the polypeptide comprises a SIRPαD1 domain having at least 85% sequence identity with SEQ ID NO:51 (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity), wherein each of X1, X2, X3, X4, X5, X6, X7, X8, and X9 is not a wild-type amino acid.

[0297] In some embodiments, the polypeptide of this aspect of the present disclosure contains no more than ten amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1. In some embodiments, the polypeptide of this aspect of the present disclosure contains no more than seven amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1.

[0298] In some embodiments, the peptide binds CD47 with a binding affinity of at least 10 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1. In some embodiments, the peptide binds CD47 with a binding affinity of at least 100 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1. In some embodiments, the peptide binds CD47 with a binding affinity of at least 1000 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof binds CD47 with a binding affinity of less than 1 x 10^6. -8 M, less than 5 x 10 -9 M, less than 1 x 10 -9 M, less than 5x10 -10 M, less than 1 x 10 -10 M or less than 1 x 10 -11 M of K D Binding to CD47. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof is in a Kc ratio between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Combined with CD47.

[0299] In another aspect, this disclosure is characterized by a polypeptide comprising a variant of the SIRPαD1 domain having the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRELIYNQX4EGX5FPRVTTVSDX6TKRNNMDFSIRIGX7ITPADAGTYYCVKFRKGSPDDVEFKSGAGTELS VRAKPS (SEQ ID NO:222), wherein 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; and wherein said variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1.

[0300] In some embodiments, the polypeptide comprises the sequence SEQ ID NO:222, wherein X1 is V, L, or I. In any of the above embodiments of this aspect of the present disclosure, X2 is A, I, or L. In any of the above embodiments, X3 is I, T, S, or F. In any of the above embodiments, X4 is K or R. In any of the above embodiments, X5 is H or P. In any of the above embodiments, X6 is L, T, or G. In any of the above embodiments, X7 is N or A.

[0301] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:222, wherein X1 is V or I. In any of the above embodiments of this aspect of the present disclosure, X2 is A or I. In any of the above embodiments, X3 is I or F. In any of the above embodiments, X4 is K or R. In any of the above embodiments, X5 is H or P. In any of the above embodiments, X6 is L or T. In any of the above embodiments, X7 is N or A.

[0302] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:222, wherein X7 is A. In any of the above embodiments of this aspect of the present disclosure, X7 is A and X1 is V or I. In any of the above embodiments of this aspect of the present disclosure, X7 is A and X2 is A or I. In any of the above embodiments, X7 is A and X3 is I or F. In any of the above embodiments, X7 is A and X4 is K or R. In any of the above embodiments, X7 is A and X5 is H or P. In any of the above embodiments, X7 is A and X6 is L or T.

[0303] In some embodiments, the polypeptide comprises the sequence SEQ ID NO:222, wherein X7 is A. In any of the above embodiments of this aspect of the present disclosure, X7 is A and X1 is I. In any of the above embodiments of this aspect of the present disclosure, X7 is A and X2 is I. In any of the above embodiments, X7 is A and X3 is F. In any of the above embodiments, X7 is A and X4 is R. In any of the above embodiments, X7 is A and X5 is P. In any of the above embodiments, X7 is A and X6 is T.

[0304] In some embodiments, the polypeptide comprises a SIRPαD1 domain having at least 85% sequence identity with 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), wherein each of X1, X2, X3, X4, X5, X6, and X7 is not a wild-type amino acid.

[0305] In some embodiments, the polypeptide of this aspect of the present disclosure contains no more than ten amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1. In some embodiments, the polypeptide of this aspect of the present disclosure contains no more than seven amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1.

[0306] In some embodiments, the peptide binds CD47 with a binding affinity of at least 10 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1. In some embodiments, the peptide binds CD47 with a binding affinity of at least 100 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1. In some embodiments, the peptide binds CD47 with a binding affinity of at least 1000 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1. In some embodiments, the fragment comprises a peptide 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 more than about 100 amino acids. The fragment retains its ability to bind to CD47. Preferably, the SIRPαD1 domain variant peptide or fragment thereof binds to CD47 with a higher affinity than the SIRPα peptide. For example, in some embodiments, the SIRPαD1 domain variant peptide or fragment thereof binds to CD47 with an affinity of less than 1 x 10⁻⁶. -8 M, less than 5 x 10 -9 M, less than 1 x 10 -9 M, less than 5 x 10 -10 M, less than 1 x 10 -10 M or less than 1x10 -11 M of K D Binding to CD47. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof is in a Kc ratio between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Combined with CD47.

[0307] In another aspect, this disclosure is characterized by a polypeptide comprising a variant of the SIRPαD1 domain having the following sequence: EEELQX1IQPDKSVSVAAGESAILHCTX2TSLX3PVGPIQWFRGAGPARELIYNQX4EGX5FPRVTTVSEX6TKRENMDFSISISX7ITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVR AKPS (SEQ ID NO:212), wherein 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; and wherein said variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2.

[0308] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:212, wherein X1 is V, L, or I. In any of the above embodiments of this aspect of the present disclosure, X2 is V, I, or L. In any of the above embodiments, X3 is I, T, S, or F. In any of the above embodiments, X4 is K or R. In any of the above embodiments, X5 is H or P. In any of the above embodiments, X6 is S, T, or G. In any of the above embodiments, X7 is N or A.

[0309] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:212, wherein X1 is V or I. In any of the above embodiments of this aspect of the present disclosure, X2 is V or I. In any of the above embodiments, X3 is I or F. In any of the above embodiments, X4 is K or R. In any of the above embodiments, X5 is H or P. In any of the above embodiments, X6 is S or T. In any of the above embodiments, X7 is N or A.

[0310] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:212, wherein X7 is A. In any of the above embodiments of this aspect of the present disclosure, X7 is A and X1 is V or I. In any of the above embodiments of this aspect of the present disclosure, X7 is A and X2 is V or I. In any of the above embodiments, X7 is A and X3 is I or F. In any of the above embodiments, X7 is A and X4 is K or R. In any of the above embodiments, X7 is A and X5 is H or P. In any of the above embodiments, X7 is A and X6 is S or T.

[0311] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:212, wherein X7 is A. In any of the above embodiments of this aspect of the present disclosure, X7 is A and X1 is I. In any of the above embodiments of this aspect of the present disclosure, X7 is A and X2 is I. In any of the above embodiments, X7 is A and X3 is F. In any of the above embodiments, X7 is A and X4 is R. In any of the above embodiments, X7 is A and X5 is P. In any of the above embodiments, X7 is A and X6 is T.

[0312] In some embodiments, the polypeptide comprises a SIRPαD1 domain having at least 85% sequence identity with 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), wherein each of X1, X2, X3, X4, X5, X6, and X7 is not a wild-type amino acid.

[0313] In some embodiments, the polypeptide of this aspect of the present disclosure contains no more than ten amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2. In some embodiments, the polypeptide of this aspect of the present disclosure contains no more than seven amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2.

[0314] In some embodiments, the peptide binds CD47 with a binding affinity of at least 10 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2. In some embodiments, the peptide binds CD47 with a binding affinity of at least 100 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2. In some embodiments, the peptide binds CD47 with a binding affinity of at least 1000 times that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2. In some embodiments, the fragment comprises a peptide 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 more than about 100 amino acids. The fragment retains its ability to bind to CD47. Preferably, the SIRPαD1 domain variant peptide or fragment thereof binds to CD47 with a higher affinity than the SIRPα peptide. For example, in some embodiments, the SIRPαD1 domain variant peptide or fragment thereof binds to CD47 with an affinity of less than 1 x 10⁻⁶. -8 M, less than 5 x 10-9 M, less than 1 x 10 -9 M, less than 5 x 10 -10 M, less than 1 x 10 -10 M or less than 1x10 -11 M of K D Binding to CD47. In some embodiments, the SIRPαD1 domain variant peptide or a fragment thereof is in a Kc ratio between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Combined with CD47.

[0315] In some embodiments, this document describes peptides comprising variants of the SIRPαD1 domain according to the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9X 10 X 11 X 12 ADAGTYYCX 13 KFRKGSPDDVEFKSG AGTELSVRAKPS (SEQ ID NO:218), wherein 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 For any amino acid; X 11 For any amino acid; X 12 For any amino acid; and X 13 It is V or I; and the SIRPαD1 domain variant therein contains at least two amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence according to SEQ ID NO:1.

[0316] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:212, where X1 is A and X9 is A. In any of the above embodiments of this aspect of the present disclosure, X9 is N. In any of the above embodiments of this aspect of the present disclosure, X 10X9 is N and X10 is P. In any of the above embodiments of this aspect of the present disclosure, X9 is N and X11 is any amino acid other than S, T, or C. In any of the above embodiments of this aspect of the present disclosure, X... 11 For T. In any of the above embodiments of this aspect of the present disclosure, X 11 It is an amino acid other than T. In any of the above embodiments of this aspect of the present disclosure, X 12 For P. In any of the above embodiments of this aspect of the present disclosure, X9 is N and X 12 It can be any amino acid except P.

[0317] In some embodiments, this document describes polypeptides comprising variants of the SIRPαD1 domain according to the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9ITX 10 ADAGTYYCX 11 KFRKGSPDDVEFKSGAG TELSVRAKPS (SEQ ID NO:219), wherein 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 It is any amino acid except P; and X 11 It is V or I; and the SIRPαD1 domain variant therein contains at least two amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence according to SEQ ID NO:1.

[0318] In another aspect of this disclosure, compositions comprising a SIRPαD1 domain variant polypeptide or a fragment thereof having the amino acid sequence SEQ ID NO:48 are disclosed. In some embodiments, the SIRPαD1 domain variant polypeptide or a fragment thereof binds to CD47 with a higher affinity than the SIRPα polypeptide. In some embodiments, the SIRPαD1 domain variant polypeptide binds to CD47 with an affinity of less than 1 x 10⁻⁶. -8 M, or less than 1 x 10 -9 M, less than 1 x 10 -10 M or less than 1x10 -11 M of K DBinding to CD47. In some embodiments, the aforementioned SIRPαD1 domain variant peptide is attached to or fused with a second peptide. In some embodiments, the second peptide includes, but is not limited to, an Fc peptide, an Fc variant, or the aforementioned fragment.

[0319] Without limiting the foregoing, in some embodiments, the SIRPαD1 domain variant peptide is selected from any one of SEQ ID NO:53-87 and 213 shown in Table 6.

[0320] Table 6. SIRPα variant peptides

[0321]

[0322]

[0323]

[0324]

[0325] In some embodiments, the polypeptide comprises a SIRPαD1 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) with any of the variants provided in Table 6.

[0326] In some embodiments, the polypeptide comprises a SIRPαD1 domain having at least 85% sequence identity with SEQ ID NO:80, 81 or 85 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).

[0327] Fc domain variants and fusion peptides containing them

[0328] In some embodiments, this document discloses polypeptides comprising a variant of the signal regulatory protein α (SIRP-α)D1, the variant comprising a SIRPαD1 domain or a fragment thereof, the SIRPαD1 domain having an amino acid mutation at residue 80 relative to the wild-type SIRPαD1 domain (e.g., the wild-type SIRPαD1 domain described in SEQ ID NO:1 or 2); and having at least one additional amino acid mutation at residues selected from the group consisting of the wild-type SIRPαD1 domain (e.g., the wild-type SIRPαD1 domain described in SEQ ID NO:1 or 2): residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92.

[0329] In some embodiments, this document also discloses an Fc domain variant dimer comprising two Fc domain variants, each Fc domain variant being independently selected from (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region containing mutations S228P, E233P, F234V, L235A, delG236, and N297A.

[0330] Antibodies targeting cell surface antigens can trigger immunostimulatory and effector functions associated with binding to Fc receptors (FcRs) on immune cells. Many Fc receptors exist that are specific to certain classes of antibodies, including IgG (γ receptor), IgE (η receptor), IgA (α receptor), and IgM (μ receptor). Binding of the Fc region to Fc receptors on the cell surface can trigger numerous biological responses, including phagocytosis of antibody-coated particles (antibody-dependent cell-mediated phagocytosis or ADCP), clearance of immune complexes, cytotoxic cell lysis of antibody-coated cells (antibody-dependent cell-mediated cytotoxicity or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. Additionally, the binding of the C1 component of complement to antibodies can activate the complement system. Activation of complement is important for the lysis of cellular pathogens. However, complement activation can also stimulate inflammatory responses and may be involved in autoimmune hypersensitivity reactions or other immune disorders. Variant Fc regions with the ability to reduce or eliminate binding to certain Fc receptors can be used to develop therapeutic antibodies and Fc-fusion peptide constructs that function by targeting, activating or neutralizing ligand function without damaging or destroying local cells or tissues.

[0331] In some embodiments, the SIRPαD1 peptide construct comprises a non-naturally occurring SIRPαD1 domain variant linked to an Fc domain variant that forms an Fc domain having eliminated or reduced effector functions.

[0332] In some embodiments, the Fc domain variant refers to a polypeptide chain that includes 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 any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, and IgD. Additionally, in some embodiments, the Fc domain variant is any IgG isotype (e.g., IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, and IgG4). In some embodiments, the Fc domain variant contains up to ten amino acid modifications (e.g., insertions, deletions, and / or substitutions) relative to the wild-type Fc domain monomer sequence (e.g., 1-10, 1-8, 1-6, 1-4 amino acid substitutions, additions, or insertions, deletions, or combinations thereof) that alter the interaction between the Fc domain and the Fc receptor.

[0333] As used herein, the term "Fc domain dimer" refers to a dimer of two Fc domains. In a wild-type Fc domain dimer, the two wild-type Fc domains dimerize through interactions between the two CH3 antibody constant domains and through one or more disulfide bonds formed between the hinge domains of the two dimerized Fc domains.

[0334] 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 mutated to lack effector function, such as a "dead Fc domain dimer variant". In some embodiments, each Fc domain 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 Fc receptors such as Fcγ receptor (FcγR), Fcα receptor (FcαR), or Fcε (FcεR)).

[0335] In some embodiments, a SIRPαD1 domain variant (e.g., any of the variants described in Tables 2, 5, and 6) is fused to an Fc domain variant of an immunoglobulin or a fragment of an Fc domain variant. In some embodiments, an Fc domain variant of an immunoglobulin or a fragment of an Fc domain variant is capable of forming an Fc domain dimer with another Fc domain variant. In some embodiments, an Fc domain variant of an immunoglobulin or a fragment of an Fc domain variant is not capable of forming an Fc domain dimer with another Fc domain variant. In some embodiments, an Fc domain variant or a fragment of an Fc domain variant is fused to a peptide of the present disclosure to increase the serum half-life of the peptide. In some embodiments, an Fc domain variant or a fragment of an Fc domain variant fused to a peptide of the present disclosure dimers with a second Fc domain variant to form an Fc domain dimer variant that binds to an Fc receptor; alternatively, the Fc domain variant binds to an Fc receptor. In some implementations, Fc domain variants or fragments of Fc domain variants fused to the peptide to increase the peptide's serum half-life do not induce any immune system-related responses.

[0336] In some embodiments, the SIRPα peptide or construct provided herein comprises a SIRPαD1 domain or a variant thereof linked to a first Fc domain variant and an antibody-variable domain linked to a second Fc domain variant, wherein the first and second Fc domain variants are combined to form an Fc domain dimer variant (e.g., a heterodimeric Fc domain dimer variant). Fc domain dimers are protein structures found at the C-terminus of immunoglobulins. An Fc domain dimer comprises two Fc domains dimerized by an interaction between constant antibody domains at CH3. Wild-type Fc domain dimers form minimal structures that bind Fc receptors such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and FcγRIV.

[0337] Fc domain dimers do not directly participate in antibody binding to their targets, but can participate in various effector functions, such as antibody-dependent cytotoxicity. In some embodiments, the Fc domain in the SIRPα peptide or construct of this disclosure comprises 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 cytolysis (CDC), reduced antibody-dependent cell-mediated phagocytosis (ADCP), or any combination thereof. In some embodiments, the SIRPα peptide or construct of this disclosure is characterized by reduced binding to the human Fc receptor (e.g., minimal binding or no binding) and reduced binding to complement protein C1q (e.g., minimal binding or no binding). In some embodiments, the SIRPα construct of this disclosure is characterized by reduced binding to human FcγRI, FcγRIIA, FcγRIIB, FcγRIIIB, or any combination thereof and C1q (e.g., minimal binding or no binding). To alter or reduce the function of antibody-dependent effectors, such as ADCC, CDC, ADCP, or any combination thereof, in some embodiments, the Fc domain in the SIRPα construct of this disclosure is of the IgG class and contains one or more amino acid substitutions at E233, L234, L235, G236, G237, D265, D270, N297, E318, K320, K322, A327, A330, P331, or P329 (according to Kabat's EU index number (Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991))).

[0338] In some embodiments, peptide constructs containing the non-natural Fc region described herein exhibit reduced or eliminated binding to at least one of the Fcγ receptors CD16a, CD32a, CD32b, CD32c, and CD64, compared to peptide constructs containing the natural Fc region. In some cases, the peptide constructs described herein exhibit reduced or eliminated binding to the CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors.

[0339] CDC refers to a form of cytotoxicity in which the complement cascade is activated by the binding of complement component C1q to the antibody Fc domain. In some embodiments, peptide constructs containing the non-natural Fc region described herein exhibit at least a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater reduction in C1q binding compared to peptide constructs containing the wild-type Fc region. In some cases, peptide constructs containing the non-natural Fc region described herein exhibit reduced CDC compared to peptide constructs containing the wild-type Fc region. In some embodiments, peptide constructs containing the non-natural Fc region described herein exhibit at least a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater reduction in CDC compared to peptide constructs containing the wild-type Fc region. In some cases, peptide constructs containing non-natural Fc domain variants or Fc domain dimer variants as described herein exhibit negligible CDC compared to peptide constructs containing wild-type Fc regions.

[0340] In some embodiments, the Fc domain variants or Fc domain dimer variants described herein are minimally glycosylated or have reduced glycosylation relative to the wild-type sequence. In some embodiments, deglycosylation is achieved by mutating N297A or by mutating N297 to any amino acid other than N. In some embodiments, deglycosylation is achieved by disrupting the motif N-Xaa1-Xaa2-Xaa3, where N = asparagine; Xaa1 = any amino acid other than P (proline); Xaa2 = T (threonine), S (serine), or C (cysteine); and Xaa3 = any amino acid other than P (proline). In one embodiment, the N-Xaa1-Xaa2-Xaa3 motif refers to residues 297-300 as specified by Kabat et al., 1991. In some implementations, mutations in any one or more of N, Xaa1, Xaa2, or Xaa3 result in deglycosylation of Fc domain variants or Fc domain dimer variants.

[0341] In some embodiments, variants of the constant region of antibody IgG (e.g., Fc domain variants or Fc domain dimer variants) have reduced ability to specifically bind to the Fcγ receptor or reduced ability to induce phagocytosis. In some embodiments, variants of the constant region of antibody IgG (e.g., Fc domain variants or Fc domain dimer variants) have both 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 to lack effector function, typically a "dead" Fc domain variant. For example, in some embodiments, the Fc domain variant includes specific amino acid substitutions known to minimize the interaction between the Fc domain dimer and the Fcγ receptor. In some embodiments, the Fc domain variant is derived from an IgG1 antibody and includes one or more of the amino acid substitutions L234A, L235A, G237A, and N297A (as designated according to the EU numbering system of Kabat et al., 1991). In some embodiments, such IgG1 Fc domain variants contain one or more additional mutations. Non-limiting examples of such mutations in 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 wild-type human IgG1 sequences. In some embodiments, such IgG1 Fc domain variants include one or more additional deletions. For example, in some embodiments, the C-terminal lysine of the Fc domain IgG1 heavy chain constant region provided in SEQ ID NO:88 of Table 7 is deleted to increase the homogeneity of the peptide, for example, when the peptide 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 wild-type human IgG1 sequences (see, for example, SEQ ID NO:161 below). In some implementations, the IgG1 Fc domain variant has a sequence according to any one of SEQ ID NO:135, SEQ ID NO:136 or SEQ ID NO:137.

[0342] SEQ ID NO:161:

[0343] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0344] In some embodiments, the Fc domain variant is derived from an IgG2 or IgG4 antibody and includes amino acid substitutions of A330S, P331S, or both A330S and P331S. The aforementioned amino acid positions are defined according to Kabat et al. (1991). For a given antibody, the Kabat number of the amino acid residues can be determined by comparing homologous regions of the antibody sequence with “standard” Kabat numbered sequences. In some embodiments, the Fc domain variant comprises a human IgG2 Fc domain sequence containing one or more of the amino acid substitutions A330S, P331S, and N297A (as specified in the EU numbering system of 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 in human IgG2 Fc domain variants include V234A, G237A, P238S, V309L, and H268A (as specified in the EU numbering system of 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, 3, or fewer mutations compared to wild-type human IgG2 sequences. In some embodiments, such IgG2 Fc domain variants include one or more additional deletions. For example, in some embodiments, the C-terminal lysine residue of the IgG2 heavy chain constant region of the Fc domain provided in SEQ ID NO:89 of Table 7 is deleted to increase the homogeneity of the peptide, for example, when the peptide 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 wild-type human IgG2 sequences (see, for example, SEQ ID NO:162 below).

[0345] SEQ ID NO:162:

[0346] ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPI EKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0347] 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 mutation 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 amino acid substitutions of S228P, E233P, F234V, L235A, and delG236 (as specified according to the EU numbering system of Kabat et al. (1991)). In some embodiments, the Fc domain variant contains a human IgG4 Fc sequence containing one or more of the amino acid substitutions of S228P, E233P, F234V, L235A, delG236, and N297A (as specified according to the EU numbering system of Kabat et al. (1991)).

[0348] In some embodiments, the Fc domain variant comprises at least one of the mutations L234A, L235A, G237A, or N297A in the IgG1 Fc region, or at least one of the mutations A330S, P331S, or N297A in the IgG2 Fc region. In some embodiments, the Fc domain variant comprises at least two of the mutations L234A, L235A, G237A, or N297A in the IgG1 Fc region, or at least two of the mutations A330S, P331S, or N297A in the IgG2 Fc region. In some embodiments, the Fc domain variant comprises at least three of the mutations L234A, L235A, G237A, or N297A in the IgG1 Fc region, or is composed of mutations A330S, P331S, and N297A in the IgG2 Fc region. In some embodiments, the Fc domain variant is composed of mutations L234A, L235A, G237A, and N297A.

[0349] In some embodiments, the Fc domain variant exhibits reduced binding to the subject's Fc receptor compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain variant exhibits eliminated binding to the subject's 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 eliminated phagocytosis compared to the wild-type human IgG Fc region.

[0350] SEQ ID NO:88 and SEQ ID NO:89 provide the amino acid sequences of the constant regions of the Fc domain IgG1 and IgG2 heavy chains. In some embodiments, the Fc domain variants are any variants of SEQ ID NO:90-95 as shown in Table 7.

[0351] Table 7. Amino acid sequences of Fc domain variants

[0352]

[0353]

[0354] Antibody-dependent cell-mediated cytotoxicity (also referred to herein as ADCC) refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells and neutrophils), enabling these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill the target cells. Antibody-dependent cell-mediated phagocytosis (also referred herein as ADCP) refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain phagocytes (e.g., macrophages), enabling these phagogenic effector cells to specifically bind to target cells carrying antigens and subsequently phagocytose and digest the target cells. Ligand-specific high-affinity IgG antibodies against the surface of target cells can stimulate cytotoxic or phagocytes and can be used for this killing. In some embodiments, peptide constructs containing Fc domain variants or Fc domain dimer variants as described herein exhibit reduced ADCC or ADCP compared to peptide constructs containing wild-type Fc regions. In some embodiments, peptide 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 greater reductions in ADCC or ADCP compared to peptide constructs containing a wild-type Fc region. In some embodiments, peptide constructs containing the Fc domain variants or Fc domain dimer variants described herein exhibit eliminated ADCC or ADCP compared to peptide constructs containing a wild-type Fc region.

[0355] Complement-directed cytotoxicity (also referred to herein as CDC) refers to a form of cytotoxicity in which the complement cascade is activated by the binding of complement component C1q to the antibody Fc domain. In some embodiments, peptide constructs containing the Fc domain variants or Fc domain dimer variants described herein exhibit at least a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater reduction in C1q binding compared to peptide constructs containing the wild-type Fc region. In some cases, peptide constructs containing the Fc domain variants or Fc domain dimer variants described herein exhibit reduced CDC compared to peptide constructs containing the wild-type Fc region. In some embodiments, peptide constructs containing the Fc domain variants or Fc domain dimer variants described herein exhibit a reduction in CDC of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater compared to peptide constructs containing a wild-type Fc region. In some cases, peptide constructs containing the Fc domain variants or Fc domain dimer variants described herein exhibit negligible CDC compared to peptide constructs containing a wild-type Fc region.

[0356] The Fc domain variants or Fc domain dimer variants described herein include those that exhibit reduced binding to the Fcγ receptor compared to the wild-type human IgG Fc region. For example, in some embodiments, the Fc domain variants or Fc domain dimer variants exhibit less binding to the Fcγ receptor compared to the binding exhibited by the wild-type human IgG Fc region, as described in the examples. In some cases, the binding of the Fc domain variants or Fc domain dimer variants to the Fcγ receptor is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (complete elimination of effector function). In some embodiments, the reduced binding is against any one or more Fcγ receptors, such as CD16a, CD32a, CD32b, CD32c, or CD64.

[0357] In some cases, the Fc domain variants or Fc domain dimer variants disclosed herein exhibit reduced phagocytosis compared to the wild-type human IgG Fc region. These Fc domain variants or Fc domain dimer variants exhibit reduced phagocytosis compared to the wild-type human IgG Fc region, with phagocytic activity reduced by, for example, 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 eliminated phagocytosis compared to the wild-type human IgG Fc region.

[0358] In some embodiments, the Fc domain variants or Fc domain dimer variants disclosed herein are coupled to one or more fusion couplers. In some cases, the fusion coupler is a therapeutic portion. In some cases, the fusion coupler is selected to enable targeting, purification, screening, display, etc., of the expressed protein. In some embodiments, the fusion coupler also affects the degree of binding to the Fc receptor or the degree of reduction in phagocytosis. As described herein, in some embodiments, when the Fc domain variant or Fc domain dimer variant is coupled to a fusion coupler, it forms a peptide construct as described below.

[0359] In some embodiments, the fusion partner is linked to an Fc domain variant or an Fc domain dimer variant sequence via a linker sequence. In some embodiments, the linker sequence typically contains a small number of amino acids, such as fewer than ten amino acids, although longer linkers are also used. In some cases, the linker has a length of less than 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.

[0360] In some embodiments, a fusion coupler is a targeting or signaling sequence that directs an Fc domain variant or Fc domain dimer variant protein and any associated fusion coupler to a desired cellular location or extracellular medium. In some embodiments, certain signaling sequences target proteins secreted into growth media or located in the periplasmic space between the inner and outer cell membranes. In some embodiments, a fusion coupler is a sequence encoding a peptide or protein capable of purification or screening. Such fusion couplers 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 immobilized metal affinity chromatography (IMAC) systems (e.g., Ni+2 affinity columns), GST fusions, MBP fusions, Strep-tags, BSP biotinylated target sequences of the bacterial enzyme BirA, and antibody-targeted epitope tags (e.g., c-myc tags, flag-tags, etc.).

[0361] In some embodiments, such tags can be used for purification, screening, or both. For example, in some embodiments, Fc domain variants or Fc domain dimer variants are purified using a His-tag by immobilizing them onto a Ni+2 affinity column, and then, after purification, the same His-tag is used to immobilize the antibody onto a Ni+2-coated plate for ELISA or other binding assays, as described elsewhere herein. In some embodiments, fusion couplers enable the use of selection methods to screen for Fc domain variants or Fc domain dimer variants as described herein.

[0362] Various fusion couplers capable of enabling multiple selection methods can be used. For example, phage display can be used by fusing members of a library of Fc domain variants or Fc domain dimer variants with a gene III protein. In some embodiments, the fusion coupler Fc domain variant or Fc domain dimer variant is tagged. Alternatively, in some embodiments, the fusion coupler binds to a specific sequence on an expression vector, enabling the fusion coupler and the associated Fc domain variant or Fc domain dimer variant to be covalently or non-covalently linked to the nucleic acid encoding them.

[0363] In some embodiments, when the fusion partner is a therapeutic portion, the therapeutic portion is, for example, a peptide, protein, antibody, siRNA, or small molecule. Non-limiting examples of therapeutic antibodies conjugated to Fc domain variants or Fc domain dimer variants of this disclosure include, but are not limited to, antibodies that recognize CD47. Non-limiting examples of therapeutic peptides conjugated to Fc domain variants or Fc domain dimer variants of this disclosure include, but are not limited to, CD47-binding peptides, including SIRPα peptides. In such cases, the CD47-binding peptide is attached to or fused to an Fc domain variant or Fc domain dimer variant of this disclosure. Examples of CD47-binding peptides include, but are not limited to, anti-CD47 antibodies or fragments thereof, and ligands of CD47 such as SIRPα or fragments thereof. Other examples of CD47-binding peptides include, but are not limited to, naturally occurring forms of SIRPα and its mutants.

[0364] In some embodiments, this document discloses peptides comprising Fc domain dimer variants, wherein the Fc domain dimer variant comprises two Fc domain variants, each Fc domain variant being independently selected from (i) a human IgG1 Fc region composed of L234A, L235A, G237A, and N297A mutations; (ii) a human IgG2 Fc region composed of A330S, P331S, and N297A mutations; or (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations. 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 Fc domain variant in the Fc domain dimer is a human IgG1 Fc region composed of mutants L234A, L235A, G237A, and N297A. In some embodiments, at least one Fc domain variant in the Fc domain dimer is a human IgG2 Fc region composed of mutants A330S, P331S, and N297A. In some embodiments, the Fc domain dimer variant exhibits eliminated or reduced binding to the Fcγ receptor compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain dimer variant exhibits eliminated or reduced binding to the CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain dimer variant exhibits eliminated or reduced binding to C1q compared to the wild-type human IgG Fc fusion. In some embodiments, at least one Fc domain variant of the Fc domain dimer is a human IgG4 Fc region comprising the mutants S228P, E233P, F234V, L235A, delG236, and N297A. In some embodiments, the Fc domain dimer variants exhibit eliminated or reduced binding to the Fcγ receptor compared to the wild-type human IgG4 Fc region. In some embodiments, the Fc domain dimer variants exhibit eliminated or reduced binding to the CD16a and CD32b Fcγ receptors compared to the wild-type human IgG4 Fc region. In some embodiments, the Fc domain dimer variants have a binding density greater than about 5 x 10⁻⁶. -6 M of K D Binds to Fcγ receptor.

[0365] In some embodiments, the Fc domain dimer variant also comprises a CD47-binding peptide. In some embodiments, the Fc domain dimer variant exhibits eliminated or reduced binding to the Fcγ receptor compared to the wild-type human IgG Fc region. In some embodiments, the CD47-binding peptide does not induce acute anemia in rodents and non-human primates. In some embodiments, the CD47-binding peptide does not induce acute anemia in humans.

[0366] In some embodiments, the CD47-binding polypeptide is a signal regulatory protein α (SIRP-α) polypeptide or a fragment thereof. In some embodiments, the SIRPα polypeptide comprises a SIRPαD1 domain variant containing the following amino acid sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMDFSIRIGX8ITPADAGTYYCX9KFRKGSPDDVEFKSGAGTELS VRAKPS (SEQ ID NO: 221), wherein X1 is V or I; X2 is A or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is H or P; X7 is L or T; X8 is any amino acid except N; and X9 is V or I. In some embodiments, the SIRPα polypeptide comprises a variant of the SIRPαD1 domain, wherein X1 is V or I; X2 is A or I; X3 is I or F; X4 is E; X5 is K or R; X6 is H or P; X7 is L or T; X8 is not N; and X9 is V.

[0367] In some embodiments, this document discloses a polypeptide comprising: a SIRPαD1 domain variant, wherein the SIRPαD1 domain variant is a non-naturally occurring high-affinity SIRPαD1 domain, wherein the SIRPαD1 domain variant binds to human CD47 with an affinity at least 10 times that of the naturally occurring D1 domain; and an Fc domain variant, wherein the Fc domain variant is linked to a second polypeptide comprising a second Fc domain variant to form an Fc domain dimer variant, wherein the Fc domain dimer variant has eliminated or reduced effector function. In some embodiments, the non-naturally occurring high-affinity SIRPαD1 domain contains an amino acid mutation at residue 80.

[0368] In some embodiments, this document discloses a SIRPαD1 domain variant, wherein the SIRPαD1 domain variant has a K0 value of less than 250 nM. D Combining CD47 from the first species; and wherein the SIRPαD1 domain variant has a K0 of less than 250 nM. DCombined with CD47 from the second species; and K from CD47 of the first species. D and K from CD47 of the second species D Within 100 times each other; wherein the first and second species are selected from the group consisting of: humans, rodents, and non-human primates. In some embodiments, the SIRPαD1 domain variant binds to CD47 from at least three different species. In some embodiments, the non-human primate is a cynomolgus monkey.

[0369] In some embodiments, this document discloses a polypeptide comprising (a) K at less than 250 nM. D The polypeptide binds to the SIRPαD1 domain of human CD47; and (b) an Fc domain or a variant thereof linked to the N-terminus or C-terminus of the SIRPαD1 domain, wherein the polypeptide does not cause acute anemia in rodents and non-human primates. In some embodiments, the polypeptide is a non-naturally occurring variant of human SIRP-α. In some embodiments, in vivo administration of the polypeptide results in a decrease in hemoglobin of less than 50% within the first week following administration. In some embodiments, in vivo administration of the polypeptide in humans results in a decrease in hemoglobin of less than 50% within the first week following administration. In some embodiments, the peptide 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 composed of L234A, L235A, G237A, and N297A mutations; (ii) a human IgG2 Fc region composed of A330S, P331S, and N297A mutations; or (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations. In some embodiments, the Fc domain variant is a human IgG1 Fc region composed of mutated L234A, L235A, G237A, and N297A. In some embodiments, the Fc domain variant is a human IgG2 Fc region composed of mutated A330S, P331S, and N297A.

[0370] The SIRPα construct of this disclosure includes a SIRPα domain or a variant thereof, the C-terminus of which is attached 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 peptide and the Fc domain or a variant thereof. In some embodiments, the peptide of this disclosure comprising a variant of the SIRPαD1 domain is fused with an Fc domain variant that cannot form a dimer. In some embodiments, the peptide of this disclosure is fused with an Fc domain or a variant thereof that is capable of forming a dimer with another Fc domain or a variant thereof, such as a heterodimer. In some embodiments, the peptide of this invention is fused with an Fc domain or a variant thereof, and the fusion protein forms a homodimer. In some embodiments, the peptide of this disclosure is fused with a first Fc domain or a variant thereof, and a different protein or peptide (e.g., an antibody variable region) is fused with a second Fc domain or a variant thereof. In some embodiments, the SIRPαD1 domain or a variant thereof is linked to a first Fc domain or a variant thereof, and the therapeutic protein (e.g., a cytokine, interleukin, antigen, steroid, anti-inflammatory agent, or immunomodulator) is linked to a second Fc domain or a variant thereof. In some embodiments, the first and second Fc domains or variants thereof form a heterodimer.

[0371] Without limiting the foregoing, in some embodiments, a SIRPαD1 domain variant peptide (e.g., any of the variants described in Tables 2, 5, and 6) is fused with an Fc peptide or an Fc variant peptide (such as an Fc domain or a variant thereof). Examples of peptides comprising a SIRPαD1 domain variant peptide and a fused Fc domain variant peptide include, but are not limited to, SEQ ID NOs: 96-137, 214, and 216 shown in Table 8.

[0372] Table 8. Peptides containing SIRPαD1 domain variants fused with Fc domain variants

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381] In some embodiments, the polypeptide comprises a SIRPαD1 variant domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with any variant provided in Table 8.

[0382] In some embodiments, the polypeptide comprises a variant of the SIRPαD1 domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with SEQ ID NO: 98-104, 107-113, 116-122, or 135-137 in Table 8.

[0383] In some embodiments, the peptide comprises (a) a SIRP-α D1 variant, wherein the SIRP-α D1 domain variant comprises the amino acid sequence, EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSLX 11 PVGPIQWFRGAGPX 12 RX 13 LI YNQX 14 X 15 GX 16 FPRVTTVSX 17 X 18 TX 19 RX 20 NMDFX 21 IX 22 IX 23 X 24 ITX 25 ADAGTYYCX 26 KX 27 RKGSPDX 28 X 29 EX 30 KSGAGTELSVRX 31 KPS (SEQ ID NO:47), wherein 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 For A, V, I, or L; X 11 For I, T, S, or F; X 12 For A or G; X 13For E, V, or L; X 14 For K or R; X 15 For E or Q; X 16 For H, P, or R; X 17 For D or E; X 18 For S, L, T or G; X 19 For K or R; X 20 For E or N; X 21 For S or P; X 22 For S or R; X 23 For S or G; X 24 For any amino acid; X 25 For any amino acid; X 26 For V or I; X 27 For F, L, or V; X 28 D or does not exist; X 29 For T or V; X 30 For F or V; and X 31 The SIRPαD1 domain variant is A or G; and the SIRPαD1 domain variant contains at least two amino acid substitutions relative to the wild-type SIRPαD1 domain having a sequence according to any one of SEQ ID NO: 1 to 10; and (b) an Fc domain dimer variant having two Fc domain variants, wherein each Fc domain variant is independently (i) a human IgG1 Fc region containing the N297A mutation; (ii) a human IgG1 Fc region containing the L234A, L235A, and G237A mutations; (iii) a human IgG1 Fc region containing the L234A, L235A, G237A, and N297A mutations; (iv) a human IgG2 Fc region containing the N297A mutation; (v) a human IgG2 Fc region containing the A330S and P331S mutations; (vi) a human IgG2 Fc region containing the A330S, P331S, and N297A mutations. Fc region; (vii) human IgG4 Fc region containing S228P, E233P, F234V, L235A and delG236 mutations; or (viii) human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236 and N297A mutations.

[0384] In some embodiments, the peptide comprises a SIRPαD1 domain variant, wherein the SIRPαD1 domain variant comprises an amino acid sequence according to SEQ ID NO:47; and an Fc domain dimer having two Fc domains, wherein one of the Fc domains is an Fc domain variant comprising a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations.

[0385] Dimerization of Fc domains

[0386] In some embodiments, a SIRPαD1 domain variant polypeptide (e.g., any of the variants described in Tables 2, 5, and 6) is fused to a first Fc domain (e.g., an Fc domain variant) at its N-terminus or C-terminus. In some embodiments, the first Fc domain is a non-dimerizing variant. In some embodiments, the first Fc domain dimers 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.

[0387] 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, for example, from a first subunit comprising a SIRPαD1 domain variant polypeptide fused to a first Fc domain and a second subunit comprising a second Fc domain (e.g., without a SIRPαD1 domain variant polypeptide or any other polypeptide). In some embodiments, the construct has a single SIRPαD1 domain variant polypeptide (e.g., a single arm) linked to the Fc domain dimer. In some embodiments, the construct has two SIRPαD1 domain variant polypeptides (e.g., two arms) linked to the Fc domain dimer. In some embodiments, K D A variant of the SIRPαD1 domain, approximately 500 nM in size, is particularly suitable for two-arm constructs. In some embodiments, K... D A variant of the SIRPαD1 domain, approximately 50 nM in size, is particularly suitable for two-arm constructs. In some embodiments, K... D A variant of the SIRPαD1 domain of approximately 5 nM can be used in both two-arm and one-arm constructs. In some implementations, K D A variant of the SIRPαD1 domain, approximately 500 pM in size, can be used in both two-arm and one-arm constructs. In some implementations, K... D A variant of the SIRPαD1 domain, approximately 100 pM in size, can be used in both two-arm and one-arm constructs. In some implementations, K... D A variant of the SIRPαD1 domain, approximately 50 pM in size, can be used in both two-arm and one-arm constructs. In some implementations, K... D A SIRPαD1 domain variant of approximately 10 pM can be used for both two-arm and one-arm constructs.

[0388] In some embodiments, heterodimerization of the Fc domains is promoted by introducing different but compatible substitutions, such as "knob-into-hole" residue pairs and charge residue pairs, into the two Fc domains. Knob and hole interactions favor heterodimer formation, while knob-knob and hole-hole interactions hinder homodimer formation due to steric conflict and the absence of favorable interactions. A hole is a gap created when a native amino acid in a protein is replaced by a different amino acid with a smaller side chain volume. A hole is a protrusion created when a native 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 in the CH3 antibody constant domain of the Fc domain and participates in the dimerization of the two Fc domains. In some embodiments, a hole is created in one CH3 antibody constant domain to accommodate a hole in another CH3 antibody constant domain, such that the hole and hole amino acids promote or favor the heterodimerization of the two Fc domains. In some embodiments, a mortise is generated in one CH3 antibody constant domain to better accommodate the original amino acid in the other CH3 antibody constant domain. In some embodiments, a mortise is generated in one CH3 antibody constant domain to form additional interactions with the original amino acid in the other CH3 antibody constant domain.

[0389] In some embodiments, a mortar is 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 constant domain of a CH3 antibody. Similarly, in some embodiments, a pestle is constructed by replacing an amino acid with a smaller side chain, such as the T366W mutation in the constant domain of a CH3 antibody, with an amino acid with a larger side chain. In some embodiments, one Fc domain contains the pestle mutation T366W, while another Fc domain contains the mortar mutations T366S, L358A, and Y407V. In some embodiments, a polypeptide of this disclosure containing a variant of the SIRPαD1 domain is fused with an Fc domain containing the pestle mutation T366W to limit unwanted pestle-pepper homodimer formation. Examples of pestle-mortar amino acid pairs include, but are not limited to, those in Table 9, and Table 10 provides examples of pestle-mortar Fc domain variants and SIRPα-Fc fusions.

[0390] Table 9. Amino acid pairs in mortar and pestle

[0391]

[0392] Table 10. Exemplary Fc domain variants and SIRPαD1 domain variant-Fc domain variant fusion peptides

[0393]

[0394]

[0395] In addition to the mortar and pestle strategy, some embodiments also employ electrostatic reversal to control the dimerization of the Fc domain. Electrostatic reversal refers to the use of favorable electrostatic interactions between peptides, protein domains, and oppositely charged amino acids in a protein to control the formation of more ordered protein molecules. Specifically, to use electrostatic reversal to control the dimerization of the Fc domain, one or more amino acid residues constituting the CH3-CH3 interface are replaced with positively or negatively charged amino acid residues, making the interaction electrostatically favorable or unfavorable depending on the specific charged amino acid introduced. In some embodiments, positively charged amino acids (such as lysine, arginine, or histidine) at the interface are replaced with negatively charged amino acids (such as aspartic acid or glutamic acid). In some embodiments, negatively charged amino acids at the interface are replaced with positively charged amino acids. In some embodiments, charged amino acids are introduced into one or both of the interacting CH3 antibody constant domains. In some implementations, introducing charged amino acids into the CH3 antibody constant domain of the interaction between two Fc domains promotes the selective formation of heterodimers of the Fc domains, such as through an electrostatic reversal effect resulting from the interaction between the charged amino acids. Examples of electrostatic reversal amino acid pairs include, but are not limited to, those listed in Table 11.

[0396] Table 11. Electrostatically Oriented Amino Acid Pairs

[0397]

[0398] Other methods for controlling the heterodimerization of the Fc domain can be used, especially in the context of constructing bispecific antibodies.

[0399] In some embodiments, relative to the sequence of human IgG1, the first Fc domain and the second Fc domain each include one or more of the following amino acid substitutions: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T and K409I.

[0400] In some embodiments, the Fc domain comprises: (a) one of the following amino acid substitutions relative to wild-type human IgG1: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T or K409I; or (b)(i) relative to human IgG1 (ii) N297A mutation in the Fc region; (iii) L234A, L235A, and G237A mutations relative to the human IgG1 Fc region; (iv) N297A mutation relative to the human IgG2 Fc region; (v) A330S and P331S mutations relative to the human IgG2 Fc region; (vi) A330S, P331S, and N297A mutations relative to the human IgG2 Fc region; (vii) S228P, E233P, F234V, L235A, and delG236 mutations relative to the human IgG4 Fc region; or (viii) S228P, E233P, F234V, L235A, delG236, and N297A mutations relative to the human IgG4 Fc region.In some implementations, the Fc domain variant comprises: (a) one of the following amino acid substitutions relative to wild-type human IgG1: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L 368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, or K409I; and (b) further includes (i) an N297A mutation relative to the human IgG1 Fc region; (ii) a mutation relative to human IgG1 (iii) L234A, L235A, and G237A mutations in the Fc region; (iv) N297A mutations in the human IgG1 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.

[0401] 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.

[0402] connector

[0403] In some embodiments, this document discloses a polypeptide comprising a variant of the signal regulatory protein α (SIRP-α)D1, the variant comprising a SIRPαD1 domain or a fragment thereof, the SIRPαD1 domain having an amino acid mutation at residue 80 relative to the wild-type SIRPαD1 domain; and having at least one additional amino acid mutation at residues selected from the group consisting of the wild-type SIRPαD1 domain: residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92.

[0404] In some embodiments, this document also discloses peptides comprising Fc variants, wherein the Fc variants comprise Fc domain dimers comprising two Fc domain variants, wherein each Fc domain variant is independently selected from (i) a human IgG1 Fc region composed of L234A, L235A, G237A, and N297A mutations; (ii) a human IgG2 Fc region composed of A330S, P331S, and N297A mutations; or (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations.

[0405] In this disclosure, a linker is used to describe a bond or connection between peptide or protein domains or associated non-protein motifs. In some embodiments, the linker is a bond or connection between an Fc domain (or a variant thereof) and a SIRPαD1 domain variant. In some embodiments, the linker connects the C-terminus of the SIRPαD1 domain variant and the N-terminus of the Fc domain variant, such that the two peptides are linked to each other in tandem.

[0406] In some embodiments, the linker is a simple covalent bond, such as a peptide bond, a synthetic polymer bond, or any kind of bond generated by a chemical reaction (e.g., chemical conjugation). 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 the peptide bond. In some embodiments, the peptide bond is formed by synthetic means through conventional organic chemical reactions or by natural generation by the host cell, wherein nucleic acid molecules encoding DNA sequences of two tandem proteins (e.g., an Fc domain variant and a SIRPαD1 domain variant) can be directly transcribed and translated into a continuous polypeptide encoding the two proteins via essential molecular mechanisms in the host cell (e.g., DNA polymerase and ribosomes).

[0407] When the linker is a synthetic polymer, in some embodiments, the polymer is functionalized at each end with reactive chemical functional groups to react with the terminal amino acids at the linker ends of the two proteins.

[0408] When the linker (besides the peptide bond mentioned above) is prepared by a chemical reaction, 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 protein, respectively. In some embodiments, the two functional groups then react through synthetic chemistry to form a chemical bond, thereby linking the two proteins together.

[0409] spacers

[0410] In some embodiments of this disclosure, the linker between the Fc domain monomer and the SIRPαD1 variant peptide of this disclosure is an amino acid spacer comprising about 1-200 amino acids. Suitable peptide spacers include peptide linkers containing flexible amino acid residues such as glycine and serine. Examples of linker sequences are provided in Table 12. In some embodiments, the spacer contains motifs such as GS, GG, GGS, GGG, GGGGS (SEQ ID NO:163), GGSG (SEQ ID NO:164), or SGGG (SEQ ID NO:165), such as multiple or repeated motifs. In some embodiments, the spacer contains 2 to 12 amino acids, including GS motifs such as GS, GSGS (SEQ ID NO:166), GSG SGS (SEQ ID NO:167), GSGSGSGS (SEQ ID NO:168), GSGSGSGSGS (SEQ ID NO:169), or GSGSGSGSGSGS (SEQ ID NO:170). In some embodiments, the spacer contains 3 to 12 amino acids, including the GGS motif, such as GGS, GGSGGS (SEQ ID NO: 171), GGSGGSGGS (SEQ ID NO: 172), and GGSGGSGGSGGS (SEQ ID NO: 173). In some embodiments, the spacer contains 4 to 12 amino acids, including the GGSG (SEQ ID NO: 164) motif, such as GGSG (SEQ ID NO: 164), GGSGGGSG (SEQ ID NO: 174), or GGSGGGSGGGSG (SEQ ID NO: 175). In some embodiments, the spacer contains the GGGGS (SEQ ID NO: 163) motif, such as GGG GSGGGGSGGGGS (SEQ ID NO: 176).In some embodiments, the spacer contains 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), GGSAGGSGSGSSGG SSGASGTGTAGGTGSGSGTGSG (SEQ ID NO:190), AAANSSIDLISVPV DSR (SEQ ID NO:189). NO:191) or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGG SGGGS (SEQ ID NO:192).

[0411] In some embodiments, the spacer contains the motif of EAAAK (SEQ ID NO: 193), or multiple or repeated motifs. In some embodiments, the spacer contains a proline-rich sequence such as (XP)n, or multiple or repeated motifs, where X is any amino acid (e.g., A, K, or E) and n is 1-5, and the motif of PAPAP (SEQ ID NO: 194).

[0412] Table 12. Connector Sequence

[0413] SEQ ID NO: amino acid sequence 163 GGGGS 164 GGSG 165 SGGG 166 GSGS 167 GSGSGS 168 GSGSGSGS 169 GSGSGSGSGS 170 GSGSGSGSGSGS 171 GGSGGS 172 GGSGGSGGS 173 GGSGGSGGSGGS 174 GGSGGGSG 175 GGSGGGSGGGGSG 176 GGGGSGGGGSGGGGS 177 AAS 178 AAAL 179 AAAK 180 AAAR 181 EGKSSGSGSESKST 182 GSAGSAAGSGEF 183 AEAAAKEAAAKA 184 KESGSVSSEQLAQFRSLD 185 GGGGAGGGG 186 GENLYFQSGG 187 SACYCELS 188 RSIAT 189 RPACKIPNDLKQKVMNH 190 GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG 191 AAANSSIDLISVPVDSR 192 GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS 193 EAAAK 194 PAPAP

[0414] In some embodiments, the length of the peptide spacer and amino acids used is adjusted according to the desired 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 to avoid aggregate formation. In some embodiments, the spacer is A or AAAL (SEQ ID NO: 178).

[0415] Vector, host cell, and protein production

[0416] In some embodiments, this document discloses a polypeptide comprising a variant of the signal regulatory protein α (SIRP-α)D1, the variant comprising a SIRPαD1 domain or a fragment thereof, the SIRPαD1 domain having an amino acid mutation at residue 80 relative to the wild-type SIRPαD1 domain; and having at least one additional amino acid mutation at residues selected from the group consisting of the wild-type SIRPαD1 domain: residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92.

[0417] In some embodiments, this document also discloses peptides comprising Fc variants, wherein the Fc variants comprise Fc domain dimers having two Fc domain monomers, wherein each Fc domain monomer is 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 comprising mutants S228P, E233P, F234V, L235A, delG236, and N297A.

[0418] In some embodiments, the polypeptides of this disclosure are produced by host cells. A host cell is defined as a medium containing the necessary cellular components (e.g., organelles) required for the expression of the polypeptides and fusion polypeptides described herein by means of their corresponding nucleic acids. In some embodiments, the nucleic acids are contained in a nucleic acid vector introduced into the host cell via transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc. In some embodiments, the choice of nucleic acid vector depends on the host cell to be used. In some embodiments, the host cell is of prokaryotic (e.g., bacterial) or eukaryotic (e.g., mammalian) origin.

[0419] In some embodiments, peptides, such as peptide constructs comprising a SIRPαD1 domain variant (e.g., any variants provided in Tables 2, 5, and 6) and a fusion partner (e.g., an Fc variant), are generated by culturing host cells transformed with an expression vector containing nucleic acids encoding the peptide construct (e.g., an Fc variant, a linker, and a fusion partner) under appropriate conditions that induce or cause expression of the peptide construct. In some embodiments, the conditions suitable for expression vary depending on the chosen expression vector and host cell. In some embodiments, a variety of suitable host cells are used, including but not limited to mammalian cells, bacteria, insect cells, and yeast. For example, cells available from the U.S. Center for Type Culture Collection. The cell line catalog describes various cell lines that can be used with this disclosure. In some embodiments, variants of the Fc domain of this disclosure are expressed in cells optimized to prevent the glycosylation of proteins expressed by such cells, through genetic engineering of the cell line or modification of cell culture conditions such as the addition of chiffonines or the use of naturally non-glycosylated hosts such as prokaryotes (Escherichia coli, etc.), and in some cases, without the need to modify the glycosylated sequence in the Fc.

[0420] Nucleic acid vector construction and host cells

[0421] The nucleic acid sequence encoding the amino acid sequence of the disclosed polypeptide 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 molecule encoding the disclosed polypeptide is obtained using standard techniques (e.g., gene synthesis). Alternatively, standard techniques (e.g., QuikChange) can be used. TM Mutagenesis involves mutating nucleic acid molecules encoding the wild-type SIRPαD1 domain to include specific amino acid substitutions. In some cases, nucleic acid molecules are synthesized using a nucleotide synthesizer or PCR technology.

[0422] In some embodiments, nucleic acids encoding a polypeptide construct (e.g., a polypeptide construct containing a SIRPαD1 domain variant (e.g., any variants provided in Tables 2, 5, and 6) and a fusion partner (such as the Fc variant)) are incorporated into an expression vector to express a protein. A variety of expression vectors can be used for protein expression. Expression vectors may comprise self-replicating extrachromosomal vectors or vectors integrated into the host genome. Vectors may also include various components or elements. For example, in some embodiments, vector components include, but are not limited to, transcriptional and translational regulatory sequences, such as promoter sequences, ribosome binding sites, signal sequences, transcription initiation and termination sequences, translation initiation and termination sequences, 3' and 5' untranslated regions (UTRs) and enhancer or activator sequences; origin of replication; selection marker genes; and nucleic acid sequences encoding the polypeptide of interest and transcription termination sequences. In some embodiments, the expression vector contains a protein operatively linked to a control or regulatory sequence, a selection marker, any fusion partner, additional elements, or any combination thereof. The term "operatively linked" means that the nucleic acid is in a functional relationship with another nucleic acid sequence. Typically, these expression vectors contain transcriptional and translational regulatory nucleic acids operatively linked to nucleic acids encoding Fc variants, and are generally adapted for use in host cells for protein expression. Selection genes or markers (such as, but not limited to, antibiotic resistance genes or fluorescent protein genes) can be used to select host cells containing the expression vector, for example, by antibiotic or fluorescent expression. A variety of selection genes can be used.

[0423] In some embodiments, the components or elements of the vector are optimized to make the expression vector compatible with the host cell type. Expression vectors that can be used in this disclosure include, but are not limited to, those capable of expressing proteins in mammalian cells, bacteria, insect cells, yeast, and in vitro systems.

[0424] In some embodiments, mammalian cells are used as host cells to produce the polypeptides of this disclosure. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK 293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NSO, Sp2 / O, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NSO (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 to produce the polypeptides of this disclosure. Examples of *E. coli* strains include, but are not limited to, *E. coli* 294 (… 31,446), Escherichia coli λ1776 ( 31,537, Escherichia coli BL21(DE3)( BAA-1025) and Escherichia coli RV308 ( 31,608).

[0425] Different host cells possess characteristic and specific mechanisms for the post-translational processing and modification (e.g., glycosylation) of protein products. In some implementations, appropriate cell lines or host systems are selected to ensure proper modification and processing of the expressed peptides. Once the vector is introduced into the host cells for protein production, the host cells are cultured in conventional nutrient media modified to induce promoters, select transformants, or amplify genes encoding desired sequences.

[0426] In some embodiments, the polypeptide construct, such as a polypeptide construct containing a SIRPαD1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and a fusion partner (such as the Fc variant), is expressed in a mammalian expression system, including systems in which the expression construct is introduced into mammalian cells using viruses such as retroviruses or adenoviruses. In some embodiments, human, mouse, rat, hamster, or primate cells are used. Suitable cells also include known research cells, including but not limited to Jurkat T cells, NIH3T3, CHO, COS, and 293 cells. Alternatively, in some embodiments, the protein is expressed in bacterial cells. Bacterial expression systems are well known in the art and include Escherichia coli, Bacillus subtilis, Streptococcus cremoris, and Streptococcus lividans. In some cases, peptide constructs containing Fc domain variants are generated in insect cells (such as, but not limited to, Sf9 and Sf21 cells) or yeast cells (such as, but not limited to, organisms from the genera *Saccharomyces*, *Pichia*, *Kluyveromyces*, *Hansenula*, and *Yarrowia*). In some cases, peptide constructs containing Fc domain variants are expressed in vitro using cell-free translation systems. In vitro translation systems derived from two types of prokaryotic (e.g., *Escherichia coli*) and eukaryotic (e.g., wheat germ, rabbit reticulocyte) cells are available, and in some embodiments, the selection is based on the expression level and functional characteristics of the protein of interest. For example, as those skilled in the art will understand, some display techniques, such as ribosome display, require in vitro translation. Furthermore, in some embodiments, Fc domain variants are generated by chemical synthesis methods, such as, but not limited to, liquid-phase peptide synthesis and solid-phase peptide synthesis. When using non-glycosylated systems such as bacterial extracts for in vitro transcription, Fc will not be glycosylated even in the presence of natural glycosylation sites, so inactivation of Fc will be obtained equivalently.

[0427] In some embodiments, the peptide construct comprises a non-natural amino acid, amino acid analog, amino acid mimic, or any combination thereof that functions in a manner similar to naturally occurring amino acids. Naturally encoded amino acids typically refer to the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) as well as pyrrolidone and selenocysteine. Amino acid analogs are compounds having the same basic chemical structure as naturally occurring amino acids (e.g., carbon atoms bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. In some embodiments, such analogs have modified R groups (such as ortholeucine) or modified peptide backbones, but generally retain the same basic chemical structure as naturally occurring amino acids.

[0428] Protein production, recycling, and purification

[0429] In some embodiments, the host cells used to produce the polypeptides of this disclosure are grown in a culture medium suitable for culturing the selected host cells. Examples of suitable culture media for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco Modified Eagle Medium (DMEM), and Expi 293. TM Expression medium, DMEM supplemented with fetal bovine serum (FBS), and RPMI-1640. Examples of suitable culture media for bacterial host cells include Luria broth (LB) with necessary supplements, such as selectants, for example, ampicillin. In some embodiments, host cells are cultured at suitable temperatures (e.g., from about 20°C to about 39°C, for example, from about 25°C to about 37°C, preferably 37°C) and CO2 levels (e.g., from about 5% to 10%). In some embodiments, the pH of the medium is from about pH 6.8 to pH 7.4, for example, pH 7.0, which depends primarily on the host organism. If an inducible promoter is used in the expression vector, protein expression can be induced under conditions suitable for promoter activation.

[0430] In some embodiments, protein recovery involves disrupting host cells, such as through osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris is removed by centrifugation or filtration. The protein can then be further purified. In some embodiments, the peptides of this disclosure are purified by a variety of protein purification methods, such as by chromatography (e.g., ion exchange chromatography, affinity chromatography, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard technique used for protein purification. For example, in some embodiments, the protein is separated and purified by appropriately selecting and combining affinity columns such as protein A columns (e.g., POROS protein A chromatography) with chromatographic columns (e.g., POROS HS-50 cation exchange chromatography), filtration, ultrafiltration, desalting, and dialysis procedures. In some embodiments, the peptide is conjugated with a labeled sequence (such as a peptide) to facilitate purification. An example of a labeled amino acid sequence is a six-histidine peptide (His6-tag (SEQ ID NO:223)) that can bind to a nickel-functionalized agarose affinity column with micromolar affinity. Alternatively, a hemagglutinin “HA” tag corresponding to an epitope derived from influenza hemagglutinin protein can be used.

[0431] In some embodiments, peptides of this disclosure, such as peptide constructs containing SIRPαD1 domain variants (e.g., any variants provided in Tables 2, 5, and 6) and fusion couplers (e.g., Fc variants), are generated from the cells of a subject (e.g., a human), for example in the context of gene therapy, by administering a vector containing a nucleic acid molecule encoding the peptide of this disclosure, such as a viral vector (e.g., a retroviral vector, adenovirus vector, poxvirus vector (e.g., vaccinia virus vector, such as modified Ankara vaccinia (MVA)), adeno-associated virus vector, and alpha virus vector). Once the vector is in the subject's cells (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.), it can be used to express the peptide disclosed herein. In some cases, the peptide is secreted by the cells. In some embodiments, no further action is required if the treatment of the disease or condition is the desired outcome. In some embodiments, if protein collection is required, blood is collected from the subject and the protein is purified from the blood by various methods.

[0432] Methods of treating cancer

[0433] This document provides a method for treating cancer in an individual (e.g., a human individual) 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 (e.g., at least one chemotherapeutic agent, such as at least two, at least three, or at least four chemotherapeutic agents). This document also provides a method for treating cancer in an individual (e.g., a human individual) comprising administering to the individual an effective amount of (a) a peptide comprising a SIRPαD1 domain variant (e.g., the SIRPαD1 domain variant described herein) and an Fc domain variant (e.g., the Fc domain variant described herein) and (b) a chemotherapeutic agent (e.g., at least one chemotherapeutic agent, such as at least two, at least three, or at least four chemotherapeutic agents). In some embodiments, the method further comprises administering to the individual an effective amount of a therapeutic antibody (e.g., at least one therapeutic antibody, such as at least two, at least three, or at least four therapeutic antibodies). Additionally or alternatively, in some embodiments, the method further comprises administering to the individual an effective amount of an immunotherapeutic agent (e.g., at least one immunotherapeutic agent, such as at least two, at least three, or at least four immunotherapeutic agents). Alternatively or alternatively, in some embodiments, the method includes administering the peptide and chemotherapeutic agent in combination with one or more other treatment modalities (including, but not limited to, radiotherapy, surgery, cryoablation, and bone marrow transplantation).

[0434] Combination therapies including chemotherapy agents and exemplary chemotherapy agents

[0435] Exemplary chemotherapeutic agents that can be used in the methods for treating cancer described herein include, but are not limited to, methotrexate ( Amethopterin, cyclophosphamide Abiraterone, abemaciclib, atratramine, thalidomide acridine formamide, Actinomycin, Actinomycin-D, Afatinib, 17-N-allylamino-17-demethoxygerdomylmycin, Alectinib, Alpelisib, Aminopterin, Acridine, Anlotinib, Anthracyclines, Antitumor Drugs, Apartinib, 5-Azacitidine, 6-Mercaptopurine, 6-Thioguanine, Cytarabine, Axitinib, Azacitidine, Azathioprine, BL22, Bendamustine, Binimetinib, Biricodar, Bleomycin, Bortezomib, Bosutinib Brigatinib, lichenin, busulfan, cabozantinib, calyculin, camptothecin, capecitabine, carboplatin, carmustine, ceritinib, chlorambucil, cisplatin, cladribine, clofarapine, cobimetinib, crizotinib, cytarabine, dabrafenib, dacarbazine, dacomitinib, dasatinib, daunorubicin, dexamethasone, dichloroacetic acid, disccodermolide, docetaxel, doxorubicin. Encorafenib, epirubicin, entrectinib, enzalutamide, epothilone, erdafitinib, eribulin, erlotinib, estradiol, etoposide, everolimus, exatecan, escitaline, mirtrol, fluorouracil, fludarabine, fluorouracil (such as 5-fluorouracil), leucovorin, phosphostel, fruquintinib, ganciclovir, gefitinib, gemcitabine, gilteritinib, goserelin, hexamethylpyrimidine Hydroxycarbamide, hydroxyurea, IT-101, ibrutinib, icotinib, idarubicin, idelalisib, ifosfamide, imatinib, irinoimiquimod, irinotecan, ilofofen, ivosidenib, ixabepilone, laniquidar, lapatinib, larotrectinib, lenalidomide, lenvatinib, lorlatinib, roxamustine, letopecan, equine phosphoramide, masorophytin.Nitrogen mustard, melphalan, mercaptopurine, methotrexate, methylprednisolone, mitomycin, mitotan, mitoxantrone, nerabine, neratinib, niraparib, nilotinib, nintedanib, oblimersen, olaparib, osimertinib, oxaliplatin, nedaplatin, phenelzine, pyrplatin, PAC-1, paclitaxel, palbociclib, pazopanib, pemetrexed, pegfilgrastim, pentostatin, piperobromane, picotinone, procainox, prednisone, panatinib, mebenzylhydrazine, proteasome inhibitors (e.g., bortezomib), pyrotinib, raltitrexed, rebeccamycin Regrafenib, Ribociclib, Rubitecan, Rucaparib, Ruxolitinib, SN-38, Salinosporamide A. Saplatin, Sirolimus, Sonidegib, Sorafenib, Streptozotocin, Sunitinib, Sorghum extract, Talazoparib, Tariquidar, Taxane, Tegafur / Uracil, Tesseromoximide, Teniposide, Temozolomide, Testrolide, Thiotepapi, Thioguanine, Topotecan, Trabectedin, Trametinib, Retinoic acid, Trifluorouridine, Triplatintetranitrate, Tri(2-chloroethyl)amine, Trasatabine, Uramustine, Pentorubicin, Vandetanib, Vemurafenib, Venetoclax (ABT-199), Navitocx (ABT-263), Vincristine, Vincristine, Vinorelbine, Vemoderil, Vorinostat, Aflibercept Azoquine, etc.

[0436] In some embodiments, methods of treating cancer include combining an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) with a specific class of chemotherapeutic agents. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is a peptide described herein (e.g., a fusion peptide comprising a SIRPαd1 domain variant and an Fc variant; a fusion peptide comprising a SIRPγ variant, a SIRPβ1 variant, or a SIRPβ2 variant and an Fc variant). For example, in some embodiments, methods of treating cancer include combining a peptide described herein (e.g., a fusion peptide) with an adrenal inhibitor (including, but not limited to, the adrenal inhibitors described herein). For example, in some embodiments, methods of treating cancer include combining a peptide described herein with an anthracycline (including, but not limited to, the anthracyclines described herein). In some embodiments, methods of treating cancer include combining a peptide described herein with an alkylating agent (including, but not limited to, the alkylating agents described herein). In some embodiments, methods of treating cancer include combining a peptide described herein with an androgen inhibitor (including, but not limited to, the androgen inhibitors described herein). In some embodiments, the method of treating cancer includes the combined administration of the peptide described herein with antimetabolites such as purine analogs (including, but not limited to, the antimetabolites described herein, such as purine analogs). In some embodiments, the method of treating cancer includes the combined administration of the peptide described herein with antitumor antibiotics (including, but not limited to, the antitumor antibiotics described herein). In some embodiments, the method of treating cancer includes the combined administration of the peptide described herein with a BLC-2 inhibitor (including, but not limited to, the BLC-2 inhibitors described herein). In some embodiments, the method of treating cancer includes combining the peptide described herein with a BTK inhibitor (including, but not limited to, the BTK inhibitor described herein). In some embodiments, the method of treating cancer includes combining the peptide described herein with a CDK 4 / 6 inhibitor (including, but not limited to, the CDK 4 / 6 inhibitor described herein). In some embodiments, the method of treating cancer includes combining the peptide described herein with a colony-stimulating factor (including, but not limited to, the colony-stimulating factor described herein). In some embodiments, the method of treating cancer includes combining the peptide described herein with a corticosteroid (including, but not limited to, the corticosteroid described herein). In some embodiments, the method of treating cancer includes combining the peptide described herein with an EGFR inhibitor (including, but not limited to, the EGFR inhibitor described herein). In some embodiments, the method of treating cancer includes combining the peptide described herein with a gonadotropin-releasing hormone (GnRH) agonist (including, but not limited to, the GnRH agonist described herein).In some embodiments, the method of treating cancer includes combining the peptide described herein with a mitotic inhibitor / microtubule inhibitor (including, but not limited to, the mitotic inhibitor / microtubule inhibitor described herein). In some embodiments, the method of treating cancer includes combining the peptide described herein with an mTOR kinase inhibitor (including, but not limited to, the mTOR kinase inhibitor described herein). In some embodiments, the method of treating cancer includes combining the peptide described herein with a proteasome inhibitor (including, but not limited to, the proteasome inhibitor described herein). In some embodiments, the method of treating cancer includes combining the peptide described herein with a signal transduction inhibitor, such as a protein-tyrosine kinase inhibitor, PAK4 inhibitor, or PI3K inhibitor (including, but not limited to, the signal transduction inhibitor described herein). In some embodiments, the method of treating cancer includes combining the peptide described herein with a topoisomerase inhibitor (including, but not limited to, the topoisomerase inhibitor described herein). In some embodiments, the method of treating cancer includes combining the peptide described herein with a tyrosine kinase inhibitor (including, but not limited to, the tyrosine kinase inhibitor described herein). In some embodiments, methods of treating cancer include combining the peptide described herein 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, methods of treating cancer include combining the peptide described herein with an apoptosis modulator, such as by modulating the activity of Bcl-2, Mcl1, Bcl-1x, etc. (including, but not limited to, the apoptosis modulators described herein, such as by modulating the activity of Bcl-2, Mcl1, Bcl-1x, etc.). In some embodiments, methods of treating cancer include combining the peptide described herein with a platinum-based agent (including, but not limited to, the platinum-based agent described herein). In some embodiments, methods of treating cancer include combining the peptide described herein with an inhibitor of NTRK1, NTRK2, and / or NTRK3, an ALK inhibitor, a ROS inhibitor, an FLT3 inhibitor, a BRAF inhibitor, a MEK1 and / or MEK2 inhibitor, or a HER2, HER3, and / or HER2 inhibitor. 4. Inhibitors, RET / PTC inhibitors, BCR-ABL inhibitors, c-KIT inhibitors, PDGFR-α and / or PDGFR-β inhibitors, FGFR1, FGFR2, FGFR3 and / or FGFR4 inhibitors, Smoothened 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 described in further detail below.

[0437] In some embodiments, the chemotherapeutic agent is a small molecule anticancer agent (such as a small molecule inhibitor). In some embodiments, methods of treating cancer include the combined administration of the peptides described herein with small molecule inhibitors of VEGFR and / or PDGFR, small molecule EGFR, small molecule ALK, small molecule CDK4 / 6, small molecule PARP, small molecule PAK4, small molecule mTOR, small molecule KRAS, small molecule TRK, small molecule BCL2, small molecule B-raf, small molecule IDH, small molecule PI3K, small molecule DDR (DNA damage response) inhibitors, or small molecule hypomethylating agents. In other cases, small molecules, such as IDO / TDO inhibitors, AhR inhibitors, arginase inhibitors, A2aR inhibitors, TLR agonists, STING agonists, or Rig-1 agonists, regulate cellular signaling pathways in cells expressing CD47.

[0438] In some embodiments, methods of treating cancer include combining the administration of the peptide described herein (e.g., a fusion peptide comprising a SIRPαd1 domain variant and an Fc variant) with at least one, at least two, at least three, or at least four chemotherapeutic agents. In some embodiments involving the administration of two or more chemotherapeutic agents, the two or more chemotherapeutic agents are from different classes (as described above) and / or exert their anticancer effects through different mechanisms of action.

[0439] Further details regarding exemplary pharmaceutical compositions and formulations, exemplary dosages, and exemplary routes of administration of the fusion peptides described herein are provided in WO 2017 / 027422 and U.S. Patent No. 10,259,859, the contents of which are each incorporated herein by reference in their entirety.

[0440] Combination therapies including therapeutic antibodies and exemplary therapeutic antibodies

[0441] In some embodiments, the methods of treating cancer provided herein include administering to an individual an effective amount of a therapeutic antibody (e.g., at least one therapeutic antibody, such as at least two, three, or four therapeutic antibodies), in combination with an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., a fusion peptide described herein) and a chemotherapeutic agent described herein (e.g., at least one chemotherapeutic agent, such as at least two, three, or four chemotherapeutic agents). In some embodiments, the therapeutic antibody is conjugated with a drug (i.e., an antibody-drug conjugate or “ADC”).

[0442] Exemplary therapeutic antibodies (e.g., therapeutic monoclonal antibodies) used in the methods described herein include, but are not limited to, 3F8, 8H9, abagovomab, abciximab, abituzumab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afelimomab, afutuzumab, alacizumab pegol, ALD518, alemtuzumab, alirocumab, attumomab pentetate, amatuximab, and anatumomab. mafenatox, Anetumabravtansine, Anifrolumab, IMA-638, Apolizumab, Arcitumomab, Ascrivacumab, Aselizumab, Atezolizumab, Attinumab, Atlizumab, Tocilizumab, Atorlimumab, Avelumab, Bapin The following are listed: euzumab, basiliximab, bavituximab, beectumomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, bivatuzumab mertansine, blinatumomab, blobsozumab, bococizumab, and brentuximab.vedotin), briakinumab, brodalumab, brolucizumab, brontictuzumab, cabiralizumab (FPA008), camrelizumab, canakinumab, cantuzumabmertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catutoxumab, cBR96-doxorubicin immunoconjugate, CC49, cedelizumab, cetholizumab pegol), cetuximab, Ch.14.18, citatuzumab bogatox, cixutumumab, clazakizumab, claliximab, clivatuzumab tetraxetan, codrituzumab, and coltuximab. ravtansine, Conatumumab, Concizumab, Crenzumab, CR6261, Dacetuzumab, Daclizumab, Dalotuzumab, Dapirolizumabpegol, Daratumumab, Dectrekumab, Demcizumab, Denintuzumab mafodotin, Denosumab, Derlotuximab biotin, Detumomab, Dinutuximab, Diridavumab, Dorlimomabaritox, drozitumab, duligotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edrecolomab, edalizumab, efungumab, eldelumab, elgemtumab, ellotuzumab, elsilimomab, emactuzumab (RG7155), emmibetuzumab, enavatuzumab, enfortumab vedotin, enlimomabpegol, emoblituzumab, enokizumab, enoticumab, ensituximab, epipitumomabcituxetan, epratuzumab, erlizumab, ertumaxomab, etaracizumab, estradiol, evoloxumab, exbivirumab, faolesomab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab mab), Ficlatuzumab, Figitumumab, Frivumab, Flanvotumab, Fletikumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumabozogamicin, Gevokizumab, Girentuximab, Glembatumumabvedotin, golimumab, gomiliximab, guselkumab, ibazumab, irimomumab-tecitan, icrucumab, edarucizumab, icgovomab, IMAB362, imarumab, imciromab, imatrazumab, inclacumab, indatuximab ravtansine, vitaminumab vedotin, infliximab, intetumumab, inolimomab, Inotuzumabozogamicin, ipilimumab, itatuximab, itolizumab, ickelizumab, keliximab mab), latozumab, lambrolizumab, laparizumab, lebrikizumab, lemalesomab, lenzilumab, lerdelimumab, lexatumumab, libivirumab, vedotin, ligelizumab, lilotomab satetraxetan, lintuzumab, lirilumab, lodelcizumab, lokivetmab, and lorvotuzumabmertansine, Lucatumumab, Lulizumabpegol, Lumiliximab, Lumretuzumab, MSB0010718C (avelumab), Mapatumumab, Margetuximab, Maslimomab, Mavrilimumab, Matu (e.g., zumab), MEDI6469, MEDI0680, MEDI6383, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Mogamulizumab, Morolimumab, Motavizumab, Moxetumomab-Pardotoxin) pasudotox, Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomabestafenatox, Narnatumab, Natalizumab, Nebacumab, Necitumumab, Nemolizumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentan, obiltoxaximab, obinutuzumab, oxcaratuzumab, oxcrelizumab, oxulimomab, alfatumumab, olalatumab, oxoxizumab, oxalizumab, onartuzumab, ontuxizumab, oxicinumab, oxportuzumabMonatox, Oregovomab, Orticumab, Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab, Pankomab, Panobacumab Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Polatuzumab vedotin), Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO140, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab b) Refanezumab, Regavirumab, Reslizumab, Rilotumumab, Rinucumab, Rituximab, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Sacituzumabgovitecan), Samalizumab, SAR650984 (Isatuximab), Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, Sibrotuzumab, SGN-CD19A, SGN-CD33A, Sifalimumab, Siltuximab, Simtuzumab, Sintilimab, Siplizumab, Sirukumab, Sofituzumab vedotin), solanezumab, solitomab, sopoxib, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tabatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tarextumab, tefibazumab, telimomabaritox, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, TGN1412, ticilimumab (tremelimumab), tildrakizumab, tigatuzumab, TNX-650, tocilizumab (atlizumab) Toralizumab, Toripalimab, Tosatoxumab, Tositumomab, Tovetumab, Tralokinumab, Trastuzumab, Trastuzumab-emtansine, TRBS07, Tregalizumab, Tremelimumab, Tucotuzumab-interleukin celmoleukin, tuvirumab, ubutuximab, ullocuplumab, urtolumab, urtoxazumab, utstekinumab, utomilumab (PF-05082566), vandortuzumab vedotin, vantictumab, vanucizumab, vapaxiximab, varlilumab, valatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, volecithin, volerolizumab (RG7888), martin-vosetuzumabbiosimilars of any of the aforementioned therapeutic antibodies, including mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, or zolimomab aritox.

[0443] Other exemplary therapeutic antibodies (e.g., therapeutic monoclonal antibodies) that may be used in the methods described herein are, but are not limited to, anti-CD20 antibodies, anti-EGFR antibodies, anti-Her2 / Neu(ERBB2) antibodies, anti-EPCAM antibodies, anti-GL2 antibodies, anti-GD2, anti-GD3, anti-CD2, anti-CD3, anti-CD4, anti-CD8, and anti-CD4 antibodies. I9, anti-CD22, anti-CD30, anti-CD33, anti-CD39, anti-CD45, anti-CD47, anti-CD52, anti-CD56, anti-CD70, anti-CD73, anti-CD117, anti-SIRPα antibody, anti-LILRB1, anti-LILRB2, anti-LILRB4 antibody, anti-PD-1 antibody (e.g., anti-PD-1 antagonist antibody), anti-PD-L1 antibody (e.g., anti-PD-L1 antagonist antibody), anti-PD-L2 antibody, or any antibody designed to bind to tumor cells, cells infected by viruses or bacteria, immune cells, or healthy normal cells, or any kind of cytokine, chemokine, or hormone.

[0444] In some implementations, the therapeutic antibodies used in the methods described herein are, for example, antibodies that bind to 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-β, TGFβ2, TGFβ1, TFPI, etc. Tenosin C, TEM1, TAG-72, T-cell receptor, STEAP1, sphingosine 1-phosphate, SOST, SLAMF7, BCL-2, selectin P, SDC1, osteosclerosing protein, RTN4, RON, rhesus monkey factor, RHD, respiratory syncytial virus, RANKL, rabies virus glycoprotein, platelet-derived growth factor receptor β, phosphatidylserine, sodium phosphate cotransporter, PDGF-Rα, PDCD1, PD-1, PD-L1, PCSK9, o xLDL, OX-40, NRP1, Notch receptor 4, Notch receptor 3, Notch receptor 2, Notch receptor 1, NOGO-A, NGF, neurotrophic regulatory protease 1, NCA-90 (granulocyte antigen), NARP-1, N-hydroxyacetylneuraminic acid, myostatin, myelin-associated glycoprotein, mucin CanAg, MUC1, MSLN, MS4A1, MIF, mesothelin, MCP-1, LTA, LOXL2, lipoteichoic acid, LI NGO-1, LFA-1 (CD11a), Lewis-Y antigen, L-selectin (CD62L), KIR2D, ITGB2 (CD18), ITGA2, interferon α / β receptor, interferon receptor, interferon γ-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, IL 20, IgG, IgG4, IGF-I, IGF-I receptor, IgEFc region, IFN-γ, IFN-α, ICAM-1 (CD54), human TNF, human dispersive 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 α chain, phosphatidylinositol polysaccharide 3, GD3 ganglioside, GD2, ganglioside GD2, Frizzled receptor, folate receptor 1, folate hydrolase, fibronectin extradomain-B, fibrin IIβ chain, FAP, respiratory syncytial virus F protein, ERBB3, episialin, EpCAM, endotoxin, EGFR, EGFL7, Shiga toxin type 2, Shiga toxin type 1, DRS, DPP4, DLL4, dabigatran, cytomegalovirus glycoprotein B, CTLA-4, CSF2, CSF1R, agglutination factor A, CLDN18.2, ch4DS, CFD, CEA-associated 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, CD3ε, 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 Antibodies against CCR5, CCR4, CCL11 (eosinophil chemokine-I), cardiac myosin, carbonic anhydrase 9 (CA-IX), canine IL31, CA-125, C5, C242 antigen, CXC chemokine receptor type 4, β-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-β-amyloid.

[0445] In some implementations, the therapeutic antibody used in the methods described herein binds to an antigen expressed by cancer cells (e.g., expressed on the surface of cancer cells). Exemplary antigens expressed by cancer are known in the art and include, but are not limited to, 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, nectin4, NaPi2b, LIV-1A, guanylate cyclase C, DLL3, EGFR, HER2, VEGF, VEGFR, integrin αVβ3, integrin α5β1, MET, IGF1R, TRAILR1, TRAILR2, RANKL, FAP, tendinin, Le y EpCAM, CEA, gpA33, PSMA, TAG72, mucin, CAIX, EPHA3, folate receptor α, GD2, GD3, and 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 peptide described herein is administered in combination with a chemotherapeutic agent (e.g., at least one chemotherapeutic agent) and a monoclonal antibody that binds to CD123 (also known as IL-3 receptor α), such as tastuzumab (also known as CSL362 and JNJ-56022473).

[0446] In some embodiments, the therapeutic antibody (e.g., a therapeutic monoclonal antibody) used in the methods described 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), and CD160. 6 (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).

[0447] Combination therapies including immunotherapeutic agents and exemplary immunotherapeutic agents

[0448] In some embodiments, the methods of treating cancer provided herein include administering to an individual an effective amount of an immunotherapeutic agent (e.g., at least one immunotherapeutic agent, such as at least two, at least three, or at least four immunotherapeutic agents), i.e., an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., a peptide described herein) and a chemotherapeutic agent described herein (e.g., at least one chemotherapeutic agent, such as at least two, at least three, or at least four chemotherapeutic agents).

[0449] In some embodiments, an immunotherapeutic agent refers to any therapeutic agent that targets the immune system and promotes therapeutic redirection of the immune system, such as regulators of co-stimulatory pathways, cancer vaccines, recombinant modified immune cells, etc. Exemplary and non-limiting immunotherapeutic agents are described below. In some embodiments, an immunotherapeutic agent is an antibody or contains an antibody. Exemplary targets of immunotherapeutic antibodies are known in the art and include, but are not limited to, BDCA2, BDCA4, ILT7, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, Siglec-3, Siglec-7, Siglec-9, Siglec-10, Siglec-15, FGL-1, CD200, CD200R, CSF-1R, CD24, CD40, CD40L, CD163, CD206, DEC205, CD47, CD123, arginase, IDO, TDO, AhR, EP2, COX-2, CCR2, CCR-7, CXCR1, CX3 CR1, CXCR2, CXCR3, CXCR4, CXCR7, TGF-βRI, TGF-βRII, 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.

[0450] Approved or late-stage clinical trials of immunotherapeutic agents include, but are not limited to, ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, and duruvamab. In some embodiments, a combination of an agent that blocks the interaction between CD47 and SIRPα (such as the peptides described herein) and an inhibitor of the PD-L1 / PD-1 pathway, such as an antibody, a small molecule, or a peptide that blocks the interaction between PD-L1 and PD-1 (e.g., by binding to PD-1 or PD-L1), is administered. In some embodiments, the inhibitor of the PD-L1 / PD-1 pathway is an antisense polynucleotide. In some embodiments, the inhibitor of the PD-L1 / PD-1 pathway is an anti-PD-L1 or anti-PD-1 antagonist antibody (e.g., the anti-PD-1 or anti-PD-L1 antagonist antibody described elsewhere herein). As demonstrated herein, the combination administration of an agent that blocks the interaction between CD47 and SIRPα (such as the peptides described herein) with an inhibitor of the PD-L1 / PD-1 pathway can result in synergistic antitumor activity. In some embodiments, the immunotherapeutic agent is or comprises a vaccine, oncolytic virus, adoptive cell therapy, cytokine, or small molecule immunotherapeutic agent. Examples of such immunotherapeutic agents are known in the art. For example, adoptive cell therapies and therapeutic agents may include, but are not limited to, chimeric antigen receptor T-cell therapy (CAR-T), tumor-infiltrating lymphocytes (TILs), TCR-engineered T cells, TCR-engineered NK cells, and macrophage products. Vaccines may include, but are not limited to, polynucleotide vaccines, peptide vaccines, or cell-based (e.g., tumor- or dendritic cell-based) vaccines. Various cytokines that can be used to treat cancer are known and include, 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. Small molecule immunotherapeutic agents may include, but are not limited to, IDO / TDO inhibitors, AhR inhibitors, arginase inhibitors, A2αR inhibitors, TLR agonists, STING agonists, and Rig-1 agonists.

[0451] In some embodiments, an agent that blocks the interaction between CD47 and SIRPα (such as the peptides described herein) and a chemotherapeutic agent (e.g., at least one chemotherapeutic agent) are administered in combination with other agents described herein (e.g., therapeutic antibodies, small molecule inhibitors, immunotherapeutic agents, etc.), said other agents being from different classes and / or exerting their anticancer effects through different mechanisms of action. For example, in some embodiments, a method of treating cancer includes the combined administration of an agent that blocks the interaction between CD47 and SIRPα (such as the peptides described herein) 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 anti-HER2 antibodies). In some embodiments, an agent that blocks the interaction between CD47 and SIRPα (such as the peptides 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 contemplated.

[0452] In some embodiments, an agent that blocks the interaction between CD47 and SIRPα (such as the peptide described herein) is administered in combination with one or more agents, including but not limited to, antidiarrheal agents, antiemetics, analgesics, opioids and / or nonsteroidal anti-inflammatory agents.

[0453] Combination therapy that includes other treatment methods

[0454] In some embodiments, an agent that blocks the interaction between CD47 and SIRPα (such as the peptides described herein) is administered in combination with at least one chemotherapeutic agent and one or more additional therapeutic modalities. In some embodiments, one or more additional therapeutic modalities include radiotherapy (e.g., gamma rays, X-rays, and / or targeted delivery of radioisotopes to tumor cells, microwaves, UV radiation), or gene therapy. For example, therapeutic genes for gene therapy include, but are not limited to, antisense forms of cell proliferation inducers (oncogenes), cell proliferation inhibitors (tumor suppressor factors), or programmed cell death inducers (pro-apoptotic genes). In some embodiments, any one or more of the combined therapies described herein are administered in conjunction with surgery (e.g., resection).

[0455] Exemplary therapy combinations

[0456] In some implementations, methods of treating cancer include the combined administration of an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) with nivolumab and one or more agents selected from the following: lenalidomide, ibrutinib, palbociclib, enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin, cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine, gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin, docetaxel, estradiol, paclitaxel, vinblastine, etoposide, irinotecan, teniposide, topotecan, prednisone, methylprednisolone, and dexamethasone. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is the peptide described herein (e.g., a fusion peptide comprising a SIRPαd1 domain variant and an Fc variant).

[0457] In some implementations, methods of treating cancer include the combined administration of an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) 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, estradiol, paclitaxel, vinblastine, etoposide, irinotecan, teniposide, topotecan, prednisone, methylprednisolone, and dexamethasone. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is the peptide described herein (e.g., a fusion peptide comprising a SIRPαd1 domain variant and an Fc variant).

[0458] In some implementations, methods of treating cancer include the combined administration of an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) with trastuzumab and one or more agents selected from the following: lenalidomide, ibrutinib, palbociclib, enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin, cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine, gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin, docetaxel, estradiol, paclitaxel, vinblastine, etoposide, irinotecan, teniposide, topotecan, prednisone, methylprednisolone, and dexamethasone. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is the peptide described herein (e.g., a fusion peptide comprising a SIRPαd1 domain variant and an Fc variant).

[0459] In some implementations, methods of treating cancer include the combined administration of an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) with bevacizumab and one or more agents selected from the following: lenalidomide, ibrutinib, palbociclib, enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin, cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine, gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin, docetaxel, estradiol, paclitaxel, vinblastine, etoposide, irinotecan, teniposide, topotecan, prednisone, methylprednisolone, and dexamethasone. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is the peptide described herein (e.g., a fusion peptide comprising a SIRPαd1 domain variant and an Fc variant).

[0460] In some implementations, methods of treating cancer include the combined administration of an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) with rituximab and one or more agents selected from the following: lenalidomide, ibrutinib, palbociclib, enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin, cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine, gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin, docetaxel, estradiol, paclitaxel, vinblastine, etoposide, irinotecan, teniposide, topotecan, prednisone, methylprednisolone, and dexamethasone. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is the peptide described herein (e.g., a fusion peptide comprising a SIRPαd1 domain variant and an Fc variant).

[0461] In some implementations, methods of treating cancer include the combined administration of an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) with pertuzumab and one or more agents selected from the following: lenalidomide, ibrutinib, palbociclib, enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin, cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine, gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin, docetaxel, estradiol, paclitaxel, vinblastine, etoposide, irinotecan, teniposide, topotecan, prednisone, methylprednisolone, and dexamethasone. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is the peptide described herein (e.g., a fusion peptide comprising a SIRPαd1 domain variant and an Fc variant).

[0462] In some implementations, methods of treating cancer include the combined administration of an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) with denosumab and one or more agents selected from the following: lenalidomide, ibrutinib, palbociclib, enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib, enzalutamide, cyclophosphamide, carboplatin, cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine, gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin, docetaxel, estradiol, paclitaxel, vinblastine, etoposide, irinotecan, teniposide, topotecan, prednisone, methylprednisolone, and dexamethasone. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is the peptide described herein (e.g., a fusion peptide comprising a SIRPαd1 domain variant and an Fc variant).

[0463] Exemplary cancer

[0464] In some implementation schemes, the cancers treated by the methods provided herein are breast cancer, lung cancer, lung adenocarcinoma, squamous cell lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), head and neck cancer, mesothelioma, brain cancer, brain tumor, abdominal cancer, colon cancer, colorectal cancer, esophageal cancer, parapharyngeal cancer, gastrointestinal cancer, glioma, liver cancer, stomach cancer, oral cancer, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, kidney cancer, bladder cancer, renal pelvis cancer, pancreatic cancer, retinoblastoma, cervical cancer, uterine cancer, Wilms' tumor, multiple myeloma, skin cancer, lymphoma, leukemia, blood cancer, thyroid cancer, bone cancer, adenoid cystic tumor, chondrosarcoma, islet cell tumor, neuroendocrine tumor, prostate cancer, glioblastoma, endometrial carcinoma, leiomyosarcoma, gallbladder cancer, hepatocellular carcinoma, melanoma, or solid tumors.

[0465] In some implementations, the cancer treated by the methods provided herein is a blood cancer. In some implementations, the blood cancer is multiple myeloma or leukemia, including but not limited to, acute or chronic myeloid leukemia, acute or chronic lymphocytic 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... Hematologic malignancies (JMML), large granular lymphoblastic (LGL) leukemia, plasmacytoma, blastic plasmacytoid dendritic cell tumor (BPDCN), B-prolymphocytic leukemia (B-PLL), T-prolymphocytic leukemia (T-PLL), multiple myeloma (MM), and non-Hodgkin's lymphoma (such as diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, mantle cell lymphoma (MCL), peripheral T-cell lymphoma (PTCL), lymphoplasmacytic lymphoma, and Waldenström macroglobulinemia). Marginal zone lymphoma (MZL) and follicular lymphoma (FL).

[0466] Treatment methods for leukemia

[0467] In some implementations, treatment is provided for individuals (e.g., human individuals) with 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 tumor (BPDCN), B-prolymphocytic leukemia (B-PLL), T-prolymphocytic leukemia (T-PLL), multiple myeloma (MM), and non-Hodgkin's lymphoma (such as diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, mantle cell lymphoma (MCL), peripheral T-cell lymphoma (PTCL), lymphoplasmacytic lymphoma, Waldenström megacolon). A method for treating globulinemia, marginal zone lymphoma (MZL), and follicular lymphoma (FL), the method comprising administering to an individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) and (b) a Bcl2 inhibitor. In some embodiments, the Bcl2 inhibitor is venetoc (also known as ABT-199), ABT-737, navitoc (also known as ABT-263), BCL201, or AZD-0466. In some embodiments, the agent is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPαD1 domain variant (e.g., the SIRPαD1 domain variant described herein) and an Fc domain variant (e.g., the Fc domain variant described herein). In some embodiments, the polypeptide (e.g., the fusion polypeptide) comprises a SIRPαD1 domain variant, the SIRPαD1 domain variant comprising SEQ 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, wherein the numbering is based on the Kabat EU index; (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations, wherein the numbering is based on the Kabat EU index; (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is based on the Kabat EU index; or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is based on the Kabat EU index. In some embodiments, the polypeptide (e.g., a fusion polypeptide) administered to an individual contains or SEQ ID NO:136 or SEQ ID NO:85. The amino acid sequence of NO:135.In some embodiments, the peptide (e.g., a fusion peptide) forms a homodimer. In some embodiments, the peptide (e.g., a fusion peptide) and a Bcl2 inhibitor (e.g., venetoclax) are administered simultaneously, in parallel, or sequentially.

[0468] Bcl2 inhibitors are a class of anticancer drugs that are thought to exert their cytotoxic effects by competing with pro-apoptotic Bcl2 to occupy BH3 docking slots on the surface of anti-apoptotic family members. By binding to one or more Bcl2 family members, these inhibitors induce apoptosis and restore apoptosis in tumor cells by mimicking the activity of natural antagonists of BCL-2 and other related proteins.

[0469] Venetoclax (also known as GDC-0199, ABT-199, and RG7601) is an exemplary selective Bcl2 inhibitor used in the methods described herein. Venetoclax is a pale to deep yellow solid with the empirical formula C0. 45 H 50 ClN7O7S has a molecular weight of 868.44 g / mol. Venetoclax has very low water solubility. Chemically, venetoclax is described as 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-yl)methyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide), and its chemical structure is as follows:

[0470]

[0471] Venetoclax has a CAS registration number of 1257044-40-8. Venetoclax is administered orally and sold under the brand names Venclexta and Venclyxto. Complete information on the preparation, dispensing, dosage, and administration schedule of venetoclax can be found in the local packaging insert (for the United States, see, for example, www.accessdata.fda.gov / drugsatfda_docs / label / 2016 / 208573s000lbl.pdf; for Europe, see, for example, www.ema.europa.eu / en / medicines / human / EPAR / venclyxto#product-information-section). In some embodiments, venetoclax is administered according to the dosage and frequency recommended in the local packaging insert.

[0472] ABT-737 is another exemplary selective Bcl2 inhibitor used in the methods described herein. ABT-737 simultaneously inhibits both Bcl2 and Bcl-xL, as shown in the empirical formula C. 42 H 45 ClN6O5S2 has a molecular weight of 813.43 g / mol. ABT-737 has a CAS registry number of 852-808-04-9. Chemically, ABT-737 is described as 4-{4-[(4'-chloro-2-biphenyl)methyl]-1-piperazinyl}-N-[(4-{[(2R)-4-(dimethylamino)-1-(phenylthio)-2-butyl]amino}-3-nitrophenyl)sulfonyl]benzamide, and its chemical structure is as follows:

[0473]

[0474] Another exemplary selective Bcl2 inhibitor used in the methods described herein is navittox (also known as ABT-263). Navittox inhibits Bcl2, Bcl-xL, and Bcl-w, with the empirical formula C. 47 H 55 ClF3N5O6S3, with a molecular weight of 974.6 g / mol. Navitoc's CAS registry number is 923564-51-6. ABT-737 is chemically described as 4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-morpholin-4-yl-1-phenylthiobut-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide and has the following chemical structure. Further details about Navitoc are provided, for example, in Tse et al. (2008) Cancer Res. 68(9):3421-3429.

[0475]

[0476] Another exemplary selective Bcl2 inhibitor used in the methods described herein is S55746 (also known as BCL201 and Servier-1). S55746 occupies the hydrophobic channel of BCL-2. Its selective characteristics do not show significant binding to MCL-1, BFL-1 (BCL2A1 / A1), and it has poor affinity for BCL-XL. S55746 has no cytotoxic activity against BCL-XL-dependent cells such as platelets (see, for example, Casara et al. (2008) Oncotarget. 9(28):29975-20088). The empirical formula for S55746 is C43 H 42 N4O6 has a molecular weight of 710.82 g / mol. S55746 has the CAS registry number 1448584-12-0. S55746 is chemically described as (S)-N-(4-hydroxyphenyl)-3-(6-(3-(morpholinomethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)benzo[d][1,3]dioxacyclopenten-5-yl)-N-phenyl-5,6,7,8-tetrahydroindoleazine-1-carboxamide, and its chemical structure is as follows:

[0477]

[0478] Treatment methods for solid tumors

[0479] In some embodiments, a method of treating a solid tumor in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) and (b) a platinum-based chemotherapeutic agent. In some embodiments, the solid tumor is colon cancer (e.g., colon carcinoma), 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, triplatinum tetranitrate, phenanthreneplatin, pyridine, and / or saxaplatin. 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 SIRPαD1 domain variant (e.g., the SIRPαD1 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 variant of the SIRPαD1 domain, which comprises 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, numbered according to the Kabat EU index; (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations, numbered according to the Kabat EU index; (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to the Kabat EU index; or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, numbered according to the Kabat EU index. In some embodiments, the polypeptide (e.g., a fusion polypeptide) administered to an individual contains the amino acid sequence of SEQ ID NO:136 or SEQ ID NO:135. In some embodiments, the peptide (e.g., a fusion peptide) forms a homodimer. In some embodiments, the peptide (e.g., a fusion peptide) and a platinum-based chemotherapeutic agent (e.g., cisplatin) are administered simultaneously, in parallel, or sequentially.

[0480] Platinum-based drugs (such as carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatinum tetranitrate, phenanthreneplatin, pyridine, and saxaplatin) are widely used antitumor drugs that can cause DNA cross-linking into single adducts, interstrand cross-links, intrastrand cross-links, or DNA-protein cross-links. Platinum-based drugs typically act on the adjacent N-7 position of guanine to form 1,2 intrastrand cross-links (Poklar et al. (1996). Proc. Natl. Acad. Sci. USA 93(15):7606-11; Rudd et al. (1995). Cancer Chemother. Pharmacol. 35(4):323–6). The resulting cross-links inhibit DNA repair and / or DNA synthesis in cancer cells.

[0481] Cisplatin is an exemplary platinum coordination compound used in the methods described herein. The chemical name of cisplatin is diaminedichloroplatinum, and its structural formula is as follows:

[0482]

[0483] Cisplatin is an inorganic, water-soluble platinum complex with the molecular formula Pt(NH3)2Cl2 and a molecular weight of 300.046. After hydrolysis, it reacts with DNA to produce intra- and inter-strand cross-links. These cross-links appear to impair DNA replication and transcription. The cytotoxicity of cisplatin is associated with cell arrest in the G2 phase of the cell cycle. Cisplatin, assigned CAS registry number 15663-27-1, can be used as a... Commercially available cisplatin products include CDDP, CISPLAN, CISPLAT, PLATIKEM, PLATIONCO, PRACTICIS, PLATICIS, BLASTOLEM, CISMAX, CISPLAN, CISPLATINUM, CISTEEN, DUPLAT, KEMOPLAT, ONCOPLATIN-AQ, PLATINEX, PLATIN, and TEVAPLATIN. Complete information on cisplatin preparation, dispensing, dosage, and administration schedules can be found in the local packaging insert (for the United States, see, for example, www.accessdata.fda.gov / drugsatfda_docs / label / 2011 / 018057s080lbl.pdf and www.accessdata.fda.gov / drugsatfda_docs / label / 2015 / 018057s083lbl.pdf). In some implementations, cisplatin is administered according to the dosage and frequency recommended in the local packaging insert.

[0484] Carboplatin is another exemplary platinum coordination compound used in the methods described herein. The chemical name of carboplatin is [1,1-cyclobutane-dicarboxylic acid (2-)-O,O]-diammineplatinum, (SP-4-2), and the structural formula of carboplatin is as follows:

[0485]

[0486] Carboplatin is a water-soluble platinum complex with the molecular formula C6H. 12 N₂O₄Pt has a molecular weight of 373.26. Carboplatin has been assigned CAS registry number 41575-94-4, and its mechanism of action is similar to that of cisplatin. Carboplatin is generally more commonly used in prescriptions than cisplatin.

[0487] Carboplatin can be used as Available commercially. Complete information on carboplatin preparation, reconstitution, dosage, and administration schedules can be found in the local packaging insert (for the United States, see, for example, www.accessdata.fda.gov / drugsatfda_docs / label / 2010 / 020452s005lbl.pdf and www.accessdata.fda.gov / drugsatfda_docs / label / 2012 / 077139Orig1s016lbl.pdf). In some embodiments, carboplatin is administered according to the dosage and frequency recommended in the local packaging insert.

[0488] In some embodiments, a method of treating a solid tumor in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-HER2 antibody, and (c) an anti-PDL1 antibody. In some embodiments, the anti-HER2 antibody is trastuzumab (CAS Registry No. 180288-69-1). In some embodiments, the anti-PDL1 antibody is atezolizumab (CAS Registry No. 1380723-44-3), avelumab (CAS Registry No. 1537032-82-8), or duruvarumab (CAS Registry No. 1428935-60-7). In some embodiments, the agent is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPαD1 domain variant (e.g., the SIRPαD1 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 variant of the SIRPαD1 domain, which comprises 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, numbered according to the Kabat EU index; (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations, numbered according to the Kabat EU index; (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to the Kabat EU index; or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, numbered according to the Kabat EU index. In some embodiments, the polypeptide (e.g., a fusion polypeptide) administered to an individual 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), an anti-HER2 antibody, and an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody) 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. + Solid tumor. In some embodiments, the solid tumor is colon cancer (e.g., HER2). +(colon cancer).

[0489] Treatment methods for gastric cancer or gastroesophageal junction (GEJ) cancer

[0490] In some embodiments, a method of treating gastric cancer or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-HER2 antibody, (c) an anti-VEGFR2 antibody, and (d) paclitaxel. In some embodiments, the anti-HER2 antibody is trastuzumab (CAS Registry No. 180288-69-1). In some embodiments, the anti-VEGFR2 antibody is ramucirumab (CAS Registry No. 947687-13-0). In some embodiments, the agent is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPαD1 domain variant (e.g., the SIRPαD1 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 variant of the SIRPαD1 domain, which comprises 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, numbered according to the Kabat EU index; (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations, numbered according to the Kabat EU index; (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to the Kabat EU index; or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, numbered according to the Kabat EU index. In some embodiments, the polypeptide (e.g., a fusion polypeptide) administered to an individual contains the amino acid sequence of SEQ ID NO:136 or SEQ ID NO:135. In some embodiments, the peptide (e.g., a fusion peptide) forms a homodimer. In some embodiments, the peptide (e.g., a fusion peptide), an anti-HER2 antibody, an anti-VEGFR2 antibody, and paclitaxel are administered simultaneously, in parallel, or sequentially. In some embodiments, the peptide (e.g., a fusion peptide) is administered to the individual at a dose of 10 mg / kg once weekly or 15 mg / kg once weekly. In some embodiments, the individual receiving treatment has gastric cancer or GEJ adenocarcinoma. In some embodiments, the individual receiving treatment has HER2... +Stomach cancer or HER2 + GEJ cancer (e.g., gastric cancer or GEJ cancer with HER2 overexpression). In some implementations, HER2... + Stomach cancer or HER2 + GEJ cancer is advanced and / or metastatic. In some embodiments, the individual receiving treatment has gastric cancer or GEJ cancer that progressed during or after prior treatment containing an anti-HER2 antibody (e.g., trastuzumab). In some embodiments, the individual receiving treatment has gastric cancer or GEJ cancer that progressed during or after prior treatment containing an anti-HER2 antibody (e.g., trastuzumab) and fluoropyrimidine. In some embodiments, the individual receiving treatment has gastric cancer or GEJ cancer that progressed during or after prior treatment containing an anti-HER2 antibody (e.g., trastuzumab) and a platinum-based chemotherapy agent. In some embodiments, the individual receiving treatment has gastric cancer or GEJ cancer that progressed during or after prior treatment containing an anti-HER2 antibody (e.g., trastuzumab) and / or fluoropyrimidine and / or a platinum-based chemotherapy agent. + (Gastric cancer or GEJ cancer). In some embodiments, the individual has failed prior treatment with anti-HER2 antibodies, anti-HER2 antibodies and fluoropyrimidine, or anti-HER2 antibodies and platinum-based chemotherapy agents (e.g., relapse or no response thereafter). In some embodiments, the fluoropyrimidine is fluorouracil (also known as 5-fluorouracil). In some embodiments, treatment with peptides, anti-HER2 antibodies, anti-VEGFR2 antibodies, and paclitaxel does not cause side effects. In some embodiments, treatment with peptides, anti-HER2 antibodies, anti-VEGFR2 antibodies, and paclitaxel causes only low-grade side effects.

[0491] 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) 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 No. 1374853-91-4). In some embodiments, the anti-HER2 antibody is trastuzumab (CAS Registry No. 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 SIRPαD1 domain variant (e.g., the SIRPαD1 domain variant described herein) and an Fc domain variant (e.g., the Fc domain variant described herein). In some embodiments, the polypeptide (e.g., the fusion polypeptide) comprises a SIRPαD1 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, numbered according to the Kabat EU index; (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations, numbered according to the Kabat EU index; (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to the Kabat EU index; or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, numbered according to the Kabat EU index. In some embodiments, the polypeptide (e.g., a fusion polypeptide) administered to an individual contains the amino acid sequence of SEQ ID NO:136 or SEQ ID NO:135. In some implementations, the polypeptide (e.g., a fusion polypeptide) forms a homodimer.In some embodiments, the peptide (e.g., a fusion peptide), anti-PD-1 antibody, anti-HER2 antibody, 5-fluorouracil, and platinum-based chemotherapy agent are administered simultaneously, concurrently, or sequentially. In some embodiments, the peptide (e.g., a fusion peptide), anti-PD-1 antibody, anti-HER2 antibody, capecitabine, and platinum-based chemotherapy agent are administered simultaneously, concurrently, or sequentially. In some embodiments, the individual receiving treatment has HER2-overexpressing gastric cancer or HER2-overexpressing GEJ cancer. In some embodiments, the gastric cancer or GEJ cancer is advanced and / or metastatic. In some embodiments, the individual has not received prior treatment for gastric cancer or GEJ cancer.

[0492] Treatment methods for head and neck cancer

[0493] In some embodiments, a method of treating head and neck cancer (e.g., head and neck squamous cell carcinoma or HNSCC) in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) a PD-1 inhibitor, (c) an antimetabolite, and (d) a platinum-based agent. In some embodiments, the PD-1 inhibitor is a small molecule inhibitor, an antisense nucleotide, or a peptide. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is pembrolizumab, nivolumab, pildizilzumab, cimipril, or BMS-936559. In some embodiments, the anti-PD-1 antibody is pembrolizumab (CAS Registry No. 1374853-91-4). In some embodiments, the antimetabolite is 5-fluorouracil, 6-mercaptopurine, capecitabine, cytarabine, fluorouridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, or clarithrexate. In some embodiments, the antimetabolite is 5-fluorouracil. In some embodiments, the platinum-based chemotherapeutic agent is carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatinum tetranitrate, phenanthreneplatin, pyridine, or saplatin. 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 SIRPαD1 domain variant (e.g., the SIRPαD1 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 SIRPαD1 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, numbered according to the Kabat EU index; (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations, numbered according to the Kabat EU index; (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to the Kabat EU index; or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, numbered according to the Kabat EU index. In some embodiments, the polypeptide (e.g., a fusion polypeptide) administered to an individual contains the amino acid sequence of SEQ ID NO:136 or SEQ ID NO:135. In some implementations, the polypeptide (e.g., a fusion polypeptide) forms a homodimer.In some embodiments, the peptide (e.g., a fusion peptide), a PD-1 inhibitor (e.g., an anti-PD-1 antibody, such as pembrolizumab), an antimetabolite (e.g., 5-fluorouracil), and a platinum-based chemotherapy agent (e.g., cisplatin or carboplatin) are administered simultaneously, concurrently, or sequentially. In some embodiments, the peptide (e.g., a fusion peptide) is administered to the individual at a dose of 10 mg / kg once weekly or 15 mg / kg once weekly. In some embodiments, the individual receiving treatment has HNSCC. In some embodiments, the HNSCC is advanced and / or metastatic. 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 the peptide, PD-1 inhibitor (e.g., pembrolizumab), antimetabolite (e.g., 5-fluorouracil), and platinum-based chemotherapy agent (e.g., cisplatin or carboplatin) does not cause side effects. In some implementations, treatment with peptides, PD-1 inhibitors (e.g., pembrolizumab), antimetabolites (e.g., 5-fluorouracil), and platinum-based chemotherapy agents (e.g., cisplatin or carboplatin) results in only low-grade side effects.

[0494] Combination cancer therapy including anti-TROP2 antibodies

[0495] In some embodiments, a method of treating cancer in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) and (b) an anti-TROP2 antibody. In some embodiments, the anti-TROP2 antibody is RS7, described in U.S. Patent No. 10,179,171, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the anti-TROP2 antibody is conjugated with a drug (i.e., an antibody-drug conjugate or “ADC”). In some embodiments, the anti-TROP2 ADC is goxatozumab (also known as hRS7-SN38 or IMMU-132), described in US2017 / 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 SIRPαD1 domain variant (e.g., the SIRPαD1 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 variant of the SIRPαD1 domain, which comprises 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, numbered according to the Kabat EU index; (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations, numbered according to the Kabat EU index; (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to the Kabat EU index; or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, numbered according to the Kabat EU index. In some embodiments, the polypeptide (e.g., a fusion polypeptide) administered to an individual 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 anti-TROP2 antibody are administered simultaneously, in parallel, or sequentially. In some embodiments, the cancer is a solid tumor, gastric cancer, nasopharyngeal carcinoma, gallbladder cancer, cervical cancer, extranodal NK / T-cell lymphoma, lung cancer, laryngeal squamous cell carcinoma, colon cancer, hilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrioid endometrial cancer, or ovarian cancer. In some embodiments, the cancer is characterized by TROP2 overexpression. In some embodiments, the cancer is not characterized by TROP2 overexpression.

[0496] Methods to increase target cell phagocytosis

[0497] In some embodiments, a method for increasing the phagocytic activity of target cells (e.g., cancer cells) is provided, 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, 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 goxatozumab (also known as hRS7-SN38 or IMMU-132), described in US2017 / 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 SIRPαD1 domain variant (e.g., the SIRPαD1 domain variant described herein) and an Fc domain variant (e.g., the Fc domain variant described herein). In some embodiments, the polypeptide (e.g., the fusion polypeptide) comprises a SIRPαD1 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, numbered according to the Kabat EU index; (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations, numbered according to the Kabat EU index; (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to the Kabat EU index; or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, numbered according to the Kabat EU index. In some embodiments, the polypeptide (e.g., a fusion polypeptide) administered to an individual 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 target cell is a cancer cell. In some embodiments, the cancer cell is a solid tumor cell, gastric cancer cell, nasopharyngeal cancer cell, gallbladder cancer cell, cervical cancer cell, extranodal NK / T cell lymphoma cell, lung cancer cell, laryngeal squamous cell carcinoma cell, colon cancer cell, hilar cholangiocarcinoma cell carcinoma cell, pancreatic cancer cell, oral squamous cell carcinoma cell, endometrioid endometrial cancer cell, or ovarian cancer cell.

[0498] In some embodiments, a method for enhancing phagocytosis of target cells is provided, the method comprising contacting the target cell with an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) and (b) a second agent capable of enhancing phagocytosis. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPαD1 domain variant (e.g., the SIRPαD1 domain variant described herein) and an Fc domain variant (e.g., the Fc domain variant described herein). In some embodiments, the polypeptide (e.g., the fusion polypeptide) comprises a SIRPαD1 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, numbered according to the Kabat EU index; (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations, numbered according to the Kabat EU index; (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to the Kabat EU index; or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations, numbered according to the Kabat EU index. In some embodiments, the polypeptide (e.g., a fusion polypeptide) administered to an individual contains the amino acid sequence of SEQ ID NO:136 or SEQ ID NO:135. In some embodiments, the peptide (e.g., a fusion peptide) forms a homodimer. In some embodiments, the second agent enhances phagocytosis, for example, by blocking the "don't eat me" signal. Exemplary agents include, but are not limited to, anti-LILRB2 antibodies, anti-LILRB1 antibodies, anti-SIGLEC-10 antibodies, anti-CD24 antibodies, anti-SIRPα antibodies, anti-PD1 antibodies (e.g., anti-PD-1 antagonist antibodies), and anti-PD-L1 antibodies (e.g., anti-PD-L1 antagonist antibodies). In some embodiments, the second agent enhances phagocytosis, for example, by enhancing the "eat me" signal. Exemplary agents include, but are not limited to, BTK activators, TLR agonists, agents that promote the interaction between Mac-1 and SLAMF7, and agents that promote the interaction between calreticulin and LRP1. Other exemplary agents that enhance phagocytosis include, for example, agents that regulate podocyte adhesion, agents that regulate lamin A expression levels, activators of SHP-1 phosphatase activity, and activators of myosin IIa assembly.In some embodiments, the method includes contacting target cells with (a) a polypeptide (e.g., a fusion polypeptide) containing a SIRPαD1 domain variant (e.g., the SIRPαD1 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 includes contacting target cells with (a) the fusion polypeptide and (b) a BTK activator. In some embodiments, the method includes contacting target cells with (a) the fusion polypeptide and (b) a TLR agonist.

[0499] In some embodiments, the method includes contacting target cells with (a) a polypeptide (e.g., a fusion polypeptide) containing a SIRPαD1 domain variant (e.g., the SIRPαD1 domain variant described herein) and an Fc domain variant (e.g., the Fc domain variant described herein) and (b) two or more agents (e.g., including but not limited to the two or more agents described herein) capable of enhancing phagocytosis. In some embodiments, the method includes contacting target cells with (a) a polypeptide (e.g., a fusion polypeptide) containing a SIRPαD1 domain variant (e.g., the SIRPαD1 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 includes contacting target cells with (a) a fusion peptide, (b) an anti-LILBR2 antibody, and (c) an anti-PD1 antibody (e.g., an anti-PD-1 antagonist antibody). In some embodiments, the method includes contacting target cells with (a) a fusion peptide, (b) an anti-LILBR2 antibody, and (c) an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody).

[0500] 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, contacting target cells with (a) a polypeptide containing a SIRPαD1 domain variant (e.g., the SIRPαD1 domain variant described herein) and an Fc domain variant (e.g., the Fc domain variant described herein) and (b) with one or more agents capable of enhancing phagocytosis, compared to contacting target cells with one or more agents capable of enhancing phagocytosis (i.e., in the absence of a polypeptide containing a SIRPαD1 domain variant (e.g., the SIRPαD1 domain variant described herein) and an Fc domain variant (e.g., the Fc domain variant described herein), increases 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 greater than 99%.

[0501] reagent kits and products

[0502] In another embodiment of the invention, an article or kit comprising a polypeptide (e.g., the fusion polypeptide described herein) is provided, said polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant. In some embodiments, the SIRPαD1 domain variant comprises an amino acid sequence selected from the group consisting of SEQ ID NO:81 and SEQ ID NO:85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region containing L234A, L235A, G237A, and N297A mutations, numbered according to the Kabat EU index; (ii) a human IgG2 Fc region containing A330S, P331S, and N297A mutations, numbered according to the Kabat EU index; (iii) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, and delG236 mutations, numbered according to the Kabat EU index; or (iv) a human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, 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 comprises the amino acid sequence of SEQ ID NO:135 or SEQ ID NO:136. In some embodiments, the kit or product is used according to the treatment methods provided herein.

[0503] In some embodiments, the kit or product also contains an anti-BCL2 antibody. In some embodiments, the anti-BCL2 inhibitor is venetoclax. In some embodiments, the kit includes a packaging insert or label having instructions for treating an individual (such as a human individual) with cancer (e.g., leukemia, including (but not limited to) acute or chronic lymphocytic leukemia, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), chronic myeloid leukemia (CML), hairy cell leukemia, chronic myelomonocytic leukemia (CMML), juvenile dementia. Instructions for use to treat or delay the progression of myelomonocytic leukemia (JMML), large granular lymphocytic (LGL) leukemia, blastic plasmacytoid dendritic cell tumor (BPDCN), B-prolymphocytic leukemia (B-PLL), T-prolymphocytic leukemia (T-PLL), multiple myeloma (MM), and non-Hodgkin's lymphoma (such as diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, mantle cell lymphoma (MCL), peripheral T-cell lymphoma (PTCL), lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma (MZL), and follicular lymphoma (FL)).

[0504] In some embodiments, the kit or product further comprises a platinum-based chemotherapy agent. In some embodiments, the platinum-based chemotherapy agent is carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatinum tetranitrate, phenanthreneplatin, pyridineplatin, or saxaplatin. In some embodiments, the kit includes a packaging insert or label with instructions for treating or delaying the progression of a solid tumor (e.g., colon cancer, coloncarcinoma, lung cancer, head and neck cancer, esophageal cancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer, testicular cancer, endometrial cancer, liver cancer, gastric cancer, brain tumor, mesothelioma, or neuroblastoma) in an individual (such as a human individual) using a combination of a peptide (e.g., a fusion peptide) and a platinum-based chemotherapy agent (e.g., cisplatin).

[0505] 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 duruvarumab). In some embodiments, the kit includes a packaging insert or label with instructions for treating an individual (such as a human individual) with cancer (e.g., a solid tumor) or delaying its progression using a peptide (e.g., a fusion peptide) in combination with an anti-HER2 antibody (e.g., trastuzumab) and a PD-L1 inhibitor (e.g., atezolizumab, avelumab, or duruvarumab). In some embodiments, the cancer (e.g., a solid tumor) is colon cancer, lung cancer, head and neck cancer, esophageal cancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer, testicular cancer, endometrial cancer, liver cancer, gastric cancer, gastroesophageal junction cancer, brain tumor, mesothelioma, or neuroblastoma. In some embodiments, the cancer (e.g., a solid tumor) is HER2. + Cancer. In some implementations, the cancer is colon cancer (e.g., HER2). + (colon cancer).

[0506] 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 packaging insert or label with instructions for treating, for example, an individual (such as a human individual) with gastric or gastroesophageal junction (GEJ) cancer or delaying its progression using a peptide (e.g., a fusion peptide) in combination with an anti-HER2 antibody (e.g., trastuzumab), an anti-VEGFR2 antibody (e.g., ramucirumab), and paclitaxel, according to the methods described herein.

[0507] 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 packaging insert or label with instructions for treating an individual (e.g., a human individual) with gastric or gastroesophageal junction (GEJ) cancer or delaying its progression using a peptide (e.g., a fusion peptide) in combination with 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 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 implementations, the kit includes a packaging insert or label with instructions for treating an individual (such as a human individual) with gastric or gastroesophageal junction (GEJ) cancer or delaying its progression using a combination of a peptide (e.g., a fusion peptide) 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).

[0508] In some implementations, the kit or product also contains a PD-1 inhibitor (e.g., an anti-PD-1 antibody, such as pembrolizumab, nivolumab, pildizilzumab, cimiprizumab, or BMS936559), an antimetabolite (e.g., 5-fluorouracil, 6-mercaptopurine, capecitabine, cytarabine, fluorouridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, celestate), and a platinum-based agent (e.g., cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatinum tetranitrate, phenanthreneplatin, pyrplatin, or saxaplatin). In some implementations, the kit includes a packaging insert or label with instructions for treating an individual (such as a human individual) with head and neck cancer (e.g., squamous cell carcinoma of the head and neck) or delaying its progression using, for example, a combination of a peptide (e.g., a fusion peptide) with a PD-1 inhibitor (e.g., pembrolizumab, nivolumab, pildizumab, cimipril, or BMS936559), an antimetabolites (e.g., 5-fluorouracil, 6-mercaptopurine, capecitabine, cytarabine, fluorouridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, photoaminoprexate) and a platinum-based agent (e.g., cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatinum tetranitrate, phenanthreneplatin, pyrplatin, or sapaplatin).

[0509] 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 Nos. 10,179,171) or goxatozumab. In some embodiments, the kit comprises a packaging insert or label having information on treating an individual (such as a human individual) with TROP2 using a combination of a peptide (e.g., a fusion peptide) and an anti-TROP2 antibody (e.g., cisplatin). + Instructions for use regarding cancer (e.g., solid tumors, gastric cancer, nasopharyngeal carcinoma, gallbladder cancer, cervical cancer, extranodal NK / T-cell lymphoma, lung cancer, laryngeal squamous cell carcinoma, colon cancer, hilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrioid endometrial cancer, or ovarian cancer) or delaying its progression.

[0510] In some embodiments, the peptide (e.g., a fusion peptide) and one or more additional anticancer agents (e.g., as outlined in the embodiments above) are provided together in the kit. In some embodiments, the peptide (e.g., a fusion peptide) and one or more additional anticancer agents are provided in the same container or separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. Containers can be formed from a variety of materials, such as glass, plastics (e.g., polyvinyl chloride or polyolefins), or metal alloys (e.g., stainless steel or Hastelloy). In some embodiments, the container contains the formulation and a label held on or associated with the container indicating instructions for use. The product or kit may also include other materials desired from a commercial and user perspective, including additional buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for use. In some embodiments, the product also contains one or more other agents (e.g., chemotherapeutic agents, antitumor agents, therapeutic antibodies, etc.). Suitable containers for one or more agents include, for example, bottles, vials, bags, and syringes.

[0511] This specification is considered sufficient to enable those skilled in the art to practice the invention. In addition to those shown and described herein, various modifications of the invention will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

[0512] Example

[0513] This disclosure will be more fully understood by referring to the following embodiments. However, these embodiments should not be construed as limiting the scope of this disclosure. It should be understood that the embodiments and implementations described herein are for illustrative purposes only and will suggest to those skilled in the art various modifications or changes based on these embodiments and implementations, and will be included within the spirit and scope of this application and the appended claims.

[0514] Example 1A: Antitumor activity of drug A in combination with venetoclax in an acute leukemia model.

[0515] In this embodiment, the antitumor activity of drug A, namely an exemplary peptide containing SIRPαd1 domain variants and Fc variants, in combination with venetoclax was evaluated in an RS4;11 xenograft model.

[0516] Materials and methods

[0517] RS4; 11 Xenograft Model

[0518] RS4;11 cells (described in Stong et al. (1985) Blood. 65(1):21-31) were administered at 5 x 10⁻⁶ cells per mouse. 6 A concentration of [number] cells was injected into the right side of NOD-SCID female mice using a 1:1 ratio of Corning gel and RPMI 1640. Tumors were monitored until the average size of all tumors reached 190 mm. 3 Mice were randomly assigned to four groups: a PBS control group, a venetoclax (Selleckchem) group, a drug A group, and a venetoclax / drug A combination group, with 10 mice in each group. The formulation ratio of venetoclax was DMSO:ethanol:Cremophor EL:5% glucose solution (D5W) at a volume ratio of 2.5:5:10:20:67.5. Mice treated with venetoclax were administered 250 μg of venetoclax orally via tube feeding twice, with an interval of 3 days. Mice treated with drug A were administered 10 mg / kg of venetoclax intraperitoneally four times, with an interval of 3–4 days. Mice treated with venetoclax / drug A were administered 250 μg of venetoclax orally via tube feeding twice, with an interval of 3 days, and drug A was administered at a dose of 10 mg / kg one day after venetoclax administration, four times, with an interval of 3–4 days. The tumor was measured in two dimensions using calipers, and the tumor volume was ...

Claims

1. Use of peptides containing SIRPαD1 domain variants and Fc domain variants in the preparation of medicaments for the treatment of gastric cancer or gastroesophageal junction (GEJ) cancer in individuals; The SIRPαD1 domain variant contains the amino acid sequence of SEQ ID NO:85; The Fc domain variants mentioned therein are human IgG1 Fc regions containing L234A, L235A, G237A, and N297A mutations, where the numbering is based on Kabat's EU index; The drug is intended to be administered to the individual in combination with (a) an anti-HER2 antibody, (b) an anti-VEGFR2 antibody, and (c) paclitaxel; The individual in question has received at least one prior treatment for gastric cancer or GEJ cancer; and the individual in question is a human being.

2. The use according to claim 1, wherein 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 chemotherapy agent.

3. The use according to claim 1 or 2, wherein the anti-HER2 antibody is trastuzumab.

4. The use according to claim 1 or 2, wherein the anti-VEGFR2 antibody is ramucirumab.

5. The use according to claim 1 or 2, wherein the gastric cancer or GEJ cancer is HER2. + Stomach cancer or HER2 + GEJ cancer.

6. The use according to claim 1 or 2, wherein the peptide comprising the SIRPαD1 domain variant and the Fc domain variant is administered once weekly at a dose of 10 mg / kg.

7. The use according to claim 1 or 2, wherein the peptide comprising the SIRPαD1 domain variant and the Fc domain variant is administered once weekly at a dose of 15 mg / kg.

8. The use according to claim 1 or 2, wherein the Fc domain variant comprises the amino acid sequence of SEQ ID NO:

91.

9. The use according to any one of claims 1 or 2, wherein the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO:

136.

10. The use according to claim 1 or 2, wherein the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant forms a homodimer.

Images