Methods of treating cancer by SIRP alpha-Fc fusion in combination with immune checkpoint inhibitors

Abstract

Methods for treating cancer (e.g., non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), HER2-positive gastric / gastroesophageal junction (GEJ) cancer, de novo or transformed diffuse large B-cell lymphoma (DLBCL), or indolent lymphoma) in an individual are provided, the methods comprising administering to the individual (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) an anti-cancer antibody (e.g., an anti-PD1 antibody, an anti-HER2 antibody, or an anti-CD20 antibody). Related kits and pharmaceutical compositions are also provided.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 855,821, filed May 31, 2019, and U.S. Provisional Application No. 63 / 022,187, filed May 8, 2020, the contents of each of which are hereby incorporated by reference in their entirety.

[0002] Submission of Sequence Listing in ASCII Text File The content of the following submission in ASCII text file is hereby incorporated by reference in its entirety: Sequence Listing in Computer - Readable Format (CRF) (file name: 757972001040SEQLIST.TXT, date of record: May 26, 2020, size: 333 KB).

[0003] The present invention relates to a method of treating cancer, which includes administering a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant and an Fc domain variant in combination with a therapeutic antibody.

Background Art

[0004] Many cancers have a poor prognosis even when treated with available therapeutic agents. For example, metastatic non-small cell lung cancer (NSCLC) patients who have been administered PD-1 and PD-L1 checkpoint inhibitors that did not respond to prior platinum-based therapies have a median overall survival of about 1 year (Garon et al., New Engl J Med (2015) 372:2018-28; Herbst et al., Lancet (2016) 387:1540-50; Fehrenbacher et al., Lancet (2016) 387(10030):1837-46), and more than half of NSCLC patients with advanced disease have an overall 5-year survival rate of 17.7% (U.S. Cancer Statistics Working Group, available at website www.cdc.gov / uscs). Similarly, the overall 5-year survival rate of gastric cancer patients in the United States is 30.4% (US Cancer Statistics Working Group). In patients with recurrent indolent lymphoma, subsequent relapses usually occur with an increasingly aggressive histology and a 30% risk of transformation within one series up to 10 years (Montoto et al., J Clin Oncol (2007) 25(17):2426-33). Furthermore, in the case of patients with recurrent aggressive histotypes, cure is rare and new salvage regimens are needed (Larouche et al., J Clin Oncol (2010) 28(12):2094-100). CD20-positive non-Hodgkin lymphoma (NHL) is the 10th most common cancer in the world and the 10th leading cause of cancer death, with 199,670 deaths annually worldwide (World Health Organization 2016(a), available at website globocan.iarc.fr / Pages / fact_sheets_cancer.aspx). It is estimated that there are more than 35,000 metastatic HNSCC patients in the United States, and new cases span more than 50,000 at all stages diagnosed in 2019. The 5-year survival rate is 84% for patients diagnosed with localized disease but decreases to only 39% for patients diagnosed with metastatic disease.There is a need in the art for new therapies that serve as additional treatment options and improve the outcome of such patients.

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

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

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

[0008] A method of treating non-small cell lung cancer (NSCLC) in an individual is provided, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) an anti-PD-1 antibody, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), and the individual is human. In some embodiments, the individual's NSCLC is progressing during prior immune checkpoint inhibitor (CPI) therapy and / or has a PD-L1 tumor proportion score (TPS) of less than 50%.

[0009] Also provided is a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant for use in the manufacture of a medicament for treating NSCLC in an individual, wherein the medicament is for use in combination with an anti-PD1 antibody (such as formulated for use), wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), and the individual is human. In some embodiments, the individual's NSCLC is progressing during prior immune checkpoint inhibitor (CPI) therapy and / or has a PD-L1 tumor proportion score (TPS) of less than 50%.

[0010] Compositions (such pharmaceutical compositions) comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant for use in combination with an anti-PD1 antibody for treating NSCLC in an individual (e.g., for use in a method of treating NSCLC in an individual) are also provided, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), and the individual is human. In some embodiments, the individual's NSCLC is progressing during prior immune checkpoint inhibitor (CPI) therapy and / or has a PD-L1 tumor proportion score (TPS) of less than 50%.

[0011] In some embodiments, the prior CPI (immune checkpoint inhibitor therapy) comprised one or more agents selected from the group consisting of nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab. In some embodiments, the anti-PD-1 antibody blocks the interaction between PD-1 and PD-L1. In some embodiments, the anti-PD-1 antibody is pembrolizumab. In some embodiments, pembrolizumab is administered to the individual at a dose of 200 mg every 3 weeks (Q3W) by intravenous (IV) infusion.

[0012] Methods for treating head and neck squamous cell carcinoma (HNSCC) of an individual are also provided, the methods comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) an anti-PD-1 antibody, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), wherein the individual's HNSCC has progressed during or after prior platinum therapy, and the individual is human.

[0013] Also provided are polypeptides comprising an SIRPα D1 domain variant and an Fc domain variant for use in the manufacture of a medicament for treating HNSCC in an individual, wherein the medicament is for use in combination with an anti-PD1 antibody (such as formulated for use), wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), wherein the HNSCC in the individual has progressed during or after prior platinum therapy, and the individual is human.

[0014] A composition (such a pharmaceutical composition) comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant for use in combination with an anti-PD1 antibody for treating HNSCC of an individual (e.g., for use in a method of treating HNSCC of an individual), wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), wherein the HNSCC of the individual has progressed during or after prior platinum therapy, and the individual is human.

[0015] In some embodiments, an individual having HNSCC has received prior treatment with an immune checkpoint inhibitor (e.g., an immune checkpoint inhibitor described herein). In some embodiments, such an individual is considered / mentioned as having "experienced a checkpoint inhibitor". In some embodiments, an individual having HNSCC has not received prior treatment with an immune checkpoint inhibitor. In some embodiments, such an individual is considered / mentioned as having "not experienced a checkpoint inhibitor". In some embodiments, the prior platinum therapy included one or more therapeutic agents selected from the group consisting of cisplatin, carboplatin, and oxaliplatin. In some embodiments, the anti-PD-1 antibody blocks the interaction between PD-1 and PD-L1. In some embodiments, the anti-PD-1 antibody is pembrolizumab. In some embodiments, pembrolizumab is administered to the individual at a dose of 200 mg every three weeks (Q3W) by intravenous (IV) infusion (such as that formulated for administration).

[0016] A method for treating an individual's HER2-positive gastric / gastroesophageal junction (GEJ) cancer is provided, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) an anti-HER2 antibody, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), wherein the individual's gastric / GEJ cancer has progressed after prior treatment with a fluoropyrimidine-based therapy and / or prior treatment with an anti-HER2 antibody, and the individual is human.

[0017] Also provided is a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant for use in the manufacture of a medicament for treating HER2-positive gastric / gastroesophageal junction (GEJ) cancer of an individual, wherein the medicament is for use in combination with an anti-HER2 antibody (such as one formulated for use), where the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; and the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (where numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows the Kabat EU index), where the gastric / GEJ cancer of the individual has progressed after prior treatment with a fluoropyrimidine-based therapy and / or prior treatment with an anti-HER2 antibody, and the individual is human.

[0018] A composition (such a pharmaceutical composition) comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant for use in combination with an anti-HER2 antibody for treating HER2-positive gastric / gastroesophageal junction (GEJ) cancer in an individual (e.g., for use in a method of treating HER2-positive gastric / gastroesophageal junction (GEJ) cancer in an individual), wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), wherein the individual's gastric / GEJ cancer has progressed after prior treatment with a fluoropyrimidine-based therapy and / or prior treatment with an anti-HER2 antibody, and the individual is human.

[0019] In some embodiments, the prior treatment with a fluoropyrimidine-based therapy or the prior treatment with an anti-HER2 antibody comprises one or more therapeutic agents selected from the group consisting of trastuzumab, pertuzumab, 5-fluorouracil, capecitabine, margetuximab, and FOLFOX. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, trastuzumab is administered to the individual at an initial dose of 8 mg / kg and subsequent doses of 6 mg / kg (such as those formulated for administration), and trastuzumab is administered to the individual every 3 weeks (Q3W) by IV infusion.

[0020] A method for treating an individual's aggressive non-Hodgkin lymphoma (NHL) is provided, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) an anti-CD20 antibody, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), wherein the individual's aggressive NHL is relapsed and / or refractory to prior treatment of aggressive NHL and there is no available curative therapy, and the individual is human.

[0021] Also provided is a polypeptide comprising an SIRPα D1 domain variant and an Fc domain variant for use in the manufacture of a medicament for treating aggressive non-Hodgkin lymphoma (NHL) in an individual, wherein the medicament is for use in combination with an anti-CD20 antibody (such as formulated for use), wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; and the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), wherein the aggressive NHL in the individual is relapsed and / or refractory to prior treatment of aggressive NHL, there is no available curative therapy, and the individual is human.

[0022] Polypeptides comprising a SIRPα D1 domain variant and an Fc domain variant for use in combination with an anti-CD20 antibody for treating an individual's aggressive non-Hodgkin lymphoma (NHL) (e.g., for use in a method of treating aggressive NHL), wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; and the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), wherein the individual's aggressive NHL is relapsed and / or refractory to prior treatment of aggressive NHL and there is no available curative therapy, and the individual is human.

[0023] In some embodiments, the aggressive NHL is diffuse large B-cell lymphoma (DLBCL), such as de novo DLBCL or transformed DLBCL. In some embodiments, prior treatment of the aggressive NHL is rituximab, cyclophosphamide, doxorubicin, vincristine, gemcitabine, lenalidomide, prednisone, prednisolone, etoposide, procarbazine, epirubicin, bendamustine, cisplatin, oxaliplatin, cytarabine, ifosfamide, carboplatin, dexamethasone, mesna, carmustine, melphalan, solumedrol, methylglyoxal bis(guanylhydrazone), thiotepa, methotrexate, ibrutinib, obinutuzumab, tisagenlecleucel, axicabtagene, brentuximab vedotin, and combinations thereof. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, rituximab is administered to an individual (such as that formulated for administration) by IV infusion at a dose of 375 mg / m 2 and is administered to the individual (such as that formulated for administration) once a week for 4 weeks and then once a month.

[0024] A method of treating non-Hodgkin's lymphoma in an individual is provided, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) an anti-CD20 antibody, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), wherein the non-Hodgkin's lymphoma in the individual is relapsed and / or refractory to a prior treatment of non-Hodgkin's lymphoma, and the individual is human.

[0025] Also provided is a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant for use in the manufacture of a medicament for treating an individual's indolent lymphoma, wherein the medicament is for use in combination with an anti-CD20 antibody (such as formulated for use), wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), wherein the individual's indolent lymphoma is recurrent and / or refractory to prior treatment of indolent lymphoma, and the individual is human.

[0026] A composition (such a pharmaceutical composition) comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant for use in combination with an anti-CD20 antibody for treating non-Hodgkin's lymphoma in an individual (e.g., for use in a method of treating non-Hodgkin's lymphoma in an individual), wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; and the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), wherein the non-Hodgkin's lymphoma in the individual is relapsed and / or refractory to a prior treatment of non-Hodgkin's lymphoma, and the individual is human.

[0027] In some embodiments, the indolent lymphoma is indolent non-Hodgkin lymphoma (NHL). In some embodiments, the indolent NHL is marginal zone lymphoma or follicular lymphoma. In some embodiments, the prior treatment of indolent lymphoma consists of rituximab, cyclophosphamide, doxorubicin, vincristine, gemcitabine, lenalidomide, prednisone, prednisolone, etoposide, procarbazine, epirubicin, bendamustine, cisplatin, oxaliplatin, cytarabine, ifosfamide, carboplatin, dexamethasone, mesna, carmustine, melphalan, solumedrol, methylglyoxal bis(guanylhydrazone), thiotepa, methotrexate, ibrutinib, obinutuzumab, tisagenlecleucel, axicabtagene, brentuximab vedotin, fludarabine mitoxantrone, everolimus, bortezomib, navitoclax, and combinations thereof. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, rituximab is administered to an individual (such as those formulated for administration) by IV infusion at a dose of 375 mg / m 2 and is administered to the individual (such as those formulated for administration) once a week for 4 weeks and then once a month. In some embodiments, a polypeptide comprising an SIRPα D1 domain variant and an Fc domain variant (or a pharmaceutical product manufactured using such a polypeptide or a pharmaceutical composition comprising such a polypeptide) is administered to an individual (such as those formulated for administration) once a week (QW) at a dose of 10 mg / kg or 15 mg / kg, for example, by IV infusion.

[0028] In some embodiments of any of the methods of this specification, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, the Fc domain variant is a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, numbered according to the Kabat EU index. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 136. In some embodiments, the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant forms a homodimer.

[0029] In some embodiments, a polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant (or a pharmaceutical product produced using such a polypeptide or a pharmaceutical composition comprising such a polypeptide, etc.) is administered to an individual once a week (QW) at a dose of 10 mg / kg (such as those formulated for administration). In some embodiments, a polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant (a pharmaceutical product produced therefrom or a pharmaceutical composition comprising such a polypeptide) is administered to an individual by IV infusion (such as those formulated for administration).

[0030] In an individual (e.g., a human individual) in need thereof, a kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant (or a pharmaceutical product manufactured using such a polypeptide or a pharmaceutical composition comprising such a polypeptide) for use in combination with pembrolizumab to treat non-small cell lung cancer (NSCLC) according to the methods described herein is also provided. In some embodiments, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (where numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows the Kabat EU index). In some embodiments, the individual's NSCLC is progressing during prior immune checkpoint inhibitor (CPI) therapy and / or has a PD-L1 tumor proportion score (TPS) of less than 50%. In some embodiments, the kit further comprises instructions for administering pembrolizumab at a dose of 200 mg every 3 weeks (Q3W) by intravenous infusion. In some embodiments, the kit further comprises instructions for administering the polypeptide (e.g., the fusion polypeptide) at a dose of 10 mg / kg once a week, e.g., by intravenous infusion.

[0031] In an individual (e.g., a human individual) in need thereof, a kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant for use in combination with pembrolizumab to treat head and neck squamous cell carcinoma (HNSCC) according to the methods described herein (or a pharmaceutical product manufactured using such a polypeptide or a pharmaceutical composition comprising such a polypeptide, etc.) is also provided. In some embodiments, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), wherein the individual's HNSCC has progressed during or after prior platinum therapy. In some embodiments, the individual has received prior treatment with an immune checkpoint inhibitor. In some embodiments, the individual has not received prior treatment with an immune checkpoint inhibitor. In some embodiments, the kit further comprises instructions for administering pembrolizumab at a dose of 200 mg every 3 weeks (Q3W) by intravenous infusion. In some embodiments, the kit further comprises instructions for administering the polypeptide (e.g., the fusion polypeptide) at a dose of 10 mg / kg once a week, e.g., by intravenous infusion.

[0032] In an individual (e.g., a human individual) in need thereof, a kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier for use in combination with trastuzumab to treat HER2-positive gastric / gastroesophageal junction (GEJ) cancer according to the methods described herein is also provided herein. In some embodiments, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), wherein the individual's HER2-positive gastric / GEJ cancer has progressed after prior fluoropyrimidine-based therapy or prior therapy with an anti-HER2 antibody. In some embodiments, the kit further comprises instructions for administering trastuzumab at an initial dose of 8 mg / kg and each subsequent dose at a dose of 6 mg / kg, and trastuzumab is administered to the individual by IV infusion every 3 weeks (Q3W). In some embodiments, the kit further comprises instructions for administering the polypeptide (e.g., the fusion polypeptide) at a dose of 10 mg / kg once a week, e.g., by IV infusion.

[0033] In an individual in need thereof (e.g., a human individual), a kit comprising a polypeptide comprising an SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier for use in combination with rituximab to treat aggressive non-Hodgkin lymphoma (NHL) according to the methods described herein is also provided. In some embodiments, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (where numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows the Kabat EU index), wherein the individual's aggressive NHL is relapsed and / or refractory to prior treatment of aggressive NHL and there is no available curative therapy. In some embodiments, the aggressive NHL is diffuse large B-cell lymphoma (DLBCL), e.g., de novo DLBCL or transformed DLBCL. In some embodiments, the aggressive NHL is mantle cell lymphoma. In some embodiments, the kit further comprises instructions for administering rituximab at a dose of 375 mg / m 2 by IV infusion, and rituximab is administered to the individual once a week for 4 weeks and then once a month. In some embodiments, the kit further comprises instructions for administering the polypeptide (e.g., the fusion polypeptide) at a dose of 10 mg / kg or 15 mg / kg once a week, e.g., by IV infusion.

[0034] In an individual in need thereof (e.g., a human individual), a kit comprising a polypeptide comprising an SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier for use in treating indolent lymphoma in combination with rituximab according to the methods described herein is also provided. In some embodiments, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), wherein the individual's indolent lymphoma is relapsed and / or refractory to prior treatment of indolent lymphoma. In some embodiments, the indolent lymphoma is indolent non-Hodgkin lymphoma (NHL). In some embodiments, the indolent NHL is marginal zone lymphoma or follicular lymphoma. In some embodiments, the kit further comprises instructions for administering rituximab at a dose of 375 mg / m 2 by IV infusion, wherein rituximab is administered to the individual once a week for 4 weeks and then once a month. In some embodiments, the kit further comprises instructions for administering the polypeptide (e.g., the fusion polypeptide) at a dose of 10 mg / kg or 15 mg / kg once a week, e.g., by IV infusion.

[0035] In some embodiments of any of the kits, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, the Fc domain variant is a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, numbered according to the Kabat EU index. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 136. In some embodiments, the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant forms a homodimer. In some embodiments, the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant (or a pharmaceutical manufactured therefrom or a pharmaceutical composition comprising such a polypeptide) further comprises instructions for administration to an individual at a dose of 10 mg / kg once a week (QW). In some embodiments, the kit comprises instructions for administering to an individual by IV infusion a polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant (a pharmaceutical manufactured therefrom or a pharmaceutical composition comprising such a polypeptide).

Brief Description of the Drawings

[0036]

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Mode for Carrying Out the Invention

[0037] Definition The terms "about" or "approximately" mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art and depend in part on how the value is measured or determined, i.e., on the limitations of the measuring system. For example, "about" can mean within one standard deviation, or within one or more than one standard deviation, in accordance with the convention in the relevant art. Alternatively, "about" can mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, especially with respect to biological systems or processes, the term can mean within an order of magnitude of the value, preferably within five-fold, more preferably within two-fold. Unless otherwise specified, when a particular value is recited in the present application and claims, the term "about" is considered to mean within an acceptable error range for the particular value.

[0038] The terms used herein are for the purpose of describing particular instances only and are not intended to be limiting. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Further, to the extent that the terms "including", "includes", "having", "has", "with", or variations thereof are used in either the detailed description or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising".

[0039] As used herein, terms such as "treatment", "treating", etc. refer to administering an agent or performing a procedure in order to obtain an effect. In some embodiments, the effect is prophylactic in that it completely or partially prevents a disease or its symptoms. In some embodiments, the effect is therapeutic in that it affects partial or complete cure of a disease or a symptom of the disease.

[0040] As used herein, the term "antibody" refers to intact antibodies, antibody fragments, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody-like proteins when they exhibit the desired biological activity (e.g., epitope binding).

[0041] As used herein, the term "antibody variable domain" refers to the portions of the light and heavy chains of an antibody that include the amino acid sequences of the complementarity-determining regions (CDRs, e.g., CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3) and the framework regions (FRs).

[0042] As used herein, the term "linker" refers to a connection between two elements, e.g., between protein domains. In some embodiments, the linker 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 1-200 amino acid sequence) that occurs between two polypeptides or polypeptide domains to provide space or flexibility (or both space and flexibility) between the two polypeptides or polypeptide domains. In some embodiments, the amino acid spacer is part of the primary sequence of the polypeptide (e.g., is attached to a polypeptide or polypeptide domain spaced via the polypeptide backbone).

[0043] As used herein, the term "effective amount" refers to the amount of a polypeptide or a pharmaceutical composition comprising a polypeptide described herein, e.g., a polypeptide having the SIRPα D1 domain or a variant thereof, that is sufficient and effective to achieve the desired therapeutic effect when treating a patient having a disease such as cancer, e.g., a solid tumor or a hematological cancer. In some embodiments, an effective amount of the polypeptide will avoid adverse side effects.

[0044] As used herein, the term "pharmaceutical composition" refers to a pharmaceutical or pharmaceutical preparation that contains an active ingredient as well as an excipient or diluent (or both an excipient and a diluent), and the active ingredient can be administered by a suitable method of administration. In some embodiments, the pharmaceutical compositions disclosed herein contain pharmaceutically acceptable ingredients that are compatible with the polypeptide. In some embodiments, the pharmaceutical composition is in the form of a tablet or capsule for oral administration, or in an aqueous form for intravenous or subcutaneous administration, for example by injection.

[0045] As used herein, the terms "subject", "individual", and "patient" are used synonymously to refer to a vertebrate, such as a mammal. Mammals include, but are not limited to, mice, monkeys, humans, livestock, sports animals, and pets. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro. None of the terms require the supervision of a medical professional.

[0046] As used herein, the term "affinity" or "binding affinity" refers to the strength of the binding interaction between two molecules. Generally, binding affinity refers to the total strength of the non-covalent interactions between a molecule and its binding partner (such as the SIRPα D1 domain variant and CD47). Unless otherwise specified, binding affinity refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of the binding pair. The binding affinity between two molecules is generally described by the dissociation constant (KD) or the association constant (KA). Two molecules with low binding affinity for each other generally bind slowly and tend to dissociate easily, exhibiting a large KD. Two molecules with high affinity for each other generally bind easily and tend to remain bound for a long time, exhibiting a small KD. In some embodiments, the KD of two interacting molecules is determined using known methods and techniques, such as surface plasmon resonance (SPR). KD can be calculated as the ratio of koff / kon.

[0047] As used herein, "K" DThe term "less than" refers to a numerically small K D value and a binding affinity that increases compared to the recited KD value. As used herein, the term "KD less than" refers to a binding affinity that decreases compared to a numerically larger KD value and the recited KD value.

[0048] As used herein, "in combination with" refers to the administration of another therapy in addition to one therapy. Thus, "in combination with" refers to the administration of another therapy before, during, or after the administration of one therapy to an individual.

[0049] Summary Provided herein is a method of treating cancer in an individual (e.g., a human individual) comprising administering to the individual (a) a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) a therapeutic antibody. In some embodiments, the polypeptide comprises any one of the SIRPα D1 domain variants described herein (unless otherwise specified).

[0050] In some embodiments, provided is a method of treating non-small cell lung cancer (NSCLC) in an individual, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) an anti-PD-1 antibody, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), and the individual is human. In some embodiments, the individual's NSCLC is progressing during prior immune checkpoint inhibitor (CPI) therapy and / or has a PD-L1 tumor proportion score (TPS) of less than 50%. In some embodiments, the individual has not received prior CPI therapy. In some embodiments, the individual is PD-L1 negative. In some embodiments, the individual is PD-L1 positive.

[0051] In some embodiments, methods of treating a subject with head and neck squamous cell carcinoma (HNSCC) are also provided, the methods comprising administering to the subject an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) an anti-PD-1 antibody, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), wherein the subject's HNSCC has progressed during or after prior platinum therapy, and the subject is human. In some embodiments, the subject has received prior immune checkpoint inhibitor (CPI) therapy (e.g., treatment with an immune checkpoint inhibitor as described herein). In some embodiments, the subject has not received prior CPI therapy. In some embodiments, the subject is PD-L1 negative. In some embodiments, the subject is PD-L1 positive.

[0052] In some embodiments, if an individual has a cancer that does not express the PD-L1 biomarker or expresses PD-L1 at very low levels, the individual is "PD-L1 negative." In some embodiments, if the expression of PD-L1 (such as protein expression) is not detected on (or in) tumor cells (TC) in a sample of the individual, if the expression of PD-L1 (such as protein expression) is not detected on (or in) tumor-infiltrating immune cells (IC) in a sample of the individual, or if the expression of PD-L1 (e.g., protein expression) is detected at very low levels on (or in) TC and / or IC in a sample of the individual, the individual is "PD-L1 negative" or has "PD-L1 negative cancer." In some embodiments, in a sample obtained from an individual who expresses PD-L1, when determined using an assay (e.g., an assay described herein) for determining the PD-L1 status of the individual, for example, if 0%, less than about 1%, less than about 5%, or less than about 10% of tumor cells (TC) and / or tumor-infiltrating immune cells (IC), the individual is PD-L1 negative. In some embodiments, an individual or tumor may be considered PD-L1 negative because it has no T cell infiltration. Such assays are known to and routinely used by medical professionals.

[0053] In some embodiments, an individual is "PD-L1 positive" if the individual has cancer that expresses the PD-L1 biomarker (e.g., as shown to express in a diagnostic test). In some embodiments, such an individual is "PD-L1 positive" or has cancer that is "PD-L1 positive cancer". In some embodiments, if PD-L1 expression (e.g., protein expression) is detected on (or in) tumor cells (TC) in a sample of the individual, or if PD-L1 expression (e.g., protein expression) is detected on (or in) tumor-infiltrating immune cells (IC) in a sample of the individual, then the individual is "PD-L1 positive" or has "PD-L1 positive cancer". In some embodiments, the TC and / or IC of the individual express low levels of the PD-L1 biomarker. In some embodiments, the TC and / or IC of the individual express high levels of the PD-L1 biomarker. In some embodiments, when determining the PD-L1 status of an individual using an assay (e.g., an assay described herein), the individual is "PD-L1 positive" or has cancer that is "PD-L1 positive cancer" if the PD-L1 biomarker is present (e.g., detected) at greater than 0% of the sample, at least 1% of the sample, at least 5% of the sample, or at least 10% of the sample of the individual (e.g., a sample of the individual that includes the individual's TC and / or IC). Such assays are known to and routinely used by medical professionals. In some embodiments, an individual is "PD-L1" positive or has "PD-L1 positive cancer" if the individual's tumor proportion score (TPS) is 50% or greater (i.e., greater than 50% of the viable tumor cells in the sample of the individual express PD-L1, e.g., at any level).

[0054] In some embodiments, provided is a method of treating an individual's HER2-positive gastric / gastroesophageal junction (GEJ) cancer, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) an anti-HER2 antibody, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), wherein the individual's gastric / GEJ cancer has progressed after prior treatment with a fluoropyrimidine-based therapy and / or prior treatment with an anti-HER2 antibody, and the individual is human.

[0055] In some embodiments, methods are provided for treating an individual's aggressive non-Hodgkin lymphoma or NHL (e.g., de novo or transformed diffuse large B-cell lymphoma (DLBCL) or mantle cell lymphoma), the methods comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) an anti-CD20 antibody, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), wherein the individual's aggressive NHL is relapsed and / or refractory to prior treatment of the aggressive NHL and there is no available curative therapy, and the individual is human. In some embodiments, the relapsed / refractory aggressive NHL is relapsed / refractory DLBCL (e.g., de novo or transformed DLBCL). In some embodiments, the relapsed / refractory aggressive NHL is relapsed / refractory mantle cell lymphoma (MCL).

[0056] In some embodiments, a method of treating a non-Hodgkin lymphoma in an individual is provided, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) an anti-CD20 antibody, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index), wherein the non-Hodgkin lymphoma in the individual is relapsed and / or refractory to a prior treatment of non-Hodgkin lymphoma, and the individual is human. In some embodiments, the non-Hodgkin lymphoma (such as relapsed / refractory non-Hodgkin lymphoma) is a non-Hodgkin lymphoma (NHL), for example, relapsed / refractory indolent NHL. In some embodiments, the indolent NHL (such as relapsed / refractory indolent NHL) is follicular lymphoma (such as relapsed / refractory follicular lymphoma). In some embodiments, the indolent NHL (such as relapsed / refractory indolent NHL) is marginal zone lymphoma (such as relapsed / refractory marginal zone lymphoma).

[0057] Further details regarding the method of treatment and the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant are described below. It is described in U.S. Patent No. 10,259,859, the content of which is incorporated herein by reference in its entirety. Signal regulatory protein alpha (SIRP-α) D1 domain and variants thereof

[0058] In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein alpha (SIRP-α) D1 variant comprising a SIRPα D1 domain or a fragment thereof, wherein the SIRPα D1 domain has an amino acid mutation at residue 80 relative to the wild-type SIRPα D1 domain; and at least one additional amino acid mutation relative to the wild-type SIRPα D1 domain at a residue selected from the group consisting of residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92.

[0059] Also disclosed herein is, in some embodiments, a polypeptide comprising an Fc domain variant, wherein the Fc domain variant dimer comprises two Fc domain variants, and each Fc domain variant is independently selected from (i) a human IgG1 Fc region comprising the mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region comprising the mutations A330S, P331S and N297A; or (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A.

[0060] Signal regulatory protein alpha (“SIRP-α” or “SIRP-alpha”) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily that is widely expressed on the membranes of myeloid cells. SIRPα interacts with CD47, a protein that is widely expressed in many cell types in the body. The interaction between SIRPα and CD47 prevents the engulfment of “self” cells that can be recognized by the immune system in other ways. High expression of CD47 in tumor cells has been observed to act as a negative prognostic factor for survival in acute myeloid leukemia and some solid tumor cancers.

[0061] Native SIRPα is composed of three highly homologous immunoglobulin (Ig)-like extracellular domains (D1, D2, and D3). The SIRPα D1 domain (the "D1 domain") refers to the membrane-distal extracellular domain of SIRPα and mediates the binding of SIRPα to CD47. As used herein, the term "SIRPα polypeptide" refers to any SIRPα polypeptide or fragment thereof that can bind to CD47. There are at least 10 variants of wild-type human SIRPα. Table 1 shows the amino acid sequences of the D1 domains of 10 naturally occurring wild-type human SIRPα D1 domain variants (SEQ ID NOs: 1-10). In some embodiments, the SIRPα polypeptide comprises the SIRPα D1 domain. In some embodiments, the SIRPα polypeptide comprises a wild-type D1 domain such as those provided in SEQ ID NOs: 1-10. In some embodiments, the SIRPα polypeptide comprises the D2 or D3 domain (or both the D2 and D3 domains) of wild-type human SIRPα (see Table 3). TIFF0007710997000001.tif248170TIFF0007710997000002.tif105170

[0062] As used herein, the term "SIRPα D1 domain variant" refers to a polypeptide comprising the CD47-binding portion of an SIRPα D1 domain or an SIRPα polypeptide that has a higher affinity for CD47 than wild-type SIRPα. SIRPα D1 domain variants contain at least one amino acid substitution, deletion, or insertion (or combinations thereof) relative to wild-type SIRPα.

[0063] In some embodiments, the SIRPα D1 domain variants disclosed herein include the SIRPα D1 domain or variants thereof. In some embodiments, the SIRPα D1 domain variants include one or more amino acid substitutions, insertions, additions, or deletions relative to the wild-type D1 domain shown in SEQ ID NOs: 1-10. Table 2 lists exemplary amino acid substitutions in each SIRPα D1 domain variant (SEQ ID NOs: 13-22). In some embodiments, the SIRPα D1 domain polypeptide or SIRPα D1 domain variant includes a fragment of the D1 domain. In some embodiments, the SIRPα polypeptide fragment or SIRPα D1 domain variant fragment includes an amino acid sequence less than 10 amino acids in length, about 10 amino acids in length, about 20 amino acids in length, about 30 amino acids in length, about 40 amino acids in length, about 50 amino acids in length, about 60 amino acids in length, about 70 amino acids in length, about 80 amino acids in length, about 90 amino acids in length, about 100 amino acids in length, or about 100 amino acids or more in length. In some embodiments, the SIRPα D1 domain fragment retains the ability to bind to CD47.

[0064] In some embodiments, the polypeptides of the disclosure that include an SIRPα D1 domain variant bind to CD47 with a higher binding affinity than the wild-type human SIRPα D1 domain. In some embodiments, the SIRPα D1 domain variant binds to human CD47 with an affinity that is at least 1-fold (e.g., at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 5-fold or more) higher than the affinity of the naturally occurring D1 domain. In some embodiments, the SIRPα D1 domain variant binds to human CD47 with an affinity that is at least 1-fold (e.g., at least 10-fold, 100-fold, 1000-fold, or 1000-fold or more) higher than the affinity of the naturally occurring D1 domain.

[0065] As used herein, the terms “optimized affinity” or “optimized binding affinity” refer to an optimized strength of the binding interaction between a polypeptide disclosed herein, such as a SIRPα D1 domain variant, and CD47. For example, in some embodiments, the polypeptide binds to CD47 on cancer cells predominantly or with a higher affinity and does not substantially bind or binds with a lower affinity to CD47 on non-cancer cells. In some embodiments, the binding affinity between the polypeptide and CD47 is optimized such that the interaction does not cause or reduces clinically relevant toxicity as compared to variants that bind with maximal affinity. In some embodiments, to achieve the optimized binding affinity between the polypeptides provided herein and CD47, polypeptides comprising a SIRPα D1 domain variant are developed to have a binding affinity to CD47 that is lower than what is maximally achievable. In some embodiments, the SIRPα D1 domain variants disclosed herein cross-react with murine, non-human primate (NHP), and human CD47.

[0066] As used herein, the term “immunogenicity” refers to the property of a protein (e.g., a therapeutic protein) to elicit an immune response in a host as if it were a foreign antigen. The immunogenicity of a protein can be assayed in vitro by various methods, such as an in vitro T cell proliferation assay.

[0067] As used herein, the term “minimal immunogenicity” refers to the immunogenicity of a protein (e.g., a therapeutic protein) that has been modified (e.g., by amino acid substitution) to be lower (e.g., at least 10%, 25%, 50%, or 100% lower) than the immunogenicity before the amino acid substitution was introduced (e.g., the unmodified protein). In some embodiments, the protein (e.g., a therapeutic protein) is modified to have minimal immunogenicity and elicits little or no host immune response even if it is a foreign antigen.

[0068] In some embodiments, the SIRPα D1 domain variant exhibits minimal immunogenicity. In some embodiments, the SIRPα polypeptide of the disclosure administered to a subject has the same amino acid sequence as the SIRPα polypeptide in the subject's biological sample, except for amino acid changes that increase the affinity for the SIRPα D1 domain variant. In some embodiments, the polypeptide variants disclosed herein reduce the risk of side effects as compared to anti-CD47 antibodies or wild-type SIRPα. In some embodiments, the polypeptide variants disclosed herein reduce the risk of anemia as compared to anti-CD47 antibodies or wild-type SIRPα. In some embodiments, the polypeptide variants disclosed herein do not cause acute anemia in rodent or non-human primate (NHP) tests.

[0069] Table 2 shows the specific amino acid substitutions of the SIRPα D1 domain variant for each D1 domain sequence. In some embodiments, the SIRPα D1 domain variant comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more) of the substitutions listed in Table 2. In some embodiments, the SIRPα D1 domain variant comprises up to 14 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant comprises up to 10 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant comprises up to 7 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant of the disclosure has at least 90% (e.g., at least 92%, 95%, 97% or greater than 97%) amino acid sequence identity to the sequence of the wild-type D1 domain.

[0070] In some embodiments, the SIRPα D1 domain variant is a chimeric SIRPα D1 domain variant that includes two or more wild-type D1 domains or portions thereof (e.g., one wild-type D1 domain or portion thereof and another wild-type D1 domain or portion thereof). In some embodiments, the chimeric SIRPα D1 domain variant includes at least two portions of a wild-type D1 domain or its variant (e.g., 3, 4, 5, or more portions), where each of the portions is derived from a different wild-type D1 domain. In some embodiments, the chimeric SIRPα D1 domain variant further includes one or more amino acid substitutions listed in Table 2. TIFF0007710997000003.tif250170TIFF0007710997000004.tif247170TIFF0007710997000005.tif208170

[0071] In some embodiments, the polypeptide includes a SIRPα D1 domain variant that includes the following sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGNITPADAGTYYCX 12 KX 13 RKGSPDDVEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 13), X1 is L, I, or V, X2 is V, L, or I, X3 is A or V, X4 is A, I, or L, X5 is I, T, S, or F, X6 is E, V, or L, X7 is K or R, X8 is E or Q, X9 is H, P, or R, X 10 is L, T, or G, X 11 is K or R, X 12 is V or I, X 13 is F, L, or V, X 14is F or V, and the variant contains at least one amino acid substitution relative to the wild-type SIRPα D1 domain containing the sequence of SEQ ID NO: 1.

[0072] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant comprising the following sequence: EEGX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGNITPADAGTYYCX 12 KX 13 RKGSPDDVEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 16), X1 is L, I, or V, X2 is V, L, or I, X3 is A or V, X4 is A, I, or L, X5 is I, T, S, or F, X6 is E, V, or L, X7 is K or R, X8 is E or Q, X9 is H, P, or R, X 10 is L, T, or G, X 11 is K or R, X 12 is V or I, X 13 is F, L, or V, X 14 is F or V, and the variant contains at least one amino acid substitution relative to the wild-type SIRPα D1 domain containing the sequence of SEQ ID NO: 4.

[0073] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant comprising the following sequence: EEEX1QX2IQPDKFVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGNITPADAGTYYCX 12 KX 13 RKGSPDDVEX 14KSGAGTELSVRAKPS (SEQ ID NO: 17), where X1 is L, I, or V; X2 is V, L, or I; X3 is A or V; X4 is A, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K or R; X8 is E or Q; X9 is H, P, or R; X 10 is L, T, or G; X 11 is K or R; X 12 is V or I; X 13 is F, L, or V; X 14 is F or V, and the variant contains at least one amino acid substitution relative to the wild-type SIRPα D1 domain containing the sequence of SEQ ID NO: 5. In some embodiments, the polypeptide comprises a SIRPα D1 domain variant comprising the following sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFPIRIGNITPADAGTYYCX 12 KX 13 RKGSPDDVEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 18), where X1 is L, I, or V; X2 is V, L, or I; X3 is A or V; X4 is A, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K or R; X8 is E or Q; X9 is H, P, or R; X 10 is L, T, or G; X 11 is K or R; X 12 is V or I; X 13 is F, L, or V; X 14 is F or V, and the variant contains at least one amino acid substitution relative to the wild-type SIRPα D1 domain containing the sequence of SEQ ID NO: 6.

[0074] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant comprising the following sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRISNITPADAGTYYCX 12 KX 13 RKGSPDDVEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 21), X1 is L, I, or V, X2 is V, L, or I, X3 is A or V, X4 is A, I, or L, X5 is I, T, S, or F, X6 is E, V, or L, X7 is K or R, X8 is E or Q, X9 is H, P, or R, X 10 is L, T, or G, X 11 is K or R, X 12 is V or I, X 13 is F, L, or V, X 14 is F or V, and the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO: 9.

[0075] In any of the foregoing embodiments, the polypeptide comprises a SIRPα D1 domain variant comprising any one of the sequences of SEQ ID NOs: 13, 16-18, and 21, wherein X1 is L, I, or V. In any of the foregoing embodiments, X2 is V, L, or I. In any of the foregoing embodiments, X3 is A or V. In any of the foregoing embodiments, X4 is A, I, or L. In any of the foregoing embodiments, X5 is I, T, S, or F. In any of the foregoing embodiments, X6 is E, V, or L. In any of the foregoing embodiments, X7 is K or R. In any of the foregoing embodiments, X8 is E or Q. In any of the foregoing embodiments, X9 is H, P, or R. In any of the foregoing embodiments, X10 is L, T, or G. In any of the foregoing embodiments, X 11 is K or R. In any of the foregoing embodiments, X 12 is V or I. In any of the foregoing embodiments, X 13 is F, L, V. In any of the foregoing embodiments, X 14 is F or V. In some embodiments, the polypeptide of this aspect of the disclosure comprises six or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain comprising any one of the sequences of SEQ ID NOs: 1, 4-6, and 9.

[0076] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain comprising any one of the sequences of SEQ ID NOs: 1, 4-6, and 9. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain comprising any one of the sequences of SEQ ID NOs: 1, 4-6, and 9. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain comprising any one of the sequences of SEQ ID NOs: 1, 4-6, and 9. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a KD of less than 1×10 -8 M, less than 5×10 -9 M, less than 1×10 -9 M, less than 5×10 -10 M, less than 1x10 -10 M, or less than 1x10 -11 M. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K D of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM.

[0077] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant comprising the following sequence: EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 14), X1 is L, I, or V, X2 is V, L, or I, X3 is A or V, X4 is V, I, or L, X5 is I, T, S, or F, X6 is E, V, or L, X7 is K or R, X8 is E or Q, X9 is H, P, or R, X 10 is S, T, or G, X 11 is K or R, X 12 is V or I, X 13 is F, L, or V, X 14 is F or V, and the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO: 2.

[0078] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant comprising the following sequence: EEEX1QX2IQPDKSVSVAAGESX3ILLCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14KSGAGTELSVRAKPS (SEQ ID NO: 15), where X1 is L, I, or V; X2 is V, L, or I; X3 is A or V; X4 is V, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K or R; X8 is E or Q; X9 is H, P, or R; X 10 is S, T, or G; X 11 is K or R; X 12 is V or I; X 13 is F, L, or V; X 14 is F or V, and the variant contains at least one amino acid substitution relative to the wild-type SIRPα D1 domain containing the sequence of SEQ ID NO: 3.

[0079] In some embodiments, the polypeptide contains a SIRPα D1 domain variant containing the following sequence: EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14 KSGAGTELSVRGKPS (SEQ ID NO: 19), where X1 is L, I, or V; X2 is V, L, or I; X3 is A or V; X4 is V, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K or R; X8 is E or Q; X9 is H, P, or R; X 10 is S, T, or G; X 11 is K or R; X 12 is V or I; X 13 is F, L, or V; X 14 is F or V, and the variant contains at least one amino acid substitution relative to the wild-type SIRPα D1 domain containing the sequence of SEQ ID NO: 7.

[0080] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant comprising the following sequence: EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 22), X1 is L, I, or V, X2 is V, L, or I, X3 is A or V, X4 is V, I, or L, X5 is I, T, S, or F, X6 is E, V, or L, X7 is K or R, X8 is E or Q, X9 is H, P, or R, X 10 is S, T, or G, X 11 is K or R, X 12 is V or I, X 13 is F, L, or V, X 14 is F or V, and the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO: 10.

[0081] In this aspect of the present disclosure, in any of the foregoing embodiments, the polypeptide comprises any one of the sequences of SEQ ID NOs: 14, 15, 19, and 22, where X1 is L, I, or V. In any of the foregoing embodiments, X2 is V, L, or I. In any of the foregoing embodiments, X3 is A or V. In any of the foregoing embodiments, X4 is V, I, or L. In any of the foregoing embodiments, X5 is I, T, S, or F. In any of the foregoing embodiments, X6 is E, V, or L. In any of the foregoing embodiments, X7 is K or R. In any of the foregoing embodiments, X8 is E or Q. In any of the foregoing embodiments, X9 is H, P, or R. In any of the foregoing embodiments, X 10 is S, T, or G. In any of the foregoing embodiments, X 11 is K or R. In any of the foregoing embodiments, X 12 is V or I. In any of the foregoing embodiments, X 13 is F, L, or V. In any of the foregoing embodiments, X 14 is F or V. In some embodiments, the polypeptide of this aspect of the present disclosure comprises six or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain comprising any one of the sequences of SEQ ID NOs: 2, 3, 7, and 10.

[0082] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M or a K of less than 1×10 D M. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K D of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM.

[0083] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14KSGAGTELSVRAKPS (SEQ ID NO: 20), where X1 is L, I, or V; X2 is V, L, or I; X3 is A or V; X4 is A, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K or R; X8 is E or Q; X9 is H, P, or R; X 10 is S, T, or G; X 11 is K or R; X 12 is V or I; X 13 is F, L, or V; X 14 is F or V, and the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 8.

[0084] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 20, where X1 is L, I, or V. In any of the foregoing embodiments of this aspect of the present disclosure, X2 is V, L, or I. In any of the foregoing embodiments, X3 is A or V. In any of the foregoing embodiments, X4 is A, I, or L. In any of the foregoing embodiments, X5 is I, T, S, or F. In any of the foregoing embodiments, X6 is E, V, or L. In any of the foregoing embodiments, X7 is K or R. In any of the foregoing embodiments, X8 is E or Q. In any of the foregoing embodiments, X9 is H, P, or R. In any of the foregoing embodiments, X 10 is S, T, or G. In any of the foregoing embodiments, X 11 is K or R. In any of the foregoing embodiments, X 12 is V or I. In any of the foregoing embodiments, X 13 is F, L, or V. In any of the foregoing embodiments, X 14 is F or V. In some embodiments, the polypeptide of this aspect of the present disclosure comprises no more than 6 amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 8.

[0085] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 8. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 8. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 8. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M, or less than 1×10 D M. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K D of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM.

[0086] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSLX 11 PVGPIQWFRGAGPX 12 RX 13 LIYNQX 14 X 15 GX 16 FPRVTTVSX 17 X 18 TX 19 RX 20 NMDFX 21 IX 22 IX23 NITPADAGTYYCX 24 KX 25 RKGSPDX 26 X 27 EX 28 KSGAGTELSVRX 29 KPS (SEQ ID NO: 23), X1 is E or G; X2 is L, I, or V; X3 is V, L, or I; X4 is S or F; X5 is L or S; X6 is S or T; X7 is A or V; X8 is I or T; X9 is H or R; X 10 is A, V, I, or L; X 11 is I, T, S, or F; X 12 is A or G; X 13 is E, V, or L; X 14 is K or R; X 15 is E or Q; X 16 is H, P, or R; X 17 is D or E; X 18 is S, L, T, or G; X 19 is K or R; X 20 is E or D; X 21 is S or P; X 22 is S or R; X 23 is S or G; X 24 is V or I; X 25 is F, L, V; X 26 is D or absent; X 27 is T or V; X 28 is F or V; X 29 is A or G; wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 1-10.

[0087] X2 is L, I, or V. In any of the foregoing embodiments, X3 is V, L, or I. In any of the foregoing embodiments, X4 is S or F. In any of the foregoing embodiments, X5 is L or S. In any of the foregoing embodiments, X6 is S or T. In any of the foregoing embodiments, X7 is A or V. In any of the foregoing embodiments, X8 is I or T. In any of the foregoing embodiments, X9 is H or R. In any of the foregoing embodiments, X 10 is A, V, I, or L. In any of the foregoing embodiments, X 11 is I, T, S, or F. In any of the foregoing embodiments, X 12 is A or G. In any of the foregoing embodiments, X 13 is E, V, or L. In any of the foregoing embodiments, X 14 is K or R. In any of the foregoing embodiments, X 15 is E or Q. In any of the foregoing embodiments, X 16 is H, P, or R. In any of the foregoing embodiments, X 17 is D or E. In any of the foregoing embodiments, X 18 is S, L, T, or G. In any of the foregoing embodiments, X 19 is K or R. In any of the foregoing embodiments, X 20 is E or D. In any of the foregoing embodiments, X 21 is S or P. In any of the foregoing embodiments, X 22 is S or R. In any of the foregoing embodiments, X 23 is S or G. In any of the foregoing embodiments, X 24 is V or I. In any of the foregoing embodiments, X 25 is F, L, V. In any of the foregoing embodiments, X 26 is D or does not exist. In any of the foregoing embodiments, X 27is T or V. In any of the foregoing embodiments, X 28 is F or V. In any of the foregoing embodiments, X 29 is A or G. In some embodiments, the polypeptide of this aspect of the present disclosure comprises no more than 6 amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 1-10.

[0088] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 1-10. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 1-10. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 1-10. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M or less than 1×10 D M. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K D of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM.

[0089] In some embodiments, the polypeptides of the present disclosure comprising the SIRPα D1 domain variant further comprise the D2 domain having the sequence of SEQ ID NO: 24, the D3 domain having the sequence of SEQ ID NO: 25, or both the D2 domain having the sequence of SEQ ID NO: 24 and the D3 domain having the sequence of SEQ ID NO: 25 of wild-type human SIRPα shown in Table 3. In some embodiments, the SIRPα D1 domain variant further comprises a fragment or variant of the D2 domain, or a fragment or variant of the D3 domain. In some embodiments, the SIRPα D1 domain variant further comprises a fragment or variant of the D2 domain and a fragment or variant of the D3 domain. In some embodiments, the SIRPα D1 domain variant is linked to the D2 or D3 domain via a linker. In some embodiments, the SIRPα D1 domain variant is linked to the D2 and D3 domains via a linker. TIFF0007710997000006.tif51170

[0090] In some embodiments, the polypeptides of the present disclosure comprising the SIRPα D1 domain variant are attached to an Fc domain variant in order to improve the pharmacokinetic properties of the polypeptide, for example, to increase the serum half-life. In some embodiments, the SIRPα D1 domain variant is attached to an Fc domain variant that cannot dimerize. In some embodiments, the Fc domain variant serves to increase the serum half-life of the polypeptides described herein. In some embodiments, the polypeptides of the present disclosure comprising the SIRPα D1 domain variant do not include any of the sequences of SEQ ID NOs: 26-36 shown in Table 4. TIFF0007710997000007.tif190170

[0091] In some embodiments, the polypeptides and polypeptide constructs described herein are utilized in vitro for binding assays such as immunoassays. For example, in some embodiments, the polypeptides and polypeptide constructs described herein are utilized in solution or are bound to a solid support. In some embodiments, the polypeptides utilized in immunoassays are detectably labeled in various ways.

[0092] In some embodiments, the polypeptides and polypeptide constructs described herein are bound to various carriers and used to detect the presence of specific antigen-expressing cells. Examples of carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamide, agarose, and magnetite. The nature of the carrier can be either soluble or insoluble.

[0093] A variety of different labels and labeling methods are known. Examples of labels include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds. A variety of techniques are available for conjugating labels to the polypeptides disclosed herein.

[0094] In some embodiments, the polypeptide is conjugated to a low molecular weight hapten. These haptens are then specifically detected by a second reaction. For example, in some embodiments, the hapten biotin is used with avidin, or the haptens dinitrophenol, pyridoxal, or fluorescein are detected with specific anti-hapten antibodies (e.g., anti-dinitrophenol antibody, anti-pyridoxal antibody, and anti-fluorescein antibody, respectively).

[0095] SIRPα D1 domain variant with modified glycosylation pattern In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein alpha (SIRP-α) D1 variant comprising a SIRPα D1 domain or a fragment thereof having an amino acid mutation at residue 80 relative to the wild-type SIRPα D1 domain; and at least one additional amino acid mutation at a residue selected from the group consisting of residues 6, 27, 31, 47, 53, 54, 56, 66, and 92 relative to the wild-type SIRPα D1 domain.

[0096] In some embodiments, also disclosed herein is a polypeptide comprising an Fc domain variant, wherein the Fc domain variant dimer comprises two Fc domain variants, and each Fc domain variant is independently selected from (i) a human IgG1 Fc region comprising the mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region comprising the mutations A330S, P331S and N297A; or (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A.

[0097] In some embodiments, the polypeptide in the compositions disclosed herein comprises a SIRPα D1 domain variant with reduced or minimal glycosylation. Each D1 domain of the 10 wild-type human SIRPα proteins (SEQ ID NOs: 1-10 in Table 1) contains a single potential N-linked glycosylation site at amino acid N80 in the sequence N80ITP. Expression of the SIRPα D1 domain in Chinese hamster ovary (CHO) cells results in a major band of 16 kDa (non-glycosylated) and minor high molecular weight bands that are removed by Endo Hf. Endo Hf is a recombinant protein fusion of endoglycosidase H and maltose-binding protein. Endo Hf cleaves within the high mannose chitobiose core and some hybrid oligosaccharides from N-linked glycoproteins. This means that the proline at amino acid position 83 can reduce the efficiency of glycosylation, resulting in proteins with different degrees of glycosylation and thus heterogeneity. In the case of drug development, heterogeneity can pose challenges in process development. Thus, in some embodiments, amino acid N80 of the SIRPα D1 variant is mutated to Ala to investigate the possibility of generating a homogeneous non-glycosylated form of the SIRPα D1 domain variant. In some embodiments, to generate a non-glycosylated, SIRPα D1 domain variant, amino acid N80 in the SIRPα D1 domain variant is replaced by any natural and non-natural amino acid, such as any amino acid like N80A and N80Q. In some embodiments, the SIRPα D1 domain variant comprises an N80A mutation and at least one additional mutation (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more additional mutations). In some embodiments, the additional mutation is in the CD47 binding site. In some embodiments, the additional mutation is within the hydrophobic core of the D1 domain.

[0098] In some embodiments, the polypeptide in the compositions disclosed herein includes a SIRPα D1 domain variant with increased glycosylation relative to the wild-type SIRPα D1 domain. In another option to enhance the homogeneity of the final product, the efficiency of glycosylation at amino acid N80 is increased to generate a SIRPα D1 domain variant with increased glycosylation relative to the wild-type. In some embodiments, the amino acid P83 in the sequence NITP83 affects the degree of glycosylation at amino acid N80. In some embodiments, changing P83 to any amino acid increases the efficiency of glycosylation at N80. In some embodiments, the amino acid P83 in the SIRPα D1 domain variant is replaced by any amino acid, such as natural and non-natural amino acids, for example, P83V, P83A, P83I, and P83L. In some embodiments, the polypeptides of the present disclosure are expressed in cells optimized to not glycosylate proteins expressed, for example, by genetic engineering of cell lines (e.g., genetically engineered yeast or mammalian hosts), or by changing cell culture conditions such as the addition of kifunensine, or using natural non-glycosylating hosts such as prokaryotes (e.g., E. coli).

[0099] Table 5 shows specific amino acid substitutions of the SIRPα D1 domain variant for each D1 domain variant array. In some embodiments, the SIRPα D1 domain variant comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more) of the substitutions listed in Table 5. In some embodiments, the SIRPα D1 domain variant is not glycosylated or minimally glycosylated. In some embodiments, the SIRPα D1 domain variant is fully glycosylated or nearly fully glycosylated. In some embodiments, the SIRPα D1 domain variant comprises up to 14 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant comprises up to 10 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant comprises up to 7 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant of the present disclosure has at least 90% (e.g., at least 92%, 95%, 97% or more than 97%) amino acid sequence identity to the sequence of the wild-type D1 domain.

[0100] In some embodiments, the SIRPα D1 domain variant is a chimeric SIRPα D1 domain variant that comprises two or more wild-type D1 domains or portions thereof (e.g., one wild-type D1 domain or portion thereof and another wild-type D1 domain or portion thereof). In some embodiments, the chimeric SIRPα D1 domain variant comprises at least two portions (e.g., 3, 4, 5, or more portions) of the wild-type D1 domain or variant thereof, where each of the portions is derived from a different wild-type D1 domain. In some embodiments, the chimeric SIRPα D1 domain variant further comprises one or more amino acid substitutions listed in Table 5. TIFF0007710997000008.tif224170TIFF0007710997000009.tif244170TIFF0007710997000010.tif211170TIFF0007710997000011.tif243170TIFF0007710997000012.tif254170TIFF0007710997000013.tif216170TIFF0007710997000014.tif112170

[0101] In some embodiments, the polypeptide comprises an SIRPα D1 domain variant having the following sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGX 12I TX 13 ADAGTYYCX 14 KX 15 RKGSPDDVEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 37), X1 is L, I, or V; X2 is V, L, or I; X3 is A, or V; X4 is A, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K, or R; X8 is E, or Q; X9 is H, P, or R; X 10 is L, T, or G; X 11 is K, or R; X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; X 14 is V, or I; X 15 is F, L, or V; X 16 is F or V; the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1.

[0102] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEGX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSPDDVEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 40), X1 is L, I, or V; X2 is V, L, or I; X3 is A or V; X4 is A, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K or R; X8 is E or Q; X9 is H, P, or R; X 10 is L, T, or G; X 11 is K or R; X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; X 14 is V or I; X 15 is F, L, or V; and X 16 is F or V; the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 4.

[0103] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEEX1QX2IQPDKFVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGX12I TX 13 ADAGTYYCX 14 KX 15 RKGSPDDVEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 41), X1 is L, I, or V; X2 is V, L, or I; X3 is A, or V; X4 is A, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K, or R; X8 is E, or Q; X9 is H, P, or R; X 10 is L, T, or G; X 11 is K, or R; X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; X 14 is V, or I; X 15 is F, L, or V; X 16 is F or V; The variant contains at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 5.

[0104] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFPIRIGX 12I TX 13 ADAGTYYCX 14 KX 15 RKGSPDDVEX 16KSGAGTELSVRAKPS (SEQ ID NO: 42), where X1 is L, I, or V; X2 is V, L, or I; X3 is A or V; X4 is A, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K or R; X8 is E or Q; X9 is H, P, or R; X 10 is L, T, or G; X 11 is K or R; X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; X 14 is V or I; X 15 is F, L, or V; X 16 is F or V; the variant contains at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 6.

[0105] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRISX 12I TX 13 ADAGTYYCX 14 KX 15 RKGSPDDVEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 45), where X1 is L, I, or V; X2 is V, L, or I; X3 is A or V; X4 is A, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K or R; X8 is E or Q; X9 is H, P, or R; X 10 is L, T, or G; X 11is K or R; X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; X 14 is V or I; X 15 is F, L, or V; X 16 is F or V; The variant contains at least one amino acid substitution with respect to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 9.

[0106] In any of the foregoing embodiments of this aspect of the present disclosure, the polypeptide comprises a SIRPα D1 domain variant having any one of the sequences of SEQ ID NOs: 37, 40-42, and 45, and X1 is L, I, or V. In any of the foregoing embodiments, X2 is V, L, or I. In any of the foregoing embodiments, X3 is A or V. In any of the foregoing embodiments, X4 is A, I, or L. In any of the foregoing embodiments, X5 is I, T, S, or F. In any of the foregoing embodiments, X6 is E, V, or L. In any of the foregoing embodiments, X7 is K or R. In any of the foregoing embodiments, X8 is E or Q. In any of the foregoing embodiments, X9 is H, P, or R. In any of the foregoing embodiments, X 10 is L, T, or G. In any of the foregoing embodiments, X 11 is K or R. In any of the foregoing embodiments, X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. In any of the foregoing embodiments, X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. In any of the foregoing embodiments, X 14 is V or I. In any of the foregoing embodiments, X 15is F, L, V. In any of the foregoing embodiments, X 16 is F, or V.

[0107] In some embodiments, the polypeptides provided herein comprise 10 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 1, 4-6, and 9. In some embodiments, the polypeptides provided herein comprise 7 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 1, 4-6, and 9.

[0108] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 1, 4-6, and 9. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 1, 4-6, and 9. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 1, 4-6, and 9. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M, or less than 1×10 D M. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K D of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM.

[0109] In some embodiments, the polypeptide comprises an SIRPα D1 domain variant having the following sequence: EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSPDTEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 38), X1 is L, I, or V; X2 is V, L, or I; X3 is A or V; X4 is V, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K or R; X8 is E or Q; X9 is H, P, or R; X 10 is S, T, or G; X 11 is K or R; X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; X 14 is V or I; X 15 is F, L, or V; and X 16 is F or V; the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2.

[0110] In some embodiments, the polypeptide comprises an SIRPα D1 domain variant having the following sequence: EEEX1QX2IQPDKSVSVAAGESX3ILLCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISX 12 ITX 13 ADAGTYYCX14 KX 15 RKGSPDTEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 39), X1 is L, I, or V; X2 is V, L, or I; X3 is A or V; X4 is V, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K or R; X8 is E or Q; X9 is H, P, or R; X 10 is S, T, or G; X 11 is K or R; X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; X 14 is V or I; X 15 is F, L, or V; and X 16 is F or V; the variant contains at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 3.

[0111] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSPDTEX 16 KSGAGTELSVRGKPS (SEQ ID NO: 43), 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 10is S, T, or G; X 11 is K or R; X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; X 14 is V or I; X 15 is F, L, or V; and X 16 is F or V; the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 7.

[0112] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSPDTEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 46), X1 is L, I, or V; X2 is V, L, or I; X3 is A or V; X4 is V, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K or R; X8 is E or Q; X9 is H, P, or R; X 10 is S, T, or G; X 11 is K or R; X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; X 14 is V or I; X 15 is F, L, or V; and X16 is F or V; the variant contains at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 10.

[0113] In any of the foregoing embodiments of this aspect of the present disclosure, the polypeptide contains a SIRPα D1 domain variant having any one of the sequences of SEQ ID NOs: 38, 39, 43, and 46, and X1 is L, I, or V. In any of the foregoing embodiments, X2 is V, L, or I. In any of the foregoing embodiments, X3 is A or V. In any of the foregoing embodiments, X4 is V, I, or L. In any of the foregoing embodiments, X5 is I, T, S, or F. In any of the foregoing embodiments, X6 is E, V, or L. In any of the foregoing embodiments, X7 is K or R. In any of the foregoing embodiments, X8 is E or Q. In any of the foregoing embodiments, X9 is H, P, or R. In any of the foregoing embodiments, X 10 is S, T, or G. In any of the foregoing embodiments, X 11 is K or R. In any of the foregoing embodiments, X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. In any of the foregoing embodiments, X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. In any of the foregoing embodiments, X 14 is V or I. In any of the foregoing embodiments, X 15 is F, L, or V. In any of the foregoing embodiments, X 16 is F or V.

[0114] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having 10 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having 7 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 2, 3, 7, and 10.

[0115] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M or less than 1×10 D M. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K D of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM.

[0116] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSPDTEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 44), X1 is L, I, or V; X2 is V, L, or I; X3 is A or V; X4 is A, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K or R; X8 is E or Q; X9 is H, P, or R; X 10 is S, T, or G; X 11 is K or R; X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; X 14 is V or I; X 15 is F, L, or V; and X 16 is F or V; the variant contains at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 8.

[0117] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 44, and X1 is L, I, or V. In any of the foregoing embodiments of this aspect of the present disclosure, X2 is V, L, or I. In any of the foregoing embodiments, X3 is A or V. In any of the foregoing embodiments, X4 is A, I, or L. In any of the foregoing embodiments, X5 is I, T, S, or F. In any of the foregoing embodiments, X6 is E, V, or L. In any of the foregoing embodiments, X7 is K or R. In any of the foregoing embodiments, X8 is E or Q. In any of the foregoing embodiments, X9 is H, P, or R. In any of the foregoing embodiments, X 10 is S, T, or G. In any of the foregoing embodiments, X 11 is K or R. In any of the foregoing embodiments, X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. In any of the foregoing embodiments, X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. In any of the foregoing embodiments, X 14 is V or I. In any of the foregoing embodiments, X 15 is F, L, or V. In any of the foregoing embodiments, X 16 is F or V.

[0118] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having 10 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 8. In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having 7 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 8.

[0119] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 8. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 8. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 8. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M, or less than 1×10 D M. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K D of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM.

[0120] In another aspect, the disclosure features a polypeptide comprising a SIRPα D1 domain variant having the following sequence: EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSLX 11 PVGPIQWFRGAGPX 12 RX 13 LIYNQX 14 X 15 GX 16 FPRVTTVSX 17 X 18 TX 19 RX 20 NMDFX 21 IX 22 IX 23 X 24 ITX25 ADAGTYYCX 26 KX 27 RKGSPDX 28 X 29 EX 30 KSGAGTELSVRX 31 KPS (SEQ ID NO: 47), X1 is E or G; X2 is L, I, or V; X3 is V, L, or I; X4 is S or F; X5 is L or S; X6 is S or T; X7 is A or V; X8 is I or T; X9 is H, R, or L; X 10 is A, V, I, or L; X 11 is I, T, S, or F; X 12 is A or G; X 13 is E, V, or L; X 14 is K or R; X 15 is E or Q; X 16 is H, P, or R; X 17 is D or E; X 18 is S, L, T, or G; X 19 is K or R; X 20 is E or N; X 21 is S or P; X 22 is S or R; X 23 is S or G; X 24 is any amino acid; X 25 is any amino acid; X 26 is V or I; X 27 is F, L, V; X 28 is D or absent; X 29 is T or V; X 30 is F or V; X 31 is A or G; The variant contains at least one amino acid substitution relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 1 to 10.

[0121] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 47, and X1 is E or G. In any of the foregoing embodiments of this aspect of the disclosure, X2 is L, I, or V. In any of the foregoing embodiments, X3 is V, L, or I. In any of the foregoing embodiments, X4 is S or F. In any of the foregoing embodiments, X5 is L or S. In any of the foregoing embodiments, X6 is S or T. In any of the foregoing embodiments, X7 is A or V. In any of the foregoing embodiments, X8 is I or T. In any of the foregoing embodiments, X9 is H or R. In any of the foregoing embodiments, X 10 is A, V, I, or L. In any of the foregoing embodiments, X 11 is I, T, S, or F. In any of the foregoing embodiments, X 12 is A or G. In any of the foregoing embodiments, X 13 is E, V, or L. In any of the foregoing embodiments, X 14 is K or R. In any of the foregoing embodiments, X 15 is E or Q. In any of the foregoing embodiments, X 16 is H, P, or R. In any of the foregoing embodiments, X 17 is D or E. In any of the foregoing embodiments, X 18 is S, L, T, or G. In any of the foregoing embodiments, X 19 is K or R. In any of the foregoing embodiments, X 20 is E or N. In any of the foregoing embodiments, X 21 is S or P. In any of the foregoing embodiments, X 22 is S or R. In any of the foregoing embodiments, X 23 is S or G. In any of the foregoing embodiments, X 24is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. In any of the foregoing embodiments, X 25 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. In any of the foregoing embodiments, X 26 is V or I. In any of the foregoing embodiments, X 27 is F, L, V. In any of the foregoing embodiments, X 28 is D or absent. In any of the foregoing embodiments, X 29 is T or V. In any of the foregoing embodiments, X 30 is F or V. In any of the foregoing embodiments, X 31 is A or G.

[0122] In some embodiments, the polypeptide of this aspect of the disclosure comprises 10 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 1-10. In some embodiments, the polypeptide of this aspect of the disclosure comprises 7 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 1-10.

[0123] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 1-10. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 1-10. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NOs: 1-10. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof is < 1 × 10 -8 M, < 5 × 10 -9 M, < 1 × 10 -9 M, < 5 × 10-10 less than M, 1x10 -10 less than or equal to M or 1x10 -11 K less than M D and binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM D and binds to CD47.

[0124] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVX2VAAGEX3AX4LX5CTX6TSLX7PVGPIQWFRGAGPX8RX9LIYNQX 10 X 11 GX 12 FPRVTTVSX 13 X 14 TKRX 15 NMDFSIX 16 IX 17 X 18 ITPADAGTYYCX 19 KFRKGX 20 X 21 X 22 DX 23 EFKSGAGTELSVRAKPS (SEQ ID NO: 48), X1 is V or I; X2 is L or S; X3 is T or S; X4 is T or I; X5 is R or H; X6 is A, V, or I; X7 is I, R, Y, K or F; X8 is G or A; X9 is E or V; X 10 is K or R; X 11 is E, D or Q; X 12 is H or P; X 13 is D or E; X 14 is S, L or T; X 15 is N or E; X 16 is R or S; X 17is G or S; X 18 is N or A; X 19 is V or I; X 20 is S, I or M; X 21 is P or absent; X 22 is D or P; X 23 is V or T, or a fragment thereof.

[0125] In another aspect, the disclosure features a polypeptide comprising a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9ITPADAGTYYCX 10 KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 49), X1 is V, L, or I; X2 is A, I, V, or L; X3 is I, F, S, or T; X4 is E, V, or L; X5 is K or R; X6 is E or Q; X7 is H, P, or R; X8 is L, T, S, or G; X9 is A; X 10 is V or I; wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1.

[0126] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 49, and X1 is V, L, or I. In any of the foregoing embodiments of this aspect of the present disclosure, X2 is A, I, V, or L. In any of the foregoing embodiments, X3 is I, F, S, or T. In any of the foregoing embodiments, X4 is E, V, or L. In any of the foregoing embodiments, X5 is K or R. In any of the foregoing embodiments, X6 is E or Q. In any of the foregoing embodiments, X7 is H, P, or R. In any of the foregoing embodiments, X8 is L, T, S, or G. In any of the foregoing embodiments, X9 is A. In any of the foregoing embodiments, X 10 is V or I.

[0127] In some embodiments, the polypeptide comprises an SIRPα D1 domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 49, and each of X1, X2, X3, X4, X5, X6, X7, X8, X9, and X 10 is not a wild-type amino acid.

[0128] In some embodiments, the polypeptide of this aspect of the present disclosure comprises no more than 10 amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1. In some embodiments, the polypeptide of this aspect of the present disclosure comprises no more than 7 amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1.

[0129] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, or less than 1×10 -11 M. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K D of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D In another aspect, the present disclosure features a polypeptide comprising a SIRPα D1 domain variant having the following sequence:

[0130] EEELQX1IQPDKSVSVAAGESAILHCTX2TSLX3PVGPIQWFRGAGPARX4LIYNQX5X6GX7FPRVTTVSEX8TKRENMDFSISISX9ITPADAGTYYCX KFRKGSPDTEFKSGAGTELSVRAKPS (SEQ ID NO: 50), where X1 is V or I; X2 is V or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is E or Q; X7 is H or P; X8 is S or T; X9 is N or A; X 10 is as defined above.10 is V or I; wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 2.

[0131] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 50, and X1 is V or I. In any of the foregoing embodiments of this aspect of the present disclosure, X2 is V or I. In any of the foregoing embodiments, X3 is I or F. In any of the foregoing embodiments, X4 is E or V. In any of the foregoing embodiments, X5 is K or R. In any of the foregoing embodiments, X6 is E or Q. In any of the foregoing embodiments, X7 is H or P. In any of the foregoing embodiments, X8 is S or R. In any of the foregoing embodiments, X9 is N or A. In any of the foregoing embodiments, X 10 is V or I.

[0132] In some embodiments, the polypeptide comprises a SIRPα D1 domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the sequence of SEQ ID NO: 50, and each of X1, X2, X3, X4, X5, X6, X7, X8, X9, and X 10 is not a wild-type amino acid.

[0133] In some embodiments, the polypeptide of this aspect of the present disclosure comprises no more than 10 amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 2. In some embodiments, the polypeptide of this aspect of the present disclosure comprises no more than 7 amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 2.

[0134] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 2. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M, or less than 1×10 D M. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K D of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM.

[0135] In another aspect, the disclosure features a polypeptide comprising a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMDFSIRIGX8ITPADAGTYYCX9KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 221), where X1 is V or I; X2 is A or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is H or P; X7 is L or T; X8 is N or A; X9 is V or I; provided that the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1.

[0136] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 51 and X1 is V or I. In any of the foregoing embodiments of this aspect of the present disclosure, X2 is A or I. In any of the foregoing embodiments, X3 is I or F. In any of the foregoing embodiments, X4 is E or V. In any of the foregoing embodiments, X5 is K or R. In any of the foregoing embodiments, X6 is H or P. In any of the foregoing embodiments, X7 is L or T. In any of the foregoing embodiments, X8 is N or A. In any of the foregoing embodiments, X9 is V or I. In some embodiments, X4 is not V.

[0137] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 51, and X8 is A. In any of the foregoing embodiments in this aspect of the present disclosure, X8 is A, and X1 is V or I. In any of the foregoing embodiments in this aspect of the present disclosure, X8 is A, and X2 is A or I. In any of the foregoing embodiments, X8 is A, and X3 is I or F. In any of the foregoing embodiments, X8 is A, and X4 is E or V. In some embodiments, X4 is not V. In any of the foregoing embodiments, X8 is A, and X5 is K or R. In any of the foregoing embodiments, X8 is A, and X6 is H or P. In any of the foregoing embodiments, X8 is A, and X7 is A or V. In any of the foregoing embodiments, X8 is A, and X9 is V or I.

[0138] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 51, and X8 is A. In any of the foregoing embodiments in this aspect of the present disclosure, X8 is A, and X1 is V or I. In any of the foregoing embodiments in this aspect of the present disclosure, X8 is A, and X2 is A or I. In any of the foregoing embodiments, X8 is A, and X3 is I or F. In any of the foregoing embodiments, X8 is A, and X4 is E or V. In some embodiments, X4 is not V. In any of the foregoing embodiments, X8 is A, and X5 is K or R. In any of the foregoing embodiments, X8 is A, and X6 is H or P. In any of the foregoing embodiments, X8 is A, and X7 is A or V. In any of the foregoing embodiments, X8 is A, and X9 is V or I.

[0139] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having at least 85% sequence identity to 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), and each of X1, X2, X3, X4, X5, X6, X7, X8, and X9 is not a wild-type amino acid.

[0140] In some embodiments, the polypeptide of this aspect of the disclosure comprises 10 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1. In some embodiments, the polypeptide of this aspect of the disclosure comprises 7 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1.

[0141] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K of less than 1×10 -8 M, less than 5×10 -9 M, less than 1×10 -9 M, less than 5×10 -10 M, less than 1x10 -10 M, or less than 1x10 -11 M of K DIt binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof has a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM D It binds to CD47.

[0142] In another aspect, the present disclosure features a polypeptide comprising a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRELIYNQX4EGX5FPRVTTVSDX6TKRNNMDFSIRIGX7ITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 222), where X1 is V, L, or I; X2 is A, I, or L; X3 is I, T, S, or F; X4 is K or R; X5 is H or P; X6 is L, T, or G; X7 is N or A; wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence set forth in SEQ ID NO: 1.

[0143] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 222, where X1 is V, L, or I. In any of the foregoing embodiments of this aspect of the present disclosure, X2 is A, I, or L. In any of the foregoing embodiments, X3 is I, T, S, or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In the foregoing embodiments, X6 is L, T, or G. In any of the foregoing embodiments, X7 is N or A.

[0144] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 222, where X1 is V or I. In any of the foregoing embodiments in this aspect of the present disclosure, X2 is A or I. In any of the foregoing embodiments, X3 is I or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In the foregoing embodiments, X6 is L or T. In any of the foregoing embodiments, X7 is N or A.

[0145] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 222, where X7 is A. In any of the foregoing embodiments in this aspect of the present disclosure, X7 is A and X1 is V or I. In any of the foregoing embodiments in this aspect of the present disclosure, X7 is A and X2 is A or I. In any of the foregoing embodiments, X7 is A and X3 is I or F. In any of the foregoing embodiments, X7 is A and X4 is K or R. In any of the foregoing embodiments, X7 is A and X5 is H or P. In any of the foregoing embodiments, X7 is A and X6 is L or T.

[0146] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 222, where X7 is A. In any of the foregoing embodiments in this aspect of the present disclosure, X7 is A and X1 is I. In any of the foregoing embodiments in this aspect of the present disclosure, X7 is A and X2 is I. In any of the foregoing embodiments, X7 is A and X3 is F. In any of the foregoing embodiments, X7 is A and X4 is R. In any of the foregoing embodiments, X7 is A and X5 is P. In any of the foregoing embodiments, X7 is A and X6 is T.

[0147] In some embodiments, the polypeptide comprises an SIRPα D1 domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 222, and each of X1, X2, X3, X4, X5, X6, and X7 is not a wild-type amino acid.

[0148] In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than 10 amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1. In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than 7 amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1.

[0149] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1. In some embodiments, the fragment comprises a polypeptide less than 10 amino acids in length, about 10 amino acids in length, about 20 amino acids in length, about 30 amino acids in length, about 40 amino acids in length, about 50 amino acids in length, about 60 amino acids in length, about 70 amino acids in length, about 80 amino acids in length, about 90 amino acids in length, about 100 amino acids in length, or about 100 amino acids or more in length. The fragment retains the ability to bind to CD47. Preferably, the SIRPα D1 domain variant polypeptide and fragments thereof bind to CD47 with a higher affinity than the SIRPα polypeptide binds to CD47. For example, in some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof is 1×10 -8Less than M, 5x10 -9 Less than M, 1x10 -9 Less than M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than M D binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof has a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM D binds to CD47.

[0150] In another aspect, the disclosure features a polypeptide comprising a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVSVAAGESAILHCTX2TSLX3PVGPIQWFRGAGPARELIYNQX4EGX5FPRVTTVSEX6TKRENMDFSISISX7ITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS (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 of G; X7 is N or A; wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 2.

[0151] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, where X1 is V, L, or I. In any of the foregoing embodiments in this aspect of the present disclosure, X2 is V, I, or L. In any of the foregoing embodiments, X3 is I, T, S, or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In the foregoing embodiments, X6 is S, T, or G. In any of the foregoing embodiments, X7 is N or A.

[0152] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, where X1 is V or I. In any of the foregoing embodiments in this aspect of the present disclosure, X2 is V or I. In any of the foregoing embodiments, X3 is I or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In the foregoing embodiments, X6 is S or T. In any of the foregoing embodiments, X7 is N or A.

[0153] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, where X7 is A. In any of the foregoing embodiments in this aspect of the present disclosure, X7 is A and X1 is V or I. In any of the foregoing embodiments in this aspect of the present disclosure, X7 is A and X2 is V or I. In any of the foregoing embodiments, X7 is A and X3 is I or F. In any of the foregoing embodiments, X7 is A and X4 is K or R. In any of the foregoing embodiments, X7 is A and X5 is H or P. In any of the foregoing embodiments, X7 is A and X6 is S or T.

[0154] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, where X7 is A. In any of the foregoing embodiments of this aspect of the present disclosure, X7 is A and X1 is I. In any of the foregoing embodiments of this aspect of the present disclosure, X7 is A and X2 is I. In any of the foregoing embodiments, X7 is A and X3 is F. In any of the foregoing embodiments, X7 is A and X4 is R. In any of the foregoing embodiments, X7 is A and X5 is P. In any of the foregoing embodiments, X7 is A and X6 is T.

[0155] In some embodiments, the polypeptide comprises an SIRPα D1 domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 212, and each of X1, X2, X3, X4, X5, X6, and X7 is not a wild-type amino acid.

[0156] In some embodiments, the polypeptide of this aspect of the present disclosure comprises 10 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 2. In some embodiments, the polypeptide of this aspect of the present disclosure comprises 7 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 2.

[0157] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold higher than that of the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 2. In some embodiments, the fragment comprises a polypeptide having a length of less than 10 amino acids, a length of about 10 amino acids, a length of about 20 amino acids, a length of about 30 amino acids, a length of about 40 amino acids, a length of about 50 amino acids, a length of about 60 amino acids, a length of about 70 amino acids, a length of about 80 amino acids, a length of about 90 amino acids, a length of about 100 amino acids, or a length of about 100 amino acids or more. The fragment retains the ability to bind to CD47. Preferably, the SIRPα D1 domain variant polypeptide and fragments thereof bind to CD47 with a higher affinity than the SIRPα polypeptide binds to CD47. For example, in some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K -8 of less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M or less than 1×10 D M. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a K D of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM.

[0158] In some embodiments described herein, a polypeptide comprising a SIRPα D1 domain variant having the sequence set forth below: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9X 10 X 11 X 12 ADAGTYYCX 13 KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 218), X1 is V, L, or I; X2 is A, V, L, or I; X3 is I, S, T, or F; X4 is E, L, or V; X5 is K or R; X6 is E or Q; X7 is H, R, or P; X8 is S, G, L, or T; X9 is any amino acid; X 10 is any amino acid; X 11 is any amino acid; X 12 is any amino acid; X 13 is V or I; wherein the SIRPα D1 domain variant comprises at least two amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence set forth in SEQ ID NO: 1.

[0159] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, wherein X1 where X9 is A. In any of the foregoing embodiments of this aspect of the present disclosure, X9 is N. In any of the foregoing embodiments of this aspect of the present disclosure, X 10 is I. In any of the foregoing embodiments of this aspect of the present disclosure, X9 is N and X 10 is P. In any of the foregoing embodiments of this aspect of the present disclosure, X9 is N and X 11 is any amino acid other than S, T, or C. In any of the foregoing embodiments of this aspect of the present disclosure, X 11 is T. In any of the foregoing embodiments of this aspect of the present disclosure, X 11is any amino acid other than T. In any of the foregoing embodiments in this aspect of the present disclosure, X 12 is P. In any of the foregoing embodiments in this aspect of the present disclosure, X9 is N, and X 12 is any amino acid other than P.

[0160] In some embodiments described herein, a polypeptide comprising a SIRPα D1 domain variant having the sequences described below: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9ITX 10 ADAGTYYCX 11 KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 219), X1 is V, L, or I; X2 is A, V, L, or I; X3 is I, S, T, or F; X4 is E, L, or V; X5 is K or R; X6 is E or Q; X7 is H, R, or P; X8 is S, G, L, or T; X9 is N; X 10 is any amino acid other than P; X 11 is V or I; wherein the SIRPα D1 domain variant comprises at least two amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence set forth in SEQ ID NO: 1.

[0161] In another aspect of the present disclosure, a composition comprising a SIRPα D1 domain variant polypeptide having the amino acid sequence of SEQ ID NO: 48, or a fragment thereof, is disclosed herein. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a higher affinity compared to the affinity of the SIRPα polypeptide for binding to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide is less than 1×10 -8 M, or less than 1×10 -9 M, less than 1×10 -10 M, or less than 1×10 -11K less than M D binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide described above is attached to or fused with a second polypeptide. In some embodiments, the second polypeptide includes, but is not limited to, an Fc polypeptide, an Fc variant, or a fragment as described above.

[0162] Without limiting the foregoing, in some embodiments, the SIRPα D1 domain variant polypeptide is selected from any one of SEQ ID NOs: 53-87 and 213 shown in Table 6. TIFF0007710997000015.tif245170TIFF0007710997000016.tif251170TIFF0007710997000017.tif250170TIFF0007710997000018.tif81170

[0163] In some embodiments, the polypeptide includes an SIRPα D1 domain variant having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to any variant shown in Table 6.

[0164] In some embodiments, the polypeptide includes an SIRPα D1 domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 (SEQ ID NO: 223)%, or 100% sequence identity) to SEQ ID NO: 80, 81, or 85 in Table 6. Fc domain variant and fusion polypeptide comprising an Fc domain variant

[0165] In some embodiments, disclosed herein is a signal regulatory protein alpha (SIRP-α) D1 variant polypeptide comprising a SIRPα D1 domain or a fragment thereof having an amino acid mutation at residue 80 relative to the wild-type SIRPα D1 domain; and at least one additional amino acid mutation relative to the wild-type SIRPα D1 domain at a residue selected from the group consisting of residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92.

[0166] In some embodiments, also disclosed herein is an Fc domain variant dimer, wherein the Fc domain variant dimer comprises two Fc domain variants, and each Fc domain variant is independently selected from (i) a human IgG1 Fc region comprising the mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region comprising the mutations A330S, P331S and N297A; or (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A.

[0167] Antibodies that target cell surface antigens can elicit immune stimulation and effector functions associated with the engagement of Fc receptors (FcRs) on immune cells. There are multiple Fc receptors specific for different classes of antibodies, such as IgG (gamma receptor), IgE (epsilon receptor), IgA (alpha receptor), and IgM (mu receptor). Binding of the antibody to the Fc receptor for the Fc region on the cell surface can trigger multiple biological responses, such as phagocytosis of antibody-coated particles (antibody-dependent cell-mediated phagocytosis, or ADCP), clearance of immune complexes, lysis of antibody-coated cells by killer cells (antibody-dependent cell-mediated cytotoxicity, or ADCC), and release of inflammatory mediators, placental transfer, and regulation of immunoglobulin production. Furthermore, the complement C1 component can bind to the antibody, leading to activation of the complement system. Activation of the complement is important for lysis of pathogenic cells. However, activation of the complement can also stimulate an inflammatory response and may be involved in autoimmune hypersensitivity or other immune disorders. Variant Fc regions with reduced or removed ability to bind to specific Fc receptors are useful for the development of therapeutic antibodies and Fc fusion polypeptide constructs that act by targeting, activating, or neutralizing ligand functions without damaging or destroying local cells or tissues.

[0168] In some embodiments, the SIRPα D1 polypeptide construct comprises a non-native SIRPα D1 domain variant linked to an Fc domain variant that forms an Fc domain having reduced or removed effector function.

[0169] In some embodiments, the Fc domain variant refers to a polypeptide chain comprising the second and third antibody constant domains (e.g., CH2 and CH3). In some embodiments, the Fc domain variant also includes a hinge domain. In some embodiments, the Fc domain variant is of any immunoglobulin antibody isotype such as IgG, IgE, IgM, IgA, and IgD. Further, in some embodiments, the Fc domain variant is of any IgG subtype (e.g., IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, and IgG4). In some embodiments, the Fc domain variant comprises 10 amino acid modifications (e.g., insertions, deletions, and / or substitutions) that alter the interaction between the Fc domain and the Fc receptor relative to the wild-type Fc domain monomer sequence (e.g., 1-10, 1-8, 1-6, 1-4 amino acid substitutions, additions or insertions, deletions, or combinations thereof).

[0170] As used herein, the term "Fc domain dimer" refers to a dimer of two Fc domains. In a wild-type Fc domain dimer, the two wild-type Fc domains dimerize by the interaction between the two CH3 antibody constant domains and one or more disulfide bonds formed between the hinge domains of the two dimerized Fc domains.

[0171] 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, e.g., a "dead Fc domain dimer variant". In some embodiments, each of the Fc domains of the Fc domain dimer variant comprises an amino acid substitution in the CH2 antibody constant domain to reduce the interaction or binding between the Fc domain dimer variant and an Fc receptor such as the Fc gamma receptor (FcγR), Fc alpha receptor (FcαR), or Fc epsilon (FcεR).

[0172] In some embodiments, the SIRPα D1 domain variant (e.g., any of the variants described in Tables 2, 5, and 6) is fused to an Fc domain variant of an immunoglobulin or a fragment of an Fc domain variant. In some embodiments, the Fc domain variant of an immunoglobulin or a fragment of an Fc domain variant can form an Fc domain dimer with another Fc domain variant. In some embodiments, the Fc domain variant of an immunoglobulin or a fragment of an Fc domain variant cannot form an Fc domain dimer with another Fc domain variant. In some embodiments, the Fc domain variant or the D fragment of an Fc domain variant is fused to a polypeptide of the present disclosure to increase the serum half-life of the polypeptide. In some embodiments, the Fc domain variant or the fragment of an Fc domain variant fused to a polypeptide of the present disclosure dimerizes with a second Fc domain variant to form an Fc domain dimer variant that binds to an Fc receptor, or the Fc domain variant binds to an Fc receptor. In some embodiments, the Fc domain variant or the fragment of an Fc domain variant fused to a polypeptide to increase the serum half-life of the polypeptide does not induce any immune system-related response.

[0173] In some embodiments, the SIRPα polypeptide or construct provided herein comprises a SIRPα D1 domain or variant thereof bound to a first Fc domain variant, and an antibody variable domain bound to a second Fc domain variant, wherein the first and second Fc domain variants combine to form an Fc domain dimer variant (e.g., a heterodimeric Fc domain dimer variant). An Fc domain dimer is a protein structure found at the C-terminus of an immunoglobulin. An Fc domain dimer contains two Fc domains that are dimerized by interactions between the CH3 antibody constant domains. A wild-type Fc domain dimer forms the minimal structure that binds to Fc receptors, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and FcγRIV.

[0174] The Fc domain is not directly involved in the binding of the antibody to its target, but can be involved in various effector functions such as the involvement of the antibody in antibody-dependent cell-mediated cytotoxicity. In some embodiments, the Fc domain in the SIRPα polypeptide or construct of the present disclosure results in a decrease in effector function such as a decrease in antibody-dependent cell-mediated cytotoxicity (ADCC), a decrease in complement-dependent cytotoxicity (CDC), a decrease in antibody-dependent cell-mediated phagocytosis (ADCP), or any combination thereof, including amino acid substitutions, additions or insertions, deletions, or any combination thereof. In some embodiments, the SIRPα polypeptide or construct of the present disclosure is characterized by a decrease in binding to human Fc receptors (e.g., minimal or no binding) and a decrease in binding to complement protein C1q (e.g., minimal or no binding). In some embodiments, the SIRPα construct of the present disclosure is characterized by a decrease in binding to human FcγRI, FcγRIIA, FcγRIIB, FcγRIIIB, or any combination thereof, and C1q (e.g., minimal or no binding). In some embodiments, to alter or reduce antibody-dependent effector functions such as ADCC, CDC, ADCP, or any combination thereof, in some embodiments, the Fc domain in the SIRPα construct of the present disclosure is of the IgG class and contains one or more amino acid substitutions at E233, L234, L235, G236, G237, D265, D270, N297, E318, K320, K322, A327, A330, P331, or P329 (numbering according to the Kabat EU index) (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991))).

[0175] In some embodiments, the polypeptide construct comprising the non-natural Fc region described herein exhibits a decrease or elimination of binding to at least one of the Fcγ receptors CD16a, CD32a, CD32b, CD32c, and CD64 as compared to a polypeptide construct comprising a natural Fc region. In some cases, the polypeptide constructs described herein exhibit a decrease or elimination of binding to the CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors.

[0176] CDC refers to a form of cytotoxicity in which the complement cascade is activated by the complement component C1q that binds to the antibody Fc domain. In some embodiments, the polypeptide construct comprising the non-natural Fc region described herein shows a decrease in C1q binding of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more as compared to a polypeptide construct comprising a wild-type Fc region. In some cases, the polypeptide construct comprising the non-natural Fc region described herein exhibits a decrease in CDC as compared to a polypeptide construct comprising a wild-type Fc region. In some embodiments, the polypeptide construct comprising the non-natural Fc region described herein shows a decrease in CDC of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more as compared to a polypeptide construct comprising a wild-type Fc region. In some cases, the polypeptide construct comprising the non-natural Fc domain variant or Fc domain dimer variant described herein exhibits negligible CDC as compared to a polypeptide construct comprising a wild-type Fc region.

[0177] In some embodiments, the Fc domain variant or Fc domain dimer variant described herein is minimally glycosylated or has reduced glycosylation relative to the wild-type sequence. In some embodiments, deglycosylation is achieved by the N297A mutation 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 designated according to Kabat et al., (1991). In some embodiments, mutation of any one or more of N, Xaa1, Xaa2, or Xaa3 results in deglycosylation of the Fc domain variant or Fc domain dimer variant.

[0178] In some embodiments, variants of the antibody IgG constant region (e.g., Fc domain variants or Fc domain dimer variants) have a reduced ability to specifically bind to Fcγ receptors or a reduced ability to induce phagocytosis. In some embodiments, variants of the antibody IgG constant region (e.g., Fc domain variants or Fc domain dimer variants) have a reduced ability to specifically bind to Fcγ receptors and a reduced ability to induce phagocytosis. For example, in some embodiments, the Fc domain variant is mutated to not include effector functions typical of a "dead" Fc domain variant. For example, in some embodiments, the Fc domain variant includes specific amino acid substitutions known to minimize the interaction between the Fc domain dimer and the Fcγ receptor. In some embodiments, the Fc domain variant is derived from an IgG1 antibody and includes one or more of the amino acid substitutions L234A, L235A, G237A, and N297A (designated according to the EU numbering system by Kabat et al., (1991)). In some embodiments, one or more additional mutations are included in such IgG1 Fc domain variants. Non-limiting examples of such additional mutations to the human IgG1 Fc domain variant include E318A and K322A. In some cases, the human IgG1 Fc domain variant has a total of up to 12, 11, 10, 9, 8, 7, 6, 5, or 4 or fewer mutations compared to the wild-type human IgG1 sequence. In some embodiments, one or more additional deletions are included in such IgG1 Fc domain variants. For example, in some embodiments, the C-terminal lysine of the Fc domain IgG1 heavy chain constant region provided in SEQ ID NO: 88 in Table 7 is deleted, for example, to enhance the homogeneity of the polypeptide when the polypeptide is produced in bacteria or mammalian cells. In some cases, the human IgG1 Fc domain variant has a total of up to 12, 11, 10, 9, 8, 7, 6, 5, or 4 or fewer deletions compared to the wild-type human IgG1 sequence (see, for example, SEQ ID NO: 161 below).In some embodiments, the IgG1 Fc domain variant has the sequence set forth in any one of SEQ ID NO: 135, SEQ ID NO: 136, or SEQ ID NO: 137. SEQ ID NO: 161 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0179] In some embodiments, the Fc domain variant is derived from an IgG2 or IgG4 antibody and includes the amino acid substitutions A330S, P331S, or both A330S and P331S. The foregoing amino acid positions are defined according to Kabat et al., (1991). The Kabat numbering of amino acid residues can be determined for a given antibody by alignment in the homology region of the antibody's sequence with the "standard" Kabat numbering sequence. In some embodiments, the Fc domain variant includes a human IgG2 Fc domain sequence that includes one or more of the A330S, P331S, and N297A amino acid substitutions (designated according to the EU numbering system by Kabat et al., (1991)). In some embodiments, one or more additional mutations are included in such an IgG2 Fc domain variant. Non-limiting examples of such additional mutations to the human IgG2 Fc domain variant include V234A, G237A, P238S, V309L, and H268A (designated according to the EU numbering system by Kabat et al., (1991)). Optionally, the human IgG2 Fc domain variant has a total of up to 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 or fewer mutations compared to the wild-type human IgG2 sequence. In some embodiments, one or more additional deletions are included in such an IgG2 Fc domain variant. For example, in some embodiments, the C-terminal lysine of the Fc domain IgG2 heavy chain constant region provided in SEQ ID NO: 89 in Table 7 is deleted to enhance the homogeneity of the polypeptide, for example, when the polypeptide is produced in bacteria or mammalian cells. Optionally, the human IgG2 Fc domain variant has a total of up to 12, 11, 10, 9, 8, 7, 6, 5, or 4, or fewer deletions compared to the wild-type human IgG2 sequence (see, for example, SEQ ID NO: 162 below). SEQ ID NO: 162: ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0180] When the Fc domain variant is an IgG4 Fc domain variant, in some embodiments, such an Fc domain variant comprises the S228P mutation (designated according to Kabat et al., (1991)). Optionally, the human IgG4 Fc domain variant has a total of up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations compared to the wild-type human IgG4 sequence. In some embodiments, the Fc domain variant comprises a human IgG4 Fc sequence comprising one or more of the S228P, E233P, F234V, L235A, and delG236 amino acid substitutions (designated according to the EU numbering system by Kabat et al., (1991)). In some embodiments, the Fc domain variant comprises a human IgG4 Fc sequence comprising one or more of the S228P, E233P, F234V, L235A, delG236, and N297A amino acid substitutions (designated according to the EU numbering system by Kabat et al., (1991)).

[0181] 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 consists of at least three of the mutations L234A, L235A, G237A, or N297A in the IgG1 Fc region, or is composed of the mutations A330S, P331S, or N297A in the IgG2 Fc region. In some embodiments, the Fc domain variant consists of the mutations L234A, L235A, G237A, and N297A.

[0182] In some embodiments, the Fc domain variant exhibits a decrease in binding to the target Fc receptor as compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain variant exhibits a removal of binding to the target Fc receptor as compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain variant exhibits a decrease in phagocytosis as compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain variant exhibits a removal of phagocytosis as compared to the wild-type human IgG Fc region.

[0183] SEQ ID NO: 88 and SEQ ID NO: 89 provide the amino acid sequences of the Fc domain IgG1 and IgG2 heavy chain constant regions. In some embodiments, the Fc domain variant is any of the variants of SEQ ID NOs: 90-95 as shown in Table 7. TIFF0007710997000019.tif249170TIFF0007710997000020.tif13170

[0184] As used herein, antibody-dependent cell-mediated cytotoxicity, also referred to as ADCC, refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells and neutrophils), thereby enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells. As used herein, antibody-dependent cell-mediated phagocytosis, also referred to as ADCP, refers to a process in which secreted Ig binds to Fc receptors (FcRs) present on certain phagocytic cells (e.g., macrophages), thereby enabling these phagocytic effector cells to specifically bind to antigen-bearing target cells and subsequently engulf and digest the target cells. Ligand-specific high-affinity IgG antibodies directed against the surface of target cells can stimulate cytotoxic cells or phagocytic cells and can be used for such killing. In some embodiments, a polypeptide construct comprising an Fc domain variant or Fc domain dimer variant described herein exhibits reduced ADCC or ADCP as compared to a polypeptide construct comprising a wild-type Fc region. In some embodiments, a polypeptide construct comprising an Fc domain variant or Fc domain dimer variant described herein exhibits a decrease in ADCC or ADCP of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more as compared to a polypeptide construct comprising a wild-type Fc region. In some embodiments, a polypeptide construct comprising an Fc domain variant or Fc domain dimer variant described herein exhibits ablation of ADCC or ADCP as compared to a polypeptide construct comprising a wild-type Fc region.

[0185] As used herein, complement-directed cytotoxicity, also referred to as CDC, refers to a form of cytotoxicity in which the complement cascade is activated by complement component C1q that binds to the antibody Fc domain. In some embodiments, a polypeptide construct comprising an Fc domain variant or an Fc domain dimer variant described herein exhibits a reduction in C1q binding of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more as compared to a polypeptide construct comprising a wild-type Fc region. Optionally, a polypeptide construct comprising an Fc domain variant or an Fc domain dimer variant described herein exhibits reduced CDC as compared to a polypeptide construct comprising a wild-type Fc region. In some embodiments, a polypeptide construct comprising an Fc domain variant or an Fc domain dimer variant described herein exhibits a decrease in CDC of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more as compared to a polypeptide construct comprising a wild-type Fc region. In some embodiments, a polypeptide construct comprising an Fc domain variant or an Fc domain dimer variant described herein exhibits negligible CDC as compared to a polypeptide construct comprising a wild-type Fc region.

[0186] Fc domain variants or Fc domain dimer variants as described herein include those that exhibit a reduced binding to Fcγ receptors as compared to the wild-type human IgG Fc region. For example, in some embodiments, the Fc domain variant or Fc domain dimer variant exhibits a reduced binding to Fcγ receptors as compared to the binding to Fcγ receptors exhibited by the wild-type human IgG Fc region as described in the examples. In some cases, the Fc domain variant or Fc domain dimer variant has a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (where effector function is completely removed) reduction in binding to Fcγ receptors. In some embodiments, the reduction in binding is for any one or more Fcγ receptors, such as CD16a, CD32a, CD32b, CD32c, or CD64.

[0187] In some cases, the Fc domain variants or Fc domain dimer variants disclosed herein exhibit a decrease in phagocytosis as compared to their wild-type human IgG Fc regions. Such Fc domain variants or Fc domain dimer variants exhibit a decrease in phagocytosis as compared to their wild-type human IgG Fc regions, where the decrease in phagocytic activity is, for example, by a factor of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some cases, the Fc domain variant or Fc domain dimer variant exhibits a removal of phagocytosis as compared to its wild-type human IgG Fc region.

[0188] In some embodiments, the Fc domain variants or Fc domain dimer variants disclosed herein are conjugated to one or more fusion partners. Optionally, the fusion partner is a therapeutic moiety. Optionally, the fusion partner is selected to enable targeting, purification, screening, presentation, etc. of the expressed protein. In some embodiments, the fusion partner also affects the degree of binding to Fc receptors or the degree of reduction of opsonization. As described herein, in some embodiments, when an Fc domain variant or Fc domain dimer variant is conjugated to a fusion partner, a polypeptide construct as described below is formed.

[0189] In some embodiments, the fusion partner is linked to the Fc domain variant or Fc domain dimer variant sequence via a linker sequence. In some embodiments, the linker sequence generally comprises a few amino acids, such as less than 10 amino acids, although longer linkers are also utilized. Optionally, the linker has a length of 10, 9, 8, 7, 6, or 5 amino acids or less. Optionally, the linker has a length of at least 10, 11, 12, 13, 14, 15, 20, 25, 30, or 35, or more amino acids. Optionally, in some embodiments, a cleavable linker is used.

[0190] In some embodiments, the fusion partner is a targeting or signal sequence that directs the Fc domain variant or Fc domain dimer variant protein and any associated fusion partner to a desired cellular location or extracellular medium. In some embodiments, certain signaling sequences target proteins that are secreted either into the growth medium or into the periplasmic space located between the inner and outer membranes of the cell. In some embodiments, the fusion partner is a sequence encoding a peptide or protein that enables purification or screening. Such fusion partners include, but are not limited to, polyhistidine tags (His tags) (e.g., His6 (SEQ ID NO: 223) and His10 (SEQ ID NO: 224)), or immobilized metal affinity chromatography (IMAC) systems (e.g., Ni+2 affinity columns), GST fusions, MBP fusions, Strep tags, the BSP biotinylation target sequence of the bacterial enzyme BirA, and other tags for use with epitope tags that are targets of antibodies (e.g., c-myc tag, flag tag, etc.).

[0191] In some embodiments, such tags are useful for purification, screening, or both. For example, in some embodiments, the Fc domain variant or Fc domain dimer variant is purified by immobilizing it on a Ni+2 affinity column, and then after purification, the same His tag is used to immobilize the antibody on a Ni+2-coated plate and perform an ELISA or other binding assay as described elsewhere herein. In some embodiments, the fusion partner enables the use of a selection method for screening Fc domain variants or Fc domain dimer variants as described herein.

[0192] A variety of fusion partners are available that enable various selection methods. For example, phage display can be used by fusing members of an Fc domain variant or Fc domain dimer variant library to the gene III protein. In some embodiments, the fusion partner is an Fc domain variant or Fc domain dimer variant that is labeled. Alternatively, in some embodiments, the fusion partner binds to a specific sequence on an expression vector, thereby enabling the fusion partner and the associated Fc domain variant or Fc domain dimer variant to be linked to the nucleic acid encoding them by covalent or non-covalent bonds.

[0193] In some embodiments, when the fusion partner is a therapeutic moiety, the therapeutic moiety is, for example, a peptide, protein, antibody, siRNA, or small molecule. Non-limiting examples of therapeutic antibodies that bind to the Fc domain variants or Fc domain dimer variants of the present disclosure include, but are not limited to, antibodies that recognize CD47. Non-limiting examples of therapeutic polypeptides that bind to the Fc domain variants or Fc domain dimer variants of the present disclosure include, but are not limited to, CD47-binding polypeptides such as the SIRPα polypeptide. In such cases, the CD47-binding polypeptide is attached or fused to the Fc domain variant or Fc domain dimer variant of the present disclosure. Examples of CD47-binding polypeptides include, but are not limited to, anti-CD47 antibodies or fragments thereof, as well as ligands of CD47 such as SIRPα or fragments thereof. Additional examples of CD47-binding polypeptides include, but are not limited to, naturally occurring forms of SIRPα and variants thereof.

[0194] In some embodiments, also disclosed herein is a polypeptide comprising an Fc domain dimer variant, wherein the Fc domain dimer variant comprises two Fc domain variants, and each Fc domain variant is independently selected from (i) a human IgG1 Fc region consisting of the mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of the mutations A330S, P331S and N297A; or (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A. In some embodiments, the Fc domain variants are identical (i.e., homodimer). In some embodiments, the Fc domain variants are different (i.e., heterodimer). In some embodiments, at least one of the Fc domain variants in the Fc domain dimer is a human IgG1 Fc region consisting of the mutations L234A, L235A, G237A, and N297A. In some embodiments, at least one of the Fc domain variants in the Fc domain dimer is a human IgG2 Fc region consisting of the mutations A330S, P331S, and N297A. In some embodiments, the Fc domain dimer variant exhibits removal or reduction of binding to Fcγ receptors as compared to the wild-type of the human IgG Fc region. In some embodiments, the Fc domain dimer variant exhibits removal or reduction of binding to the CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors as compared to the wild-type of the human IgG Fc region. In some embodiments, the Fc domain dimer variant exhibits removal or reduction of binding to C1q as compared to the wild-type of the human IgG Fc fusion. In some embodiments, at least one of the Fc domain variants in the Fc domain dimer variant is a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A. In some embodiments, the Fc domain dimer variant exhibits removal or reduction of binding to Fcγ receptors as compared to the wild-type human IgG4 Fc region.In some embodiments, the Fc domain dimer variant exhibits removal or reduction of binding to CD16a and CD32b Fcγ receptors as compared to the wild-type of its human IgG4 Fc region. In some embodiments, the Fc domain dimer variant has a K -6 greater than about 5×10 D M and binds to the Fcγ receptor.

[0195] In some embodiments, the Fc domain dimer variant further comprises a CD47-binding polypeptide. In some embodiments, the Fc domain dimer variant exhibits removal or reduction of binding to the Fcγ receptor as compared to the wild-type of the human IgG Fc region. In some embodiments, the CD47-binding polypeptide does not cause acute anemia in rodents and non-human primates. In some embodiments, the CD47-binding polypeptide does not cause acute anemia in humans.

[0196] 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 SIRPα D1 domain variant having the following amino acid sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMDFSIRIGX8ITPADAGTYYCX9KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 221), where X1 is V or I; X2 is A or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is H or P; X7 is L or T; X8 is any amino acid other than N; and X9 is V or I. In some embodiments, the SIRPα polypeptide comprises a SIRPα D1 domain variant, where X1 is V or I; X2 is A or I; X3 is I or F; X4 is E; X5 is K or R; X6 is H or P; X7 is L or T; X8 is not N; and X9 is V.

[0197] In some embodiments disclosed herein, a polypeptide comprising an SIRPα D1 domain variant, wherein the SIRPα D1 domain variant is a non-naturally occurring high-affinity SIRPα D1 domain, the SIRPα D1 domain variant binds to human CD47 with an affinity that is at least 10-fold higher than the affinity of the naturally occurring D1 domain and the Fc domain variant, and the Fc domain variant is linked to a second polypeptide comprising a second Fc domain variant to form an Fc domain dimer variant, and the Fc domain dimer variant has an effector function removed or reduced. In some embodiments, the non-naturally occurring high-affinity SIRPα D1 domain comprises an amino acid mutation at residue 80.

[0198] In some embodiments, disclosed herein is an SIRPα D1 domain variant that binds to a first type of CD47 with a KD of less than 250 nM, the SIRPα D1 domain variant binds to a second type of CD47 with a KD of less than 250 nM, the KD of the first type of CD47 and the KD of the second type of CD47 are within 100-fold of each other, and the first and second types are selected from the group consisting of human, rodent, and non-human primate. In some embodiments, the SIRPα D1 domain variant binds to CD47 of at least three different species. In some embodiments, the non-human primate is a cynomolgus monkey.

[0199] In some embodiments, disclosed herein is a polypeptide comprising (a) an SIRPα D1 domain that binds human CD47 with a KD of less than 250 nM and (b) an Fc domain or variant thereof linked to the N-terminus or C-terminus of the SIRPα D1 domain, wherein the polypeptide does not cause acute anemia in rodents and non-human primates. In some embodiments, the polypeptide is a non-naturally occurring variant of human SIRP-α. In some embodiments, administration of the polypeptide in vivo results in less than a 50% decrease in hemoglobin during the first week after administration. In some embodiments, administration of the polypeptide in humans results in less than a 50% decrease in hemoglobin during the first week after administration. In some embodiments, the polypeptide further comprises at least one Fc domain dimer variant, wherein the Fc domain dimer variant comprises an Fc domain variant selected from (i) a human IgG1 Fc region consisting of the mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of the mutations A330S, P331S and N297A; or (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A. In some embodiments, the Fc domain variant is a human IgG1 Fc region consisting of the mutations L234A, L235A, G237A, and N297A. In some embodiments, the Fc domain variant is a human IgG2 Fc region consisting of the mutations A330S, P331S and N297A.

[0200] The SIRPα constructs of the present disclosure include an SIRPα domain or a variant thereof having a C-terminus that is linked to the N-terminus of an Fc domain or a variant thereof via a linker using conventional genetic or chemical means, such as chemical conjugation. In some embodiments, a linker (e.g., a spacer) is inserted between the polypeptide and the Fc domain or a variant thereof. In some embodiments, the polypeptides of the present disclosure that include an SIRPα D1 domain variant are fused to an Fc domain variant that is unable to form a dimer. In some embodiments, the polypeptides of the present disclosure are fused to an Fc domain or a variant thereof that is capable of forming a dimer, such as a heterodimer, with another Fc domain or a variant thereof. In some embodiments, the polypeptides of the present invention are fused to an Fc domain or a variant thereof, and this fusion protein forms a homodimer. In some embodiments, the polypeptides of the present disclosure are fused to a first Fc domain or a variant thereof, and a different protein or peptide (e.g., an antibody variable region) is fused to a second Fc domain or a variant thereof. In some embodiments, an SIRPα D1 domain or a variant thereof is linked to a first Fc domain or a variant thereof, and a therapeutic protein (e.g., a cytokine, an interleukin, an antigen, a steroid, an anti-inflammatory agent, or an immunomodulatory agent) is linked to a second Fc domain or a variant thereof. In some embodiments, the first and second Fc domains or variants thereof form a heterodimer.

[0201] Without the foregoing limitations, in some embodiments, an SIRPα D1 domain variant polypeptide (e.g., any of the variants described in Tables 2, 5, and 6) is fused to an Fc polypeptide, or an Fc variant polypeptide such as an Fc domain or a variant thereof. Examples of polypeptides that include an SIRPα D1 domain variant polypeptide and a fused Fc domain variant polypeptide include, but are not limited to, SEQ ID NOs: 96-137, 214, and 216 shown in Table 8. TIFF0007710997000021.tif249170TIFF0007710997000022.tif248170TIFF0007710997000023.tif249170TIFF0007710997000024.tif249170TIFF0007710997000025.tif249170TIFF0007710997000026.tif249170TIFF0007710997000027.tif249170TIFF0007710997000028.tif181170

[0202] In some embodiments, the polypeptide comprises an SIRPα D1 variant domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to any of the variants shown in Table 8.

[0203] In some embodiments, the polypeptide comprises an SIRPα D1 domain variant having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NOs: 98 - 104, 107 - 113, 116 - 122, or 135 - 137 of Table 8.

[0204] In some embodiments, the polypeptide comprises (a) a signal regulatory protein alpha (SIRP-α) D1 variant and (b) an fc domain dimer variant, wherein the SIRPα D1 domain variant comprises the following amino acid sequence: EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSLX 11 PVGPIQWFRGAGPX 12 RX 13 LIYNQX 14 X 15 GX 16 FPRVTTVSX 17 X 18TX 19 RX 20 NMDFX 21 IX 22 IX 23 X 24 ITX 25 ADAGTYYCX 26 KX 27 RKGSPDX 28 X 29 EX 30 KSGAGTELSVRX 31 KPS (SEQ ID NO: 47), X1 is E, or G; X2 is L, I, or V; X3 is V, L, or I; X4 is S, or F; X5 is L, or S; X6 is S, or T; X7 is A, or V; X8 is I, or T; X9 is H, R, or L; X 10 is A, V, I, or L; X 11 is I, T, S, or F; X 12 is A, or G; X 13 is E, V, or L; X 14 is K, or R; X 15 is E, or Q; X 16 is H, P, or R; X 17 is D, or E; X 18 is S, L, T, or G; X 19 is K, or R; X 20 is E, or N; X 21 is S, or P; X 22 is S, or R; X 23 is S, or G; X 24 is any amino acid; X 25 is any amino acid; X 26 is V, or I; X 27 is F, L, or V; X 28 is D or absent; X 29 is T, or V; X 30 is F, or V; X 31is A or G; wherein the SIRPα D1 domain variant comprises at least two amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence set forth in any one of SEQ ID NOs: 1-10, and each Fc domain variant independently is (i) a human IgG1 Fc region comprising the N297A mutation; (ii) a human IgG1 Fc region comprising the L234A, L235A, and G237A mutations; (iii) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations; (iv) a human IgG2 Fc region comprising the N297A mutation; (v) a human IgG2 Fc region comprising the A330S and P331S mutations; (vi) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations; (vii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations; or (viii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations.

[0205] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant and an Fc domain dimer having two Fc domains, the SIRPα D1 domain variant comprises the amino acid sequence according to SEQ ID NO: 47, and one of the Fc domains is an Fc domain variant comprising a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations.

[0206] Dimerization of the Fc domain In some embodiments, the SIRPα D1 domain variant polypeptide (e.g., any of the variants described in Tables 2, 5, and 6) is fused to a first Fc domain (e.g., an Fc domain variant) at either the N-terminus or the C-terminus. In some embodiments, the first Fc domain is a variant that cannot form a dimer. In some embodiments, the first Fc domain forms a dimer with a second Fc domain. In some embodiments, the first Fc domain and the second Fc domain comprise amino acid substitutions that promote heterodimerization between the first domain Fc domain and the second domain Fc domain.

[0207] In some embodiments, each of the two Fc domains in the Fc domain dimer comprises an amino acid substitution that promotes heterodimerization of the two monomers. In some embodiments, the SIRPα construct is formed from a first subunit such as, for example, a SIRPα D1 domain variant polypeptide fused to a first Fc domain and a second subunit such as a second Fc domain (e.g., without including a SIRPα D1 domain variant polypeptide or any other polypeptide). In some embodiments, the construct has a single SIRPα D1 domain variant polypeptide linked to an Fc domain dimer (e.g., a single arm). In some embodiments, the construct has two SIRPα D1 domain variant polypeptides linked to an Fc domain dimer (e.g., a dual arm). In some embodiments, a SIRPα D1 domain variant having a KD of about 500 nM is particularly useful in a dual arm construct. In some embodiments, a SIRPα D1 domain variant having a KD of about 50 nM is particularly useful in a dual arm construct. In some embodiments, a SIRPα D1 domain variant having a KD of about 5 nM is useful in both dual arm constructs and single arm constructs. In some embodiments, a SIRPα D1 domain variant having a KD of about 500 pM is useful in both dual arm constructs and single arm constructs. In some embodiments, a SIRPα D1 domain variant having a KD of about 100 pM is useful in both dual arm constructs and single arm constructs. In some embodiments, a SIRPα D1 domain variant having a KD of about 50 pM is useful in both dual arm constructs and single arm constructs. In some embodiments, a SIRPα D1 domain variant having a KD of about 10 pM is useful in both dual arm constructs and single arm constructs.

[0208] In some embodiments, the heterodimerization of the Fc domain is facilitated by introducing different but compatible substitutions into the two Fc domains, such as "knob-into-hole" residue pairs and charge residue pairs. The knob and hole interactions are favorable for the formation of heterodimers, but the interactions between knobs and between holes prevent the formation of homodimers due to steric clashes and the loss of favorable interactions. A hole refers to a cavity created when the original amino acid in a protein is replaced with a different amino acid having a small side-chain volume. A knob refers to a protrusion created when the original amino acid in a protein is replaced with a different amino acid having a large side-chain volume. For example, in some embodiments, the amino acids to be substituted are in the CH3 antibody constant domain of the Fc domain and are involved in the dimerization of the two Fc domains. In some embodiments, the hole of one CH3 antibody constant domain is created to accommodate the knob of another CH3 antibody constant domain such that the knob and hole amino acids act to promote or be favorable for the heterodimerization of the two Fc domains. In some embodiments, the hole of one CH3 antibody constant domain is created to better accommodate the original amino acid in another CH3 antibody constant domain. In some embodiments, the knob of one CH3 antibody constant domain is created to form additional interactions with the original amino acid in another CH3 antibody constant domain.

[0209] In some embodiments, the hole is constructed by replacing an amino acid having a larger side chain, such as tyrosine or tryptophan, with an amino acid having a smaller side chain, such as alanine, valine, or threonine, for example, the Y407V mutation in the CH3 antibody constant domain. Similarly, in some embodiments, the knob is constructed by replacing an amino acid having a smaller side chain with an amino acid having a larger side chain, such as the T366W mutation in the CH3 antibody constant domain. In some embodiments, one Fc domain contains the knob mutation T366W and the other Fc domain contains the hole mutations T366S, L358A, and Y407V. In some embodiments, the polypeptides of the disclosure comprising the SIRPα D1 domain variant are fused to an Fc domain containing the knob mutation T366W to limit unwanted knob - knob homodimer formation. Examples of knob - into - hole amino acid pairs are listed in Table 9, but are not limited thereto. Examples of knob - into - hole Fc domain variants and SIRPα - Fc fusions are shown in Table 10. TIFF0007710997000029.tif45170TIFF0007710997000030.tif245170TIFF0007710997000031.tif238170

[0210] In addition to the knob-into-hole strategy, in some embodiments, electrostatic steering is also used to control dimerization of the Fc domain. Electrostatic steering refers to controlling the formation of higher-order protein molecules by taking advantage of favorable electrostatic interactions between peptides, protein domains, and oppositely charged amino acids within a protein. In particular, to control dimerization of the Fc domain using electrostatic steering, one or more amino acid residues that make up the CH3-CH3 interface are replaced with positively or negatively charged amino acid residues such that the interaction is either electrostatically favorable or unfavorable, depending on the particular charged amino acids introduced. In some embodiments, positively charged amino acids at the interface, such as lysine, arginine, or histidine, are replaced with negatively charged amino acids such as aspartic acid or glutamic acid. In some embodiments, negatively charged amino acids at the interface are replaced with positively charged amino acids. In some embodiments, the charged amino acids are introduced into one or both of the interacting CH3 antibody constant domains. In some embodiments, introducing charged amino acids into the interacting CH3 antibody constant domains of two Fc domains promotes the selective formation of heterodimers of the Fc domain, such that it is controlled by the electrostatic steering effect resulting from the interaction between the charged amino acids. Examples of electrostatic steering amino acid pairs are shown in Table 11, but are not limited thereto. TIFF0007710997000032.tif77170

[0211] In particular, in the context of constructing bispecific antibodies, other methods are available for controlling heterodimerization of the Fc domain.

[0212] In some embodiments, the first Fc domain and the second Fc domain each comprise one or more of the amino acid substitutions of T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, and K409I with respect to the sequence of human IgG1.

[0213] In some embodiments, the Fc domain comprises: (a) with respect to wild-type human IgG1, one of the following amino acid substitutions: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, or K409I; or (b) (i) an N297A mutation with respect to the human IgG1 Fc region; (ii) L234A, L235A, and G237A mutations with respect to the human IgG1 Fc region; (iii) L234A, L235A, G237A, and N297A mutations with respect to the human IgG1 Fc region; (iv) an N297A mutation with respect to the human IgG2 Fc region; (v) A330S and P331S mutations with respect to the human IgG2 Fc region; (vi) A330S, P331S, and N297A mutations with respect to the human IgG2 Fc region; (vii) S228P, E233P, F234V, L235A, and delG236 mutations with respect to the human IgG4 Fc region; or (viii) S228P, E233P, F234V, L235A, delG236, and N297A mutations with respect to the human IgG4 Fc region.In some embodiments, the Fc domain variant comprises: (a) with respect to wild-type human IgG1, one of the following amino acid substitutions: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, or K409I; and further (b) (i) the N297A mutation with respect to the human IgG1 Fc region; (ii) the L234A, L235A, and G237A mutations with respect to the human IgG1 Fc region; (iii) the L234A, L235A, G237A, and N297A mutations with respect to the human IgG1 Fc region; (iv) the N297A mutation with respect to the human IgG2 Fc region; (v) the A330S and P331S mutations with respect to the human IgG2 Fc region; (vi) the A330S, P331S, and N297A mutations with respect to the human IgG2 Fc region; (vii) the S228P, E233P, F234V, L235A, and delG236 mutations with respect to the human IgG4 Fc region; or (viii) the S228P, E233P, F234V, L235A, delG236, and N297A mutations with respect to the human IgG4 Fc region.

[0214] In some embodiments, the first Fc domain and the second Fc domain comprise different amino acid substitutions. In some embodiments, the first Fc domain comprises T366W. In some embodiments, the second Fc domain comprises T366S, L368A, and Y407V. In some embodiments, the first Fc domain comprises D399K. In some embodiments, the second Fc domain comprises K409D.

[0215] Linker In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein α (SIRP-α) D1 variant comprising a SIRPα D1 domain or a fragment thereof having an amino acid mutation at residue 80 relative to the wild-type SIRPα D1 domain; and at least one additional amino acid mutation relative to the wild-type SIRPα D1 domain at a residue selected from the group consisting of residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92.

[0216] In some embodiments, also disclosed herein is a polypeptide comprising an Fc variant, wherein the Fc variant comprises an Fc domain dimer comprising two Fc domain variants, and each Fc domain variant is independently selected from (i) a human IgG1 Fc region comprising the mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region comprising the mutations A330S, P331S and N297A; or (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A.

[0217] In the present disclosure, a linker is used to describe a linkage or connection between a polypeptide or protein domain or an associated non-protein moiety. In some embodiments, the linker is a linkage or connection between an Fc domain (or a variant thereof) and a SIRPα D1 domain variant. In some embodiments, the linker connects the C-terminus of the SIRPα D1 domain variant and the N-terminus of the Fc domain variant such that two polypeptides are joined to each other in a tandem series.

[0218] In some embodiments, the linker is any type of bond formed from a simple covalent bond, such as a peptide bond, a synthetic polymer, or a chemical reaction, such as a chemical conjugate. When the linker is a peptide bond, in some embodiments, the carboxylic acid group at the C-terminus of a protein domain reacts with the amino group at the N-terminus of another protein domain in a condensation reaction to form a peptide bond. In some embodiments, the peptide bond is formed from synthetic means by conventional organic chemical reactions or by natural production from a host cell, and nucleic acid molecules encoding the DNA sequences of both proteins in a tandem series (e.g., an Fc domain variant and an SIRPα D1 domain variant) can be directly transcribed and translated in a host cell by the necessary molecular machinery (e.g., DNA polymerase and ribosome) into a continuous polypeptide encoding both proteins.

[0219] When the linker is a synthetic polymer, in some embodiments, the polymer is functionalized with reactive chemical functional groups at each end to react with the terminal amino acids at the connecting ends of the two proteins.

[0220] When the linker (excluding the above peptide bond) is made from a chemical reaction, in some embodiments, chemical functional groups (e.g., an amine, a carboxylic acid, an ester, an azide, or other functional groups) are synthetically attached to the C-terminus of one protein and the N-terminus of another protein, respectively. Next, in some embodiments, the two functional groups react via synthetic chemical means to form a chemical bond, thus connecting the two proteins together.

[0221] Spacer In some embodiments of the present disclosure, the linker between the Fc domain monomer and the SIRPα D1 variant polypeptide of the present disclosure is an amino acid spacer such as about 1 to 200 amino acids. Suitable peptide spacers include peptide linkers containing flexible amino acid residues such as glycine and serine. Examples of linker sequences are shown in Table 12. In some embodiments, the spacer comprises a motif of GS, GG, GGS, GGG, GGGGS (SEQ ID NO: 163), GGSG (SEQ ID NO: 164), or SGGG (SEQ ID NO: 165), for example, a plurality or repeating motifs. In some embodiments, the spacer comprises a motif of GS, for example, 2 to 12 amino acids such as GS, GSGS (SEQ ID NO: 166), GSGSGS (SEQ ID NO: 167), GSGSGSGS (SEQ ID NO: 168), GSGSGSGSGS (SEQ ID NO: 169), or GSGSGSGSGSGS (SEQ ID NO: 170). In some embodiments, the spacer comprises a motif of GGS, for example, 3 to 12 amino acids including GGS, GGSGGS (SEQ ID NO: 171), GGSGGSGGS (SEQ ID NO: 172), and GGSGGSGGSGGS (SEQ ID NO: 173). In some embodiments, the spacer comprises a motif of GGSG (SEQ ID NO: 164), for example, 4 to 12 amino acids such as GGSG (SEQ ID NO: 164), GGSGGGSG (SEQ ID NO: 174), or GGSGGGSGGGSG (SEQ ID NO: 175). In some embodiments, the spacer comprises a motif of GGGGS (SEQ ID NO: 163), for example, GGGGSGGGGSGGGGS (SEQ ID NO: 176).In some embodiments, the spacer includes amino acids other than glycine and serine, such as AAS (SEQ ID NO: 177), AAAL (SEQ ID NO: 178), AAAK (SEQ ID NO: 179), AAAR (SEQ ID NO: 180), EGKSSGSGSESKST (SEQ ID NO: 181), GSAGSAAGSGEF (SEQ ID NO: 182), AEAAAKEAAAKA (SEQ ID NO: 183), KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), GGGGAGGGG (SEQ ID NO: 185), GENLYFQSGG (SEQ ID NO: 186), SACYCELS (SEQ ID NO: 187), RSIAT (SEQ ID NO: 188), RPACKIPNDLKQKVMNH (SEQ ID NO: 189), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 190), AAANSSIDLISVPVDSR (SEQ ID NO: 191), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 192).

[0222] In some embodiments, the spacer includes a motif of EAAAK (SEQ ID NO: 193), such as a plurality of or repeating motifs. In some embodiments, the spacer includes a motif of a proline-rich sequence, such as (XP)n, where X is any amino acid (e.g., A, K, or E), n is from 1 to 5, and PAPAP (SEQ ID NO: 194). TIFF0007710997000033.tif192170

[0223] In some embodiments, the length of the peptide spacer and the amino acids used are adjusted according to the degree of flexibility desired for the two proteins involved and the final protein fusion polypeptide. In some embodiments, the length of the spacer is adjusted to ensure proper protein folding and avoid aggregate formation. In some embodiments, the spacer is A or AAAL (SEQ ID NO: 178).

[0224] Vectors, Host Cells, and Protein Production In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein alpha (SIRP-α) D1 variant comprising a SIRPα D1 domain or a fragment thereof having an amino acid mutation at residue 80 relative to the wild-type SIRPα D1 domain; and at least one additional amino acid mutation relative to the wild-type SIRPα D1 domain at a residue selected from the group consisting of residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92.

[0225] Also disclosed herein are, in some embodiments, polypeptides comprising an Fc variant, wherein the Fc variant comprises an Fc domain dimer having two Fc domain monomers, and each Fc domain monomer is independently selected from (i) a human IgG1 Fc region comprising the mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region comprising the mutations A330S, P331S and N297A; or (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A.

[0226] In some embodiments, the polypeptides of the disclosure are produced from host cells. Host cells refer to vehicles containing the necessary cellular components, such as organelles, required to express the polypeptides and fusion polypeptides described herein from their corresponding nucleic acids. In some embodiments, the nucleic acid is contained in a nucleic acid vector that is introduced into the host cell by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, or the like. In some embodiments, the selection of the nucleic acid vector depends on the host cell to be used. In some embodiments, the host cell is of either prokaryotic (e.g., bacterial) or eukaryotic (e.g., mammalian) origin.

[0227] In some embodiments, a polypeptide construct comprising a polypeptide, such as a SIRPα D1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and a fusion partner such as an Fc variant, is produced by culturing a host cell transformed with a nucleic acid, preferably an expression vector, comprising a nucleic acid encoding the polypeptide construct (e.g., Fc variant, linker, and fusion partner) under conditions appropriate to induce or cause expression of the polypeptide construct. In some embodiments, the conditions appropriate for expression vary depending on the selected expression vector and host cell. In some embodiments, a variety of suitable host cells are used, including but not limited to mammalian cells, bacteria, insect cells, and yeast. For example, various cell lines found to be useful in the present disclosure are described in the ATCC® cell line catalog available from the American Type Culture Collection. In some embodiments, the Fc domain variants of the present disclosure are expressed in cells optimized to not glycosylate the proteins expressed by such cells, either by genetic engineering of the cell line or by changing cell culture conditions such as the addition of kifunensine, or by using a native non-glycosylated host such as a prokaryote (e.g., E. coli), and in some cases, modification of the glycosylation sequences within the Fc is not necessary.

[0228] Construction of Nucleic Acid Vectors and Host Cells The nucleic acid sequences encoding the amino acid sequences of the polypeptides of the present disclosure can be prepared by various methods. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. In some embodiments, the nucleic acid molecules encoding the polypeptides of the present disclosure are obtained using standard techniques, such as gene synthesis. Alternatively, nucleic acid molecules encoding the wild-type SIRPα D1 domain are mutated to contain specific amino acid substitutions using standard techniques, such as QuikChange™ mutagenesis. In some cases, the nucleic acid molecules are synthesized using a nucleotide synthesizer or PCR techniques.

[0229] In some embodiments, a nucleic acid encoding a polypeptide construct, such as a fusion partner including a SIRPα D1 domain variant (e.g., any variant shown in Tables 2, 5, and 6) and an Fc variant, is incorporated into an expression vector to express the protein. Various expression vectors can be utilized for protein expression. The expression vector can include an extrachromosomal vector that self-replicates or a vector that is incorporated into the host genome. The vector can also include various components or elements. For example, in some embodiments, vector components include, but are not limited to, transcriptional and translational regulatory sequences, such as promoter sequences, ribosome binding sites, signal sequences, transcription start and stop sequences, translation start and stop sequences, 3' and 5' untranslated regions (UTRs), and enhancer or activator sequences; an origin of replication; a selectable marker gene; as well as a nucleic acid sequence encoding the polypeptide of interest and a transcription termination sequence. In some embodiments, the expression vector includes a protein operably linked to a control or regulatory sequence, a selectable marker, any fusion partner, additional elements, or any combination thereof. The term "operably linked" means that the nucleic acid is placed in a functional relationship with another nucleic acid sequence. Generally, these expression vectors include transcriptional and translational regulatory nucleic acids operably linked to a nucleic acid encoding an Fc variant, and are typically appropriate for the host cells used to express the protein. Selectable genes or markers, such as 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. Various selectable genes are available.

[0230] In some embodiments, the components or elements of the vector are optimized such that the expression vector is compatible with the host cell type. Expression vectors found to be useful in the present disclosure include, but are not limited to, those enabling protein expression in mammalian cells, bacteria, insect cells, yeast, and in vitro systems.

[0231] In some embodiments, mammalian cells are used as host cells for producing the polypeptides of the present disclosure. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NS0, Sp2 / 0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells. In some embodiments, E. coli cells are used as host cells for producing the polypeptides of the present disclosure. Examples of E. coli strains include, but are not limited to, E. coli 294 (ATCC® 31,446), E. coli λ1776 (ATCC® 31,537, E. coli BL21(DE3) (ATCC® BAA-1025)), and E. coli RV308 (ATCC® 31,608).

[0232] Different host cells have characteristic and specific mechanisms for post-translational processing and modification (e.g., glycosylation) of protein products. In some embodiments, an appropriate cell line or host system is selected to ensure proper modification and processing of the expressed polypeptide. When a vector is introduced into a host cell for protein production, the host cell is cultured in a conventional nutrient medium that has been appropriately modified for induction of the promoter, selection of transformants, or amplification of the gene encoding the desired sequence.

[0233] In some embodiments, a polypeptide construct, e.g., a polypeptide construct comprising a fusion partner such as an SIRPα D1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and an Fc variant, is expressed in a mammalian expression system, such as a system in which an expression construct is introduced into mammalian cells using a virus such as a retrovirus or an adenovirus. In some embodiments, cells of human, mouse, rat, hamster, or primate origin are utilized. Suitable cells include, but are not limited to, known research cells such as Jurkat T cells, NIH3T3, CHO, COS, and 293 cells. Alternatively, in some embodiments, the protein is expressed in bacterial cells. Bacterial expression systems are well known in the art and include Escherichia coli (E. coli), Bacillus subtilis, Streptococcus cremoris, and Streptococcus lividans. In some cases, a polypeptide construct comprising an Fc domain variant is produced in insect cells such as Sf9 and Sf21 cells, or in yeast cells such as organisms of the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, and Yarrowia. In some cases, a polypeptide construct comprising an Fc domain variant is expressed in vitro using a cell-free translation system. In vitro translation systems derived from both prokaryotic (e.g., E. coli) and eukaryotic (e.g., wheat germ, rabbit reticulocyte) cells are available and, in some embodiments, are selected based on the expression level and functional properties of the protein of interest. For example, as will be understood by those skilled in the art, in vitro translation is required for some display technologies, such as ribosome display. Further, in some embodiments, the Fc domain variant is produced by chemical synthesis methods such as liquid phase peptide synthesis and solid phase peptide synthesis.In the case of in vitro transcription using a non-glycosylated system such as a bacterial extract, even if a natural glycosylation site exists, Fc is not glycosylated, so inactivation of Fc can be obtained equivalently.

[0234] In some embodiments, the polypeptide construct includes unnatural amino acids, amino acid analogs, amino acid mimetics, or any combination thereof that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids generally refer to the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), and pyrrolysine and selenocysteine. An amino acid analog is a compound having the same basic chemical structure as a naturally occurring amino acid, for example, a carbon bonded to hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. In some embodiments, such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but generally retain the same basic chemical structure as natural amino acids.

[0235] Production, recovery, and purification of proteins In some embodiments, the host cells used to produce the polypeptides of the present disclosure are grown in a medium suitable for culturing the selected host cells. Examples of suitable media for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Expi293™ Expression Medium, DMEM supplemented with fetal bovine serum (FBS), and RPMI-1640. Examples of suitable media for bacterial host cells include Luria Broth (LB) supplemented with necessary supplements such as a selective agent, e.g., ampicillin. In some embodiments, the host cells are cultured at a suitable temperature, such as about 20°C to about 39°C, for example, about 25°C to about 37°C, preferably 37°C, and a CO2 level of 5% to 10%. In some embodiments, the pH of the medium is about pH 6.8 to pH 7.4, for example, pH 7.0, mainly depending on the host organism. When an inducible promoter is used in the expression vector, protein expression is induced under conditions suitable for activation of the promoter.

[0236] In some embodiments, protein recovery involves disruption of the host cells, e.g., by 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 polypeptides of the present disclosure are purified by various methods of protein purification, e.g., chromatography (e.g., ion exchange chromatography, affinity chromatography, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. For example, in some embodiments, the protein is isolated and purified by appropriately selecting and combining an affinity column such as a Protein A column (e.g., POROS Protein A chromatography), a chromatography column (e.g., POROS HS-50 cation exchange chromatography), filtration, ultrafiltration, desalting, and dialysis procedures. In some embodiments, the polypeptide is conjugated to a marker sequence, such as a peptide, to facilitate purification. An example of a marker amino acid sequence is a hexahistidine peptide (His6-tag (SEQ ID NO: 223)) that can bind to a nickel-functionalized agarose affinity column with micromolar affinity. Alternatively, a hemagglutinin "HA" tag corresponding to an epitope derived from the influenza hemagglutinin protein can be used.

[0237] In some embodiments, a polypeptide construct comprising a polypeptide of the disclosure, such as a SIRPα D1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and a fusion partner such as an Fc variant, is produced by a cell of a subject (e.g., a human) by administering, for example, in the context of gene therapy, a vector such as a viral vector (e.g., a retroviral vector, an adenoviral vector, a poxviral vector (e.g., a vaccinia virus vector, e.g., modified vaccinia Ankara (MVA)), an adeno-associated viral vector, and an alphavirus vector) comprising a nucleic acid molecule encoding the polypeptide of the disclosure. The vector can be used for the expression of the polypeptides disclosed herein when it enters the cells of the subject (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.). Optionally, the polypeptide is secreted from the cell. In some embodiments, no further action is required if the treatment of a disease or disorder is the desired outcome. In some embodiments, if protein collection is desired, blood is collected from the subject and the protein is purified from the blood by various methods.

[0238] Method for treating cancer Provided herein is a method for treating cancer in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant as described herein) and an Fc domain variant (e.g., an Fc domain variant as described herein), and (b) a therapeutic antibody.

[0239] Lung cancer In some embodiments, a method of treating lung cancer (e.g., non-small cell lung cancer or "NSCLC") in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein), and (b) a therapeutic antibody that interferes with the interaction between PD-1 and PD-L1, wherein the individual has progressed (e.g., shown disease progression) during (or after) prior treatment for lung cancer (e.g., NSCLC). In some embodiments, the prior treatment was an immune checkpoint inhibitor (CPI) therapy. Additionally or alternatively, in some embodiments, the individual has a PD-L1 tumor proportion score (TPS) of less than 50%. In some embodiments, the individual has not received prior CPI therapy. In some embodiments, the polypeptide (e.g., the fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (where numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows the Kabat EU index). In some embodiments, the polypeptide (e.g., the fusion polypeptide) administered to the individual comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85.In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to the individual comprises an Fc domain variant that is a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, numbered according to the Kabat EU index. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to the individual comprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., fusion polypeptide) forms a homodimer. In some embodiments, the therapeutic antibody that blocks the interaction between PD-1 and PD-L1 is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is pembrolizumab.

[0240] In some embodiments, pembrolizumab is administered subcutaneously. In some embodiments, pembrolizumab is administered by intravenous infusion. In some embodiments, pembrolizumab is administered according to the instructions on its label. In some embodiments, pembrolizumab is administered to an individual at a dose of approximately 200 mg every three weeks (Q3W) (e.g., via IV infusion). In some embodiments, pembrolizumab is administered to an individual for up to 24 months. In some embodiments, pembrolizumab is administered to an individual for at least 24 months. In some embodiments, dose modifications of pembrolizumab are made according to the prescribing information in each country. Complete information regarding the preparation, formulation, dosage, and administration schedule of pembrolizumab can be found in the prescribing information in each country (see, e.g., www.accessdata.fda.gov / drugsatfda_docs / label / 2016 / 125514s012lbl.pdf in the United States; see, e.g., www.ema.europa.eu / en / documents / product-information / keytruda-epar-product-information_en.pdf in Europe). In some embodiments, a polypeptide (e.g., a fusion polypeptide) is administered to an individual (e.g., via intravenous infusion) at a dose of, e.g., 10.0 mg / kg weekly (i.e., once every 7 days or “qw”).

[0241] In some embodiments, the cancer is NSCLC. In some embodiments, the NSCLC is locally advanced NSCLC. In some embodiments, the NSCLC is metastatic NSCLC. In some embodiments, the individual has metastatic NSCLC and did not show disease progression within 8 weeks from the start of prior treatment with a PD-1 or PD-L1 inhibitor. In some embodiments, the individual has a PD-L1 tumor proportion score (TPS) score greater than 1%. In some embodiments, the individual has locally advanced NSCLC or metastatic NSCLC with a TPS score of less than 50%. In some embodiments, the individual progressed after systemic therapy for their metastatic disease. In some embodiments, the individual has locally advanced NSCLC or metastatic NSCLC with a TPS score greater than 1% and the individual progressed during prior checkpoint inhibitor (CPI) therapy for NSCLC.

[0242] In some embodiments, prior CPI therapy for lung cancer (e.g., NSCLC) in a progressing individual was a therapy comprising treatment with nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, semaprelimab, tislelizumab (also known as BGB-A317), toripalimab, sintilimab, camrelizumab (also known as SHR-1210 or INCSHR-1210), spartalizumab (also known as PDR001), TSR-042, and / or FAZ053. In some embodiments, for example, when determined by Response Evaluation In Solid Tumor (RECIST) criteria (e.g., version 1.0 or 1.1) or modified RECIST criteria (e.g., Therasse et al. (2000) J. Natl Cancer Inst. 92:205-216; Eisenhauer et al. (2009) Eur J. Cancer. 45:229-247; and Jang et al. (2013) Chin J. Cancer Res. 25(6):689-694), World Health Organization (WHO) criteria (e.g., WHO. Handbook for Reporting Results of Cancer Treatment. Geneva: World Health Organization Offset Publication; 1979. p. 48; and Miller et al. (1981) Cancer. 47:207-214), or any set of response assessment criteria described in Hwang et al. (2017) "Response Evaluation of Chemotherapy for Lung Cancer" Tuberc Respir Dis (Seoul). 80(2):136-142, and the references cited therein, if an individual showed progression (PD), the individual was considered to have progressed during prior CPI therapy for lung cancer (e.g., NSCLC). In some embodiments, the individual is resistant to standard therapy (e.g., curative therapy) for lung cancer (e.g., NSCLC).In some embodiments, there is no standard therapy (e.g., curative therapy) available for treating lung cancer (e.g., NSCLC).

[0243] In some embodiments, the individual's programmed death-ligand 1 tumor proportion score (TPS) is determined using an in vitro diagnostic immunohistochemistry (IHC) assay for the detection of PD-L1 in formalin-fixed, paraffin-embedded (FFPE) human tissue sections. In some embodiments, the IHC assay is the PD-L1 IHC 22C3 PharmDx. The PD-L1 IHC 22C3 PharmDx is a qualitative immunohistochemical assay in which a mouse monoclonal anti-PD-L1 antibody (clone 22C3) is used to detect PD-L1 (i.e., human PD-L1) in formalin-fixed paraffin-embedded (FFPE) lung cancer tissue (e.g., NSCLC tissue) obtained from an individual (e.g., a patient) using a DAKO (trademark) AUTOSTAINER LINK 48 automated staining system with an ENVISION (trademark) FLEX visualization system. In some embodiments, the TPS is a measure of PD-L1 protein expression in a lung cancer (e.g., NSCLC) tissue sample from an individual (e.g., a patient). In some embodiments, the TPS is the percentage of viable tumor cells showing partial or complete membrane staining at any intensity. If viable tumor cells with a TPS of 50% or greater exhibit membrane staining at any intensity, the specimen is considered PD-L1 positive. In some embodiments, tumor-associated immune cells (such as infiltrating lymphocytes or macrophages) are not included in the scoring for determining the TPS. In some embodiments, the labeling of the lung cancer (e.g., NSCLC) sample is performed by a pathologist. In some embodiments, the staining is determined via an optical microscope at magnifications of 10x to 40x. Further details regarding the PD-L1 IHC 22C3 PharmDx assay, the reagents and equipment for performing the assay, the interpretation of assay results, and the TPS scoring are provided in www(dot)accessdata(dot)fda(dot)gov / cdrh_docs / pdf15 / p150013b(dot)pdf and Reck et al. (2016) "Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer" NEJM.375:1823-1833.

[0244] Head and neck cancer In some embodiments, a method of treating a head and neck cancer (e.g., head and neck squamous cell carcinoma or "HNSSC") in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein), and (b) a therapeutic antibody that interferes with the interaction between PD-1 and PD-L1, wherein the individual has progressed (e.g., shown disease progression) during or after a prior treatment for head and neck cancer (e.g., HNSSC). In some embodiments, the prior treatment was a platinum-containing treatment. In some embodiments, the polypeptide (e.g., the fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index). In some embodiments, the polypeptide (e.g., the fusion polypeptide) administered to the individual comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In some embodiments, the polypeptide (e.g., the fusion polypeptide) administered to the individual comprises an Fc domain variant that is a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, the numbering following 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 (e.g., fusion polypeptide) administered to a subject comprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., fusion polypeptide) forms a homodimer. In some embodiments, the therapeutic antibody that blocks the interaction between PD-1 and PD-L1 is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is pembrolizumab. In some embodiments, the HNSCC is PD-L1 negative. In some embodiments, the HNSCC is PD-L1 positive. Further details regarding "PD-L1 negative" and "PD-L1 positive" are described elsewhere in this specification.

[0245] In some embodiments, pembrolizumab is administered subcutaneously. In some embodiments, pembrolizumab is administered by intravenous infusion. In some embodiments, pembrolizumab is administered in accordance with the instructions on its label. In some embodiments, pembrolizumab is administered to an individual at a dose of approximately 200 mg every three weeks (Q3W) (e.g., via IV infusion). In some embodiments, pembrolizumab is administered to an individual for up to 24 months. In some embodiments, pembrolizumab is administered to an individual for at least 24 months. In some embodiments, the dosage of pembrolizumab is changed in accordance with the prescribing information in each country. Complete information regarding the preparation, formulation, dosage, and administration schedule of pembrolizumab may be described in the prescribing information in each country (see, e.g., www.accessdata.fda.gov / drugsatfda_docs / label / 2016 / 125514s012lbl.pdf in the United States; see, e.g., www.ema.europa.eu / en / documents / product-information / keytruda-epar-product-information_en.pdf in Europe). In some embodiments, a polypeptide (e.g., a fusion polypeptide) is administered to an individual (e.g., via intravenous infusion) at a dose of, for example, 10.0 mg / kg weekly (i.e., once every 7 days or “qw”).

[0246] In some embodiments, the individual has recurrent HNSCC. In some embodiments, the HNSCC is metastatic HNSCC. In some embodiments, the individual has received prior treatment with an immune checkpoint inhibitor (「CPI」), i.e., the individual is a 「CPI-experienced」 individual. In some embodiments, the individual has recurrent HNSCC or metastatic HNSCC and has not shown disease progression within 8 weeks from the start of prior treatment with CPI. In some embodiments, the prior CPI therapy was or included a PD-1 or PD-L1 inhibitor. In some embodiments, the prior CPI therapy was or included treatment with nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, semiprimab, tislelizumab (also known as BGB-A317), toripalimab, sintilimab, camrelizumab (also known as SHR-1210 or INCSHR-1210), spartalizumab (also known as PDR001), TSR-042, and / or FAZ053. In some embodiments, the individual has not received prior treatment with CPI (e.g., the individual is 「immune checkpoint inhibitor naive」 or 「CPI naive」).

[0247] In some embodiments, prior platinum-containing therapy that the individual underwent during treatment or after treatment was a therapy that included treatment with carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and / or satraplatin. In some embodiments, for example, when determined by a set of response criteria described in Response Evaluation In Solid Tumor (RECIST) criteria (e.g., version 1.0 or 1.1) or modified RECIST criteria (e.g., Therasse et al. (2000) J. Natl Cancer Inst. 92:205-216; Eisenhauer et al. (2009) Eur J. Cancer. 45:229-247; and Jang et al. (2013) Chin J. Cancer Res. 25(6):689-694), World Health Organization (WHO) criteria (e.g., WHO. Handbook for Reporting Results of Cancer Treatment. Geneva: World Health Organization Offset Publication; 1979. p. 48; and Miller et al. (1981) Cancer. 47:207-214), or Wray et al. (2016) "Therapy Response Assessment and Patient Outcomes in Head and Neck Squamous Cell Carcinoma: FDG PET Hopkins Criteria Versus Residual Neck Node Size and Morphologic Features" Am J. Roentgenology. 207:641-647), if the individual shows progression (PD), the individual is considered to have progressed during or after prior platinum-containing therapy for head and neck cancer (e.g., HNSCC). In some embodiments, the individual is resistant to standard therapy (e.g., curative therapy) for head and neck cancer (e.g., HNSCLC).In some embodiments, there is no standard therapy (e.g., curative therapy) available for treating head and neck cancer (e.g., HNSCC).

[0248] Gastric / gastroesophageal (GEJ) cancer In some embodiments, a method of treating a stomach / gastroesophageal junction (GEJ) cancer (e.g., HER2-positive gastric or GEJ adenocarcinoma) in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein), and (b) a therapeutic anti-HER2 antibody, wherein the individual has progressed (e.g., demonstrated disease progression) during or after a prior treatment regimen for the stomach / GEJ cancer (e.g., stomach / GEJ adenocarcinoma), and the prior treatment regimen was an anti-HER2 antibody therapy and / or a fluoropyrimidine-based therapy. In some embodiments, the polypeptide (e.g., the fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index). In some embodiments, the polypeptide (e.g., the fusion polypeptide) administered to the individual comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In some embodiments, the polypeptide (e.g., the fusion polypeptide) administered to the individual comprises an Fc domain variant that is a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering follows the Kabat EU index.In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to an individual comprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., fusion polypeptide) forms a homodimer. In some embodiments, the therapeutic anti-HER2 antibody administered to an individual in combination with the fusion polypeptide is trastuzumab.

[0249] In some embodiments, trastuzumab is administered subcutaneously. In some embodiments, trastuzumab is administered by intravenous infusion. In some embodiments, trastuzumab is administered according to the instructions on its label. In some embodiments, trastuzumab is administered to an individual every three weeks (Q3W) (e.g., via intravenous infusion). In some embodiments, the first (i.e., the 1st) dose of trastuzumab is about 8 mg / kg, and all subsequent doses (i.e., after the 1st dose) are about 6 mg / kg. In some embodiments, dose modifications of trastuzumab are made according to the prescribing information in each country. Complete information regarding the preparation, formulation, dosage, and administration schedule of trastuzumab may be described in the prescribing information in each country (in the United States, for example, see www.accessdata.fda.gov / drugsatfda_docs / label / 2017 / 103792s5337lbl.pdf; in Europe, for example, see www.ema.europa.eu / en / documents / product-information / herceptin-epar-product-information_en.pdf). In some embodiments, the polypeptide (e.g., fusion polypeptide) is administered to an individual (e.g., via intravenous infusion) once a week (i.e., once every 7 days or “qw”), for example, at a dose of 10.0 mg / kg.

[0250] In some embodiments, the HER2 status (i.e., HER2 positive status or HER2 negative status) of gastric cancer or GEJ cancer is determined via immunohistochemistry (IHC) or in situ hybridization (ISH, e.g., fluorescence ISH or “FISH”). In some embodiments, the HER2 status of gastric cancer or GEJ cancer is determined according to the criteria described in Abrahao-Machado et al. (2016) World J. Gastroenterol. 22(19):4619-4625 and the references cited therein. In some embodiments, HER2 positive gastric cancer or HER2 positive GEJ cancer is HER2 positive gastric / HER2 positive GEJ adenocarcinoma. In some embodiments, HER2 positive gastric cancer / HER2 positive GEJ cancer (e.g., adenocarcinoma) is metastatic gastric / GEJ cancer (e.g., metastatic adenocarcinoma). In some embodiments, the individual has metastatic gastric / GEJ cancer (e.g., adenocarcinoma) and showed at least a stable (SD) effect (i.e., an effect better than progressive (PD)) as the best effect of prior treatment.

[0251] In some embodiments, the individual progressed (e.g., demonstrated disease progression) during or after prior treatment with an anti-HER2 antibody. In some embodiments, the prior anti-HER2 antibody therapy included treatment with trastuzumab, pertuzumab, and / or margetuximab. Additionally or alternatively, in some embodiments, the individual progressed (e.g., demonstrated disease progression) during or after prior treatment with a fluoropyrimidine-based therapy. In some embodiments, the prior fluoropyrimidine-based therapy included treatment with, for example, capecitabine, floxuridine, 4-fluorouracil, 5-fluorouracil, carmofur, doxifluridine, ftorafur (tegafur), UFT (i.e., a 1:4 molar combination of ftorafur and uracil), S-1 (a combination of ftorafur, gimeracil, and oteracil), and / or FOLFOX (a combination of folic acid, 5-fluorouracil, and oxaliplatin). In some embodiments, the individual progressed during or after treatment with an anti-HER2 antibody and a fluoropyrimidine-based therapy. In some embodiments, the anti-HER2 antibody and the fluoropyrimidine-based therapy were administered in combination (e.g., both agents were part of a single treatment regimen). In some embodiments, the anti-HER2 antibody and the fluoropyrimidine-based therapy were each administered in separate treatment regimens (e.g., two separate prior treatments or two separate prior treatment lines).

[0252] In some embodiments, for example, when evaluated by any series of effect determinations such as Response Evaluation In Solid Tumor (RECIST) criteria (e.g., version 1.0 or 1.1) or modified RECIST criteria (e.g., Therasse et al. (2000) J. Natl Cancer Inst. 92:205-216; Eisenhauer et al. (2009) Eur J. Cancer. 45:229-247; and Jang et al. (2013) Chin J. Cancer Res. 25(6):689-694), World Health Organization (WHO) criteria (e.g., WHO. Handbook for Reporting Results of Cancer Treatment. Geneva: World Health Organization Offset Publication; 1979. p. 48; and Miller et al. (1981) Cancer. 47:207-214), or as described in Kurokawa et al. (2013) Ann Surg Oncol. 20(9):3009-3014; Yanagawa et al. (2012) J Nucl Med. 53(6):872-880; Lordick et al. (2016) Ann Oncol. 27(suppl 5):v50-v57; or Kim et al. (2015) Oncology. 88:69-75, if an individual shows progression (PD), that individual is considered to have progressed during prior anti-HER2 antibody therapy and / or prior fluoropyrimidine-based therapy for gastric / GEJ cancer (e.g., HER2-positive gastric / GEJ cancer). In some embodiments, the individual is resistant to standard therapy (e.g., curative therapy) for gastric / GEJ cancer (e.g., HER2-positive gastric / HER2-positive GEJ cancer). In some embodiments, there is no standard therapy (e.g., curative therapy) available for treating gastric / GEJ cancer (e.g., HER2-positive gastric / HER2-positive GEJ cancer).

[0253] Lymphoma (i) Aggressive non-Hodgkin lymphoma In some embodiments, provided is a method of treating aggressive non-Hodgkin lymphoma or "NHL" (e.g., diffuse large B-cell lymphoma ("DLBCL", e.g., de novo DLBCL or transformed DLBCL or mantle cell lymphoma (MCL)) in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein), and (b) a therapeutic anti-CD20 antibody, wherein the aggressive NHL is a relapsed and / or refractory aggressive NHL (e.g., the individual has relapsed during or after prior treatment for aggressive NHL and / or was refractory to prior treatment for aggressive NHL), and wherein there is no available therapy (e.g., curative therapy) for the aggressive NHL (e.g., DLBCL such as de novo DLBCL, transformed DLBCL, or mantle cell lymphoma). In some embodiments, the polypeptide (e.g., the fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index).In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to the individual comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to the individual comprises an Fc domain variant that is a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, numbering 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 (e.g., fusion polypeptide) administered to the individual comprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., fusion polypeptide) forms a homodimer. In some embodiments, the therapeutic anti-CD20 antibody is rituximab. In some embodiments, the aggressive NHL is diffuse large B-cell lymphoma (DLBCL), e.g., de novo DLBCL or transformed DLBCL. In some embodiments, the aggressive NHL is mantle cell lymphoma (MCL).

[0254] In some embodiments, rituximab is administered subcutaneously. In some embodiments, rituximab is administered by intravenous infusion. In some embodiments, rituximab is administered according to the instructions on its label. In some embodiments, rituximab is administered to the individual at a dose of about 375 mg / m 2 2 (e.g., via intravenous infusion). In some embodiments, the first four doses of rituximab (i.e., doses 1-4) are administered to the individual once a week (e.g., every 7 days, or "qw") for the first four weeks (e.g., 28 days) of treatment at a dose of about 375 mg / m 2administered, and the next eight doses (i.e., doses 5-12) are administered to the individual once every four weeks (e.g., every 28 days or “q4w”). In some embodiments, the first four doses of rituximab (i.e., doses 1-4) are administered to the individual once a week (e.g., once every 7 days or “qw”) for the first four weeks (e.g., 28 days) of treatment, at a dose of, for example, about 375 mg / m 2 administered, and the next four doses (i.e., doses 5-8) are administered to the individual once every four weeks (e.g., every 28 days or “q4w”). In some embodiments, dose modifications of rituximab are made in accordance with the national prescribing information. Complete information regarding the preparation, dispensing, dosage, and administration schedule of rituximab may be described in the national prescribing information (see, e.g., www.accessdata.fda.gov / drugsatfda_docs / label / 2012 / 103705s5367s5388lbl.pdf in the United States; see, e.g., www.ema.europa.eu / en / documents / product-information / mabthera-epar-product-information_en.pdf in Europe). In some embodiments, the polypeptide (e.g., fusion polypeptide) is administered to the individual once a week (i.e., once every 7 days or “qw”), for example, at a dose of 10.0 mg / kg or 15.0 mg / kg, for example, via intravenous infusion.

[0255] In some embodiments, if an individual has no history of lymphoma, the individual is diagnosed with de novo DLBCL (e.g., de novo relapsed and / or refractory DLBCL). In some embodiments, if an individual has a history of lymphoma, such as indolent lymphoma, e.g., marginal zone lymphoma, lymphoplasmacytic lymphoma, small lymphocytic lymphoma / chronic lymphocytic leukemia, follicular lymphoma, or lymphocyte-predominant Hodgkin lymphoma, the individual is diagnosed with transformed DLBCL. In some embodiments, if an individual is found to have one or more of the following chromosomal abnormalities: t(11;14), t(14;18), the individual is diagnosed with mantle cell lymphoma (MCL). In some embodiments, if overexpression of SOX11 is detected in a sample of the individual's leukemic cells, the individual is diagnosed with mantle cell lymphoma (MCL). Other criteria for diagnosing de novo DLBCL, transformed DLBCL, and MCL are known in the art and are routinely used by those of skill in the art. See, for example, Balsas et al. (2017) Blood. 130(4):501-513; National Guideline Alliance (UK), Non-Hodgkin’s Lymphoma: Diagnosis and Management. London: National Institute for Health and Care Excellence (UK); 2016 Jul. (NICE Guideline, No. 52.) 3, Staging; Dreyling et al. Am Soc Clin Oncol Educ Book. 2014:191-8; and others.

[0256] In some embodiments, an individual achieved the lowest stable (SD) treatment effect for a prior treatment, but for example, if the effect stopped within any of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after discontinuation of the prior treatment (e.g., showed progression of the disease), the individual is considered to have relapsed after a prior treatment for aggressive NHL (e.g., de novo DLBCL, transformed DLBCL, or MCL). In some embodiments, if an individual did not respond to a prior treatment (e.g., if at least a stable (SD) treatment effect could not be achieved during or after the prior treatment), the individual is considered refractory to the prior treatment for aggressive NHL. In some embodiments, the treatment effect for the treatment of aggressive NHL is determined according to Cheson et al. (2014) "Recommendations for Initial Evaluation, Staging and Response Assessment of Hodgkin and Non-Hodgkin Lymphoma: The Lugano Classification" J. Clin Oncol. 32:3059-3067.

[0257] In some embodiments, the individual was refractory to treatment with, or had relapsed from, at least one prior therapy for aggressive NHL (e.g., at least one prior approved standard therapy, at least two prior approved standard therapies, at least three approved standard therapies, etc.). Standard treatments for DLBCL (e.g., de novo or transformed DLBCL) and MCL include, but are not limited to, for example, rituximab, RCHP (i.e., rituximab, cyclophosphamide, doxorubicin, and prednisone), R-CHOP (i.e., rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone); R-CHOEP (i.e., rituximab, cyclophosphamide, doxorubicin, vincristine, etoposide, and prednisone, typically administered in 6 cycles on a 21-day cycle); EPOCH-R (i.e., rituximab, cyclophosphamide, doxorubicin, vincristine, etoposide, and prednisone, typically administered as a 4-day continuous infusion); R-GCVP (i.e., rituximab, gemcitabine, cyclophosphamide, vincristine, and prednisolone); R-CEPP (i.e., rituximab, cyclophosphamide, etoposide, procarbazine, and prednisone); R-CEOP (i.e., rituximab, cyclophosphamide, epirubicin, vincristine, and prednisone); R-CVP (i.e., rituximab, cyclophosphamide, vincristine, and prednisone); rituximab and bendamustine; rituximab and lenalidomide; DHAP (i.e., dexamethasone, high-dose cytarabine, and cisplatin); RDHAP (i.e., DHAP combined with rituximab); ICE (i.e., ifosfamide, carboplatin, and etoposide); RICE (i.e., ICE combined with rituximab); DICE (i.e., ICE combined with dexamethasone); DICE and mesna; BEAM (i.e., carmustine, etoposide, cytarabine, and melphalan); R-BEAM (i.e., BEAM combined with rituximab); ESHAP (i.e., etoposide, solumedrol, high-dose cytarabine, and cisplatin);R-ESHAP (i.e., ESHAP combined with rituximab); MIME (i.e., methylglyoxal bis(guanylhydrazone), ifosfamide, methotrexate, and etoposide); parsaclisib (also known as INCB050465); MATRIX (i.e., methotrexate, cytarabine, thiotepa, and rituximab); hyper-CVAD or HCVAD (i.e., high-dose fractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone) RHCVAD (i.e., HCVAD combined with rituximab); RHCVAD / MA (i.e., alternating administration of RHCVAD with methotrexate and cytarabine); DHAP (i.e., dexamethasone, cisplatin, and cytarabine); R-DHAP (i.e., DHAP combined with rituximab); ibrutinib; rituximab, obinutuzumab, CVAD (i.e., cyclophosphamide, doxorubicin, vincristine, and prednisone); RCVAD (i.e., CVAD combined with rituximab); GemOx (i.e., gemcitabine and oxaliplatin); R-GemOx (i.e., GemOx combined with rituximab); DHAX (i.e., dexamethasone, cytarabine, and oxaliplatin); R-DHAX (i.e., DHAX combined with rituximab); GIFOX (i.e., gemcitabine, ifosfamide, and oxaliplatin); RGIFOX (i.e., GIFOX combined with rituximab); bortezomib and GIFOX; ASCT (i.e., autologous stem cell transplantation) HD-ASCT (i.e., ASCT combined with high-dose therapy, e.g., high-dose chemotherapy); CAR T cell therapy (e.g., tisagenlecleucel or axicabtagene);Examples include brentuximab vedotin and lenalidomide. In some embodiments, prior treatment for aggressive NHL included any two or more of the prior treatments (administered together in a single treatment regimen or separately in distinct treatment regimens). In some embodiments, there are no treatment options (e.g., curative therapy options) available to an individual having aggressive NHL (e.g., relapsed / refractory aggressive NHL). In some embodiments, the individual has DLBCL (e.g., de novo DLBCL or transformed DLBCL) or MCL for which curative therapy is not available. In some embodiments, the individual has DLBCL (e.g., de novo DLBCL or transformed DLBCL) or MCL that has relapsed or is refractory following standard approved therapy (e.g., curative therapy).;

[0258] (ii) indolent lymphoma In some embodiments, a method for treating non-Hodgkin lymphoma in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) an SIRPα D1 domain variant (e.g., an SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., a polypeptide (e.g., a fusion polypeptide) comprising an Fc domain variant described herein) and (b) a therapeutic anti-CD20 antibody, wherein the non-Hodgkin lymphoma is relapsed and / or refractory non-Hodgkin lymphoma (e.g., the individual has relapsed during and / or after at least one prior treatment for non-Hodgkin lymphoma, e.g., an approved standard therapy, and / or is refractory to at least one prior treatment for non-Hodgkin lymphoma, e.g., an approved standard therapy). In some embodiments, the individual has relapsed during and / or after two or more approved standard therapies (e.g., 2, 3, or more standard therapies) for non-Hodgkin lymphoma and / or is refractory to two or more standard therapies (e.g., 2, 3, or more standard therapies, e.g., curative therapies) for non-Hodgkin lymphoma. In some embodiments, the polypeptide (e.g., the fusion polypeptide) comprises an SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (wherein the numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (wherein the numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (wherein the numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein the numbering follows the Kabat EU index).In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to the individual comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to the individual comprises an Fc domain variant that is a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, numbered according to the Kabat EU index. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to the individual comprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., fusion polypeptide) forms a homodimer. In some embodiments, the therapeutic anti-CD20 antibody is rituximab.

[0259] In some embodiments, rituximab is administered subcutaneously. In some embodiments, rituximab is administered by intravenous infusion. In some embodiments, rituximab is administered according to the instructions on its label. In some embodiments, rituximab is administered to the individual at a dose of about 375 mg / m 2 (e.g., via intravenous infusion). In some embodiments, the first four doses of rituximab (i.e., doses 1-4) are administered to the individual once a week (e.g., every 7 days or "qw") for the first four weeks (e.g., 28 days) of treatment at a dose of about 375 mg / m 2 (e.g.,). The next eight doses (i.e., doses 5-12) are administered to the individual once every four weeks (e.g., every 28 days or "q4w"). In some embodiments, the first four doses of rituximab (i.e., doses 1-4) are administered to the individual once a week (e.g., every 7 days or "qw") for the first four weeks (e.g., 28 days) of treatment at a dose of about 375 mg / m 2The first four doses (i.e., doses 1-4) are administered weekly (i.e., once every 7 days or "qw") to the individual, and the next four doses (i.e., doses 5-8) are administered to the individual once every four weeks (e.g., every 28 days or "q4w"). In some embodiments, the dosage modification of rituximab is performed in accordance with the prescribing information of each country. Complete information regarding the preparation, formulation, dosage, and administration schedule of rituximab may be described in the prescribing information of each country (in the United States, for example, see www.accessdata.fda.gov / drugsatfda_docs / label / 2012 / 103705s5367s5388lbl.pdf; in Europe, for example, see www.ema.europa.eu / en / documents / product-information / mabthera-epar-product-information_en.pdf). In some embodiments, the polypeptide (e.g., a fusion polypeptide) is administered to the individual (e.g., via intravenous infusion) at, for example, a dose of 10.0 mg / kg or 15.0 mg / kg once a week (i.e., once every 7 days or "qw").

[0260] In some embodiments, the indolent lymphoma is indolent non-Hodgkin lymphoma (NHL). In some embodiments, the indolent NHL is marginal zone lymphoma (MZL). In some embodiments, the NHL is follicular lymphoma (FL). Details regarding the diagnosis and classification of marginal zone lymphoma and follicular lymphoma (as well as DLBCL, and mantle cell lymphoma) are described, for example, in Ayyappan et al. (2018) Curr Oncol Rep. 20(4):33; Dreyling et al. (2013) ESMO Consensus Guidelines: Marginal Cell Lymphoma, Mantle Cell Lymphoma, Peripheral T-cell Lymphoma」 Ann Oncol. 24(4):857-877; Vose, JM (2017) 「Mantle cell lymphoma: 2017 update on diagnosis, risk-stratification, and clinical management」 Am J. Hematol. 92(8):806-813; Ciobanu et al. (2013) 「Indolent Lymphoma: Diagnosis and Prognosis in Medical Practice」 Maedica (Buchar) 8(4)338-342, and elsewhere.

[0261] In some embodiments, an individual achieved the lowest stable (SD) treatment effect for a prior treatment, but for example, if the effect ceased within any of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after discontinuation of the prior treatment (e.g., indicating progression of the disease), the individual is considered to have relapsed after a prior treatment for indolent lymphoma (e.g., indolent NHL). In some embodiments, if an individual did not respond to a prior treatment (e.g., if at least a stable (SD) treatment effect could not be achieved during or after the prior treatment), the individual is considered refractory to the prior treatment for indolent lymphoma (e.g., indolent NHL). In some embodiments, the treatment effect for the treatment of indolent lymphoma (e.g., indolent NHL) is determined according to Cheson et al. (2014) "Recommendations for Initial Evaluation, Staging and Response Assessment of Hodgkin and Non-Hodgkin Lymphoma: The Lugano Classification" J. Clin Oncol. 32:3059-3067.

[0262] In some embodiments, the individual was refractory to or had relapsed following treatment with at least one prior treatment for indolent lymphoma (e.g., indolent NHL), such as at least one prior approved standard treatment, at least two prior approved standard treatments, at least three approved standard treatments, etc. Standard treatments for indolent lymphoma (e.g., indolent NHL such as marginal zone lymphoma or follicular lymphoma) include, but are not limited to, for example, standard treatments for aggressive NHL (e.g., DLBCL or mantle cell lymphoma). Details of these are described elsewhere in this specification. Other standard treatments for indolent lymphoma (e.g., indolent NHL) include, but are not limited to, for example, fludarabine, FR (i.e., fludarabine and rituximab), FCR (i.e., FR combined with cyclophosphamide); FCM (i.e., fludarabine, cyclophosphamide, mitoxantrone); FCMR (i.e., FCM combined with rituximab); ibritumomab tiuxetan; tositumomab; vorinostat; everolimus; bortezomib; navitoclax (also known as ABT-263); high-dose therapy (HDT); autologous stem cell transplantation; and allogeneic stem cell transplantation. In some embodiments, prior treatment for indolent lymphoma (e.g., indolent NHL) includes any two or more of the preceding standard treatments (such as the treatment of aggressive NHL described elsewhere in this specification). In some embodiments, two or more standard treatments for indolent lymphoma (e.g., indolent NHL) (such as standard treatments for aggressive NHL) were given together in a single treatment regimen. In some embodiments, two or more standard treatments for indolent NHL (such as standard treatments for aggressive NHL) were given in separate treatment regimens. In some embodiments, there are no available treatment options (e.g., curative treatment options) for an individual having indolent lymphoma (e.g., indolent NHL) (e.g., relapsed / refractory indolent NHL).

[0263] In some embodiments of any of the treatment methods provided herein, the fusion polypeptide is supplied for use (e.g., for intravenous administration) in 100 mg / 5 ml type I clear glass vials sealed with a 20 mm Teflon-coated rubber septum stopper and an aluminum seal. In some embodiments, the fusion polypeptide is supplied for use (e.g., for intravenous administration) in 400 mg / 20 ml type I clear glass vials sealed with a 20 mm Teflon-coated rubber septum stopper and an aluminum seal. In some embodiments, the fusion polypeptide is stored at 2-8 °C (36-46 °F) in the original container until use (e.g., for intravenous administration).

[0264] In some embodiments of any of the treatment methods described herein, the polypeptide (e.g., a fusion polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant) is administered subcutaneously. In some embodiments, the polypeptide (e.g., a fusion polypeptide) is administered via intravenous infusion. In some embodiments, the polypeptide (e.g., a fusion polypeptide) is administered to an individual (e.g., a human individual) at a dose of 0.3 mg / kg, 1.0 mg / kg, 3.0 mg / kg, 10.0 mg / kg, 15.0 mg / kg, or 30.0 mg / kg (such as any range between these values) (e.g., via intravenous infusion). In some embodiments, the polypeptide (e.g., a fusion polypeptide) is administered to an individual (e.g., a human individual) at a dose of 0.3 mg / kg, 1.0 mg / kg, 3.0 mg / kg, 10.0 mg / kg, 15.0 mg / kg, or 30.0 mg / kg (including any range between these values) once a week (i.e., once every 7 days or "qw") (e.g., via intravenous infusion). In some embodiments, the polypeptide (e.g., a fusion polypeptide) is administered to an individual at a dose of 0.3 mg / kg, 1.0 mg / kg, 3.0 mg / kg, 10.0 mg / kg, 15.0 mg / kg, or 30.0 mg / kg (including any range between these values) every other week (i.e., once every 14 days, or "q2w" or "QoW") (e.g., via intravenous infusion). In some embodiments, the polypeptide (e.g., a fusion polypeptide) and the therapeutic antibody (e.g., an anti-PD1 antibody (pembrolizumab), an anti-HER2 antibody (trastuzumab), or an anti-CD20 antibody (rituximab)) are sequentially administered on the day when their administration schedules coincide. In some embodiments, the polypeptide (e.g., a fusion polypeptide) is administered before the therapeutic antibody (e.g., about 30 minutes before). In some embodiments, if the polypeptide is not administered, the therapeutic antibody is administered about 24 hours after it was not administered.

[0265] In some embodiments, the therapeutic effect of the treatment methods provided herein on an individual having NSCLC, HNSCC, gastric cancer, or GEJ cancer is determined, for example, by the RECIST version 1.1 criteria described in Therasse et al. (2000) J. Natl Cancer Inst. 92:205-216; Eisenhauer et al. (2009) Eur J. Cancer. 45:229-247, immune-related response criteria derived from RECIST 1.1 (irRECIST) (e.g., source Nishino, et al. (2013) "Developing a Common Language for Tumor Response to Immunotherapy: Immune-Related Response Criteria Using Unidimensional Measurements" Clinical Cancer Research 19(14):3936-43). In some embodiments, the treatment response of an individual having aggressive lymphoma (e.g., aggressive NHL such as DLBCL or MCL) to the treatment methods provided herein is determined, for example, in accordance with Cheson et al. (2014) "Recommendations for Initial Evaluation, Staging and Response Assessment of Hodgkin and Non-Hodgkin Lymphoma: The Lugano Classification" J. Clin Oncol. 32:3059-3067. In some embodiments, the therapeutic effect of the treatment methods provided herein on an individual having indolent lymphoma (e.g., indolent NHL such as FL or MZL) is determined in accordance with the Lugano criteria (see Cheson et al, 2014).

[0266] In some embodiments, an individual undergoing treatment for HNSCC, NSCLC, gastric cancer, or GEJ cancer has at least one measurable lesion as defined by RECIST version 1.1 criteria (e.g., as described in Therasse et al. (2000) J. Natl Cancer Inst. 92:205-216; Eisenhauer et al. (2009) Eur J. Cancer. 45:229-247). In some embodiments, an individual undergoing treatment for lymphoma (e.g., aggressive lymphoma such as DLBCL or MCL or indolent lymphoma such as FL or MZL) has at least one measurable lesion as defined by Lugano criteria (e.g., as described in Cheson et al. (2014) “Recommendations for Initial Evaluation, Staging and Response Assessment of Hodgkin and Non-Hodgkin Lymphoma: The Lugano Classification”. Clin Oncol. 32:3059-3067).

[0267] In some embodiments, an individual undergoing treatment according to the methods herein has adequate bone marrow function, kidney function, liver function, and heart function. In some embodiments, the individual has an Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) score of 0 or 1 (see, e.g., www.npcrc.org / files / news / ECOG_performance_status.pdf). In some embodiments, the individual does not have symptomatic central nervous system (CNS) metastases or leptomeningeal disease requiring steroids. In some embodiments, an individual undergoing treatment of lung cancer (e.g., NSCLC) according to the methods herein does not have an ALK or EGFR genomic tumor abnormality. In some embodiments, an individual undergoing treatment according to the methods herein does not have a history of (non-infectious) pneumonia requiring steroids or does not currently have pneumonia. In some embodiments, an individual undergoing treatment according to the methods herein does not have high-grade lymphoma (e.g., Burkitt lymphoma, lymphoblastic lymphoma, or Richter transformation), chronic lymphocytic leukemia, or plasma cell leukemia. In some embodiments, the individual has not received high-dose chemotherapy requiring allogeneic stem cell rescue. In some embodiments, an individual undergoing treatment of lung cancer (e.g., NSCLC), head and neck cancer (e.g., HNSCC), or gastric / GEJ cancer (e.g., HER2-positive gastric / GEJ adenocarcinoma) according to the methods herein has not received more than 25% prior irradiation to the bone marrow. In some embodiments, the individual has not received radiation therapy within two weeks of the start of treatment. In some embodiments, the individual has not received prior treatment with an anti-CD47 or anti-SIRPα agent. In some embodiments, the individual has not received systemic anti-cancer therapy within four weeks (six weeks in the case of mitomycin C or nitrosourea) of starting treatment. In some embodiments, the individual does not have intolerance to any excipients in an antibody or injected therapeutic protein(s) or formulation(s) containing a therapeutic protein or has not had a severe allergic or anaphylactic reaction.In some embodiments, the individual has not discontinued treatment due to grade 3 or higher immune-related adverse events (AEs) from prior treatment with an anti-PD-1, anti-PD-L1, or anti-PD-L2 agent, or an agent aimed at modulating another immune cell target (e.g., CTLA-1, OX40, 41BB, etc.). In some embodiments, the individual has not received an experimental antibody or live vaccine (e.g., but not limited to, measles, mumps, rubella, varicella-zoster, yellow fever, rabies, Bacillus Calmette-Guerin (BCG), typhoid, and intranasal influenza vaccine, etc.). In some embodiments, the individual has not received current active treatment for primary diagnosis (e.g., lung cancer (NSCLC), head and neck cancer (HNSCC), gastric / GEJ cancer (HER2-positive gastric / GEJ adenocarcinoma), aggressive lymphoma (de novo DLBCL or transformed DLBCL or mantle cell lymphoma), or indolent lymphoma (e.g., indolent NHL such as marginal zone lymphoma or follicular lymphoma)). In some embodiments, the individual has not received a blood product transfusion within 14 days of treatment initiation. In some embodiments, the individual does not have a history of active autoimmune disorders (e.g., but not limited to, Crohn's disease, rheumatoid arthritis, scleroderma, systemic lupus erythematosus, Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia, etc.) and other conditions that compromise or impair the immune system (excluding hypogammaglobulinemia). In some embodiments, the individual does not have active, uncontrolled, clinically significant bacterial, fungal, or viral infections such as those related to hepatitis B (HBV), hepatitis C (HCV), known human immunodeficiency virus (HIV), or acquired immunodeficiency syndrome (AIDS)-related diseases. In some embodiments, the individual does not have active graft-versus-host disease (GVHD) or has not received immunosuppressive therapy for GVHD. In some embodiments, the individual has not had any of myocardial infarction, severe / unstable angina, coronary / peripheral artery bypass graft, symptomatic congestive heart failure, cerebrovascular accident, transient ischemic attack, deep vein thrombosis, or symptomatic pulmonary embolism in the past 12 months.In some embodiments, the individual has not been diagnosed with any other malignancy within the past three years prior to the start of treatment, except for, for example, appropriately treated non-melanoma skin cancer, and in situ cancer that has received a potentially curative therapy (e.g., breast cancer, cervical intraepithelial neoplasia). Kit and Manufactured Product

[0268] In another embodiment of the invention, there is provided a manufactured product or kit comprising a polypeptide comprising an SIRPα D1 domain variant and an Fc domain variant (e.g., a fusion polypeptide described herein). In some embodiments, the SIRPα D1 domain variant comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 81 and SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (where numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (where numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (where numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (where numbering follows the Kabat EU index). In some embodiments, the 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 polypeptide forms a homodimer. In some embodiments, the kit or manufactured product is for use according to the treatment methods provided herein.

[0269] In some embodiments, the kit or product further comprises an anti-PD1 antibody. In some embodiments, the anti-PD1 antibody is pembrolizumab. In some embodiments, the kit comprises an accompanying document or label containing instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-PD1 antibody (e.g., pembrolizumab) to treat or delay the progression of lung cancer (e.g., NSCLC such as metastatic NSCLC) in an individual according to the methods herein. In some embodiments, the kit comprises an accompanying document or label containing instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-PD1 antibody (e.g., pembrolizumab) to treat or delay the progression of lung cancer (e.g., NSCLC such as metastatic NSCLC) in an individual who has received prior treatment for NSCLC according to the methods herein. In some embodiments, the individual has progressed (e.g., shown disease progression) during (or after) prior treatment for lung cancer (e.g., prior immune checkpoint inhibitor therapy). In some embodiments, the individual has a PD-L1 tumor proportion score (TPS) of less than 50%. In some embodiments, the kit comprises an accompanying document or label containing instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-PD1 antibody (e.g., pembrolizumab) to treat or delay the progression of HNSCC in an individual according to the methods herein. In some embodiments, the kit comprises an accompanying document or label containing instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-PD1 antibody (e.g., pembrolizumab) for use in treating or delaying the progression of HNSCC in an individual who has received prior immune checkpoint inhibitor therapy (e.g., for HNSCC). In some embodiments, the kit comprises an accompanying document or label containing instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-PD1 antibody (e.g., pembrolizumab) for use in treating or delaying the progression of HNSCC in an individual who has not received prior immune checkpoint inhibitor therapy (e.g., for HNSCC).In some embodiments, the kit comprises a package insert or label containing instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-PD1 antibody (e.g., pembrolizumab) to treat or delay the progression of head and neck cancer (e.g., HNSCC, metastatic HNSCC, etc.) in an individual who has progressed (e.g., shown disease progression) during (or after) prior platinum therapy (e.g., platinum-containing therapy). In some embodiments, the kit or product further comprises instructions for administering pembrolizumab at a dose of 200 mg every three weeks (Q3W) by intravenous infusion. In some embodiments, the kit or product further comprises instructions for administering a polypeptide (e.g., a fusion polypeptide) at a dose of 10 mg / kg weekly (QW) by intravenous infusion.

[0270] In some embodiments, the kit or product further comprises an anti-HER2 antibody. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the kit comprises instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-HER2 antibody (e.g., trastuzumab) to treat or delay the progression of HER2-positive gastric cancer or HER2-positive GEJ cancer (e.g., HER2-positive gastric adenocarcinoma or HER2-positive GEJ adenocarcinoma) in an individual according to the methods provided herein. In some embodiments, the kit comprises instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-HER2 antibody (e.g., trastuzumab) to treat or delay the progression of HER2-positive gastric cancer or HER2-positive GEJ cancer (e.g., HER2-positive gastric adenocarcinoma or HER2-positive GEJ adenocarcinoma) in an individual who has progressed (e.g., shown disease progression) during (or after) prior treatment of gastric cancer or GEJ cancer according to the methods provided herein. In some embodiments, the prior treatment regimen included an anti-HER2 antibody and / or a prior fluoropyrimidine-based treatment regimen. In some embodiments, the kit or product further comprises instructions for administering trastuzumab via intravenous infusion once every three weeks (q3W), wherein the initial dose of trastuzumab is 8 mg / kg and each subsequent dose of trastuzumab (i.e., after the initial dose) is 6 mg / kg. In some embodiments, the kit or product further comprises instructions for administering a polypeptide (e.g., a fusion polypeptide) by intravenous infusion at a dose of 10 mg / kg per week (QW).

[0271] In some embodiments, the kit or article of manufacture further comprises an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, the kit includes a package insert or label containing instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-CD20 antibody (e.g., rituximab) to treat or delay the progression of aggressive non-Hodgkin lymphoma or "NHL" (e.g., de novo DLBCL or transformed DLBCL, or mantle cell lymphoma) in an individual according to the methods of the present specification. In some embodiments, the kit includes a package insert or label containing instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-CD20 antibody (e.g., rituximab) to treat or delay the progression of aggressive non-Hodgkin lymphoma or "NHL" (e.g., de novo DLBCL or transformed DLBCL, or mantle cell lymphoma) in an individual who has relapsed or is refractory to a prior treatment regimen (e.g., a prior standard / curative treatment) for aggressive NHL, or in an individual for whom there is no available treatment regimen (e.g., curative treatment) for aggressive NHL according to the methods of the present specification. In some embodiments, the kit includes a package insert or label containing instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-CD20 antibody (e.g., rituximab) to treat or delay the progression of indolent lymphoma (e.g., indolent NHL such as marginal zone lymphoma or follicular lymphoma) in an individual according to the methods of the present specification. In some embodiments, the kit includes a package insert or label containing instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-CD20 antibody (e.g., rituximab) to treat or delay the progression of indolent lymphoma (e.g., indolent NHL such as marginal zone lymphoma or follicular lymphoma) in an individual who has relapsed or is refractory to a prior treatment (e.g., a prior standard treatment, e.g., curative treatment) for indolent lymphoma, or in an individual for whom there is no available treatment regimen (e.g., curative treatment) for indolent lymphoma (e.g., indolent NHL).In some embodiments, the kit or product comprises rituximab at a dose of 375 mg / m² for the first 4 weeks (e.g., 28 days) of treatment. 2 It is administered to an individual once a week (e.g., once every 7 days or "qw") for the first 4 weeks, and then rituximab is administered at a dose of 375 mg / m² 2 either for up to 4 additional administrations after the first 4 administrations, or for up to 8 additional administrations after the first 4 administrations, once every 4 weeks (e.g., every 28 days or "q4w"). In some embodiments, the kit or product further comprises instructions for administering a polypeptide (e.g., a fusion polypeptide) intravenously at a dose of 10 mg / kg or 15 mg / kg per week (QW).

[0272] In some embodiments of any of the kits or products provided herein, the polypeptide (e.g., a fusion polypeptide) and the therapeutic antibody (e.g., pembrolizumab, trastuzumab, or rituximab) are in the same container or in separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The container can be formed from a variety of materials, such as glass, plastic (such as polyvinyl chloride or polyolefin), or alloy (such as stainless steel or hastelloy). In some embodiments, the container holds the formulation, and the label on the container or a label associated with the container can indicate instructions for use. The product or kit can further include other buffers, diluents, filters, needles, syringes, and other materials desirable from a commercial and user perspective, including an accompanying document with instructions for use. In some embodiments, the product further comprises one or more of another agent (e.g., a chemotherapeutic agent and an anti-tumor agent). Suitable containers for the one or more agents include, for example, bottles, vials, bags, and syringes.

[0273] This specification is considered to be sufficient to enable a person skilled in the art to practice the present invention. In addition to the modifications shown and described herein, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description and are included within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Examples

[0274] Example 1: Phase 1 trial of Drug A in combination with an established anti-cancer antibody in patients with progressive malignancies. In this example, a Phase 1 clinical trial is described to evaluate the safety, efficacy, pharmacodynamics (PD), and pharmacokinetics (PK) of Drug A in combination with pembrolizumab, trastuzumab, or rituximab in patients with progressive malignancies. Drug A is a fusion protein consisting of a high-affinity CD47-binding SIRPα D1 domain variant fused to a human immunoglobulin Fc domain variant modified to abolish binding to Fc gamma receptors (Figure 1).

[0275] Purpose of the trial Main purpose The main purpose of this trial was to evaluate the safety and durability of Drug A administered once weekly and / or every two weeks in combination with pembrolizumab, trastuzumab, or rituximab in patients with progressive malignancies such as non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), HER2-overexpressing gastric cancer, and non-Hodgkin lymphoma (NHL).

[0276] Secondary purposes The secondary purposes of this trial were as follows. · To determine the anti-tumor effect of Drug A in combination with an anti-cancer therapeutic agent in patients with progressive malignancies. · To determine the overall safety profile of Drug A in combination with other anti-cancer therapeutic agents. · To clarify the characteristics of the maximum tolerated dose (MTD) / optimal biological dose (OBD) of Drug A in combination with an anti-cancer therapeutic agent. · To clarify the pharmacokinetic characteristics of single and multiple doses of Drug A in combination with pembrolizumab, trastuzumab, or rituximab, which are anti-cancer drugs. · To evaluate the immunogenicity of Drug A. Exploratory objectives

[0277] The exploratory objective of this study was to investigate the pharmacodynamic effects of Drug A in combination with anti-cancer drugs in patients with progressive malignancies.

[0278] Patients Selection criteria All patients met the following criteria for the dose-escalation phase of the study. · Progressive / metastatic solid tumor malignancies or relapsed / refractory CD20 + Histological or cytological diagnosis of B-cell non-Hodgkin lymphoma: o Resistant to standard therapy or curative therapy not available. Or o Meeting the following dose-expansion criteria (a - d) · Lesions may be measurable or non-measurable.

[0279] All patients met the following criteria for the dose-expansion phase of this study. a) Locally advanced or metastatic NSCLC (TPS ≥ 1%) progressing on prior checkpoint therapy, or locally advanced or metastatic NSCLC (TPS < 50%) progressing after systemic therapy for metastatic disease; patients were suitable for treatment with pembrolizumab; or b) Recurrent or metastatic HNSCC in which the disease progressed during or after platinum-containing chemotherapy and was suitable for treatment with pembrolizumab; or c) HER2-overexpressing metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma that progressed after systemic therapy with a fluoropyrimidine-containing regimen and / or therapy with an anti-HER2 antibody for metastatic disease and was suitable for treatment with trastuzumab. d) Relapsed or refractory, de novo or transformed diffuse large B-cell lymphoma (DLBCL), or mantle cell lymphoma for which curative therapy was not available; also, indolent lymphomas (marginal zone, follicular) that relapsed or were refractory to approved standard therapies. · Patients had at least one measurable lesion as defined by RECIST version 1.1 or Lugano criteria (2014).

[0280] Furthermore, all patients met the following criteria. · Adequate bone marrow function, including: o Absolute neutrophil count (ANC) ≥ 1,500 / mm 3 (≥ 1.5 x 10 9 / L); for non-Hodgkin lymphoma only, ANC ≥ 1,000 / mm 3 (≥ 1.0 x 10 9 / L); o Platelets ≥ 75,000 / mm 3 (≥ 75 x 10 9 / L); for non-Hodgkin lymphoma only: platelets ≥ 50,000 / mm 3 (≥ 50 x 10 9 / L); o Hemoglobin ≥ 9 g / dL (≥ 90 g / L); for non-Hodgkin lymphoma only: hemoglobin ≥ 8 g / dL (≥ 80 g / L). · Adequate renal function, including: o Serum creatinine ≤ 1.5 x upper limit of normal (ULN) or estimated creatinine clearance ≥ 60 mL / min (calculated using the facility's method standard). · Adequate liver function, including: o Total serum bilirubin ≤ 1.5 x ULN (≤ 3.0 x ULN if the patient has a record of Gilbert syndrome); o Aspartate and alanine transaminases (AST and ALT) ≤ 3.0 x ULN; in the presence of liver lesions secondary to the tumor, ≤ 5.0 x ULN. o Alkaline phosphatase ≤ 2.5 x ULN (≤ 5.0 x ULN in cases of bone or liver metastases). · The QT interval was corrected for heart rate Fridericia (QTcF) intervals of 480 milliseconds or less (based on the average of 3 ECGs). · 18 years of age or older. · Eastern Cooperative Oncology Group (ECOG) Performance Status of 0 or 1. · Except for AEs that do not constitute a safety risk at the discretion of the principal investigator of the trial, the acute effects of prior treatment for baseline severity or grade ≤ 1 (NCI CTCAE v.4.03) were resolved. · Archived (or fresh) metastatic biopsy samples available prior to the start of the trial (expansion phase only). · Negative serum pregnancy test at screening (for women with childbearing potential).

[0281] Exclusion Criteria Patients with any of the following characteristics were not included in this trial: · Patients with known symptomatic CNS metastases or leptomeningeal disease requiring steroids. Patients previously diagnosed with brain metastases were eligible if they had completed treatment, recovered from the acute effects of radiotherapy or surgery prior to trial enrollment, discontinued corticosteroid treatment for these metastases, were clinically stable for at least 4 weeks from anti-seizure medications, and were neurologically stable prior to enrollment. · Patients with ALK or EGFR genomic tumor abnormalities (NSCLC, part 2 expansion only), or a history of (non-infectious) pneumonia requiring steroids, or patients currently suffering from pneumonia. · Patients with high-grade lymphoma (Burkitt lymphoma, lymphoblastic lymphoma, Richter syndrome, etc.), CLL or plasma cell leukemia · Prior high-dose chemotherapy requiring allogeneic stem cell rescue. ·Prior irradiation to >25% of the bone marrow (only for non-lymphoma patients), or patients who received prior radiotherapy within 2 weeks from the start of the test treatment. Note: Participants are assumed to have recovered from all toxicities related to radiation, not require corticosteroids, and not have radiation pneumonitis. For palliative radiotherapy for non-CNS diseases (radiotherapy for less than 2 weeks), a 1-week washout was allowed. ·Prior treatment with anti-CD47 or anti-SIRPα agents. ·Systemic anti-cancer therapy within 4 weeks from the start of the test treatment (6 weeks for mitomycin C or nitrosourea). If systemic anti-cancer therapy was performed within 4 weeks, patients were included if the drug's elimination half-life had passed 4 - 5 times. ·Prior treatment by the following (only in the expansion phase): o PD-1 or PD-L1 inhibitors (NSCLC; HNSCC) for metastatic diseases with disease progression within 8 weeks from the start for diseases that progress. o Trastuzumab for metastatic diseases where the best effect is disease progression (gastric / GEJ cancer). ·Patients who are intolerant to antibodies or injected therapeutic proteins or have severe allergic or anaphylactic reactions; patients who have severe allergic or anaphylactic reactions to any of the substances (such as excipients) contained in the test drug; or patients who discontinued treatment due to grade 3 or higher immune-related AEs from prior treatment with anti-PD-1, anti-PD-L1, or anti-PD-L2 agents, or agents aimed at regulating another immune cell target (e.g., CTLA-1, OX40, 41BB, etc.). ·Any experimental antibodies or live vaccines in the past 28 days prior to the first administration of the test drug. Live attenuated vaccines were not permitted. ·Current active therapy for primary diagnosis ·Blood product transfusion within 14 days from day 1 of cycle 1. ·History of (active) autoimmune diseases (including but not limited to Crohn's disease, rheumatoid arthritis, scleroderma, systemic lupus erythematosus, Graves' disease, etc.) and other conditions that weaken or impair the immune system (excluding hypogammaglobulinemia). · History of autoimmune hemolytic anemia or autoimmune thrombocytopenia. · Patients with active, uncontrolled, clinically significant bacterial, fungal, or viral infections, such as hepatitis B (HBV), hepatitis C (HCV), known human immunodeficiency virus (HIV), or diseases related to acquired immunodeficiency syndrome (AIDS). · Patients with active graft-versus-host disease (GVHD) or during immunosuppression for GVHD. · Any of the following within the past 12 months: myocardial infarction, severe / unstable angina, coronary / peripheral artery bypass graft, symptomatic congestive heart failure, cerebrovascular accident, transient ischemic attack, deep vein thrombosis, or symptomatic pulmonary embolism. · Current active treatment in another interventional clinical trial. · Diagnosed with any other malignancy within the past 3 years prior to enrollment, except for appropriately treated non-melanoma skin cancer or in situ cancer (e.g., breast cancer, cervical intraepithelial neoplasia) that has received potentially curative therapy. · Other severe acute or chronic medical or psychiatric conditions, such as recent (within the past 1 year) or active suicidal ideation or behavior, or laboratory abnormalities that increase the risk associated with participation in the trial or administration of the investigational drug, or interfere with the interpretation of trial results, such that in the judgment of the responsible investigator of the trial, the patient is inappropriate for participation in this trial. · Men and women of childbearing potential who are not using highly effective contraception or who have not agreed to continue highly effective contraception for at least 90 days after the last dose of the investigational drug. · Pregnant or lactating patients.

[0282] Investigational treatment As shown in Figure 2, there were three treatment groups in this trial (Drug A + pembrolizumab; Drug A + trastuzumab; Drug A + rituximab). This trial included an initial dose escalation part and then a dose expansion part. Each dose escalation group had approximately 12 patients with progressive malignancies. Each expansion group had approximately 20 - 40 patients at multiple sites.

[0283] Drug A was administered as an IV infusion once weekly (or every two weeks) over approximately 60 minutes externally. To ensure accurate delivery of the investigational drug, the use of an infusion pump was the preferred method of administration, although gravity drip was permitted.

[0284] Drug A was supplied in 100 mg / 5 mL or 400 mg / 20 mL Type 1 clear glass vials, sealed with a 20 mm Teflon-coated rubber septum stopper and an anti-tamper aluminum seal. Each single-use vial delivers 100 mg of Drug A (5 mL) or 400 mg of Drug A (20 mL) for intravenous (IV) administration.

[0285] The time from the administration on Day 1 to the next administration on Day 1 was defined as one cycle. In the absence of treatment delays, the cycle was 21 days for weekly administration and 28 days for administration every two weeks.

[0286] All test treatments were conducted externally. Patients were observed at the clinic for at least 2 hours after injecting Drug A on Day 1 of Cycle 1 (C1D1) and then as clinically indicated.

[0287] Pretreatment with Drug A was not required. The guidelines in the package insert for the combination therapy of pembrolizumab, trastuzumab, and rituximab were followed.

[0288] In the dose escalation and expansion phases, the combination partner therapies were administered according to the instructions in their labels. o Trastuzumab: The initial dose of 8 mg / kg is administered as an intravenous infusion over 90 minutes, followed by an intravenous injection (IV) of 6 mg / kg every 3 weeks over 30 - 90 minutes. o Pembrolizumab: 200 mg of IV is administered as an intravenous infusion over 30 minutes every 3 weeks for up to 24 months. o Rituximab: 375 mg / m 2It was intravenously injected 4 times once a week, and then administered 8 times once a month (starting at a rate of 50 mg / h and increasing by 50 mg / h every 30 minutes, up to a maximum of 400 mg / h if there was no infusion toxicity). If the patient was tolerant to the first infusion of rituximab, subsequent infusions started at a rate of 100 mg / h and, if there was no toxicity, were increased by 100 mg / h at 30-minute intervals up to a maximum of 400 mg / h.

[0289] On the dosing days when the dosing schedules matched, the combination partner was started approximately 30 minutes after the end of drug A therapy. On such days, if drug A was not administered due to toxicity, the partner drug was administered 24 hours after the non-administration. If drug A was permanently discontinued, the patient discontinued the treatment phase of the trial. If the partner drug was permanently discontinued and the patient was deriving clinical benefit from drug A in the opinion of the principal investigator of the clinical trial, the patient continued single-agent drug A for up to 24 months.

[0290] Dose escalation component For drug A, the initial dose escalation component was started at one dose level below the single-agent maximum tolerated dose (MTD) or maximum administered dose (MAD) (3 - 6 patients at each dose level), taking into account the observed single-agent drug A dose-limiting toxicity (DLT) profile and known safety profile of the proposed combination agent (Table A). If the dose of drug A was safe and well tolerated in combination, the dose level of drug A was increased to MTD or MAD using the combination agent.

[0291] The MTD of drug A as a single agent was not reached. The MAD of drug A as a single agent was 30 mg / kg administered IV every other week (QOW or Q2W). TIFF0007710997000034.tif50170

[0292] Expansion phase Drug A was administered once a week (QW) as an intravenous (IV) infusion at a dose of 10 mg / kg over approximately 60 minutes in an outpatient patient base.

[0293] During the expansion phase, as described above, combination partner therapy was administered according to the description of its label.

[0294] The drug A + pembrolizumab expansion cohort included up to 20 patients with metastatic NSCLC and up to 20 patients with recurrent or metastatic head and neck squamous cell carcinoma.

[0295] The drug A + trastuzumab expansion cohort included up to 20 patients with HER2 overexpression (HER2-positive) gastric cancer.

[0296] The drug A + rituximab expansion cohort included approximately 10 patients with relapsed or refractory diffuse large B-cell lymphoma and approximately 10 patients with ...

Claims

**Claim 1** A medicament for treating HER2-positive gastric / gastroesophageal junction (GEJ) cancer in an individual by a combination therapy, comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, wherein said medicament is administered in combination with trastuzumab, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant comprises the amino acid sequence of SEQ ID NO: 136; and the gastric / GEJ cancer of the individual has progressed after prior treatment with a fluoropyrimidine-based therapy and / or prior treatment with an anti-HER2 antibody, and the individual is a human, the medicament. **Claim 2** The medicament according to claim 1, wherein the prior treatment with a fluoropyrimidine-based therapy or the prior treatment with an anti-HER2 antibody comprises one or more therapeutic agents selected from the group consisting of trastuzumab, pertuzumab, 5-fluorouracil, capecitabine, margetuximab, and FOLFOX. **Claim 3** The medicament according to claim 1 or 2, wherein trastuzumab is administered to the individual at an initial dose of 8 mg / kg and each subsequent dose at a dose of 6 mg / kg, and trastuzumab is administered to the individual by IV infusion every 3 weeks (Q3W). **Claim 4** The medicament according to any one of claims 1 to 3, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant forms a homodimer. **Claim 5** The medicament according to any one of claims 1 to 4, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered to the individual once a week (QW) at a dose of 10 mg / kg. **Claim 6** The medicament according to any one of claims 1 to 4, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered to the individual once a week (QW) at a dose of 15 mg / kg. **Claim 7** The medicament according to claim 5 or 6, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered to the individual by IV infusion.

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