Methods of treating cancer with SIRP alpha fc fusion in combination with immune checkpoint inhibitor

Abstract

To provide methods of 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.SOLUTION: Provided are methods that comprise administering to an 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, anti-HER2 antibody, or an anti-CD20 antibody). Also provided are related kits pharmaceutical compositions.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to 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 incorporated by reference in their entirety herein.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 757972001040SEQLIST.TXT, Recorded: May 26, 2020, Size: 333KB).

[0003] The present invention relates to methods of treating cancer comprising administering a polypeptide (eg, a fusion polypeptide) comprising a SIRPα D1 domain variant and an Fc domain variant in combination with a therapeutic antibody. [Background technology]

[0004] Many cancers have poor prognoses even when treated with available therapeutic agents. For example, non-small cell lung cancer (NSCLC) patients with metastatic disease receiving PD-1 and PD-L1 checkpoint inhibitors who have failed prior platinum-based therapy have a median overall survival of approximately 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 stage disease have an overall 5-year survival rate of 17.7% (US Cancer Statistics Working Group, available at the website www(dot)cdc(dot)gov / uscs). Similarly, the overall 5-year survival rate for 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 increasingly aggressive histology and a 30% risk of transformation by 10 years in one series (Montoto et al., J Clin Oncol (2007) 25(17):2426-33). Furthermore, for patients with recurrent aggressive histology, cure is rare and new salvage regimens are necessary (Larouche et al., J Clin Oncol (2010) 28(12):2094-100). CD20-positive non-Hodgkin lymphoma (NHL) is the 10th most common cancer and the 10th leading cause of cancer death worldwide, with 199,670 deaths per year worldwide (World Health Organization 2016(a), available at the website globocan(dot)iarc(dot)fr / Pages / fact_sheets_cancer(dot)aspx). There are an estimated 35,000 people with metastatic HNSCC in the United States, with more than 50,000 new cases of all stages diagnosed in 2019. The 5-year survival rate is 84% ​​for patients diagnosed with localized disease but drops to just 39% for those diagnosed with metastatic disease.There is a need in the art for novel therapies to provide additional treatment options and improve outcomes for such patients.

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

[0006] Macrophage-mediated destruction of cancer cells requires both disruption of "don't eat me" signals (e.g., CD47-SIRPα) and activation of "eat me" signals. Either component alone is not sufficient to trigger a maximal phagocytic response against tumor cells. As mentioned above, CD47 provides the basal "don't eat me" signal through its interaction with SIRPα on macrophages. Pro-phagocytic "eat me" signals can be provided to the same macrophages by binding to their activating Fc gamma receptors. For example, pro-phagocytic "eat me" signals can be provided by 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] Methods of treating non-small cell lung cancer (NSCLC) in an individual are 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 comprises (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, and the individual is a human. In some embodiments, the individual's NSCLC has progressed on 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 (such as formulated for use) in combination with an anti-PD1 antibody, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; and the Fc domain variant is selected from the group consisting of: (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, and the individual is a human. In some embodiments, the individual's NSCLC has progressed on prior immune checkpoint inhibitor (CPI) therapy and / or has a PD-L1 tumor proportion score (TPS) of less than 50%.

[0010] Also provided is 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 NSCLC in an individual (e.g., for use in a method of treating NSCLC 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 numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, and the individual is a human. In some embodiments, the individual's NSCLC has progressed on 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) included 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 three weeks (Q3W) by intravenous (IV) infusion.

[0012] Also provided is a method of treating head and neck squamous cell carcinoma (HNSCC) 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 comprises (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the individual's HNSCC has progressed during or after prior platinum therapy, and the individual is human.

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

[0014] Also provided is 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 in an individual (e.g., for use in a method of treating HNSCC in an individual), 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 L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the individual's HNSCC has progressed during or after prior platinum therapy, and the individual is human.

[0015] In some embodiments, the individual with 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 / referred to as "checkpoint inhibitor experienced." In some embodiments, the individual with HNSCC has not received prior treatment with an immune checkpoint inhibitor. In some embodiments, such an individual is considered / referred to as "checkpoint inhibitor naive." 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 (e.g., formulated for administration) to the individual at a dose of 200 mg every three weeks (Q3W) by intravenous (IV) infusion.

[0016] Methods of treating HER2-positive gastric / gastroesophageal junction (GEJ) cancer in an individual are 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 comprises (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the 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 a 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 in an individual, the medicament being for use (e.g., formulated for use) in combination with 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; and the Fc domain variant comprises: (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the 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 a human.

[0018] Also provided is a composition (such 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; and the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the 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 a 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, the trastuzumab is administered to the individual (e.g., formulated for administration) at an initial dose of 8 mg / kg and at a dose of 6 mg / kg for each dose thereafter, and the trastuzumab is administered to the individual by IV infusion every three weeks (Q3W).

[0020] Methods of treating aggressive non-Hodgkin's lymphoma (NHL) in an individual are 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 comprises (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the individual's aggressive NHL has relapsed and / or is refractory to prior treatment for aggressive NHL and no curative therapy is available, and the individual is a human.

[0021] 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 aggressive non-Hodgkin's lymphoma (NHL) in an individual, wherein the medicament is for use (such as formulated for use) in combination with 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; and the Fc domain variant is selected from the group consisting of: (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the individual's aggressive NHL has relapsed and / or is refractory to prior treatment for aggressive NHL and no curative therapy is available, and the individual is a human.

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

[0023] In some embodiments, the aggressive NHL is diffuse large B-cell lymphoma (DLBCL), e.g., de novo DLBCL or transformed DLBCL. In some embodiments, prior treatment of the aggressive NHL consisted 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, and combinations thereof. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, rituximab is administered by IV infusion at a dose of 375 mg / m 2 and rituximab is administered to the individual (e.g., formulated for administration) once a week for four weeks and then once a month thereafter.

[0024] Methods of treating indolent lymphoma in an individual are 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 comprises (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the individual's indolent lymphoma has relapsed and / or is refractory to prior treatment for the indolent lymphoma, and the individual is a 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 indolent lymphoma in an individual, wherein the medicament is for use (such as formulated for use) in combination with 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; and the Fc domain variant is selected from the group consisting of: (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the individual's indolent lymphoma has relapsed and / or is refractory to prior treatment for the indolent lymphoma, and the individual is a human.

[0026] Also provided are 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-CD20 antibody for treating an indolent lymphoma in an individual (e.g., for use in a method of treating an indolent 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 L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the individual's indolent lymphoma has relapsed and / or is refractory to prior treatment for the indolent lymphoma, and the individual is a human.

[0027] In some embodiments, the indolent lymphoma is an indolent non-Hodgkin's lymphoma (NHL). In some embodiments, the indolent NHL is a marginal zone lymphoma or a follicular lymphoma. In some embodiments, prior treatment for indolent lymphoma consisted 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 at a dose of 375 mg / m by IV infusion. 2 and rituximab is administered (e.g., formulated for administration) to the individual once a week for four weeks and once a month thereafter. In some embodiments, a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant (or a medicament made using such a polypeptide or a pharmaceutical composition comprising such a polypeptide) is administered (e.g., formulated for administration) to the individual at a dose of 10 mg / kg or 15 mg / kg once a week (QW), for example, by IV infusion.

[0028] In some embodiments of any of the methods herein, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, the Fc domain variant is a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, numbering according to EU index of Kabat. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide 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 a SIRPα D1 domain variant and an Fc domain variant (or a medicament made using such a polypeptide or a pharmaceutical composition comprising such a polypeptide) is administered (e.g., formulated for administration) to an individual at a dose of 10 mg / kg once per week (QW). In some embodiments, a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant (or a medicament made therefrom or a pharmaceutical composition comprising such a polypeptide) is administered (e.g., formulated for administration) to an individual by IV infusion.

[0030] Also provided are kits comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant (or a medicament 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) in an individual (e.g., a human individual) in need thereof, according to the methods described 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 L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations (wherein numbering is according to EU index of Kabat); or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein numbering is according to EU index of Kabat). In some embodiments, the individual's NSCLC has progressed on 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 three weeks (Q3W) by IV infusion. In some embodiments, the kit further comprises instructions for administering the polypeptide (e.g., fusion polypeptide) at a dose of 10 mg / kg, e.g., once weekly by IV infusion.

[0031] Also provided are kits comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant (or a medicament, etc., manufactured using such a polypeptide or a pharmaceutical composition comprising such a polypeptide) for use in combination with pembrolizumab to treat head and neck squamous cell carcinoma (HNSCC) in an individual (e.g., a human individual) in need thereof, according to the methods described 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 L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations (wherein numbering is according to EU index of Kabat); or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations. Fc region (wherein numbering is according to EU index of Kabat), where 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 three weeks (Q3W) by IV infusion. In some embodiments, the kit further comprises instructions for administering the polypeptide (e.g., fusion polypeptide) at a dose of 10 mg / kg, e.g., once weekly by IV infusion.

[0032] Also provided herein are kits comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharma- ceutically acceptable carrier for use in combination with trastuzumab to treat HER2-positive gastric / gastroesophageal junction (GEJ) cancer, according to the methods described herein, in an individual (e.g., a human individual) in need thereof. 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 L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations (wherein numbering is according to EU index of Kabat); or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations. Fc region (wherein numbering is according to EU index of Kabat), where the individual's HER2-positive gastric / GEJ cancer has progressed after prior fluoropyrimidine-based therapy or prior treatment 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, where trastuzumab is administered to the individual by IV infusion every three weeks (Q3W). In some embodiments, the kit further comprises instructions for administering the polypeptide (e.g., fusion polypeptide) once a week at a dose of 10 mg / kg, e.g., by IV infusion.

[0033] Also provided are kits comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharma- ceutically acceptable carrier for use in combination with rituximab to treat aggressive non-Hodgkin's lymphoma (NHL) in an individual (e.g., a human individual) in need thereof, according to the methods described 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 L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations (wherein numbering is according to EU index of Kabat); or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations. Fc region (wherein numbering is according to EU index of Kabat), where the individual's aggressive NHL has relapsed and / or is refractory to prior treatment for aggressive NHL and no curative therapy is available. 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 is administered at a dose of 375 mg / m by IV infusion. 2 wherein the rituximab is administered to the individual once a week for four weeks, then once a month. In some embodiments, the kit further includes instructions for administering the polypeptide (e.g., fusion polypeptide) at a dose of 10 mg / kg or 15 mg / kg, e.g., by IV infusion, once a week.

[0034] Also provided are kits comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharma- ceutically acceptable carrier for use in combination with rituximab to treat indolent lymphoma according to the methods described herein in an individual (e.g., a human individual) in need thereof. 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 L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations (wherein numbering is according to EU index of Kabat); or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations. Fc region (wherein numbering is according to EU index of Kabat), and wherein the individual's indolent lymphoma has relapsed and / or is refractory to prior treatment for the indolent lymphoma. In some embodiments, the indolent lymphoma is indolent non-Hodgkin's lymphoma (NHL). In some embodiments, the indolent NHL is marginal zone lymphoma or follicular lymphoma. In some embodiments, the kit is administered at a dose of 375 mg / m by IV infusion. 2 wherein the rituximab is administered to the individual once a week for four weeks, then once a month. In some embodiments, the kit further includes instructions for administering the polypeptide (e.g., fusion polypeptide) at a dose of 10 mg / kg or 15 mg / kg, e.g., by IV infusion, once a week.

[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 L234A, L235A, G237A, and N297A mutations, numbering according to EU index of Kabat. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide 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 a SIRPα D1 domain variant and an Fc domain variant (or a medicament made therefrom or a pharmaceutical composition comprising such a polypeptide) further comprises instructions for administering to an individual at a dose of 10 mg / kg once per week (QW). In some embodiments, the kit comprises instructions for administering the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant (or a medicament made therefrom or a pharmaceutical composition comprising such a polypeptide) to an individual by IV infusion. [Brief description of the drawings]

[0036] [Figure 1]A shows Drug A as a monomer and B shows Drug A as a homodimer. The SIRPα D1 domain variant selectively binds CD47 with picomolar binding affinity and blocks the interaction with SIRPα. The molecular weight of Drug A is half the size of a typical antibody and can deliver twice the molar concentration to tumors. The Fc domain is modified to abolish binding to Fc gamma receptors to minimize toxicity. Drug A exhibits antibody-like pharmacokinetics. [Diagram 2] FIG. 1 shows a schematic diagram of the clinical trial described in Example 1. [Figure 3A] Figure 3 shows the clinical activity of Drug A + Trastuzumab in a HER2-positive gastric / GEJ cancer expansion cohort. Figure 3A provides the change in tumor diameter as a percentage from baseline. ORR = overall response rate. DCR = disease control rate; mPFS = median progression-free survival; CI = confidence interval. The change in tumor diameter as a percentage from baseline for each patient during the study is provided in Figure 3B. Figures 3A-3B: Patients received at least one dose of Drug A during the expansion phase, had a baseline assessment, and included at least one post-baseline disease assessment. The upper dashed line indicates the objective progression threshold. The lower dashed line indicates the overall response rate threshold. The treatment duration for each enrolled patient who received at least one dose of Drug A during the expansion phase is shown in Figure 3C. Tumors were scored according to the new Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (EA Eisenhauer, et al., European Journal of Cancer 45 (2009) 228-247). [Figure 3B]Figure 3 shows the clinical activity of Drug A + Trastuzumab in a HER2-positive gastric / GEJ cancer expansion cohort. Figure 3A provides the change in tumor diameter as a percentage from baseline. ORR = overall response rate. DCR = disease control rate; mPFS = median progression-free survival; CI = confidence interval. The change in tumor diameter as a percentage from baseline for each patient during the study is provided in Figure 3B. Figures 3A-3B: Patients received at least one dose of Drug A during the expansion phase, had a baseline assessment, and included at least one post-baseline disease assessment. The upper dashed line indicates the objective progression threshold. The lower dashed line indicates the overall response rate threshold. The treatment duration for each enrolled patient who received at least one dose of Drug A during the expansion phase is shown in Figure 3C. Tumors were scored according to the new Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (EA Eisenhauer, et al., European Journal of Cancer 45 (2009) 228-247). [Figure 3C] Figure 3 shows the clinical activity of Drug A + Trastuzumab in a HER2-positive gastric / GEJ cancer expansion cohort. Figure 3A provides the change in tumor diameter as a percentage from baseline. ORR = overall response rate. DCR = disease control rate; mPFS = median progression-free survival; CI = confidence interval. The change in tumor diameter as a percentage from baseline for each patient during the study is provided in Figure 3B. Figures 3A-3B: Patients received at least one dose of Drug A during the expansion phase, had a baseline assessment, and included at least one post-baseline disease assessment. The upper dashed line indicates the objective progression threshold. The lower dashed line indicates the overall response rate threshold. The treatment duration for each enrolled patient who received at least one dose of Drug A during the expansion phase is shown in Figure 3C. Tumors were scored according to the new Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (EA Eisenhauer, et al., European Journal of Cancer 45 (2009) 228-247). [Figure 4A]Figure 4 shows the clinical activity of Drug A + Pembrolizumab in the HNSCC expansion cohort. Figure 4A provides the change in tumor size, shown as a percentage from baseline. ORR = overall response rate. DCR = disease control rate; mPFS = median progression-free survival; CI = confidence interval; CP = immune checkpoint therapy; CPS = combined positive score for PD-L1 staining. Figure 4B provides the change in tumor size, shown as a percentage from baseline, for each patient during the study period. Figures 4A-4B: Included were patients who received at least one dose of Drug A during the expansion phase, had a baseline assessment, and had at least one post-baseline disease assessment. The upper dashed line indicates the threshold for objective progression. The lower dashed line indicates the threshold for overall response rate. The treatment duration for each enrolled patient who received at least one dose of Drug A during the expansion phase is shown in Figure 4C. Tumors were scored according to the revised Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (EA Eisenhauer, et al., European Journal of Cancer 45 (2009) 228-247). [Figure 4B] Figure 4 shows the clinical activity of Drug A + Pembrolizumab in the HNSCC expansion cohort. Figure 4A provides the change in tumor size, shown as a percentage from baseline. ORR = overall response rate. DCR = disease control rate; mPFS = median progression-free survival; CI = confidence interval; CP = immune checkpoint therapy; CPS = combined positive score for PD-L1 staining. Figure 4B provides the change in tumor size, shown as a percentage from baseline, for each patient during the study period. Figures 4A-4B: Included were patients who received at least one dose of Drug A during the expansion phase, had a baseline assessment, and had at least one post-baseline disease assessment. The upper dashed line indicates the threshold for objective progression. The lower dashed line indicates the threshold for overall response rate. The treatment duration for each enrolled patient who received at least one dose of Drug A during the expansion phase is shown in Figure 4C. Tumors were scored according to the revised Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (EA Eisenhauer, et al., European Journal of Cancer 45 (2009) 228-247). [Figure 4C] Figure 4 shows the clinical activity of Drug A + Pembrolizumab in the HNSCC expansion cohort. Figure 4A provides the change in tumor size, shown as a percentage from baseline. ORR = overall response rate. DCR = disease control rate; mPFS = median progression-free survival; CI = confidence interval; CP = immune checkpoint therapy; CPS = combined positive score for PD-L1 staining. Figure 4B provides the change in tumor size, shown as a percentage from baseline, for each patient during the study period. Figures 4A-4B: Included were patients who received at least one dose of Drug A during the expansion phase, had a baseline assessment, and had at least one post-baseline disease assessment. The upper dashed line indicates the threshold for objective progression. The lower dashed line indicates the threshold for overall response rate. The treatment duration for each enrolled patient who received at least one dose of Drug A during the expansion phase is shown in Figure 4C. Tumors were scored according to the revised Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (EA Eisenhauer, et al., European Journal of Cancer 45 (2009) 228-247). [Figure 5A-5C] Figure 5 shows the clinical activity of Drug A + Pembrolizumab in the NSCLC expansion cohort. Figure 5A provides the change in tumor size, shown as a percentage from baseline. DCR = disease control rate; mPFS = median progression-free survival; CI = confidence interval; TPS = tumor proportion score for PD-L1 staining. Figure 5B provides the change in tumor size, shown as a percentage from baseline, for each patient during the study period. Figures 5A-5B: Patients received at least one dose of Drug A during the expansion phase, had a baseline assessment, and included at least one post-baseline disease assessment. The upper dashed line indicates the objective progression threshold. The lower dashed line indicates the partial response rate threshold. The treatment duration for each enrolled patient who received at least one dose of Drug A during the expansion phase is shown in Figure 5C. Tumors were assessed according to the new Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (EA Eisenhauer, et al., European Journal of Cancer 45 (2009) 228-247). [Figure 6A]FIG. 1 shows pharmacokinetic and CD47 target occupancy parameters of Drug A administered in combination with pembrolizumab or trastuzumab during the study period. As shown in A, Drug A PK (Drug A serum concentrations) was within the predicted 95% interval based on the established population PK model. The steady-state half-life of Drug A (10 mg / kg QW) was predicted to be approximately 16 days. Drug A PK time points included intensive sampling in cycles 1 and 3, and pre-dose and end of infusion in all subsequent cycles. [Figure 6B] Figure 1 shows pharmacokinetic and CD47 target occupancy parameters of Drug A administered in combination with pembrolizumab or trastuzumab over the course of the study. When Drug A is administered in combination with pembrolizumab or trastuzumab, it is shown that near complete CD47 target occupancy is maintained by CD4+ T cells expressing CD47 throughout the entire dosing interval of Drug A. TO = target occupancy. [Figure 7A] Figure 1 shows antitumor shrinkage effects in patients treated with drug A in combination with pembrolizumab or trastuzumab. Positive rates of intratumoral CD68+ (left), CD163+ (center), and CD8+ (right) cells in paired biopsies before and after treatment are provided for five NSCLC patients treated with drug A + pembrolizumab, six HNSCC patients treated with drug A + pembrolizumab, and one gastric cancer patient treated with drug A + trastuzumab. [Figure 7B] Figure 1 shows antitumor shrinkage effects in patients treated with drug A in combination with pembrolizumab or trastuzumab. Tumor-associated macrophages and infiltrating lymphocytes increased after treatment. Images from biopsies of NSCLC PD-L1(+) patients treated with drug A + pembrolizumab show staining for CD68 and CD8 before and during treatment (C3 = cycle 3). The graph on the right shows the change in tumor size as a percentage from baseline for each patient over the course of the study. The upper dashed line indicates the threshold for objective progression, and the lower dashed line indicates the threshold for partial response. The CD68 and CD163 markers indicate macrophages, and the CD8 marker indicates T lymphocytes. [Figure 7C] Figure 1 shows antitumor shrinkage effects in patients treated with Drug A in combination with pembrolizumab or trastuzumab. Images from biopsies of HNSCC PD-L1(+) patients treated with Drug A + pembrolizumab show staining for CD68 and CD8 before and during treatment (cycle 3). The graph on the right shows the change in tumor size as a percentage from baseline for each patient over the course of the study. The upper dashed line indicates the threshold for objective progression, and the lower dashed line indicates the threshold for partial response. The CD68 and CD163 markers indicate macrophages, and the CD8 marker indicates T lymphocytes. [Figure 8] FIG. 1 provides a schematic diagram of the proposed mechanism for the anti-tumor activity of Drug A when administered in combination with anti-cancer therapeutic antibodies such as pembrolizumab, trastuzumab, or rituximab. [Figure 9A] Schematic diagram showing the clinical activity of drug A in combination with pembrolizumab in evaluable >2L HNSCC patients, including both checkpoint inhibitor-naïve and checkpoint inhibitor-experienced patients. CPS = combined positive score (i.e., the number of PD-L1 positive staining cells (tumor cells, lymphocytes, and macrophages) divided by the total number of viable tumor cells multiplied by 100). [Figure 9B] FIG. 1 provides a schematic diagram showing best overall response and duration of response in >2L HNSCC patients treated with drug A and pembrolizumab. [Figure 10A] FIG. 1 is a schematic showing the clinical activity of drug A in combination with pembrolizumab in evaluable >2L NSCLC patients. Patients marked with * showed >80% change. TPS = tumor proportion score. [Figure 10B] FIG. 1 provides a schematic diagram showing best overall response and duration of response in >2L NSCLC patients treated with drug A and pembrolizumab. [Figure 11A]Schematic diagram showing the clinical activity of drug A in combination with trastuzumab in patients with evaluable >2L HER2+ gastric or HER2+ GEJ cancer. One patient with clinical progression is not shown. The HER2 score reflects the HER2 expression level of tumor cells. [Figure 11B] FIG. 1 is a schematic showing best overall response and duration of response in >2L HER2+ gastric or HER2+ GEJ cancer patients treated with drug A and trastuzumab. [Figure 12] FIG. 1 is a schematic showing the clinical activity of Drug A in combination with Rituximab in evaluable patients with relapsed / refractory non-Hodgkin's lymphoma. Patients marked with "^" have an 80% or greater increase in measurable disease compared to baseline. Patients marked with "*" achieved a complete response or metabolic complete response. Patients marked with "mPR" achieved a metabolic partial response. Two patients with clinical progression (one each at 10 mg / kg and 15 mg / kg Drug A) are not shown. One patient in metabolic CR (10 mg / kg Drug A) is not shown in the plot. [Figure 13A] FIG. 1 is a schematic diagram showing best overall response and duration of response in patients with relapsed / refractory non-Hodgkin's lymphoma treated with 10 mg / kg of Drug A and rituximab. [Figure 13B] 1 is a schematic diagram showing best overall response and duration of response in relapsed / refractory non-Hodgkin's lymphoma patients treated with 15 mg / kg of Drug A and Rituximab. One patient is not shown as best response, day 8 of clinical progression study. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] definition The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, depending in part on the method of measuring or determining the value, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, as is customary in the art. Alternatively, "about" can mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, more preferably within 2-fold, of a value. Unless otherwise stated, when a particular value is described in this application and claims, the term "about" is deemed to mean within an acceptable error range for the particular value.

[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 indicates otherwise. Furthermore, to the extent that "including," "include," "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 similar manner as the term "comprising."

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

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

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

[0042] As used herein, the term "linker" refers to a link between two elements, e.g., protein domains. In some embodiments, a linker can be a covalent bond or a spacer. The term "spacer" refers to a moiety (e.g., a polyethylene glycol (PEG) polymer) or 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, an amino acid spacer is part of the primary sequence of a polypeptide (e.g., attached to spaced polypeptides or polypeptide domains via the polypeptide backbone).

[0043] As used herein, the term "effective amount" refers to an amount of a polypeptide or pharmaceutical composition comprising a polypeptide described herein, e.g., a polypeptide having a SIRPα D1 domain or a variant thereof, that is sufficient and effective to achieve a desired therapeutic effect in treating a patient with a disease such as cancer, e.g., a solid tumor or a 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 product or formulation that includes an active ingredient and an excipient or diluent (or both an excipient and a diluent), such that the active ingredient may be administered by a suitable method of administration. In some embodiments, the pharmaceutical compositions disclosed herein include pharma- ceutical acceptable ingredients that are compatible with the polypeptide. In some embodiments, the pharmaceutical composition is in tablet or capsule form for oral administration, or in aqueous form for intravenous or subcutaneous administration, e.g., by injection.

[0045] As used herein, the terms "subject," "individual," and "patient" are used interchangeably to refer to vertebrates, e.g., mammals. Mammals include, but are not limited to, murines, monkeys, humans, farm animals, sports animals, and pets. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro. Neither term requires the supervision of a medical professional.

[0046] As used herein, the term "affinity" or "binding affinity" refers to the strength of the binding interaction between two molecules. In general, binding affinity refers to the strength of the sum of non-covalent interactions between a molecule and its binding partner (such as SIRPα D1 domain variant and CD47). Unless otherwise specified, binding affinity refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair. The binding affinity between two molecules is generally described by a dissociation constant (KD) or an association constant (KA). Two molecules that have low binding affinity to each other generally bind slowly, tend to dissociate easily, and exhibit a large KD. Two molecules that have high affinity to each other generally bind easily, tend to remain bound for a long time, and exhibit 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 As used herein, the term "less than a KD" refers to a numerically greater KD value and a binding affinity that is decreased compared to the listed KD value.

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

[0049] overview Provided herein are methods 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, 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 selected from the group consisting of (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, and the individual is a human. In some embodiments, the individual's NSCLC has progressed on 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, there is also provided a method of treating head and neck squamous cell carcinoma (HNSCC) 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 comprises (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the individual's HNSCC has progressed during or after prior platinum therapy, and the individual is human. In some embodiments, the individual has received prior immune checkpoint inhibitor (CPI) therapy (e.g., treatment with an immune checkpoint inhibitor described herein). 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.

[0052] In some embodiments, an individual is "PD-L1 negative" if they have a cancer that does not express the PD-L1 biomarker or that expresses very low levels of PD-L1. In some embodiments, an individual is "PD-L1 negative" or has a "PD-L1 negative cancer" if no expression (e.g., protein expression) of PD-L1 is detected on (or in) tumor cells (TC) in the individual's sample, if no PD-L1 expression (e.g., protein expression) is detected on (or in) tumor infiltrating immune cells (IC) in the individual's sample, or if PD-L1 expression (e.g., protein expression) is detected at very low levels on (or in) TCs and / or ICs in the individual's sample. In some embodiments, an individual is PD-L1 negative if, for example, 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) in a sample obtained from the individual express PD-L1 as determined using an assay for determining the individual's PD-L1 status (e.g., an assay described herein). In some embodiments, an individual or tumor may be considered PD-L1 negative because they do not have a T cell infiltrate. Such assays are known and routinely used by medical professionals.

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

[0054] In some embodiments, a method of treating HER2-positive gastric / gastroesophageal junction (GEJ) cancer in an individual is provided, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) an anti-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 comprises (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the 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 a human.

[0055] In some embodiments, a method of treating aggressive non-Hodgkin's lymphoma or NHL (e.g., de novo or transformed diffuse large B-cell lymphoma (DLBCL) or mantle cell 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 comprises (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations. (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat, where the individual's aggressive NHL has relapsed and / or is refractory to prior treatment for aggressive NHL, no curative therapy is available, and the individual is a 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 indolent 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 selected from the group consisting of (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the individual's indolent lymphoma has relapsed and / or is refractory to a prior treatment for indolent lymphoma, and the individual is a human. In some embodiments, the indolent lymphoma (such as relapsed / refractory indolent lymphoma) is a non-Hodgkin's lymphoma (NHL), e.g., relapsed / refractory indolent NHL. In some embodiments, the indolent NHL (e.g., relapsed / refractory NHL) is a follicular lymphoma (e.g., relapsed / refractory follicular lymphoma). In some embodiments, the indolent NHL (eg, relapsed / refractory NHL) is marginal zone lymphoma (eg, relapsed / refractory marginal zone lymphoma).

[0057] Further details regarding therapeutic methods and polypeptides comprising SIRPα D1 domain variants and Fc domain variants are described below and in U.S. Patent No. 10,259,859, the contents of which are incorporated herein by reference in their entirety. Signal Regulatory Protein-α (SIRP-α) D1 Domain and Its Variants

[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 comprising 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 residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92.

[0059] Also disclosed herein, in some embodiments, is a polypeptide comprising an Fc domain variant, wherein the Fc domain variant dimer comprises two Fc domain variants, each Fc domain variant independently selected from: (i) a human IgG1 Fc region consisting of 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.

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

[0061] Native SIRPα is composed of three highly homologous immunoglobulin (Ig)-like extracellular domains (D1, D2, and D3). The SIRPα D1 domain ("D1 domain") refers to the membrane-distal extracellular domain of SIRPα and mediates binding of SIRPα to CD47. As used herein, the term "SIRPα polypeptide" refers to any SIRPα polypeptide or fragment thereof capable of binding to CD47. There are at least ten wild-type human SIRPα variants. Table 1 shows the amino acid sequences of the D1 domain of ten naturally occurring wild-type human SIRPα D1 domain variants (SEQ ID NOs: 1-10). In some embodiments, the SIRPα polypeptide comprises a 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). TIFF2025003971000002.tif248170TIFF2025003971000003.tif105170

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

[0063] In some embodiments, the SIRPα D1 domain variant disclosed herein comprises a SIRPα D1 domain or a variant thereof. In some embodiments, the SIRPα D1 domain variant comprises one or more amino acid substitutions, insertions, additions, or deletions relative to the wild-type D1 domain set forth 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 comprises a fragment of the D1 domain. In some embodiments, the SIRPα polypeptide fragment or SIRPα D1 domain variant fragment comprises an amino acid sequence less than 10 amino acids in length, about 10 amino acids in length, about 20 amino acids in length, about 30 amino acids in length, about 40 amino acids in length, about 50 amino acids in length, about 60 amino acids in length, about 70 amino acids in length, about 80 amino acids in length, about 90 amino acids in length, about 100 amino acids in length, or more than about 100 amino acids in length. In some embodiments, the SIRPα D1 domain fragment retains the ability to bind to CD47.

[0064] In some embodiments, a polypeptide of the disclosure comprising a SIRPα D1 domain variant binds to CD47 with a higher binding affinity than the wild-type human SIRPα D1 domain. In some embodiments, a 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) greater than that of the naturally occurring D1 domain. In some embodiments, a 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 greater) greater than that of the naturally occurring D1 domain.

[0065] As used herein, the term "optimized affinity" or "optimized binding affinity" refers to an optimized strength of binding interaction between a polypeptide disclosed herein, such as a SIRPα D1 domain variant, and CD47. For example, in some embodiments, the polypeptide binds primarily or with higher affinity to CD47 on cancer cells and does not substantially bind or binds with lower affinity to CD47 on non-cancer cells. In some embodiments, the binding affinity between the polypeptide and CD47 is optimized such that the interaction does not cause clinically relevant toxicity or reduces toxicity compared to a variant that binds with maximum affinity. In some embodiments, to achieve an optimized binding affinity between a polypeptide provided herein and CD47, a polypeptide comprising a SIRPα D1 domain variant is developed to have a lower binding affinity to CD47 than is maximally achievable. In some embodiments, the SIRPα D1 domain variant disclosed herein cross-reacts with rodent, 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 in a variety of ways, such as in vitro T cell proliferation assays.

[0067] As used herein, the term "minimally immunogenic" refers to the immunogenicity of a protein (e.g., a therapeutic protein) that has been modified, e.g., by an amino acid substitution, to be less immunogenic (e.g., at least 10%, 25%, 50%, or 100% less) than the immunogenicity before the amino acid substitution was introduced (e.g., the unmodified protein). In some embodiments, a protein (e.g., a therapeutic protein) is modified to have minimal immunogenicity and provokes no or little 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 present disclosure administered to a subject has the same amino acid sequence as the affinity of the SIRPα polypeptide in the subject's biological sample, except for the amino acid changes that increase the affinity of the SIRPα D1 domain variant. In some embodiments, the polypeptide variants disclosed herein reduce the risk of side effects compared to anti-CD47 antibodies or wild-type SIRPα. In some embodiments, the polypeptide variants disclosed herein reduce the risk of anemia compared to anti-CD47 antibodies or wild-type SIRPα. In some embodiments, the polypeptide variants disclosed herein do not cause acute anemia in rodent or non-human primate (NHP) studies.

[0069] Table 2 shows the specific amino acid substitutions of the SIRPα D1 domain variants for each D1 domain sequence. In some embodiments, the SIRPα D1 domain variants include 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 variants include up to 14 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variants include up to 10 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variants include up to 7 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variants of the present disclosure have at least 90% (e.g., at least 92%, 95%, 97%, or more than 97%) amino acid sequence identity to the sequence of the wild-type D1 domain.

[0070] 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 of variants thereof (e.g., a portion of one wild-type D1 domain or a variant thereof and a portion of another wild-type D1 domain or a variant thereof). In some embodiments, the chimeric SIRPα D1 domain variant comprises at least two portions (e.g., three, four, five, or more portions) of the wild-type D1 domain or a 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 2. TIFF2025003971000004.tif250170TIFF2025003971000005.tif247170TIFF2025003971000006.tif208170

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

[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 X1 is L, I, or V, X2 is V, L, or I, X3 is A or V, X4 is A, I, or L, X5 is I, T, S, or F, X6 is E, V, or L, X7 is K or R, X8 is E or Q, X9 is H, P, or R, and X 10 is L, T, or G, and X 11 is K or R, and X 12 is V or I, and X 13 is F, L, or V, and X 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: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 14X1 is L, I, or V, X2 is V, L, or I, X3 is A or V, X4 is A, I, or L, X5 is I, T, S, or F, X6 is E, V, or L, X7 is K or R, X8 is E or Q, X9 is H, P, or R, and X 10 is L, T, or G, and X 11 is K or R, and X 12 is V or I, and X 13 is F, L, or V, and X 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: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 X1 is L, I, or V, X2 is V, L, or I, X3 is A or V, X4 is A, I, or L, X5 is I, T, S, or F, X6 is E, V, or L, X7 is K or R, X8 is E or Q, X9 is H, P, or R, and X 10 is L, T, or G, and X 11 is K or R, and X 12 is V or I, and X 13 is F, L, or V, and X 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: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 X1 is L, I, or V, X2 is V, L, or I, X3 is A or V, X4 is A, I, or L, X5 is I, T, S, or F, X6 is E, V, or L, X7 is K or R, X8 is E or Q, X9 is H, P, or R, and X 10 is L, T, or G, and X 11 is K or R, and X 12 is V or I, and X 13 is F, L, or V, and X 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 the sequence of any one 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 above embodiments, X 11 is K or R. In any of the above embodiments, X 12 is V or I. In any of the above embodiments, X 13 is F, L, V. In any of the above 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 a wild-type SIRPα D1 domain comprising the sequence of any one of SEQ ID NOs: 1, 4-6, and 9.

[0076] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a 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 at least 100-fold higher binding affinity than a 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 at least 1000-fold higher binding affinity than a 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 at least 1×10 -8 Less than M, 5×10 -9 Less than M, 1×10 -9 Less than M, 5×10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a KD 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.

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

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

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

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

[0081] In any of the foregoing embodiments of this aspect of the disclosure, the polypeptide comprises the sequence of any one 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 above embodiments, X 11 is K or R. In any of the above embodiments, X 12 is V or I. In any of the above embodiments, X 13 is F, L, or V. In any of the above 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 a wild-type SIRPα D1 domain comprising the sequence of any one of SEQ ID NOs: 2, 3, 7, and 10.

[0082] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with at least 1×10 -8 Under M, 5x10 -9 Less than M, 1x10 -9 Under M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D and binds to CD47.

[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 14X1 is L, I, or V, X2 is V, L, or I, X3 is A or V, X4 is A, I, or L, X5 is I, T, S, or F, X6 is E, V, or L, X7 is K or R, X8 is E or Q, X9 is H, P, or R, and X 10 is S, T, or G, and X 11 is K or R, and X 12 is V or I, and X 13 is F, L, or V, and X 14 is F or V, and the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO:8.

[0084] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:20, wherein X1 is L, I, or V. In any of the foregoing embodiments in this aspect of the 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 above embodiments, X 11 is K or R. In any of the above embodiments, X 12 is V or I. In any of the above embodiments, X 13 is F, L, or V. In any of the above 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 a wild-type SIRPα D1 domain having the sequence of SEQ ID NO:8.

[0085] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 8. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 8. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 8. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1×10 -8 Less than M, 5×10 -9 Less than M, 1×10 -9 Less than M, 5×10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D and binds to CD47.

[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 XKPS (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; 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 a wild-type SIRPα D1 domain having the sequence of any one 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 preceding embodiments, X 15 is E or Q. In any of the preceding embodiments, X 16 is H, P, or R. In any of the foregoing embodiments, X 17 is D or E. In any of the preceding 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 preceding embodiments, X 20 is E or D. In any of the preceding embodiments, X 21 is S or P. In any of the preceding 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 preceding embodiments, X 25 is F, L, or V. In any of the preceding embodiments, X 26 In any of the preceding embodiments, X is D or absent. 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 disclosure comprises six or fewer amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 1-10.

[0088] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having a sequence of any one of SEQ ID NOs: 1-10. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having a sequence of any one of SEQ ID NOs: 1-10. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having a sequence of any one of SEQ ID NOs: 1-10. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1×10 -8 Under M, 5x10 -9 Less than M, 1x10 -9 Under M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D and binds to CD47.

[0089] In some embodiments, the polypeptide of the present disclosure comprising a SIRPα D1 domain variant further comprises a D2 domain having the sequence of SEQ ID NO: 24, a D3 domain having the sequence of SEQ ID NO: 25, or a D2 domain having the sequence of SEQ ID NO: 24 and a D3 domain having the sequence of SEQ ID NO: 25 of wild-type human SIRPα as shown in Table 3. In some embodiments, the SIRPα D1 domain variant further comprises a fragment or variant of the D2 domain, or a fragment or variant of the D3 domain. In some embodiments, the SIRPα D1 domain variant further comprises a fragment or variant of the D2 domain and a fragment or variant of the D3 domain. In some embodiments, the SIRPα D1 domain variant is linked to the D2 or D3 domain via a linker. In some embodiments, the SIRPα D1 domain variant is linked to the D2 and D3 domains via a linker. TIFF2025003971000007.tif51170

[0090] In some embodiments, the polypeptides of the present disclosure comprising a SIRPα D1 domain variant are attached to an Fc domain variant to improve the pharmacokinetic properties of the polypeptide, e.g., to increase serum half-life. In some embodiments, the SIRPα D1 domain variant is attached to an Fc domain variant that cannot dimerize. In some embodiments, the Fc domain variant serves to increase serum half-life of the polypeptides described herein. In some embodiments, the polypeptides of the present disclosure comprising a SIRPα D1 domain variant do not comprise any of the sequences of SEQ ID NOs: 26-36 shown in Table 4. TIFF2025003971000008.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 liquid phase or bound to a solid phase 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, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier can be either soluble or insoluble.

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

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

[0095] SIRPα D1 domain variants with altered glycosylation patterns 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 residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92.

[0096] In some embodiments, also disclosed herein are polypeptides comprising an Fc domain variant, wherein the Fc domain variant dimer comprises two Fc domain variants, each Fc domain variant independently selected from: (i) a human IgG1 Fc region consisting of 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.

[0097] In some embodiments, the polypeptide in the compositions disclosed herein comprises a SIRPα D1 domain variant with reduced or minimal glycosylation. The D1 domain of each of the ten 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 (unglycosylated) and a minor band of higher molecular weight that is removed by Endo Hf. Endo Hf is a recombinant protein fusion of endoglycosidase H and maltose binding protein. Endo Hf cleaves within the high mannose chitobiose core and some hybrid oligosaccharides from N-linked glycoproteins. This means that the proline at amino acid position 83 may reduce the efficiency of glycosylation resulting in proteins with different degrees of glycosylation and therefore heterogeneity. For drug development, heterogeneity can pose challenges in process development. Therefore, to investigate the possibility of generating a homogeneous non-glycosylated form of the SIRPα D1 domain variant, in some embodiments, the amino acid N80 of the SIRPα D1 variant is mutated to Ala. In some embodiments, to generate a non-glycosylated, SIRPα D1 domain variant, the amino acid N80 in the SIRPα D1 domain variant is replaced by any amino acid, such as any natural and non-natural amino acid, for example, 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 in the hydrophobic core of the D1 domain.

[0098] In some embodiments, the polypeptide in the composition disclosed herein comprises a SIRPα D1 domain variant with increased glycosylation relative to the wild-type SIRPα D1 domain. Another option to increase the homogeneity of the final product is to increase the efficiency of glycosylation at amino acid N80 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 substituted by any amino acid, including natural and unnatural amino acids, for example, P83V, P83A, P83I, and P83L. In some embodiments, the polypeptides of the disclosure are expressed in cells that have been optimized not to glycosylate the expressed protein, for example, by genetic engineering of the cell line (e.g., a genetically engineered yeast or mammalian host), or by altering cell culture conditions, such as the addition of kifunensine, or by using a naturally non-glycosylating host, such as prokaryote (e.g., E. coli).

[0099] Table 5 shows the specific amino acid substitutions of the SIRPα D1 domain variants for each D1 domain variant sequence. In some embodiments, the SIRPα D1 domain variants include 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 variants are not glycosylated or are minimally glycosylated. In some embodiments, the SIRPα D1 domain variants are fully glycosylated or nearly fully glycosylated. In some embodiments, the SIRPα D1 domain variants include up to 14 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variants include up to 10 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variants include up to 7 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, a SIRPα D1 domain variant of the disclosure has at least 90% (eg, at least 92%, 95%, 97% or greater than 97%) amino acid sequence identity to the sequence of the wild-type D1 domain.

[0100] In some embodiments, the SIRPα D1 domain variant is a chimeric SIRPα D1 domain variant that comprises two or more portions of a wild-type D1 domain or variant thereof (e.g., a portion of one wild-type D1 domain or variant thereof and a portion of another wild-type D1 domain or variant thereof). In some embodiments, the chimeric SIRPα D1 domain variant comprises at least two portions (e.g., three, four, five, or more portions) of a 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. TIFF2025003971000009.tif224170TIFF2025003971000010.tif244170TIFF2025003971000011.tif211170TIFF20250039710 00012.tif243170TIFF2025003971000013.tif254170TIFF2025003971000014.tif216170TIFF2025003971000015.tif112170

[0101] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGX 12I TX 13 ADAGTYYCX 14 KX 15 RKGSPDDVEX 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; 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 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; 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 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; 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: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 16X1 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; 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:6.

[0105] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNMDFSIRISX 12I TX 13 ADAGTYYCX 14 KX 15 RKGSPDDVEX 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; 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 comprises at least one amino acid substitution relative 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 disclosure, the polypeptide comprises a SIRPα D1 domain variant having a sequence of any one 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, or V. In any of the preceding embodiments, X 16 is F or V.

[0107] In some embodiments, the polypeptides provided herein comprise no more than 10 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 1, 4-6, and 9. In some embodiments, the polypeptides provided herein comprise no more than 7 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 1, 4-6, and 9.

[0108] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 1, 4-6, and 9. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 1, 4-6, and 9. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one 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 at least 1×10 -8 Less than M, 5×10 -9 Less than M, 1×10 -9 Less than M, 5×10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D and binds to CD47.

[0109] 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 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; 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 a SIRPα D1 domain variant having the following sequence: EEEX1QX2IQPDKSVSVAAGESX3ILLCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISX 12 ITX 13 ADAGTYYCX14 KX 15 RKGSPDTEX 16 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; 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: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 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; 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 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; 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 comprises 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 disclosure, the polypeptide comprises a SIRPα D1 domain variant having a sequence of any one of SEQ ID NOs: 38, 39, 43, and 46, 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 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 no more than 10 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having no more than 7 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 2, 3, 7, and 10.

[0115] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with at least 1×10 -8 Under M, 5x10 -9 Less than M, 1x10 -9 Under M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D and binds to CD47.

[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 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; 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: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 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 no more than 10 amino acid substitutions relative to a 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 no more than 7 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 8.

[0119] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 8. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 8. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 8. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1×10 -8 Less than M, 5×10 -9 Less than M, 1×10 -9 Less than M, 5×10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D and binds to CD47.

[0120] In another aspect, the disclosure features a polypeptide that includes 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 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 not present; X 29 is T or V;X 30 is F or V;X 31 is A or G; the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 1-10.

[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 preceding embodiments, X 15 is E or Q. In any of the preceding embodiments, X 16 is H, P, or R. In any of the foregoing embodiments, X 17 is D or E. In any of the preceding 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 preceding embodiments, X 20 is E or N. In any of the preceding embodiments, X 21 is S or P. In any of the preceding 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 preceding embodiments, X 27 is F, L, or V. In any of the preceding embodiments, X 28 In any of the above embodiments, X is D or absent. 29 is T or V. In any of the above embodiments, X 30 is F or V. In any of the above embodiments, X 31 is A or G.

[0122] In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 10 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 1-10. In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 7 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 1-10.

[0123] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having a sequence of any one of SEQ ID NOs: 1-10. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having a sequence of any one of SEQ ID NOs: 1-10. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having a sequence of any one of SEQ ID NOs: 1-10. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1×10 -8 Under M, 5x10 -9 Less than M, 1x10 -9 Under M, 5x10-10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D 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 X7 is I, R, Y, K or F; X8 is G or A; X9 is E or V; 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; 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 not present; 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 that includes a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9ITPADAGTYYCX 10 X1 is V, L, or I; X2 is A, I, V, or L; X3 is I, F, S, or T; X4 is E, V, or L; X5 is K or R; X6 is E or Q; X7 is H, P, or R; X8 is L, T, S, or G; X9 is A; 10 is V or I; where the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of any one 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 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 a SIRPα D1 domain comprising at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO:49, and is selected from the group consisting of X1, X2, X3, X4, X5, X6, X7, X8, X9, and X10. 10 Each of is not a wild-type amino acid.

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

[0129] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 1. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 1. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with at least 1×10 -8 Less than M, 5×10 -9 Less than M, 1×10 -9 Less than M, 5×10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D and binds to CD47.

[0130] In another aspect, the disclosure features a polypeptide that includes a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVSVAAGESAILHCTX2TSLX3PVGPIQWFRGAGPARX4LIYNQX5X6GX7FPRVTTVSEX8TKRENMDFSISISX9ITPADAGTYYCX 10 X1 is V or I; X2 is V or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is E or Q; X7 is H or P; X8 is S or T; X9 is N or A;10 is V or I; where the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of any one 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 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 comprising at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO:50, and is selected from the group consisting of X1, X2, X3, X4, X5, X6, X7, X8, X9, and X10. 10 Each of is not a wild-type amino acid.

[0133] In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 10 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 2. In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 7 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 2.

[0134] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 2. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 2. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 2. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with at least 1×10 -8 Less than M, 5×10 -9 Less than M, 1×10 -9 Less than M, 5×10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D and binds to CD47.

[0135] In another aspect, the disclosure features a polypeptide that includes a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMDFSIRIGX8ITPADAGTYYCX9KFRKGSPDDVEFKSGAGTELSVRAKPS (sequence number 221), wherein X1 is V or I; X2 is A or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is H or P; X7 is L or T; X8 is N or A; and X9 is V or I; wherein the variant comprises at least one amino acid substitution relative to a wild-type SIRPα D1 domain having the sequence of any one 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 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 of this aspect of the disclosure, X8 is A and X1 is V or I. In any of the foregoing embodiments of this aspect of the disclosure, X8 is A and X2 is A or I. In any of the foregoing embodiments, X8 is A and X3 is I or F. In any of the foregoing embodiments, X8 is A and X4 is E or V. In some embodiments, X4 is not V. In any of the foregoing embodiments, X8 is A and X5 is K or R. In any of the foregoing embodiments, X8 is A and X6 is H or P. In any of the foregoing embodiments, X8 is A and X7 is A or V. In any of the foregoing embodiments, X8 is A and X9 is V or I.

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

[0139] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant comprising at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 51, 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 polypeptides of this aspect of the disclosure comprise no more than 10 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NO: 1. In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 7 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NO: 1.

[0141] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 1. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 1. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with at least 1×10 -8 Less than M, 5×10 -9 Less than M, 1×10 -9 Less than M, 5×10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than M DIn some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D and binds to CD47.

[0142] In another aspect, the disclosure features a polypeptide that includes a SIRPα D1 domain variant having the following sequence: X4 is K or R; X5 is H or P; X6 is L, T, or G; and X7 is N or A; wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence set forth in SEQ ID NO:1.

[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 disclosure, X2 is A, I, or L. In any of the foregoing embodiments, X3 is I, T, S, or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In any of the foregoing embodiments, X6 is L, T, or G. In any of the foregoing embodiments, X7 is N or A.

[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 of this aspect of the disclosure, X2 is A or I. In any of the foregoing embodiments, X3 is I or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In any of the foregoing embodiments, X6 is L or T. In any of the foregoing embodiments, X7 is N or A.

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

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

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

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

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

[0150] In another aspect, the disclosure features a polypeptide that includes a SIRPα D1 domain variant having the following sequence: X4 is K or R; X5 is H, P, or R; X6 is G, S, T; and X7 is N or A; wherein the variant comprises at least one amino acid substitution relative to a wild-type SIRPα D1 domain having the sequence of any one 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 of this aspect of the disclosure, X2 is V, I, or L. In any of the foregoing embodiments, X3 is I, T, S, or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In any of the foregoing embodiments, X6 is S, T, or G. In any of the foregoing embodiments, X7 is N or A.

[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 of this aspect of the disclosure, X2 is V or I. In any of the foregoing embodiments, X3 is I or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In any of the foregoing embodiments, X6 is S or T. In any of the foregoing embodiments, X7 is N or A.

[0153] 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 disclosure, X7 is A and X1 is V or I. In any of the foregoing embodiments of this aspect of the disclosure, X7 is A and X2 is V or I. In any of the foregoing embodiments, X7 is A and X3 is I or F. In any of the foregoing embodiments, X7 is A and X4 is K or R. In any of the foregoing embodiments, X7 is A and X5 is H or P. In any of the foregoing embodiments, X7 is A and X6 is S or T.

[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 disclosure, X7 is A and X1 is I. In any of the foregoing embodiments of this aspect of the disclosure, X7 is A and X2 is I. In any of the foregoing embodiments, X7 is A and X3 is F. In any of the foregoing embodiments, X7 is A and X4 is R. In any of the foregoing embodiments, X7 is A and X5 is P. In any of the foregoing embodiments, X7 is A and X6 is T.

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

[0156] In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 10 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 2. In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 7 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 2.

[0157] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 2. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 2. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NOs: 2. In some embodiments, the fragment comprises a polypeptide less than 10 amino acids in length, about 10 amino acids in length, about 20 amino acids in length, about 30 amino acids in length, about 40 amino acids in length, about 50 amino acids in length, about 60 amino acids in length, about 70 amino acids in length, about 80 amino acids in length, about 90 amino acids in length, about 100 amino acids in length, or more than about 100 amino acids in length. The fragment retains the ability to bind to CD47. Preferably, the SIRPα D1 domain variant polypeptides and fragments thereof bind to CD47 with higher affinity than the SIRPα polypeptide binds to CD47. For example, in some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is administered at a concentration of 1×10 -8 Under M, 5x10 -9 Less than M, 1x10 -9 Under M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D and binds to CD47.

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

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

[0160] In some embodiments described herein, the polypeptide comprises a SIRPα D1 domain variant having the sequence set forth below: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9ITX 10 ADAGTYYCX 11 X1 is V, L, or I; X2 is A, V, L, or I; X3 is I, S, T, or F; X4 is E, L, or V; X5 is K or R; X6 is E or Q; X7 is H, R, or P; X8 is S, G, L, or T; X9 is N; 10 is any 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 disclosure, disclosed herein is a composition comprising a SIRPα D1 domain variant polypeptide having an amino acid sequence of SEQ ID NO: 48, or a fragment thereof. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a higher affinity than the affinity with which the SIRPα polypeptide binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide is at least 1×10 -8 Less than M or 1×10 -9 Less than M, 1×10 -10 Less than M or 1×10 -11K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide is attached or fused to a second polypeptide. In some embodiments, the second polypeptide includes, but is not limited to, an Fc polypeptide, an Fc variant, or a fragment of the foregoing.

[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. TIFF2025003971000016.tif245170TIFF2025003971000017.tif251170TIFF2025003971000018.tif250170TIFF2025003971000019.tif81170

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

[0164] 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 (SEQ ID NO: 223)%, or 100% sequence identity) to SEQ ID NO: 80, 81, or 85 in Table 6. Fc domain variants and fusion polypeptides comprising Fc domain variants

[0165] 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 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 are Fc domain variant dimers, wherein the Fc domain variant dimer comprises two Fc domain variants, each Fc domain variant independently selected from: (i) a human IgG1 Fc region consisting of 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.

[0167] Antibodies targeting cell surface antigens can trigger 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 receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibodies to Fc receptors of 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 implantation, and control of immunoglobulin production. In addition, the complement system can be activated by the binding of the C1 component of complement to antibodies. Complement activation is important for the lysis of cellular pathogens. However, complement activation can also stimulate inflammatory responses and may be involved in autoimmune hypersensitivity or other immune disorders. Variant Fc regions that have reduced or eliminated ability to bind to specific Fc receptors are useful for the development of therapeutic antibody and Fc fusion polypeptide constructs that act by targeting, activating, or neutralizing ligand function without damaging or destroying local cells or tissues.

[0168] In some embodiments, a SIRPα D1 polypeptide construct comprises a non-native SIRPα D1 domain variant linked to an Fc domain variant forming an Fc domain with eliminated or reduced effector function.

[0169] In some embodiments, an Fc domain variant refers to a polypeptide chain that includes a second and a third antibody constant domain (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. Additionally, in some embodiments, the Fc domain variant is of any IgG subtype (e.g., IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, and IgG4). In some embodiments, the Fc domain variant includes as many as 10 amino acid modifications (e.g., insertions, deletions, and / or substitutions) that alter the interaction between the Fc domain and an 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 which the two wild-type Fc domains dimerize via 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 that has been mutated to not contain effector function, e.g., a "dead Fc domain dimer variant." In some embodiments, each of the Fc domains of the Fc domain dimer variant includes an amino acid substitution in the CH2 antibody constant domain to reduce interaction or binding between the Fc domain dimer variant and an Fc receptor, such as an Fcγ receptor (FcγR), an Fcα receptor (FcαR), or an Fcε (FcεR).

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

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

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

[0175] In some embodiments, a polypeptide construct comprising a non-native Fc region described herein exhibits reduced or ablated binding to at least one of the Fcγ receptors CD16a, CD32a, CD32b, CD32c, and CD64, as compared to a polypeptide construct comprising a native Fc region. In some cases, a polypeptide construct described herein exhibits reduced or ablated 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 complement component C1q binding to an antibody Fc domain. In some embodiments, the polypeptide constructs comprising the non-native Fc regions described herein exhibit at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduced C1q binding compared to polypeptide constructs comprising wild-type Fc regions. In some cases, the polypeptide constructs comprising the non-native Fc regions described herein exhibit reduced CDC compared to polypeptide constructs comprising wild-type Fc regions. In some embodiments, the polypeptide constructs comprising the non-native Fc regions described herein exhibit at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduced CDC compared to polypeptide constructs comprising wild-type Fc regions. In some cases, a polypeptide construct comprising a non-naturally occurring Fc domain variant or an Fc domain dimer variant described herein exhibits negligible CDC compared to a polypeptide construct comprising a wild-type Fc region.

[0177] In some embodiments, the Fc domain variants or Fc domain dimer variants described herein are minimally glycosylated or have reduced glycosylation relative to the wild-type sequence. In some embodiments, deglycosylation is achieved by mutation of N297A or by mutating N297 to any amino acid other than N. In some embodiments, deglycosylation is achieved by disrupting the motif N-Xaa1-Xaa2-Xaa3, 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, mutations to any one or more of N, Xaa1, Xaa2, or Xaa3 result in deglycosylation of the Fc domain variant or Fc domain dimer variant.

[0178] In some embodiments, the antibody IgG constant region variant (e.g., Fc domain variant or Fc domain dimer variant) has a reduced ability to specifically bind to Fcγ receptors or a reduced ability to induce phagocytosis. In some embodiments, the antibody IgG constant region variant (e.g., Fc domain variant or Fc domain dimer variant) has 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 so that it does not contain effector functions typical of "dead" Fc domain variants. For example, in some embodiments, the Fc domain variant contains specific amino acid substitutions known to minimize the interaction between Fc domain dimers and Fcγ receptors. In some embodiments, the Fc domain variant is derived from an IgG1 antibody and contains 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 for human IgG1 Fc domain variants include E318A and K322A. In some cases, the human IgG1 Fc domain variants have a total of up to 12, 11, 10, 9, 8, 7, 6, 5, or 4 or less 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 of Table 7 is deleted, for example to increase the homogeneity of the polypeptide when the polypeptide is produced in bacteria or mammalian cells. In some cases, the human IgG1 Fc domain variants have a total of up to 12, 11, 10, 9, 8, 7, 6, 5, or 4 or less 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 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

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

[0180] Where the Fc domain variant is an IgG4 Fc domain variant, in some embodiments such an Fc domain variant comprises a S228P mutation (designated according to Kabat et al., (1991)). In some cases, the human IgG4 Fc domain variant has a total of up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations compared to the wild-type human IgG4 sequence. In some embodiments, the Fc domain variant comprises a human IgG4 Fc sequence comprising one or more of the following amino acid substitutions: S228P, E233P, F234V, L235A, and delG236 (designated according to the EU numbering system according to Kabat et al., (1991)). In some embodiments, the Fc domain variant comprises a human IgG4 Fc sequence containing one or more of the following amino acid substitutions: S228P, E233P, F234V, L235A, delG236, and N297A (designated according to the EU numbering system according to 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 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 variants exhibit reduced binding to an Fc receptor of interest compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain variants exhibit ablated binding to an Fc receptor of interest compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain variants exhibit reduced phagocytosis compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain variants exhibit ablated phagocytosis 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 variant of SEQ ID NO: 90-95, as shown in Table 7. TIFF2025003971000020.tif249170TIFF2025003971000021.tif13170

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

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

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

[0187] In some cases, the Fc domain variant or Fc domain dimer variant disclosed herein exhibits reduced phagocytosis compared to its wild-type human IgG Fc region. Such Fc domain variants or Fc domain dimer variants exhibit reduced phagocytosis compared to its wild-type human IgG Fc region, where the reduction in phagocytic activity is, for example, by a factor of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some cases, the Fc domain variant or Fc domain dimer variant exhibits ablation of phagocytosis 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 linked to one or more fusion partners. In some cases, the fusion partner is a therapeutic moiety. In some cases, the fusion partner is selected to allow for targeting, purification, screening, 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 in phagocytosis. As described herein, in some embodiments, when the Fc domain variants or Fc domain dimer variants are linked to a fusion partner, they form the polypeptide constructs described below.

[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 small number of amino acids, such as less than 10 amino acids, although longer linkers are also utilized. In some cases, the linker has a length of 10, 9, 8, 7, 6, or 5 amino acids or less. In some cases, the linker has a length of at least 10, 11, 12, 13, 14, 15, 20, 25, 30, or 35 or more amino acids. Optionally, in some embodiments, a cleavable linker is used.

[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 partners to a desired cellular location or extracellular medium. In some embodiments, certain signaling sequences target proteins to be secreted either to the growth medium or to the periplasmic space located between the inner and outer membranes of the cell. In some embodiments, the fusion partner is a sequence that encodes a peptide or protein that allows for purification or screening. Such fusion partners include, but are not limited to, polyhistidine tags (His tags) (e.g., His6 (SEQ ID NO: 223) and His10 (SEQ ID NO: 224)) or other tags for use with immobilized metal affinity chromatography (IMAC) systems (e.g., Ni+2 affinity columns), GST fusions, MBP fusions, Strep tags, BSP biotinylation target sequence for the bacterial enzyme BirA, and epitope tags targeted by antibodies (e.g., c-myc tags, Flag tags, 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 allows for the use of selection methods to screen for Fc domain variants or Fc domain dimer variants as described herein.

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

[0193] In some embodiments, when the fusion partner is a therapeutic moiety, the therapeutic moiety is, for example, a peptide, a protein, an antibody, an siRNA, or a small molecule. Non-limiting examples of therapeutic antibodies that are attached 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 are attached to the Fc domain variants or Fc domain dimer variants of the present disclosure include, but are not limited to, CD47 binding polypeptides, such as SIRPα polypeptides. In such cases, the CD47 binding polypeptide is attached or fused to the Fc domain variants or Fc domain dimer variants of the present disclosure. Examples of CD47 binding polypeptides include, but are not limited to, anti-CD47 antibodies or fragments thereof, and 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, each Fc domain variant being independently selected from: (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region consisting of mutations S228P, E233P, F234V, L235A, delG236, and N297A. In some embodiments, the Fc domain variants are identical (i.e., homodimers). In some embodiments, the Fc domain variants are different (i.e., heterodimers). In some embodiments, at least one of the Fc domain variants in the Fc domain dimer is a human IgG1 Fc region consisting of the mutations L234A, L235A, G237A, and N297A. In some embodiments, at least one of the Fc domain variants in the Fc domain dimer is a human IgG2 Fc region consisting of the mutations A330S, P331S, and N297A. In some embodiments, the Fc domain dimer variant exhibits ablated or reduced binding to Fcγ receptors compared to the wild type of the human IgG Fc region. In some embodiments, the Fc domain dimer variant exhibits ablated or reduced binding to CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors compared to the wild type of the human IgG Fc region. In some embodiments, the Fc domain dimer variant exhibits ablated or reduced binding to C1q 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 ablated or reduced binding to Fcγ receptors compared to a wild-type human IgG4 Fc region.In some embodiments, the Fc domain dimer variant exhibits ablated or reduced binding to CD16a and CD32b Fcγ receptors compared to the wild type of its human IgG4 Fc region. In some embodiments, the Fc domain dimer variant exhibits ablated or reduced binding to CD16a and CD32b Fcγ receptors compared to the wild type of its human IgG4 Fc region. -6 K exceeds M D It binds to Fcγ receptors.

[0195] In some embodiments, the Fc domain dimer variant further comprises a CD47 binding polypeptide. In some embodiments, the Fc domain dimer variant exhibits ablated or reduced binding to Fcγ receptors compared to the wild type of 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 alpha (SIRP-alpha) polypeptide or a fragment thereof. In some embodiments, the SIRPalpha polypeptide comprises a SIRPalpha D1 domain variant comprising the following amino acid sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMDFSIRIGX8ITPADAGTYYCX9KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 221), where X1 is V or I; X2 is A or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is H or P; X7 is L or T; X8 is any amino acid except N; and X9 is V or I. In some embodiments, the SIRPα polypeptide comprises a SIRPα D1 domain variant, wherein X1 is V or I; X2 is A or I; X3 is I or F; X4 is E; X5 is K or R; X6 is H or P; X7 is L or T; X8 is not N; and X9 is V.

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

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

[0199] In some embodiments, disclosed herein is a polypeptide comprising: (a) a 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 mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236, and N297A. In some embodiments, the Fc domain variant is a human IgG1 Fc region consisting of mutations L234A, L235A, G237A, and N297A. In some embodiments, the Fc domain variant is a human IgG2 Fc region consisting of mutations A330S, P331S, and N297A.

[0200] The SIRPα construct of the present disclosure comprises a SIRPα domain or variant thereof with its C-terminus linked to the N-terminus of the Fc domain or variant thereof via a linker using conventional genetic or chemical means, e.g., chemical conjugation. In some embodiments, a linker (e.g., a spacer) is inserted between the polypeptide and the Fc domain or variant thereof. In some embodiments, a polypeptide of the present disclosure comprising a SIRPα D1 domain variant is fused to an Fc domain variant that cannot form a dimer. In some embodiments, a polypeptide of the present disclosure is fused to an Fc domain or variant thereof that can form a dimer, e.g., a heterodimer, with another Fc domain or variant thereof. In some embodiments, a polypeptide of the present disclosure is fused to an Fc domain or variant thereof, and the fusion protein forms a homodimer. In some embodiments, a polypeptide of the present disclosure is fused to a first Fc domain or variant thereof, and a different protein or peptide (e.g., an antibody variable region) is fused to a second Fc domain or variant thereof. In some embodiments, the SIRPα D1 domain or variant thereof is linked to a first Fc domain or variant thereof and a therapeutic protein (e.g., a cytokine, interleukin, antigen, steroid, anti-inflammatory agent, or immunomodulatory agent) is linked to a second Fc domain or variant thereof. In some embodiments, the first and second Fc domains or variants thereof form a heterodimer.

[0201] Without limiting the foregoing, in some embodiments, a SIRPα D1 domain variant polypeptide (e.g., any of the variants described in Tables 2, 5, and 6) is fused to an Fc polypeptide, or an Fc variant polypeptide, such as an Fc domain or variant thereof. Examples of polypeptides that include SIRPα D1 domain variant polypeptides and fused Fc domain variant polypeptides include, but are not limited to, SEQ ID NOs: 96-137, 214, and 216 shown in Table 8. TIFF2025003971000022.tif249170TIFF2025003971000023.tif248170TIFF2025003971000024.tif249170TIFF2025003971000025.tif249170 TIFF2025003971000026.tif249170TIFF2025003971000027.tif249170TIFF2025003971000028.tif249170TIFF2025003971000029.tif181170

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

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

[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 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 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; X1 is E, G, 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; 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 a wild-type SIRPα D1 domain having a sequence as set forth in any one of SEQ ID NOs: 1-10, and each Fc domain variant independently comprises: (i) a human IgG1 Fc region comprising an N297A mutation; (ii) a human IgG1 Fc region comprising L234A, L235A, and G237A mutations; (iii) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations; (iv) a human IgG2 Fc region comprising an N297A mutation; (v) a human IgG2 Fc region comprising A330S and P331S mutations; (vi) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations. an Fc region; (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, wherein the SIRPα D1 domain variant comprises an amino acid sequence according to SEQ ID NO: 47, and one of the Fc domains is an Fc domain variant comprising a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations.

[0206] Fc domain dimerization In some embodiments, a SIRPα D1 domain variant polypeptide (e.g., any of the variants described in Tables 2, 5, and 6) is fused to a first Fc domain (e.g., an Fc domain variant) at either the N-terminus or C-terminus. In some embodiments, the first Fc domain is a variant that is unable to 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., does not include 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 with a KD of about 500 nM is particularly useful in a dual arm construct. In some embodiments, a SIRPα D1 domain variant with a KD of about 50 nM is particularly useful in a dual arm construct. In some embodiments, SIRPα D1 domain variants with a KD of about 5 nM are useful in dual and single arm constructs. In some embodiments, SIRPα D1 domain variants with a KD of about 500 pM are useful in dual and single arm constructs. In some embodiments, SIRPα D1 domain variants with a KD of about 100 pM are useful in dual and single arm constructs. In some embodiments, SIRPα D1 domain variants with a KD of about 50 pM are useful in dual and single arm constructs. In some embodiments, SIRPα D1 domain variants with a KD of about 10 pM are useful in dual and single arm constructs.

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

[0209] In some embodiments, holes are constructed by replacing amino acids with larger side chains, such as tyrosine or tryptophan, with amino acids with smaller side chains, such as alanine, valine, or threonine, for example, a Y407V mutation in the CH3 antibody constant domain. Similarly, in some embodiments, knobs are constructed by replacing amino acids with smaller side chains with amino acids with larger side chains, for example, a T366W mutation in the CH3 antibody constant domain. In some embodiments, one Fc domain contains the knob mutation T366W and the other Fc domain contains the hole mutations T366S, L358A, and Y407V. In some embodiments, a polypeptide of the present disclosure comprising a SIRPα D1 domain variant is fused to an Fc domain containing the knob mutation T366W to limit undesired homodimer formation between the knobs. Examples of knob-into-hole amino acid pairs are provided, but are not limited to, in Table 9. Examples of knob-into-hole Fc domain variants and SIRPα-Fc fusions are provided in Table 10. TIFF2025003971000030.tif45170TIFF2025003971000031.tif245170TIFF2025003971000032.tif238170

[0210] In addition to the knobs-into-holes strategy, in some embodiments, electrostatic steering is also used to control the dimerization of Fc domains. Electrostatic steering refers to the use of favorable electrostatic interactions between oppositely charged amino acids in peptides, protein domains, and proteins to control the formation of higher-order protein molecules. In particular, to control the dimerization of Fc domains 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 so that the interaction is electrostatically favored or unfavorable depending on the specific charged amino acid introduced. In some embodiments, a positively charged amino acid at the interface, such as lysine, arginine, or histidine, is replaced with a negatively charged amino acid, such as aspartic acid or glutamic acid. In some embodiments, a negatively charged amino acid at the interface is replaced with a positively charged amino acid. In some embodiments, a charged amino acid is introduced into one or both of the interacting CH3 antibody constant domains. In some embodiments, the introduction of charged amino acids into the interacting CH3 antibody constant domains of two Fc domains promotes the selective formation of heterodimers of Fc domains as controlled by electrostatic steering effects resulting from interactions between charged amino acids. Examples of electrostatic steering amino acid pairs are shown in Table 11, but are not limited thereto. TIFF2025003971000033.tif77170

[0211] Other methods are available that can be used to control heterodimerization of Fc domains, particularly in the context of constructing bispecific antibodies.

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

[0213] In some embodiments, the Fc domain has: (a) one of the following amino acid substitutions relative to wild-type human IgG1: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S36 4A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, or K409I; or (b)(i) human IgG1 (ii) L234A, L235A, and G237A mutations for a human IgG1 Fc region; (iii) L234A, L235A, G237A, and N297A mutations for a human IgG1 Fc region; (iv) N297A mutation for a human IgG2 Fc region; (v) A330S and P331S mutations for a human IgG2 Fc region; (vi) A330S, P331S, and N297A mutations for a human IgG2 Fc region; (vii) S228P, E233P, F234V, L235A, and delG236 mutations for a human IgG4 Fc region; or (viii) S228P, E233P, F234V, L235A, delG236, and N297A mutations for a human IgG4 Fc region.In some embodiments, the Fc domain variant comprises: (a) one of the following amino acid substitutions relative to wild-type human IgG1: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S353S, S354D ... (b) (i) human IgG1 (ii) L234A, L235A, and G237A mutations for a human IgG1 Fc region; (iii) L234A, L235A, G237A, and N297A mutations for a human IgG1 Fc region; (iv) N297A mutation for a human IgG2 Fc region; (v) A330S and P331S mutations for a human IgG2 Fc region; (vi) A330S, P331S, and N297A mutations for a human IgG2 Fc region; (vii) S228P, E233P, F234V, L235A, and delG236 mutations for a human IgG4 Fc region; or (viii) S228P, E233P, F234V, L235A, delG236, and N297A mutations for a 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 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 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 are polypeptides comprising an Fc variant, wherein the Fc variant comprises an Fc domain dimer comprising two Fc domain variants, each Fc domain variant independently selected from: (i) a human IgG1 Fc region consisting of 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 consisting of the mutations S228P, E233P, F234V, L235A, delG236, and N297A.

[0217] In the present disclosure, linker is used to describe a link or connection between a polypeptide or protein domain or related non-protein moiety. In some embodiments, the linker is a link or connection between an Fc domain (or 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 the two polypeptides are linked to each other in a tandem series.

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

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

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

[0221] Spacer In the present disclosure, in some embodiments, 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 1-200 amino acids. Suitable peptide spacers include peptide linkers that include flexible amino acid residues, such as glycine and serine. Examples of linker sequences are shown in Table 12. In some embodiments, the spacer includes a GS, GG, GGS, GGG, GGGGS (SEQ ID NO: 163), GGSG (SEQ ID NO: 164), or SGGG (SEQ ID NO: 165) motif, such as multiple or repeated motifs. In some embodiments, the spacer includes a GS motif, such as 2-12 amino acids, such as GS, GSGS (SEQ ID NO: 166), GSGSGS (SEQ ID NO: 167), GSGSGSGS (SEQ ID NO: 168), GSGSGSGSGS (SEQ ID NO: 169), or GSGSGSGSGSGS (SEQ ID NO: 170). In some embodiments, the spacer comprises 3-12 amino acids including a GGS motif, e.g., GGS, GGSGGS (SEQ ID NO: 171), GGSGGSGGS (SEQ ID NO: 172), and GGSGGSGGSGGS (SEQ ID NO: 173). In some embodiments, the spacer comprises 4-12 amino acids including a GGSG motif (SEQ ID NO: 164), e.g., GGSG (SEQ ID NO: 164), GGSGGGSG (SEQ ID NO: 174), or GGSGGGSGGGSG (SEQ ID NO: 175). In some embodiments, the spacer comprises a GGGGS (SEQ ID NO: 163) motif, e.g., GGGGSGGGGSGGGGGS (SEQ ID NO: 176).In some embodiments, the spacer includes amino acids other than glycine and serine, e.g., AAS (SEQ ID NO: 177), AAAL (SEQ ID NO: 178), AAAK (SEQ ID NO: 179), AAR (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, e.g., multiple or repeated motifs, of EAAAK (SEQ ID NO: 193). In some embodiments, the spacer includes a motif, e.g., multiple or repeated motifs of proline-rich sequences, such as (XP)n, where X is any amino acid (e.g., A, K, or E) and n is from 1 to 5, and PAPAP (SEQ ID NO: 194). TIFF2025003971000034.tif192170

[0223] In some embodiments, the length of the peptide spacer and the amino acids used are adjusted depending on the two proteins involved and the degree of flexibility desired for the final protein fusion polypeptide. In some embodiments, the length of the spacer is adjusted to ensure proper protein folding and avoid the formation of aggregates. 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 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, in some embodiments, is a polypeptide comprising an Fc variant, wherein the Fc variant comprises an Fc domain dimer having two Fc domain monomers, each Fc domain monomer independently selected from: (i) a human IgG1 Fc region consisting of 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.

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

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

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

[0229] In some embodiments, the nucleic acid encoding the polypeptide construct, for example, a polypeptide construct including a fusion partner such as a SIRPα D1 domain variant (e.g., any of the variants shown in Tables 2, 5, and 6) and an Fc variant, is incorporated into an expression vector to express the protein. A variety of expression vectors are available for protein expression. Expression vectors can include self-replicating extrachromosomal vectors or vectors that integrate into a host genome. A vector can also include a variety of 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, transcriptional start and stop sequences, translational start and stop sequences, 3' and 5' untranslated regions (UTRs), and enhancer or activator sequences; an origin of replication; a selectable marker gene; and a nucleic acid sequence encoding a polypeptide of interest, and a transcription termination sequence. In some embodiments, the expression vector includes a protein operably linked to a control or regulatory sequence, a selectable marker, any fusion partner, additional elements, or any combination thereof. The term "operably linked" means that a nucleic acid is placed in a functional relationship with another nucleic acid sequence. Generally, these expression vectors contain transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the Fc variant, and are typically appropriate for the host cell used to express the protein. A selection gene or marker, such as, but not limited to, an antibiotic resistance gene or a fluorescent protein gene, can be used to select host cells containing the expression vector, for example, by antibiotic or fluorescent expression. A variety of selection genes are available.

[0230] In some embodiments, vector components or elements are optimized so that the expression vector is compatible with the host cell type. Expression vectors that find use in the present disclosure include, but are not limited to, those that allow for protein expression in mammalian cells, bacteria, insect cells, yeast, and in vitro systems.

[0231] In some embodiments, mammalian cells are used as host cells to produce 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 to produce 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 correct modification and processing of the expressed polypeptide. Once the vector is introduced into the host cell for protein production, the host cell is cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0233] In some embodiments, the polypeptide construct, for example, a polypeptide construct comprising a fusion partner such as a SIRPα D1 domain variant (e.g., any of the variants provided in Tables 2, 5, and 6) and an Fc variant, is expressed in a mammalian expression system, such as a system in which the expression construct is introduced into a mammalian cell using a virus such as a retrovirus or an adenovirus. In some embodiments, human, mouse, rat, hamster, or primate cells are utilized. Suitable cells also include known research cells, such as, but not limited to, Jurkat T cells, NIH3T3, CHO, COS, and 293 cells. Alternatively, in some embodiments, the protein is expressed in bacterial cells. Bacterial expression systems are well known in the art and include Escherichia coli (E. coli), Bacillus subtilis, Streptococcus cremoris, and Streptococcus lividans. In some cases, the polypeptide constructs comprising the Fc domain variants are produced in insect cells, such as, but not limited to, Sf9 and Sf21 cells, or yeast cells, such as organisms of the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, and Yarrowia. In some cases, the polypeptide constructs comprising the Fc domain variants are 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 appreciated by those skilled in the art, in vitro translation is required for some display technologies, such as ribosome display. Additionally, in some embodiments, the Fc domain variants are produced by chemical synthesis methods, such as, but not limited to, solution phase peptide synthesis and solid phase peptide synthesis.In vitro transcription using a non-glycosylated system such as bacterial extracts results in an equally inactivated Fc, since the Fc is not glycosylated even though native glycosylation sites are present.

[0234] In some embodiments, the polypeptide construct comprises unnatural amino acids, amino acid analogs, amino acid mimetics, or any combination thereof that function similarly to naturally occurring amino acids. Naturally encoded amino acids generally refer to the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, e.g., carbons attached to hydrogen, carboxyl groups, amino groups, and R groups, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. In some embodiments, such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but generally retain the same basic chemical structure as naturally occurring amino acids.

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

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

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

[0238] Cancer Treatment Methods Provided herein are methods of treating cancer in an individual (e.g., a human individual), the methods comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein); and (b) a 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., exhibited disease progression) during (or after) prior treatment for lung cancer (e.g., NSCLC). In some embodiments, the prior treatment was 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, a polypeptide (eg, a fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat. In some embodiments, a polypeptide (eg, a fusion polypeptide) administered to an 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 L234A, L235A, G237A, and N297A mutations, numbering according to EU index of Kabat. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide (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 its label instructions. In some embodiments, pembrolizumab is administered to an individual (e.g., via IV infusion) at a dose of about 200 mg every three weeks (Q3W). 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 individual's package insert. Complete information regarding the preparation, formulation, dosage, and administration schedule of pembrolizumab may be found in the local package insert (in the United States, see, e.g., www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2016 / 125514s012lbl(dot)pdf; in Europe, see, e.g., www(dot)ema(dot)europa(dot)eu / en / documents / product-information / keytruda-epar-product-information_en(dot)pdf). In some embodiments, the polypeptide (e.g., fusion polypeptide) is administered to an individual (e.g., via intravenous infusion) weekly (i.e., once every 7 days or "qw"), e.g., at a dose of 10.0 mg / kg.

[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 has not shown disease progression within 8 weeks of initiation 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 of more than 1%. In some embodiments, the individual has locally advanced or metastatic NSCLC with a TPS score of less than 50%. In some embodiments, the individual has progressed after systemic therapy for their metastatic disease. In some embodiments, the individual has locally advanced or metastatic NSCLC with a TPS score of more than 1% and the individual has progressed during prior checkpoint inhibitor (CPI) therapy for the NSCLC.

[0242] In some embodiments, the prior CPI therapy for lung cancer (e.g., NSCLC) in the individual with advanced disease was therapy that included treatment with nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, 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 tumor is evaluated using, for example, Response Evaluation In Solid Tumor (RECIST) criteria (e.g., version 1.0 or version 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 Hwang et al. (2017) "Response Evaluation of Chemotherapy for Lung Cancer" Tuberc Respir Dis (Seoul). 80(2): 136-142, and references cited therein. An individual is considered to have progressed during prior CPI therapy for lung cancer (e.g., NSCLC) if the individual exhibits progressive disease (PD) as determined by any set of response criteria described therein. In some embodiments, the individual is resistant to a standard therapy (e.g., curative therapy) for lung cancer (e.g., NSCLC).In some embodiments, there is no standard therapy (eg, curative therapy) available to treat lung cancer (eg, NSCLC).

[0243] In some embodiments, the individual's PD-L1 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 PD-L1 IHC 22C3 Pharm Dx. PD-L1 IHC 22C3 Pharm Dx is a qualitative immunohistochemistry 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., patient) using a DAKO™ AUTOSTAINER LINK 48 automated staining system with an ENVISION™ 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., patient). In some embodiments, TPS is the percentage of viable tumor cells that exhibit partial or complete membrane staining at any intensity. If viable tumor cells with a TPS of 50% or more exhibit membrane staining at any intensity, the specimen shall be considered PD-L1 positive. In some embodiments, tumor-associated immune cells (such as infiltrating lymphocytes or macrophages) are not included in the scoring to determine TPS. In some embodiments, labeling of lung cancer (e.g., NSCLC) samples is performed by a pathologist. In some embodiments, staining is assessed via light microscopy at 10x to 40x magnification. Further details regarding the PD-L1 IHC 22C3 Pharm Dx assay, reagents and equipment for performing the assay, interpretation of the assay results, and TPS scoring are provided at www(dot)accessdata(dot)fda(dot)gov / cdrh_docs / pdf15 / p150013b(dot)pdf and in 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 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., exhibited 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., fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat. In some embodiments, a polypeptide (e.g., a fusion polypeptide) administered to an 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, a polypeptide (e.g., a fusion polypeptide) administered to an individual comprises an Fc domain variant that is a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, numbering according to EU index of Kabat.In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide (e.g., a fusion polypeptide) administered to the individual comprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., a fusion polypeptide) forms a homodimer. In some embodiments, the 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 provided elsewhere herein.

[0245] In some embodiments, pembrolizumab is administered subcutaneously. In some embodiments, pembrolizumab is administered by intravenous infusion. In some embodiments, pembrolizumab is administered according to its label instructions. In some embodiments, pembrolizumab is administered to an individual (e.g., via IV infusion) at a dose of about 200 mg every three weeks (Q3W). 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 individual's package insert. Complete information regarding the preparation, formulation, dosage, and administration schedule of pembrolizumab may be found in the local package insert (in the United States, see, e.g., www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2016 / 125514s012lbl(dot)pdf; in Europe, see, e.g., www(dot)ema(dot)europa(dot)eu / en / documents / product-information / keytruda-epar-product-information_en(dot)pdf). In some embodiments, the polypeptide (e.g., fusion polypeptide) is administered to an individual (e.g., via intravenous infusion) weekly (i.e., once every 7 days or "qw"), e.g., at a dose of 10.0 mg / kg.

[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 or metastatic HNSCC and has not shown disease progression within 8 weeks of initiating prior treatment with a 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 is or included treatment with nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, 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 a CPI (e.g., the individual is "immune checkpoint inhibitor naive" or "CPI naive").

[0247] In some embodiments, the prior platinum-containing therapy during or after which the individual progressed was a therapy that included treatment with carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and / or satraplatin. In some embodiments, the tumor is evaluated using, for example, Response Evaluation In Solid Tumor (RECIST) criteria (e.g., version 1.0 or version 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 An individual is considered to have progressed on or after prior platinum-containing therapy for head and neck cancer (e.g., HNSCC) if the individual exhibits progressive disease (PD) as determined by the set of response criteria described in "Primary Phase 3-D Plasma Carcinoma: FDG PET Hopkins Criteria Versus Residual Neck Node Size and Morphologic Features" Am J. Roentgenology. 207:641-647. 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 (eg, a curative therapy) available to treat head and neck cancer (eg, HNSCC).

[0248] Stomach / gastroesophageal (GEJ) cancer In some embodiments, a method of treating gastric / gastroesophageal (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., exhibited disease progression) during or after a prior therapy for gastric / GEJ cancer (e.g., gastric / GEJ adenocarcinoma), and the prior therapy was an anti-HER2 antibody therapy and / or a fluoropyrimidine-based therapy. In some embodiments, the polypeptide (e.g., fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat. In some embodiments, a polypeptide (e.g., a fusion polypeptide) administered to an 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, a polypeptide (e.g., a fusion polypeptide) administered to an individual comprises an Fc domain variant that is a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, numbering according to EU index of Kabat.In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide (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-HER2 antibody administered to the 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 its label instructions. In some embodiments, trastuzumab is administered to an individual (e.g., via intravenous infusion) every three weeks (Q3W). In some embodiments, the first (i.e., first) dose of trastuzumab is about 8 mg / kg, and all subsequent doses (i.e., after the first dose) are about 6 mg / kg. In some embodiments, dose modification of trastuzumab is performed according to the package insert of each country. Complete information regarding the preparation, formulation, dosage, and administration schedule of trastuzumab may be found in the respective country's package insert (in the United States, see, e.g., www.accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2017 / 103792s5337lbl(dot) pdf; in Europe, see, e.g., www(dot)ema(dot)Europa(dot)eu / en / documents / product-information / herceptin-epar-product-information_en(dot)pdf). In some embodiments, the polypeptide (e.g., fusion polypeptide) is administered to an individual (e.g., via intravenous infusion) weekly (i.e., once every 7 days or "qw"), e.g., at a dose of 10.0 mg / kg.

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

[0251] In some embodiments, the individual has progressed (e.g., has shown 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 has progressed (e.g., has shown 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 folinic acid, 5-fluorouracil, and oxaliplatin). In some embodiments, the individual has 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 lines of treatment).

[0252] In some embodiments, the tumor is evaluated using, for example, Response Evaluation In Solid Tumor (RECIST) criteria (e.g., version 1.0 or version 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 the criteria set forth in Kurokawa et al. (2013) Ann Surg. An 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) if the individual exhibits progressive disease (PD) as assessed by any of the suite of response measures described in 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. 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 (eg, curative therapy) available to treat gastric / GEJ cancer (eg, HER2-positive gastric / HER2-positive GEJ cancer).

[0253] Lymphoma (i) Aggressive non-Hodgkin's lymphoma In some embodiments, a method of treating aggressive non-Hodgkin's 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) 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-CD20 antibody, wherein the aggressive NHL is relapsed and / or refractory aggressive NHL (e.g., the individual has relapsed during or after a prior treatment for aggressive NHL and / or has been refractory to a prior treatment for aggressive NHL), wherein the aggressive NHL (e.g., de novo DLBCL or transformed DLBCL or mantle cell lymphoma (MCL)) is There is no treatment (e.g., curative treatment) available for DLBCL, such as DLBCL, transformed DLBCL, or mantle cell lymphoma. In some embodiments, the polypeptide (e.g., 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 selected from the group consisting of: (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. Fc region (wherein numbering is according to EU index of Kabat); or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) containing the S228P, E233P, F234V, L235A, delG236, and N297A mutations.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 L234A, L235A, G237A, and N297A mutations, numbering according to the EU index of Kabat. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide (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 its label instructions. In some embodiments, rituximab is administered at a dose of about 375 mg / m 2 In some embodiments, the first four doses of rituximab (i.e., doses 1-4) are administered to an individual (e.g., at a dose of about 375 mg / m) once a week (e.g., once every 7 days, or "qw") for the first four weeks (e.g., 28 days) of treatment. 2In some embodiments, the first four doses of rituximab (i.e., doses 1-4) are administered to an individual once a week (e.g., once every 7 days, or "qw") for the first four weeks (e.g., 28 days) of treatment (e.g., a dose of about 375 mg / m 2 The first dose is administered once every 28 days (at 10:00 am) and the next four doses (i.e., doses 5-8) are administered once every 4 weeks (e.g., every 28 days or "q4w") to the individual. In some embodiments, dose modifications of rituximab are made according to the national package insert. Complete information regarding the preparation, dispensing, dosage, and administration schedule of rituximab may be found in the national package insert (in the United States, see, e.g., www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2012 / 103705s5367s5388lbl(dot)pdf; in Europe, see, e.g., www(dot)ema(dot)europa(dot)eu / en / documents / product-information / mabthera-epar-product-information_en(dot)pdf). In some embodiments, a polypeptide (e.g., a fusion polypeptide) is administered to an individual (e.g., via intravenous infusion) weekly (i.e., once every 7 days or "qw"), for example, at a dose of 10.0 mg / kg or 15.0 mg / kg.

[0255] In some embodiments, an individual is diagnosed with de novo DLBCL (e.g., de novo relapsed and / or refractory DLBCL) if the individual has no history of lymphoma. In some embodiments, an individual is diagnosed with transformed DLBCL if the individual has a history of lymphoma, such as, for example, indolent lymphoma, for example, marginal zone lymphoma, lymphoplasmacytic lymphoma, small lymphocytic lymphoma / chronic lymphocytic leukemia, follicular lymphoma, or lymphocyte-predominant Hodgkin's lymphoma. In some embodiments, an individual is diagnosed with mantle cell lymphoma (MCL) if the individual is found to have one or more of the following chromosomal abnormalities: t(11;14), t(14;18). In some embodiments, an individual is diagnosed with mantle cell lymphoma (MCL) if overexpression of SOX11 is detected in a sample of the individual's leukemia cells. Other criteria for diagnosing de novo DLBCL, transformed DLBCL, and MCL are known in the art and are routinely used by those skilled in the art (see, e.g., 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 is considered to have relapsed after prior treatment for aggressive NHL (e.g., de novo DLBCL, transformed DLBCL, or MCL) if the individual achieved the lowest stable (SD) therapeutic response to the prior treatment, but stopped responding (e.g., exhibited disease progression) within any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after cessation of the prior treatment. In some embodiments, an individual is considered to be refractory to prior treatment for aggressive NHL if the individual did not respond to the prior treatment (e.g., failed to achieve at least a stable (SD) therapeutic response during or after the prior treatment). In some embodiments, therapeutic response to 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 has been refractory to or has relapsed from at least one prior therapy for aggressive NHL (e.g., at least one prior approved standard of care, at least two prior approved standard of care, at least three approved standard of care, etc.). Standard of care therapies for DLBCL (e.g., de novo or transformed DLBCL) and MCL include, but are not limited to, 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); 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 rivaroxaban); vincristine, 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 in combination with rituximab); IC E (i.e., ifosfamide, carboplatin, and etoposide); RICE (i.e., ICE in combination with rituximab); DICE (i.e., ICE in combination with dexamethasone); DICE and mesna; BEAM (i.e., carmustine, etoposide, cytarabine, and melphalan); R-BEAM (i.e., BEAM in combination with rituximab); ESHAP (i.e., etoposide, solumedrol, high-dose cytarabine, and cisplatin);R-ESHAP (i.e., ESHAP in combination with rituximab); MIME (i.e., methylglyoxal bis(guanylhydrazone), ifosfamide, methotrexate, and etoposide); palsaclisib (also known as INCB050465); MATRIX (i.e., methotrexate, cytarabine, thiotepa, and rituximab); HyperCVAD or HCVAD (i.e., hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone) RHCVAD (i.e., HCVAD in combination with rituximab); RHCVAD / MA (i.e., RHCVAD alternating with methotrexate and cytarabine); DHAP (i.e., dexamethasone, cisplatin, and cytarabine); R-DHAP (i.e., DHAP in combination with rituximab); Ibrutinib (i.e., rituximab); nibs; rituximab, obinutuzumab, CVAD (i.e., cyclophosphamide, doxorubicin, vincristine, and prednisolone); RCVAD (i.e., CVAD in combination with rituximab); GemOx (i.e., gemcitabine and oxaliplatin); R-GemOx (i.e., GemOx in combination with rituximab); DHAX (i.e., dexamethasone, cytarabine, and oxaliplatin); R-DHAX (i.e., DHAX in combination with rituximab); GIFOX (i.e., gemcitabine, ifosfamide, and oxaliplatin); RGIFOX (i.e., GIFOX in combination with rituximab); GIFOX with bortezomib; ASCT (i.e., autologous stem cell transplant) HD-ASCT (i.e., ASCT in combination with high-dose therapy, e.g., high-dose chemotherapy); CAR T-cell therapy (e.g., tisagenlecleucel or axicabtagene);In some embodiments, the prior treatment for aggressive NHL included any two or more of the preceding therapies (given together in a single treatment regimen or given in separate treatment regimens). In some embodiments, there are no treatment options (e.g., curative therapy options) available for individuals with 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 no curative therapy is available. In some embodiments, the individual has DLBCL (e.g., de novo DLBCL or transformed DLBCL) or MCL that has relapsed or been refractory after standard approved therapy (e.g., curative therapy).

[0258] (ii) Indolent lymphoma In some embodiments, a method of treating indolent lymphoma in an individual (e.g., a human individual) is provided, the method comprising administering to an individual an effective amount of (a) a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant as described herein), The method includes administering to the individual (e.g., a polypeptide (e.g., a fusion polypeptide) comprising an Fc domain variant (e.g., an Fc domain variant described herein), and (b) a therapeutic anti-CD20 antibody, wherein the indolent lymphoma is relapsed and / or is refractory indolent lymphoma (e.g., the individual has relapsed during or after at least one prior treatment, e.g., an approved standard of care, for the indolent lymphoma, and / or is refractory to at least one prior treatment, e.g., an approved standard of care, for the indolent lymphoma). In some embodiments, the individual has relapsed during or after two or more approved standards of care (e.g., two, three, or more standards of care) for the indolent lymphoma, and / or is refractory to two or more standards of care (e.g., two, three, or more standards of care, e.g., curative therapies) for the indolent lymphoma. In some embodiments, the polypeptide (e.g., fusion polypeptide) comprises a SIRPα polypeptide comprising the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat.In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to the individual comprises 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 L234A, L235A, G237A, and N297A mutations, numbering according to EU index of Kabat. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide (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 its label instructions. In some embodiments, rituximab is administered at a dose of about 375 mg / m 2 In some embodiments, the first four doses of rituximab (i.e., doses 1-4) are administered to an individual (e.g., at a dose of about 375 mg / m) once a week (e.g., once every 7 days, or "qw") for the first four weeks (e.g., 28 days) of treatment. 2 In some embodiments, the first four doses of rituximab (i.e., doses 1-4) are administered to an individual once a week (e.g., once every 7 days, or "qw") for the first four weeks (e.g., 28 days) of treatment (e.g., a dose of about 375 mg / m 2The first dose is administered once every 28 days (at 10:00 am) and the next four doses (i.e., doses 5-8) are administered once every 4 weeks (e.g., every 28 days or "q4w") to the individual. In some embodiments, dose modifications of rituximab are made according to the national package insert. Complete information regarding the preparation, dispensing, dosage, and administration schedule of rituximab may be found in the national package insert (in the United States, see, e.g., www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2012 / 103705s5367s5388lbl(dot)pdf; in Europe, see, e.g., www(dot)ema(dot)europa(dot)eu / en / documents / product-information / mabthera-epar-product-information_en(dot)pdf). In some embodiments, a polypeptide (e.g., a fusion polypeptide) is administered to an individual (e.g., via intravenous infusion) weekly (i.e., once every 7 days or "qw"), for example, at a dose of 10.0 mg / kg or 15.0 mg / kg.

[0260] In some embodiments, the indolent lymphoma is an indolent non-Hodgkin's lymphoma (NHL). In some embodiments, the indolent NHL is a marginal zone lymphoma (MZL). In some embodiments, the NHL is a 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 others.

[0261] In some embodiments, an individual is considered to have relapsed after prior treatment for indolent lymphoma (e.g., indolent NHL) if the individual achieved the lowest stable (SD) therapeutic response to the prior treatment, but stopped responding (e.g., exhibited disease progression) within any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the cessation of the prior treatment. In some embodiments, an individual is considered to be refractory to prior treatment for indolent lymphoma (e.g., indolent NHL) if the individual did not respond to the prior treatment (e.g., failed to achieve at least a stable (SD) therapeutic response during or after the prior treatment). In some embodiments, therapeutic response to 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 has been refractory to or has relapsed from at least one prior therapy (e.g., at least one prior approved standard therapy, at least two prior approved standard therapy, at least three approved standard therapy, etc.) for indolent lymphoma (e.g., indolent NHL, such as marginal zone lymphoma or follicular lymphoma). Standard therapies for indolent lymphoma (e.g., indolent NHL, such as marginal zone lymphoma or follicular lymphoma) include, but are not limited to, standard therapies for aggressive NHL (e.g., DLBCL or mantle cell lymphoma). Details of these are provided elsewhere herein. Other standard therapies 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 in combination with cyclophosphamide); FCM (i.e., fludarabine, cyclophosphamide, mitoxantrone); FCMR (i.e., FCM in combination with rituximab); ibritumomab tiuxetan; tositumomab; vorinostat; everolimus; bortezomib; navitoclax (also known as ABT-263); high-dose therapy (HDT); autologous stem cell transplant; and allogeneic stem cell transplant. In some embodiments, prior treatment of indolent lymphoma (e.g., indolent NHL) includes any two or more of the preceding standard therapies (such as treatment of aggressive NHL described elsewhere herein). In some embodiments, two or more standard therapies (such as standard therapies for aggressive NHL) for indolent lymphoma (e.g., indolent NHL) are given together in a single treatment regimen. In some embodiments, two or more standard therapies (such as standard therapies for aggressive NHL) for indolent NHL are given in separate treatment regimens. In some embodiments, there are no treatment options (e.g., curative treatment options) available for individuals with indolent lymphoma (e.g., indolent NHL) (e.g., relapsed / refractory indolent NHL).

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

[0264] In some embodiments of any of the methods of treatment 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 (e.g., via intravenous infusion) 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). In some embodiments, the polypeptide (e.g., a fusion polypeptide) is administered (e.g., via intravenous infusion) weekly (i.e., once every 7 days or "qw") 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). In some embodiments, the polypeptide (e.g., fusion polypeptide) is administered (e.g., via intravenous infusion) 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"). In some embodiments, the polypeptide (e.g., 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 administered sequentially on days when the administration schedules of the polypeptide (e.g., fusion polypeptide) and the therapeutic antibody coincide. In some embodiments, the polypeptide (e.g., fusion polypeptide) is administered before (e.g., about 30 minutes before) the therapeutic antibody. In some embodiments, if administration of the polypeptide is not performed, the therapeutic antibody is administered about 24 hours after administration of the polypeptide.

[0265] In some embodiments, the therapeutic response of an individual having NSCLC, HNSCC, gastric cancer or GEJ cancer to the treatment methods provided herein is assessed by immune-related response criteria derived from RECIST version 1.1 (irRECIST), 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 (e.g., adapted from 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 therapeutic response of an individual with an aggressive lymphoma (e.g., aggressive NHL, such as DLBCL or MCL) to the therapeutic methods provided herein is determined, for example, 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. In some embodiments, the therapeutic response of an individual with an indolent lymphoma (e.g., indolent NHL, such as FL or MZL) to the therapeutic methods provided herein is determined according to the Lugano criteria (see Cheson et al, 2014).

[0266] In some embodiments, individuals being treated for HNSCC, NSCLC, gastric cancer, or GEJ cancer have 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, individuals being treated for lymphoma (e.g., aggressive lymphoma (such as DLBCL or MCL) or indolent lymphoma (such as FL or MZL) have at least one measurable lesion as defined by the 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, individuals treated according to the methods herein have adequate bone marrow, renal, hepatic, and cardiac function. In some embodiments, individuals have an Eastern Cooperative Oncology Group (ECOG) performance status (PS) score of 0 or 1 (see, e.g., www(dot)npcrc(dot)org / files / news / ECOG)_performance_status(dot)pdf). In some embodiments, individuals do not have symptomatic central nervous system (CNS) metastases or leptomeningeal disease requiring steroids. In some embodiments, individuals treated according to the methods herein for lung cancer (e.g., NSCLC) do not have ALK or EGFR genomic tumor aberrations. In some embodiments, individuals treated according to the methods herein do not have a history of (non-infectious) pneumonia requiring steroids or currently have pneumonia. In some embodiments, an individual being treated according to the methods herein does not have high-grade lymphoma (e.g., Burkitt's lymphoma, lymphoblastic lymphoma, or Richter's 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 being treated according to the methods herein for lung cancer (e.g., NSCLC), head and neck cancer (e.g., HNSCC), or gastric / GEJ cancer (e.g., HER2-positive gastric / GEJ adenocarcinoma) has not received prior irradiation to greater than 25% of the bone marrow. In some embodiments, the individual has not received radiation therapy within 2 weeks of initiating 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 4 weeks of initiating treatment (6 weeks for mitomycin C or nitrosoureas). In some embodiments, the individual does not have an intolerance or has suffered a severe allergic or anaphylactic reaction to the antibody or the infused therapeutic protein(s) or any of the excipients in the formulation(s) comprising the therapeutic protein.In some embodiments, the individual has not discontinued treatment due to a grade 3 or higher immune-related adverse event (AE) 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 (such as, but not limited to, measles, mumps, rubella, varicella zoster, yellow fever, rabies, Bacillus Calmette-Guerin (BCG), typhoid, and intranasal influenza vaccines). In some embodiments, the individual has not received a primary diagnosis (e.g., lung cancer (NSCLC), head and neck cancer (HNSCC), gastric / GEJ cancer (HER2-positive gastric / GEJ adenocarcinoma), aggressive lymphoma (de novo The individual is not currently receiving active treatment for 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 transfusion of blood products within 14 days of starting treatment. In some embodiments, the individual has no history of active autoimmune disorders (e.g., but not limited to, Crohn's disease, rheumatoid arthritis, scleroderma, systemic lupus erythematosus, Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia, etc.) and other conditions that compromise or impair the immune system (except for hypogammaglobulinemia). In some embodiments, the individual does not have an active, uncontrolled, clinically significant bacterial, fungal, or viral infection, such as Hepatitis B (HBV), Hepatitis C (HCV), known human immunodeficiency virus (HIV), or acquired immune deficiency syndrome (AIDS)-related illness. In some embodiments, the individual does not have active graft-versus-host disease (GVHD) or is not receiving immunosuppressive therapy for GVHD. In some embodiments, the individual does not have any of the following in 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.In some embodiments, the individual has not been diagnosed with any other malignancy within the past three years prior to initiating treatment, except for, for example, adequately treated non-melanoma skin cancer, carcinoma in situ (e.g., breast cancer, cervical intraepithelial neoplasia) that has undergone potentially curative therapy. Kits and manufactured products

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

[0269] In some embodiments, the kit or article of manufacture further comprises an anti-PD1 antibody. In some embodiments, the anti-PD1 antibody is pembrolizumab. In some embodiments, the kit comprises a package insert or label comprising instructions for using the 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 a package insert or label comprising instructions for using the 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 been previously treated for NSCLC according to the methods herein. In some embodiments, the individual has progressed (e.g., exhibited disease progression) during (or after) a prior treatment (e.g., prior immune checkpoint inhibitor therapy) for lung cancer. In some embodiments, the individual has a PD-L1 tumor proportion score (TPS) of less than 50%. In some embodiments, the kit includes a package insert or label containing instructions for using the polypeptide (e.g., 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 includes a package insert or label containing instructions for using the polypeptide (e.g., fusion polypeptide) in combination with an anti-PD1 antibody (e.g., pembrolizumab) to treat or delay the progression of HNSCC in an individual who has received prior immune checkpoint inhibitor therapy (e.g., for HNSCC). In some embodiments, the kit includes a package insert or label containing instructions for using the polypeptide (e.g., fusion polypeptide) in combination with an anti-PD1 antibody (e.g., pembrolizumab) to treat or delay 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 includes a package insert or label including instructions for using the polypeptide (e.g., fusion polypeptide) in combination with an anti-PD1 antibody (e.g., pembrolizumab) to treat or delay progression of head and neck cancer (e.g., HNSCC, metastatic HNSCC, etc.) in individuals who have progressed (e.g., shown disease progression) during (or after) prior platinum therapy (e.g., platinum-containing therapy). In some embodiments, the kit or article of manufacture further includes instructions for administering pembrolizumab at a dose of 200 mg every three weeks (Q3W) by IV infusion. In some embodiments, the kit or article of manufacture further includes instructions for administering the polypeptide (e.g., fusion polypeptide) at a dose of 10 mg / kg weekly (QW) by intravenous infusion.

[0270] In some embodiments, the kit or article of manufacture further comprises an anti-HER2 antibody. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the kit comprises a package insert or label comprising instructions for using the polypeptide (e.g., 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 a package insert or label comprising instructions for using the polypeptide (e.g., 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., exhibited disease progression) during (or after) a prior treatment for gastric cancer or GEJ cancer according to the methods provided herein. In some embodiments, the prior therapy included an anti-HER2 antibody and / or a prior fluoropyrimidine-based therapy. In some embodiments, the kit or article of manufacture further includes instructions for administering trastuzumab via intravenous infusion once every three weeks (q3W), where 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 article of manufacture further includes instructions for administering the polypeptide (e.g., fusion polypeptide) at a weekly (QW) dose of 10 mg / kg by intravenous infusion.

[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 comprises a package insert or label containing instructions for using the 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's lymphoma or "NHL" (e.g., de novo or transformed DLBCL, or mantle cell lymphoma) in an individual according to the methods herein. In some embodiments, the kit comprises a package insert or label containing instructions for using the 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's 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 prior therapies (e.g., prior standard / curative therapies) for aggressive NHL, or in an individual for whom there is no available treatment (e.g., curative therapy) for aggressive NHL, in accordance with the methods herein. In some embodiments, the kit comprises a package insert or label containing instructions for using the 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., an indolent NHL such as marginal zone lymphoma or follicular lymphoma) in an individual, in accordance with the methods herein. In some embodiments, the kit comprises a package insert or label containing instructions for using the polypeptide (e.g., a fusion polypeptide) in combination with an anti-CD20 antibody (e.g., rituximab) to treat or delay the progression of an indolent lymphoma (e.g., an 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., a curative therapy) for the indolent lymphoma (e.g., an indolent NHL) or in an individual who has no available treatment (e.g., a curative therapy) for the indolent lymphoma (e.g., an indolent NHL).In some embodiments, the kit or article of manufacture provides rituximab at a dose of 375 mg / m for the first 4 weeks (e.g., 28 days) of treatment. 2 and administering rituximab to the individual once a week (e.g., once every 7 days or "qw") at a dose of 375 mg / m for up to four additional doses after the first four doses, or for up to eight additional doses after the first four doses. 2 In some embodiments, the kit or article of manufacture further comprises instructions for administering the polypeptide (e.g., fusion polypeptide) at a weekly (QW) dose of 10 mg / kg or 15 mg / kg by intravenous infusion.

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

[0273] The present specification is deemed sufficient to enable one skilled in the art to practice the invention. In addition to those 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 study of Drug A in combination with an established anti-cancer antibody in patients with advanced malignancies. This example describes a phase 1 clinical trial evaluating the safety, efficacy, pharmacodynamics (PD) and pharmacokinetics (PK) of Drug A in combination with pembrolizumab, trastuzumab or rituximab in patients with advanced 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] Test Purpose Main Objective The primary objective of this study was to evaluate the safety and tolerability of Drug A administered weekly and / or every 2 weeks in combination with pembrolizumab, trastuzumab, or rituximab in patients with advanced malignancies, including non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), HER2-overexpressing gastric cancer, and non-Hodgkin lymphoma (NHL).

[0276] Secondary Objectives The secondary objectives of this study are to: To determine the antitumor efficacy of drug A in combination with anticancer therapeutic agents in patients with advanced malignancies. To determine the overall safety profile of Drug A in combination with other anti-cancer therapeutics. To characterize the maximum tolerated dose (MTD) / optimal biological dose (OBD) of drug A in combination with anticancer therapeutics. To characterize the pharmacokinetics of single and multiple doses of drug A in combination with the anticancer therapeutics pembrolizumab, trastuzumab, or rituximab. To evaluate the immunogenicity of drug A. exploratory purpose

[0277] The exploratory objective of this study was to investigate the pharmacodynamic effects of drug A in combination with anticancer therapeutic agents in patients with advanced malignancies.

[0278] patient Selection Criteria All patients met the following criteria for the dose escalation phase of the study: Advanced / metastatic solid tumor malignancies or relapsed / refractory CD20 + Histologic or cytologic diagnosis of B-cell non-Hodgkin lymphoma: o The disease was resistant to standard therapy or curative therapy was not available; or o Meets the following dose escalation criteria (a-d) · Lesions may be measurable or non-measurable.

[0279] All patients met the following criteria for the dose-expansion phase of the study: a) Locally advanced or metastatic NSCLC (TPS ≥ 1%) that has progressed on prior checkpoint therapy or locally advanced or metastatic NSCLC (TPS < 50%) that has progressed after systemic therapy for metastatic disease; patients were suitable for treatment with pembrolizumab; or b) recurrent or metastatic HNSCC with disease progression on or after platinum-containing chemotherapy and suitable for treatment with pembrolizumab; or c) HER2-overexpressing metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma that has 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 no curative therapy was available; also, indolent lymphomas (marginal zone, follicular) that have relapsed or are refractory to approved standard therapies. Patients had at least one measurable lesion as defined by RECIST version 1.1 or the Lugano criteria (2014).

[0280] In addition, all patients met the following criteria: Adequate bone marrow function, including: Absolute neutrophil count (ANC) ≥ 1,500 / mm 3 (≧1.5x10 9 / L); non-Hodgkin lymphoma only, ANC ≥ 1,000 / mm 3 (≧1.0x10 9 / L); o Platelets ≥75,000 / mm 3 (≧75x10 9 / L); Non-Hodgkin's lymphoma only: platelets ≥ 50,000 / mm 3 (≧50x10 9 / L); o Hemoglobin ≥ 9g / dL (≥ 90g / L);Non-Hodgkin's lymphoma only: Hemoglobin ≥ 8g / dL (≥ 80g / 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 facility method standards). Proper liver function, including: o Total serum bilirubin ≤ 1.5 x ULN (≤ 3.0 x ULN if patient had documented Gilbert syndrome); o Aspartate and alanine transaminase (AST and ALT) ≤ 3.0 x ULN; if there was liver disease secondary to the tumor, ≤ 5.0 x ULN. o Alkaline phosphatase ≤ 2.5 x ULN (≤ 5.0 x ULN for bone or liver metastases). · QT interval was corrected to a heart rate Fridericia (QTcF) interval of 480 ms or less (based on the average of three ECGs). ·Age 18 or older. Eastern Cooperative Oncology Group (ECOG) Performance Status of 0 or 1. Resolved acute effect of prior treatment on baseline severity or grade ≤ 1 (NCI CTCAE v.4.03), except for AEs that do not constitute a safety risk per the investigator's judgment. ·Archival (or fresh) metastatic biopsy sample available prior to study initiation (expansion phase only). · A negative serum pregnancy test at screening (for women of childbearing potential).

[0281] Exclusion criteria Patients with any of the following characteristics were not included in the study: Patients with known symptomatic CNS metastases or leptomeningeal disease requiring steroids. Patients with previously diagnosed brain metastases were eligible if they had completed treatment and recovered from the acute effects of radiation therapy or surgery prior to study enrollment, had discontinued corticosteroid treatment for these metastases, were clinically stable for at least 4 weeks off anticonvulsants, and were neurologically stable prior to enrollment. Patients with ALK or EGFR genomic tumor aberrations (NSCLC, Part 2 expansion only) or with a history of or current pneumonia requiring steroids (non-infectious) pneumonia. Patients with high-grade lymphoma (Burkitt's lymphoma, lymphoblastic lymphoma, Richter's syndrome, etc.), CLL or plasma cell leukemia · Upfront high-dose chemotherapy requiring allogeneic stem cell rescue. Prior irradiation to >25% of bone marrow (non-lymphoma patients only) or prior radiation therapy within 2 weeks of starting study treatment. Note: Participants must have recovered from all radiation-related toxicities, not require corticosteroids, and not have radiation pneumonitis. Palliative radiation therapy for non-CNS disease (radiotherapy for ≤2 weeks) allowed for a 1-week washout. Prior treatment with anti-CD47 or anti-SIRPα agents. Systemic anticancer therapy within 4 weeks of starting study treatment (6 weeks for mitomycin C or nitrosoureas). Patients were included if systemic anticancer therapy was administered within 4 weeks or more, or if 4–5 elimination half-lives of the drug had elapsed. Prior treatment (expansion phase only) with: oPD-1 or PD-L1 inhibitors for metastatic disease with disease progression within 8 weeks of initiation (NSCLC; HNSCC). o Trastuzumab for metastatic disease (gastric / GEJ cancer) with best response being disease progression. Patients with intolerance or a severe allergic or anaphylactic reaction to the antibody or infused therapeutic protein; patients with a severe allergic or anaphylactic reaction to any of the substances contained in the study drug (e.g., excipients); or patients who discontinued treatment due to grade ≥ 3 immune-related 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.). Any experimental antibody or live vaccine within the past 28 days prior to the first dose of study drug. Live attenuated vaccines were not allowed. Current active therapy for primary diagnosis -Transfusion of blood products within 14 days of 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) 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 Acquired Immune Deficiency Syndrome (AIDS)-related illness. ·Patients with active graft-versus-host disease (GVHD) or immunosuppressed for GVHD. - In the past 12 months, any of the following: myocardial infarction, severe / unstable angina, coronary artery / 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 treatment clinical trial. - Have been diagnosed with any other malignancy within the past 3 years prior to enrollment, except for adequately treated non-melanoma skin cancer or carcinoma in situ that has received potentially curative therapy (e.g., breast cancer, cervical intraepithelial neoplasia). Recent (within the past year) or active suicidal thoughts or behavior, or other severe acute or chronic medical or psychiatric condition, such as laboratory abnormalities that increased the risks associated with study participation or administration of the study drug, or that interfered with interpretation of the study results, made the patient unsuitable for participation in the study, in the opinion of the investigator. Men and women of childbearing potential who are not using highly effective contraception or who do not agree to continue using highly effective contraception for at least 90 days after the last dose of study drug. -Pregnant or breastfeeding patients.

[0282] Study treatment As shown in Figure 2, the study had three treatment arms (drug A + pembrolizumab; drug A + trastuzumab; drug A + rituximab). The study included an initial dose escalation portion and then a dose expansion portion. Each dose escalation arm had approximately 12 patients with advanced malignancies. Each expansion arm had approximately 20-40 patients across multiple sites.

[0283] Drug A was administered weekly (or every 2 weeks) as an IV infusion over approximately 60 minutes in an outpatient setting. To ensure accurate delivery of the study drug, use of an infusion pump was the preferred method of administration, although gravity drip was acceptable.

[0284] Drug A is supplied in 100 mg / 5 mL or 400 mg / 20 mL Type 1 clear glass vials sealed with 20 mm Teflon coated rubber septum stoppers and tamper evident aluminum seals. Each single use vial delivers 100 mg of Drug A (5 mL) or 400 mg of Drug A (20 mL) and is intended for intravenous (IV) administration.

[0285] A cycle was defined as the time from dose on Day 1 to the next dose on Day 1. If there were no treatment delays, the cycle was 21 days for weekly dosing and 28 days for every 2 weeks dosing.

[0286] All study treatments were administered on an outpatient basis. Patients were observed in the clinic for at least 2 hours after infusion of Drug A on Day 1 of Cycle 1 (C1D1) and thereafter as clinically indicated.

[0287] No premedication with drug A was required. The guidelines in the package insert for the combination therapy of pembrolizumab, trastuzumab, and rituximab were followed.

[0288] During the dose escalation and expansion phases, combination partner therapy was administered according to its label instructions. o Trastuzumab: Initial dose of 8 mg / kg administered as an intravenous infusion over 90 minutes, then 6 mg / kg administered as an intravenous infusion (IV) over 30 to 90 minutes every 3 weeks. o Pembrolizumab: 200 mg IV as an intravenous infusion over 30 minutes every 3 weeks for up to 24 months. Rituximab: 375 mg / m 2was administered intravenously once weekly for four doses, then once monthly for eight doses (initial rate of 50 mg / h, increased by 50 mg / h every 30 minutes to a maximum of 400 mg / h in the absence of infusion toxicity). If the first infusion of rituximab was tolerated, subsequent infusions were initiated at a rate of 100 mg / h, increased by 100 mg / h every 30 minutes to a maximum of 400 mg / h in the absence of toxicity.

[0289] On dosing days with a matched dosing schedule, the combination partner was started approximately 30 minutes after the end of drug A therapy. On such days, if a drug A dose was missed because of toxicity, the partner drug was administered 24 hours after the missed dose. If drug A was permanently discontinued, patients discontinued the treatment phase of the study. If the partner drug was permanently discontinued, patients continued on single-agent drug A for up to 24 months if, in the investigator's opinion, the patient was deriving clinical benefit from drug A.

[0290] Dose Escalation Components For drug A, the initial dose escalation component was initiated 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 drug A single agent dose-limiting toxicity (DLT) profile and the known safety profile of the proposed combination (Table A). If the drug A dose was safe and well tolerated in combination, the drug A dose level was escalated to the MTD or MAD with the combination.

[0291] The MTD of drug A as a single agent has not been reached. The MAD of drug A as a single agent is 30 mg / kg IV every other week (QOW or Q2W). TIFF2025003971000035.tif50170

[0292] Expansion Stage Drug A was administered once weekly (QW) at a dose of 10 mg / kg as an intravenous (IV) infusion over approximately 60 minutes on an outpatient basis.

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

[0294] The Drug A plus pembrolizumab expansion arm 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 plus trastuzumab expansion group included up to 20 patients with HER2-overexpressing (HER2-positive) gastric cancer.

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

[0297] Each patient received drug A until disease progression, unacceptable toxicity, withdrawal of consent, or end of study. Patients received study treatment in the study after radiographic progression if, in the investigator's estimation, they were deriving clinical benefit from study treatment.

[0298] Efficacy analysis Overall response rate (ORR), disease control rate (DCR), duration of response (DoR), progression-free survival (PFS), and overall survival (OS) were analyzed in the intention-to-treat and evaluable populations.

[0299] Objective tumor response was assessed using the revised Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Tumor response assessments included all known or suspected sites of disease. Imaging included CT or MRI scans of the chest, abdomen, and pelvis; brain CT or MRI scans for patients with known or suspected brain metastases; and bone scans and / or bone radiographs for patients with known or suspected bone metastases. In addition, for patients with lymphoma, examinations included PET scans and bone marrow evaluations. The same imaging techniques used to characterize each lesion identified and reported at baseline were used for subsequent tumor response assessments.

[0300] Antitumor activity was assessed by radiation tumor response assessments performed at baseline, during treatment, when disease progression was suspected (e.g., worsening symptoms), and at the time of withdrawal from the study (if not performed in the previous 6 weeks).

[0301] Response assessment was performed using RECIST version 1.1 or, when relevant, the Lugano criteria (Cheson et al., J. Clin. Oncol (2014) 32:27:3059-3068.).

[0302] Changes in tumor diameter were classified as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD), the latter incorporating the appearance of new lesions. In the expansion cohort, a secondary analysis was performed using immune-related RECIST 1.1. To facilitate this secondary analysis, diameters of all targets and new measurable lesions (longest for nonnodal lesions and shortest for nodal lesions) were collected only in the expansion cohort. Furthermore, in the expansion cohort, both progression and response were required to be confirmed by imaging at least 4 weeks from the date of first recording.

[0303] PK / PD and biomarker analysis Drug concentrations of Drug A were measured using a validated method. Serum concentrations of Drug A were analyzed using a validated ligand-binding ELISA. PK parameters were determined from the respective concentration-time data using standard non-compartmental methods. Actual sample collection times were used for parameter calculations. For Drug A, the maximum concentration (C max ), time to maximum concentration (T max ), area under the concentration-time curve (AUC last , AUC inf , and / or AUC τ PK parameters such as clearance (CL), volume of distribution (Vz), and terminal elimination half-life (t 1 / 2 ), and accumulation rate (R ac Additional PK parameters such as PK, β-blockers, and β-blockers were calculated.

[0304] PK / PD analyses were performed to explore exposure-effect relationships using appropriate model-based methods to support OBD determination. Pharmacodynamic data (receptor occupancy and immunophenotype) were summarized graphically with descriptive statistics by time and dose.

[0305] PK / PD analyses using appropriate model-based methods were investigated to better understand the exposure-effect relationship. Pre- and post-dose levels of CD47 target occupancy were analyzed and immunophenotyping of circulating leukocyte populations was performed. CD47 target occupancy in peripheral blood T lymphocytes and erythrocytes was measured by flow cytometry. Infiltrating leukocyte populations and immune-modulating molecules in tumor biopsy tissues before and after treatment, as well as specific cytokines and chemokines in serum before and after treatment were analyzed. Exploratory molecular analyses in peripheral blood and tumor biopsy samples (such as but not limited to additional immune markers) were performed before and after treatment.

[0306] CD8, CD68, CD163, and PD-L1 on tumor tissues were measured by immunohistochemistry (IHC) assays. CD8, CD68, and CD163 positive percentages were obtained by image analysis. PD-L1 (clone 22C3) tumor proportion scores (TPS) and combined positive scores (CPS) were obtained by pathologist review. HER2 levels were determined using HERCEPTEST™. RNA expression from paired tumor biopsies was determined using the NANOSTRINGIO360™ expression panel. Cell type abundance and pathway profiling analysis using predefined gene signatures was performed using NANOSTRINGnSOLVER™ analysis software.

[0307] Blood samples were collected at the baseline visit and retained for pharmacogenomic analyses related to drug response, for example, investigation of SIRPα gene polymorphisms, putative safety biomarkers, drug metabolizing enzyme genes, drug transporter protein genes, or genes thought to be related to the mechanism of drug action.

[0308] result Patient characteristics Eighty-two patients with advanced solid tumor malignancies were enrolled in this study. The baseline characteristics of the patients are shown in Table B. TIFF2025003971000036.tif133170

[0309] safety Drug A in combination with trastuzumab or pembrolizumab was well tolerated, with most treatment-related adverse events (TRAEs) being low grade and infrequent. Treatment-related adverse events occurring in ≥2 patients are shown in Table C for the Drug A + trastuzumab combination and Table D for the Drug A + pembrolizumab combination. The most frequent TRAE with the Drug A + trastuzumab combination was fatigue (26.7%). The most frequent TRAE with the Drug A + pembrolizumab combination was increased AST (15.4%). TIFF2025003971000037.tif74170TIFF2025003971000038.tif134170

[0310] Grade 3 severity TRAEs were infrequent.One serious treatment-rel...

Claims

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

2. 2. The method of claim 1, wherein the NSCLC in the individual has progressed on prior immune checkpoint inhibitor (CPI) therapy and / or has a PD-L1 tumor proportion score (TPS) of less than 50%.

3. 3. The method of claim 2, wherein the prior immune checkpoint inhibitor therapy comprises one or more agents selected from the group consisting of nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab.

4. 1. A method of treating head and neck squamous cell carcinoma (HNSCC) 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; the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; The Fc domain variant comprises: (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat; The method, wherein the HNSCC in the individual has progressed during or after prior platinum therapy, and the individual is a human.

5. 5. The method of claim 4, wherein the individual has undergone prior treatment with an immune checkpoint inhibitor.

6. 5. The method of claim 4, wherein the individual has not received prior treatment with an immune checkpoint inhibitor.

7. The method of any one of claims 4 to 6, wherein the prior platinum therapy comprises one or more therapeutic agents selected from the group consisting of cisplatin, carboplatin, and oxaliplatin.

8. The method of any one of claims 1 to 7, wherein the anti-PD-1 antibody is pembrolizumab.

9. 9. The method of claim 8, wherein pembrolizumab is administered to the individual at a dose of 200 mg every three weeks (Q3W) by intravenous (IV) infusion.

10. 1. A method of treating HER2-positive gastric / gastroesophageal junction (GEJ) cancer in an individual, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, and (b) an anti-HER2 antibody; the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; The Fc domain variant comprises: (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat; wherein the gastric / GEJ cancer in 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.

11. 11. The method of claim 10, wherein said prior treatment with a fluoropyrimidine-based therapy or said 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.

12. The method of claim 10 or 11, wherein the anti-HER2 antibody is trastuzumab.

13. 13. The method of claim 12, wherein trastuzumab is administered to the individual at an initial dose of 8 mg / kg and each dose thereafter at a dose of 6 mg / kg, and wherein trastuzumab is administered to the individual by IV infusion every three weeks (Q3W).

14. 1. A method of treating an aggressive non-Hodgkin's lymphoma (NHL) in an individual's body, 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; the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; The Fc domain variant comprises: (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat; wherein the individual's aggressive NHL has relapsed and / or is refractory to prior treatment for aggressive NHL, there is no curative therapy available, and the individual is a human.

15. 15. The method of claim 14, wherein the aggressive NHL is diffuse large B-cell lymphoma (DLBCL).

16. The method of claim 15, wherein the DLBCL is de novo DLBCL or transformed DLBCL.

17. 15. The method of claim 14, wherein the aggressive NHL is mantle cell lymphoma (MCL).

18. 18. The method of any one of claims 14 to 17, wherein the prior treatment of aggressive NHL comprises 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.

19. 1. A method of treating indolent lymphoma 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-CD20 antibody; the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; The Fc domain variant comprises: (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat; wherein the indolent lymphoma in the individual is relapsed and / or refractory to prior treatment for indolent lymphoma, and the individual is a human.

20. 20. The method of claim 19, wherein the indolent lymphoma is an indolent non-Hodgkin's lymphoma (NHL).

21. 21. The method of claim 20, wherein the indolent NHL is marginal zone lymphoma or follicular lymphoma.

22. 22. The method of any one of claims 19 to 21, wherein the prior treatment of indolent lymphoma comprises 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, rituximab, obinutuzumab, tisagenlecleucel, axicabtagene, brentuximab vedotin, fludarabine mitoxantrone, everolimus, bortezomib, navitoclax, and combinations thereof.

23. The method of any one of claims 14 to 22, wherein the anti-CD20 antibody is rituximab.

24. Rituximab was administered by IV infusion at a dose of 375 mg / m ２ and rituximab is administered to the individual once a week for four weeks, then once a month thereafter.

25. The method of any one of claims 1 to 24, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO:

85.

26. The method of any one of claims 1 to 24, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO:

81.

27. 27. The method of any one of claims 1 to 26, wherein the Fc domain variant is a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, numbering according to the EU index of Kabat.

28. 28. The method of claim 27, wherein the Fc domain variant comprises the amino acid sequence of SEQ ID NO:

91.

29. The method of any one of claims 1 to 25 or 27 to 28, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant comprises the amino acid sequence of SEQ ID NO:

136.

30. The method of any one of claims 1 to 24 or 26 to 28, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant comprises the amino acid sequence of SEQ ID NO:

135.

31. The method of any one of claims 1 to 30, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant forms a homodimer.

32. 32. The method of any one of claims 1 to 31, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered to the individual at a once weekly (QW) dose of 10 mg / kg.

33. 32. The method of any one of claims 14 to 31, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered to the individual at a once weekly (QW) dose of 15 mg / kg.

34. 34. The method of claim 32 or 33, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered to the individual by IV infusion.

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

36. 36. The kit of claim 35, wherein the NSCLC in the individual has progressed on prior immune checkpoint inhibitor (CPI) therapy and / or has a PD-L1 Tumor Proportion Score (TPS) of less than 50%.

37. 1. A kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharma- ceutically acceptable carrier for use in combination with pembrolizumab to treat head and neck squamous cell carcinoma (HNSCC) in an individual in need thereof, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; The Fc domain variant comprises: (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat; The kit, wherein the HNSCC in the individual has progressed during or after prior platinum therapy, and the individual is a human.

38. 38. The kit of any one of claims 34-37, further comprising instructions to administer pembrolizumab at a dose of 200 mg every three weeks (Q3W) by IV infusion.

39. 1. A kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharma- ceutically acceptable carrier for use in combination with trastuzumab to treat HER2-positive gastric / gastroesophageal junction (GEJ) cancer in an individual in need thereof, comprising: the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; The Fc domain variant comprises: (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat; wherein the HER2 gastric / GEJ cancer of the individual has progressed after prior fluoropyrimidine-based therapy or prior treatment with an anti-HER2 antibody, and the individual is a human.

40. 40. The kit of claim 39, further comprising instructions to administer trastuzumab at an initial dose of 8 mg / kg and each subsequent dose at a dose of 6 mg / kg, wherein trastuzumab is administered to the individual by IV infusion every three weeks (Q3W).

41. 1. A kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharma- ceutically acceptable carrier for use in combination with rituximab to treat aggressive non-Hodgkin's lymphoma (NHL) in an individual in need thereof, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; The Fc domain variant comprises: (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat; wherein the individual's aggressive NHL has relapsed and / or is refractory to prior treatment for aggressive NHL, there is no curative therapy available, and the individual is a human.

42. 42. The kit of claim 41, wherein the aggressive NHL is diffuse large B-cell lymphoma (DLBCL).

43. The kit of claim 42, wherein the DLBCL is de novo DLBCL or transformed DLBCL.

44. 42. The kit of claim 41, wherein the aggressive NHL is mantle cell lymphoma.

45. 1. A kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharma- ceutically acceptable carrier for use in combination with rituximab to treat indolent lymphoma in an individual in need thereof, comprising: the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; The Fc domain variant comprises: (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index of Kabat; wherein the indolent lymphoma of the individual is relapsed or refractory to prior treatment for indolent lymphoma, and the individual is a human.

46. 46. ​​The kit of claim 45, wherein the indolent lymphoma is indolent non-Hodgkin's lymphoma (NHL).

47. 47. The kit of claim 46, wherein the indolent NHL is marginal zone lymphoma or follicular lymphoma.

48. Dose 375 mg / m via IV infusion ２ 48. The kit of any one of claims 41-47, further comprising instructions for administering rituximab to the individual once a week for four weeks, then once a month.

49. The kit of any one of claims 35 to 48, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO:

85.

50. The kit of any one of claims 35 to 48, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO:

81.

51. 51. The kit of any one of claims 35 to 50, wherein the Fc domain variant is a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, numbering according to the EU index of Kabat.

52. The kit of claim 51 , wherein the Fc domain variant comprises the amino acid sequence of SEQ ID NO:

91.

52. The kit of any one of claims 35 to 49 or 51, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant comprises the amino acid sequence of SEQ ID NO:

136.

53. The kit of any one of claims 35 to 48, 50 or 51, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant comprises the amino acid sequence of SEQ ID NO:

135.

54. The kit of any one of claims 35 to 53, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant forms a homodimer.

55. 55. The kit of any one of claims 35-54, further comprising instructions for administering the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant to the individual at a dose of 10 mg / kg once weekly (QW).

56. 55. The kit of any one of claims 41-54, further comprising instructions for administering the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant to the individual at a dose of 15 mg / kg once weekly (QW).

57. 57. The kit of claim 55 or 56, further comprising instructions for administering the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant to the individual by IV infusion.

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