Methods of treating cancer with SIRP [alpha] Fc fusions in combination with immune checkpoint inhibitors

Abstract

The present invention relates to methods of treating cancer using SIRP [alpha] Fc fusions in combination with immune checkpoint inhibitors. Specifically, the present invention provides a method of treating cancer (e.g., non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), HER2 positive gastric cancer / gastroesophageal junction (GEJ) cancer, primary or transformed diffuse large B cell lymphoma (DLBCL) or inert lymphoma) in a subject comprising administering to the subject (a) a polypeptide comprising a SIRP [alpha] D1 domain variant and an Fc domain variant, and (b) an anti-cancer antibody (e.g., an anti-PD1 antibody, an anti-HER2 antibody, or an anti-CD20 antibody). A related kit pharmaceutical composition is also provided.

Description

[0001] This application is a divisional application of the invention application with a filing date of May 28, 2020, Chinese application number 202080047746.1, and invention name “Method for treating cancer using SIRPαFc fusion combined with immune checkpoint inhibitors”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 855,821, filed on May 31, 2019, and U.S. Provisional Application No. 63 / 022,187, filed on May 8, 2020, the contents of each of which are incorporated herein by reference in their entirety.

[0004] Submit sequence listing as ASCII text file

[0005] The following submission of ASCII text file is incorporated herein by reference in its entirety: Computer Readable Form (CRF) of Sequence Listing (File Name: 757972001040SEQLIST.TXT, Record Date: May 26, 2020, Size: 333KB). Technical Field

[0006] The present invention relates to a method for treating cancer, comprising administering a polypeptide (eg, a fusion polypeptide) comprising a SIRPαD1 domain variant and an Fc domain variant in combination with a therapeutic antibody. Background Art

[0007] Many cancers have a poor prognosis even when treated with available therapeutic agents. For example, patients with non-small cell lung cancer (NSCLC) with metastatic disease who have received PD-1 and PD-L1 checkpoint inhibitors who have failed in previous platinum-based therapies have a median overall survival rate of about one year (Garon et al., New Engl J Med (2015) 372: 2018-28; Herbst et al., Lancet (2016) 387: 1540-50; Fehrenbacher et al., Lancet (2016) 387 (10030): 1837-46), and more than half of NSCLC patients with advanced disease have an overall 5-year survival rate of 17.7% (US Cancer Statistics Working Group, available at www (dot) cdc (dot) gov / uscs). Similarly, the overall 5-year survival rate of gastric cancer patients in the United States is 30.4% (US Cancer Statistics Working Group). In patients with relapsed indolent lymphoma, subsequent relapses often occur in a series with increasing aggressive histology and a 30% risk of transformation over 10 years (Montoto et al., J Clin Oncol (2007) 25(17):2426-33). In addition, for patients with relapsed aggressive histology, cure is rare and new salvage regimens are needed (Larouche et al., J Clin Oncol (2010) 28(12):2094-100). CD20-positive non-Hodgkin lymphoma (NHL) is the 10th most common cancer and the 10th leading cause of cancer death worldwide, causing 199,670 deaths per year worldwide (World Health Organization 2016(a), available at globocan(dot)iarc(dot)fr / Pages / fact_sheets_cancer(dot)aspx). It is estimated that more than 35,000 people in the United States have metastatic HNSCC, with more than 50,000 new cases of all stages diagnosed in 2019. Five-year survival is 84% ​​for patients diagnosed with localized disease, but drops to only 39% for patients diagnosed with metastatic disease. New treatments are needed in the art to provide additional treatment options and improve outcomes for these patients.

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

[0009] Macrophage-mediated destruction of cancer cells requires the destruction of "don't eat me" signals (e.g., CD47-SIRPα) and the activation of "eat me" signals. Neither component alone is sufficient to elicit a maximal phagocytic response against tumor cells. As described above, CD47 provides a basic "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 activated Fcγ receptors. For example, pro-phagocytic "eat me" signals can be provided by binding of anti-tumor antibodies to Fc receptors on macrophages.

[0010] All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are hereby incorporated by reference in their entirety to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the invention

[0011] A method of treating non-small cell lung cancer (NSCLC) in an individual is provided, comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) an anti-PD-1 antibody, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein the numbering is according to the 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 the 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 the EU index of Kabat; and wherein 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%,

[0012] Also provided is a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant for use in the preparation of a medicament for treating NSCLC in an individual, wherein the medicament is for (such as formulated for) use in combination with an anti-PD1 antibody, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein the numbering is according to the 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 the EU index of Kabat; or (iv) human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236 and N297A mutations, wherein numbering is according to the EU index of Kabat; and wherein the individual is a human. In some embodiments, the individual's NSCLC has progressed on a previous immune checkpoint inhibitor (CPI) therapy and / or has a PD-L1 tumor proportion score (TPS) of less than 50%.

[0013] Also provided is a composition (such as a pharmaceutical composition) comprising a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, the composition being used in combination with an anti-PD1 antibody to treat NSCLC in an individual (e.g., a method for treating NSCLC in an individual), wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is according to the 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 the EU index of Kabat; or (iv) human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236 and N297A mutations, wherein numbering is according to the EU index of Kabat; and wherein the individual is a human. In some embodiments, the individual's NSCLC has progressed on a previous immune checkpoint inhibitor (CPI) therapy and / or has a PD-L1 tumor proportion score (TPS) of less than 50%.

[0014] In some embodiments, the prior CPI (immune checkpoint inhibitor therapy) comprises one or more agents selected from the group consisting of nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplizumab. 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 an individual at a dose of 200 mg once every 3 weeks (Q3W) by intravenous (IV) infusion.

[0015] Also provided is a method of treating head and neck squamous cell carcinoma (HNSCC) in an individual, comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) an anti-PD-1 antibody, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein the numbering is according to the 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 as in Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering is according to EU index as in Kabat; wherein the individual's HNSCC has progressed on or after prior platinum therapy, and wherein the individual is human.

[0016] Also provided is a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant for use in the preparation of a medicament for treating HNSCC in an individual, wherein the medicament is for (such as formulated for) use in combination with an anti-PD1 antibody, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein the numbering is according to the 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 as in Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering is according to EU index as in Kabat; wherein the individual's HNSCC has progressed on or after prior platinum therapy, and wherein the individual is human.

[0017] Also provided is a composition (such as a pharmaceutical composition) comprising a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, the composition being used in combination with an anti-PD1 antibody to treat HNSCC in an individual (e.g., a method for treating HNSCC in an individual), wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is according to the 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 as in Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering is according to EU index as in Kabat; wherein the individual's HNSCC has progressed on or after prior platinum therapy, and wherein the individual is human.

[0018] In some embodiments, individuals with HNSCC receive previous immune checkpoint inhibitors (e.g., immune checkpoint inhibitors described herein) therapy. In some embodiments, such individuals are considered / referred to as "experienced checkpoint inhibitors". In some embodiments, individuals with HNSCC have not received previous immune checkpoint inhibitor therapy. In some embodiments, such individuals are considered / referred to as "not experienced checkpoint inhibitors". In some embodiments, previous platinum therapy includes one or more therapeutic agents selected from the group consisting of: cisplatin, carboplatin, and oxaliplatin. In some embodiments, anti-PD-1 antibodies block the interaction between PD-1 and PD-L1. In some embodiments, anti-PD-1 antibodies are pembrolizumab. In some embodiments, pembrolizumab is administered (such as formulated for administration) to individuals at a dose of 200 mg once every 3 weeks (Q3W) by intravenous (IV) infusion.

[0019] A method for treating HER2-positive gastric cancer / gastroesophageal junction (GEJ) cancer in an individual is provided, comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) an anti-HER2 antibody, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is according to the 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 the EU index as in Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering is according to the EU index as in Kabat; wherein the individual's gastric cancer / GEJ cancer has progressed following prior treatment with a fluoropyrimidine-based therapy and / or prior treatment with an anti-HER2 antibody, and wherein the individual is a human.

[0020] Also provided is a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant for use in the preparation of a medicament for treating HER2-positive gastric cancer / gastroesophageal junction (GEJ) cancer in an individual, wherein the medicament is for (such as formulated for) use in combination with an anti-HER2 antibody, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein the numbering is according to the 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 the EU index as in Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering is according to the EU index as in Kabat; wherein the individual's gastric cancer / GEJ cancer has progressed following prior treatment with a fluoropyrimidine-based therapy and / or prior treatment with an anti-HER2 antibody, and wherein the individual is a human.

[0021] Also provided is a composition (such as a pharmaceutical composition) comprising a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, the composition being used in combination with an anti-HER2 antibody to treat HER2-positive gastric cancer / gastroesophageal junction (GEJ) cancer in an individual (e.g., a method for treating HER2-positive gastric cancer / gastroesophageal junction (GEJ) cancer in an individual), wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein the numbering is according to the 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 as in Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering is according to EU index as in Kabat; wherein the individual's gastric cancer / GEJ cancer has progressed following prior treatment with a fluoropyrimidine-based therapy and / or prior treatment with an anti-HER2 antibody, and wherein the individual is a human.

[0022] In some embodiments, the treatment with fluoropyrimidine-based therapy or the treatment with anti-HER2 antibody previously included one or more therapeutic agents selected from the group consisting of trastuzumab, pertuzumab, 5-fluorouracil, capecitabine, magetuximab and FOLFOX. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, trastuzumab is administered (such as formulated for administration) to an individual at an initial dose of 8 mg / kg and a dose of 6 mg / kg for each subsequent dose, and wherein trastuzumab is administered to an individual once every 3 weeks (Q3W) by IV infusion.

[0023] A method of treating aggressive non-Hodgkin lymphoma (NHL) in an individual is provided, comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) an anti-CD20 antibody, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is according to the 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 as in Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering is according to EU index as in Kabat; wherein the individual's aggressive NHL is relapsed and / or refractory to previous treatment for the aggressive NHL and there is no available curative therapy, and wherein the individual is human.

[0024] Also provided is a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant for use in the preparation of a medicament for treating aggressive non-Hodgkin's lymphoma (NHL) in an individual, wherein the medicament is for (such as formulated for) use in combination with an anti-CD20 antibody, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein the numbering is according to the 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 as in Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering is according to EU index as in Kabat; wherein the individual's aggressive NHL is relapsed and / or refractory to previous treatment for the aggressive NHL and there is no available curative therapy, and wherein the individual is a human.

[0025] Also provided is a composition (such as a pharmaceutical composition) comprising a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, the composition being used in combination with an anti-CD20 antibody to treat aggressive non-Hodgkin lymphoma (NHL) in an individual (e.g., a method for treating aggressive NHL), wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is according to the 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 as in Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering is according to EU index as in Kabat; wherein the individual's aggressive NHL is relapsed and / or refractory to previous treatment for the aggressive NHL and there is no available curative therapy, and wherein the individual is human.

[0026] In some embodiments, the aggressive NHL is diffuse large B-cell lymphoma (DLBCL), such as denovo DLBCL or transformed DLBCL. In some embodiments, previous treatments for aggressive NHL include rituximab, cyclophosphamide, doxorubicin, vincristine, gemcitabine, lenalidomide, prednisone, prednisolone, etoposide, procarbazine, epirubicin, bendamustine, cisplatin, oxaliplatin, cytarabine, ifosfamide, carboplatin, dexamethasone, mesna, carmustine, melphalan, methylprednisolone, methyl-glyoxal-bis(guanylhydrazone), thiotepa, methotrexate, ibrutinib, obinituzumab, 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 375 mg / m 2 A dose of rituximab is administered (such as formulated for administration) to the individual, wherein rituximab is administered (such as formulated for administration) to the individual once a week for four weeks and once a month thereafter.

[0027] A method for treating an indolent lymphoma in an individual is provided, comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) an anti-CD20 antibody, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein the numbering is according to the 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 as in Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering is according to EU index as in Kabat; wherein the individual's indolent lymphoma is relapsed and / or refractory to previous treatment for the indolent lymphoma, and wherein the individual is a human.

[0028] Also provided are polypeptides comprising a SIRPαD1 domain variant and an Fc domain variant for use in the preparation of a medicament for treating an indolent lymphoma in an individual, wherein the medicament is used in combination with an anti-CD20 antibody (e.g., formulated for use), wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is according to the 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 as in Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering is according to EU index as in Kabat; wherein the individual's indolent lymphoma is relapsed and / or refractory to previous treatment for the indolent lymphoma, and wherein the individual is a human.

[0029] Also provided are compositions (such as pharmaceutical compositions) comprising a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant for use in combination with an anti-CD20 antibody to treat an indolent lymphoma in an individual (e.g., a method for treating an indolent lymphoma in an individual), wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is according to the 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 as in Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering is according to EU index as in Kabat; wherein the individual's indolent lymphoma is relapsed and / or refractory to previous treatment for the indolent lymphoma, and wherein the individual is a human.

[0030] 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, the previous treatment of the indolent lymphoma includes rituximab, cyclophosphamide, doxorubicin, vincristine, gemcitabine, lenalidomide, prednisone, prednisolone, etoposide, procarbazine, epirubicin, bendamustine, cisplatin, oxaliplatin, cytarabine, ifosfamide, carboplatin, dexamethasone, mesna, carmustine, melphalan, methylprednisolone, methyl-glyoxal-bis(guanylhydrazone), thiotepa, methotrexate, ibrutinib, obinutuzumab, tesalet, acataquin, vebutuximab, fludarabine, mitoxantrone, everolimus, bortezomib, navicla, and combinations thereof. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, rituximab is administered by IV infusion at 375 mg / m 2 In some embodiments, a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant (such as a medicament made using such a polypeptide or a pharmaceutical composition comprising such a polypeptide) is administered (such as formulated for administration) to an individual at a dose of 10 mg / kg or 15 mg / kg once a week (QW), for example, by IV infusion.

[0031] In some embodiments of any of the methods herein, the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NOL: 81. In some embodiments, the Fc domain variant is a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 136. In some embodiments, the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant forms a homodimer.

[0032] In some embodiments, polypeptides comprising SIRPαD1 domain variants and Fc domain variants (such as medicaments manufactured using such polypeptides or pharmaceutical compositions comprising such polypeptides) are administered (such as formulated for administration) to an individual once a week (QW) at a dose of 10 mg / kg. In some embodiments, polypeptides comprising SIRPαD1 domain variants and Fc domain variants (or medicaments manufactured therefrom or pharmaceutical compositions comprising such polypeptides) are administered (such as formulated for administration) to an individual by IV infusion.

[0033] Also provided is a kit comprising polypeptides containing SIRPαD1 domain variants and Fc domain variants (such as medicaments prepared using such polypeptides or pharmaceutical compositions comprising such polypeptides), which is used in combination with pembrolizumab according to the methods described herein for treating non-small cell lung cancer (NSCLC) in an individual (e.g., a human individual) in need thereof. In some embodiments, the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein the numbering is according to the EU index as in Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein the numbering is according to the EU index as in Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A and delG236 mutations, wherein the numbering is according to the EU index as in 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 as in Kabat. In some embodiments, the individual's NSCLC has progressed in a previous immune checkpoint inhibitor (CPI) therapy and / or has a PD-L1 tumor proportion score (TPS) of less than 50%. In some embodiments, the kit also includes instructions for administering pembrolizumab once every 3 weeks (Q3W) at a dose of 200 mg by IV infusion. In some embodiments, the kit also includes instructions for administering a polypeptide (e.g., a fusion polypeptide) once a week, for example, at a dose of 10 mg / kg by IV infusion.

[0034] Also provided is a kit comprising polypeptides containing SIRPαD1 domain variants and Fc domain variants (such as medicaments prepared using such polypeptides or pharmaceutical compositions comprising such polypeptides), which is used in combination with pembrolizumab according to the methods described herein for treating head and neck squamous cell carcinoma (HNSCC) in an individual (e.g., a human individual) in need thereof. In some embodiments, the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising 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. Fc region, wherein numbering is according to the EU index of Kabat; and the HNSCC of the individual has progressed in a previous platinum therapy or after platinum therapy. In some embodiments, the individual has received a previous immune checkpoint inhibitor therapy. In some embodiments, the individual has not received a previous immune checkpoint inhibitor therapy. In some embodiments, the kit also includes instructions for administering pembrolizumab once every 3 weeks (Q3W) at a dose of 200 mg by IV infusion. In some embodiments, the kit also includes instructions for administering a polypeptide (e.g., a fusion polypeptide) once a week at a dose of 10 mg / kg, for example, by IV infusion.

[0035] Also provided herein is a kit comprising a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, the kit being used in combination with trastuzumab for treating HER2-positive gastric cancer / gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual) in need thereof according to the methods described herein. In some embodiments, the SIRPαD1 domain variant comprises an amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 comprising S228P, E233P, F234V, L235A, and delG236 mutations Fc region, wherein numbering is according to the EU index of Kabat; or (iv) human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236 and N297A mutations, wherein numbering is according to the EU index of Kabat; and the individual's HER2-positive gastric cancer / GEJ cancer has progressed after previous fluoropyrimidine-based therapy or previous anti-HER2 antibody treatment. In some embodiments, the kit also includes instructions for administering trastuzumab with an initial dose of 8 mg / kg and a dose of 6 mg / kg for each subsequent dose, and wherein trastuzumab is administered to an individual once every 3 weeks (Q3W) by IV infusion. In some embodiments, the kit also includes instructions for administering a polypeptide (e.g., a fusion polypeptide) once a week, for example, by IV infusion at a dose of 10 mg / kg.

[0036] A kit is provided comprising a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier for use in combination with rituximab for treating aggressive non-Hodgkin lymphoma (NHL) in an individual (e.g., a human individual) in need thereof according to the methods described herein. In some embodiments, the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising 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. Fc region, wherein numbering is according to the EU index of Kabat; and the individual's aggressive NHL is relapsed and / or refractory to previous treatment for aggressive NHL and there is no available curative therapy. In some embodiments, the aggressive NHL is diffuse large B-cell lymphoma (DLBCL), such as primary DLBCL or transformed DLBCL. In some embodiments, the aggressive NHL is mantle cell lymphoma. In some embodiments, the kit further comprises 375 mg / m 2 In some embodiments, the kit further comprises instructions for administering rituximab at a dose of 10 mg / kg or 15 mg / kg once a week to the individual, wherein rituximab is administered to the individual once a week for four weeks and once a month thereafter. In some embodiments, the kit further comprises instructions for administering the polypeptide (e.g., fusion polypeptide) once a week, for example, by IV infusion at a dose of 10 mg / kg or 15 mg / kg.

[0037] Also provided is a kit comprising a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier for use in combination with rituximab for treating an indolent lymphoma in an individual (e.g., a human individual) in need thereof according to the methods described herein. In some embodiments, the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising 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. Fc region, wherein numbering is according to the EU index of Kabat; and the individual's indolent lymphoma is relapsed or refractory to previous treatment for indolent lymphoma. In some embodiments, the indolent lymphoma is indolent non-Hodgkin lymphoma (NHL). In some embodiments, the indolent NHL is marginal zone lymphoma or follicular lymphoma. In some embodiments, the kit further comprises 375 mg / m by IV infusion. 2 In some embodiments, the kit further comprises instructions for administering rituximab at a dose of 10 mg / kg or 15 mg / kg once a week to the individual, wherein rituximab is administered to the individual once a week for four weeks and once a month thereafter. In some embodiments, the kit further comprises instructions for administering the polypeptide (e.g., fusion polypeptide) once a week, for example, by IV infusion at a dose of 10 mg / kg or 15 mg / kg.

[0038] In some embodiments of any kit, the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:85. In some embodiments, the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NOL:81. In some embodiments, the Fc domain variant is a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein the numbering is according to the EU index of Kabat. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO:91. In some embodiments, the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO:136. In some embodiments, the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant comprises the amino acid sequence of SEQ ID NO:135. In some embodiments, the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant forms a homodimer. In some embodiments, instructions for administering a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant (or a medicament prepared therefrom or a pharmaceutical composition comprising such a polypeptide) to an individual at a dose of 10 mg / kg once a week (QW) are also included. In some embodiments, the kit comprises instructions for administering a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant (or a medicament prepared therefrom or a pharmaceutical composition comprising such a polypeptide) to an individual by IV infusion. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1A-1B Describes that drug A is a monomer ( Figure 1A ) and homodimers ( Figure 1B ). SIRPα D1 domain variants selectively bind CD47 with picomolar binding affinity to block its interaction with SIRPα. Drug A has a molecular weight that is half the size of a typical antibody, allowing for two-fold molar delivery to tumors. The Fc domain is modified to eliminate binding to Fcγ receptors, thereby minimizing toxicity. Drug A exhibits antibody-like pharmacokinetics.

[0040] Figure 2 A schematic diagram of the clinical study described in Example 1 is provided.

[0041] Figure 3A-3C Clinical activity of drug A plus trastuzumab was demonstrated in an expansion cohort of HER2-positive gastric / GEJ cancer. Figure 3A The change in tumor size is provided, shown as a percentage relative to baseline. ORR = overall response rate; DCR = disease control rate; mPFS = median progression-free survival; CI = confidence interval. Figure 3BThe percent change in tumor size from baseline during the study is provided for each patient. Figure 3A-3B : Includes patients who received at least one dose of drug A during the expansion phase and had a baseline assessment and at least one post-baseline disease assessment; the top dotted line indicates the threshold for objective progression; the bottom dotted line indicates the threshold for overall response. Figure 3C The duration of treatment for each enrolled patient who received at least one dose of drug A in the extension phase is provided. Tumors were assessed according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (EAEisenhauer et al., European Journal of Cancer 45 (2009) 228-247.)

[0042] Figure 4A-4C Clinical activity of drug A + pembrolizumab in the HNSCC expansion cohort was shown. Figure 4A The change in tumor size is provided, shown as a percentage relative to 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 of PD-L1 staining. Figure 4B The percent change in tumor size from baseline during the study is provided for each patient. Figure 4A-4B : Includes patients who received at least one dose of drug A during the expansion phase and had a baseline assessment and at least one post-baseline disease assessment; the top dotted line indicates the threshold for objective progression; the bottom dotted line indicates the threshold for overall response. Figure 4C The duration of treatment for each enrolled patient who received at least one dose of drug A in the extension phase is provided. Tumors were assessed according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (EAEisenhauer et al., European Journal of Cancer 45 (2009) 228-247.)

[0043] Figure 5A-5C Clinical activity of drug A + pembrolizumab in the NSCLC expansion cohort was shown. Figure 5A The change in tumor size is provided, shown as a percentage relative to baseline. DCR = disease control rate; mPFS = median progression-free survival; CI = confidence interval; TPS = tumor proportion score stained for PD-L1. Figure 5B The percent change in tumor size from baseline during the study is provided for each patient. Figure 5A-Figure 5B : Includes patients who received at least one dose of drug A during the expansion phase and had a baseline assessment and at least one post-baseline disease assessment; the top dotted line indicates the threshold for objective progression; the bottom dotted line indicates the threshold for partial response. Figure 5CThe duration of treatment for each enrolled patient who received at least one dose of drug A in the extension phase is provided. Tumors were assessed according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (EAEisenhauer et al., European Journal of Cancer 45 (2009) 228-247.)

[0044] Figure 6A-6B Pharmacokinetic and CD47 target occupancy parameters for Drug A administered in combination with pembrolizumab or trastuzumab during the study are provided. Fig. 6A As shown, based on the established population PK model, drug A PK (serum concentration of drug A) is within the predicted 95% interval. The steady-state half-life of drug A (10 mg / kg QW) is predicted to be approximately 16 days. Drug A PK time points include intensive sampling in cycles 1 and 3, and only before dosing and end of infusion in each cycle thereafter. Figure 6B It was shown that when Drug A was administered in combination with either pembrolizumab or trastuzumab, near complete CD47 target occupancy was maintained in CD4+ T cells expressing CD47 throughout the Drug A dosing interval. TO = target occupancy.

[0045] Figure 7A-7C Antitumor responses of patients treated with drug A in combination with either pembrolizumab or trastuzumab are shown. Fig. 7A The percentages of positive intratumoral CD68+ (left), CD163+ (center), and CD8+ (right) cells in paired biopsies before and after treatment are presented for 5 NSCLC patients treated with Agent A + pembrolizumab, 6 HNSCC patients treated with Agent A + pembrolizumab, and 1 gastric cancer patient treated with Agent A + trastuzumab. Tumor-associated macrophages and infiltrating lymphocytes increased after treatment. Figure 7B Images from a biopsy of a NSCLC PD-L1(-) patient treated with Drug A + Pembrolizumab showing staining for CD68 and CD8 before and during treatment (C3 = Cycle 3) are provided in . Figure 7C Images of biopsies from HNSCC PD-L1(+) patients treated with Drug A + Pembrolizumab showing staining for CD68 and CD8 before and during treatment (cycle 3) are provided in . Figure 7B-Figure 7C Middle: The graph on the right shows the percentage change in tumor size from baseline for each patient during the study; the top dashed line indicates the threshold for objective progression; the bottom dashed line indicates the threshold for partial response. The CD68 and CD163 markers indicate macrophages; the CD8 marker indicates T lymphocytes.

[0046] Figure 8A schematic diagram of the proposed mechanism of anti-tumor activity of Drug A when administered in combination with anti-cancer therapeutic antibodies such as pembrolizumab, trastuzumab, or rituximab is provided.

[0047] Fig.9A A schematic diagram showing the clinical activity of drug A in combination with pembrolizumab in response-evaluable ≥2L HNSCC patients (including two checkpoint inhibitor-naive patients among those who experienced checkpoint inhibitors) is provided. CPS = combined positive score (i.e., the number of PD-L1 positively stained cells (tumor cells, lymphocytes, and macrophages) divided by the total number of viable tumor cells, multiplied by 100).

[0048] Fig. 9B A schematic diagram showing the best overall response and duration of response in patients with ≥2L HNSCC treated with Agent A and pembrolizumab is provided.

[0049] Fig. 10A A graphical representation showing the clinical activity of Drug A in combination with pembrolizumab in response evaluable patients with ≥2L NSCLC is provided. Patients indicated with * showed a change greater than 80%. TPS = Tumor Proportion Score. Fig. 10B A schematic diagram showing the best overall response and duration of response in patients with ≥2L NSCLC treated with Agent A and pembrolizumab is provided.

[0050] Fig.11A The results showed that the combination of drug A and trastuzumab was effective in response-evaluable patients with ≥2L HER2 + Gastric cancer or HER2 + Schematic representation of clinical activity in GEJ cancer patients. One patient did not show clinical progression. The HER2 score reflects the HER2 expression level of tumor cells. Fig. 11B A study was provided showing that patients with ≥2L HER2 treated with Agent A and trastuzumab + Gastric cancer or HER2 + Schematic representation of best overall response and duration of response in patients with GEJ cancer.

[0051] Fig.12 A schematic diagram showing the clinical activity of the combination of Drug A and Rituximab in response-evaluable patients with relapsed / refractory non-Hodgkin's lymphoma is provided. Patients represented by "^" showed an increase of more than 80% in measurable lesions compared to baseline. Patients represented by "*" achieved a complete response or a metabolic complete response. Patients represented by "mPR" achieved a metabolic partial response. Two patients (1 each at 10 mg / kg and 15 mg / kg Drug A) did not show clinical progression. One patient with a metabolic CR (10 mg / kg Drug A) is not represented in the figure.

[0052] Fig.13A A graphical representation showing best overall response and duration of response in patients with relapsed / refractory non-Hodgkin lymphoma treated with 10 mg / kg of Drug A and rituximab is provided. Fig. 13B A graphical representation showing best overall response and duration of response in patients with relapsed / refractory non-Hodgkin lymphoma treated with 15 mg / kg of Drug A and rituximab is provided. One patient who had clinical progression study day 8 as best response is not shown. DETAILED DESCRIPTION

[0053] definition

[0054] The term "about" or "approximately" means within an acceptable error range for a particular value determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" may mean within 1 or more than 1 standard deviation. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly for biological systems or methods, the term may refer to within the order of magnitude of a numerical value, preferably within 5 times, more preferably within 2 times. Where specific values ​​are described in the present application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable error range for a specific value.

[0055] The terminology used herein is for the purpose of describing particular instances only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including," "includes," "having," "has," "with," or variations thereof are used in the detailed description or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0056] As used herein, the terms "treatment", "treating" and the like refer to the administration of an agent or the performance of a procedure for the purpose of obtaining an effect. In some embodiments, the effect is prophylactic, in terms of completely or partially preventing a disease or its symptoms. In some embodiments, the effect is therapeutic, in terms of effecting a partial or complete cure of a disease or disease symptom.

[0057] As used herein, the term "antibody" refers to intact antibodies; antibody fragments, provided 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.

[0058] As used herein, the term "antibody variable domain" refers to the portion of the light and heavy chains of an antibody, which includes 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).

[0059] As used herein, the term "joint" refers to the connection between two elements (e.g., protein domains). In some embodiments, the joint can be a covalent bond or a spacer. The term "spacer" refers to a part (e.g., polyethylene glycol (PEG) polymer) or an amino acid sequence (e.g., 1-200 amino acid sequence) that is present between two polypeptides or polypeptide domains to provide space or flexibility (or space and flexibility) between two polypeptides or polypeptide domains. In some embodiments, the amino acid spacer is a part of the primary sequence of a polypeptide (e.g., a polypeptide or polypeptide domain connected to the interval by a polypeptide backbone).

[0060] As used herein, the term "effective amount" refers to the amount of a polypeptide or a pharmaceutical composition containing a polypeptide described herein (e.g., a polypeptide having a SIRPαD1 domain or a variant thereof) sufficient and effective to achieve the desired therapeutic effect in treating a patient with a disease (such as cancer, e.g., a solid tumor or a blood cancer). In some embodiments, an effective amount of a polypeptide will avoid adverse side effects.

[0061] As used herein, the term "pharmaceutical composition" refers to a medicine or pharmaceutical preparation that contains an active ingredient and an excipient or diluent (or both an excipient and a diluent) and enables the active ingredient to be administered by a suitable administration method. In some embodiments, the pharmaceutical composition disclosed herein includes a pharmaceutically acceptable component that is compatible with the polypeptide. In some embodiments, the pharmaceutical composition is in the form of a tablet or capsule for oral administration or an aqueous form for intravenous or subcutaneous administration (e.g., by injection).

[0062] As used herein, the terms "subject," "individual," and "patient" are used interchangeably to refer to vertebrates, such as mammals. Mammals include, but are not limited to, rodents, apes, humans, farm animals, sports animals, and pets. Also included are tissues, cells, and progeny of biological entities obtained in vivo or cultured in vitro. None of these terms require the supervision of a medical professional.

[0063] As used herein, the term "affinity" or "binding affinity" refers to the strength of the binding interaction between two molecules. Typically, binding affinity refers to the strength of the sum of non-covalent interactions between a molecule and its binding partner (such as SIRPαD1 domain variants and CD47). Unless otherwise specified, binding affinity refers to intrinsic binding affinity, which reflects a 1: 1 interaction between binding pair members. The binding affinity between two molecules is generally described by a dissociation constant (KD) or an association constant (KA). Two molecules with low binding affinity to each other generally bind slowly, tend to dissociate easily, and exhibit a large KD. Two molecules with high affinity to each other generally bind easily, tend to remain bound longer, 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.

[0064] As used herein, the term "less than K D " refers to the smaller K D As used herein, the term "greater than KD" refers to a numerically larger KD value and a decreased binding affinity relative to the KD value.

[0065] As used herein, "in combination" refers to administering one therapeutic modality in addition to another therapeutic modality. Thus, "in combination" refers to administering one therapeutic modality before, during, or after the other therapeutic modality is administered to an individual.

[0066] Overview

[0067] Provided herein are methods for 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 SIRPαD1 domain variant and an Fc domain variant and (b) a therapeutic antibody. In some embodiments, the polypeptide comprises any of the SIRPαD1 domain variants described herein (unless otherwise specified).

[0068] In some embodiments, a method of treating non-small cell lung cancer (NSCLC) in an individual is provided, comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) an anti-PD-1 antibody, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is according to the 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 the EU index of Kabat; or (iv) human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236 and N297A mutations, wherein numbering is according to the EU index of Kabat; and wherein the individual is a person. In some embodiments, the individual's NSCLC has progressed in a previous 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 previous CPI therapy. In some embodiments, the individual is PD-L1 negative. In some embodiments, the individual is PD-L1 positive.

[0069] In some embodiments, a method of treating head and neck squamous cell carcinoma (HNSCC) in an individual is provided, comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) an anti-PD-1 antibody, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations. Fc region, wherein numbering is according to the EU index of Kabat; (iii) human IgG4 Fc region comprising S228P, E233P, F234V, L235A and delG236 mutations, wherein numbering is according to the EU index of Kabat; or (iv) human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236 and N297A mutations, wherein numbering is according to the EU index of Kabat; wherein the HNSCC of the individual has progressed in the previous platinum therapy or after the platinum therapy, and wherein the individual is a person. In some embodiments, the individual has received previous immune checkpoint inhibitor (CPI) therapy (for example, treated with immune checkpoint inhibitors as described herein). In some embodiments, the individual has not received previous CPI therapy. In some embodiments, the individual is PD-L1 negative. In some embodiments, the individual is PD-L1 positive.

[0070] In some embodiments, an individual is "PD-L1 negative" if the individual has a cancer that does not express the PD-L1 biomarker or expresses very low levels of PD-L1. In some embodiments, an individual is "PD-L1 negative" or has a "PD-L1 negative cancer" if PD-L1 expression (e.g., protein expression) is not detected on (or in) tumor cells (TCs) in a sample from an individual, if PD-L1 expression (e.g., protein expression) is not detected on (or in) tumor infiltrating immune cells (ICs) in a sample from an individual, or if PD-L1 expression (e.g., protein expression) is detected at very low levels on (or in) TCs and / or ICs in a sample from an individual. In some embodiments, 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 an individual express PD-L1, as determined using an assay for determining the PD-L1 status of an individual (e.g., an assay described herein), the individual is PD-L1 negative. In some embodiments, an individual or tumor may be considered PD-L1 negative because it has no T cell infiltration. Such assays are known and routinely used by medical professionals.

[0071] 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) a PD-L1 biomarker. In some embodiments, the individual is "PD-L1 positive" or has a cancer that is a "PD-L1 positive cancer". In some embodiments, if PD-L1 expression (e.g., protein expression) is detected on (or in) tumor cells (TCs) in a sample from an individual, or if PD-L1 expression (e.g., protein expression) is detected on (or in) tumor infiltrating immune cells (ICs) in a sample from an individual, then the individual is "PD-L1 positive" or has a "PD-L1 positive cancer". In some embodiments, the TCs and / or ICs of an individual express low levels of PD-L1 biomarkers. In some embodiments, the TCs and / or ICs of an individual express high levels of PD-L1 biomarkers. In some embodiments, if the PD-L1 biomarker is present (e.g., detected) in more than 0% of the samples, in at least 1% of the samples, in at least 5% of the samples, or in at least 10% of the samples from the individual (e.g., samples from the individual containing the individual's TCs and / or ICs), as determined using an assay for determining the PD-L1 status of the individual (e.g., an assay described herein), the individual is "PD-L1 positive" or has a cancer that is a "PD-L1 positive cancer". Such assays are known and routinely used by medical professionals. In some embodiments, if the individual's tumor proportion score (TPS) is ≥50% (i.e., if ≥50% of the live tumor cells in the sample from the individual express PD-L1, e.g., at any level), the individual is "PD-L1" positive or has a "PD-L1 positive cancer".

[0072] In some embodiments, a method of treating HER2-positive gastric cancer / gastroesophageal junction (GEJ) cancer in an individual is provided, comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) an anti-HER2 antibody, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is according to the 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 the EU index as in Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering is according to the EU index as in Kabat; wherein the individual's gastric cancer / GEJ cancer has progressed following prior treatment with a fluoropyrimidine-based therapy and / or prior treatment with an anti-HER2 antibody, and wherein the individual is a human.

[0073] In some embodiments, a method of treating an aggressive non-Hodgkin lymphoma or "NHL" (e.g., primary or transformed diffuse large B-cell lymphoma (DLBCL) or mantle cell lymphoma) in an individual is provided, comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) an anti-CD20 antibody, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1 comprising L234A, L235A, G237A, and N297A mutations. Fc region, wherein numbering is according to the EU index of Kabat; (ii) human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein numbering is according to the EU index of Kabat; (iii) human IgG4 Fc region comprising S228P, E233P, F234V, L235A and delG236 mutations, wherein numbering is according to the EU index of Kabat; or (iv) human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236 and N297A mutations, wherein numbering is according to the EU index of Kabat; wherein the individual's aggressive NHL is relapsed and / or refractory to previous treatment for aggressive NHL and there is no available curative therapy, and wherein the individual is a human. In some embodiments, the relapsed / refractory aggressive NHL is relapsed / refractory DLBCL (e.g., primary or transformed DLBCL). In some embodiments, the relapsed / refractory aggressive NHL is relapsed / refractory mantle cell lymphoma (MCL).

[0074] In some embodiments, a method of treating an indolent lymphoma in an individual is provided, comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) an anti-CD20 antibody, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is according to the 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 the EU index of Kabat; or (iv) human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236 and N297A mutations, wherein numbering is according to the EU index of Kabat; wherein the individual's indolent lymphoma is relapsed and / or refractory to previous treatment for indolent lymphoma, and wherein the individual is a human. In some embodiments, indolent lymphoma (such as relapsed / refractory indolent lymphoma) is non-Hodgkin lymphoma (NHL), e.g., relapsed / refractory indolent NHL. In some embodiments, indolent NHL (e.g., relapsed / refractory NHL) is follicular lymphoma (e.g., relapsed / refractory follicular lymphoma). In some embodiments, indolent NHL (e.g., relapsed / refractory NHL) is marginal zone lymphoma (e.g., relapsed / refractory marginal zone lymphoma).

[0075] Further details regarding treatment methods and polypeptides comprising SIRPαD1 domain variants and Fc domain variants are described below. See also US Patent No. 10,259,859, the contents of which are incorporated herein by reference in their entirety.

[0076] Signal regulatory protein α (SIRP-α) D1 domain and its variants

[0077] In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein α (SIRP-α) D1 variant, wherein the variant comprises a SIRPαD1 domain or a fragment thereof, wherein the SIRPαD1 domain comprises an amino acid mutation at residue 80 relative to the wild-type SIRPαD1 domain; and comprises at least one additional amino acid mutation at a residue selected from the group consisting of: residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66 and residue 92 relative to the wild-type SIRPαD1 domain.

[0078] In some embodiments, the present invention also discloses a polypeptide comprising an Fc domain variant, wherein the Fc domain variant dimer comprises two Fc domain variants, wherein each Fc domain variant is independently selected from (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236 and N297A.

[0079] Signal regulatory protein alpha ("SIRP-α" or "SIRP-alpha") is a transmembrane glycoprotein belonging to the Ig superfamily that is widely expressed on the membranes of myeloid cells. SIRPα interacts with CD47, a protein widely expressed on many cell types in the body. The interaction of SIRPα with CD47 prevents the phagocytosis of "self" cells that would otherwise be recognized by the immune system. It has been observed that high CD47 expression on tumor cells can serve as a negative prognostic factor for survival in acute myeloid leukemia and several solid tumor cancers.

[0080] Natural SIRPα contains three highly homologous immunoglobulin (Ig)-like extracellular domains—D1, D2, and D3. The SIRPαD1 domain ("D1 domain") refers to the membrane-distal extracellular domain of SIRPα and mediates the binding of SIRPα to CD47. As used herein, the term "SIRPα polypeptide" refers to any SIRPα polypeptide or fragment thereof that is capable of binding to CD47. There are at least ten variants of wild-type human SIRPα. Table 1 shows the amino acid sequences of the D1 domain of ten naturally occurring wild-type human SIRPαD1 domain variants (SEQ ID NOs: 1-10). In some embodiments, the SIRPα polypeptide comprises the SIRPαD1 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).

[0081] Table 1. Sequences of wild-type SIRPαD1 domains

[0082]

[0083]

[0084] As used herein, the term "SIRPαD1 domain variant" refers to a polypeptide comprising a SIRPαD1 domain or a CD47 binding portion of a SIRPα polypeptide, which has a higher affinity for CD47 than wild-type SIRPα. The SIRPαD1 domain variant comprises at least one amino acid substitution, deletion or insertion (or a combination thereof) relative to wild-type SIRPα.

[0085] In some embodiments, the SIRPαD1 domain variant disclosed herein comprises a SIRPαD1 domain or a variant thereof. In some embodiments, the SIRPαD1 domain variant comprises one or more amino acid substitutions, insertions, additions or deletions relative to the wild-type D1 domain shown in SEQ ID NO: 1-10. Table 2 lists exemplary amino acid substitutions (SEQ ID NO: 13-22) in each SIRPαD1 domain variant. In some embodiments, the SIRPαD1 domain polypeptide or SIRPαD1 domain variant comprises a fragment of the D1 domain. In some embodiments, the SIRPα polypeptide fragment or SIRPαD1 domain variant fragment comprises an amino acid sequence of 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 SIRPαD1 domain fragment retains the ability to bind CD47.

[0086] In some embodiments, the polypeptides of the present disclosure comprising SIRPαD1 domain variants bind to CD47 with a higher binding affinity than the wild-type human SIRPαD1 domain. In some embodiments, the SIRPαD1 domain variants bind to human CD47 with an affinity of at least 1 times (e.g., at least 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 5 times, or more than 5 times) the affinity of the naturally occurring D1 domain. In some embodiments, the SIRPαD1 domain variants bind to human CD47 with an affinity of at least 1 times (e.g., at least 10 times, 100 times, 1000 times, or more than 1000 times) the affinity of the naturally occurring D1 domain.

[0087] As used herein, the term "optimized affinity" or "optimized binding affinity" refers to the optimized binding interaction strength between the polypeptides disclosed herein (including SIRPαD1 domain variants) and CD47. For example, in some embodiments, the polypeptide binds primarily or with a higher affinity to CD47 on cancer cells, but does not substantially bind or binds with a lower affinity to CD47 on non-cancerous cells. In some embodiments, the binding affinity between the polypeptide and CD47 is optimized so that the interaction does not cause clinically relevant toxicity or reduces toxicity compared to variants bound with maximum affinity. In some embodiments, in order to achieve the optimized binding affinity between the polypeptides provided herein and CD47, polypeptides comprising SIRPαD1 domain variants are developed to have a binding affinity for CD47 that is lower than the maximum achievable binding affinity. In some embodiments, the SIRPαD1 domain variants disclosed herein cross-react with rodent, non-human primate (NHP) and human CD47.

[0088] As used herein, the term "immunogenicity" refers to the property of a protein (e.g., a therapeutic protein) that evokes an immune response in a host, as if it were a foreign antigen. The immunogenicity of a protein can be determined in vitro in a variety of different ways, such as by an in vitro T cell proliferation assay.

[0089] As used herein, the term "minimal immunogenicity" refers to a protein (e.g., a therapeutic protein) that is modified (e.g., by amino acid substitution) and has a lower immunogenicity (e.g., at least 10%, 25%, 50%, or 100%) than the immunogenicity (e.g., unmodified protein) before the amino acid substitution was introduced. In some embodiments, the protein (e.g., a therapeutic protein) is modified to have minimal immunogenicity and does not elicit or elicits very little host immune response even if it is a foreign antigen.

[0090] In some embodiments, SIRPαD1 domain variants show minimal immunogenicity. In some embodiments, the SIRPα polypeptide of the present disclosure administered to a subject has the same amino acid sequence as the SIRPα polypeptide in the subject's biological sample, except for amino acid changes that increase the affinity of the SIRPαD1 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.

[0091] Table 2 lists specific amino acid substitutions relative to each D1 domain sequence in the SIRPαD1 domain variants. In some embodiments, the SIRPαD1 domain variants include one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more) substitutions listed in Table 2. In some embodiments, the SIRPαD1 domain variants comprise up to fourteen amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPαD1 domain variants comprise up to ten amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPαD1 domain variants comprise up to seven amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPαD1 domain variants of the present disclosure have at least 90% (e.g., at least 92%, 95%, 97% or greater than 97%) amino acid sequence identity with the sequence of the wild-type D1 domain.

[0092] In some embodiments, the SIRPαD1 domain variant is a chimeric SIRPαD1 domain variant, which comprises a portion of two or more wild-type D1 domains or variants thereof (e.g., a portion of one wild-type D1 domain or variant thereof and a portion of another wild-type D1 domain or variant thereof). In some embodiments, the chimeric SIRPαD1 domain variant comprises at least two portions (e.g., three, four, five or more portions) of a wild-type D1 domain or variant thereof, wherein each portion is from a different wild-type D1 domain. In some embodiments, the chimeric SIRPαD1 domain variant further comprises one or more amino acid substitutions listed in Table 2.

[0093] Table 2. Amino Acid Substitutions in SIRPαD1 Domain Variants

[0094]

[0095]

[0096]

[0097]

[0098] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant comprising the following sequence:

[0099] EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11RNNMDFSIRIGNITPADAGTYYCX 12 KX 13 RKGSPDDVEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 13), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is A, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10 L, T or G; X 11 K or R; X 12 V or I; X 13 is F, L or V; and X 14 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain comprising the sequence of SEQ ID NO: 1.

[0100] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant comprising the following sequence:

[0101] EEGX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPG RX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGNITPADAGTYYCX 12 KX 13 RKGSPDDVEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 16), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is A, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10 L, T or G; X 11 K or R; X 12 V or I; X 13 is F, L or V; and X 14 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain comprising the sequence of SEQ ID NO:4.

[0102] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant comprising the following sequence:

[0103] EEEX1QX2IQPDKFVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGNITPADAGTYYCX 12 KX 13 RKGSPDDVEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 17), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is A, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10 L, T or G; X 11 K or R; X 12 V or I; X 13 is F, L or V; and X 14 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain comprising the sequence of SEQ ID NO:5.

[0104] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant comprising the following sequence:

[0105] EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFPIRIGNITPADAGTYYCX 12 KX 13 RKGSPDDVEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 18), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is A, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10 L, T or G; X 11 K or R; X 12 V or I; X 13 is F, L or V; and X 14 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain comprising the sequence of SEQ ID NO:6.

[0106] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant comprising the following sequence:

[0107] EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRISNITPADAGTYYCX 12 KX 13 RKGSPDDVEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 21), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is A, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10 L, T or G; X 11 K or R; X 12 V or I; X 13 is F, L or V; and X 14 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain comprising the sequence of SEQ ID NO:9.

[0108] In any of the above embodiments, the polypeptide comprises a SIRPαD1 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 above embodiments, X2 is V, L, or I. In any of the above embodiments, X3 is A or V. In any of the above embodiments, X4 is A, I, or L. In any of the above embodiments, X5 is I, T, S, or F. In any of the above embodiments, X6 is E, V, or L. In any of the above embodiments, X7 is K or R. In any of the above embodiments, X8 is E or Q. In any of the above embodiments, X9 is H, P, or R. In any of the above embodiments, X 10 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 no more than six amino acid substitutions relative to the wild-type SIRPαD1 domain comprising the sequence of any one of SEQ ID NOs: 1, 4-6, and 9.

[0109] In some embodiments, the polypeptide binds to CD47 with at least 10 times greater binding affinity than a wild-type SIRPαD1 domain comprising 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 times greater binding affinity than a wild-type SIRPαD1 domain comprising 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 times greater binding affinity than a wild-type SIRPαD1 domain comprising the sequence of any one of SEQ ID NOs: 1, 4-6, and 9. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof binds to CD47 with less than 1x10 -8 M, less than 5x10 -9 M, less than 1x10 -9 M, less than 5x10 -10 M, less than 1x10 -10 M or less than 1x10 -11 M of K D Binding to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof binds to CD47 with a KD of between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM.

[0110] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant comprising the following sequence:

[0111] EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPA RX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 14), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is V, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10 S, T or G; X 11 K or R; X12 V or I; X 13 is F, L or V; and X 14 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain comprising the sequence of SEQ ID NO: 2.

[0112] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant comprising the following sequence:

[0113] EEEX1QX2IQPDKSVSVAAGESX3ILLCTX4TSLX5PVGPIQWFRGAGPAR X6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 15), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is V, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10 S, T or G; X 11 K or R; X 12 V or I; X 13 is F, L or V; and X 14 is ForV; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain comprising the sequence of SEQ ID NO:3.

[0114] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant comprising the following sequence:

[0115] EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPA RX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14KSGAGTELSVRGKPS (SEQ ID NO: 19), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is V, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10 S, T or G; X 11 K or R; X 12 V or I; X 13 is F, L or V; and X 14 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain comprising the sequence of SEQ ID NO:7.

[0116] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant comprising the following sequence:

[0117] EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPA RX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 22), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is V, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10 S, T or G; X 11 K or R; X 12 V or I; X 13 is F, L or V; and X 14 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain comprising the sequence of SEQ ID NO: 10.

[0118] In any of the above embodiments of this aspect of the disclosure, the polypeptide comprises the sequence of any of SEQ ID NOs: 14, 15, 19, and 22, wherein X1 is L, I, or V. In any of the above embodiments, X2 is V, L, or I. In any of the above embodiments, X3 is A or V. In any of the above embodiments, X4 is V, I, or L. In any of the above embodiments, X5 is I, T, S, or F. In any of the above embodiments, X6 is E, V, or L. In any of the above embodiments, X7 is K or R. In any of the above embodiments, X8 is E or Q. In any of the above embodiments, X9 is H, P, or R. In any of the above 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 no more than six amino acid substitutions relative to the wild-type SIRPαD1 domain comprising the sequence of any one of SEQ ID NOs: 2, 3, 7, and 10.

[0119] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof binds to CD47 with an affinity that is less than 1x10 -8 M, less than 5x10 -9 M, less than 1x10 -9 M, less than 5x10 -10 M, less than 1x10 -10 M or less than 1x10 -11 M of K DBinds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

[0120] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having the following sequence:

[0121] EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 20), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is A, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10 S, T or G; X 11 K or R; X 12 V or I; X 13 is F, L or V; and X 14 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 8.

[0122] In one embodiment, the polypeptide comprises the sequence of SEQ ID NO:20, wherein X1 is L, I or V. In any of the above embodiments of this aspect of the disclosure, X2 is V, L or I. In any of the above embodiments, X3 is A or V. In any of the above embodiments, X4 is A, I or L. In any of the above embodiments, X5 is I, T, S or F. In any of the above embodiments, X6 is E, V or L. In any of the above embodiments, X7 is K or R. In any of the above embodiments, X8 is E or Q. In any of the above embodiments, X9 is H, P or R. In any of the above embodiments, X 10 is S, T or G. In any of the above embodiments, X11 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 no more than six amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:8.

[0123] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10 times greater than that of a wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 8. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100 times greater than that of a wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 8. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000 times greater than that of a wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 8. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof binds to CD47 with an affinity that is less than 1x10 -8 M, less than 5x10 -9 M, less than 1x10 -9 M, less than 5x10 -10 M, less than 1x10 -10 M or less than 1x10 -11 M of K D Binds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

[0124] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having the following sequence:

[0125] EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSX 11 PVGPIQWFR GAGPX 12 RX 13 SXWh 14 X15GX 16 FPRVTTVSX 17X 18 TX 19 RX 20 NMDFX 21 IX 22 IX 23 NITPADAGTYYCX 24 KX 25 RKGSPDX 26 X 27 EX 28 KSGAGTELSVRX 29 KPS (SEQ ID NO: 23), wherein X1 is E or G; X2 is L, I or V; X3 is V, L or I; X4 is S or F; X5 is L or S; X6 is S or T; X7 is A or V; X8 is I or T; X9 is H or R; X 10 A, V, I or L; X 11 I, T, S or F; X 12 A or G; X 13 E, V or L; X 14 K or R; X 15 is E or Q; X 16 H, P or R; X 17 D or E; X 18 S, L, T or G; X 19 K or R; X 20 E or D; X 21 S or P; X 22 S or R; X 23 S or G; X 24 V or I; X 25 F, L, V; X 26 D or not present; X 27 T or V; X 28 is F or V; and X 29 is A or G; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of any one of SEQ ID NOs: 1-10.

[0126] In any of the above embodiments of this aspect of the disclosure, X2 is L, I or V. In any of the above embodiments, X3 is V, L or I. In any of the above embodiments, X4 is S or F. In any of the above embodiments, X5 is L or S. In any of the above embodiments, X6 is S or T. In any of the above embodiments, X7 is A or V. In any of the above embodiments, X8 is I or T. In any of the above embodiments, X9 is H or R. In any of the above embodiments, X 10 is A, V, I or L. In any of the above embodiments, X 11is I, T, S or F. In any of the above embodiments, X 12 is A or G. In any of the above embodiments, X 13 is E, V or L. In any of the above embodiments, X 14 is K or R. In any of the above embodiments, X 15 is E or Q. In any of the above embodiments, X 16 is H, P or R. In any of the above embodiments, X 17 is D or E. In any of the above embodiments, X 18 is S, L, T or G. In any of the above embodiments, X 19 is K or R. In any of the above embodiments, X 20 is E or D. In any of the above embodiments, X 21 is S or P. In any of the above embodiments, X 22 is S or R. In any of the above embodiments, X 23 is S or G. In any of the above embodiments, X 24 is V or I. In any of the above embodiments, X 25 is F, L, V. In any of the above embodiments, X 26 is D or absent. In any of the above embodiments, X 27 is T or V. In any of the above embodiments, X 28 is F or V. In any of the above embodiments, X 29 is A or G. In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than six amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of any one of SEQ ID NOs: 1-10.

[0127] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NOs: 1-10. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NOs: 1-10. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NOs: 1-10. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof binds to CD47 with an affinity that is less than 1x10 -8 M, less than 5x10 -9 M, less than 1x10 -9 M, less than 5x10 -10 M, less than 1x10 -10M or less than 1x10 -11 M of K D Binds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

[0128] In some embodiments, the polypeptides of the present disclosure including SIRPα D1 domain variants further comprise a D2 domain having a sequence of SEQ ID NO: 24, a D3 domain having a sequence of SEQ ID NO: 25, or a D2 domain having a sequence of SEQ ID NO: 24 and a D3 domain having a sequence of SEQ ID NO: 25 of the wild-type human SIRPα shown in Table 3. In some embodiments, the SIRPα D1 domain variants further comprise a fragment or variant of the D2 domain or a fragment or variant of the D3 domain. In some embodiments, the SIRPα D1 domain variants further comprise a fragment or variant of the D2 domain and a fragment or variant of the D3 domain. In some embodiments, the SIRPα D1 domain variants are connected to the D2 or D3 domains via a linker. In some embodiments, the SIRPα D1 domain variants are connected to the D2 and D3 domains via a linker.

[0129] Table 3. Amino acid sequences of SIRPα D2 and D3 domains

[0130]

[0131]

[0132] In some embodiments, the polypeptides of the present disclosure comprising SIRPαD1 domain variants are attached to Fc domain variants to improve the pharmacokinetic properties of the polypeptides, such as increasing serum half-life. In some embodiments, SIRPαD1 domain variants are attached to Fc domain variants that cannot dimerize. In some embodiments, Fc domain variants are used to increase the serum half-life of polypeptides described herein. In some embodiments, the polypeptides of the present disclosure comprising SIRPαD1 domain variants do not include the sequence of any one of SEQ ID NOs: 26-36 shown in Table 4.

[0133] Table 4.

[0134]

[0135]

[0136] In some embodiments, the polypeptides and polypeptide constructs described herein are used in vitro for binding assays, such as immunoassays. For example, in some embodiments, the polypeptides and polypeptide constructs described herein are used in liquid phase or combined with solid phase carriers. In some embodiments, the polypeptides used in immunoassays are detectably labeled in various ways.

[0137] In some embodiments, polypeptides and polypeptide constructs described herein are combined with 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 soluble or insoluble.

[0138] Various different labels and labeling methods are known. Examples of labels include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds. Various techniques can be used to bind labels to the polypeptides disclosed herein.

[0139] In some embodiments, the polypeptide is coupled to a low molecular weight hapten. These haptens are then specifically detected by a second reaction. For example, in some embodiments, the hapten biotin is used together with avidin, or the hapten dinitrophenol, pyridoxal, or fluorescein is detected with a specific anti-hapten antibody (e.g., anti-dinitrophenol antibody, anti-pyridoxal antibody, and anti-fluorescein antibody, respectively).

[0140] SIRPαD1 domain variants with altered glycosylation patterns

[0141] In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein α (SIRP-α) D1 variant, the variant comprising a SIRPαD1 domain or a fragment thereof, the SIRPαD1 domain having an amino acid mutation at residue 80 relative to the wild-type SIRPαD1 domain; and having at least one additional amino acid mutation at a residue selected from the group consisting of: residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66 and residue 92 relative to the wild-type SIRPαD1 domain.

[0142] In some embodiments, the present invention also discloses a polypeptide comprising an Fc domain variant, wherein the Fc domain variant dimer comprises two Fc domain variants, wherein each Fc domain variant is independently selected from (i) a human IgG1 Fc region consisting of L234A, L235A, G237A and N297A mutations; (ii) a human IgG2 Fc region consisting of A330S, P331S and N297A mutations; or (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236 and N297A mutations.

[0143] In some embodiments, the polypeptides in the compositions disclosed herein comprise SIRPαD1 domain variants 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 SIRPαD1 domain in Chinese hamster ovary (CHO) cells produces a major band of 16 kDa (non-glycosylated) and a secondary band of higher molecular weight removed by Endo Hf. Endo Hf is a recombinant protein fusion of endoglycosidase H and maltose binding protein. Endo Hf cuts within a high mannose chitobiose core and some hybrid oligosaccharides from N-linked glycoproteins. This means that the proline at amino acid position 83 can reduce the efficiency of glycosylation, resulting in proteins with varying degrees of glycosylation and therefore heterogeneity. For drug development, heterogeneity can cause challenges in process development. Therefore, in order to study the possibility of producing a homogeneous, non-glycosylated form of SIRPαD1 domain variants, in some embodiments, the amino acid N80 of the SIRPαD1 variant is mutated to Ala. In some embodiments, in order to prepare a non-glycosylated SIRPαD1 domain variant, the amino acid N80 in the SIRPαD1 domain variant is replaced by any amino acid, including any naturally and non-naturally occurring amino acids, such as N80A and N80Q. In some embodiments, the SIRPαD1 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 additional mutations or more). In some embodiments, the additional mutation is in the CD47 binding site. In some embodiments, the additional mutation is in the hydrophobic core of the D1 domain.

[0144] In some embodiments, the polypeptide in the composition disclosed herein comprises a SIRPαD1 domain variant with increased glycosylation relative to the wild-type SIRPαD1 domain. Another option to increase the homogeneity of the final product is to enhance the glycosylation efficiency at amino acid N80 and produce a SIRPαD1 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 glycosylation efficiency at N80. In some embodiments, the amino acid P83 in the SIRPαD1 domain variant is replaced by any amino acid, including natural and non-natural amino acids, such as P83V, P83A, P83I and P83L. In some embodiments, polypeptides of the present disclosure are expressed in cells that are optimized not to glycosylate proteins expressed by such cells, for example, through genetic engineering of the cell line (e.g., genetically engineered yeast or mammalian hosts) or modification of cell culture conditions such as the addition of kifunine or by using naturally non-glycosylating hosts such as prokaryotes (E. coli, etc.).

[0145] Table 5 lists specific amino acid substitutions in SIRPαD1 domain variants relative to each D1 domain variant sequence. In some embodiments, the SIRPαD1 domain variant includes one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more) substitutions listed in Table 5. In some embodiments, the SIRPαD1 domain variant is not glycosylated or minimally glycosylated. In some embodiments, the SIRPαD1 domain variant is fully glycosylated or almost fully glycosylated. In some embodiments, the SIRPαD1 domain variant comprises up to fourteen amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPαD1 domain variant comprises up to ten amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPαD1 domain variant comprises up to seven amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variants disclosed herein have 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.

[0146] In some embodiments, the SIRPαD1 domain variant is a chimeric SIRPαD1 domain variant, which comprises a portion of two or more wild-type D1 domains or variants thereof (e.g., a portion of a wild-type D1 domain or variant thereof and a portion of another wild-type D1 domain or variant thereof). In some embodiments, the chimeric SIRPαD1 domain variant comprises at least two portions (e.g., three, four, five or more portions) of a wild-type D1 domain or variant thereof, wherein each portion is from a different wild-type D1 domain. In some embodiments, the chimeric SIRPαD1 domain variant further comprises one or more amino acid substitutions listed in Table 5.

[0147] Table 5. Amino Acid Substitutions in SIRPαD1 Domain Variants

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having the following sequence:

[0157] EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGX 12 ITX 13 ADAGTY YCX 14 KX 15 RKGSPDDVEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 37), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is A, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X10 L, T or G; X 11 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 V or I; X 15 is F, L or V; and X 16 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1.

[0158] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having the following sequence:

[0159] EEGX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPG RX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGX 12 ITX 13 ADAGTY YCX 14 KX 15 RKGSPDDVEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 40), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is A, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10 L, T or G; X 11 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 V or I; X 15 is F, L or V; and X 16 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 4.

[0160] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having the following sequence:

[0161] EEEX1QX2IQPDKFVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGX 12 ITX 13 ADAGTY YCX 14 KX 15 RKGSPDDVEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 41), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is A, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10 L, T or G; X 11 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 V or I; X 15 is F, L or V; and X 16 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 5.

[0162] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having the following sequence:

[0163] EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFPIRIGX 12 ITX 13 ADAGTY YCX 14 KX 15 RKGSPDDVEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 42), and wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is A, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X10 L, T or G; X 11 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 V or I; X 15 is F, L or V; and X 16 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 6.

[0164] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having the following sequence:

[0165] EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRISX 12 ITX 13 ADAGTYY CX 14 KX 15 RKGSPDDVEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 45), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is A, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10 L, T or G; X 11 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 V or I; X 15 is F, L or V; and X 16 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:9.

[0166] In any of the above embodiments of this aspect of the disclosure, the polypeptide comprises a SIRPαD1 domain variant having a sequence of any one of SEQ ID NOs: 37, 40-42, and 45, wherein X1 is L, I, or V. In any of the above embodiments, X2 is V, L, or I. In any of the above embodiments, X3 is A or V. In any of the above embodiments, X4 is A, I, or L. In any of the above embodiments, X5 is I, T, S, or F. In any of the above embodiments, X6 is E, V, or L. In any of the above embodiments, X7 is K or R. In any of the above embodiments, X8 is E or Q. In any of the above embodiments, X9 is H, P, or R. In any of the above embodiments, X 10 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 N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y. In any of the above embodiments, X 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 above embodiments, X 14 is V or I. In any of the above embodiments, X 15 is F, L, V. In any of the above embodiments, X 16 It is F or V.

[0167] In some embodiments, the polypeptides provided herein comprise no more than ten amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NOs: 1, 4-6, and 9. In some embodiments, the polypeptides provided herein comprise no more than seven amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NOs: 1, 4-6, and 9.

[0168] In some embodiments, the polypeptide binds to CD47 with at least 10 times greater binding affinity than a wild-type SIRPαD1 domain having a 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 times greater binding affinity than a wild-type SIRPαD1 domain having a 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 times greater binding affinity than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NOs: 1, 4-6, and 9. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof binds to CD47 with less than 1x10 -8 M, less than 5x10 -9M, less than 1x10 -9 M, less than 5x10 -10 M, less than 1x10 -10 M or less than 1x10 -11 M of K D Binds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

[0169] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having the following sequence:

[0170] EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPA RX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISX 12 ITX 13 ADAGTYY CX 14 KX 15 RKGSPDTEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 38), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is V, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10 S, T or G; X 11 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 V or I; X 15 is F, L or V; and X 16 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 2.

[0171] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having the following sequence:

[0172] EEEX1QX2IQPDKSVSVAAGESX3ILLCTX4TSLX5PVGPIQWFRGAGPAR X6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISX 12 ITX 13 ADAGTYYC X 14 KX 15 RKGSPDTEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 39), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is V, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10 S, T or G; X 11 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 V or I; X 15 is F, L or V; and X 16 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 3.

[0173] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having the following sequence:

[0174] EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPA RX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISX 12 ITX 13 ADAGTYY CX 14 KX 15 RKGSPDTEX 16KSGAGTELSVRGKPS (SEQ ID NO: 43), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is V, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10 S, T or G; X 11 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 V or I; X 15 is F, L or V; and X 16 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 7.

[0175] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having the following sequence:

[0176] EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPA RX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISX 12 ITX 13 ADAGTYY CX 14 KX 15 RKGSPDTEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 46), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is V, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10 S, T or G; X 11 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 V or I; X 15 is F, L or V; and X 16is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 10.

[0177] In any of the above embodiments of this aspect of the disclosure, the polypeptide comprises a SIRPαD1 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 above embodiments, X2 is V, L, or I. In any of the above embodiments, X3 is A or V. In any of the above embodiments, X4 is V, I, or L. In any of the above embodiments, X5 is I, T, S, or F. In any of the above embodiments, X6 is E, V, or L. In any of the above embodiments, X7 is K or R. In any of the above embodiments, X8 is E or Q. In any of the above embodiments, X9 is H, P, or R. In any of the above 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 N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y. In any of the above embodiments, X 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 above embodiments, X 14 is V or I. In any of the above embodiments, X 15 is F, L or V. In any of the above embodiments, X 16 It is F or V.

[0178] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having no more than ten amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having no more than seven amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NOs: 2, 3, 7, and 10.

[0179] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof binds to CD47 with an affinity that is less than 1x10 -8 M, less than 5x10 -9 M, less than 1x10 -9 M, less than 5x10 -10 M, less than 1x10 -10 M or less than 1x10 -11 M of K D Binds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

[0180] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having the following sequence:

[0181] EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPA RX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISX 12 ITX 13 ADAGTYY CX 14 KX 15 RKGSPDTEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 44), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is A, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10S, T or G; X 11 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 V or I; X 15 is F, L or V; and X 16 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 8.

[0182] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:44, wherein X1 is L, I or V. In any of the above embodiments of this aspect of the disclosure, X2 is V, L or I. In any of the above embodiments, X3 is A or V. In any of the above embodiments, X4 is A, I or L. In any of the above embodiments, X5 is I, T, S or F. In any of the above embodiments, X6 is E, V or L. In any of the above embodiments, X7 is K or R. In any of the above embodiments, X8 is E or Q. In any of the above embodiments, X9 is H, P or R. In any of the above 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 N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y. In any of the above embodiments, X 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 above embodiments, X 14 is V or I. In any of the above embodiments, X 15 is F, L or V. In any of the above embodiments, X 16 It is F or V.

[0183] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having no more than ten amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of SEQ ID NO: 8. In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having no more than seven amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of SEQ ID NO: 8.

[0184] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10 times greater than that of a wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 8. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100 times greater than that of a wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 8. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000 times greater than that of a wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 8. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof binds to CD47 with an affinity that is less than 1x10 -8 M, less than 5x10 -9 M, less than 1x10 -9 M, less than 5x10 -10 M, less than 1x10 -10 M or less than 1x10 -11 M of K D Binds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

[0185] In another aspect, the disclosure features a polypeptide comprising a SIRPαD1 domain variant having the following sequence:

[0186] EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSX 11 PVGPIQWFR GAGPX 12 RX 13 SXWh 14 X15GX 16 FPRVTTVSX 17 X 18 TX 19 RX 20 NMDFX 21 IX 22 IX 23 X 24 ITX 25 ADAGTYYCX 26 KX 27 RKGSPDX 28 X 29EX 30 KSGAGTELSVRX 31 KPS (SEQ ID NO: 47), wherein X1 is E or G; X2 is L, I or V; X3 is V, L or I; X4 is S or F; X5 is L or S; X6 is S or T; X7 is A or V; X8 is I or T; X9 is H, R or L; X 10 A, V, I or L; X 11 I, T, S or F; X 12 A or G; X 13 E, V or L; X 14 K or R; X 15 is E or Q; X 16 H, P or R; X 17 D or E; X 18 S, L, T or G; X 19 K or R; X 20 E or N; X 21 S or P; X 22 S or R; X 23 S or G; X 24 is any amino acid; X 25 is any amino acid; X 26 V or I; X 27 F, L, V; X 28 D or not present; X 29 T or V; X 30 is F or V; and X 31 is A or G; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of any one of SEQ ID NOs: 1-10.

[0187] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:47, wherein X1 is E or G. In any of the above embodiments of this aspect of the disclosure, X2 is L, I or V. In any of the above embodiments, X3 is V, L or I. In any of the above embodiments, X4 is S or F. In any of the above embodiments, X5 is L or S. In any of the above embodiments, X6 is S or T. In any of the above embodiments, X7 is A or V. In any of the above embodiments, X8 is I or T. In any of the above embodiments, X9 is H or R. In any of the above embodiments, X 10 is A, V, I or L. In any of the above embodiments, X 11 is I, T, S or F. In any of the above embodiments, X 12 is A or G. In any of the above embodiments, X 13 is E, V or L. In any of the above embodiments, X14 is K or R. In any of the above embodiments, X 15 is E or Q. In any of the above embodiments, X 16 is H, P or R. In any of the above embodiments, X 17 is D or E. In any of the above embodiments, X 18 is S, L, T or G. In any of the above embodiments, X 19 is K or R. In any of the above embodiments, X 20 is E or N. In any of the above embodiments, X 21 is S or P. In any of the above embodiments, X 22 is S or R. In any of the above embodiments, X 23 is S or G. In any of the above embodiments, X 24 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y. In any of the above 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 above embodiments, X 26 is V or I. In any of the above embodiments, X 27 is F, L, V. In any of the above embodiments, X 28 is D or absent. In any of the above embodiments, X 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 It is A or G.

[0188] In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than ten amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NOs: 1-10. In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than seven amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NOs: 1-10.

[0189] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NOs: 1-10. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NOs: 1-10. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NOs: 1-10. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof binds to CD47 with an affinity that is less than 1x10 -8 M, less than 5x10 -9 M, less than 1x10 -9 M, less than 5x10 -10 M, less than 1x10 -10 M or less than 1x10 -11 M of K D Binds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

[0190] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having the following sequence:

[0191] 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 23EFKSGAGTELSVRAKPS (SEQ ID NO: 48), wherein X1 is V or I; X2 is L or S; X3 is T or S; X4 is T or I; X5 is R or H; X6 is A, V or I; X7 is I, R, Y, K or F; X8 is G or A; X9 is E or V; X 10 K or R; X 11 is E, D or Q; X 12 H or P; X 13 D or E; X 14 S, L or T; X 15 N or E; X 16 R or S; X 17 G or S; X 18 N or A; X 19 V or I; X 20 S, I or M; X 21 P or not present; X 22 is D or P; and X 23 is V or T, or a fragment thereof.

[0192] In another aspect, the disclosure features a polypeptide comprising a SIRPαD1 domain variant having the following sequence:

[0193] EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9ITPADAGTYYCX 10 KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 49), wherein X1 is V, L or I; X2 is A, I, V or L; X3 is I, F, S or T; X4 is E, V or L; X5 is K or R; X6 is E or Q; X7 is H, P or R; X8 is L, T, S or G; X9 is A; and X 10 is V or I; and wherein the variant comprises at least one amino acid substitution relative to a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 1.

[0194] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:49, wherein X1 is V, L or I. In any of the above embodiments of this aspect of the disclosure, X2 is A, I, V or L. In any of the above embodiments, X3 is I, F, S or T. In any of the above embodiments, X4 is E, V or L. In any of the above embodiments, X5 is K or R. In any of the above embodiments, X6 is E or Q. In any of the above embodiments, X7 is H, P or R. In any of the above embodiments, X8 is L, T, S or G. In any of the above embodiments, X9 is A. In any of the above embodiments, X 10 It is V or I.

[0195] In some embodiments, the polypeptide comprises a SIRPαD1 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, wherein X 1、 X 2、 X 3、 X 4、 X 5、 X 6、 X 7、 X 8、 X 9、 and X 10 Each of the amino acids is not a wild-type amino acid.

[0196] In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than ten amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 1. In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than seven amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 1.

[0197] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 1. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof binds to CD47 with an affinity that is less than 1x10 -8 M, less than 5x10 -9M, less than 1x10 -9 M, less than 5x10 -10 M, less than 1x10 -10 M or less than 1x10 -11 M of K D Binds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

[0198] In another aspect, the disclosure features a polypeptide comprising a SIRPαD1 domain variant having the following sequence:

[0199] EEELQX1IQPDKSVSVAAGESAILHCTX2TSLX3PVGPIQWFRGAGPAR X4LIYNQX5X6GX7FPRVTTVSEX8TKRENMDFSISISX9ITPADAGTYYCX 10 KFRKGSPDTEFKSGAGTELSVRAKPS, (SEQ ID NO: 50), wherein X1 is V or I; X2 is V or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is E or Q; X7 is H or P; X8 is S or T; X9 is N or A; and X 10 is V or I; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of any one of SEQ ID NO: 2.

[0200] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:50, wherein X1 is V or I. In any of the above embodiments of this aspect of the disclosure, X2 is V or I. In any of the above embodiments, X3 is I or F. In any of the above embodiments, X4 is E or V. In any of the above embodiments, X5 is K or R. In any of the above embodiments, X6 is E or Q. In any of the above embodiments, X7 is H or P. In any of the above embodiments, X8 is S or R. In any of the above embodiments, X9 is N or A. In any of the above embodiments, X 10 It is V or I.

[0201] In some embodiments, the polypeptide comprises a SIRPαD1 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, wherein X 1、 X 2、 X 3、 X 4、 X 5、 X 6、 X 7、 X 8、 X 9、 and X 10 Each of the amino acids is not a wild-type amino acid.

[0202] In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than ten amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 2. In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than seven amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 2.

[0203] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 2. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof binds to CD47 with an affinity that is less than 1x10 -8 M, less than 5x10 -9 M, less than 1x10 -9 M, less than 5x10 -10 M, less than 1x10 -10 M or less than 1x10 -11 M of K DBinds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

[0204] In another aspect, the disclosure features a polypeptide comprising a SIRPαD1 domain variant having the following sequence:

[0205] EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMDFSIRIGX8ITPADAGTYYCX9KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO:221), wherein X1 is V or I; X2 is A or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is H or P; X7 is L or T; X8 is N or A; and X9 is V or I; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of any one of SEQ ID NO:1.

[0206] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:51, wherein X1 is V or I. In any of the above embodiments of this aspect of the disclosure, X2 is A or I. In any of the above embodiments, X3 is I or F. In any of the above embodiments, X4 is E or V. In any of the above embodiments, X5 is K or R. In any of the above embodiments, X6 is H or P. In any of the above embodiments, X7 is L or T. In any of the above embodiments, X8 is N or A. In any of the above embodiments, X9 is V or I. In any of the above embodiments, X4 is not V.

[0207] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:51, wherein X8 is A. In any of the above embodiments of this aspect of the disclosure, X8 is A and X1 is V or I. In any of the above embodiments of this aspect of the disclosure, X8 is A and X2 is A or I. In any of the above embodiments, X8 is A and X3 is I or F. In any of the above embodiments, X8 is A and X4 is E or V. In some embodiments, X4 is not V. In any of the above embodiments, X8 is A and X5 is K or R. In any of the above embodiments, X8 is A and X6 is H or P. In any of the above embodiments, X8 is A and X7 is A or V. In any of the above embodiments, X8 is A and X9 is V or I.

[0208] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:51, wherein X8 is A. In any of the above embodiments of this aspect of the disclosure, X8 is A and X1 is I. In any of the above embodiments of this aspect of the disclosure, X8 is A and X2 is I. In any of the above embodiments, X8 is A and X3 is F. In any of the above embodiments, X8 is A and X4 is V. In any of the above embodiments, X8 is A and X5 is R. In any of the above embodiments, X8 is A and X6 is P. In any of the above embodiments, X8 is A and X7 is T. In any of the above embodiments, X8 is A and X9 is I.

[0209] In some embodiments, the polypeptide comprises a SIRPαD1 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: 51, wherein X 1、 X 2、 X 3、 X 4、 X 5、 X 6、 X 7、 X 8、 and each of X9 is not a wild-type amino acid.

[0210] In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than ten amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 1. In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than seven amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 1.

[0211] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000 times greater than a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 1. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof binds to CD47 with an affinity that is less than 1x10 -8 M, less than 5x10 -9 M, less than 1x10 -9 M, less than 5x10 -10 M, less than 1x10 -10 M or less than 1x10 -11 M of K D Binds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

[0212] In another aspect, the disclosure features a polypeptide comprising a SIRPαD1 domain variant having the following sequence:

[0213] EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGR ELIYNQX4EGX5FPRVTTVSDX6TKRNNMDFSIRIGX7ITPADAGTYYCVKFR KGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO:222), wherein X1 is V, L or I; X2 is A, I or L; X3 is I, T, S or F; X4 is K or R; X5 is H or P; X6 is L, T or G; X7 is N or A; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1.

[0214] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 222, wherein X1 is V, L or I. In any of the above embodiments of this aspect of the disclosure, X2 is A, I or L. In any of the above embodiments, X3 is I, T, S or F. In any of the above embodiments, X4 is K or R. In any of the above embodiments, X5 is H or P. In any of the above embodiments, X6 is L, T or G. In any of the above embodiments, X7 is N or A.

[0215] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 222, wherein X1 is V or I. In any of the above embodiments of this aspect of the disclosure, X2 is A or I. In any of the above embodiments, X3 is I or F. In any of the above embodiments, X4 is K or R. In any of the above embodiments, X5 is H or P. In any of the above embodiments, X6 is L or T. In any of the above embodiments, X7 is N or A.

[0216] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 222, wherein X7 is A. In any of the above embodiments of this aspect of the disclosure, X7 is A and X1 is V or I. In any of the above embodiments of this aspect of the disclosure, X7 is A and X2 is A or I. In any of the above embodiments, X7 is A and X3 is I or F. In any of the above embodiments, X7 is A and X4 is K or R. In any of the above embodiments, X7 is A and X5 is H or P. In any of the above embodiments, X7 is A and X6 is L or T.

[0217] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 222, wherein X7 is A. In any of the above embodiments of this aspect of the disclosure, X7 is A and X1 is I. In any of the above embodiments of this aspect of the disclosure, X7 is A and X2 is I. In any of the above embodiments, X7 is A and X3 is F. In any of the above embodiments, X7 is A and X4 is R. In any of the above embodiments, X7 is A and X5 is P. In any of the above embodiments, X7 is A and X6 is T.

[0218] In some embodiments, the polypeptide comprises a SIRPαD1 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, wherein X 1、 X 2、 X 3、 X 4、 X 5、 X 6、and each of X7 is not a wild-type amino acid.

[0219] In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than ten amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 1. In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than seven amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 1.

[0220] In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 10 times greater than that of a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 100 times greater than that of a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 1000 times greater than that of a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 1. In some embodiments, the fragment includes a polypeptide having a length of less than 10 amino acids, a length of about 10 amino acids, a length of about 20 amino acids, a length of about 30 amino acids, a length of about 40 amino acids, a length of about 50 amino acids, a length of about 60 amino acids, a length of about 70 amino acids, a length of about 80 amino acids, a length of about 90 amino acids, a length of about 100 amino acids, or a length of more than about 100 amino acids. The fragment retains the ability to bind to CD47. Preferably, the SIRPαD1 domain variant polypeptide and its fragment binds to CD47 with a higher affinity than the SIRPα polypeptide binds to CD47. For example, in some embodiments, the SIRPαD1 domain variant polypeptide or its fragment binds to CD47 with an affinity of less than 1×10 -8 M, less than 5x10 -9 M, less than 1x10 -9 M, less than 5x10 -10 M, less than 1x10 -10 M or less than 1x10 -11 M of K D Binds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

[0221] In another aspect, the disclosure features a polypeptide comprising a SIRPαD1 domain variant having the following sequence:

[0222] EEELQX1IQPDKSVSVAAGESAILHCTX2TSLX3PVGPIQWFRGAGPARE LIYNQX4EGX5FPRVTTVSEX6TKRENMDFSISISX7ITPADAGTYYCVKFRK GSPDTEFKSGAGTELSVRAKPS (SEQ ID NO: 212), wherein X1 is V, L or I; X2 is V, I or L; X3 is I, T, S or F; X4 is K or R; X5 is H, P or R; X6 is S, T or G; X7 is N or A; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of any one of SEQ ID NO: 2.

[0223] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, wherein X1 is V, L or I. In any of the above embodiments of this aspect of the disclosure, X2 is V, I or L. In any of the above embodiments, X3 is I, T, S or F. In any of the above embodiments, X4 is K or R. In any of the above embodiments, X5 is H or P. In any of the above embodiments, X6 is S, T or G. In any of the above embodiments, X7 is N or A.

[0224] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, wherein X1 is V or I. In any of the above embodiments of this aspect of the disclosure, X2 is V or I. In any of the above embodiments, X3 is I or F. In any of the above embodiments, X4 is K or R. In any of the above embodiments, X5 is H or P. In any of the above embodiments, X6 is S or T. In any of the above embodiments, X7 is N or A.

[0225] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, wherein X7 is A. In any of the above embodiments of this aspect of the disclosure, X7 is A and X1 is V or I. In any of the above embodiments of this aspect of the disclosure, X7 is A and X2 is V or I. In any of the above embodiments, X7 is A and X3 is I or F. In any of the above embodiments, X7 is A and X4 is K or R. In any of the above embodiments, X7 is A and X5 is H or P. In any of the above embodiments, X7 is A and X6 is S or T.

[0226] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, wherein X7 is A. In any of the above embodiments of this aspect of the disclosure, X7 is A and X1 is I. In any of the above embodiments of this aspect of the disclosure, X7 is A and X2 is I. In any of the above embodiments, X7 is A and X3 is F. In any of the above embodiments, X7 is A and X4 is R. In any of the above embodiments, X7 is A and X5 is P. In any of the above embodiments, X7 is A and X6 is T.

[0227] In some embodiments, the polypeptide comprises a SIRPαD1 domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to SEQ ID NO: 212, wherein X 1、 X 2、 X 3、 X 4、 X 5、 X 6、 and each of X7 is not a wild-type amino acid.

[0228] In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than ten amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 2. In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than seven amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 2.

[0229] In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 10 times greater than that of a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 100 times greater than that of a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 1000 times greater than that of a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 2. In some embodiments, the fragment includes a polypeptide having a length of less than 10 amino acids, a length of about 10 amino acids, a length of about 20 amino acids, a length of about 30 amino acids, a length of about 40 amino acids, a length of about 50 amino acids, a length of about 60 amino acids, a length of about 70 amino acids, a length of about 80 amino acids, a length of about 90 amino acids, a length of about 100 amino acids, or a length of more than about 100 amino acids. The fragment retains the ability to bind to CD47. Preferably, the SIRPαD1 domain variant polypeptide and its fragment binds to CD47 with a higher affinity than the SIRPα polypeptide binds to CD47. For example, in some embodiments, the SIRPαD1 domain variant polypeptide or its fragment binds to CD47 with an affinity of less than 1×10 -8 M, less than 5x10 -9 M, less than 1x10 -9 M, less than 5x10 -10 M, less than 1x10 -10 M or less than 1x10 -11 M of K D Binds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

[0230] In some embodiments, described herein are polypeptides comprising a SIRPαD1 domain variant having a sequence according to: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQ WFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9X 10 X 11 X 12 ADAGTYYCX 13KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 218), wherein X1 is V, L or I; X2 is A, V, L or I; X3 is I, S, T or F; X4 is E, L or V; X5 is K or R; X6 is E or Q; X7 is H, R or P; X8 is S, G, L or T; X9 is any amino acid; X 10 is any amino acid; X 11 is any amino acid; X 12 is any amino acid; and X 13 is V or I; and wherein the SIRPαD1 domain variant comprises at least one amino acid substitution relative to a wild-type SIRPαD1 domain having a sequence according to SEQ ID NO:1.

[0231] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, wherein X1, wherein X9 is A. In any of the above embodiments of this aspect of the disclosure, X9 is N. In any of the above embodiments of this aspect of the disclosure, X 10 is I. In any of the above embodiments of this aspect of the present disclosure, X9 is N and X 10 is P. In any of the above embodiments of this aspect of the present disclosure, X9 is N and X 11 is any amino acid except S, T or C. In any of the above embodiments of this aspect of the disclosure, X 11 is T. In any of the above embodiments of this aspect of the disclosure, X 11 is an amino acid other than T. In any of the above embodiments of this aspect of the disclosure, X 12 is P. In any of the above embodiments of this aspect of the present disclosure, X9 is N and X 12 is any amino acid except P.

[0232] In some embodiments, described herein are polypeptides comprising a SIRPα D1 domain variant having a sequence according to: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPI QWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9ITX 10 ADAGTYYCX 11 KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 219), wherein X1 is V, L or I; X2 is A, V, L or I; X3 is I, S, T or F; X4 is E, L or V; X5 is K or R; X6 is E or Q; X7 is H, R or P; X8 is S, G, L or T; X9 is N; X 10is any amino acid except P; and X 11 is V or I; and wherein the SIRPαD1 domain variant comprises at least two amino acid substitutions relative to the wild-type SIRPαD1 domain having a sequence according to SEQ ID NO:1.

[0233] In another aspect of the present disclosure, disclosed herein is a composition comprising a SIRPαD1 domain variant polypeptide or a fragment thereof having an amino acid sequence of SEQ ID NO: 48. In some embodiments, the SIRPαD1 domain variant polypeptide or a fragment thereof binds to CD47 with a higher affinity than the affinity of the SIRPα polypeptide to bind CD47. In some embodiments, the SIRPαD1 domain variant polypeptide binds to CD47 with an affinity of less than 1x10 -8 M, or less than 1x10 -9 M, less than 1x10 -10 M or less than 1x10 -11 M of K D Binding to CD47. In some embodiments, the above SIRPαD1 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.

[0234] Without limiting the foregoing, in some embodiments, the SIRPαD1 domain variant polypeptide is selected from any one of SEQ ID NOs: 53-87 and 213 shown in Table 6.

[0235] Table 6. SIRPα variant polypeptides

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to any variant provided in Table 6.

[0243] In some embodiments, the polypeptide comprises at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9 ... (SEQ ID NO:223) % or 100% sequence identity) to the SIRPαD1 domain.

[0244] Fc domain variants and fusion polypeptides containing the same

[0245] In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein α (SIRP-α) D1 variant, the variant comprising a SIRPαD1 domain or a fragment thereof, the SIRPαD1 domain having an amino acid mutation at residue 80 relative to the wild-type SIRPαD1 domain; and having at least one additional amino acid mutation at a residue selected from the group consisting of: residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66 and residue 92 relative to the wild-type SIRPαD1 domain.

[0246] In some embodiments, the present invention also discloses an Fc domain variant dimer, wherein the Fc domain variant dimer comprises two Fc domain variants, wherein each Fc domain variant is independently selected from (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236 and N297A.

[0247] Antibodies targeting cell surface antigens can trigger immunostimulatory and effector functions associated with Fc receptor (FcR) engagement on immune cells. There are many Fc receptors that are specific for specific classes of antibodies, including IgG (γ receptors), IgE (η receptors), IgA (α receptors) and IgM (μ receptors). The binding of the Fc region to the Fc receptors on the cell surface can trigger many biological responses, including phagocytosis of antibody-coated particles (antibody-dependent cell-mediated phagocytosis or ADCP), clearance of immune complexes, lysis of antibody-coated cells by killer cells (antibody-dependent cell-mediated cytotoxicity or ADCC) and control of the release of inflammatory mediators, placental transfer and immunoglobulin production. In addition, the binding of the C1 component of complement to antibodies can activate the complement system. The activation of complement is important for the lysis of cell pathogens. However, the activation of complement can also stimulate inflammatory responses and can also be involved in autoimmune hypersensitivity reactions or other immune disorders. Variant Fc regions with reduced or abolished ability to bind certain Fc receptors can be used to develop therapeutic antibodies and Fc-fusion polypeptide constructs that act by targeting, activating or neutralizing ligand function without damaging or destroying local cells or tissues.

[0248] In some embodiments, the SIRPαD1 polypeptide construct comprises a non-naturally occurring SIRPαD1 domain variant linked to an Fc domain variant, which forms an Fc domain with abolished or reduced effector function.

[0249] In some embodiments, Fc domain variant refers to a polypeptide chain including the second and third antibody constant domains (e.g., CH2 and CH3). In some embodiments, Fc domain variant also includes a hinge domain. In some embodiments, Fc domain variant is any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA and IgD. In addition, in some embodiments, Fc domain variant is any IgG subtype (e.g., IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3 and IgG4). In some embodiments, Fc domain variant comprises up to ten amino acid modifications (e.g., insertions, deletions and / or substitutions) (e.g., 1-10, 1-8, 1-6, 1-4 amino acid substitutions, additions or insertions, deletions or combinations thereof) relative to wild-type Fc domain monomer sequences, which changes the interaction between Fc domains and Fc receptors.

[0250] As used herein, the term "Fc domain dimer" refers to a dimer of two Fc domains. In a wild-type Fc domain dimer, two wild-type Fc domains dimerize through the interaction between two CH3 antibody constant domains and one or more disulfide bonds formed between the hinge domains of the two dimerized Fc domains.

[0251] As used herein, the term "Fc domain dimer variant" comprises at least one Fc domain variant. In some embodiments, the Fc domain dimer variant comprises an Fc domain variant that is mutated to lack effector function, such as a "dead Fc domain dimer variant". In some embodiments, each Fc domain in the Fc domain dimer variant comprises an amino acid substitution in the CH2 antibody constant domain to reduce the interaction or binding between the Fc domain dimer variant and an Fc receptor (such as an Fcγ receptor (FcγR), an Fcα receptor (FcαR) or an Fcε (FcεR)).

[0252] In some embodiments, a SIRPαD1 domain variant (e.g., any of the variants described in Tables 2, 5, and 6) is fused to an Fc domain variant of an immunoglobulin or a fragment of an Fc domain variant. In some embodiments, an Fc domain variant of an immunoglobulin or a fragment of an Fc domain variant is capable of forming an Fc domain dimer with another Fc domain variant. In some embodiments, an Fc domain variant of an immunoglobulin or a fragment of an Fc domain variant cannot form an Fc domain dimer with another Fc domain variant. In some embodiments, an Fc domain variant or a fragment of an Fc domain variant is fused to a polypeptide of the present invention to increase the serum half-life of the polypeptide. In some embodiments, an Fc domain variant or a fragment of an Fc domain variant fused to a polypeptide of the present invention dimerizes with a second Fc domain variant to form an Fc domain dimer variant that binds to an Fc receptor, alternatively, an Fc domain variant binds to an Fc receptor. In some embodiments, the Fc domain variant or fragment of the Fc domain variant fused to the polypeptide to increase the serum half-life of the polypeptide does not induce any immune system related response.

[0253] In some embodiments, the SIRPα polypeptide or construct provided herein comprises a SIRPαD1 domain or a variant thereof connected to a first Fc domain variant and an antibody variable domain connected to a second Fc domain variant, wherein the first and second Fc domain variants are combined to form an Fc domain dimer variant (e.g., a heterodimer Fc domain dimer variant). The Fc domain dimer is a protein structure found at the C-terminus of an immunoglobulin. The Fc domain dimer includes two Fc domains dimerized by interactions between CH3 antibody constant domains. The wild-type Fc domain dimer forms a minimal structure that binds to an Fc receptor, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and FcγRIV.

[0254] Fc domain dimers are not directly involved in the binding of antibodies to their targets, but may participate in various effector functions, such as antibody participation in antibody-dependent cellular toxicity. 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, which results in reduced effector functions, such as reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced complement-dependent cytolysis (CDC), reduced antibody-dependent cell-mediated phagocytosis (ADCP), or any combination thereof. In some embodiments, the SIRPα polypeptide or construct of the present disclosure is characterized by reduced binding to human Fc receptors (e.g., minimal binding or absence of binding) and reduced binding to complement protein C1q (e.g., minimal binding or absence of binding). In some embodiments, the SIRPα construct of the present disclosure is characterized by reduced binding to human FcγRI, FcγRIIA, FcγRIIB, FcγRIIIB, or any combination thereof and C1q (e.g., minimal binding or absence of binding). To alter or reduce antibody-dependent effector functions, such as ADCC, CDC, ADCP, or any combination thereof, in some embodiments, the Fc domain in the SIRPα constructs of the present disclosure is of IgG class and comprises one or more amino acid substitutions at E233, L234, L235, G236, G237, D265, D270, N297, E318, K320, K322, A327, A330, P331, or P329 (numbered according to the EU index of Kabat (Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991))).

[0255] In some embodiments, the polypeptide constructs comprising the non-native Fc regions described herein exhibit reduced or eliminated binding to at least one of the Fcγ receptors CD16a, CD32a, CD32b, CD32c, and CD64 compared to polypeptide constructs comprising native Fc regions. In some cases, the polypeptide constructs described herein exhibit reduced or eliminated binding to CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors.

[0256] CDC refers to a form of cytotoxicity in which the complement cascade is activated by the binding of complement component C1q to the Fc domain of an antibody. In some embodiments, a polypeptide construct comprising a non-natural Fc region as described herein exhibits at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in C1q binding compared to a polypeptide construct comprising a wild-type Fc region. In some cases, a polypeptide construct comprising a non-natural Fc region as described herein exhibits reduced CDC compared to a polypeptide construct comprising a wild-type Fc region. In some embodiments, a polypeptide construct comprising a non-natural Fc region as described herein exhibits at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in CDC compared to a polypeptide construct comprising a wild-type Fc region. In some cases, a polypeptide construct comprising a non-naturally occurring Fc domain variant or Fc domain dimer variant described herein exhibits negligible CDC compared to a polypeptide construct comprising a wild-type Fc region.

[0257] 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 mutation of N297 to any amino acid other than N. In some embodiments, deglycosylation is achieved by destroying the motif N-Xaa1-Xaa2-Xaa3, wherein N=asparagine; Xaa1=any amino acid except P (proline); Xaa2=T (threonine), S (serine) or C (cysteine); and Xaa3=any amino acid except P (proline). In one embodiment, the N-Xaa1-Xaa2-Xaa3 motif refers to residues 297-300 specified according to Kabat et al., 1991. In some embodiments, mutation of any one or more of N, Xaa1, Xaa2, or Xaa3 results in aglycosylation of the Fc domain variant or Fc domain dimer variant.

[0258] In some embodiments, variants of antibody IgG constant regions (e.g., Fc domain variants or Fc domain dimer variants) have the ability of specific binding to Fcγ receptors reduced or have the ability of inducing phagocytosis reduced. In some embodiments, variants of antibody IgG constant regions (e.g., Fc domain variants or Fc domain dimer variants) have the ability of specific binding to Fcγ receptors reduced and have the ability of inducing phagocytosis reduced. For example, in some embodiments, Fc domain variants are mutated to lack effector functions, typically "dead" Fc domain variants. For example, in some embodiments, Fc domain variants include specific amino acid substitutions known to minimize the interaction between Fc domain dimers and Fcγ receptors. In some embodiments, Fc domain variants are from IgG1 antibodies and include one or more of amino acid substitutions L234A, L235A, G237A and N297A (according to Kabat et al., 1991 EU numbering system designation). In some embodiments, one or more additional mutations are included in such IgG1 Fc domain variants. Non-limiting examples of such additional mutations of human IgG1 Fc domain variants include E318A and K322A. In some cases, human IgG1 Fc domain variants have up to 12, 11, 10, 9, 8, 7, 6, 5 or 4 or fewer mutations compared to wild-type human IgG1 sequences. In some embodiments, one or more additional deletions are included in such IgG1 Fc domain variants. For example, in some embodiments, the C-terminal lysine of the Fc domain IgG1 heavy chain constant region provided in SEQ ID NO:88 in Table 7 is deleted, for example, to increase the homogeneity of the polypeptide when the polypeptide is produced in bacteria or mammalian cells. In some cases, human IgG1 Fc domain variants have up to 12, 11, 10, 9, 8, 7, 6, 5 or 4 or fewer deletions compared to wild-type human IgG1 sequences (see, for example, SEQ ID NO:161 below). In some embodiments, the IgG1 Fc domain variant has a sequence according to any one of SEQ ID NO:135, SEQ ID NO:136, or SEQ ID NO:137.

[0259] SEQ ID NO:161:

[0260] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0261] In some embodiments, Fc domain variants are from IgG2 or IgG4 antibodies and include amino acid substitutions A330S, P331S or both A330S and P331S. The above amino acid positions are defined according to Kabat et al. (1991). For a given antibody, the kabat numbering of amino acid residues can be determined by comparing the homology region of the antibody sequence with the "standard" Kabat numbering sequence. In some embodiments, Fc domain variants include human IgG2 Fc domain sequences, which include one or more of A330S, P331S and N297A amino acid substitutions (according to Kabat, et al. (1991) EU numbering system designation). In some embodiments, one or more additional mutations are included in such IgG2 Fc domain variants. The non-limiting examples of such additional mutations of human IgG2 Fc domain variants include V234A, G237A, P238S, V309L and H268A (according to Kabat, et al. (1991) EU numbering system designation). In some cases, human IgG2 Fc domain variants have up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or fewer mutations compared to wild-type human IgG2 sequences. In some embodiments, one or more additional deletions are included in such IgG2 Fc domain variants. For example, in some embodiments, the C-terminal lysine of the Fc domain IgG2 heavy chain constant region provided in SEQ ID NO:89 in Table 7 is deleted, for example, to increase the homogeneity of the polypeptide when the polypeptide is produced in bacteria or mammalian cells. In some cases, human IgG2 Fc domain variants have up to 12, 11, 10, 9, 8, 7, 6, 5 or 4 or fewer deletions compared to wild-type human IgG2 sequences (see, for example, SEQ ID NO:162 below).

[0262] SEQ ID NO:162:

[0263] ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPI EKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0264] When the Fc domain variant is an IgG4 Fc domain variant, in some embodiments, such Fc domain variant comprises an S228P mutation (as specified according to Kabat, et al. (1991)). In some cases, the human IgG4 Fc domain variant has up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations in total 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 S228P, E233P, F234V, L235A and delG236 amino acid substitutions (as specified according to the EU numbering system of Kabat, et al. (1991)). In some embodiments, the Fc domain variant comprises a human IgG4 Fc sequence comprising one or more of S228P, E233P, F234V, L235A, delG236 and N297A amino acid substitutions (as specified according to the EU numbering system of Kabat, et al. (1991)).

[0265] In some embodiments, the Fc domain variant comprises at least one of the mutations L234A, L235A, G237A or N297A in the IgG1 Fc region or at least one of the mutations A330S, P331S or N297A in the IgG2 Fc region. In some embodiments, the Fc domain variant comprises at least two of the mutations L234A, L235A, G237A or N297A in the IgG1 Fc region or at least two of the mutations A330S, P331S or N297A in the IgG2 Fc region. In some embodiments, the Fc domain variant comprises at least three of the mutations L234A, L235A, G237A or N297A in the IgG1 Fc region or consists of the mutations A330S, P331S and N297A in the IgG2 Fc region. In some embodiments, the Fc domain variant consists of the mutations L234A, L235A, G237A and N297A.

[0266] In some embodiments, the Fc domain variants exhibit reduced binding to a subject's Fc receptor compared to a wild-type human IgG Fc region. In some embodiments, the Fc domain variants exhibit eliminated binding to a subject's Fc receptor compared to a wild-type human IgG Fc region. In some embodiments, the Fc domain variants exhibit reduced phagocytosis compared to a wild-type human IgG Fc region. In some embodiments, the Fc domain variants exhibit eliminated phagocytosis compared to a wild-type human IgG Fc region.

[0267] The amino acid sequences of the Fc domain IgG1 and IgG2 heavy chain constant regions are provided in SEQ ID NO: 88 and SEQ ID NO: 89. In some embodiments, the Fc domain variant is any variant of SEQ ID NO: 90-95 as shown in Table 7.

[0268] Table 7. Amino acid sequences of Fc domain variants

[0269]

[0270]

[0271] Antibody-dependent cell-mediated cytotoxicity (also referred to herein as ADCC) refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells and neutrophils), enabling these cytotoxic effector cells to specifically bind to target cells carrying antigens, and then kill 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 phagocytic cells (e.g., macrophages), enabling these phagocytic effector cells to specifically bind to target cells carrying antigens, and then engulf and digest target cells. Ligand-specific high-affinity IgG antibodies directed against the surface of target cells can stimulate cytotoxic or phagocytic cells and can be used for such killing. In some embodiments, polypeptide constructs comprising 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, compared with a polypeptide construct comprising a wild-type Fc region, a polypeptide construct comprising an Fc domain variant or an Fc domain dimer variant as described herein exhibits at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in ADCC or ADCP. In some embodiments, compared with a polypeptide construct comprising a wild-type Fc region, a polypeptide construct comprising an Fc domain variant or an Fc domain dimer variant as described herein exhibits eliminated ADCC or ADCP.

[0272] Complement directed cytotoxicity (also referred to herein as CDC) refers to a form of cytotoxicity in which the complement cascade is activated by the binding of complement component C1q to the Fc domain of an antibody. In some embodiments, a polypeptide construct comprising an Fc domain variant or Fc domain dimer variant as described herein exhibits at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in C1q binding compared to a polypeptide construct comprising a wild-type Fc region. In some cases, a polypeptide construct comprising an Fc domain variant or Fc domain dimer variant as described herein exhibits reduced CDC compared to a polypeptide construct comprising a wild-type Fc region. In some embodiments, compared to a polypeptide construct comprising a wild-type Fc region, a polypeptide construct comprising an Fc domain variant or an Fc domain dimer variant as described herein exhibits at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in CDC. In some cases, compared to a polypeptide construct comprising a wild-type Fc region, a polypeptide construct comprising an Fc domain variant or an Fc domain dimer variant as described herein exhibits negligible CDC.

[0273] Fc domain variants or Fc domain dimer variants herein include those that show reduced binding to Fcγ receptors compared to wild-type human IgG Fc regions. For example, in some embodiments, compared to the binding to Fcγ receptors shown by wild-type human IgG Fc regions, Fc domain variants or Fc domain dimer variants show smaller binding to Fcγ receptors, as described in the embodiments. In some cases, Fc domain variants or Fc domain dimer variants are reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (completely eliminated effector functions) for binding to Fcγ receptors. In some embodiments, the reduced binding is for any one or more Fcγ receptors, such as CD16a, CD32a, CD32b, CD32c or CD64.

[0274] In some cases, Fc domain variants or Fc domain dimer variants disclosed herein show reduced phagocytosis compared to their wild-type human IgG Fc regions. Such Fc domain variants or Fc domain dimer variants show reduced phagocytosis compared to their wild-type human IgG Fc regions, wherein phagocytic activity is reduced by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some cases, Fc domain variants or Fc domain dimer variants show eliminated phagocytosis compared to their wild-type human IgG Fc regions.

[0275] In some embodiments, the Fc domain variants or Fc domain dimer variants disclosed herein are coupled to one or more fusion partners. In some cases, the fusion partner is a therapeutic part. In some cases, the fusion partner is selected to be able to target expressed proteins, purification, screening, display, etc. In some embodiments, the fusion partner also affects the degree of binding to Fc receptors or the degree of phagocytosis reduction. As described herein, in some embodiments, when the Fc domain variants or Fc domain dimer variants are coupled to a fusion partner, it forms a polypeptide construct as described below.

[0276] In some embodiments, the fusion partner is connected to the Fc domain variant or Fc domain dimer variant sequence by a joint sequence. In some embodiments, the joint sequence generally comprises a small amount of amino acids, such as less than ten amino acids, although longer joints are also used. In some cases, the joint has a length less than 10, 9, 8, 7, 6 or 5 amino acids or shorter. In some cases, the joint has a length of at least 10, 11, 12, 13, 14, 15, 20, 25, 30 or 35 amino acids or longer. Optionally, in some embodiments, a cleavable joint is used.

[0277] In some embodiments, fusion partner is to direct Fc domain variant or Fc domain dimer variant protein and any relevant fusion partner to the target or signal sequence of desired cell position or extracellular medium.In some embodiments, some signal sequence targets secretion into the growth medium or the protein in the periplasmic space between the inner membrane and the outer membrane.In some embodiments, fusion partner is to encode the sequence of peptide or protein that can be purified or screened.Such fusion partner includes but is not limited to polyhistidine tag (His-tag) (such as His6 (SEQ ID NO:223) and His10 (SEQ ID NO:224)) or other tags for immobilized metal affinity chromatography (IMAC) system (such as Ni+2 affinity column), GST fusion, MBP fusion, Strep-tag, BSP biotinylation target sequence of bacterial enzyme BirA and epitope tag (such as c-myc tag, flag-tag etc.) of antibody targeting.

[0278] In some embodiments, such tags can be used for purification, screening, or both. For example, in some embodiments, Fc domain variants or Fc domain dimer variants are purified by fixing them to a Ni+2 affinity column using a His-tag, and then after purification, the same His-tag is used to fix the antibody to a Ni+2 coated plate for ELISA or other binding assays, as described elsewhere herein. In some embodiments, the fusion partner enables the use of a selection method to screen Fc domain variants or Fc domain dimer variants as described herein.

[0279] Various fusion partners that can realize multiple selection methods can be used. For example, by fusing the member of Fc domain variant or Fc domain dimer variant library with gene III protein, phage display can be used. In some embodiments, fusion partner Fc domain variant or Fc domain dimer variant is marked. Alternatively, in some embodiments, fusion partner is combined with a specific sequence on an expression vector so that fusion partner and related Fc domain variant or Fc domain dimer variant can be covalently or non-covalently connected with the nucleic acid encoding them.

[0280] In some embodiments, when the fusion partner is a therapeutic moiety, the therapeutic moiety is, for example, a peptide, protein, antibody, siRNA, or small molecule. Non-limiting examples of therapeutic antibodies coupled 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 coupled to the Fc domain variants or Fc domain dimer variants of the present disclosure include, but are not limited to, CD47 binding polypeptides, including 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. Other examples of CD47 binding polypeptides include, but are not limited to, naturally occurring forms of SIRPα and mutants thereof.

[0281] In some embodiments, disclosed herein are polypeptides comprising Fc domain dimer variants, wherein the Fc domain dimer variants comprise two Fc domain variants, wherein each Fc domain variant is independently selected from (i) a human IgG1 Fc region consisting of L234A, L235A, G237A, and N297A mutations; (ii) a human IgG2 Fc region consisting of A330S, P331S, and N297A mutations; or (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations. In some embodiments, the Fc domain variants are identical (i.e., homodimers). In some embodiments, the Fc domain variants are different (i.e., heterodimers). In some embodiments, at least one Fc domain variant in the Fc domain dimer is a human IgG1 Fc region consisting of mutations L234A, L235A, G237A, and N297A. In some embodiments, at least one Fc domain variant in the Fc domain dimer is a human IgG2 Fc region consisting of mutations A330S, P331S, and N297A. In some embodiments, compared to a wild-type version of the human IgG Fc region, the Fc domain dimer variant exhibits eliminated or reduced binding to Fcγ receptors. In some embodiments, compared to a wild-type version of the human IgG Fc region, the Fc domain dimer variant exhibits eliminated or reduced binding to CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors. In some embodiments, compared to a wild-type version of the human IgG Fc fusion, the Fc domain dimer variant exhibits eliminated or reduced binding to C1q. In some embodiments, at least one of the Fc domain dimer variants is a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236, and N297A. In some embodiments, the Fc domain dimer variant exhibits eliminated or reduced binding to Fcγ receptors compared to the wild-type human IgG4 Fc region. In some embodiments, the Fc domain dimer variant exhibits eliminated or reduced binding to CD16a and CD32b Fcγ receptors compared to the wild-type version of its human IgG4 Fc region. In some embodiments, the Fc domain dimer variant is greater than about 5x10 -6 M of K D Binds to Fcγ receptors.

[0282] In some embodiments, the Fc domain dimer variant further comprises a CD47 binding polypeptide. In some embodiments, the Fc domain dimer variant exhibits eliminated or reduced binding to Fcγ receptors compared to the wild-type version of the human IgG Fc region. In some embodiments, the CD47 binding polypeptide does not cause acute anemia in rodents and non-human primates. In some embodiments, the CD47 binding polypeptide does not cause acute anemia in humans.

[0283] In some embodiments, the CD47 binding polypeptide is a signal regulatory protein alpha (SIRP-α) polypeptide or a fragment thereof. In some embodiments, the SIRPα polypeptide comprises a SIRPαD1 domain variant comprising the following amino acid sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWF RGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMDFSIRIGX8ITPADAG TYYCX9KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 221), wherein X1 is V or I; X2 is A or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is H or P; X7 is L or T; X8 is any amino acid except N; and X9 is V or I. In some embodiments, the SIRPα polypeptide comprises a 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.

[0284] In some embodiments, disclosed herein is a polypeptide comprising: a SIRPαD1 domain variant, wherein the SIRPαD1 domain variant is a non-naturally occurring high-affinity SIRPαD1 domain, wherein the SIRPαD1 domain variant binds to human CD47 with an affinity at least 10 times greater than that of a naturally occurring D1 domain; and an Fc domain variant, wherein the Fc domain variant is linked to a second polypeptide comprising a second Fc domain variant to form an Fc domain dimer variant, wherein the Fc domain dimer variant has an effector function that is eliminated or reduced. In some embodiments, the non-naturally occurring high-affinity SIRPαD1 domain comprises an amino acid mutation at residue 80.

[0285] In some embodiments, a SIRPαD1 domain variant is disclosed herein, wherein the SIRPαD1 domain variant binds CD47 from a first species with a KD of less than 250nM; and wherein the SIRPαD1 domain variant binds CD47 from a second species with a KD of less than 250nM; and the KD of CD47 from the first species and the KD of CD47 from the second species are within 100 times of each other; wherein the first species and the second species are selected from the group consisting of: humans, rodents, and non-human primates. In some embodiments, the SIRPαD1 domain variant binds CD47 from at least 3 different species. In some embodiments, the non-human primate is a cynomolgus monkey.

[0286] In some embodiments, disclosed herein is a polypeptide comprising (a) a SIRPαD1 domain that binds to human CD47 with a KD of less than 250nM; and (b) an Fc domain or a variant thereof connected to the N-terminus or C-terminus of the SIRPαD1 domain, wherein the polypeptide does not cause acute anemia in rodents and non-human primates. In some embodiments, the polypeptide is a non-naturally occurring variant of human SIRP-α. In some embodiments, in vivo administration of the polypeptide results in a decrease in hemoglobin of less than 50% within the first week after administration. In some embodiments, administration of the polypeptide in humans results in a decrease in hemoglobin of less than 50% within 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 the following: (i) a human IgG1 Fc region consisting of L234A, L235A, G237A and N297A mutations; (ii) a human IgG2 Fc region consisting of A330S, P331S and N297A mutations; or (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236 and N297A mutations. In some embodiments, the Fc domain variant is a human IgG1 Fc region 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.

[0287] The SIRPα construct of the present disclosure includes a SIRPα domain or a variant thereof, the C-terminus of which is connected to the N-terminus of an Fc domain or a variant thereof by a joint using conventional genetic or chemical means (e.g., chemical conjugation). In some embodiments, a joint (e.g., a spacer) is inserted between a polypeptide and an Fc domain or a variant thereof. In some embodiments, a polypeptide of the present disclosure comprising a SIRPαD1 domain variant is fused with an Fc domain variant that cannot form a dimer. In some embodiments, the polypeptide of the present disclosure is fused with an Fc domain or a variant thereof, and the Fc domain or a variant thereof is capable of forming a dimer, such as a heterodimer, with another Fc domain or a variant thereof. In some embodiments, the polypeptide of the present disclosure is fused with an Fc domain or a variant thereof, and the fusion protein forms a homodimer. In some embodiments, the polypeptide of the present disclosure is fused with a first Fc domain or a variant thereof, and a different protein or peptide (e.g., an antibody variable region) is fused with a second Fc domain or a variant thereof. In some embodiments, the SIRPαD1 domain or a variant thereof is linked to a first Fc domain or a variant thereof and a therapeutic protein (e.g., a cytokine, an interleukin, an antigen, a steroid, an anti-inflammatory agent, or an immunomodulator) is linked to a second Fc domain or a variant thereof. In some embodiments, the first and second Fc domains or variants thereof form a heterodimer.

[0288] Without limiting the foregoing, in some embodiments, a SIRPαD1 domain variant polypeptide (e.g., any of the variants described in Tables 2, 5, and 6) is fused to an Fc polypeptide or Fc variant polypeptide (such as an Fc domain or a variant thereof). Examples of polypeptides comprising a SIRPαD1 domain variant polypeptide and a fused Fc domain variant polypeptide include, but are not limited to, SEQ ID NOs: 96-137, 214, and 216 shown in Table 8.

[0289] Table 8. Polypeptides comprising SIRPαD1 domain variants fused to Fc domain variants

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301] In some embodiments, the polypeptide comprises a SIRPαD1 variant domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to any variant provided in Table 8.

[0302] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to SEQ ID NO: 98-104, 107-113, 116-122 or 135-137 in Table 8.

[0303] In some embodiments, the polypeptide comprises (a) a signal regulatory protein alpha (SIRP-α) D1 variant, wherein the SIRPα D1 domain variant comprises an amino acid sequence,

[0304] EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSX 11 PVGPIQWFRGAG PX 12 RX 13 SXWh 14 X15GX 16 FPRVTTVSX 17 X 18 TX 19 RX 20 NMDFX 21 IX 22 IX 23 X 24 ITX 25 ADAGTYYCX 26 KX 27 RKGSPDX 28 X 29 EX 30 KSGAGTELSVRX 31KPS (SEQ ID NO: 47), wherein X1 is E or G; X2 is L, I or V; X3 is V, L or I; X4 is S or F; X5 is L or S; X6 is S or T; X7 is A or V; X8 is I or T; X9 is H, R or L; X 10 is A, V, I or L; X 11 is I, T, S or F; X 12 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 does not exist; X 29 is T or V; X 30 is F or V; and X 31 is A or G; and wherein the SIRPαD1 domain variant comprises at least two amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence according to any one of SEQ ID NOs: 1 to 10; and (b) an Fc domain dimer variant having two Fc domain variants, wherein each Fc domain variant is independently (i) a human IgG1 Fc region 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. Fc region; (vi) a human IgG2 Fc region comprising A330S, P331S and N297A mutations; (vii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A and delG236 mutations; or (viii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236 and N297A mutations.

[0305] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant, wherein the SIRPαD1 domain variant comprises an amino acid sequence according to SEQ ID NO:47; an Fc domain dimer having two Fc domains, wherein one of the Fc domains is an Fc domain variant comprising a human IgG1 Fc region, and the human IgG1 Fc region comprises L234A, L235A, G237A and N297A mutations.

[0306] Dimerization of the Fc domain

[0307] In some embodiments, the SIRPαD1 domain variant polypeptide (e.g., any of the variants described in Tables 2, 5, and 6) is fused to the first Fc domain (e.g., Fc domain variant) at the N-terminus or C-terminus. In some embodiments, the first Fc domain is a variant that cannot form a dimer. In some embodiments, the first Fc domain forms a dimer with the second Fc domain. In some embodiments, the first and second Fc domains comprise amino acid substitutions that promote heterodimerization between the first and second domain Fc domains.

[0308] In some embodiments, each of the two Fc domains in the Fc domain dimer comprises an amino acid substitution that promotes heterodimerization of two monomers. In some embodiments, the SIRPα construct is, for example, formed by a first subunit comprising a SIRPαD1 domain variant polypeptide fused to a first Fc domain and a second subunit comprising a second Fc domain (e.g., without a SIRPαD1 domain variant polypeptide or any other polypeptide). In some embodiments, the construct has a single SIRPαD1 domain variant polypeptide (e.g., a single arm) connected to an Fc domain dimer. In some embodiments, the construct has two SIRPαD1 domain variant polypeptides (e.g., two arms) connected to an Fc domain dimer. In some embodiments, a SIRPαD1 domain variant with a KD of about 500nM may be particularly useful in a two-arm construct. In some embodiments, a SIRPαD1 domain variant with a KD of about 50nM may be particularly useful in a two-arm construct. In some embodiments, a SIRPαD1 domain variant with a KD of about 5nM may be useful in a two-arm construct. In some embodiments, SIRPαD1 domain variants with a KD of about 500 pM can be used for double-armed constructs and single-armed constructs. In some embodiments, SIRPαD1 domain variants with a KD of about 100 pM can be used for double-armed constructs and single-armed constructs. In some embodiments, SIRPαD1 domain variants with a KD of about 50 pM can be used for double-armed constructs and single-armed constructs. In some embodiments, SIRPαD1 domain variants with a KD of about 10 pM can be used for double-armed constructs and single-armed constructs.

[0309] 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. Knob and hole interactions favor heterodimer formation, while knob-knob and hole-hole interactions hinder homodimer formation due to steric conflicts and lack of favorable interactions. Hole refers to the gap created when the original amino acid in a protein is replaced by a different amino acid with a smaller side chain volume. Knob refers to the protrusion created when the original amino acid in a protein is replaced by a different amino acid with a larger side chain volume. For example, in some embodiments, the replaced amino acid is in the CH3 antibody constant domain of the Fc domain and participates in the dimerization of the two Fc domains. In some embodiments, a hole is created in one CH3 antibody constant domain to accommodate the knob in another CH3 antibody constant domain, so that the knob and hole amino acids are used to promote or favor heterodimerization of the two Fc domains. In some embodiments, a hole is created in one CH3 antibody constant domain to better accommodate the original amino acid in another CH3 antibody constant domain. In some embodiments, a knob is created in one CH3 antibody constant domain to form additional interactions with the original amino acid in another CH3 antibody constant domain.

[0310] In some embodiments, a hole is constructed by replacing an amino acid with a larger side chain, such as tyrosine or tryptophan, with an amino acid with a smaller side chain, such as alanine, valine, or threonine, such as the Y407V mutation in the CH3 antibody constant domain. Similarly, in some embodiments, a knob is constructed by replacing an amino acid with a larger side chain with an amino acid with a smaller side chain, such as the T366W mutation in the CH3 antibody constant domain. In some embodiments, one Fc domain comprises a knob mutation T366W, and the other Fc domain comprises a hole mutation T366S, L358A, and Y407V. In some embodiments, a polypeptide of the present disclosure comprising a SIRPαD1 domain variant is fused to an Fc domain comprising a knob mutation T366W to limit unwanted knob-knob homodimer formation. Examples of knob-hole amino acid pairs include, but are not limited to, Table 9, and Table 10 provides examples of knob-hole Fc domain variants and SIRPα-Fc fusions.

[0311] Table 9. Knob-to-Hole Amino Acid Pairs

[0312]

[0313]

[0314] Table 10. Exemplary Fc domain variants and SIRPαD1 domain variant-Fc domain variant fusion polypeptides

[0315]

[0316]

[0317]

[0318] In addition to the knob-and-mortar strategy, in some embodiments, electrostatic steering is also used to control the dimerization of the Fc domain. 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, in order to use electrostatic steering to control the dimerization of the Fc domain, one or more amino acid residues constituting the CH3-CH3 interface are replaced with positively or negatively charged amino acid residues so that the interaction becomes electrostatically favorable or unfavorable, depending on the specific charged amino acids introduced. In some embodiments, positively charged amino acids (such as lysine, arginine, or histidine) in the interface are replaced by negatively charged amino acids (such as aspartic acid or glutamic acid). In some embodiments, negatively charged amino acids in the interface are replaced by positively charged amino acids. In some embodiments, charged amino acids are introduced into one or two of the interacting CH3 antibody constant domains. In some embodiments, the introduction of charged amino acids into the interacting CH3 antibody constant domains of the two Fc domains promotes the selective formation of heterodimers of the Fc domains, as controlled by the electrostatic steering effect generated by the interaction between the charged amino acids. Examples of electrostatic steering amino acid pairs include, but are not limited to, Table 11.

[0319] Table 11. Electrostatic steering amino acid pairs

[0320]

[0321] Other methods for controlling heterodimerization of Fc domains may be used, particularly in the context of constructing bispecific antibodies.

[0322] In some embodiments, the first Fc domain and the second Fc domain each include one or more of the following amino acid substitutions relative 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.

[0323] In some embodiments, the Fc domain comprises: (a) one of the following amino acid substitutions relative to wild-type human IgG1: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T or K409I; or (b)(i) relative to human IgG1 N297A mutation in the Fc region; (ii) L234A, L235A and G237A mutations relative to human IgG1 Fc region; (iii) L234A, L235A, G237A and N297A mutations relative to human IgG1 Fc region; (iv) N297A mutation relative to human IgG2 Fc region; (v) A330S and P331S mutations relative to human IgG2 Fc region; (vi) A330S, P331S and N297A mutations relative to human IgG2 Fc region; (vii) S228P, E233P, F234V, L235A and delG236 mutations relative to human IgG4 Fc region; or (viii) S228P, E233P, F234V, L235A, delG236 and N297A mutations relative to 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, S364A, T366E, L368T, L 368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T or K409I; and (b) further comprising (i) an N297A mutation relative to the Fc region of human IgG1; (ii) an N297A mutation relative to the Fc region of human IgG1 (iii) L234A, L235A and G237A mutations relative to human IgG1 Fc region; (iv) N297A mutation relative to human IgG2 Fc region; (v) A330S and P331S mutations relative to human IgG2 Fc region; (vi) A330S, P331S and N297A mutations relative to human IgG2 Fc region; (vii) S228P, E233P, F234V, L235A and delG236 mutations relative to human IgG4 Fc region; or (viii) S228P, E233P, F234V, L235A, delG236 and N297A mutations relative to human IgG4 Fc region.

[0324] In some embodiments, the first and second Fc domains 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.

[0325] Connectors

[0326] In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein α (SIRP-α) D1 variant, the variant comprising a SIRPαD1 domain or a fragment thereof, the SIRPαD1 domain having an amino acid mutation at residue 80 relative to the wild-type SIRPαD1 domain; and having at least one additional amino acid mutation at a residue selected from the group consisting of: residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66 and residue 92 relative to the wild-type SIRPαD1 domain.

[0327] In some embodiments, the present invention also discloses a polypeptide comprising an Fc variant, wherein the Fc variant comprises an Fc domain dimer, wherein the Fc domain dimer comprises two Fc domain variants, wherein each Fc domain variant is independently selected from (i) a human IgG1 Fc region consisting of L234A, L235A, G237A and N297A mutations; (ii) a human IgG2 Fc region consisting of A330S, P331S and N297A mutations; or (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236 and N297A mutations.

[0328] In the present disclosure, a linker is used to describe a linkage or connection between a polypeptide or protein domain or an associated non-protein moiety. In some embodiments, the linker is a linkage or connection between an Fc domain (or a variant thereof) and a SIRPαD1 domain variant. In some embodiments, the linker connects the C-terminus of the SIRPαD1 domain variant and the N-terminus of the Fc domain variant, such that the two polypeptides are connected to each other in series.

[0329] In some embodiments, the linker is a simple covalent bond, such as a peptide bond, a synthetic polymer, or any kind of bond produced by a chemical reaction (e.g., chemical conjugation). When the linker is a peptide bond, in some embodiments, the carboxylic acid group at the C-terminus of one protein domain reacts with the amino group at the N-terminus of another protein domain in a condensation reaction to form a peptide bond. In some embodiments, the peptide bond is formed by conventional organic chemical reactions by synthetic means, or by natural production by host cells, wherein the nucleic acid molecule encoding the DNA sequence of the two proteins in series (e.g., Fc domain variants and SIRPαD1 domain variants) can be directly transcribed and translated into continuous polypeptides encoding the two proteins by the necessary molecular machinery (e.g., DNA polymerases and ribosomes) in the host cell.

[0330] When the linker is a synthetic polymer, in some embodiments, the polymer is functionalized at each end with a reactive chemical functional group to react with the terminal amino acid at the connecting end of the two proteins.

[0331] When the linker (other than the peptide bond described above) is made by 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. In some embodiments, the two functional groups are then reacted by synthetic chemistry to form a chemical bond, thereby connecting the two proteins together.

[0332] Spacer

[0333] In the present disclosure, in some embodiments, the linker between the Fc domain monomer and the SIRPαD1 variant polypeptide of the present disclosure is an amino acid spacer comprising about 1-200 amino acids. Suitable peptide spacers include peptide linkers containing flexible amino acid residues such as glycine and serine. Examples of linker sequences are provided in Table 12. In some embodiments, the spacer contains a motif of GS, GG, GGS, GGG, GGGGS (SEQ ID NO: 163), GGSG (SEQ ID NO: 164) or SGGG (SEQ ID NO: 165), such as multiple or repeated motifs. In some embodiments, the spacer contains 2 to 12 amino acids, including a GS motif, such as GS, GSGS (SEQ ID NO: 166), GSG SGS (SEQ ID NO: 167), GSGSGSGS (SEQ ID NO: 168), GSGSGSGSGS (SEQ ID NO: 169) or GSGSGSGSGSGS (SEQ ID NO: 170). In some embodiments, the spacer contains 3 to 12 amino acids, including a GGS motif, such as GGS, GGSGGS (SEQ ID NO: 171), GGSGGSGGS (SEQ ID NO: 172), and GGSGGSGGSGGS (SEQ ID NO: 173). In some embodiments, the spacer contains 4 to 12 amino acids, including a GGSG (SEQ ID NO: 164) motif, such as GGSG (SEQ ID NO: 164), GG SGGGSG (SEQ ID NO: 174), or GGSGGGSGGGSG (SEQ ID NO: 175). In some embodiments, the spacer contains a GGGGS (SEQ ID NO: 163) motif, such as GGG GSGGGGSGGGGS (SEQ ID NO: 176).In some embodiments, the spacer contains amino acids other than glycine and serine, such as AAS (SEQ ID NO: 177), AAAL (SEQ ID NO: 178), AAAK (SEQ ID NO: 179), AAAR (SEQ ID NO: 180), EGKSSGSGSESKST (SEQ ID NO: 181), GSAGSAAGSGEF (SEQ ID NO: 182), AEAAAKEAAAKA (SEQ ID NO: 183), KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), GGGGAGGGG (SEQ ID NO: 185), GENLYFQSGG (SEQ ID NO: 186), SACYCELS (SEQ ID NO: 187), RSIAT (SEQ ID NO: 188), RPACKIPNDLKQKVMNH (SEQ ID NO: 189), GGSAGGSGSGSSGG SSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 190), AAANSSIDLISVPVDSR (SEQ ID NO: 191), NO:191) or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGG SGGGS (SEQ ID NO:192).

[0334] In some embodiments, the spacer contains a motif, such as multiple or repeated motifs, of EAAAK (SEQ ID NO: 193). In some embodiments, the spacer contains a motif, such as multiple or repeated motifs, of a proline-rich sequence such as (XP)n, wherein X is any amino acid (e.g., A, K, or E), n is 1-5, and a motif of PAPAP (SEQ ID NO: 194).

[0335] Table 12. Linker sequences

[0336]

[0337]

[0338] In some embodiments, the length of the peptide spacer and amino acids used is adjusted according to the degree of flexibility required in the two proteins involved and the final protein fusion polypeptide. In some embodiments, the length of the spacer is adjusted to ensure proper protein folding and avoid aggregate formation. In some embodiments, the spacer is A or AAAL (SEQ ID NO: 178).

[0339] Vectors, host cells and protein production

[0340] In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein α (SIRP-α) D1 variant, the variant comprising a SIRPαD1 domain or a fragment thereof, the SIRPαD1 domain having an amino acid mutation at residue 80 relative to the wild-type SIRPαD1 domain; and having at least one additional amino acid mutation at a residue selected from the group consisting of: residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66 and residue 92 relative to the wild-type SIRPαD1 domain.

[0341] In some embodiments, the present invention also discloses a polypeptide comprising an Fc variant, wherein the Fc variant comprises an Fc domain dimer having two Fc domain monomers, wherein each Fc domain monomer is independently selected from (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236 and N297A.

[0342] In some embodiments, the polypeptide of the present disclosure is produced by a host cell.Host cell refers to a medium including necessary cell components (e.g., organelles) required for expressing the polypeptide described herein and fusion polypeptide by its corresponding nucleic acid.In some embodiments, nucleic acid is contained in a nucleic acid vector introduced into a host cell by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc. In some embodiments, the selection of nucleic acid vectors depends on the host cell to be used.In some embodiments, the host cell is prokaryotic (e.g., bacterial) or eukaryotic (e.g., mammalian) origin.

[0343] In some embodiments, polypeptides, such as polypeptide constructs comprising a SIRPαD1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and a fusion partner (such as an Fc variant), are produced by culturing a host cell transformed with a nucleic acid, preferably an expression vector containing 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, conditions suitable for expression vary with the expression vector and host cell selected. In some embodiments, a variety of appropriate host cells are used, including but not limited to mammalian cells, bacteria, insect cells, and yeast. For example, in the PCR product available from the American Type Culture Collection, Various cell lines useful for the present disclosure are described in the cell line catalog. In some embodiments, the Fc domain variants of the present disclosure are expressed in cells that are optimized not to glycosylate proteins expressed by such cells, by genetic engineering of the cell line or modification of cell culture conditions such as the addition of kifunine or by using a naturally non-glycosylating host such as a prokaryotic organism (E. coli, etc.), and in some cases, modification of the glycosylation sequence in the Fc is not required.

[0344] Nucleic acid vector construction and host cells

[0345] The nucleic acid sequence encoding the amino acid sequence of the polypeptide 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 polypeptide of the present disclosure are obtained using standard techniques (e.g., gene synthesis). Alternatively, standard techniques (e.g., QuikChange TM Mutagenesis) mutates a nucleic acid molecule encoding a wild-type SIRPαD1 domain to include a specific amino acid substitution. In some cases, the nucleic acid molecule is synthesized using a nucleotide synthesizer or PCR technology.

[0346] In some embodiments, a nucleic acid encoding a polypeptide construct (e.g., a polypeptide construct comprising a SIRPαD1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and a fusion partner (such as an Fc variant)) is incorporated into an expression vector to express the protein. A variety of expression vectors can be used for protein expression. The expression vector may comprise a self-replicating extrachromosomal vector or a vector that integrates into the host genome. The vector may also include various components or elements. For example, in some embodiments, vector components include, but are not limited to, transcriptional and translational regulatory sequences, such as promoter sequences, ribosome binding sites, signal sequences, transcriptional start and stop sequences, translational start and stop sequences, 3' and 5' untranslated regions (UTRs), and enhancer or activator sequences; origin of replication; selection marker gene; and a nucleic acid sequence encoding a polypeptide of interest and a transcriptional termination sequence. In some embodiments, the expression vector comprises a protein operably linked to a control or regulatory sequence, a selective marker, any fusion partner, additional elements, or any combination thereof. The term "operably linked" refers to a nucleic acid being in a functional relationship with another nucleic acid sequence. Typically, these expression vectors comprise transcription and translation regulatory nucleic acids operably linked to nucleic acids encoding Fc variants, and are generally suitable for host cells expressing proteins. Selection genes or markers (such as, but not limited to, antibiotic resistance genes or fluorescent protein genes) can be used to select host cells containing expression vectors, for example, by antibiotic or fluorescent expression. Various selection genes can be used.

[0347] In some embodiments, components or elements of the vector are optimized so that the expression vector is compatible with the host cell type.Expression vectors useful in the present disclosure include, but are not limited to, those capable of expressing proteins in mammalian cells, bacteria, insect cells, yeast, and in vitro systems.

[0348] In some embodiments, mammalian cells are used as host cells to produce polypeptides of the present disclosure. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK 293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, 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), CRL7030, and HsS78Bst cells. In some embodiments, E. coli cells are used as host cells to produce polypeptides of the present disclosure. Examples of E. coli strains include, but are not limited to, E. coli 294 ( 31,446), Escherichia coli λ1776( 31,537, Escherichia coli BL21(DE3)( BAA-1025) and Escherichia coli RV308 ( 31,608).

[0349] 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 the 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 a conventional nutrient medium modified for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequence.

[0350] In some embodiments, polypeptide constructs, such as polypeptide constructs comprising SIRPαD1 domain variants (e.g., any variant provided in Tables 2, 5, and 6) and fusion partners (such as Fc variants), are expressed in mammalian expression systems, including systems in which expression constructs are introduced into mammalian cells using viruses such as retroviruses or adenoviruses. In some embodiments, human, mouse, rat, hamster, or primate cells are used. Suitable cells also include known research cells, including but not limited to Jurkat T cells, NIH3T3, CHO, COS, and 293 cells. Alternatively, in some embodiments, proteins are expressed in bacterial cells. Bacterial expression systems are well known in the art and include Escherichia coli, Bacillus subtilis, Streptococcus cremoris, and Streptococcus lividans. In some cases, polypeptide constructs comprising Fc domain variants are produced in insect cells (such as, but not limited to, Sf9 and Sf21 cells) or yeast cells (such as, but not limited to, organisms from the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, and Yarrowia). In some cases, polypeptide constructs comprising 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, some display technologies, such as ribosome display, require in vitro translation. In addition, in some embodiments, Fc domain variants are produced by chemical synthesis methods, such as, but not limited to, liquid phase peptide synthesis and solid phase peptide synthesis. In the case of in vitro transcription using a non-glycosylation system such as bacterial extracts, Fc will not be glycosylated even in the presence of native glycosylation sites, and thus inactivation of Fc will be equivalently achieved.

[0351] In some embodiments, the polypeptide construct comprises non-natural amino acids, amino acid analogs, amino acid mimetics, or any combination thereof that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids generally refer to 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 having the same basic chemical structure (e.g., carbon, carboxyl, amino, and R groups bound to hydrogen) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. In some embodiments, such analogs have modified R groups (such as norleucine) or modified peptide backbones, but generally retain the same basic chemical structure as naturally occurring amino acids.

[0352] Protein production, recovery and purification

[0353] In some embodiments, the host cells used to produce the polypeptides of the present disclosure are grown in a medium suitable for culturing the selected host cells. Examples of suitable culture media for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Expi293 TM Expression medium, DMEM supplemented with fetal bovine serum (FBS), and RPMI-1640. Examples of suitable culture media for bacterial host cells include Luria broth (LB) plus necessary supplements, such as selection agents, such as ampicillin. In some embodiments, host cells are cultured at a suitable temperature (such as about 20°C to about 39°C, for example, about 25°C to about 37°C, preferably 37°C) and CO2 levels (such as about 5% to 10%). In some embodiments, the pH of the culture medium is about pH 6.8 to pH 7.4, for example pH 7.0, which depends primarily on the host organism. If an inducible promoter is used in the expression vector, protein expression can be induced under conditions suitable for promoter activation.

[0354] In some embodiments, protein recovery involves destroying host cells, such as by osmotic shock, sonication or lysis. Once the cells are destroyed, cell debris is removed by centrifugation or filtration. The protein can then be further purified. In some embodiments, the polypeptide of the present disclosure is purified by various protein purification methods, such as by chromatography (e.g., ion exchange chromatography, affinity chromatography and size exclusion column chromatography), centrifugation, differential solubility or by any other standard technique for purifying proteins. For example, in some embodiments, by appropriately selecting and combining affinity columns such as protein A columns (e.g., POROS protein A chromatography) and chromatographic columns (e.g., POROS HS-50 cation exchange chromatography), filtering, ultrafiltration, desalting and dialysis procedures to separate and purify proteins. In some embodiments, polypeptides are conjugated to marker sequences (such as peptides) to facilitate purification. An example of a marker amino acid sequence is a hexa-histidine peptide (His6-tag (SEQ ID NO: 223)), which can bind to a nickel-functionalized agarose affinity column with micromolar affinity. As an alternative, a hemagglutinin "HA" tag corresponding to an epitope derived from an influenza hemagglutinin protein can be used.

[0355] In some embodiments, polypeptides of the present disclosure, e.g., polypeptide constructs comprising a SIRPαD1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and a fusion partner (such as an Fc variant) are produced by cells of a subject (e.g., a human), e.g., in the context of gene therapy, 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, such as a modified vaccinia Ankara (MVA)), an adeno-associated virus vector, and an alphavirus vector) containing a nucleic acid molecule encoding the polypeptide of the present disclosure. The vector, once within the cells of the subject (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.), can be used to express the polypeptides disclosed herein. In some cases, the polypeptide is secreted by the cells. In some embodiments, if treatment of the disease or disorder is the desired outcome, no further action is required. In some embodiments, if protein collection is desired, blood is collected from the subject and the protein is purified from the blood by various methods.

[0356] Methods of treating cancer

[0357] Provided herein is a method for treating cancer in an individual (e.g., a human individual), comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPαD1 domain variant (e.g., a SIRPαD1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein) and (b) a therapeutic antibody.

[0358] Lung cancer

[0359] In some embodiments, a method for treating lung cancer (e.g., non-small cell lung cancer or "NSCLC") in an individual (e.g., a human individual) is provided, comprising administering to the individual an effective amount of (a) a polypeptide (e.g., fusion polypeptide) comprising a SIRPαD1 domain variant (e.g., a SIRPαD1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein) and (b) a therapeutic antibody that disrupts the interaction between PD-1 and PD-L1, wherein the individual has progressed (e.g., confirmed disease progression) during (or after) a previous therapy for lung cancer (e.g., NSCLC). In some embodiments, the previous therapy is an immune checkpoint inhibitor (CPI) therapy. Additionally or alternatively, in some embodiments, the individual has a PD-L1 tumor proportion score (TPS) of less than 50%. In some embodiments, the individual has not received previous CPI therapy. In some embodiments, the polypeptide (e.g., fusion polypeptide) comprises a SIRPαD1 domain variant comprising an 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, wherein numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, wherein 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 numbering is according to the EU index of Kabat. In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to an individual comprises a SIRPαD1 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 an individual comprises an Fc domain variant, which is a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to an individual comprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., fusion polypeptide) forms a homodimer. In some embodiments, the therapeutic 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.

[0360] 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 (e.g., by IV infusion) is administered to an individual once every three weeks (Q3W) at a dose of about 200 mg. 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 local package inserts. Complete information about the preparation, formulation, dosage, and administration schedule of pembrolizumab can be found in the local package insert (for the United States, see, e.g., www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2016 / 125514s012lbl(dot)pdf; for 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 a subject (e.g., via intravenous infusion), e.g., once a week (i.e., once every 7 days or "qw") at a dose of 10.0 mg / kg.

[0361] In some embodiments, lung cancer is NSCLC. In some embodiments, NSCLC is locally advanced NSCLC. In some embodiments, NSCLC is metastatic NSCLC. In some embodiments, the individual suffers from metastatic NSCLC and has not confirmed disease progression within 8 weeks of starting therapy with a previous PD-1 or PD-L1 inhibitor. In some embodiments, the individual has a PD-L1 tumor proportion score (TPS) score of ≥1%. In some embodiments, the individual suffers from locally advanced or metastatic NSCLC with a TPS score <50%. In some embodiments, the individual progresses after systemic therapy for its metastatic disease. In some embodiments, the individual suffers from locally advanced or metastatic NSCLC with a TPS score ≥1%, and the individual progresses in a previous checkpoint inhibitor (CPI) therapy for NSCLC.

[0362] In some embodiments, the individual's prior CPI therapy for lung cancer (e.g., NSCLC) in which the disease progressed is a therapy comprising treatment with nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplizumab, 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, if the individual demonstrates progressive disease (PD), e.g., as determined by the Response Evaluation in Solid Tumors (RECIST) criteria (e.g., version 1.0 or 1.1) or modified RECIST criteria (see, 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), the World Health Organization (WHO) criteria (see, 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." Tuberculosis. In some embodiments, the individual is resistant to standard therapy (e.g., curative therapy) for lung cancer (e.g., NSCLC). In some embodiments, there is no standard therapy (e.g., curative therapy) available for treating lung cancer (e.g., NSCLC).

[0363] In some embodiments, an in vitro diagnostic immunohistochemistry (IHC) assay for detecting PD-L1 in formalin-fixed paraffin-embedded (FFPE) human tissue sections is used to assess a subject's PD-L1 tumor proportion score (TPS). 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 murine monoclonal anti-PD-L1 antibody (clone 22C3) is used to detect PD-L1 in formalin-fixed paraffin-embedded (FFPE) human tissue sections. TM DAKO FLEX Visualization System TM PD-L1 (i.e., human PD-L1) in formalin-fixed paraffin-embedded (FFPE) lung cancer tissue (e.g., NSCLC tissue) obtained from an individual (e.g., a patient) is detected on the AUTOSTAINER LINK 48 automatic staining system. In some embodiments, TPS is a measure of PD-L1 protein expression in a lung cancer (e.g., NSCLC) tissue sample from an individual (e.g., a patient). In some embodiments, TPS is the percentage of live tumor cells that show partial or complete membrane staining at any intensity. If TPS ≥ 50% of live tumor cells show membrane staining at any intensity, the sample should be considered PD-L1 positive. In some embodiments, tumor-associated immune cells (such as infiltrating lymphocytes or macrophages) are not included in the score for determining TPS. In some embodiments, the labeling of lung cancer (e.g., NSCLC) samples is performed by a pathologist. In some embodiments, staining is evaluated by optical microscopy at 10-40 times magnification. Further details regarding the PD-L1 IHC 22C3 PharmDx assay, reagents and equipment for performing the assay, interpretation of assay results, and TPS scoring are provided at www(dot)accessdata(dot)fda(dot)gov / cdrh_docs / pdf15 / p150013b(dot)pdf and Reck et al. (2016) “Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer.” NEJM.375:1823-1833.

[0364] Head and Neck Cancer

[0365] 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, comprising administering to the individual an effective amount of (a) a polypeptide (e.g., a fusion polypeptide) comprising a SIRPαD1 domain variant (e.g., a SIRPαD1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein) and (b) a therapeutic antibody that disrupts the interaction between PD-1 and PD-L1, wherein the individual has progressed (e.g., confirmed disease progression) during or after a prior therapy for head and neck cancer (e.g., HNSCC). In some embodiments, the prior therapy is a platinum-containing therapy. In some embodiments, the polypeptide (e.g., a fusion polypeptide) comprises a SIRPαD1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, wherein 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 numbering is according to the EU index of Kabat. In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to an individual comprises a SIRPαD1 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 an individual comprises an Fc domain variant, which is a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO:91. In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to an individual comprises the amino acid sequence of SEQ ID NO:136 or SEQ ID NO:135. In some embodiments, the polypeptide (e.g., fusion polypeptide) forms a homodimer. In some embodiments, the therapeutic 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, HNSCC is PD-L1 negative. In some embodiments, HNSCC is PD-L1 positive.Further details regarding "PD-L1 negative" and "PD-L1 positive" are provided elsewhere herein.

[0366] 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 (e.g., by IV infusion) is administered to an individual once every three weeks (Q3W) at a dose of about 200 mg. 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 local package inserts. Complete information about the preparation, formulation, dosage, and administration schedule of pembrolizumab can be found in the local package insert (for the United States, see, e.g., www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2016 / 125514s012lbl(dot)pdf; for 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 a subject (e.g., via intravenous infusion), e.g., once a week (i.e., once every 7 days or "qw") at a dose of 10.0 mg / kg.

[0367] In some embodiments, the individual suffers from recurrent HNSCC. In some embodiments, HNSCC is metastatic HNSCC. In some embodiments, the individual has received previous immune checkpoint inhibitor ("CPI") therapy, that is, the individual is "experienced CPI" individual. In some embodiments, the individual suffers from recurrent or metastatic HNSCC and disease progression is not confirmed within 8 weeks of the start of the previous CPI therapy. In some embodiments, the previous CPI therapy is or includes PD-1 or PD-L1 inhibitors. In some embodiments, the previous CPI is or includes treatment with nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplizumab, tislelizumab (also referred to as BGB-A317), toripalizumab, sindilimab, carrelizumab (also referred to as SHR-1210 or INCSHR-1210), spartalizumab (also referred to as PDR001), TSR-042 and / or FAZ053. In some embodiments, the individual has not received prior CPI therapy (eg, the individual is "immune checkpoint inhibitor naive" or "CPI naive").

[0368] In some embodiments, the prior platinum-containing therapy in which the individual progressed or after which the individual progressed was a therapy comprising treatment with carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthridine, picoplatin, and / or satraplatin. In some embodiments, if the individual demonstrates progressive disease (PD), e.g., as determined by the Response Evaluation in Solid Tumors (RECIST) criteria (e.g., version 1.0 or 1.1) or modified RECIST criteria (see, 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), the World Health Organization (WHO) criteria (see, 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 In some embodiments, the individual is considered to have progressed on or after a prior platinum-containing therapy for head and neck cancer (e.g., HNSCC) if the individual does not meet a set of response criteria described in "FDG PET Hopkins Criteria VersusResidual 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., HNSCC). In some embodiments, there is no standard therapy (e.g., curative therapy) available for treating head and neck cancer (e.g., HNSCC).

[0369] Gastric / Gastroesophageal (GEJ) Cancer

[0370] In some embodiments, a method of treating gastric cancer / gastroesophageal (GEJ) cancer (e.g., HER2-positive gastric adenocarcinoma or GEJ adenocarcinoma) in an individual (e.g., a human individual) is provided, comprising administering to the individual an effective amount of (a) a polypeptide (e.g., fusion polypeptide) comprising a SIRPαD1 domain variant (e.g., a SIRPαD1 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., confirmed disease progression) during or following a prior therapy for gastric cancer / GEJ cancer (e.g., gastric cancer / GEJ adenocarcinoma), and wherein the prior therapy is an anti-HER2 antibody therapy and / or a fluoropyrimidine-based therapy. In some embodiments, the polypeptide (e.g., fusion polypeptide) comprises a SIRPαD1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, wherein 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 numbering is according to the EU index of Kabat. In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to an individual comprises a SIRPαD1 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 an individual comprises an Fc domain variant, which is a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO:91. In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to an individual comprises the amino acid sequence of SEQ ID NO:136 or SEQ ID NO:135. In some embodiments, the polypeptide (e.g., fusion polypeptide) forms a homodimer. In some embodiments, the therapeutic anti-HER2 antibody administered to an individual in combination with the fusion polypeptide is trastuzumab.

[0371] 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., by intravenous infusion) once every three weeks (Q3W). In some embodiments, the initial (i.e., first) dose of trastuzumab is about 8 mg / kg, and each subsequent dose (i.e., after the first dose) is about 6 mg / kg. In some embodiments, the dosage modification of trastuzumab is performed according to the local package insert. Complete information on trastuzumab preparation, formulation, dosage, and administration schedules can be found in the local package insert (for the United States, see, e.g., www.accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2017 / 103792s5337lbl(dot)pdf; for Europe, see, e.g., www(dot)ema(dot)Europa(dot)eu / en / documents / product-information / herceptin-epar-product-information_en(dot)pd f). In some embodiments, the polypeptide (e.g., fusion polypeptide) is administered to an individual (e.g., via intravenous infusion), e.g., once a week (i.e., once every 7 days or "qw") at a dose of 10.0 mg / kg.

[0372] In some embodiments, the HER2 status (i.e., HER2 positive status or HER2 negative status) of gastric cancer or GEJ cancer is assessed by immunohistochemistry (IHC) or in situ hybridization (ISH, such as fluorescent ISH or "FISH"). In some embodiments, the HER2 status of gastric cancer or GEJ cancer is evaluated according to the standards described in Abrahao-Machado et al. (2016) World J. Gastroenterol. 22 (19): 4619-4625 and the references cited therein. In some embodiments, HER2-positive gastric cancer or HER 2-positive GEJ cancer is HER2-positive gastric adenocarcinoma / HER2-positive GEJ adenocarcinoma. In some embodiments, HER2-positive gastric cancer / HER2-positive GEJ cancer (e.g., adenocarcinoma) is metastatic gastric cancer / GEJ cancer (e.g., metastatic adenocarcinoma). In some embodiments, the individual suffers from metastatic gastric cancer / GEJ cancer (e.g., adenocarcinoma) and has demonstrated at least a stable disease (SD) response (i.e., a response superior to progressive disease (PD)) as the best response to previous therapy.

[0373] In some embodiments, the individual progresses during or after a previous therapy with an anti-HER2 antibody (e.g., confirmed disease progression). In some embodiments, previous anti-HER2 antibody therapy includes treatment with trastuzumab, pertuzumab and / or magetuximab. Additionally or alternatively, in some embodiments, the individual progresses during or after a previous therapy with a fluoropyrimidine-based therapy (e.g., confirmed disease progression). In some embodiments, previous fluoropyrimidine-based therapies include treatment with, for example, capecitabine, floxuridine, 4-fluorouracil, 5-fluorouracil, camofluridine, doxifluridine, tegafur (Tegafur), UFT (i.e., a 1:4 molar combination of tegafur and uracil), S-1 (a combination of tegafur, gimeracil and oteracil) and / or FOLFOX (a combination of folinic acid, 5-fluorouracil and oxaliplatin). In some embodiments, the individual progresses during or after treatment with an anti-HER2 antibody and a fluoropyrimidine-based therapy. In some embodiments, an anti-HER2 antibody and a fluoropyrimidine-based therapy are administered in combination (e.g., where both agents are part of a single treatment regimen). In some embodiments, an anti-HER2 antibody and a fluoropyrimidine-based therapy are each administered in a separate treatment regimen (e.g., in two separate prior therapies or two separate prior therapy lines).

[0374] In some embodiments, if the individual has demonstrated progressive disease (PD), e.g., as determined by the Response Evaluation in Solid Tumors (RECIST) criteria (e.g., version 1.0 or 1.1) or modified RECIST criteria (see, 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), the World Health Organization (WHO) criteria (see, 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 Kurokawa et al. (2013) Ann Surg. Oncol.20(9):3009-3014; Yanagawa et al. (2012) J Nucl Med.53(6):872-880; Lordick et al. (2016) Ann Oncol.27(suppl 5):v50-v57; or Kim et al. (2015) Oncology.88:69-75, the individual is considered to have progressed on a previous anti-HER2 antibody therapy and / or a previous fluoropyrimidine-based therapy for gastric cancer / GEJ cancer (e.g., HER2-positive gastric cancer / HER2-positive GEJ cancer). In some embodiments, the individual is resistant to standard therapy (e.g., curative therapy) for gastric cancer / GEJ cancer (e.g., HER2-positive gastric cancer / HER2-positive GEJ cancer). In some embodiments, there is no standard therapy (e.g., curative therapy) available for treating gastric cancer / GEJ cancer (e.g., HER2-positive gastric cancer / HER2-positive GEJ cancer).

[0375] Lymphoma

[0376] (i) Aggressive non-Hodgkin's lymphoma

[0377] In some embodiments, a method of treating an aggressive non-Hodgkin lymphoma or "NHL" (e.g., diffuse large B-cell lymphoma ("DLBCL", e.g., primary DLBCL or transformed DLBCL or mantle cell lymphoma (MCL)) in an individual (e.g., a human individual) is provided, comprising administering to the individual an effective amount of (a) a polypeptide (e.g., a fusion polypeptide) comprising a SIRPαD1 domain variant (e.g., a SIRPαD1 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., wherein the individual has relapsed during or after a previous treatment for aggressive NHL and / or is refractory to a previous treatment for aggressive NHL), and wherein there is no available therapy (e.g., curative therapy) for the aggressive NHL (e.g., DLBCL, such as primary DLBCL, transformed DLBCL, or mantle cell lymphoma). In some embodiments, the polypeptide (e.g., fusion polypeptide) comprises a SIRPαD1 domain variant comprising SEQ ID NO:81 or SEQ ID NO:85 amino acid sequence. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising 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. In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to an individual comprises a SIRPαD1 domain variant comprising SEQ ID NO:81 or SEQ ID NO:85 amino acid sequence. In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to an individual comprises an Fc domain variant, which is a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein the numbering is according to the EU index of Kabat. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO:91. In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to an individual comprises the amino acid sequence of SEQ ID NO:136 or SEQ ID NO:135. In some embodiments, the polypeptide (e.g., fusion polypeptide) forms a homodimer.In some embodiments, the therapeutic anti-CD20 antibody is rituximab. In some embodiments, the aggressive NHL is diffuse large B-cell lymphoma (DLBCL), e.g., primary DLBCL or transformed DLBCL. In some embodiments, the aggressive NHL is mantle cell lymphoma (MCL).

[0378] 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 (e.g., by intravenous infusion) at about 375 mg / m 2 In some embodiments, for the first four weeks (e.g., 28 days) of treatment, the first four doses of rituximab (i.e., doses 1-4) are administered to the individual once a week (e.g., once every 7 days or "qw") (e.g., at about 375 mg / m 2 In some embodiments, the first four doses of rituximab (i.e., doses 1-4) are administered to the individual once a week (e.g., once every 7 days or "qw") for the first four weeks (e.g., 28 days) of treatment (e.g., at about 375 mg / m 2 The dosage of rituximab is modified according to the local package insert. For complete information on the preparation, dispensing, dosage and administration schedule of rituximab, see the local package insert (for the United States, see, e.g., www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2012 / 103705s5367s5388lbl(dot)pdf; for 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, the polypeptide (eg, fusion polypeptide) is administered to a subject (eg, via intravenous infusion), eg, once a week (ie, once every 7 days or "qw") at a dose of 10.0 mg / kg or 15.0 mg / kg.

[0379] In some embodiments, if the individual has no previous history of lymphoma, the individual is diagnosed as having primary DLBCL (e.g., primary recurrent and / or refractory DLBCL). In some embodiments, if the individual has a history of lymphoma, for example, an indolent lymphoma, such as marginal zone lymphoma, lymphoplasmacytic lymphoma, small lymphocytic lymphoma / chronic lymphocytic leukemia, follicular lymphoma, or lymphocyte-dominant Hodgkin lymphoma, the individual is diagnosed as having transformed DLBCL. In some embodiments, if the individual is found to have one or more of the following chromosomal abnormalities: t(11;14), t(14;18), the individual is diagnosed as having mantle cell lymphoma (MCL). In some embodiments, if SOX11 overexpression is detected in a leukemic cell sample from an individual, the individual is diagnosed as having mantle cell lymphoma (MCL). Other criteria for diagnosing primary DLBCL, transformed DLBCL, and MCL are known in the art and are routinely used by technicians. See, for example, Balsas et al. (2017) Blood. 130(4): 501-513; National Guideline Alliance (UK). Non-Hodgkin's Lymphoma: Diagnosis and Management. London: National Institute for Health and Care Excellence (UK); 2016 Jul. (NICE Guideline, No. 52.) 3, Staging; Dreyling et al. Am Soc Clin On col Educ Book. 2014: 191-8; etc.

[0380] In some embodiments, an individual is considered to have relapsed after a prior therapy for aggressive NHL (e.g., primary DLBCL, transformed DLBCL, or MCL) if the individual achieved a treatment response of least unstable disease (SD) to the prior therapy, but ceased to respond (e.g., confirmed disease progression) within, for example, any of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after cessation of the prior therapy. In some embodiments, an individual is considered to be refractory to a prior therapy for aggressive NHL if the individual did not respond to the prior therapy (e.g., failed to achieve a treatment response of at least stable disease (SD) during or after the prior therapy). In some embodiments, the treatment response to an aggressive NHL therapy is assessed according to the criteria described in Cheson et al. (2014) “Recommendations for Initial Evaluation, Staging and Response Assessment of Hodgkin and Non-Hodgk in Lymphoma: The Lugano Classification.” J. Clin Oncol. 32:3059-3067.

[0381] In some embodiments, the individual is refractory or has relapsed after treatment with at least one prior therapy for aggressive NHL (e.g., at least one prior standard-approved therapy, at least two prior standard-approved therapies, at least three standard-approved therapies, etc.). Standard therapies for DLBCL (e.g., primary or transformed DLBCL) and MCL include, but are not limited to, for example, rituximab, RCHP (i.e., rituximab, cyclophosphamide, doxorubicin, and prednisone); R-CHOP (i.e., rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone); R-CHOEP (i.e., rituximab, cyclophosphamide, doxorubicin, vincristine, etoposide, and prednisone, which are typically administered for 6 cycles in 21-day cycles); EPOCH-R (i.e., rituximab, cyclophosphamide, doxorubicin, vincristine, etoposide, and prednisone, which are typically administered as a continuous infusion over 4 days); R-GCVP ( =Rituximab, gemcitabine, cyclophosphamide, vincristine, and prednisolone); R-CEPP (rituximab, cyclophosphamide, etoposide, procarbazine, and prednisone); R-CEOP (rituximab, cyclophosphamide, epirubicin, vincristine, and prednisone); R-CVP (rituximab, cyclophosphamide, vincristine, and prednisone); rituximab and bendamustine; rituximab and lenalidomide; DHAP (dexamethasone, high-dose cytarabine, and cisplatin); RDHAP (DHAP in combination with rituximab); ICE (ifosfamide, carboplatin, and etoposide); RICE (ICE in combination with rituximab =Combination of oxaliplatin, methylprednisolone, high-dose cytarabine, and cisplatin; ESHAP (etoposide, methylprednisolone, high-dose cytarabine, and cisplatin); R-ESHAP (ESHAP and rituximab); MIME (methyl-glyoxal-bis(guanylhydrazone), ifosfamide, methotrexate, and etoposide); parsaclisib (also known as INCB050465); MATRIX (methotrexate, cytarabine, thiotepa, and rituximab) hyper-CVAD 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; 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., the combination of GemOx and rituximab); DHAX (i.e., dexamethasone, cytarabine, and oxaliplatin); R-DHAX (i.e., the combination of DHAX and rituximab); GIFOX (i.e., gemcitabine, ifosfamide, and oxaliplatin);

[0382] RGIFOX (ie, a combination of GIFOX and rituximab); bortezomib and GIFOX; ASCT (ie, autologous stem cell transplantation) HD-ASCT (ie, a combination of ASCT and high-dose therapy (eg, high-dose chemotherapy)); CAR T-cell therapy (eg, tisagenlecleucel or axicabtagene); veloximab and lenalidomide. In some embodiments, the previous therapy for aggressive NHL includes any two or more of the previous treatments (given together in a single treatment regimen, or given in separate treatment regimens). In some embodiments, there are no available treatment options (eg, curative treatment options) for individuals with aggressive NHL (eg, relapsed / refractory aggressive NHL). In some embodiments, the individual suffers from DLBCL (eg, primary DLBCL or transformed DLBCL) or MCL for which no curative therapy is available. In some embodiments, the individual has DLBCL (eg, primary DLBCL or transformed DLBCL) or MCL that has relapsed after or is refractory to standard approved therapy (eg, curative therapy).

[0383] (ii) Indolent lymphoma

[0384] In some embodiments, a method of treating an indolent lymphoma in an individual (e.g., a human individual) is provided, comprising administering to the individual an effective amount of (a) a polypeptide (e.g., a fusion polypeptide) comprising a SIRPαD1 domain variant (e.g., a SIRPαD1 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 indolent lymphoma is a relapsed and / or refractory indolent lymphoma (e.g., wherein the individual has undergone at least one prior treatment for indolent lymphoma (e.g., In some embodiments, the individual has relapsed during or after more than one standard approved therapy for indolent lymphoma (e.g., 2, 3 or more standard therapies), and / or is refractory to more than one standard therapy for indolent lymphoma (e.g., 2, 3 or more standard therapies, e.g., curative therapy). In some embodiments, the polypeptide (e.g., fusion polypeptide) comprises a SIRPαD1 domain variant comprising SEQ ID NO:81 or SEQ ID NO:85 amino acid sequence. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A and delG236 mutations, wherein 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 numbering is according to the EU index of Kabat. In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to an individual comprises a SIRPαD1 domain variant comprising SEQ ID NO:81 or SEQ ID NO:85 amino acid sequence. In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to an individual comprises a SIRPαD1 domain variant comprising SEQ ID NO:81 or SEQ ID NO:85 amino acid sequence. In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to an individual comprises an Fc domain variant, which is a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein the numbering is according to the EU index of Kabat. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO:91. In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to an individual comprises the amino acid sequence of SEQ ID NO:136 or SEQ ID NO:135. In some embodiments, the polypeptide (e.g., fusion polypeptide) forms a homodimer.In some embodiments, the therapeutic anti-CD20 antibody is rituximab.

[0385] 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 (e.g., by intravenous infusion) at about 375 mg / m 2 In some embodiments, for the first four weeks (e.g., 28 days) of treatment, the first four doses of rituximab (i.e., doses 1-4) are administered to the individual once a week (e.g., once every 7 days or "qw") (e.g., at about 375 mg / m 2 In some embodiments, the first four doses of rituximab (i.e., doses 1-4) are administered to the individual once a week (e.g., once every 7 days or "qw") for the first four weeks (e.g., 28 days) of treatment (e.g., at about 375 mg / m 2 The dosage of rituximab is modified according to the local package insert. For complete information on the preparation, dispensing, dosage and administration schedule of rituximab, see the local package insert (for the United States, see, e.g., www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2012 / 103705s5367s5388lbl(dot)pdf; for 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, the polypeptide (eg, fusion polypeptide) is administered to a subject (eg, via intravenous infusion), eg, once a week (ie, once every 7 days or "qw") at a dose of 10.0 mg / kg or 15.0 mg / kg.

[0386] In some embodiments, the indolent lymphoma is indolent non-Hodgkin lymphoma (NHL). In some embodiments, the indolent NHL is marginal zone lymphoma (MZL). In some embodiments, the indolent NHL is follicular lymphoma (FL). Details on the diagnosis and classification of marginal zone lymphoma and follicular lymphoma (as well as DLBCL and mantle cell lymphoma) are provided in, for example, 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, etc.

[0387] In some embodiments, a subject is considered to have relapsed following a prior therapy for an indolent lymphoma (e.g., indolent NHL) if the subject achieved a treatment response of least unstable disease (SD) to the prior therapy, but ceased to respond (e.g., confirmed disease progression) within, e.g., any of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after cessation of the prior therapy. In some embodiments, a subject is considered to be refractory to a prior therapy for an indolent lymphoma (e.g., indolent NHL) if the subject did not respond to the prior therapy (e.g., failed to achieve a treatment response of at least stable disease (SD) during or after the prior therapy). In some embodiments, the treatment response to therapy for indolent lymphoma (e.g., indolent NHL) is assessed according to the criteria described in 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.

[0388] In some embodiments, the individual is refractory or has relapsed after treatment with at least one previous therapy for an indolent lymphoma (e.g., indolent NHL) (e.g., at least one previous standard-approved therapy, at least two previous standard-approved therapies, at least three standard-approved therapies, etc.). Standard therapies for indolent lymphomas (e.g., indolent NHL, such as marginal zone lymphoma or follicular lymphoma) include, but are not limited to, for example, standard therapies for aggressive NHL (e.g., DLBCL or mantle cell lymphoma), which are described in detail elsewhere herein. Other standard therapies for indolent lymphomas (e.g., indolent NHL) include, but are not limited to, 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; Tositumomab; Vorinostat; Everolimus; Bortezomib; Navitoclax (also known as ABT-263); High-dose therapy (HDT); Autologous stem cell transplantation; and allogeneic stem cell transplantation. In some embodiments, the previous therapy for indolent lymphomas (e.g., indolent NHL) includes any two or more of the previous standard therapies (including the aggressive NHL therapies described elsewhere herein). In some embodiments, two or more standard therapies for indolent lymphomas (e.g., indolent NHL) (including the standard therapy for aggressive NHL) are administered together in a single treatment regimen. In some embodiments, two or more standard therapies for indolent NHL (including standard therapies for aggressive NHL) are administered in separate treatment regimens. In some embodiments, there are no available treatment options (e.g., curative treatment options) for individuals with indolent lymphoma (e.g., indolent NHL) (e.g., relapsed / refractory indolent NHL).

[0389] In some embodiments of any of the treatment methods provided herein, the fusion polypeptide is provided for use (e.g., intravenous administration) in a 100 mg / 5 ml Type I clear glass vial, which is sealed with a 20 mm Teflon-coated rubber septum stopper and an aluminum seal. In some embodiments, the fusion polypeptide is provided for use (e.g., intravenous administration) in a 400 mg / 20 ml Type I clear glass vial, which is sealed with a 20 mm Teflon-coated rubber septum stopper and an aluminum seal. In some embodiments, the fusion polypeptide is stored in its original container at 2-8 ° C (36-46 ° F) until use (e.g., intravenous administration).

[0390] In some embodiments of any of the methods of treatment described herein, the polypeptide (e.g., a fusion polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant) is administered subcutaneously. In some embodiments, the polypeptide (e.g., a fusion polypeptide) is administered by intravenous infusion. In some embodiments, a polypeptide (e.g., a fusion polypeptide) (e.g., via intravenous infusion) is administered to an individual (e.g., a human individual) at a dose of 0.3 mg / kg, 1.0 mg / kg, 3.0 mg / kg, 10.0 mg / kg, 15.0 mg / kg, or 30.0 mg / kg (including any range between these values). In some embodiments, a polypeptide (e.g., a fusion polypeptide) (e.g., via intravenous infusion) is administered to an individual, for example, at a dose of 0.3 mg / kg, 1.0 mg / kg, 3.0 mg / kg, 10.0 mg / kg, 15.0 mg / kg, or 30.0 mg / kg (including any range between these values) once a week (i.e., once every 7 days or "qw") In some embodiments, the polypeptide (e.g., fusion polypeptide) is administered to an individual, for example, every other week (i.e., once every 14 days or "q2w" or "QoW") 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), for example, via intravenous infusion. In some embodiments, the polypeptide (e.g., fusion polypeptide) and therapeutic antibody (e.g., anti-PD1 antibody (pembrolizumab), anti-HER2 antibody (trastuzumab) or anti-CD20 antibody (rituximab)) are administered sequentially on days when the dosing schedule is consistent. In some embodiments, the polypeptide (e.g., fusion polypeptide) is administered before (e.g., about 30 minutes before) the therapeutic antibody. In some embodiments, in the case of a missed polypeptide dose, the therapeutic antibody is administered about 24 hours after the missed dose.

[0391] In some embodiments, the treatment response of an individual with NSCLC, HNSCC, gastric cancer, or GEJ cancer to the treatment methods provided herein is assessed according to RECIST version 1.1 criteria, e.g., as described by Therasse et al. (2000) J. Natl Cancer Inst. 92:205-216; Eisenhauer et al. (2009) Eur J. Cancer. 45:229-247, immune-related response criteria (irRECIST) derived from RECIST 1.1, e.g., as 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 treatment response of an individual with an aggressive lymphoma (e.g., an aggressive NHL such as DLBCL or MCL) to the treatment methods provided herein is assessed according to the Lugano criteria, e.g., as described in Cheson et al. (2014) "Recommendations for Initial Eva luation, Staging and Response Assessment of Hodgkin and Non-Hodgkin Lymphoma: The Lugano Classification." J. Clin Oncol. 32: 3059-3067. In some embodiments, the treatment response of an individual with an indolent lymphoma (e.g., an indolent NHL such as FL or MZL) is assessed according to the Lugano criteria (see Cheson et al. 2014) for the treatment methods provided herein.

[0392] In some embodiments, the individual receiving treatment for HNSCC, NSCLC, gastric cancer, or GEJ cancer has at least one measurable lesion as defined by RECIST version 1.1 criteria, e.g., as described in Therasse et al. (2000) J. Natl Cancer Inst. 92:205-216; Eisenhauer et al. (2009) Eur J. Cancer. 45:229-247. In some embodiments, the individual receiving treatment for lymphoma (e.g., an aggressive lymphoma (e.g., DLBCL or MCL) or an indolent lymphoma (e.g., FL or MZL) has at least one measurable lesion 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." J. Clin Oncol. 32:3059-3067.

[0393] In some embodiments, the individual receiving treatment according to the methods herein has adequate bone marrow function, renal function, liver function, and cardiac function. In some embodiments, the individual has an Eastern Cooperative Oncology Group (ECOG) performance status (PS) score of 0 or 1 (see, e.g.,

[0394] www(dot)npcrc(dot)org / files / news / ECOG)_performance_status(dot)pdf). In some embodiments, the individual does not have symptomatic central nervous system (CNS) metastases or leptomeningeal disease requiring steroids. In some embodiments, the individual receiving lung cancer treatment according to the methods herein (e.g., NSCLC) does not suffer from ALK or EGFR genomic tumor aberrations. In some embodiments, the individual receiving treatment according to the methods herein does not have a history of (non-infectious) pneumonia requiring steroids or currently suffers from pneumonia. In some embodiments, the individual receiving treatment according to the methods herein does not have a 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 undergone high-dose chemotherapy requiring allogeneic stem cell rescue. In some embodiments, individuals who are treated for lung cancer (e.g., NSCLC), head and neck cancer (e.g., HNSCC), or gastric cancer / GEJ cancer (e.g., HER2-positive gastric adenocarcinoma / GEJ adenocarcinoma) according to the methods herein have not previously experienced radiation to >25% of the bone marrow. In some embodiments, the individual has not received radiation therapy within 2 weeks of the start of treatment. In some embodiments, the individual has not received any previous treatment with anti-CD47 or anti-SIRPα agents. In some embodiments, the individual has not received systemic anticancer therapy within 4 weeks of the start of treatment (6 weeks for mitomycin C or nitrosourea). In some embodiments, the individual has no intolerance or severe allergic or allergic reaction to any excipient in the formulation of the antibody or infused therapeutic protein or therapeutic protein. In some embodiments, the individual has not interrupted treatment due to a grade 3 or higher immune-related adverse event (AE) previously produced by an anti-PD-1, anti-PD-L1, or anti-PD-L2 agent or a therapy intended to regulate another immune cell target (e.g., CTLA-1, OX40, 41BB, etc.). In some embodiments, the individual has not received an experimental antibody or live vaccine (e.g., including but not limited to measles, mumps, rubella, varicella / zoster, yellow fever, rabies, Bacillus Calmette-Guérin (BCG), typhoid, and intranasal influenza vaccines). In some embodiments, the individual has not undergone active therapy currently used for the initial diagnosis (e.g., lung cancer (NSCLC), head and neck cancer (HNSCC), gastric cancer / GEJ cancer (HER2-positive gastric / GEJ adenocarcinoma), aggressive lymphoma (primary DLBCL or transformed DLBCL or mantle cell lymphoma), or indolent lymphoma (e.g., indolent NHL, such as marginal zone lymphoma or follicular lymphoma). In some embodiments, the individual has not received a blood product transfusion within 14 days of the start of treatment.In some embodiments, the individual has no history of active autoimmune disorders (including, but not limited to, for example, Crohn's disease, rheumatoid arthritis, scleroderma, systemic lupus erythematosus, Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia) and other disorders that impair or damage the immune system (except hypogammaglobulinemia). In some embodiments, the individual does not have an active, uncontrolled, clinically significant bacterial, fungal or viral infection, including hepatitis B (HBV), hepatitis C (HCV), known human immunodeficiency virus (HIV) or acquired immunodeficiency syndrome (AIDS)-related diseases. In some embodiments, the individual does not have active graft-versus-host disease (GVHD) or has not undergone immunosuppressive therapy for GVHD. In some embodiments, the individual has not had any of the following within the previous 12 months: myocardial infarction, severe / unstable angina, coronary artery / peripheral artery bypass grafting, 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 last 3 years prior to the start of treatment, except, for example, non-melanoma skin cancer or carcinoma in situ (e.g., breast cancer, cervical cancer in situ) that has been adequately treated with potentially curative therapy.

[0395] Kits and Products

[0396] In another embodiment of the present invention, a product or kit comprising a polypeptide (e.g., a fusion polypeptide described herein) is provided, wherein the polypeptide comprises a SIRPαD1 domain variant and an Fc domain variant. In some embodiments, the SIRPαD1 domain variant comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 81, SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, wherein 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 numbering is according to the EU index of Kabat. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide 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 used according to the treatment methods provided herein.

[0397] In some embodiments, the kit or article 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 having instructions for treating individual lung cancer (e.g., NSCLC, including metastatic NSCLC) or delaying its progression using a polypeptide (e.g., fusion polypeptide) in combination with an anti-PD1 antibody (e.g., pembrolizumab) according to the methods herein. In some embodiments, the kit comprises a package insert or label having instructions for treating individual lung cancer (e.g., NSCLC, including metastatic NSCLC) or delaying its progression using a polypeptide (e.g., fusion polypeptide) in combination with an anti-PD1 antibody (e.g., pembrolizumab), and the individual has received a previous therapy for NSCLC. In some embodiments, the individual has progressed (e.g., confirmed disease progression) during (or after) a previous therapy for lung cancer (e.g., a previous immune checkpoint inhibitor therapy). In some embodiments, the individual has a PD-L1 tumor proportion score (TPS) of less than 50%. In some embodiments, the kit includes a package insert or label having instructions for treating HNSCC or delaying the progression of an individual using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-PD1 antibody (e.g., pembrolizumab) according to the methods herein. In some embodiments, the kit includes a package insert or label having instructions for treating HNSCC or delaying the progression of an individual using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-PD1 antibody (e.g., pembrolizumab) that has received a previous immune checkpoint inhibitor therapy (e.g., for HNSCC). In some embodiments, the kit includes a package insert or label having instructions for treating HNSCC or delaying the progression of an individual using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-PD1 antibody (e.g., pembrolizumab) that has not received a previous immune checkpoint inhibitor therapy (e.g., for HNSCC). In some embodiments, the kit comprises a package insert or label having instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an anti-PD1 antibody (e.g., pembrolizumab) to treat head and neck cancer (e.g., HNSCC, including metastatic HNSCC) or delay its progression in an individual who has progressed (e.g., confirmed disease progression) in (or after) a previous platinum therapy (e.g., platinum-containing therapy). In some embodiments, the kit or article of manufacture further comprises instructions for administering pembrolizumab once every 3 weeks (Q3W) at a dose of 200 mg by IV infusion. In some embodiments, the kit or article of manufacture further comprises instructions for administering a polypeptide (e.g., a fusion polypeptide) once a week (QW) at a dose of 10 mg / kg by IV infusion.

[0398] In some embodiments, the kit or article also includes an anti-HER2 antibody. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the kit includes a package insert or label, which has a method according to which a polypeptide (e.g., fusion polypeptide) is used in conjunction with an anti-HER2 antibody (e.g., trastuzumab) to treat an individual's HER2-positive gastric cancer or HER2-positive GEJ cancer (e.g., HER2-positive gastric adenocarcinoma or HER2-positive GEJ adenocarcinoma) or delay its progress instructions. In some embodiments, the kit includes a package insert or label, which has a polypeptide (e.g., fusion polypeptide) in conjunction with an anti-HER2 antibody (e.g., trastuzumab) to treat an individual's HER2-positive gastric cancer or HER2-positive GEJ cancer (e.g., HER2-positive gastric adenocarcinoma or HER2-positive GEJ adenocarcinoma) or delay its progress instructions, and the individual has progressed (e.g., confirmed disease progression) in a previous therapy for gastric cancer or GEJ cancer (or thereafter). In some embodiments, previous therapies include anti-HER2 antibodies and / or previous fluoropyrimidine-based therapies. In some embodiments, the kit or article further comprises instructions for administering trastuzumab once every three weeks (q3W) via intravenous infusion, wherein the initial dose of trastuzumab is 8 mg / kg, and each subsequent dose of trastuzumab (i.e., after the initial dose) is 6 mg / kg. In some embodiments, the kit or article further comprises instructions for administering the polypeptide (e.g., fusion polypeptide) once a week (QW) at a dose of 10 mg / kg by IV infusion.

[0399] In some embodiments, the kit or article 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 having instructions for treating an individual's aggressive non-Hodgkin's lymphoma or "NHL" (e.g., primary DLBCL or transformed DLBCL, or mantle cell lymphoma) or delaying its progression using a polypeptide (e.g., fusion polypeptide) in combination with an anti-CD20 antibody (e.g., rituximab) according to the methods herein. In some embodiments, the kit comprises a package insert or label having instructions for treating an individual who has relapsed or refractory to a previous therapy for aggressive NHL (e.g., previous standard therapy / curative therapy) or an individual who has no available aggressive NHL therapy (e.g., curative therapy) with a polypeptide (e.g., fusion polypeptide) in combination with an anti-CD20 antibody (e.g., rituximab) or delaying its progression. In some embodiments, the kit comprises a package insert or label having instructions for using the polypeptide (e.g., fusion polypeptide) in combination with an anti-CD20 antibody (e.g., rituximab) according to the methods herein to treat an indolent lymphoma (e.g., indolent NHL, such as marginal zone lymphoma or follicular lymphoma) or delay its progression in an individual. In some embodiments, the kit comprises a package insert or label having instructions for using the polypeptide (e.g., fusion polypeptide) in combination with an anti-CD20 antibody (e.g., rituximab) to treat an indolent lymphoma (e.g., indolent NHL) or delay its progression in an individual who has relapsed or refractory to a previous therapy (e.g., a previous standard therapy, e.g., a curative therapy) for an indolent lymphoma (e.g., indolent NHL) or an individual who has no available therapy (e.g., a curative therapy) for an indolent lymphoma (e.g., indolent NHL). In some embodiments, the kit or article of manufacture further comprises instructions for administering 375 mg / m 2 of rituximab in the first four weeks (e.g., 28 days) of treatment. 2 Rituximab is administered to individuals once a week (e.g., once every 7 days or "qw"), followed by 375 mg / m 2 Instructions for administering rituximab to an individual once every four weeks (e.g., once every 28 days or "q4w") for up to four additional doses, or up to 8 additional doses after the first four doses. In some embodiments, the kit or article of manufacture further comprises instructions for administering the polypeptide (e.g., fusion polypeptide) once a week (QW) at a dose of 10 mg / kg or 15 mg / kg by IV infusion.

[0400] In some embodiments of any kit or article provided herein, the polypeptide (e.g., fusion polypeptide) and the therapeutic antibody (e.g., pembrolizumab, trastuzumab, or rituximab) are in the same container or in separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The container can be formed of various materials, such as glass, plastics (such as polyvinyl chloride or polyolefins) or metal alloys (such as stainless steel or Hastelloy). In some embodiments, the container comprises a formulation and a label that is maintained on or associated with the container and that indicates instructions for use. The article or kit may also include other materials that are desired from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article also includes one or more other agents (e.g., chemotherapeutic agents and anti-tumor agents). Suitable containers for one or more agents include, for example, bottles, vials, bags, and syringes.

[0401] This specification is considered sufficient to enable those skilled in the art to implement the present invention. Various modifications of the present invention, except those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

[0402] Example

[0403] Example 1: Phase 1 study of drug A in combination with an established anti-cancer antibody in patients with advanced malignancies.

[0404] This example describes a Phase 1 clinical study 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 SIRPαD1 domain variant fused to a human immunoglobulin Fc domain variant modified to eliminate binding to Fcγ receptors ( Figure 1A-1B ).

[0405] Purpose

[0406] Main Purpose

[0407] The primary objective of this study is 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).

[0408] Secondary Purpose

[0409] The secondary aims of this study are to:

[0410] To evaluate the anti-tumor effect of drug A in combination with anti-cancer therapeutic agents in patients with advanced malignancies.

[0411] To evaluate the overall safety of drug A in combination with other anticancer therapeutic agents.

[0412] • Characterize the maximum tolerated dose (MTD) / optimal biological dose (OBD) of drug A in combination with anticancer therapeutics.

[0413] • Characterize the single-dose and multiple-dose pharmacokinetics of Drug A in combination with the anticancer therapeutics pembrolizumab, trastuzumab, or rituximab.

[0414] To evaluate the immunogenicity of drug A.

[0415] Exploratory purpose

[0416] The exploratory objective of this study was to explore the pharmacodynamic effects of drug A in combination with anticancer therapeutic agents in patients with advanced malignancies.

[0417] patient

[0418] Inclusion Criteria

[0419] All patients met the following criteria for the dose escalation phase of the study:

[0420] Advanced / metastatic solid tumor malignancies or relapsed / refractory CD20 + Histologic or cytologic diagnosis of B-cell non-Hodgkin lymphoma:

[0421] ο is resistant to standard therapy or has no curative therapy available; or

[0422] o Meet any of the criteria for dose expansion described below (ad)

[0423] Lesions may be measurable or non-measurable.

[0424] All patients met the following criteria for the dose expansion phase of the study:

[0425] a) Locally advanced or metastatic NSCLC (TPS ≥ 1%) that has progressed on prior checkpoint therapy or has progressed after receiving systemic therapy for metastatic disease (TPS < 50%); patients are eligible for treatment with pembrolizumab; or

[0426] b) recurrent or metastatic HNSCC with disease progression on or after platinum-containing chemotherapy and who are candidates for treatment with pembrolizumab; or

[0427] c) HER2-overexpressing metastatic gastric adenocarcinoma or gastroesophageal junction (GEJ) adenocarcinoma who have progressed following systemic therapy with a fluoropyrimidine-containing regimen and / or therapy with an anti-HER2 antibody for their metastatic disease and are suitable for treatment with trastuzumab;

[0428] d) Relapsed or refractory, primary or transformed diffuse large B-cell lymphoma (DLBCL) or mantle cell lymphoma with no available curative therapy; or indolent lymphoma (marginal zone, follicular) that is relapsed or refractory to standard approved therapy.

[0429] Patients had at least one measurable lesion defined according to RECIST version 1.1 or Lugano criteria (2014).

[0430] In addition, all patients met the following criteria:

[0431] Adequate bone marrow function, including:

[0432] 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);

[0433] Platelet count ≥ 75,000 / mm 3 (≥75x10 9 / L); non-Hodgkin lymphoma only: platelets ≥50,000 / mm 3 (≥50x10 9 / L);

[0434] oHemoglobin ≥9 g / dL (≥90 g / L); non-Hodgkin lymphoma only: hemoglobin ≥8 g / dL (≥80 g / L).

[0435] Adequate renal function, including:

[0436] o Serum creatinine ≤1.5x upper limit of normal (ULN) or estimated creatinine clearance ≥60 mL / min, calculated using the institution's standard method.

[0437] Adequate liver function, including:

[0438] oTotal serum bilirubin ≤1.5 x ULN (≤3.0 x ULN if the patient has documented Gilbert's syndrome);

[0439] o Aspartate and alanine aminotransferases (AST and ALT) ≤ 3.0 x ULN; if there is secondary liver involvement from the tumor, ≤ 5.0 x ULN;

[0440] o Alkaline phosphatase ≤2.5 x ULN (≤5.0 x ULN if bone or liver metastases).

[0441] QTcF interval ≤ 480 msec (based on the average of triplicate ECGs).

[0442] Age ≥ 18 years.

[0443] Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0 or 1.

[0444] Elimination of acute effects of any prior therapy of baseline severity or ≤ Grade 1 (NCI CTCAE v.4.03), except for AEs judged by the investigator not to pose a safety risk.

[0445] Archived (or fresh) metastatic biopsy samples available prior to study entry (expansion phase only).

[0446] Negative serum pregnancy test (for women of childbearing age) at screening.

[0447] Exclusion criteria

[0448] Patients with any of the following characteristics were not included in this study:

[0449] 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 entry, had discontinued corticosteroid therapy for these metastases, and were clinically stable on anticonvulsants for at least 4 weeks and were neurologically stable prior to enrollment.

[0450] Patients with ALK or EGFR genomic tumor aberrations (NSCLC, Part 2 expansion only), or any patient with a history of (non-infectious) pneumonitis requiring steroids or current pneumonitis.

[0451] Patients with high-grade lymphoma (including Burkitt's lymphoma, lymphoblastic lymphoma, Richter's transformation), CLL, or plasma cell leukemia

[0452] Prior high-dose chemotherapy requiring allogeneic stem cell rescue.

[0453] Prior radiation to >25% of the bone marrow (non-lymphoma patients only) or any patient who received prior radiation therapy within 2 weeks of the start of study treatment. NOTE: Participants must have recovered from all radiation-related toxicities, not require corticosteroids, and not have radiation pneumonitis. A 1-week washout is permitted for palliative radiation (≤2 weeks of radiation therapy) for non-CNS disease.

[0454] Prior treatment with any anti-CD47 or anti-SIRPα agents.

[0455] • Systemic anticancer therapy within 4 weeks of the start of study treatment (6 weeks for mitomycin C or nitrosourea). If systemic anticancer therapy was given within 4 weeks, patients were included if 4-5 times the elimination half-life of the drug had elapsed.

[0456] Previous treatment with the following (Extension Phase only):

[0457] o PD-1 or PD-L1 inhibitor (NSCLC; HNSCC) for metastatic disease with disease progression within 8 weeks of initiation.

[0458] o Trastuzumab (gastric / GEJ cancer), for metastatic disease with disease progression as best response.

[0459] Patients who are intolerant to or have severe allergic or anaphylactic reactions to the antibody or infused therapeutic protein, or to any substance contained in the study drug (including excipients); or patients who discontinued treatment due to Grade 3 or higher immune-related AEs caused by previous therapy with anti-PD-1, anti-PD-L1, or anti-PD-L2 agents or agents designed to modulate another immune cell target (e.g., CTLA-1, OX40, 41BB, etc.).

[0460] Any experimental antibody or live vaccine within the last 28 days before the first dose of study drug. Live attenuated vaccines are not permitted.

[0461] Current active treatment for initial diagnosis.

[0462] Blood products transfused within 14 days of Cycle 1 Day 1.

[0463] History of autoimmune disease (or activity) (including but not limited to: Crohn's disease, rheumatoid arthritis, scleroderma, systemic lupus erythematosus, Grave's disease) and other conditions that impair or compromise the immune system (except hypogammaglobulinemia).

[0464] History of autoimmune hemolytic anemia or autoimmune thrombocytopenia.

[0465] Patients with active, uncontrolled, clinically significant bacterial, fungal, or viral infections, including hepatitis B (HBV), hepatitis C (HCV), known human immunodeficiency virus (HIV), or acquired immunodeficiency syndrome (AIDS)-related illnesses.

[0466] - Patients with active graft-versus-host disease (GVHD) or who are undergoing immunosuppression for GVHD.

[0467] Any of the following within the previous 12 months: myocardial infarction, severe / unstable angina, coronary artery / peripheral artery bypass grafting, symptomatic congestive heart failure, cerebrovascular accident, transient ischemic attack, deep vein thrombosis, or symptomatic pulmonary embolism.

[0468] Currently undergoing active treatment in another interventional therapeutic clinical study.

[0469] Any other malignancy diagnosed within the past 3 years prior to enrollment, except for non-melanoma skin cancer or carcinoma in situ (e.g., breast cancer, cervical cancer in situ) that has been adequately treated with potentially curative therapy.

[0470] Other serious acute or chronic medical or psychiatric conditions, including recent (within the past year) or active suicidal ideation or behavior, or laboratory abnormalities, that increase the risks associated with study participation or administration of the investigational product or interfere with the interpretation of the study results and, in the judgment of the Investigator, make the patient unsuitable for entry into this study.

[0471] 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 the study product.

[0472] Pregnant or breastfeeding patients.

[0473] Study Treatment

[0474] like Figure 2 As shown, this study has three treatment groups (drug A + pembrolizumab; drug A + trastuzumab; drug A + rituximab). The study includes an initial dose escalation part, followed by a dose expansion part. Each dose escalation group has approximately 12 patients with advanced malignancies. Each expansion group has approximately 20-40 patients at multiple sites.

[0475] Drug A is administered once a week (or once every 2 weeks) as an IV infusion over approximately 60 minutes on an outpatient basis. Use of an infusion pump is the preferred method of administration to ensure accurate delivery of the study product, but gravity infusion is permitted.

[0476] Drug A is provided in 100 mg / 5 mL or 400 mg / 20 mL Type 1 clear glass vials, sealed with 20 mm Teflon-coated rubber serum stoppers and tamper-resistant 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.

[0477] A cycle is defined as the time from dosing on Day 1 to the next dosing on Day 1. If there is no treatment delay, a cycle is 21 days for weekly dosing and 28 days for 2-weekly dosing.

[0478] All trial treatments were administered on an outpatient basis. Patients were observed in the outpatient clinic for at least 2 hours following the Cycle 1 Day 1 (C1D1) infusion of Drug A and thereafter as clinically indicated.

[0479] No premedication with drug A is required. Follow the guidelines in the package insert for the pembrolizumab, trastuzumab, and rituximab combination therapy.

[0480] During the dose escalation and expansion phases, administer combination partner therapy according to label instructions:

[0481] ο Trastuzumab : The initial dose is 8 mg / kg administered as an intravenous infusion over 90 minutes, followed by 6 mg / kg administered as an intravenous infusion (IV) over 30 to 90 minutes every 3 weeks.

[0482] ο Pembrolizumab : Administer 200 mg IV as an infusion over 30 minutes every 3 weeks for up to 24 months.

[0483] ο Rituximab :375mg / m 2 , administered once weekly by intravenous infusion for 4 doses, followed by once monthly for a total of 8 doses (starting at a rate of 50 mg / hr and increasing by 50 mg / hr increments every 30 minutes to a maximum of 400 mg / hr without infusion toxicity). If the first infusion of rituximab is tolerated, subsequent infusions are started at a rate of 100 mg / hr and increased by 100 mg / hr increments at 30 minute intervals to a maximum of 400 mg / hr without toxicity.

[0484] On administration days consistent with the dosing schedule, the combination partner was started approximately 30 minutes after the end of Drug A therapy. On such administration days, if a dose of Drug A was missed due to toxicity, the partner drug was administered 24 hours after the missed dose. In the event of permanent discontinuation of Drug A, the patient discontinued the treatment phase of the study. In the event of permanent discontinuation of the partner drug, the patient continued single agent Drug A for up to 24 months if, in the opinion of the investigator, the patient received clinical benefit from Drug A.

[0485] Dose escalation components

[0486] For drug A, the initial dose escalation component starts at a dose level (3-6 patients per dose level) below the single agent maximum tolerated dose (MTD) or maximum administered dose (MAD) taking into account the observed single agent dose limiting toxicity (DLT) profile of drug A and the known safety profile of the proposed combination agent (Table A). If the drug A dose is safe and well tolerated in combination, the dose level of drug A is increased to the MTD or MAD of the combination agent.

[0487] The MTD of Drug A as a single agent was not reached. The MAD of Drug A as a single agent was 30 mg / kg, administered IV once every other week (QOW or Q2W).

[0488] Table A. Dose escalation of drug A.

[0489] queue Dosage mg / kg Route of administration Dosing frequency 1 0.3 IV Once a week 2 1.0 IV Once a week 3 3.0 IV Once a week 4 10.0 IV Once a week 5 30.0 IV Every 2 weeks

[0490] Expansion Phase

[0491] Drug A was administered once weekly (QW) on an outpatient basis as an intravenous (IV) infusion over approximately 60 minutes at a dose of 10 mg / kg.

[0492] During the extension phase, the combination partner therapy is administered according to its label instructions, as described above.

[0493] The Agent A + pembrolizumab expansion group included up to 20 patients with metastatic NSCLC and up to 20 patients with recurrent or metastatic head and neck squamous cell carcinoma.

[0494] The Agent A + trastuzumab expansion group included up to 20 patients with HER2-overexpressing (HER2-positive) gastric cancer.

[0495] The Agent 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.

[0496] Each patient received drug A until disease progression, unacceptable toxicity, withdrawal of consent, or study termination. Patients received study treatment after radiographic progression if, according to the investigator's estimate, the patient received clinical benefit from study treatment.

[0497] Efficacy analysis

[0498] Overall response rate (ORR), disease control rate (DCR), duration of response (DoR), progression-free survival (PFS), and overall survival (OS) were analyzed in the ITT population and the evaluable population.

[0499] Objective tumor response was assessed for solid tumors using Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1. Tumor assessments included all known or suspected sites of disease. Imaging included CT or MRI scans of the breast, abdomen, and pelvis; brain CT or MRI scans for patients with known or suspected brain metastases; and bone scans and / or bone X-rays for patients with known or suspected bone metastases. In addition, for patients with lymphoma, testing included PET scans and bone marrow evaluations. The same imaging techniques used to characterize each identified and reported lesion at baseline were used in the following tumor assessments.

[0500] Antitumor activity was assessed by radiographic tumor assessments performed at baseline, during treatment, whenever disease progression was suspected (eg, worsening of symptoms), and at study withdrawal if not performed within the first 6 weeks.

[0501] Response was assessed using RECIST version 1.1 or, where relevant, the Lugano criteria (Cheson et al., J. Clin. Oncol (2014) 32:27:3059-3068.).

[0502] Changes in tumor size were classified as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD), the latter of which included the appearance of new lesions. In the expansion cohort, secondary analysis was performed using immune-related RECIST 1.1. To facilitate secondary analysis, only the diameters of all target lesions and new measurable lesions were collected in the expansion cohort (longest diameter for non-nodal lesions, shortest diameter for nodal lesions). In addition, in the expansion cohort, progression and response needed to be confirmed by imaging at least 4 weeks from the first recording date.

[0503] PK / PD and biomarker analysis

[0504] Drug concentrations of drug A were measured using a validated method. Drug A serum concentrations 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, PK parameters were calculated, including maximum concentration (Cmax ), time to reach maximum concentration (T max ), area under the concentration-time curve (AUC last , AUC inf and / or AUC τ ). Other PK parameters were calculated as appropriate, including clearance (CL), volume of distribution (Vz), terminal elimination half-life (t 1 / 2 ) and cumulative ratio (R ac ).

[0505] PK / PD analyses were performed to explore exposure-response relationships using appropriate model-based approaches to aid in OBD determination. Pharmacodynamic data (receptor occupancy and immune phenotype) were summarized graphically and with descriptive statistics by time and dose.

[0506] Explore PK / PD analysis using appropriate model-based methods to better understand the exposure-response relationship. Analyze CD47 target occupancy levels before and after dosing, and perform immunophenotyping of circulating leukocyte populations. Measure CD47 target occupancy in peripheral blood T lymphocytes and erythrocytes by flow cytometry. Analyze infiltrating leukocyte populations and immunomodulatory molecules in tumor biopsies before and after treatment, as well as specific cytokines and chemokines in serum before and after treatment. Perform exploratory molecular analysis (including but not limited to additional immune markers) in peripheral blood and tumor biopsy samples before and after treatment.

[0507] CD8, CD68, CD163, and PD-L1 on tumor tissues were measured by immunohistochemistry (IHC) assay. Percent positive values ​​for CD8, CD68, and CD163 were obtained by image analysis. PD-L1 (clone 22C3) tumor proportion score (TPS) and combined positive score (CPS) were obtained by pathologist review. TM HER2 levels were measured using NANOSTRING IO360 TM Expression panels assess RNA expression from paired tumor biopsies. Using NANOSTRINGnSOLVER TM The analysis software performs cell type abundance and pathway profiling using predefined gene signatures.

[0508] Blood samples were collected at the baseline visit and retained for pharmacogenomic analysis related to drug response. For example, examination of SIRPα gene polymorphisms, putative safety biomarkers, drug-metabolizing enzyme genes, drug transporter genes, or genes thought to be related to the drug's mechanism of action.

[0509] result

[0510] Patient characteristics

[0511] Eighty-two patients with advanced solid tumor malignancies were enrolled in this study. The baseline characteristics of the patients are provided in Table B.

[0512] Table B. Baseline characteristics of patients

[0513]

[0514] Security

[0515] Drug A in combination with trastuzumab or pembrolizumab was well tolerated, and most treatment-related adverse events (TRAEs) were of low grade and frequency. Treatment-related adverse events that occurred in two or more patients with the drug A + trastuzumab combination are provided in Table C, and the drug A + pembrolizumab combination is provided in Table D. The most common TRAE in the drug A + trastuzumab combination was fatigue (26.7%). The most common TRAE in the drug A + pembrolizumab combination was increased AST (15.4%).

[0516] Table C. Treatment-related adverse events occurring in two or more patients treated with the drug A + trastuzumab combination.

[0517]

[0518] Table D. Treatment-related adverse events occurring in two or more patients treated with the drug A + pembrolizumab combination.

[0519]

[0520] TRAEs of grade 3 severity were of low frequency. In the drug A + trastuzumab combination, one treatment-related serious adverse event of febrile neutropenia was reported. In the drug A + pembrolizumab c...

Claims

1. Use of an effective amount of a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant in the preparation of a medicament for treating HER2-positive gastric cancer / gastroesophageal junction (GEJ) cancer in an individual in combination with an anti-HER2 antibody, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85, wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein numbering is according to the EU index as in Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein numbering is according to the EU index as in Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A and delG236 mutations, wherein numbering is according to the EU index as in Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236 and N297A mutations, wherein numbering is according to the EU index as in Kabat; and wherein the individual's gastric / GEJ cancer has progressed following prior treatment with a fluoropyrimidine-based therapy and / or prior treatment with an anti-HER2 antibody, and wherein the individual is a human.

2. The use according to claim 1, wherein the previous treatment with a fluoropyrimidine-based therapy or the previous treatment with an anti-HER2 antibody comprises one or more therapeutic agents selected from the group consisting of trastuzumab, pertuzumab, 5-fluorouracil, capecitabine, magetuximab and FOLFOX.

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

4. The use according to claim 3, wherein trastuzumab is administered to the individual at an initial dose of 8 mg / kg and a dose of 6 mg / kg for each subsequent dose, and wherein trastuzumab is administered to the individual once every 3 weeks (Q3W) by IV infusion.

5. Use of an effective amount of a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant in the preparation of a medicament for treating non-small cell lung cancer (NSCLC) in an individual in combination with an anti-PD-1 antibody, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85, wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein numbering is according to the EU index as in Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein numbering is according to the EU index as in Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A and delG236 mutations, wherein numbering is according to the EU index as in Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236 and N297A mutations, wherein numbering is according to the EU index as in Kabat; and wherein the individual is human.

6. The use according to claim 5, wherein the individual's NSCLC has progressed on previous immune checkpoint inhibitor (CPI) therapy and / or has a PD-L1 tumor proportion score (TPS) of less than 50%.

7. The use of claim 6, wherein the prior checkpoint inhibitor therapy comprises one or more agents selected from the group consisting of nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplizumab.

8. Use of an effective amount of a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant in the preparation of a medicament for treating head and neck squamous cell carcinoma (HNSCC) in an individual in combination with an anti-PD-1 antibody, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85, wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein numbering is according to the EU index as in Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein numbering is according to the EU index as in Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A and delG236 mutations, wherein numbering is according to the EU index as in Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236 and N297A mutations, wherein numbering is according to the EU index as in Kabat; and wherein the individual's HNSCC has progressed on or after prior platinum therapy, and wherein the individual is a human.

9. The use according to claim 8, wherein the individual has received prior immune checkpoint inhibitor therapy.

10. The use according to claim 8, wherein the individual has not received previous immune checkpoint inhibitor therapy.

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