Combination Therapies for Treating Cancer

Abstract

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

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 188,388, filed May 13, 2021, and U.S. Provisional Application No. 63 / 193,581, filed May 26, 2021, the contents of each of which are incorporated by reference in their entirety herein.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 757972001640SEQLIST.txt, Recorded: May 12, 2022, Size: 302,942 bytes).

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

[0004] Many cancers have poor prognosis even when treated with available therapeutic agents. There is a need in the art for novel therapies to provide additional treatment options and improve outcomes for cancer patients.

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

[0006] Macrophage-mediated destruction of cancer cells requires both disruption of "don't eat me" signals (e.g., CD47-SIRPα) and activation of "eat me" signals. Either component alone is not sufficient to trigger a maximal phagocytic response against tumor cells. As mentioned above, CD47 provides the basal "don't eat me" signal through its interaction with SIRPα on macrophages. Pro-phagocytic "eat me" signals can be provided to the same macrophages by binding to their activating Fc gamma receptors. For example, pro-phagocytic "eat me" signals can be provided by binding of anti-tumor antibodies to Fc receptors on macrophages.

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

[0008] In some embodiments, a method of treating cancer in an individual is provided, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, (b) an anti-HER2 antibody, (c) an anti-VEGF2 antibody, and (d) paclitaxel, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is selected from the group consisting of (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the cancer is gastric or gastroesophageal junction (GEJ) cancer, and the individual has received at least one prior treatment for the gastric or GEJ cancer. In some embodiments, the gastric or GEJ cancer is HER2 overexpressing (e.g., HER2 +) gastric cancer or HER2-overexpressing GEJ cancer. In some embodiments, the individual has been pretreated with an anti-HER2 antibody, with an anti-HER2 antibody and a fluoropyrimidine, with an anti-HER2 antibody and a platinum-based chemotherapy agent, or with a platinum-based chemotherapy agent. In some embodiments, the individual has experienced progression during or after treatment with an anti-HER2 antibody, an anti-HER2 antibody and a fluoropyrimidine, an anti-HER2 antibody and a platinum-based chemotherapy agent, or a platinum-based chemotherapy agent. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the anti-VEGF antibody is ramucirumab. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of about 10 to about 60 mg / kg once weekly (qw), e.g., 10 mg / kg, 15 mg / kg, or 30 mg / kg once weekly. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 10 mg / kg, 15 mg / kg, or 30 mg / kg every two weeks. In some embodiments, trastuzumab is administered at an initial dose of 8 mg / kg followed by 6 mg / kg every three weeks. In some embodiments, trastuzumab is administered at an initial dose of 6 mg / kg followed by 4 mg / kg every two weeks (e.g., an initial dose of 6 mg / kg followed by 4 mg / kg two weeks after the initial dose of 6 mg / kg and a 4 mg / kg dose every two weeks thereafter after the first 4 mg / kg dose). In some embodiments, paclitaxel is administered at a dose of 80 mg / m2 on days 1, 8, and 15 of a 28-day cycle. In some embodiments, the overall patient population undergoing treatment has an overall response rate (ORR) of greater than 65%, 70%, 75%, 80%, 85%, 90%, or 95% for that population.

[0009] In some embodiments, the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant forms a homodimer, hi some embodiments, the individual is a human.

[0010] In some embodiments, a kit is provided comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharma- ceutically acceptable carrier for use in combination with an anti-HER2 antibody, an anti-VEGFR2 antibody, and paclitaxel, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; and the Fc domain variant comprises: (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, and the kit comprises instructions for administering a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in combination with an anti-HER2 antibody, an anti-VEGFR2 antibody, and paclitaxel to an individual with gastric or gastroesophageal junction (GEJ) cancer who has received at least one prior therapy for gastric or gastroesophageal junction (GEJ) cancer. In some embodiments, the gastric or GEJ cancer is an anti-HER2 antibody, an anti-VEGFR2 antibody, or an anti-HER2 antibody. + Gastric cancer or HER2 +The cancer is a GEJ cancer. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the anti-VEGFR2 antibody is ramucirumab. In some embodiments, the individual has received prior treatment (or multiple prior treatments) with an anti-HER2 antibody (e.g., trastuzumab) and / or a fluoropyrimidine and / or platinum-based chemotherapy agent. In some embodiments, the individual's gastric or GEJ cancer has progressed during or after prior treatment (or multiple prior treatments) including an anti-HER2 antibody (e.g., trastuzumab) and / or a fluoropyrimidine and / or platinum-based chemotherapy agent. In some embodiments, the individual has failed (e.g., relapsed after or failed to respond to) prior treatment (or multiple prior treatments) including an anti-HER2 antibody (e.g., trastuzumab) and / or a fluoropyrimidine and / or platinum-based chemotherapy agent. In some embodiments, the prior treatment(s) included an anti-HER2 antibody and a fluoropyrimidine (e.g., administered during the same or different therapy regimens). In some embodiments, the prior treatment(s) included an anti-HER2 antibody and a platinum-based chemotherapeutic agent (e.g., administered during the same or different therapy regimens).

[0011] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be available from the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0012] [Figure 1] 1 shows the results of experiments performed to determine the effect of Drug A in combination with (a) an anti-HER2 antibody, (b) an anti-PD-L1 antibody, or (c) an anti-HER2 antibody and anti-PD-L1 on tumor growth in a mouse MC38 m:h chimeric colon cancer model. [Figure 2A] 1 shows a graph of the best percent change in measurable lesions from baseline for each patient in the study described in Example 2E. [Figure 2B]FIG. 1 is a graph showing the percent change in measurable lesions from baseline as a function of time for each patient in the study described in Example 2E. [Diagram 3] 1 shows the results of experiments performed to evaluate whether Drug B enhances the antitumor activity of anti-mouse VEGFR-2 + paclitaxel dual therapy in the CT26 syngeneic mouse tumor model. [Figure 4A] 1 shows the results of an experiment performed to evaluate whether drug B enhances the antitumor activity of trastuzumab + anti-mouse VEGFR-2 + paclitaxel triple therapy in a mouse CT26 m:h HER2-expressing chimeric tumor model. [Figure 4B] 1 shows the results of an experiment performed to evaluate whether quadruple therapy with Drug B + trastuzumab + anti-mouse VEGFR-2 + paclitaxel increases predicted survival compared to triple therapy with trastuzumab + anti-mouse VEGFR-2 + paclitaxel in a murine CT26 m:h HER2-expressing chimeric tumor model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] In the following description, example methods, parameters, etc. are set forth. It should be recognized, however, that such description is not intended to limit the scope of the present disclosure, but is instead provided as a description of example embodiments.

[0014] definition The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, depending in part on the method of measuring or determining the value, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, as is customary in the art. Alternatively, "about" can mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, more preferably within 2-fold, of a value. Unless otherwise stated, when a particular value is described in this application and claims, the term "about" is deemed to mean within an acceptable error range for the particular value.

[0015] The terms used herein are for the purpose of describing particular instances only and are not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that "including," "includes," "having," "has," "including," or variations thereof are used in either the detailed description or the claims, such terms are intended to be inclusive in a similar manner as the term "comprising."

[0016] As used herein, the terms "treatment," "treating," and the like refer to administering an agent or performing a procedure to obtain an effect. In some embodiments, the effect is prophylactic in that it completely or partially prevents a disease or its symptoms. In some embodiments, the effect is therapeutic in that it affects a partial or complete cure of a disease or a symptom of a disease.

[0017] As used herein, the term "antibody" refers to intact antibodies, antibody fragments, where they exhibit the desired biological activity (e.g., epitope binding), monoclonal antibodies; polyclonal antibodies; monospecific antibodies; multispecific antibodies (e.g., bispecific antibodies); and antibody-like proteins.

[0018] As used herein, the term "antibody variable domain" refers to the portions of the antibody light and heavy chains that contain the amino acid sequences of the complementarity determining regions (CDRs, e.g., CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3) and framework regions (FRs).

[0019] As used herein, the term "linker" refers to a link between two elements, e.g., protein domains. In some embodiments, a linker can be a covalent bond or a spacer. The term "spacer" refers to a moiety (e.g., a polyethylene glycol (PEG) polymer) or an amino acid sequence (e.g., a 1-200 amino acid sequence) that occurs between two polypeptides or polypeptide domains to provide space or flexibility (or both space and flexibility) between the two polypeptides or polypeptide domains. In some embodiments, an amino acid spacer is part of the primary sequence of a polypeptide (e.g., attached to spaced polypeptides or polypeptide domains via the polypeptide backbone).

[0020] As used herein, the term "effective amount" refers to an amount of a polypeptide or pharmaceutical composition comprising a polypeptide described herein, e.g., a polypeptide having a SIRPα D1 domain or a variant thereof, that is sufficient and effective to achieve a desired therapeutic effect in treating a patient with a disease such as cancer, e.g., a solid tumor or a hematological cancer. In some embodiments, an effective amount of the polypeptide will avoid adverse side effects.

[0021] As used herein, the term "pharmaceutical composition" refers to a pharmaceutical product or formulation that includes an active ingredient and an excipient or diluent (or both an excipient and a diluent), such that the active ingredient may be administered by a suitable method of administration. In some embodiments, the pharmaceutical compositions disclosed herein include pharma- ceutical acceptable ingredients that are compatible with the polypeptide. In some embodiments, the pharmaceutical composition is in tablet or capsule form for oral administration, or in aqueous form for intravenous or subcutaneous administration, e.g., by injection.

[0022] As used herein, the terms "subject," "individual," and "patient" are used interchangeably to refer to vertebrates, e.g., mammals. Mammals include, but are not limited to, murines, monkeys, humans, farm animals, sports animals, and pets. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro. Neither term requires the supervision of a medical professional.

[0023] As used herein, the term "affinity" or "binding affinity" refers to the strength of the binding interaction between two molecules. In general, binding affinity refers to the strength of the sum of non-covalent interactions between a molecule and its binding partner (such as a SIRPα D1 domain variant and CD47). Unless otherwise specified, binding affinity refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair. The binding affinity between two molecules is generally measured by the dissociation constant (K D ) or binding constant (K A ) Two molecules that have low binding affinity for each other generally bind slowly and tend to dissociate easily, with a large K D Two molecules that have a high affinity for each other generally bind easily, tend to remain bound for a long time, and have a small K D In some embodiments, the K of two interacting molecules D is determined using known methods and techniques, for example, surface plasmon resonance (SPR). Dcan be calculated as the ratio koff / kon.

[0024] As used herein, "less than K D The term "" refers to the numerically small K D Value and stated K D As used herein, "greater K" refers to an increased binding affinity compared to the K value. D The term "K" refers to the numerically larger D Value and stated K D It refers to a decreased binding affinity compared to the original value.

[0025] As used herein, "in conjunction with" refers to the administration of one therapy in addition to another. Thus, "in conjunction with" refers to the administration of one therapy before, during, or after administration of another therapy to an individual.

[0026] overview Provided herein are methods of treating cancer in an individual (e.g., a human individual), the methods comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) and (b) a chemotherapeutic agent (such as at least one chemotherapeutic agent, e.g., at least two, at least three, or at least four chemotherapeutic agents). In some embodiments, the methods further comprise administering to the individual an effective amount of a therapeutic antibody (such as at least one therapeutic antibody, e.g., at least two, at least three, or at least four therapeutic antibodies). Additionally or alternatively, in some embodiments, the methods further comprise administering to the individual an effective amount of an immunotherapeutic agent (such as at least one immunotherapeutic agent, e.g., at least two, at least three, or at least four immunotherapeutic agents). Additionally or alternatively, in some embodiments, the methods comprise administering the polypeptide and the chemotherapeutic agent in combination with one or more additional therapeutic modalities, such as, but not limited to, radiation therapy, surgery, cryoablation, and bone marrow transplantation.

[0027] In some embodiments, the agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is a small molecule inhibitor of the CD47-SIRPα pathway (e.g., RRX-001, etc.). See, e.g., Miller et al. (2019) "Quantitative high-throughput screening assays for the discovery and development of SIRPα-CD47 interaction inhibitors." PLoS ONE 14(7):e0218897 and Sasikumar et al. ACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; October 26-30, 2017; Philadelphia, PA; Abstract B007.

[0028] In some embodiments, an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) binds to CD47 (e.g., hCD47). In some embodiments, the agent binds to CD47 (e.g., hCD47) with a K of about 10 nM. D or better D(e.g., at least one of about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM, or less than 10 pM). In some embodiments, an agent that binds CD47 (e.g., hCD47) exhibits a CD47 receptor occupancy of at least about 50% (e.g., at least one of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%) in a human subject. In some embodiments, the agent that binds CD47 (e.g., hCD47) has an EC50 of about 80 ng / ml or less, e.g., any one of about 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 ng / ml. In some embodiments, the agent that binds CD47 (e.g., hCD47) is an anti-CD47 antibody (e.g., a therapeutic anti-CD47 antibody) or an antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment is a Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv, single chain antibody, or diabody. In some embodiments, the anti-CD47 antibody is a monospecific antibody. In some embodiments, the anti-CD47 antibody is a multispecific (e.g., bispecific) antibody. In some embodiments, the term "anti-CD47 antibody" encompasses antibody-based constructs (such as multispecific constructs) including, but not limited to, triomabs, DARTs (i.e., dual affinity retargeting antibodies), TandAbs (i.e., tandem diabodies), tandem scFvs, CrossMabs, DNLs (i.e., dock-and-lock antibodies), DVD-Igs (i.e., dual variable domain immunoglobulins), tetravalent bispecific IgGs, nanobodies, dual targeting domains, and ART-Igs (i.e., asymmetric reengineering technology-immunoglobulins).Additional details regarding exemplary antibody constructs (both monospecific and multispecific) are provided in Husain et al. (2018) Biodrugs 32(5):441-464 and Spiess et al. (2015) Molecular Immunology 67(2):95-106. In some embodiments, the anti-CD47 antibody is Hu5F9-G4, B6H12.2, BRIC126, CC-90002, SRF231, or IBI188 (Innovent Biologics) (for additional information regarding these anti-CD47 antibodies, see, e.g., Zhao et al. (2011), PNAS USA 108:18342-18347; Chao et al. (2010) Cell 142:699-713, Kim et al. (2012) Leukemia 26:2538-2545; Chao et al. (2011) Blood 118:4890-4891; Goto et al. (2014) Eur J. Cancer 50:1836-1846; and Edris et al. (2012) PNAS USA 109:6656-61).

[0029] In some embodiments, the agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) binds to SIRPα (e.g., hSIRPα). In some embodiments, the agent binds to SIRPα (e.g., hSIRPα) with a K of about 10 nM. D or better D(e.g., at least one of about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM, or less than 10 pM). In some embodiments, an agent that binds to SIRPα (e.g., hSIRPα) exhibits a SIRPα receptor occupancy of at least about 50% (e.g., at least one of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%) in a human subject. In some embodiments, the agent that binds to SIRPα (e.g., hSIRPα) has an EC50 of about 80 ng / ml or less, e.g., any one of about 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 ng / ml. In some embodiments, the agent that binds to SIRPα (e.g., hSIRPα) is an anti-SIRPα antibody (e.g., a therapeutic anti-SIRPα antibody) or an antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment is a Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv, single-chain antibody, or diabody. In some embodiments, the anti-SIRPα antibody is a monospecific antibody or a monospecific antibody construct (including but not limited to those described above). In some embodiments, the anti-SIRPα antibody is a multispecific (e.g., bispecific) antibody or a multispecific antibody construct (including but not limited to those described above). In some embodiments, the anti-SIRPα antibody is KWAR23, SE12C3, 040, or MY-1 (for additional information regarding these anti-SIRPα antibodies, see, e.g., Ring et al. (2017) PNAS USA 114(49):E10578-E10585; Murata et al. (2018) Cancer Sci 109(5):1300-1308; and Yanigata et al. (2017) JCI Insight 2:e89140).In some embodiments, the anti-SIRPα antibody is an antibody described in WO2018 / 057669, US-2018-0105600-A1; US20180312587; WO2018107058; WO2019023347; US20180037652; WO2018210795; WO2017178653; WO2018149938; WO2017068164; and WO2016063233, the contents of which are incorporated by reference in their entireties.

[0030] In some embodiments, the agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is an anti-SIRPβ or anti-SIRPγ antibody (e.g., an anti-SIRPβ or anti-SIRPγ antibody capable of binding to SIRPα), or an antigen-binding fragment thereof. In some embodiments, the agent is an antibody (or antigen-binding fragment thereof) capable of binding to two or more of SIRPα, SIRPβ, and SIRPγ. In some embodiments, such an antibody has a K D or better D(such as at least one of about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM, or less than 10 pM). In some embodiments, the antibody exhibits a SIRPα receptor occupancy of at least about 50% in a human subject (e.g., at least one of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%). In some embodiments, the antibody has an EC50 of about 80 ng / ml or less, for example, any one of about 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 ng / ml. In some embodiments, the antigen-binding fragment is a Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv, single-chain antibody, or diabody. In some embodiments, the antibody is a monospecific antibody or a monospecific antibody construct (including but not limited to those described above). In some embodiments, the antibody is a multispecific (e.g., bispecific) antibody or a multispecific antibody construct (including but not limited to those described above).

[0031] In some embodiments, the agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is a fusion polypeptide that includes a portion that binds to CD47. In some embodiments, the fusion polypeptide includes an antibody Fc region and a portion that binds to CD47. In some embodiments, the portion of the fusion polypeptide that binds to CD47 (e.g., hCD47) has a K D or better D(such as at least one of about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM, or less than 10 pM) binds to CD47 (e.g., hCD47). In some embodiments, the fusion polypeptide exhibits a CD47 receptor occupancy of at least about 50% (e.g., at least one of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%) in a human subject. In some embodiments, the fusion polypeptide has an EC50 of about 80 ng / ml or less, e.g., any one of about 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 ng / ml. In some embodiments, the fusion polypeptide comprises a WT human antibody Fc region. In some embodiments, the fusion polypeptide comprises an Fc variant (e.g., a variant of a WT human antibody Fc region) that exhibits reduced (e.g., eliminated, etc.) effector function compared to the WT Fc region. Exemplary Fc variants are described in WO2017 / 027422 and US2017 / 0107270, the contents of which are incorporated herein by reference in their entireties. In some embodiments, the moiety that binds CD47 (e.g., hCD47) is WT SIRPα (e.g., hSIRPα), or WT SIRPγ (e.g., hSIRPγ). In some embodiments, the moiety that binds CD47 (e.g., hCD47) is a CD47-binding fragment (e.g., the d1 domain) of WT SIRPα (e.g., hSIRPα) or WT SIRPγ (e.g., hSIRPγ). In some embodiments, the moiety that binds CD47 (e.g., hCD47) is a SIRPα variant, a SIRPγ variant, a SIRPβ variant, or a CD47-binding fragment thereof (e.g., the d1 domain).Exemplary SIRPγ variants, SIRPβ1 variants, and SIRPβ2 variants are described in, e.g., WO2013 / 109752; US2015 / 0071905; USP9,944,911; WO2016 / 023040; WO2017 / 027422; US2017 / 0107270; USP10,259,859; US9845345; WO2016187226; US201801554 05; WO2017177333; WO2014094122; US2015329616; US20180312563; WO2018176132; WO2018081898; WO2018081897; PCT / US2019 / 048921; US20180141986A1; and EP3287470A1, the contents of which are incorporated by reference in their entireties.

[0032] In some embodiments, the agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is a fusion polypeptide comprising an antibody Fc region and a SIRPα variant. In some embodiments, the SIRPα variant has a K D or better D(e.g., at least one of about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM, or less than 10 pM). In some embodiments, the fusion polypeptide exhibits a CD47 receptor occupancy of at least about 50% (e.g., at least one of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%) in a human subject. In some embodiments, the fusion polypeptide has an EC50 of about 80 ng / ml or less, for example, about any one of 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 ng / ml. In some embodiments, the fusion polypeptide comprises a WT human antibody Fc region. In some embodiments, the fusion polypeptide comprises an Fc variant (e.g., a variant of a WT human antibody Fc region) that exhibits reduced (e.g., eliminated, etc.) effector function compared to the WT Fc region, for example, those described in the references cited herein. In some embodiments, the fusion polypeptide is selected from the group consisting of WO2013 / 109752; US2015 / 0071905; WO2016 / 023040; WO2017 / 027422; US2017 / 0107270; USP10,259,859; US9845345; WO2016187226; US20180155405; WO201717733 3; WO2014094122; US2015329616; US20180312563; WO2018176132; WO2018081898; WO2018081897; US20180141986A1; and EP3287470A1, the contents of which are incorporated by reference in their entireties.In some embodiments, the fusion polypeptide comprising an antibody Fc region and a SIRPα variant is TTI-621, TTI-622, or IMM01 (see, e.g., Petrova et al. (2017) Clin Cancer Res 23:1086-1079; Russ et al. (2018) Blood Rev S0268-960X(17)30093-0; Zhang, X, Chen, W, Fan, J et al. Disrupting CD47-SIRPα axis alone or combined with autophagy depletion for the therapy of glioblastoma. Carcinogenesis 2018;39:689-99).

[0033] In some embodiments, the agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is a fusion polypeptide comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein).

[0034] In some embodiments, provided is a method of treating cancer (e.g., gastric or gastroesophageal cancer) in an individual (e.g., a human individual), the method comprising administering to the individual effective amounts of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα); (b) an anti-HER2 antibody; (c) an anti-VEGFR2 antibody; and (d) paclitaxel. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85, and the Fc domain variant is selected from the group consisting of: (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. Fc region (wherein numbering is according to EU index of Kabat); or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) containing the S228P, E233P, F234V, L235A, delG236, and N297A mutations.

[0035] Further details regarding therapeutic methods with polypeptides comprising SIRPα D1 domain variants and Fc domain variants are described below. See also WO2017 / 027422, U.S. Patent No. 10,259,859, and PCT / US20 / 62402, the contents of each of which are incorporated herein by reference in their entirety.

[0036] Signal Regulatory Protein Alpha (SIRPα) D1 Domain and Its Variants In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein alpha (SIRP-α) D1 variant, the polypeptide comprising a SIRPα D1 domain or a fragment thereof comprising an amino acid mutation at residue 80 relative to a wild-type SIRP-α D1 domain (e.g., a wild-type SIRPα D1 domain set forth in SEQ ID NO: 1 or 2); and at least one additional amino acid mutation at a residue selected from the group consisting of residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92 relative to the wild-type SIRPα D1 domain (e.g., a wild-type SIRPα D1 domain set forth in SEQ ID NO: 1 or 2).

[0037] Also disclosed herein, in some embodiments, is a polypeptide comprising an Fc domain variant, wherein the Fc domain variant dimer comprises two Fc domain variants, each Fc domain variant independently selected from: (i) a human IgG1 Fc region consisting of 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.

[0038] Signal regulatory protein alpha ("SIRP-α" or "SIRP-alpha") is a transmembrane glycoprotein belonging to the Ig superfamily that is widely expressed on the membrane of myeloid cells. SIRPα interacts with CD47, a protein that is widely expressed on many cell types in the body. The interaction of SIRPα with CD47 prevents the engulfment of "self" cells that may otherwise be recognized by the immune system. It has been observed that high expression of CD47 on tumor cells can act as a negative prognostic factor for survival in acute myeloid leukemia and some solid tumor cancers.

[0039] Native SIRPα is composed of three highly homologous immunoglobulin (Ig)-like extracellular domains (D1, D2, and D3). The SIRPα D1 domain ("D1 domain") refers to the membrane-distal extracellular domain of SIRPα and mediates binding of SIRPα to CD47. As used herein, the term "SIRPα polypeptide" refers to any SIRPα polypeptide or fragment thereof capable of binding to CD47. There are at least ten wild-type human SIRPα variants. Table 1 shows the amino acid sequences of the D1 domain of naturally occurring wild-type human SIRPα D1 domain variants (SEQ ID NOs: 1 and 2). In some embodiments, the SIRPα polypeptide comprises a SIRPα D1 domain. In some embodiments, the SIRPα polypeptide comprises a wild-type D1 domain, such as those provided in SEQ ID NOs: 1 and 2. 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). [Table 1]

[0040] As used herein, the term "SIRPα D1 domain variant" refers to a polypeptide comprising a SIRPα D1 domain or a CD47-binding portion of a SIRPα polypeptide that has a higher affinity for CD47 than wild-type SIRPα. A SIRPα D1 domain variant contains at least one amino acid substitution, deletion, or insertion (or a combination thereof) relative to wild-type SIRPα.

[0041] In some embodiments, the SIRPα D1 domain variant disclosed herein comprises a SIRPα D1 domain or a variant thereof. In some embodiments, the SIRPα D1 domain variant comprises one or more amino acid substitutions, insertions, additions, or deletions relative to the wild-type D1 domain set forth in SEQ ID NOs: 1 and 2. Table 2 lists exemplary amino acid substitutions in each SIRPα D1 domain variant (SEQ ID NOs: 13-14). In some embodiments, the SIRPα D1 domain polypeptide or SIRPα D1 domain variant comprises a fragment of the D1 domain. In some embodiments, the SIRPα polypeptide fragment or SIRPα D1 domain variant fragment comprises an amino acid sequence less than 10 amino acids in length, about 10 amino acids in length, about 20 amino acids in length, about 30 amino acids in length, about 40 amino acids in length, about 50 amino acids in length, about 60 amino acids in length, about 70 amino acids in length, about 80 amino acids in length, about 90 amino acids in length, about 100 amino acids in length, or more than about 100 amino acids in length. In some embodiments, the SIRPα D1 domain fragment retains the ability to bind to CD47.

[0042] In some embodiments, a polypeptide of the disclosure comprising a SIRPα D1 domain variant binds to CD47 with a higher binding affinity than the wild-type human SIRPα D1 domain. In some embodiments, a SIRPα D1 domain variant binds to human CD47 with an affinity that is at least 1-fold (e.g., at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 5-fold or more) greater than that of the naturally occurring D1 domain. In some embodiments, a SIRPα D1 domain variant binds to human CD47 with an affinity that is at least 1-fold (e.g., at least 10-fold, 100-fold, 1000-fold, or 1000-fold greater) greater than that of the naturally occurring D1 domain.

[0043] As used herein, the term "optimized affinity" or "optimized binding affinity" refers to an optimized strength of binding interaction between a polypeptide disclosed herein, such as a SIRPα D1 domain variant, and CD47. For example, in some embodiments, the polypeptide binds primarily or with higher affinity to CD47 on cancer cells and does not substantially bind or binds with lower affinity to CD47 on non-cancer cells. In some embodiments, the binding affinity between the polypeptide and CD47 is optimized such that the interaction does not cause clinically relevant toxicity or reduces toxicity compared to a variant that binds with maximum affinity. In some embodiments, to achieve an optimized binding affinity between a polypeptide provided herein and CD47, a polypeptide comprising a SIRPα D1 domain variant is developed to have a lower binding affinity to CD47 than is maximally achievable. In some embodiments, the SIRPα D1 domain variant disclosed herein cross-reacts with rodent, non-human primate (NHP), and human CD47.

[0044] As used herein, the term "immunogenicity" refers to the property of a protein (e.g., a therapeutic protein) to elicit an immune response in a host as if it were a foreign antigen. The immunogenicity of a protein can be assayed in vitro in a variety of ways, such as in vitro T cell proliferation assays.

[0045] As used herein, the term "minimally immunogenic" refers to the immunogenicity of a protein (e.g., a therapeutic protein) that has been modified, e.g., by an amino acid substitution, to be less immunogenic (e.g., at least 10%, 25%, 50%, or 100% less) than the immunogenicity before the amino acid substitution was introduced (e.g., the unmodified protein). In some embodiments, a protein (e.g., a therapeutic protein) is modified to have minimal immunogenicity and provokes no or little host immune response even if it is a foreign antigen.

[0046] In some embodiments, the SIRPα D1 domain variant exhibits minimal immunogenicity. In some embodiments, the SIRPα polypeptide of the present disclosure administered to a subject has the same amino acid sequence as the affinity of the SIRPα polypeptide in the subject's biological sample, except for the amino acid changes that increase the affinity of the SIRPα D1 domain variant. In some embodiments, the polypeptide variants disclosed herein reduce the risk of side effects compared to anti-CD47 antibodies or wild-type SIRPα. In some embodiments, the polypeptide variants disclosed herein reduce the risk of anemia compared to anti-CD47 antibodies or wild-type SIRPα. In some embodiments, the polypeptide variants disclosed herein do not cause acute anemia in rodent or non-human primate (NHP) studies.

[0047] Table 2 shows the specific amino acid substitutions of the SIRPα D1 domain variants for each D1 domain sequence. In some embodiments, the SIRPα D1 domain variants include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more) of the substitutions listed in Table 2. In some embodiments, the SIRPα D1 domain variants include up to 14 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variants include up to 10 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variants include up to 7 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variants of the present disclosure have at least 90% (e.g., at least 92%, 95%, 97%, or more than 97%) amino acid sequence identity to the sequence of the wild-type D1 domain.

[0048] In some embodiments, the SIRPα D1 domain variant is a chimeric SIRPα D1 domain variant that comprises two or more wild-type D1 domains or portions of variants thereof (e.g., a portion of one wild-type D1 domain or a variant thereof and a portion of another wild-type D1 domain or a variant thereof). In some embodiments, the chimeric SIRPα D1 domain variant comprises at least two portions (e.g., three, four, five, or more portions) of the wild-type D1 domain or a variant thereof, where each of the portions is derived from a different wild-type D1 domain. In some embodiments, the chimeric SIRPα D1 domain variant further comprises one or more amino acid substitutions listed in Table 2. [Table 2]

[0049] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant comprising the following sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGNITPADAGTYYCX 12 KX 13 RKGSPDDVEX 14 X1 is L, I, or V, X2 is V, L, or I, X3 is A or V, X4 is A, I, or L, X5 is I, T, S, or F, X6 is E, V, or L, X7 is K or R, X8 is E or Q, X9 is H, P, or R, and X 10 is L, T, or G, and X 11 is K or R, and X 12 is V or I, and X 13 is F, L, or V, and X 14is F or V, and the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO:1.

[0050] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant comprising the sequence of SEQ ID NO: 13, wherein X1 is L, I, or V. In any of the foregoing embodiments, X2 is V, L, or I. In some embodiments, X3 is A or V. In some embodiments, X4 is A, I, or L. In some embodiments, X5 is I, T, S, or F. In some embodiments, X6 is E, V, or L. In some embodiments, X7 is K or R. In some embodiments, X8 is E or Q. In some embodiments, X9 is H, P, or R. In some embodiments, X 10 is L, T, or G. In some embodiments, X 11 is K or R. In some embodiments, X 12 is V or I. In some embodiments, X 13 is F, L, or V. In some embodiments, X 14 is F or V. In some embodiments, the polypeptide of this aspect of the disclosure comprises six or fewer amino acid substitutions relative to a wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO:1.

[0051] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1×10 -8 Under M, 5x10-9 Less than M, 1x10 -9 Under M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D and binds to CD47.

[0052] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant comprising the following sequence: EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14 X1 is L, I, or V, X2 is V, L, or I, X3 is A or V, X4 is V, I, or L, X5 is I, T, S, or F, X6 is E, V, or L, X7 is K or R, X8 is E or Q, X9 is H, P, or R, and X 10 is S, T, or G, and X 11 is K or R, and X 12 is V or I, and X 13 is F, L, or V, and X 14 is F or V, and the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO:2.

[0053] In some embodiments of this aspect of the disclosure, the polypeptide comprises the sequence of SEQ ID NO: 14, where X1 is L, I, or V. In some embodiments, X2 is V, L, or I. In some embodiments, X3 is A or V. In some embodiments, X4 is V, I, or L. In some embodiments, X5 is I, T, S, or F. In some embodiments, X6 is E, V, or L. In some embodiments, X7 is K or R. In some embodiments, X8 is E or Q. In some embodiments, X9 is H, P, or R. In some embodiments, X 10 is S, T, or G. In some embodiments, X 11 is K or R. In some embodiments, X 12 is V or I. In some embodiments, X 13 is F, L, or V. In some embodiments, X 14 is F or V. In some embodiments, the polypeptide of this aspect of the disclosure comprises six or fewer amino acid substitutions relative to a wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO:2.

[0054] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1×10 -8 Under M, 5x10 -9 Less than M, 1x10 -9 Under M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than MD In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D and binds to CD47.

[0055] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSLX 11 PVGPIQWFRGAGPX 12 RX 13 LIYNQX 14 X 15 GX 16 FPRVTTVSX 17 X 18 TX 19 RX 20 NMDFX 21 IX 22 IX 23 NITPADAGTYYCX 24 KX 25 RKGSPDX 26 X 27 EX 28 KSGAGTELSVRX 29 XKPS (SEQ ID NO:23), X1 is E or G; X2 is L, I, or V; X3 is V, L, or I; X4 is S or F; X5 is L or S; X6 is S or T; X7 is A or V; X8 is I or T; X9 is H or R; 10 is A, V, I, or L; X 11 is I, T, S, or F; X 12 is A or G;X 13 is E, V, or L; X 14 is K or R; X 15 is E or Q;X 16is H, P, or R; X 17 is D or E;X 18 is S, L, T, or G; X 19 is K or R; X 20 is E or D;X 21 is S or P;X 22 is S or R; X 23 is S or G;X 24 is V or I;X 25 is F, L, V;X 26 is D or not present; X 27 is T or V;X 28 is F or V;X 29 is A or G; where the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2.

[0056] In any of the foregoing embodiments of this aspect of the disclosure, X2 is L, I, or V. In any of the foregoing embodiments, X3 is V, L, or I. In some embodiments, X4 is S or F. In some embodiments, X5 is L or S. In some embodiments, X6 is S or T. In some embodiments, X7 is A or V. In some embodiments, X8 is I or T. In some embodiments, X9 is H or R. In some embodiments, X 10 is A, V, I, or L. In some embodiments, X 11 is I, T, S, or F. In some embodiments, X 12 is A or G. In some embodiments, X 13 is E, V, or L. In some embodiments, X 14 is K or R. In some embodiments, X 15 is E or Q. In some embodiments, X 16 is H, P, or R. In some embodiments, X 17 is D or E. In some embodiments, X18 is S, L, T, or G. In some embodiments, X 19 is K or R. In some embodiments, X 20 is E or D. In some embodiments, X 21 is S or P. In some embodiments, X 22 is S or R. In some embodiments, X 23 is S or G. In some embodiments, X 24 is V or I. In some embodiments, X 25 is F, L, or V. In some embodiments, X 26 is D or absent. 27 is T or V. In some embodiments, X 28 is F or V. In some embodiments, X 29 is A or G. In some embodiments, the polypeptides of this aspect of the disclosure comprise six or fewer amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2.

[0057] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1×10 -8 Under M, 5x10 -9 Less than M, 1x10 -9 Under M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than M DIn some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D and binds to CD47.

[0058] In some embodiments, the polypeptide of the present disclosure comprising a SIRPα D1 domain variant further comprises a D2 domain having the sequence of SEQ ID NO: 24, a D3 domain having the sequence of SEQ ID NO: 25, or a D2 domain having the sequence of SEQ ID NO: 24 and a D3 domain having the sequence of SEQ ID NO: 25 of wild-type human SIRPα as shown in Table 3. In some embodiments, the SIRPα D1 domain variant further comprises a fragment or variant of the D2 domain, or a fragment or variant of the D3 domain. In some embodiments, the SIRPα D1 domain variant further comprises a fragment or variant of the D2 domain, and a fragment or variant of the D3 domain. In some embodiments, the SIRPα D1 domain variant is linked to the D2 or D3 domain via a linker. In some embodiments, the SIRPα D1 domain variant is linked to the D2 and D3 domains via a linker. [Table 3]

[0059] In some embodiments, the polypeptides of the present disclosure comprising a SIRPα D1 domain variant are attached to an Fc domain variant to improve the pharmacokinetic properties of the polypeptide, e.g., to increase serum half-life. In some embodiments, the SIRPα D1 domain variant is attached to an Fc domain variant that cannot dimerize. In some embodiments, the Fc domain variant serves to increase serum half-life of the polypeptides described herein. In some embodiments, the polypeptides of the present disclosure comprising a SIRPα D1 domain variant do not comprise any of the sequences of SEQ ID NOs: 26-36 shown in Table 4. [Table 4]

[0060] In some embodiments, the polypeptides and polypeptide constructs described herein are utilized in vitro for binding assays, such as immunoassays. For example, in some embodiments, the polypeptides and polypeptide constructs described herein are utilized in liquid phase or bound to a solid phase support. In some embodiments, the polypeptides utilized in immunoassays are detectably labeled in various ways.

[0061] In some embodiments, the polypeptides and polypeptide constructs described herein are bound to various carriers and used to detect the presence of specific antigen-expressing cells. Examples of carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier can be either soluble or insoluble.

[0062] A variety of different labels and labeling methods are known. Examples of labels include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds. A variety of techniques are available for attaching labels to the polypeptides disclosed herein.

[0063] In some embodiments, the polypeptides are conjugated to low molecular weight haptens. These haptens are then specifically detected by a second reaction. For example, in some embodiments, the hapten biotin is used with avidin, or the haptens dinitrophenol, pyridoxal, or fluorescein are detected with specific anti-hapten antibodies (e.g., anti-dinitrophenol, anti-pyridoxal, and anti-fluorescein antibodies, respectively).

[0064] SIRPα D1 domain variants with altered glycosylation patterns In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein alpha (SIRP-α) D1 variant, the polypeptide comprising a SIRPα D1 domain or a fragment thereof having an amino acid mutation at residue 80 relative to a wild-type SIRP-α D1 domain (e.g., a wild-type SIRPα D1 domain set forth in SEQ ID NO: 1 or 2); and at least one additional amino acid mutation at a residue selected from the group consisting of residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92 relative to the wild-type SIRPα D1 domain (e.g., a wild-type SIRPα D1 domain set forth in SEQ ID NO: 1 or 2).

[0065] Also disclosed herein, in some embodiments, is a polypeptide comprising an Fc domain variant, wherein the Fc domain variant dimer comprises two Fc domain variants, each Fc domain variant independently selected from: (i) a human IgG1 Fc region consisting of 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.

[0066] In some embodiments, the polypeptide in the composition disclosed herein comprises a SIRPα D1 domain variant with reduced or minimal glycosylation. The D1 domains of SEQ ID NOs: 1 and 2 in Table 1 each contain a single potential N-linked glycosylation site at amino acid N80 in the sequence N80ITP. Expression of the SIRPα D1 domain in Chinese Hamster Ovary (CHO) cells results in a major band of 16 kDa (unglycosylated) and a minor band of higher molecular weight that is removed by Endo Hf. Endo Hf is a recombinant protein fusion of endoglycosidase H and maltose binding protein. Endo Hf cleaves within the high mannose chitobiose core and some hybrid oligosaccharides from N-linked glycoproteins. This means that the proline at amino acid position 83 may reduce the efficiency of glycosylation resulting in proteins with different degrees of glycosylation and therefore heterogeneity. In the case of drug development, heterogeneity may pose a challenge in process development. Thus, to investigate the possibility of generating a homogeneous non-glycosylated form of the SIRPα D1 domain variant, in some embodiments, the amino acid N80 of the SIRPα D1 variant is mutated to Ala. In some embodiments, to generate a non-glycosylated, SIRPα D1 domain variant, the amino acid N80 in the SIRPα D1 domain variant is replaced by any amino acid, including any natural and non-natural amino acid, such as N80A and N80Q. In some embodiments, the SIRPα D1 domain variant comprises an N80A mutation and at least one additional mutation (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more additional mutations). In some embodiments, the additional mutation is in the CD47 binding site. In some embodiments, the additional mutation is in the hydrophobic core of the D1 domain.

[0067] In some embodiments, the polypeptide in the composition disclosed herein comprises a SIRPα D1 domain variant with increased glycosylation compared to the wild-type SIRPα D1 domain. Another option to increase the homogeneity of the final product is to increase the efficiency of glycosylation at amino acid N80 to generate a SIRPα D1 domain variant with increased glycosylation compared to the wild-type. In some embodiments, the amino acid P83 in the sequence NITP83 affects the degree of glycosylation at amino acid N80. In some embodiments, changing P83 to any amino acid increases the efficiency of glycosylation at N80. In some embodiments, the amino acid P83 in the SIRPα D1 domain variant is substituted by any amino acid, including natural and unnatural amino acids, for example P83V, P83A, P83I, and P83L. In some embodiments, the polypeptides of the disclosure are expressed in cells that have been optimized not to glycosylate the expressed protein, for example, by genetic engineering of the cell line (e.g., a genetically engineered yeast or mammalian host), or by altering cell culture conditions, such as the addition of kifunensine, or by using a naturally non-glycosylating host, such as a prokaryote (e.g., E. coli).

[0068] Table 5 shows the specific amino acid substitutions of the SIRPα D1 domain variants for each D1 domain variant sequence. In some embodiments, the SIRPα D1 domain variants include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more) of the substitutions listed in Table 5. In some embodiments, the SIRPα D1 domain variants are not glycosylated or are minimally glycosylated. In some embodiments, the SIRPα D1 domain variants are fully glycosylated or nearly fully glycosylated. In some embodiments, the SIRPα D1 domain variants include up to 14 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variants include up to 10 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variants include up to 7 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, a SIRPα D1 domain variant of the disclosure has at least 90% (eg, at least 92%, 95%, 97% or greater than 97%) amino acid sequence identity to the sequence of the wild-type D1 domain.

[0069] In some embodiments, the SIRPα D1 domain variant is a chimeric SIRPα D1 domain variant that comprises two or more portions of a wild-type D1 domain or variant thereof (e.g., a portion of one wild-type D1 domain or variant thereof and a portion of another wild-type D1 domain or variant thereof). In some embodiments, the chimeric SIRPα D1 domain variant comprises at least two portions (e.g., three, four, five, or more portions) of a wild-type D1 domain or variant thereof, where each of the portions is derived from a different wild-type D1 domain. In some embodiments, the chimeric SIRPα D1 domain variant further comprises one or more amino acid substitutions listed in Table 5. [Table 5-1] [Table 5-2] [Table 5-3]

[0070] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSPDDVEX 16 X1 is L, I, or V; X2 is V, L, or I; X3 is A, or V; X4 is A, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K, or R; X8 is E, or Q; X9 is H, P, or R; 10 is L, T, or G; X 11 is K or R;X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; X 14 is V or I;X 15 is F, L, or V; X 16 is F or V; the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO:1.

[0071] In some embodiments of this aspect of the disclosure, the polypeptide comprises a SIRPα D1 domain variant having the sequence of SEQ ID NO:37, wherein X1 is L, I, or V. In some embodiments, X2 is V, L, or I. In some embodiments, X3 is A or V. In some embodiments, X4 is A, I, or L. In some embodiments, X5 is I, T, S, or F. In some embodiments, X6 is E, V, or L. In some embodiments, X7 is K or R. In some embodiments, X8 is E or Q. In some embodiments, X9 is H, P, or R. In some embodiments, X 10 is L, T, or G. In some embodiments, X 11 is K or R. In some embodiments, X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. In some embodiments, X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. In some embodiments, X 14 is V or I. In some embodiments, X 15 is F, L, or V. In some embodiments, X 16 is F or V.

[0072] In some embodiments, the polypeptides provided herein contain no more than 10 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptides provided herein contain no more than 7 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1.

[0073] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1×10 -8 Under M, 5x10 -9 Less than M, 1x10 -9 Under M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D and binds to CD47.

[0074] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSPDTEX 16X1 is L, I, or V; X2 is V, L, or I; X3 is A or V; X4 is V, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K or R; X8 is E or Q; X9 is H, P, or R; 10 is S, T, or G; X 11 is K or R; X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; X 14 is V or I;X 15 is F, L, or V; and X 16 is F or V; the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO:2.

[0075] In some embodiments of this aspect of the disclosure, the polypeptide comprises a SIRPα D1 domain variant having the sequence of SEQ ID NO:38, wherein X1 is L, I, or V. In some embodiments, X2 is V, L, or I. In some embodiments, X3 is A or V. In some embodiments, X4 is V, I, or L. In some embodiments, X5 is I, T, S, or F. In some embodiments, X6 is E, V, or L. In some embodiments, X7 is K or R. In some embodiments, X8 is E or Q. In some embodiments, X9 is H, P, or R. In some embodiments, X 10 is S, T, or G. In some embodiments, X 11 is K or R. In some embodiments, X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. In some embodiments, X 13is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. In some embodiments, X 14 is V or I. In some embodiments, X 15 is F, L, or V. In some embodiments, X 16 is F or V.

[0076] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having no more than 10 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having no more than 7 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2.

[0077] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1×10 -8 Under M, 5x10 -9 Less than M, 1x10 -9 Under M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM.D and binds to CD47.

[0078] In another aspect, the disclosure features a polypeptide that includes a SIRPα D1 domain variant having the following sequence: EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSLX 11 PVGPIQWFRGAGPX 12 RX 13 LIYNQX 14 X 15 GX 16 FPRVTTVSX 17 X 18 TX 19 RX 20 NMDFX 21 IX 22 IX 23 X 24 ITX 25 ADAGTYYCX 26 KX 27 RKGSPDX 28 X 29 EX 30 KSGAGTELSVRX 31 X5 is L or S; X6 is S or T; X7 is A or V; X8 is I or T; X9 is H, R, or L; X 10 is A, V, I, or L; X 11 is I, T, S, or F; X 12 is A or G;X 13 is E, V, or L; X 14 is K or R; X 15 is E or Q;X 16 is H, P, or R; X 17 is D or E;X 18 is S, L, T, or G; X 19 is K or R; X 20 is E or N;X 21 is S or P;X22 is S or R; X 23 is S or G;X 24 is any amino acid; X 25 is any amino acid; X 26 is V or I;X 27 is F, L, V;X 28 is D or not present; X 29 is T or V;X 30 is F or V;X 31 is A or G; the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2.

[0079] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:47, and X1 is E or G. In any of the foregoing embodiments of this aspect of the disclosure, X2 is L, I, or V. In any of the foregoing embodiments, X3 is V, L, or I. In any of the foregoing embodiments, X4 is S or F. In any of the foregoing embodiments, X5 is L or S. In any of the foregoing embodiments, X6 is S or T. In any of the foregoing embodiments, X7 is A or V. In any of the foregoing embodiments, X8 is I or T. In any of the foregoing embodiments, X9 is H or R. In any of the foregoing embodiments, X 10 is A, V, I, or L. In any of the 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, 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 17is 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, or V. In any of the above embodiments, X 28 In any of the above embodiments, X is D or absent. 29 is T or V. In any of the above embodiments, X 30 is F or V. In any of the above embodiments, X 31 is A or G.

[0080] In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 10 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 7 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2.

[0081] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1×10 -8 Under M, 5x10 -9 Less than M, 1x10 -9 Under M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D and binds to CD47.

[0082] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVX2VAAGEX3AX4LX5CTX6TSLX7PVGPIQWFRGAGPX8RX9LIYNQX 10 X 11 GX 12 FPRVTTVSX 13 X 14 TKRX 15 NMDFSIX 16 IX 17 X 18 ITPADAGTYYCX 19 KFRKGX 20 X 21 X 22 DX23 X7 is I, R, Y, K or F; X8 is G or A; X9 is E or V; X1 is V or I; X2 is L or S; X3 is T or S; X4 is T or I; X5 is R or H; X6 is A, V, or I; X7 is I, R, Y, K or F; X8 is G or A; X9 is E or V; 10 is K or R; X 11 is E, D or Q; X 12 is H or P; X 13 is D or E;X 14 is S, L or T; X 15 is N or E;X 16 is R or S;X 17 is G or S;X 18 is N or A;X 19 is V or I;X 20 is S, I or M; X 21 is P or not present; X 22 is D or P;X 23 is V or T, or a fragment thereof.

[0083] In another aspect, the disclosure features a polypeptide that includes a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9ITPADAGTYYCX 10 X1 is V, L, or I; X2 is A, I, V, or L; X3 is I, F, S, or T; X4 is E, V, or L; X5 is K or R; X6 is E or Q; X7 is H, P, or R; X8 is L, T, S, or G; X9 is A; 10is V or I; where the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO:1.

[0084] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:49, and X1 is V, L, or I. In any of the foregoing embodiments of this aspect of the disclosure, X2 is A, I, V, or L. In any of the foregoing embodiments, X3 is I, F, S, or T. In any of the foregoing embodiments, X4 is E, V, or L. In any of the foregoing embodiments, X5 is K or R. In any of the foregoing embodiments, X6 is E or Q. In any of the foregoing embodiments, X7 is H, P, or R. In any of the foregoing embodiments, X8 is L, T, S, or G. In any of the foregoing embodiments, X9 is A. In any of the foregoing embodiments, X 10 is V or I.

[0085] In some embodiments, the polypeptide comprises a SIRPα D1 domain comprising at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO:49, and is selected from the group consisting of X1, X2, X3, X4, X5, X6, X7, X8, X9, and X10. 10 Each of is not a wild-type amino acid.

[0086] In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 10 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NO: 1. In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 7 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of any one of SEQ ID NO: 1.

[0087] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1×10 -8 Under M, 5x10 -9 Less than M, 1x10 -9 Under M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K under M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D and binds to CD47.

[0088] In another aspect, the disclosure features a polypeptide that includes a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVSVAAGESAILHCTX2TSLX3PVGPIQWFRGAGPARX4LIYNQX5X6GX7FPRVTTVSEX8TKRENMDFSISISX9ITPADAGTYYCX 10 X1 is V or I; X2 is V or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is E or Q; X7 is H or P; X8 is S or T; X9 is N or A; 10is V or I; where the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO:2.

[0089] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:50, and X1 is V or I. In any of the foregoing embodiments of this aspect of the disclosure, X2 is V or I. In any of the foregoing embodiments, X3 is I or F. In any of the foregoing embodiments, X4 is E or V. In any of the foregoing embodiments, X5 is K or R. In any of the foregoing embodiments, X6 is E or Q. In any of the foregoing embodiments, X7 is H or P. In any of the foregoing embodiments, X8 is S or R. In any of the foregoing embodiments, X9 is N or A. In any of the foregoing embodiments, X 10 is V or I.

[0090] In some embodiments, the polypeptide comprises a SIRPα D1 domain comprising at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO:50, and is selected from the group consisting of X1, X2, X3, X4, X5, X6, X7, X8, X9, and X10. 10 Each of is not a wild-type amino acid.

[0091] In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 10 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 7 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2.

[0092] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1×10 -8 Under M, 5x10 -9 Less than M, 1x10 -9 Under M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D and binds to CD47.

[0093] In another aspect, the disclosure features a polypeptide that includes a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMDFSIRIGX8ITPADAGTYYCX9KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO:51), wherein X1 is V or I; X2 is A or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is H or P; X7 is L or T; X8 is N or A; and X9 is V or I; wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO:1.

[0094] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:51, and X1 is V or I. In any of the foregoing embodiments of this aspect of the disclosure, X2 is A or I. In any of the foregoing embodiments, X3 is I or F. In any of the foregoing embodiments, X4 is E or V. In any of the foregoing embodiments, X5 is K or R. In any of the foregoing embodiments, X6 is H or P. In any of the foregoing embodiments, X7 is L or T. In any of the foregoing embodiments, X8 is N or A. In any of the foregoing embodiments, X9 is V or I. In some embodiments, X4 is not V.

[0095] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:51, and X8 is A. In any of the foregoing embodiments of this aspect of the disclosure, X8 is A and X1 is V or I. In any of the foregoing embodiments of this aspect of the disclosure, X8 is A and X2 is A or I. In any of the foregoing embodiments, X8 is A and X3 is I or F. In any of the foregoing embodiments, X8 is A and X4 is E or V. In some embodiments, X4 is not V. In any of the foregoing embodiments, X8 is A and X5 is K or R. In any of the foregoing embodiments, X8 is A and X6 is H or P. In any of the foregoing embodiments, X8 is A and X7 is A or V. In any of the foregoing embodiments, X8 is A and X9 is V or I.

[0096] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:51, and X8 is A. In any of the preceding embodiments of this aspect of the disclosure, X8 is A and X1 is I. In any of the preceding embodiments of this aspect of the disclosure, X8 is A and X2 is I. In any of the preceding embodiments, X8 is A and X3 is F. In any of the preceding embodiments, X8 is A and X4 is V. In any of the preceding embodiments, X8 is A and X5 is R. In any of the preceding embodiments, X8 is A and X6 is P. In any of the preceding embodiments, X8 is A and X7 is T. In any of the preceding embodiments, X8 is A and X9 is I.

[0097] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant comprising at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 51, and each of X1, X2, X3, X4, X5, X6, X7, X8, and X9 is not a wild-type amino acid.

[0098] In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 10 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 7 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1.

[0099] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is at least 1×10 -8 Under M, 5x10 -9 Less than M, 1x10 -9 Under M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K under M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM.D and binds to CD47.

[0100] In another aspect, the disclosure features a polypeptide that includes a SIRPα D1 domain variant having the following sequence: X4 is K or R; X5 is H or P; X6 is L, T, or G; and X7 is N or A; wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence set forth in SEQ ID NO:1.

[0101] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:222, where X1 is V, L, or I. In any of the foregoing embodiments of this aspect of the disclosure, X2 is A, I, or L. In any of the foregoing embodiments, X3 is I, T, S, or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In any of the foregoing embodiments, X6 is L, T, or G. In any of the foregoing embodiments, X7 is N or A.

[0102] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:222, where X1 is V or I. In any of the foregoing embodiments of this aspect of the disclosure, X2 is A or I. In any of the foregoing embodiments, X3 is I or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In any of the foregoing embodiments, X6 is L or T. In any of the foregoing embodiments, X7 is N or A.

[0103] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:222, where X7 is A. In any of the foregoing embodiments of this aspect of the disclosure, X7 is A and X1 is V or I. In any of the foregoing embodiments of this aspect of the disclosure, X7 is A and X2 is A or I. In any of the foregoing embodiments, X7 is A and X3 is I or F. In any of the foregoing embodiments, X7 is A and X4 is K or R. In any of the foregoing embodiments, X7 is A and X5 is H or P. In any of the foregoing embodiments, X7 is A and X6 is L or T.

[0104] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:222, where X7 is A. In any of the foregoing embodiments of this aspect of the disclosure, X7 is A and X1 is I. In any of the foregoing embodiments of this aspect of the disclosure, X7 is A and X2 is I. In any of the foregoing embodiments, X7 is A and X3 is F. In any of the foregoing embodiments, X7 is A and X4 is R. In any of the foregoing embodiments, X7 is A and X5 is P. In any of the foregoing embodiments, X7 is A and X6 is T.

[0105] In some embodiments, the polypeptide comprises a SIRPα D1 domain comprising at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 222, and each of X1, X2, X3, X4, X5, X6, and X7 is not a wild-type amino acid.

[0106] In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 10 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 7 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1.

[0107] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO:1. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO:1. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO:1. In some embodiments, the fragment comprises a polypeptide less than 10 amino acids in length, about 10 amino acids in length, about 20 amino acids in length, about 30 amino acids in length, about 40 amino acids in length, about 50 amino acids in length, about 60 amino acids in length, about 70 amino acids in length, about 80 amino acids in length, about 90 amino acids in length, about 100 amino acids in length, or more than about 100 amino acids in length. The fragment retains the ability to bind to CD47. Preferably, the SIRPα D1 domain variant polypeptides and fragments thereof bind to CD47 with higher affinity than the SIRPα polypeptide binds to CD47. For example, in some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is administered at a concentration of 1×10 -8 Under M, 5x10 -9 Less than M, 1x10 -9 Under M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K under M DIn some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D and binds to CD47.

[0108] In another aspect, the disclosure features a polypeptide that includes a SIRPα D1 domain variant having the following sequence: X4 is K or R; X5 is H, P, or R; X6 is S, T, or G; and X7 is N or A; wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO:2.

[0109] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:212, where X1 is V, L, or I. In any of the foregoing embodiments of this aspect of the disclosure, X2 is V, I, or L. In any of the foregoing embodiments, X3 is I, T, S, or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In any of the foregoing embodiments, X6 is S, T, or G. In any of the foregoing embodiments, X7 is N or A.

[0110] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:212, where X1 is V or I. In any of the foregoing embodiments of this aspect of the disclosure, X2 is V or I. In any of the foregoing embodiments, X3 is I or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In any of the foregoing embodiments, X6 is S or T. In any of the foregoing embodiments, X7 is N or A.

[0111] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:212, where X7 is A. In any of the foregoing embodiments of this aspect of the disclosure, X7 is A and X1 is V or I. In any of the foregoing embodiments of this aspect of the disclosure, X7 is A and X2 is V or I. In any of the foregoing embodiments, X7 is A and X3 is I or F. In any of the foregoing embodiments, X7 is A and X4 is K or R. In any of the foregoing embodiments, X7 is A and X5 is H or P. In any of the foregoing embodiments, X7 is A and X6 is S or T.

[0112] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:212, where X7 is A. In any of the foregoing embodiments of this aspect of the disclosure, X7 is A and X1 is I. In any of the foregoing embodiments of this aspect of the disclosure, X7 is A and X2 is I. In any of the foregoing embodiments, X7 is A and X3 is F. In any of the foregoing embodiments, X7 is A and X4 is R. In any of the foregoing embodiments, X7 is A and X5 is P. In any of the foregoing embodiments, X7 is A and X6 is T.

[0113] In some embodiments, the polypeptide comprises a SIRPα D1 domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 212, and each of X1, X2, X3, X4, X5, X6, and X7 is not a wild-type amino acid.

[0114] In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 10 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptides of this aspect of the disclosure comprise no more than 7 amino acid substitutions relative to a wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2.

[0115] In some embodiments, the polypeptide binds to CD47 with at least 10-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO:2. In some embodiments, the polypeptide binds to CD47 with at least 100-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO:2. In some embodiments, the polypeptide binds to CD47 with at least 1000-fold higher binding affinity than a wild-type SIRPα D1 domain having the sequence of SEQ ID NO:2. In some embodiments, the fragment comprises a polypeptide less than 10 amino acids in length, about 10 amino acids in length, about 20 amino acids in length, about 30 amino acids in length, about 40 amino acids in length, about 50 amino acids in length, about 60 amino acids in length, about 70 amino acids in length, about 80 amino acids in length, about 90 amino acids in length, about 100 amino acids in length, or more than about 100 amino acids in length. The fragment retains the ability to bind to CD47. Preferably, the SIRPα D1 domain variant polypeptides and fragments thereof bind to CD47 with a higher affinity than the SIRPα polypeptide binds to CD47. For example, in some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof is administered at a concentration of 1×10 -8 Under M, 5x10 -9 Less than M, 1x10-9 Under M, 5x10 -10 Less than M, 1x10 -10 Less than M or 1x10 -11 K less than M D In some embodiments, the SIRPα D1 domain variant polypeptide or fragment thereof binds to CD47 with a K of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM. D and binds to CD47.

[0116] Described herein, in some embodiments, is a polypeptide that includes a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9X 10 X 11 X 12 ADAGTYYCX 13 X1 is V, L, or I; X2 is A, V, L, or I; X3 is I, S, T, or F; X4 is E, L, or V; X5 is K or R; X6 is E or Q; X7 is H, R, or P; X8 is S, G, L, or T; X9 is any amino acid; 10 is any amino acid; X 11 is any amino acid; X 12 is any amino acid; X 13 is V or I; wherein the SIRPα D1 domain variant comprises at least two amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence set forth in SEQ ID NO:1.

[0117] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:212, where X1 is A and X9 is A. In any of the foregoing embodiments of this aspect of the disclosure, X9 is N. In any of the foregoing embodiments of this aspect of the disclosure, X 10 In any of the foregoing embodiments of this aspect of the disclosure, X9 is N and X10 is P. In any of the foregoing embodiments of this aspect of the disclosure, X9 is N and X 11 is any amino acid other than S, T or C. In any of the foregoing embodiments of this aspect of the disclosure, X 11 is T. In any of the foregoing embodiments of this aspect of the disclosure, X 11 is any amino acid other than T. In any of the foregoing embodiments of this aspect of the disclosure, X 12 In any of the foregoing embodiments of this aspect of the disclosure, X is N and X 12 is any amino acid other than P.

[0118] Described herein, in some embodiments, is a polypeptide that includes a SIRPα D1 domain variant having the following sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9ITX 10 ADAGTYYCX 11 X1 is V, L, or I; X2 is A, V, L, or I; X3 is I, S, T, or F; X4 is E, L, or V; X5 is K or R; X6 is E or Q; X7 is H, R, or P; X8 is S, G, L, or T; X9 is N; 10 is any other than P の Amino acid;X 11is V or I; wherein the SIRPα D1 domain variant comprises at least two amino acid substitutions compared to a wild-type SIRPα D1 domain variant having a sequence according to SEQ ID NO:1.

[0119] In another aspect of the disclosure, disclosed herein is a composition comprising a SIRPα D1 domain variant polypeptide having an amino acid sequence of SEQ ID NO: 48, or a fragment thereof. In some embodiments, the SIRPα D1 domain variant polypeptide or a fragment thereof binds to CD47 with a higher affinity than the affinity with which the SIRPα polypeptide binds to CD47. In some embodiments, the SIRPα D1 domain variant polypeptide is at least 1×10 -8 Less than M or 1×10 -9 Less than M, 1×10 -10 Less than M or 1×10 -11 K under M D In some embodiments, the SIRPα D1 domain variant polypeptide is attached or fused to a second polypeptide. In some embodiments, the second polypeptide includes, but is not limited to, an Fc polypeptide, an Fc variant, or a fragment of the foregoing.

[0120] Without limiting the foregoing, in some embodiments, the SIRPα D1 domain variant polypeptide is selected from any one of SEQ ID NOs: 53-87 and 213 shown in Table 6. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5]

[0121] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to any variant listed in Table 6.

[0122] In some embodiments, the polypeptide comprises a SIRPα D1 domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 80, 81 or 85 in Table 6.

[0123] Fc domain variants and fusion polypeptides comprising Fc domain variants In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein alpha (SIRP-α) D1 variant, the polypeptide comprising a SIRPα D1 domain or a fragment thereof having an amino acid mutation at residue 80 relative to a wild-type SIRP-α D1 domain (e.g., a wild-type SIRPα D1 domain set forth in SEQ ID NO: 1 or 2); and at least one additional amino acid mutation at a residue selected from the group consisting of residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92 relative to the wild-type SIRPα D1 domain (e.g., a wild-type SIRPα D1 domain set forth in SEQ ID NO: 1 or 2).

[0124] Also disclosed herein, in some embodiments, is an Fc domain variant dimer, wherein the Fc domain variant dimer comprises two Fc domain variants, each Fc domain variant independently selected from: (i) a human IgG1 Fc region consisting of the mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of the mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A.

[0125] Antibodies targeting cell surface antigens can trigger immune stimulation and effector functions associated with the binding of Fc receptors (FcRs) on immune cells. There are multiple Fc receptors specific for certain classes of antibodies, such as IgG (gamma receptors), IgE (eta receptors), IgA (alpha receptors), and IgM (mu receptors). Binding of antibodies to Fc receptors of the Fc region on the cell surface can trigger multiple biological responses, such as phagocytosis of antibody-coated particles (antibody-dependent cell-mediated phagocytosis, or ADCP), clearance of immune complexes, lysis of antibody-coated cells by killer cells (antibody-dependent cell-mediated cytotoxicity, or ADCC) and release of inflammatory mediators, placental implantation, and control of immunoglobulin production. In addition, the complement system can be activated by binding of the C1 component of complement to antibodies. Complement activation can be important for the lysis of cellular pathogens. However, complement activation can also stimulate inflammatory responses and may be involved in autoimmune hypersensitivity or other immune disorders. Variant Fc regions that have reduced or eliminated ability to bind to specific Fc receptors are useful for the development of therapeutic antibody and Fc fusion polypeptide constructs that act by targeting, activating, or neutralizing ligand function without damaging or destroying local cells or tissues.

[0126] In some embodiments, a SIRPα D1 polypeptide construct comprises a non-native SIRPα D1 domain variant linked to an Fc domain variant forming an Fc domain with eliminated or reduced effector function.

[0127] In some embodiments, an Fc domain variant refers to a polypeptide chain that includes a second and a third antibody constant domain (e.g., CH2 and CH3). In some embodiments, the Fc domain variant also includes a hinge domain. In some embodiments, the Fc domain variant is of any immunoglobulin antibody isotype, such as IgG, IgE, IgM, IgA, and IgD. Additionally, in some embodiments, the Fc domain variant is of any IgG subtype (e.g., IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, and IgG4). In some embodiments, the Fc domain variant includes as many as 10 amino acid modifications (e.g., insertions, deletions, and / or substitutions) that alter the interaction between the Fc domain and an Fc receptor relative to the wild-type Fc domain monomer sequence (e.g., 1-10, 1-8, 1-6, 1-4 amino acid substitutions, additions or insertions, deletions, or combinations thereof).

[0128] As used herein, the term "Fc domain dimer" refers to a dimer of two Fc domains, in which the two wild-type Fc domains dimerize via the interaction between the two CH3 antibody constant domains and one or more disulfide bonds formed between the hinge domains of the two dimerized Fc domains.

[0129] As used herein, the term "Fc domain dimer variant" includes at least one Fc domain variant. In some embodiments, the Fc domain dimer variant includes an Fc domain variant that is mutated to not contain effector function, e.g., a "dead Fc domain dimer variant." In some embodiments, each of the Fc domains in the Fc domain dimer variant includes an amino acid substitution in the CH2 antibody constant domain to reduce interaction or binding between the Fc domain dimer variant and an Fc receptor, such as an Fcγ receptor (FcγR), an Fcα receptor (FcαR), or an Fcε (FcεR).

[0130] In some embodiments, the SIRPα D1 domain variant (e.g., any of the variants listed in Tables 2, 5, and 6) is fused to an immunoglobulin Fc domain variant or a fragment of an Fc domain variant. In some embodiments, the immunoglobulin Fc domain variant or a fragment of an Fc domain variant can form an Fc domain dimer with another Fc domain variant. In some embodiments, the immunoglobulin Fc domain variant or a fragment of an Fc domain variant cannot form an Fc domain dimer with another Fc domain variant. In some embodiments, the Fc domain variant or a fragment of an Fc domain variant is fused to a polypeptide of the present disclosure to increase the serum half-life of the polypeptide. In some embodiments, the Fc domain variant or a fragment of an Fc domain variant fused to a polypeptide of the present disclosure dimerizes with a second Fc domain variant to form an Fc domain dimer variant that binds to an Fc receptor, or the Fc domain variant binds to an Fc receptor. In some embodiments, the Fc domain variant or a fragment of an Fc domain variant fused to a polypeptide to increase the serum half-life of the polypeptide does not induce any immune system-related response.

[0131] In some embodiments, the SIRPα polypeptide or construct provided herein comprises a SIRPα D1 domain or variant thereof linked to a first Fc domain variant, and an antibody variable domain linked to a second Fc domain variant, where the first and second Fc domain variants combine to form an Fc domain dimer variant (e.g., a heterodimeric Fc domain dimer variant). An Fc domain dimer is a protein structure found at the C-terminus of immunoglobulins. An Fc domain dimer comprises two Fc domains that are dimerized by the interaction between the CH3 antibody constant domains. The wild-type Fc domain dimer forms the minimal structure that binds to an Fc receptor, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and FcγRIV.

[0132] The Fc domain dimer is not directly involved in binding of the antibody to its target, but may be involved in various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity. In some embodiments, the Fc domain in the SIRPα polypeptide or construct of the present disclosure comprises amino acid substitutions, additions or insertions, deletions, or any combination thereof, that result in reduced effector functions, such as reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated phagocytosis (ADCP), or any combination thereof. In some embodiments, the SIRPα polypeptide or construct of the present disclosure is characterized by reduced binding (e.g., minimal or no binding) to human Fc receptors and reduced binding (e.g., minimal or no binding) to complement protein C1q. In some embodiments, the SIRPα constructs of the present disclosure are characterized by reduced binding (e.g., minimal or no binding) to human FcγRI, FcγRIIA, FcγRIIB, FcγRIIIB, or any combination thereof, and C1q. In some embodiments, to alter or reduce antibody-dependent effector function, such as ADCC, CDC, ADCP, or any combination thereof, in some embodiments, the Fc domain in the SIRPα construct of the disclosure is of the IgG class and contains one or more amino acid substitutions at E233, L234, L235, G236, G237, D265, D270, N297, E318, K320, K322, A327, A330, P331, or P329 (numbering according to EU index of Kabat) (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0133] In some embodiments, a polypeptide construct comprising a non-native Fc region described herein exhibits reduced or ablated binding to at least one of the Fcγ receptors CD16a, CD32a, CD32b, CD32c, and CD64, as compared to a polypeptide construct comprising a native Fc region. In some cases, a polypeptide construct described herein exhibits reduced or ablated binding to the CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors.

[0134] CDC refers to a form of cytotoxicity in which the complement cascade is activated by complement component C1q binding to an antibody Fc domain. In some embodiments, the polypeptide constructs comprising the non-native Fc regions described herein exhibit at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduced C1q binding compared to polypeptide constructs comprising wild-type Fc regions. In some cases, the polypeptide constructs comprising the non-native Fc regions described herein exhibit reduced CDC compared to polypeptide constructs comprising wild-type Fc regions. In some embodiments, the polypeptide constructs comprising the non-native Fc regions described herein exhibit at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduced CDC compared to polypeptide constructs comprising wild-type Fc regions. In some cases, a polypeptide construct comprising a non-naturally occurring Fc domain variant or an Fc domain dimer variant described herein exhibits negligible CDC compared to a polypeptide construct comprising a wild-type Fc region.

[0135] In some embodiments, the Fc domain variants or Fc domain dimer variants described herein are minimally glycosylated or have reduced glycosylation relative to the wild-type sequence. In some embodiments, deglycosylation is achieved by mutation of N297A or by mutating N297 to any amino acid other than N. In some embodiments, deglycosylation is achieved by disrupting the motif N-Xaa1-Xaa2-Xaa3, where N=asparagine; Xaa1=any amino acid other than P (proline); Xaa2=T (threonine), S (serine), or C (cysteine); and Xaa3=any amino acid other than P (proline). In one embodiment, the N-Xaa1-Xaa2-Xaa3 motif refers to residues 297-300 as designated according to Kabat et al., (1991). In some embodiments, mutations to any one or more of N, Xaa1, Xaa2, or Xaa3 result in deglycosylation of the Fc domain variant or Fc domain dimer variant.

[0136] In some embodiments, the antibody IgG constant region variant (e.g., Fc domain variant or Fc domain dimer variant) has a reduced ability to specifically bind to Fcγ receptors or a reduced ability to induce phagocytosis. In some embodiments, the antibody IgG constant region variant (e.g., Fc domain variant or Fc domain dimer variant) has a reduced ability to specifically bind to Fcγ receptors and a reduced ability to induce phagocytosis. For example, in some embodiments, the Fc domain variant is mutated so that it does not contain effector functions typical of "dead" Fc domain variants. For example, in some embodiments, the Fc domain variant contains specific amino acid substitutions known to minimize the interaction between Fc domain dimers and Fcγ receptors. In some embodiments, the Fc domain variant is derived from an IgG1 antibody and contains one or more of the amino acid substitutions L234A, L235A, G237A, and N297A (designated according to the EU numbering system by Kabat et al., (1991)). In some embodiments, one or more additional mutations are included in such IgG1 Fc domain variants. Non-limiting examples of such additional mutations for human IgG1 Fc domain variants include E318A and K322A. In some cases, the human IgG1 Fc domain variants have a total of up to 12, 11, 10, 9, 8, 7, 6, 5, or 4 or less mutations compared to the wild-type human IgG1 sequence. In some embodiments, one or more additional deletions are included in such IgG1 Fc domain variants. For example, in some embodiments, the C-terminal lysine of the Fc domain IgG1 heavy chain constant region provided in SEQ ID NO: 88 of Table 7 is deleted, for example to increase the homogeneity of the polypeptide when the polypeptide is produced in bacteria or mammalian cells. In some cases, the human IgG1 Fc domain variants have a total of up to 12, 11, 10, 9, 8, 7, 6, 5, or 4 or less deletions compared to the wild-type human IgG1 sequence (see, for example, SEQ ID NO: 161 below).In some embodiments, the IgG1 Fc domain variant has the sequence set forth in any one of SEQ ID NO:135, SEQ ID NO:136, or SEQ ID NO:137. SEQ ID NO:161: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0137] In some embodiments, the Fc domain variant is derived from an IgG2 or IgG4 antibody and comprises the amino acid substitutions A330S, P331S, or both A330S and P331S. The aforementioned amino acid positions are defined according to Kabat et al., (1991). The Kabat numbering of amino acid residues can be determined for a given antibody by alignment of the antibody's sequence at the homology region with the "standard" Kabat numbering sequence. In some embodiments, the Fc domain variant comprises a human IgG2 Fc domain sequence (designated according to the EU numbering system according to Kabat et al., (1991)) that comprises one or more of the A330S, P331S, and N297A amino acid substitutions. In some embodiments, one or more additional mutations are included in such an IgG2 Fc domain variant. Non-limiting examples of such additional mutations for human IgG2 Fc domain variants include V234A, G237A, P238S, V309L and H268A (designated according to the EU numbering system by Kabat et al., (1991)). In some cases, the human IgG2 Fc domain variant has a total of up to 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 or fewer mutations compared to the wild-type human IgG2 sequence. In some embodiments, one or more additional deletions are included in such IgG2 Fc domain variants. For example, in some embodiments, the C-terminal lysine of the Fc domain IgG2 heavy chain constant region provided in SEQ ID NO: 89 of Table 7 is deleted to increase the homogeneity of the polypeptide, for example when the polypeptide is produced in bacteria or mammalian cells. In some cases, the human IgG2 Fc domain variant has a total of up to 12, 11, 10, 9, 8, 7, 6, 5, or 4 or fewer deletions compared to the wild-type human IgG2 sequence (see, for example, SEQ ID NO: 162 below). SEQ ID NO:162: ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPI EKTISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0138] Where the Fc domain variant is an IgG4 Fc domain variant, in some embodiments such an Fc domain variant comprises a S228P mutation (designated according to Kabat et al., (1991)). In some cases, the human IgG4 Fc domain variant has a total of up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations compared to the wild-type human IgG4 sequence. In some embodiments, the Fc domain variant comprises a human IgG4 Fc sequence comprising one or more of the following amino acid substitutions: S228P, E233P, F234V, L235A, and delG236 (designated according to the EU numbering system according to Kabat et al., (1991)). In some embodiments, the Fc domain variant comprises a human IgG4 Fc sequence containing one or more of the following amino acid substitutions: S228P, E233P, F234V, L235A, delG236, and N297A (designated according to the EU numbering system according to Kabat et al., (1991)).

[0139] 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.

[0140] In some embodiments, the Fc domain variants exhibit reduced binding to an Fc receptor of interest compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain variants exhibit ablated binding to an Fc receptor of interest compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain variants exhibit reduced phagocytosis compared to the wild-type human IgG Fc region. In some embodiments, the Fc domain variants exhibit ablated phagocytosis compared to the wild-type human IgG Fc region.

[0141] SEQ ID NO: 88 and SEQ ID NO: 89 provide the amino acid sequences of the Fc domain IgG1 and IgG2 heavy chain constant regions. In some embodiments, the Fc domain variant is any variant of SEQ ID NO: 90-95, as shown in Table 7. [Table 7-1] [Table 7-2]

[0142] Antibody-dependent cell-mediated cytotoxicity, also referred to herein as ADCC, refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells and neutrophils), allowing these cytotoxic effector cells to specifically bind to and subsequently kill antigen-bearing target cells. Antibody-dependent cell-mediated phagocytosis, also referred to herein as ADCP, refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain phagocytes (e.g., macrophages), allowing these phagocyte effector cells to specifically bind to and subsequently engulf and digest antigen-bearing target cells. Ligand-specific high affinity IgG antibodies directed to the surface of target cells can stimulate cytotoxic or phagocyte cells and can be used for such killing. In some embodiments, polypeptide constructs comprising the Fc domain variants or Fc domain dimer variants described herein exhibit reduced ADCC or ADCP compared to polypeptide constructs comprising wild-type Fc regions. In some embodiments, a polypeptide construct comprising an Fc domain variant or an Fc domain dimer variant described herein exhibits at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduced ADCC or ADCP compared to a polypeptide construct comprising a wild-type Fc region. In some embodiments, a polypeptide construct comprising an Fc domain variant or an Fc domain dimer variant described herein exhibits ablated ADCC or ADCP compared to a polypeptide construct comprising a wild-type Fc region.

[0143] Complement-directed cytotoxicity, also referred to as CDC herein, refers to a form of cytotoxicity in which the complement cascade is activated by the complement component C1q binding to an antibody Fc domain. In some embodiments, a polypeptide construct comprising an Fc domain variant or an Fc domain dimer variant described herein exhibits at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduced binding of C1q compared to a polypeptide construct comprising a wild-type Fc region. In some cases, a polypeptide construct comprising an Fc domain variant or an Fc domain dimer variant described herein exhibits reduced CDC compared to a polypeptide construct comprising a wild-type Fc region. In some embodiments, a polypeptide construct comprising an Fc domain variant or an Fc domain dimer variant described herein exhibits at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduced CDC compared to a polypeptide construct comprising a wild-type Fc region. In some embodiments, a polypeptide construct comprising an Fc domain variant or an Fc domain dimer variant described herein exhibits negligible CDC compared to a polypeptide construct comprising a wild-type Fc region.

[0144] Fc domain variants or Fc domain dimer variants herein include those that exhibit reduced binding to Fcγ receptors compared to wild-type human IgG Fc region. For example, in some embodiments, the Fc domain variants or Fc domain dimer variants exhibit less binding to Fcγ receptors than that exhibited by wild-type human IgG Fc region, as described in the Examples. In some cases, the Fc domain variants or Fc domain dimer variants exhibit reduced binding to Fcγ receptors by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (effector function is completely removed). In some embodiments, the reduced binding is for any one or more Fcγ receptors, such as CD16a, CD32a, CD32b, CD32c, or CD64.

[0145] In some cases, the Fc domain variant or Fc domain dimer variant disclosed herein exhibits reduced phagocytosis compared to its wild-type human IgG Fc region. Such Fc domain variants or Fc domain dimer variants exhibit reduced phagocytosis compared to its wild-type human IgG Fc region, where the reduced phagocytic activity is, for example, by a factor of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some cases, the Fc domain variant or Fc domain dimer variant exhibits ablated phagocytosis compared to its wild-type human IgG Fc region.

[0146] In some embodiments, the Fc domain variants or Fc domain dimer variants disclosed herein are linked to one or more fusion partners. In some cases, the fusion partner is a therapeutic moiety. In some cases, the fusion partner is selected to allow for targeting, purification, screening, presentation, etc. of the expressed protein. In some embodiments, the fusion partner also affects the degree of binding to Fc receptors or the degree of reduction in phagocytosis. As described herein, in some embodiments, when the Fc domain variants or Fc domain dimer variants are linked to a fusion partner, they form the polypeptide constructs described below.

[0147] In some embodiments, the fusion partner is linked to the Fc domain variant or Fc domain dimer variant sequence via a linker sequence. In some embodiments, the linker sequence generally comprises a small number of amino acids, such as less than 10 amino acids, although longer linkers are also utilized. In some cases, the linker has a length of 10, 9, 8, 7, 6, or 5 amino acids or less. In some cases, the linker has a length of at least 10, 11, 12, 13, 14, 15, 20, 25, 30, or 35 or more amino acids. Optionally, in some embodiments, a cleavable linker is used.

[0148] In some embodiments, the fusion partner is a targeting or signal sequence that directs the Fc domain variant or Fc domain dimer variant protein and any associated fusion partners to a desired cellular location or extracellular medium. In some embodiments, certain signaling sequences target proteins to be secreted either to the growth medium or to the periplasmic space located between the inner and outer membranes of the cell. In some embodiments, the fusion partner is a sequence that encodes a peptide or protein that allows for purification or screening. Such fusion partners include, but are not limited to, polyhistidine tags (His tags) (e.g., His6 (SEQ ID NO: 223) and His10 (SEQ ID NO: 224)) or other tags for use with immobilized metal affinity chromatography (IMAC) systems (e.g., Ni+2 affinity columns), GST fusions, MBP fusions, Strep tags, BSP biotinylation target sequence for the bacterial enzyme BirA, and epitope tags targeted by antibodies (e.g., c-myc tags, Flag tags, etc.).

[0149] In some embodiments, such tags are useful for purification, screening, or both. For example, in some embodiments, the Fc domain variant or Fc domain dimer variant is purified by immobilizing it to a Ni+2 affinity column using the His tag, and then after purification, the antibody is immobilized to a Ni+2 coated plate using the same His tag and an ELISA or other binding assay as described elsewhere herein is performed. In some embodiments, the fusion partner allows for the use of selection methods to screen for Fc domain variants or Fc domain dimer variants as described herein.

[0150] A variety of fusion partners are available that allow for a variety of selection methods. For example, phage display can be used by fusing members of an Fc domain variant or Fc domain dimer variant library to gene III protein. In some embodiments, the fusion partner is a labeled Fc domain variant or Fc domain dimer variant. Alternatively, in some embodiments, the fusion partner binds to a specific sequence on an expression vector, which allows the fusion partner and the associated Fc domain variant or Fc domain dimer variant to be covalently or non-covalently linked to the nucleic acid that encodes them.

[0151] In some embodiments, when the fusion partner is a therapeutic moiety, the therapeutic moiety is, for example, a peptide, a protein, an antibody, an siRNA, or a small molecule. Non-limiting examples of therapeutic antibodies that are attached to the Fc domain variants or Fc domain dimer variants of the present disclosure include, but are not limited to, antibodies that recognize CD47. Non-limiting examples of therapeutic polypeptides that are attached to the Fc domain variants or Fc domain dimer variants of the present disclosure include, but are not limited to, CD47 binding polypeptides, such as SIRPα polypeptides. In such cases, the CD47 binding polypeptide is attached or fused to the Fc domain variants or Fc domain dimer variants of the present disclosure. Examples of CD47 binding polypeptides include, but are not limited to, anti-CD47 antibodies or fragments thereof, and ligands of CD47, such as SIRPα or fragments thereof. Additional examples of CD47 binding polypeptides include, but are not limited to, naturally occurring forms of SIRPα and variants thereof.

[0152] In some embodiments, disclosed herein is a polypeptide comprising an Fc domain dimer variant, wherein the Fc domain dimer variant comprises two Fc domain variants, each Fc domain variant being independently selected from: (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region consisting of mutations S228P, E233P, F234V, L235A, delG236, and N297A. In some embodiments, the Fc domain variants are identical (i.e., homodimers). In some embodiments, the Fc domain variants are different (i.e., heterodimers). In some embodiments, at least one of the Fc domain variants in the Fc domain dimer is a human IgG1 Fc region consisting of the mutations L234A, L235A, G237A, and N297A. In some embodiments, at least one of the Fc domain variants in the Fc domain dimer is a human IgG2 Fc region consisting of the mutations A330S, P331S, and N297A. In some embodiments, the Fc domain dimer variant exhibits ablated or reduced binding to Fcγ receptors compared to a wild-type version of the human IgG Fc region. In some embodiments, the Fc domain dimer variant exhibits ablated or reduced binding to CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors compared to a wild-type version of the human IgG Fc region. In some embodiments, the Fc domain dimer variant exhibits ablated or reduced binding to C1q compared to a wild-type version of the human IgG Fc fusion. In some embodiments, at least one of the Fc domain variants in the Fc domain dimer variant is a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A. In some embodiments, the Fc domain dimer variant exhibits ablated or reduced binding to Fcγ receptors compared to a wild-type human IgG4 Fc region.In some embodiments, the Fc domain dimer variant exhibits ablated or reduced binding to CD16a and CD32b Fcγ receptors compared to a wild-type version of the human IgG4 Fc region. ... -6 K exceeds M D It binds to Fcγ receptors.

[0153] In some embodiments, the Fc domain dimer variant further comprises a CD47 binding polypeptide. In some embodiments, the Fc domain dimer variant exhibits ablated or reduced binding to Fcγ receptors compared to a 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.

[0154] 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 SIRPα D1 domain variant comprising the following amino acid sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMDFSIRIGX8ITPADAGTYYCX9KFRKGSPDDVEFKSGAGTELSVRAKPS (sequence number 221), where X1 is V or I; X2 is A or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is H or P; X7 is L or T; X8 is any amino acid other than N; and X9 is V or I. In some embodiments, the SIRPα polypeptide comprises a SIRPα D1 domain variant, 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.

[0155] In some embodiments disclosed herein is a polypeptide comprising a SIRPα D1 domain variant, wherein the SIRPα D1 domain variant is a non-naturally occurring high affinity SIRPα D1 domain, wherein the SIRPα D1 domain variant binds to human CD47 with an affinity at least 10-fold higher than the affinity of a naturally occurring D1 domain and an Fc domain variant, and wherein the Fc domain variant is linked to a second polypeptide comprising a second Fc domain variant to form an Fc domain dimer variant, wherein the Fc domain dimer variant has eliminated or reduced effector function. In some embodiments, the non-naturally occurring high affinity SIRPα D1 domain comprises an amino acid mutation at residue 80.

[0156] In some embodiments, disclosed herein is a SIRPα D1 domain variant, the SIRPα D1 domain variant having a K of less than 250 nM. D The SIRPα D1 domain variant binds to CD47 of the first type with a K of less than 250 nM. Dbinds to the second type of CD47 and the K D and type 2 CD47 K D are within 100-fold of each other, and the first and second species are selected from the group consisting of human, rodent, and non-human primate. In some embodiments, the SIRPα D1 domain variant binds to CD47 of at least three different species. In some embodiments, the non-human primate is a cynomolgus monkey.

[0157] In some embodiments, disclosed herein are antibodies that (a) bind to human CD47 with a K of less than 250 nM. D and (b) an Fc domain or variant thereof linked to the N-terminus or C-terminus of the SIRPα D1 domain, wherein the polypeptide does not cause acute anemia in rodents and non-human primates. In some embodiments, the polypeptide is a non-naturally occurring variant of human SIRP-α. In some embodiments, administration of the polypeptide in vivo results in less than a 50% decrease in hemoglobin during the first week after administration. In some embodiments, administration of the polypeptide in humans results in less than a 50% decrease in hemoglobin during the first week after administration. In some embodiments, the polypeptide further comprises at least one Fc domain dimer variant, wherein the Fc domain dimer variant comprises an Fc domain variant selected from: (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236, and N297A. In some embodiments, the Fc domain variant is a human IgG1 Fc region consisting of mutations L234A, L235A, G237A, and N297A. In some embodiments, the Fc domain variant is a human IgG2 Fc region consisting of mutations A330S, P331S, and N297A.

[0158] The SIRPα construct of the present disclosure comprises a SIRPα domain or variant thereof with its C-terminus linked to the N-terminus of the Fc domain or variant thereof via a linker using conventional genetic or chemical means, e.g., chemical conjugation. In some embodiments, a linker (e.g., a spacer) is inserted between the polypeptide and the Fc domain or variant thereof. In some embodiments, a polypeptide of the present disclosure comprising a SIRPα D1 domain variant is fused to an Fc domain variant that cannot form a dimer. In some embodiments, a polypeptide of the present disclosure is fused to an Fc domain or variant thereof that can form a dimer, e.g., a heterodimer, with another Fc domain or variant thereof. In some embodiments, a polypeptide of the present disclosure is fused to an Fc domain or variant thereof, and the fusion protein forms a homodimer. In some embodiments, a polypeptide of the present disclosure is fused to a first Fc domain or variant thereof, and a different protein or peptide (e.g., an antibody variable region) is fused to a second Fc domain or variant thereof. In some embodiments, the SIRPα D1 domain or variant thereof is linked to a first Fc domain or variant thereof and a therapeutic protein (e.g., a cytokine, interleukin, antigen, steroid, anti-inflammatory agent, or immunomodulatory agent) is linked to a second Fc domain or variant thereof. In some embodiments, the first and second Fc domains or variants thereof form a heterodimer.

[0159] Without limiting the foregoing, in some embodiments, a SIRPα D1 domain variant polypeptide (e.g., any of the variants described in Tables 2, 5, and 6) is fused to an Fc polypeptide, or an Fc variant polypeptide, such as an Fc domain or variant thereof. Examples of polypeptides that include SIRPα D1 domain variant polypeptides and fused Fc domain variant polypeptides include, but are not limited to, SEQ ID NOs: 96-137, 214, and 216 shown in Table 8. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8] [Table 8-9] [Table 8-10]

[0160] In some embodiments, the polypeptide comprises a SIRPα D1 variant domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to any variant listed in Table 8.

[0161] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NOs: 98-104, 107-113, 116-122, or 135-137 of Table 8.

[0162] In some embodiments, the polypeptide comprises (a) a signal regulatory protein alpha (SIRP-α) D1 variant and (b) an fc domain dimer variant, wherein the SIRPα D1 domain variant comprises the amino acid sequence: EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSLX 11 PVGPIQWFRGAGPX 12 RX 13 LIYNQX 14 X 15 GX 16 FPRVTTVSX 17 X 18 TX 19 RX 20 NMDFX 21 IX 22 IX 23 X 24 ITX 25 ADAGTYYCX 26 KX 27 RKGSPDX 28 X 29 EX 30 KSGAGTELSVRX 31 X5 is L or S; X6 is S or T; X7 is A or V; X8 is I or T; X9 is H, R, or L; X 10 is A, V, I, or L; X 11 is I, T, S, or F; X 12 is A or G;X 13is E, V, or L; X 14 is K or R;X 15 is E or Q;X 16 is H, P, or R; X 17 is D or E;X 18 is S, L, T, or G; X 19 is K or R;X 20 is E or N;X 21 is S or P;X 22 is S or R;X 23 is S or G;X 24 is any amino acid; X 25 is any amino acid; X 26 is V or I;X 27 is F, L, or V; X 28 is D or not present; X 29 is T or V;X 30 is F or V;X 31 is A, or G; wherein the SIRPα D1 domain variant comprises at least two amino acid substitutions relative to a wild-type SIRPα D1 domain having a sequence set forth in any one of SEQ ID NOs: 1-10. 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; (vi) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations; and (vii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. an Fc region; or (viii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations.

[0163] In some embodiments, the polypeptide comprises a SIRPα D1 domain variant and an Fc domain dimer having two Fc domains, wherein the SIRPα D1 domain variant comprises an amino acid sequence according to SEQ ID NO: 47, and one of the Fc domains is an Fc domain variant comprising a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations.

[0164] Fc domain dimerization In some embodiments, a SIRPα D1 domain variant polypeptide (e.g., any of the variants described in Tables 2, 5, and 6) is fused to a first Fc domain (e.g., an Fc domain variant) at either the N-terminus or C-terminus. In some embodiments, the first Fc domain is a variant that is unable to form a dimer. In some embodiments, the first Fc domain forms a dimer with a second Fc domain. In some embodiments, the first Fc domain and the second Fc domain comprise amino acid substitutions that promote heterodimerization between the first domain Fc domain and the second domain Fc domain.

[0165] In some embodiments, each of the two Fc domains in the Fc domain dimer comprises an amino acid substitution that promotes heterodimerization of the two monomers. In some embodiments, the SIRPα construct is formed from a first subunit, e.g., a SIRPα D1 domain variant polypeptide, fused to a first Fc domain, and a second subunit, e.g., a second Fc domain (e.g., does not include a SIRPα D1 domain variant polypeptide, or any other polypeptide). In some embodiments, the construct has a single SIRPα D1 domain variant polypeptide linked to an Fc domain dimer (e.g., a single arm). In some embodiments, the construct has two SIRPα D1 domain variant polypeptides linked to an Fc domain dimer (e.g., a double arm). In some embodiments, the construct has a K of about 500 nM. DA SIRPα D1 domain variant having a K of about 50 nM is particularly useful in a dual-arm construct. D A SIRPα D1 domain variant having a K of about 5 nM is particularly useful in a dual-arm construct. D SIRPα D1 domain variants having a K of about 500 pM are useful in dual-arm and single-arm constructs. D SIRPα D1 domain variants having a K of about 100 pM are useful in dual-arm and single-arm constructs. D SIRPα D1 domain variants having a K of about 50 pM are useful in dual-arm and single-arm constructs. D SIRPα D1 domain variants having a K of about 10 pM are useful in dual-arm and single-arm constructs. D SIRPα D1 domain variants having the following structure are useful in dual-arm and single-arm constructs.

[0166] In some embodiments, heterodimerization of Fc domains is promoted by introducing different but compatible substitutions in the two Fc domains, such as "knob-into-hole" residue pairs and charge residue pairs. Although knob and hole interactions favor the formation of heterodimers, knob-to-knob and hole-to-hole interactions prevent the formation of homodimers due to steric clashes and loss of favorable interactions. A hole refers to a void created when an original amino acid in a protein is replaced with a different amino acid with a small side chain volume. A knob refers to a protuberance created when an original amino acid in a protein is replaced with a different amino acid with a large side chain volume. For example, in some embodiments, the substituted amino acid is in the CH3 antibody constant domain of the Fc domain and is involved in the dimerization of the two Fc domains. In some embodiments, the hole of one CH3 antibody constant domain is made to accommodate the knob of another CH3 antibody constant domain, such that the amino acids of the knob and hole act to promote or favor the heterodimerization of the two Fc domains. In some embodiments, a hole in one CH3 antibody constant domain is engineered to better accommodate an original amino acid in another CH3 antibody constant domain, in some embodiments, a knob in one CH3 antibody constant domain is engineered to form additional interactions with an original amino acid in another CH3 antibody constant domain.

[0167] In some embodiments, holes are constructed by replacing amino acids with larger side chains, such as tyrosine or tryptophan, with amino acids with smaller side chains, such as alanine, valine, or threonine, for example, a Y407V mutation in the CH3 antibody constant domain. Similarly, in some embodiments, knobs are constructed by replacing amino acids with smaller side chains with amino acids with larger side chains, for example, a T366W mutation in the CH3 antibody constant domain. In some embodiments, one Fc domain contains the knob mutation T366W and the other Fc domain contains the hole mutations T366S, L358A, and Y407V. In some embodiments, a polypeptide of the present disclosure that includes a SIRPα D1 domain variant is fused to an Fc domain that includes the knob mutation T366W to limit undesired homodimer formation between the knobs. Examples of knob-into-hole amino acid pairs are provided, but are not limited to, in Table 9. Examples of knob-into-hole Fc domain variants and SIRPα-Fc fusions are provided in Table 10. [Table 9] [Table 10-1] [Table 10-2] [Table 10-3]

[0168] In addition to the knobs-into-holes strategy, in some embodiments, electrostatic steering is also used to control the dimerization of Fc domains. Electrostatic steering refers to the use of favorable electrostatic interactions between oppositely charged amino acids in peptides, protein domains, and proteins to control the formation of higher-order protein molecules. In particular, to control the dimerization of Fc domains using electrostatic steering, one or more amino acid residues that make up the CH3-CH3 interface are replaced with positively or negatively charged amino acid residues so that the interaction is electrostatically favorable or unfavorable depending on the specific charged amino acid introduced. In some embodiments, a positively charged amino acid at the interface, such as lysine, arginine, or histidine, is replaced with a negatively charged amino acid, such as aspartic acid or glutamic acid. In some embodiments, a negatively charged amino acid at the interface is replaced with a positively charged amino acid. In some embodiments, a charged amino acid is introduced into one or both of the interacting CH3 antibody constant domains. In some embodiments, the introduction of charged amino acids into the interacting CH3 antibody constant domains of two Fc domains promotes the selective formation of heterodimers of Fc domains as controlled by the electrostatic steering effect resulting from the interaction between the charged amino acids. Examples of electrostatic steering amino acid pairs are shown in Table 11, but are not limited thereto. [Table 11]

[0169] Other methods are available that can be used to control heterodimerization of Fc domains, particularly in the context of constructing bispecific antibodies.

[0170] In some embodiments, the first Fc domain and the second Fc domain each have the following amino acids with respect to the sequence of human IgG1: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A , T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, and K409I amino acid substitutions.

[0171] In some embodiments, the Fc domain has: (a) one of the following amino acid substitutions relative to wild-type human IgG1: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S36 4A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, or K409I; or (b)(i) human IgG1 (ii) L234A, L235A, and G237A mutations for a human IgG1 Fc region; (iii) L234A, L235A, G237A, and N297A mutations for a human IgG1 Fc region; (iv) N297A mutation for a human IgG2 Fc region; (v) A330S and P331S mutations for a human IgG2 Fc region; (vi) A330S, P331S, and N297A mutations for a human IgG2 Fc region; (vii) S228P, E233P, F234V, L235A, and delG236 mutations for a human IgG4 Fc region; or (viii) S228P, E233P, F234V, L235A, delG236, and N297A mutations for a human IgG4 Fc region.In some embodiments, the Fc domain variant comprises: (a) one of the following amino acid substitutions relative to wild-type human IgG1: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S353S, S354D ... (b) (i) human IgG1 (ii) L234A, L235A, and G237A mutations for a human IgG1 Fc region; (iii) L234A, L235A, G237A, and N297A mutations for a human IgG1 Fc region; (iv) N297A mutation for a human IgG2 Fc region; (v) A330S and P331S mutations for a human IgG2 Fc region; (vi) A330S, P331S, and N297A mutations for a human IgG2 Fc region; (vii) S228P, E233P, F234V, L235A, and delG236 mutations for a human IgG4 Fc region; or (viii) S228P, E233P, F234V, L235A, delG236, and N297A mutations for a human IgG4 Fc region.

[0172] In some embodiments, the first Fc domain and the second Fc domain comprise different amino acid substitutions. In some embodiments, the first Fc domain comprises T366W. In some embodiments, the second Fc domain comprises T366S, L368A, and Y407V. In some embodiments, the first Fc domain comprises D399K. In some embodiments, the second Fc domain comprises K409D.

[0173] Linker In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein alpha (SIRP-α) D1 variant comprising a SIRPα D1 domain or a fragment thereof having an amino acid mutation at residue 80 relative to the wild-type SIRPα D1 domain; and at least one additional amino acid mutation at a residue selected from the group consisting of residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92.

[0174] Also disclosed herein, in some embodiments, is a polypeptide comprising an Fc variant, where the Fc variant comprises an Fc domain dimer comprising two Fc domain variants, each Fc domain variant independently selected from: (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region consisting of mutations S228P, E233P, F234V, L235A, delG236, and N297A.

[0175] In the present disclosure, linker is used to describe a link or connection between a polypeptide or protein domain or related non-protein moiety. In some embodiments, the linker is a link or connection between an Fc domain (or a variant thereof) and a SIRPα D1 domain variant. In some embodiments, the linker connects the C-terminus of the SIRPα D1 domain variant and the N-terminus of the Fc domain variant such that the two polypeptides are linked to each other in a tandem series.

[0176] In some embodiments, the linker is a simple covalent bond, e.g., a peptide bond, a synthetic polymer, or any type of bond generated from a chemical reaction, e.g., a chemical conjugate. When the linker is a peptide bond, in some embodiments, a carboxylic acid group at the C-terminus of one protein domain reacts with an amino group at the N-terminus of another protein domain in a condensation reaction to form a peptide bond. In some embodiments, the peptide bond is formed by synthetic means by conventional organic chemical reactions or by natural generation from a host cell, and a nucleic acid molecule encoding the DNA sequences of both proteins of the tandem series (e.g., Fc domain variant and SIRPα D1 domain variant) can be directly transcribed and translated into a consecutive polypeptide encoding both proteins in the host cell by the necessary molecular machinery (e.g., DNA polymerase and ribosomes).

[0177] When the linker is a synthetic polymer, in some embodiments the polymer is functionalized with reactive chemical groups at each end to react with the terminal amino acids at the connecting ends of the two proteins.

[0178] When a linker (other than a peptide bond as described above) is made from a chemical reaction, in some embodiments, a chemical functional group (e.g., an amine, carboxylic acid, ester, azide, or other functional group) is synthetically attached to the C-terminus of one protein and the N-terminus of another protein, respectively. Then, in some embodiments, the two functional groups react via synthetic chemistry means to form a chemical bond, thus connecting the two proteins together.

[0179] Spacer In the present disclosure, in some embodiments, the linker between the Fc domain monomer and the SIRPα D1 variant polypeptide of the present disclosure is an amino acid spacer, such as about 1-200 amino acids. Suitable peptide spacers include peptide linkers that include flexible amino acid residues, such as glycine and serine. Examples of linker sequences are shown in Table 12. In some embodiments, the spacer includes a GS, GG, GGS, GGG, GGGGS (SEQ ID NO: 163), GGSG (SEQ ID NO: 164), or SGGG (SEQ ID NO: 165) motif, such as multiple or repeated motifs. In some embodiments, the spacer includes a GS motif, such as 2-12 amino acids, such as GS, GSGS (SEQ ID NO: 166), GSGSGS (SEQ ID NO: 167), GSGSGSGS (SEQ ID NO: 168), GSGSGSGSGS (SEQ ID NO: 169), or GSGSGSGSGSGS (SEQ ID NO: 170). In some embodiments, the spacer comprises 3-12 amino acids including a GGS motif, e.g., GGS, GGSGGS (SEQ ID NO: 171), GGSGGSGGS (SEQ ID NO: 172), and GGSGGSGGSGGS (SEQ ID NO: 173). In some embodiments, the spacer comprises 4-12 amino acids including a GGSG motif (SEQ ID NO: 164), e.g., GGSG (SEQ ID NO: 164), GGSGGGSG (SEQ ID NO: 174), or GGSGGGSGGGSG (SEQ ID NO: 175). In some embodiments, the spacer comprises a GGGGS (SEQ ID NO: 163) motif, e.g., GGGGSGGGGSGGGGGS (SEQ ID NO: 176).In some embodiments, the spacer includes amino acids other than glycine and serine, e.g., AAS (SEQ ID NO: 177), AAAL (SEQ ID NO: 178), AAAK (SEQ ID NO: 179), AAR (SEQ ID NO: 180), EGKSSGSGSESKST (SEQ ID NO: 181), GSAGSAAGSGEF (SEQ ID NO: 182), AEAAAKEAAAKA (SEQ ID NO: 183), KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), GGGGAGGGG (SEQ ID NO: 185), GENLYFQSGG (SEQ ID NO: 186), SACYCELS (SEQ ID NO: 187), RSIAT (SEQ ID NO: 188), RPACKIPNDLKQKVMNH (SEQ ID NO: 189), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 190), AAANSSIDLISVPVDSR (SEQ ID NO: 191), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 192).

[0180] In some embodiments, the spacer includes a motif, e.g., multiple or repeated motifs, of EAAAK (SEQ ID NO: 193). In some embodiments, the spacer includes a motif, e.g., multiple or repeated motifs of proline-rich sequences, such as (XP)n, where X is any amino acid (e.g., A, K, or E) and n is from 1 to 5, and PAPAP (SEQ ID NO: 194). [Table 12]

[0181] In some embodiments, the length of the peptide spacer and the amino acids used are adjusted depending on the two proteins involved and the degree of flexibility desired for the final protein fusion polypeptide. In some embodiments, the length of the spacer is adjusted to ensure proper protein folding and avoid the formation of aggregates. In some embodiments, the spacer is A or AAAL (SEQ ID NO: 178).

[0182] Vectors, host cells, and protein production In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein alpha (SIRP-α) D1 variant comprising a SIRPα D1 domain or a fragment thereof having an amino acid mutation at residue 80 relative to the wild-type SIRPα D1 domain; and at least one additional amino acid mutation at a residue selected from the group consisting of residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92.

[0183] Also disclosed herein, in some embodiments, is a polypeptide comprising an Fc variant, where the Fc variant comprises an Fc domain dimer having two Fc domain monomers, each Fc domain monomer independently selected from: (i) a human IgG1 Fc region consisting of 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.

[0184] In some embodiments, the polypeptides of the present disclosure are produced from host cells. Host cells refer to vehicles that contain the necessary cellular components, e.g., organelles, required to express the polypeptides and fusion polypeptides described herein from their corresponding nucleic acids. In some embodiments, the nucleic acids are contained in nucleic acid vectors that are introduced into the host cells by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc. In some embodiments, the choice of nucleic acid vector depends on the host cell used. In some embodiments, the host cells are of either prokaryotic (e.g., bacterial) or eukaryotic (e.g., mammalian) origin.

[0185] In some embodiments, a polypeptide construct comprising a polypeptide, e.g., a SIRPα D1 domain variant (e.g., any of the variants provided in Tables 2, 5, and 6) and a fusion partner, such as an Fc variant, is produced by culturing a host cell transformed with a nucleic acid, preferably an expression vector, comprising a nucleic acid encoding the polypeptide construct (e.g., an Fc variant, a linker, and a fusion partner), under appropriate conditions to induce or cause expression of the polypeptide construct. In some embodiments, the appropriate conditions for expression vary depending on the expression vector and host cell selected. In some embodiments, a wide variety of suitable host cells are used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. For example, various cell lines that find use in the present disclosure are described in the ATCC® cell line catalog available from the American Type Culture Collection. In some embodiments, the Fc domain variants of the disclosure are expressed in cells that have been optimized to not glycosylate proteins expressed by such cells, either by genetic engineering of the cell line or alteration of cell culture conditions such as addition of kifunensine, or by using naturally non-glycosylated hosts such as prokaryotes (e.g., E. coli), in some cases not requiring modification of glycosylation sequences within the Fc.

[0186] Construction of Nucleic Acid Vectors and Host Cells Nucleic acid sequences encoding the amino acid sequences of the polypeptides of the present disclosure can be prepared by a variety of methods. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. In some embodiments, nucleic acid molecules encoding the polypeptides of the present disclosure are obtained using standard techniques, such as gene synthesis. Alternatively, a nucleic acid molecule encoding a wild-type SIRPα D1 domain is mutated to include specific amino acid substitutions using standard techniques, such as QuikChange™ mutagenesis. In some cases, the nucleic acid molecule is synthesized using a nucleotide synthesizer or PCR techniques.

[0187] In some embodiments, the nucleic acid encoding the polypeptide construct, for example, a polypeptide construct including a fusion partner such as a SIRPα D1 domain variant (e.g., any of the variants shown in Tables 2, 5, and 6) and an Fc variant, is incorporated into an expression vector to express the protein. A variety of expression vectors are available for protein expression. Expression vectors can include self-replicating extrachromosomal vectors or vectors that integrate into a host genome. A vector can also include a variety of components or elements. For example, in some embodiments, vector components include, but are not limited to, transcriptional and translational regulatory sequences, such as promoter sequences, ribosome binding sites, signal sequences, transcriptional start and stop sequences, translational start and stop sequences, 3' and 5' untranslated regions (UTRs), and enhancer or activator sequences; an origin of replication; a selectable marker gene; and a nucleic acid sequence encoding a polypeptide of interest, and a transcription termination sequence. In some embodiments, the expression vector includes a protein operably linked to a control or regulatory sequence, a selectable marker, any fusion partner, additional elements, or any combination thereof. The term "operably linked" means that a nucleic acid is placed in a functional relationship with another nucleic acid sequence. Generally, these expression vectors contain transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the Fc variant, and are typically appropriate for the host cell used to express the protein. A selection gene or marker, such as, but not limited to, an antibiotic resistance gene or a fluorescent protein gene, can be used to select host cells containing the expression vector, for example, by antibiotic or fluorescent expression. A variety of selection genes are available.

[0188] In some embodiments, vector components or elements are optimized so that the expression vector is compatible with the host cell type. Expression vectors that find use in the present disclosure include, but are not limited to, those that allow for protein expression in mammalian cells, bacteria, insect cells, yeast, and in vitro systems.

[0189] In some embodiments, mammalian cells are used as host cells to produce the polypeptides of the present disclosure. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NS0, Sp2 / 0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells. In some embodiments, E. coli cells are used as host cells to produce the polypeptides of the present disclosure. Examples of E. coli strains include, but are not limited to, E. coli 294 (ATCC® 31,446), E. coli λ1776 (ATCC® 31,537, E. coli BL21(DE3) (ATCC® BAA-1025)), and E. coli RV308 (ATCC® 31,608).

[0190] Different host cells have characteristic and specific mechanisms for post-translational processing and modification (e.g., glycosylation) of protein products. In some embodiments, an appropriate cell line or host system is selected to ensure correct modification and processing of the expressed polypeptide. Once the vector is introduced into the host cell for protein production, the host cell is cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0191] In some embodiments, the polypeptide construct, for example, a polypeptide construct comprising a fusion partner such as a SIRPα D1 domain variant (e.g., any of the variants provided in Tables 2, 5, and 6) and an Fc variant, is expressed in a mammalian expression system, such as a system in which the expression construct is introduced into a mammalian cell using a virus such as a retrovirus or an adenovirus. In some embodiments, human, mouse, rat, hamster, or primate cells are utilized. Suitable cells also include known research cells, such as, but not limited to, Jurkat T cells, NIH3T3, CHO, COS, and 293 cells. Alternatively, in some embodiments, the protein is expressed in bacterial cells. Bacterial expression systems are well known in the art and include Escherichia coli (E. coli), Bacillus subtilis, Streptococcus cremoris, and Streptococcus lividans. In some cases, the polypeptide constructs comprising the Fc domain variants are produced in insect cells, such as, but not limited to, Sf9 and Sf21 cells, or yeast cells, such as organisms of the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, and Yarrowia. In some cases, the polypeptide constructs comprising the Fc domain variants are expressed in vitro using a cell-free translation system. In vitro translation systems derived from both prokaryotic (e.g., E. coli) and eukaryotic (e.g., wheat germ, rabbit reticulocyte) cells are available and, in some embodiments, are selected based on the expression level and functional properties of the protein of interest. For example, as will be appreciated by those skilled in the art, in vitro translation is required for some display technologies, such as ribosome display. Additionally, in some embodiments, the Fc domain variants are produced by chemical synthesis methods, such as, but not limited to, solution phase peptide synthesis and solid phase peptide synthesis.In vitro transcription using a non-glycosylated system such as bacterial extracts results in an equally inactivated Fc, since the Fc is not glycosylated even though native glycosylation sites are present.

[0192] In some embodiments, the polypeptide construct comprises unnatural amino acids, amino acid analogs, amino acid mimetics, or any combination thereof that function similarly to naturally occurring amino acids. Naturally encoded amino acids generally refer to the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, e.g., carbons attached to hydrogen, carboxyl groups, amino groups, and R groups, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. In some embodiments, such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but generally retain the same basic chemical structure as naturally occurring amino acids.

[0193] Protein Production, Recovery, and Purification In some embodiments, the host cells used to produce the polypeptides of the present disclosure are grown in a medium suitable for culturing the selected host cells. For mammalian host cells, examples of suitable media include Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Expi293™ Expression Medium, DMEM supplemented with fetal bovine serum (FBS), and RPMI-1640. For bacterial host cells, examples of suitable media include Luria Broth (LB) with necessary supplements, such as a selection agent, e.g., ampicillin. In some embodiments, the host cells are cultured at a suitable temperature, such as about 20° C. to about 39° C., e.g., about 25° C. to about 37° C., preferably 37° C., and at a CO2 level, such as 5% to 10%. In some embodiments, the pH of the medium is about pH 6.8 to pH 7.4, e.g., pH 7.0, depending primarily on the host organism. When an inducible promoter is used in the expression vector, protein expression can be induced under conditions suitable for activation of the promoter.

[0194] In some embodiments, recovery of the protein involves disruption of the host cells, for example, by osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris is removed by centrifugation or filtration. The protein can then be further purified. In some embodiments, the polypeptides of the present disclosure are purified by various methods of protein purification, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard procedure method for protein purification. For example, in some embodiments, the protein is isolated and purified by appropriately selecting and combining affinity columns, such as Protein A columns (e.g., POROS Protein A chromatography), chromatography columns (e.g., POROS HS-50 cation exchange chromatography), filtration, ultrafiltration, desalting, and dialysis procedures. In some embodiments, the polypeptide is bound to a marker sequence, such as a peptide, to facilitate purification. An example of a marker amino acid sequence is a hexahistidine peptide (His6-tag (SEQ ID NO: 223)) that can bind to a nickel-functionalized agarose affinity column with micromolar affinity. Alternatively, a hemagglutinin "HA" tag can be used, which corresponds to an epitope derived from the influenza hemagglutinin protein.

[0195] In some embodiments, a polypeptide of the disclosure, e.g., a polypeptide construct comprising a fusion partner, such as a SIRPα D1 domain variant (e.g., any of the variants provided in Tables 2, 5, and 6) and an Fc variant, is produced by cells of a subject (e.g., a human) by administering a vector, such as a viral vector (e.g., a retroviral vector, an adenoviral vector, a poxvirus vector (e.g., a vaccinia virus vector, e.g., Modified Vaccinia Ankara (MVA)), an adeno-associated virus vector, and an alphavirus vector), comprising a nucleic acid molecule encoding a polypeptide of the disclosure, e.g., in the context of gene therapy. The vector can be used to express a polypeptide disclosed herein upon entry into the subject's cells (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.). In some cases, the polypeptide is secreted from the cells. In some embodiments, if treatment of a disease or disorder is the desired outcome, no further action is required. In some embodiments, if collection of protein is desired, blood is collected from the subject and the protein is purified from the blood by various methods.

[0196] Cancer Treatment Methods Provided herein are methods of treating cancer in an individual (e.g., a human individual), the methods comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein), and (b) a chemotherapeutic agent (e.g., at least one chemotherapeutic agent, e.g., at least two, at least three, or at least four chemotherapeutic agents). In some embodiments, the method further comprises administering to the individual an effective amount of a therapeutic antibody (e.g., at least one therapeutic antibody, e.g., at least two, at least three, or at least four therapeutic antibodies). Additionally or alternatively, in some embodiments, the method further comprises administering to the individual an effective amount of an immunotherapeutic agent (e.g., at least one immunotherapeutic agent, e.g., at least two, at least three, or at least four immunotherapeutic agents). Additionally or alternatively, in some embodiments, the method includes administering the polypeptide and chemotherapeutic agent in combination with one or more additional therapeutic modalities, such as, but not limited to, radiation therapy, surgery, cryoablation, and bone marrow transplantation.

[0197] Combination Therapies Including Chemotherapeutic Agents and Exemplary Chemotherapeutic Agents Exemplary chemotherapeutic agent(s) that can be used in the methods of treating cancer described herein include, but are not limited to, methotrexate (RHEUMATREX®, amethopterin), cyclophosphamide (CYTOXAN®), abiraterone, abemaciclib, altretamine, thalidomide (THALIDOMID®), acridine carboxamide, actimid®, actinomycin, actinomycin-D, afatinib, 17-N-allylamino-17-demethoxygeldanamycin, alectinib, cyclophosphamide ... Nib, alpelisib, aminopterin, amsacrine, anlotinib, anthracycline, antitumor agent, antineoplaston, apartinib, 5-azacytidine, 6-mercaptopurine, 6-thioguanine, arabinosylcytosine, axitinib, azacitidine, azathioprine, BL22, bendamustine, binimetinib, biribodar, bleomycin, bortezomib, bosutinib, brigutinib, bryostatin, busulfan, cabozantinib, calyculin, camptothecin, capecitabine, carboplatin, carmus tin, ceritinib, chlorambucil, cisplatin, cladribine, clofarabine, cobimetinib, crizotinib, cytarabine, dabrafenib, dacarbazine, dacomitinib, dasatinib, daunorubicin, dexamethasone, dichloroacetic acid, discodermolide, docetaxel, doxorubicin, encorafenib, epirubicin, entrectinib, enzalutamide, epothilone, erdafitinib, eribulin, erlotinib, estramustine, etoposide, everolimus, exatecan, exisulind, ferruginol, floxuridine, Fludarabine, fluorouracil (such as 5-fluorouracil), folinic acid, fosfestrol, fotemustine, fruquintinib, ganciclovir, gefitinib, gemcitabine, gilteritinib, goserelin, hexamethylmelamine, hydroxycarbamide, hydroxyurea, IT-101, ibrutinib, icotinib, idarubicin, idelalisib, ifosfamide, imatinib, irinoimiquimod, irinotecan, irofulven, ivosidenib, ixabepilone, laniquidar,Lapatinib, larotrectinib, lenalidomide, lenvatinib, lorlatinib, lomustine, raltotecan, mafosfamide, masoprocol, mechlorethamine, melphalan, mercaptopurine, methotrexate, methylprednisolone, mitomycin, mitotane, mitoxantrone, nelarabine, neratinib, niraparib, nilotinib, nintedanib, oblimersen, olaparib, osimertinib, oxaliplatin, nedap platin, phenanthriplatin, picoplatin, PAC-1, paclitaxel, palbociclib, pazopanib, pemetrexed, pegfilgrastim, pentostatin, pipobroman, pixantrone, plicamycin, prednisone, ponatinib, procarbazine, proteasome inhibitors (e.g., bortezomib), pyrotinib, raltitrexed, rebeccamycin, Revlimid®, regorafenib, ribociclib, rubite Can, rucaparib, ruxolitinib, SN-38, salinosporamide A, satraplatin, sirolimus, sonidegib, sorafenib, streptozocin, streptozotocin, sunitinib, swainsonine, talazoparib, tariquidar, taxane, tegafur-uracil, temsirolimus, teniposide, temozolomide, testolactone, thiotepa, thioguanine, topotecan, trabectedin, trametinib, Examples of such agents include tretinoin, trifluridine, triplatin tetranitrate, tris(2-chloroethyl)amine, troxacitabine, uracil mustard, valrubicin, vandetanib, vemurafenib, venetoclax (ABT-199), navitoclax (ABT-263), vinblastine, vincristine, vinorelbine, vismodegib, vorinostat, ziv-aflibercept (ZALTRAP®), and zosuquidar.

[0198] In some embodiments, a method of treating cancer comprises administering an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) in combination with a particular class of chemotherapeutic agent(s). In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is a polypeptide described herein (e.g., a fusion polypeptide comprising a SIRPα d1 domain variant and an Fc variant; a fusion polypeptide comprising a SIRPγ variant, a SIRPβ1 variant, or a SIRPβ2 variant and an Fc variant). For example, in some embodiments, a method of treating cancer comprises administering a polypeptide described herein (e.g., a fusion polypeptide) in combination with an adrenal inhibitor (including but not limited to an adrenal inhibitor described herein). For example, in some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with an anthracycline (including but not limited to an anthracycline described herein). In some embodiments, a method of treating cancer comprises administering a polypeptide described herein in combination with an alkylating agent (including but not limited to an alkylating agent described herein). In some embodiments, the method of treating cancer comprises administering a polypeptide described herein in combination with an androgen inhibitor (including but not limited to an androgen inhibitor described herein). In some embodiments, the method of treating cancer comprises administering a polypeptide described herein in combination with an antimetabolite, e.g., a purine analog (including but not limited to an antimetabolite described herein, e.g., a purine analog). In some embodiments, the method of treating cancer comprises administering a polypeptide described herein in combination with an antitumor antibiotic (including but not limited to an antitumor antibiotic described herein). In some embodiments, the method of treating cancer comprises administering a polypeptide described herein in combination with a BLC-2 inhibitor (including but not limited to a BLC-2 inhibitor described herein).In some embodiments, the method of treating cancer comprises administering a polypeptide described herein in combination with a BTK inhibitor (including but not limited to a BTK inhibitor described herein). In some embodiments, the method of treating cancer comprises administering a polypeptide described herein in combination with a CDK4 / 6 inhibitor (including but not limited to a CDK4 / 6 inhibitor described herein). In some embodiments, the method of treating cancer comprises administering a polypeptide described herein in combination with a colony stimulating factor (including but not limited to a colony stimulating factor described herein). In some embodiments, the method of treating cancer comprises administering a polypeptide described herein in combination with a corticosteroid (including but not limited to a corticosteroid described herein). In some embodiments, the method of treating cancer comprises administering a polypeptide described herein in combination with an EGFR inhibitor (including but not limited to an EGFR inhibitor described herein). In some embodiments, the method of treating cancer comprises administering a polypeptide described herein in combination with a gonadotropin releasing hormone (GnRH) agonist (including but not limited to a GnRH agonist described herein). In some embodiments, the method of treating cancer comprises administering a polypeptide described herein in combination with an antimitotic / microtubule inhibitor (including but not limited to an antimitotic / microtubule inhibitor described herein). In some embodiments, the method of treating cancer comprises administering a polypeptide described herein in combination with an mTOR kinase inhibitor (including but not limited to an mTOR kinase inhibitor described herein). In some embodiments, the method of treating cancer comprises administering a polypeptide described herein in combination with a proteasome inhibitor (including but not limited to a proteasome inhibitor described herein).In some embodiments, the method of treating cancer comprises administering a polypeptide described herein in combination with a signal transduction inhibitor, e.g., a protein tyrosine kinase inhibitor, a PAK4 inhibitor, a PI3K inhibitor (including but not limited to, a signal transduction inhibitor described herein). In some embodiments, the method of treating cancer comprises administering a polypeptide described herein in combination with a topoisomerase inhibitor (including but not limited to, a topoisomerase inhibitor described herein). In some embodiments, the method of treating cancer comprises administering a polypeptide described herein in combination with a tyrosine kinase inhibitor (including but not limited to, a tyrosine kinase inhibitor described herein). In some embodiments, the method of treating cancer comprises administering a polypeptide described herein in combination with a VEGF inhibitor, e.g., a VEGF1 inhibitor, a VEGF2 inhibitor, and / or a VEGF3 inhibitor (including but not limited to, a VEGF inhibitor described herein). In some embodiments, methods of treating cancer include administering a polypeptide described herein in combination with an agent that modulates apoptosis, e.g., by modulating the activity of Bcl-2, Mcl1, Bcl-1x, etc. (including but not limited to, agents that modulate apoptosis by modulating the activity of Bcl-2, Mcl1, Bcl-1x, etc., as described herein). In some embodiments, methods of treating cancer include administering a polypeptide described herein in combination with a platinum-based agent (including but not limited to, platinum-based agents described herein).In some embodiments, the method of treating cancer comprises administering a polypeptide as described herein in combination with an inhibitor of NTRK1, NTRK2, and / or NTRK3, an ALK inhibitor, a ROS inhibitor, an FLT3 inhibitor, a BRAF inhibitor, an inhibitor of MEK1 and / or MEK2, an inhibitor of HER2, HER3, and / or HER4, an inhibitor of RET / PTC, an inhibitor of BCR-ABL, a c-KIT inhibitor, an inhibitor of PDGFR-alpha and / or PDGFR-beta, an inhibitor of FGFR1, FGFR2, FGFR3, and / or FGFR4, a smoothened inhibitor, and / or an inhibitor of PARP1, PARP2, and / or PARP3 (including, but not limited to, the inhibitors described herein). In some embodiments, the inhibitor is an antisense polynucleotide (such as an siRNA or RNAi). In some embodiments, the inhibitor is a small molecule inhibitor, as described in more detail below.

[0199] In some embodiments, the chemotherapeutic agent is a small molecule anti-cancer agent (such as a small molecule inhibitor). In some embodiments, the method of treating cancer comprises administering a polypeptide as described herein in combination with a small molecule inhibitor of VEGFR and / or PDGFR, a small molecule EGFR inhibitor, a small molecule ALK inhibitor, a small molecule CDK4 / 6 inhibitor, a small molecule PARP inhibitor, a small molecule PAK4 inhibitor, a small molecule mTOR inhibitor, a small molecule KRAS inhibitor, a small molecule TRK inhibitor, a small molecule BCL2 inhibitor, a small molecule B-raf inhibitor, a small molecule IDH inhibitor, a small molecule PI3K inhibitor, a small molecule DDR (DNA damage response) inhibitor, or a small molecule hypomethylating agent. In other cases, the targeted small molecule modulates a cell signaling pathway in a cell expressing CD47, e.g., an IDO / TDO inhibitor, an AhR inhibitor, an arginase inhibitor, an A2a R inhibitor, a TLR agonist, a STING agonist, or a Rig-1 agonist.

[0200] In some embodiments, the method of treating cancer comprises administering a polypeptide described herein (e.g., a fusion polypeptide comprising a SIRPα d1 domain variant and an Fc variant) in combination with at least one, at least two, at least three, or at least four chemotherapeutic agents. In some embodiments in which more than one chemotherapeutic agent is administered, the two or more chemotherapeutic agents are from different classes (as described above) and / or exert their anti-cancer effects via different mechanisms of action.

[0201] Further details regarding exemplary pharmaceutical compositions and preparations, exemplary dosages, and exemplary routes of administration of the fusion polypeptides described herein are provided in WO2017 / 027422 and U.S. Pat. No. 10,259,859, the contents of each of which are incorporated by reference in their entirety.

[0202] Combination Therapies Including Therapeutic Antibodies and Exemplary Therapeutic Antibodies In some embodiments, the methods of treating cancer provided herein comprise administering to an individual an effective amount of a therapeutic antibody (e.g., at least one therapeutic antibody, e.g., at least two, at least three, or at least four therapeutic antibodies), i.e., in combination with an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., a fusion polypeptide described herein) and a chemotherapeutic agent described herein (e.g., at least one chemotherapeutic agent, e.g., at least two, at least three, or at least four chemotherapeutic agents). In some embodiments, the therapeutic antibody is conjugated to a drug (i.e., an antibody-drug conjugate or "ADC").

[0203] Exemplary therapeutic antibodies (e.g., therapeutic monoclonal antibodies) for use in the methods herein include, but are not limited to, 3F8, 8H9, abagovomab, abciximab, abituzumab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afelimomab, afutuzumab, alacizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, anetumab ravtansine, ravtansine, Anifrolumab, Anrukinzumab (IMA-638), Apolizumab, Arcitumomab, Ascrinvacumab, Aselizumab, Atezolizumab, Atinumab, Tocilizumab, Atorolimumab, Avelumab, Ba Pinezumab, Basiliximab, Bavituximab, Bectumomab, Begelomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Bivatuzumab-mertansine mertansine, Blinatumomab, Brosozumab, Bococizumab, Brentuximab vedotin, Briakinumab, Brodalumab, Brolucizumab, Brontuximab, Cabilalizumab (FPA008), Camrelizumab, Canakinumab, Cantuzumab mertansine, Cantuzumab ravtansine, Capracizumab, Capromab pendetidependetide, Carlumab, Catumaxomab, cBR96-doxorubicin immunoconjugate, CC49, Cedelizumab, Certolizumab pegol, Cetuximab, Ch.14.18, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan, Codrituzumab, Coltuximab Ravtansine, Conatumumab, Concizumab, Crenezumab, CR6261, Dacetuzumab, Daclizumab, Dalotuzumab, Dapirolizumab pegol, Daratumumab, Dectrekumab, Demcizumab, Denintuzumab mafodotin, Denosumab, Derlotuximab biotin biotin, Detumomab, Dinutuximab, Diridavumab, Dorlimomabaritox, drozitumab, durigotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab izumab, Efungumab, Eldelumab, Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab (RG7155), Emibetuzumab, Enavatuzumab, Enfortumab vedotin, Enlimomab pegol, Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epitumomab situxetanCituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, Ficlatuzumab Ficlatuzumab, Figitumumab, Firivumab, Flanvotumab, Fletikumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab-Ozogamicin ozogamicin, Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Guselkumab, Ibalizumab, Ibritumomab tiuxetantiuxetan, Icrucumab, Idarucizumab, Igovomab, IMAB362, Imalumab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Indusatumab vedotin, Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin ozogamicin, Ipilimumab, Iratumumab, Isatuximab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lambrolizumab, Lampalizumab, Lebrikizumab, Remalesomab, Lenzilumab, Lerdelimumab, Lexatumumab, Libivirumab, Rifastuzumab vedotin vedotin, Ligelizumab, Lilotomab satetraxetan, Lintuzumab, Lirilumab, Lodelcizumab, Lokivetomab, Lorvotuzumab mertansine, Lucatumumab, Lulizumab pegolpegol, Lumiliximab, Lumretuzumab, MSB0010718C (avelumab), Mapatumumab, Margetuximab, Maslimomab, Mavrilimumab, Matuzumab, MEDI6469, MEDI0680, MEDI 6383, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Mogamulizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox, Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox, Narnatumab, Natalizumab, Nebacumab, Necitumumab, Nemolizumab, Nelerimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentane merpentan, obiltoxaximab, obinutuzumab, ocralizumab, ocrelizumab, odulimomab, ofatumumab, olaritumab, olokizumab, omalizumab, onartuzumab, ontuxizumab, opicinumab Opicinumab, Oportuzumab monatox, Oregovomab, Orticumab, Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab, Pancomab, Panobacumab, Palsat Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Polatuzumab vedotinVedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO140, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab ), Refanezumab, Regavirumab, Reslizumab, Rilotumumab, Linucumab, Rituximab, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Sacituzumab govitecan govitecan), Samalizumab, SAR650984 (Isatuximab), Sarilumab, Satumomab pentetide pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, Sibrotuzumab, SGN-CD19A, SGN-CD33A, Sifalimumab, Siltuximab, Simtuzumab, Sintilimab, Siplizumab, Sirukumab, Sofituzumab Vedotinvedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tarextumab, tefibazumab, telimomab aritox, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, Tesidolumab, TGN1412, Ticilimumab (tremelimumab), Tildrakizumab, Tigatuzumab, TNX-650, Tocilizumab Ab) (atlizumab), toralizumab, toripalimab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, trastuzumab-emtansine, TRBS07, tregalizumab, tremelimumab, tucotuzumab-celmoleukin Celmoleukin, Tuvirumab, Ublituximab, Ulocuplumab, Urelumab, Urtoxazumab, Ustekinumab, Utomilumab (PF-05082566), Vandortuzumab vedotinvedotin, vantictumab, vanucizumab, vapaliximab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vonlerolizumab (RG7888), borsetuzumab-mafodotin These include: tetanus, tetanus, tetanus, tetanus, mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, or zolimomab aritox, or a biosimilar of any of the aforementioned therapeutic antibodies.

[0204] Other exemplary therapeutic antibodies (e.g., therapeutic monoclonal antibodies) that can be used in the methods herein are antibodies, including, but not limited to, for example, anti-CD20 antibodies, anti-EGFR antibodies, anti-Her2 / Neu (ERBB2) antibodies, anti-EPCAM antibodies, anti-GL2 antibodies, anti-GD2 antibodies, anti-GD3 antibodies, anti-CD2 antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD8 antibodies, anti-CDI9 antibodies, anti-CD22 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD39 antibodies, anti-CD45 antibodies, anti-CD47 antibodies, anti-CD52 ...CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI9 antibodies, anti-CDI These include D56 antibodies, anti-CD70 antibodies, anti-CD73 antibodies, anti-CD117 antibodies, anti-SIRPα antibodies, anti-LILRB1 antibodies, anti-LILRB2 antibodies, anti-LILRB4 antibodies, anti-PD1 antibodies (e.g., anti-PD-1 antagonist antibodies), anti-PD-L1 antibodies (e.g., anti-PD-L1 antagonist antibodies), anti-PD-L2 antibodies, and antibodies designed to bind to tumor cells, virus- or bacteria-infected cells, immune cells, or healthy normal cells, or cytokines, chemokines, or hormones of any kind.

[0205] In some embodiments, the therapeutic antibody used in the methods herein is selected from the group consisting of, for example, CS1 / SLAMF7, Trop-2, VWF, vimentin, VEGFR2, VEGFR-1, VEGF, VEGF-A, TYRP1 (glycoprotein 75), TWEAK receptor, tumor-specific glycosylation of MUC1, tumor antigen CTAA16.88, TRAIL-R2, TRAIL-R1, TNF-alpha, TGF-beta, TGFbeta2, TGFbeta1, TFPI, tenascin-C, TEM1, TAG-72, T cell receptor, STEAP1, sphingosine-1-phosphate, S OST, SLAMF7, BCL-2, selectin P, SDC1, sclerostin, RTN4, RON, Rh factor, RHD, respiratory syncytial virus, RANKL, rabies virus glycoprotein, platelet-derived growth factor receptor beta, phosphatidylserine, sodium phosphate cotransporter, PDGF-R alpha, PDCD1, PD-1, PD-L1, PCSK9, oxLDL, OX-40, NRP1, Notch receptor 4, Notch receptor 3, Notch receptor 2, Notch receptor 1, NOGO-A, NGF, neuronal apoptosis-regulating protease 1 (neural apoptosis-regulated proteinase 1), NCA-90 (granulocyte antigen), NARP-1, N-glycolylneuraminic acid, myostatin, myelin-associated glycoprotein, mucin CanAg, MUC1, MSLN, MS4A1, MIF, mesothelin, MCP-1, LTA, LOXL2, lipoteichoic acid, LINGO-1, LFA-1 (CD11a), Lewis-Y antigen, L-selectin (CD62L), KIR2D, ITGB2 (CD18), ITGA2, interferon alpha / beta receptor, interferon receptor, interferon gamma-inducible protein, integrin αvβ3, integrin αIIβ3, integrin α7β7, integrin α5β1, integrin α4β7, integrin α4, insulin-like growth factor I receptor, influenza A hemagglutinin, ILGF2, IL9, IL6, IL4, IL3 IRA, IL23, ILI 7A, IL-6 receptor, IL-6, IL-S, IL-4, IL-23, IL-22, IL-I, IL-I 7A, IL-I 7, IL-13, IL-I 2, IL-I, IL20, IGHE, IgG4, IGF-I, IGF-I receptor, IgE Fc region, IFN-gamma, IFN-alpha, ICAM-1 (CD54), human TNF, human scatter factor receptor kinase, Hsp90, HNGF, HLA-DR, HIV-1, histone complex, HHGFR, HGF, HER3, HER2, HER2 / neu, HER1, hepatitis B surface antigen, hemagglutinin, GUCY2C, GPNMB, GMCSF receptor alpha chain, glypican 3, GD3 ganglioside, GD2, ganglioside GD2, Frizzled receptor, folate receptor 1, folate hydrolase, fibronectin extra domain-B, fibrin II, beta chain, FAP, respiratory syncytial virus F protein, ERBB3, episialin, EpCAM, endotoxin, EGFR, EGFL7, E. coli shiga toxin type 2 type-2), Shiga toxin-producing E. coli type I, DRS, DPP4, DLL4, dabigatran, cytomegalovirus glycoprotein B, CTLA-4, CSF2, CSF1R, clumping factor A, CLDN18.2, ch4DS, CFD, CEA-related antigen, CEA, CD80, CD79B, CD74, CD73, CD70, CD6, CD56, CD52, CD51, CD5, CD44 v6, CD41, CD40 ligand, CD40, CD4, CD39, CD38, CD37, CD33, CD30 (TNFRSF8), CD123, CD138, CD3 epsilon, CD3, CD28, CD274, CD27, CD2S (IL-2 receptor chain), CD23 (IgE receptor), CD221, CD22, CD200, CD20, CD2, CD19, CD137, CD154, CD152, CD15, CD147 (basigin), CD140a, CD125, CD11, CD-18, CCR5, CCR4, CCL11 (eotaxin-I), cardiac myosin, carbonic anhydrase 9 (CA-IX), Canis lupus familiarisThe antibody binds to IL31, CA-125, C5, C242 antigen, CXC chemokine receptor type 4, beta-amyloid, BAFF, B7-H3, B lymphoma cells, AOC3 (VAP-I), anthrax toxin, protective antigen, angiopoietin 3, angiopoietin 2, alpha-fetoprotein, AGS-22M6, adenocarcinoma antigen, ACVR2B, activin receptor-like kinase I, 5T4, 5AC, 4-IBB, or 1-40-beta-amyloid.

[0206] In some embodiments, a therapeutic antibody used in the methods herein binds to an antigen expressed by a cancer cell (eg, expressed on the surface of a cancer cell). Exemplary antigens expressed by cancer are known in the art and include, but are not limited to, for example, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD56, CD70, CD74, CD79b, CD123, CD138, CS1 / SLAMF7, Trop-2, 5T4, BCMA, mucin 1, mucin 16, PTK7, PD-L1, STEAP1, endothelin B receptor, mesothelin, EGFRvIII, ENPP3, SLC44A4, GNMB, nectin 4, NaPi2b, LIV-1A, guanyl cyclase C, DLL3, EGFR, HER2, VEGF, VEGFR, integrin αVβ3, integrin α5β1, MET, IGF1R, TRAILR1, TRAILR2, RANKL, FAP, tenascin, Le y, EpCAM, CEA, gpA33, PSMA, TAG72, mucin, CAIX, EPHA3, folate receptor alpha, GD2, GD3, MHC / peptide complexes containing peptides from NY-ESO-1 / LAGE, SSX-2, MAGE family proteins, MAGE-A3, gp100 / pmel17, Melan-A / MART1, gp75 / TRP1, tyrosinase, TRP2, CEA, PSA, TAG-72, immature laminin receptor, MOK / RAGE-1, WT-1, SAP-1, BING-4, EpCAM, MUC1, PRAME, survivin, BRCA1, BRCA2, CDK4, CML66, MART-2, p53, Ras, β-catenin, TGF-βRII, HPV E6, or HPV E7. For example, in some embodiments, a polypeptide described herein is administered in combination with a chemotherapeutic agent (e.g., at least one chemotherapeutic agent) and a monoclonal antibody that binds to CD123 (e.g., also known as IL-3 receptor alpha), such as talaxuzumab (also known as CSL362 and JNJ-56022473).

[0207] In some embodiments, the therapeutic antibody (e.g., therapeutic monoclonal antibody) used in the methods herein is an antibody that binds to an antigen expressed by NK cells. Exemplary antigens expressed by NK cells include, but are not limited to, NKR-P1A (KLRB1), CD94 (NKG2A), KLRG1, KIR2DL5A, KIR2DL5B, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, KIR2DS1, CD94 (NKG2C / E), NKG2D, CD160 (BY55), C D16 (FcγRIIIA), NKp46 (NCR1), NKp30 (NCR3), NKp44 (NCR2), DNAM1 (CD226), CRTAM, CD27, NTB-A (SLAMF6), PSGL1, CD96 (Tactile), CD100 (SEMA4D), NKp80 (KLRF1, CLEC5C), SLAMF7 (CRACC, CS1, CD319), and CD244 (2B4, SLAMF4).

[0208] Combination Therapies Including Immunotherapeutic Agents and Exemplary Immunotherapeutic Agents In some embodiments, the methods of treating cancer provided herein include administering to an individual an effective amount of an immunotherapeutic agent (e.g., at least one immunotherapeutic agent, e.g., at least two, at least three, or at least four immunotherapeutic agents), i.e., in combination with an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., a polypeptide described herein) and a chemotherapeutic agent described herein (e.g., at least one chemotherapeutic agent, e.g., at least two, at least three, or at least four chemotherapeutic agents).

[0209] In some embodiments, immunotherapeutic agents refer to any therapeutic agent that targets the immune system and promotes therapeutic redirection of the immune system, such as modulators of costimulatory pathways, cancer vaccines, recombinant modified immune cells, etc. Exemplary and non-limiting immunotherapeutic agents are described below. In some embodiments, the immunotherapeutic agent is or includes an antibody. Exemplary targets of immunotherapeutic antibodies are known in the art and include, but are not limited to, BDCA2, BDCA4, ILT7, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, Siglec-3, Siglec-7, Siglec-9, Siglec-10, Siglec-15, FGL-1, CD200, CD200R, CSF-1R, CD24, CD40, CD40L, CD163, CD206, DEC205, CD47, CD123, arginase, IDO, TDO, AhR, EP2, COX-2, CCR2, CCR-7, CXCR1, CX3CR1, CXCR2, CXCR3, CXCR4, CXCR7, TGF-β RI, TGF-β These include RII, c-Kit, CD244, L-selectin / CD62L, CD11b, CD11c, CD68, 41BB, CTLA4, PD1, PD-L1, PD-L2, TIM-3, BTLA, VISTA, LAG-3, CD28, OX40, GITR, CD137, CD27, HVEM, CCR4, CD25, CD103, KIRg1, Nrp1, CD278, Gpr83, TIGIT, CD154, CD160, TNFR2, PVRIG, DNAM, and ICOS.

[0210] Immunotherapeutic agents that are approved or in late stage clinical trials include, but are not limited to, ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, and the like. In certain embodiments, an agent that blocks the interaction between CD47 and SIRPα (such as a polypeptide described herein) is administered in combination with an inhibitor of the PD-L1 / PD-1 pathway, e.g., an antibody, small molecule, or polypeptide that blocks the interaction between PD-L1 and PD-1 (e.g., by binding to PD-1 or PD-L1). In some embodiments, the inhibitor of the PD-L1 / PD-1 pathway is an antisense polynucleotide. In some embodiments, the inhibitor of the PD-L1 / PD-1 pathway is an anti-PD-1 or anti-PD-L1 antagonist antibody (e.g., an anti-PD-1 or anti-PD-L1 antagonist antibody described elsewhere herein). As shown herein, the combined administration of an agent that blocks the interaction between CD47 and SIRPα (such as a polypeptide described herein) with an inhibitor of the PD-L1 / PD-1 pathway can result in synergistic anti-tumor activity. In some embodiments, the immunotherapeutic agent is or includes a vaccine, an oncolytic virus, an adoptive cell therapy, a cytokine, or a small molecule immunotherapeutic agent. Examples of such immunotherapeutic agents are known in the art. For example, adoptive cell therapy and therapeutic agents can include, but are not limited to, chimeric antigen receptor T cell therapy (CAR-T), tumor infiltrating lymphocytes (TIL), TCR engineered T cells, TCR engineered NK cells, and macrophage cell products. Vaccines can include, but are not limited to, polynucleotide vaccines, polypeptide vaccines, or cell-based (e.g., tumor or dendritic cell-based) vaccines. Various cytokines are known to be useful in the treatment of cancer, including, but not limited to, IL-2, IL-15, IL-7, IL-10, IL-12, IL21, TNFa, IFN, GM-CSF, and engineered cytokine mutants.Small molecule immunotherapeutics can include, but are not limited to, IDO / TDO inhibitors, AhR inhibitors, arginase inhibitors, A2a R inhibitors, TLR agonists, STING agonists, and Rig-1 agonists.

[0211] In some embodiments, where an agent that blocks the interaction between CD47 and SIRPα (such as a polypeptide described herein) and a chemotherapeutic agent (e.g., at least one chemotherapeutic agent) are administered in combination with an additional agent(s) described herein (e.g., a therapeutic antibody, a small molecule inhibitor, an immunotherapeutic agent, etc.), the additional agent(s) are of a different class and / or exert their anti-cancer effect through a different mechanism of action. For example, in some embodiments, a method of treating cancer includes administering an agent that blocks the interaction between CD47 and SIRPα (such as a polypeptide described herein) in combination with a chemotherapeutic agent (including but not limited to those described herein) and a therapeutic antibody (including but not limited to those described herein, e.g., an anti-HER2 antibody). In some embodiments, an agent that blocks the interaction between CD47 and SIRPα (such as a polypeptide described herein) is administered in combination with a chemotherapeutic agent (including but not limited to those described herein) and a small molecule inhibitor (including but not limited to those described herein). Other combinations are also contemplated.

[0212] In some embodiments, an agent that blocks the interaction between CD47 and SIRPα (such as a polypeptide described herein) is administered in combination with one or more agents, including, but not limited to, antidiarrheals, antiemetics, analgesics, opioids and / or nonsteroidal anti-inflammatory agents.

[0213] Combination therapy including additional therapeutic modality(s) In some embodiments, an agent that blocks the interaction between CD47 and SIRPα (such as a polypeptide described herein) is administered in combination with at least one chemotherapeutic agent and one or more additional therapeutic modalities. In some embodiments, the one or more additional therapeutic modalities include radiation therapy (e.g., gamma rays, X-rays, and / or direct delivery of radioisotopes to tumor cells, microwave, UV radiation, or gene therapy. For example, therapeutic genes for gene therapy include, but are not limited to, antisense versions of inducers of cell proliferation (oncogenes), inhibitors of cell proliferation (tumor suppressors), or inducers of programmed cell death (proapoptotic genes). In some embodiments, any one or more combination therapies described herein are administered in conjunction with surgery (e.g., resection).

[0214] Exemplary Therapeutic Combinations In some embodiments, the method of treating cancer includes administering to an individual in need of treatment an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) in combination with trastuzumab and one or more agents selected from the following: lenalidomide, ibrutinib, palbociclib, enzalutamide, pemetrexed, nilotinib, abiraterone, imatinib, palbociclib, erlotinib, bortezomib ... Tezomib, enzalutamide, cyclophosphamide, carboplatin, cisplatin, oxaliplatin, 5-fluorouracil, 6-mercaptopurine, cytarabine, gemcitabine, methotrexate, bleomycin, daunorubicin, doxorubicin, docetaxel, estramustine, paclitaxel, vinblastine, etoposide, irinotecan, teniposide, topotecan, prednisone, methylprednisolone, and dexamethasone. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is a polypeptide described herein (e.g., a fusion polypeptide comprising a SIRPα D1 domain variant and an Fc variant).

[0215] In some embodiments, methods of treating cancer include administering an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) in combination with trastuzumab, ramucirumab, and paclitaxel. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is a polypeptide described herein (e.g., a fusion polypeptide comprising a SIRPα D1 domain variant and an Fc variant).

[0216] In some embodiments, a method of treating cancer in an individual is provided, the method comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, (b) an anti-HER2 antibody, (c) an anti-VEGF2 antibody, and (d) paclitaxel, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; the Fc domain variant is selected from the group consisting of (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region (wherein numbering is according to EU index of Kabat) comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, and the cancer is gastric cancer or gastroesophageal junction (GEJ) cancer, and the individual has received at least one prior treatment for gastric cancer or GEJ cancer. In some embodiments, the polypeptide (e.g., fusion polypeptide) 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 dimer (e.g., a homodimer). In some embodiments, the gastric cancer or GEJ cancer is characterized by HER2 overexpression (e.g., HER2 +) gastric cancer or HER2-overexpressing GEJ cancer. In some embodiments, the individual has been prior treated with an anti-HER2 antibody, with an anti-HER2 antibody and a fluoropyrimidine, or with an anti-HER2 antibody and a platinum-based chemotherapy agent. In some embodiments, the individual has progressed (e.g., experienced disease progression) during or after prior treatment with trastuzumab and a fluoropyrimidine-containing chemotherapy (e.g., fluorouracil). In some embodiments, the individual has progressed (e.g., experienced disease progression) during or after prior treatment with trastuzumab and a platinum-containing chemotherapy. In some embodiments, the individual has progressed (e.g., experienced disease progression) during or after prior treatment with trastuzumab, a fluoropyrimidine-containing chemotherapy (e.g., fluorouracil), and a platinum-containing chemotherapy. In some embodiments, the individual has not been prior treated with an anti-CD47 agent or an anti-SIRPα agent. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the anti-VEGF antibody is ramucirumab. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered to an individual at the dosage and frequency described below. In some embodiments, the anti-HER2 antibody (e.g., trastuzumab) is administered to an individual at the dosage and frequency described below. In some embodiments, the anti-HER2 antibody (e.g., trastuzumab) is administered to an individual according to the dosage and frequency indicated on the prescription label. For example, in the United States, details regarding the dosage and frequency of administration of trastuzumab can be found at www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2010 / 103792s5250lbl(dot)pdf.

[0217] In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of about 10 to about 60 mg / kg once per week (qw). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of about 10 to about 60 mg / kg once every two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 10 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 15 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 20 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 30 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 40 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 50 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 60 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 10 mg / kg once per two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 15 mg / kg once per two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 20 mg / kg once every two weeks (q2w).In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 30 mg / kg once every two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 40 mg / kg once every two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 50 mg / kg once every two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 60 mg / kg once every two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant comprises a SIRPα variant comprising SEQ ID NO: 81 or SEQ ID NO: 85, wherein the Fc domain variant is selected from the group consisting of: (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations (wherein numbering is according to EU index of Kabat); or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations. In some embodiments, the polypeptide (e.g., fusion polypeptide) 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 dimer (e.g., a homodimer). In some embodiments, a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered to an individual in combination with (a) an anti-HER2 antibody, (b) an anti-VEGF2 antibody, and (c) paclitaxel.In some embodiments, the cancer is gastric cancer or gastroesophageal junction (GEJ) cancer. In some embodiments, the individual has had at least one prior treatment for gastric cancer or GEJ cancer. In some embodiments, the gastric cancer or GEJ cancer is HER2 overexpressing (e.g., HER2. +) gastric cancer or HER2-overexpressing GEJ cancer. In some embodiments, the individual has been prior treated with an anti-HER2 antibody, with an anti-HER2 antibody and a fluoropyrimidine, or with an anti-HER2 antibody and a platinum-based chemotherapy agent. In some embodiments, the individual has progressed (e.g., experienced disease progression) during or after prior treatment with an anti-HER2 antibody (e.g., trastuzumab) and a fluoropyrimidine-containing chemotherapy (e.g., fluorouracil). In some embodiments, the individual has progressed (e.g., experienced disease progression) during or after prior treatment with an anti-HER2 antibody (e.g., trastuzumab) and a platinum-containing chemotherapy. In some embodiments, the individual has progressed (e.g., experienced disease progression) during or after prior treatment with an anti-HER2 antibody (e.g., trastuzumab), a fluoropyrimidine-containing chemotherapy (e.g., fluorouracil), and a platinum-containing chemotherapy. In some embodiments, the individual has not been prior treated with an anti-CD47 agent or an anti-SIRPα agent. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the anti-VEGF antibody is ramucirumab. In some embodiments, the anti-VEGFR-2 antibody (e.g., ramucirumab) is administered to the individual according to the dosage and frequency indicated on the prescription label. For example, in the United States, details regarding the dosage and frequency of administration of ramucirumab can be found at www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2020 / 125477s034lbl(dot)pdf. In some embodiments, the anti-HER2 antibody (e.g., trastuzumab) is administered to the individual at the dosage and frequency described below. In some embodiments, the anti-HER2 antibody (e.g., trastuzumab) is administered to the individual according to the dosage and frequency indicated on the prescription label. For example, in the United States, details regarding the dosage and frequency of administration of trastuzumab can be found at www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2010 / 103792s5250lbl(dot)pdf.

[0218] In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 10 mg / kg once weekly in combination with trastuzumab at an initial dose of 8 mg / kg followed by 6 mg / kg once every three weeks (e.g., an initial dose of 8 mg / kg in week 1 followed by a 6 mg / kg dose in week 2 and 6 mg / kg doses once every three weeks thereafter after the first 6 mg / kg dose). In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 10 mg / kg once weekly in combination with trastuzumab at an initial dose of 8 mg / kg followed by 6 mg / kg once every three weeks and ramucirumab at an 8 mg / kg dose once every other week (e.g., on days 1 and 15 of each 28 day cycle). In some embodiments, a method of treating cancer in an individual includes administering a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant at a dose of 10 mg / kg once weekly, followed by an initial dose of 8 mg / kg, followed by trastuzumab at 6 mg / kg once every three weeks, ramucirumab at an 8 mg / kg dose once every other week (e.g., on days 1 and 15 of each 28 day cycle), and ramucirumab at 80 mg / kg once weekly for three weeks during each four week cycle (e.g., on days 1, 8, and 15 of each 28 day cycle). 2The method includes administering to an individual a dose of paclitaxel in combination with In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant comprises a SIRPα variant comprising SEQ ID NO: 81 or SEQ ID NO: 85, wherein the Fc domain variant is selected from the group consisting of: (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations (wherein numbering is according to EU index of Kabat); or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations. In some embodiments, the polypeptide (e.g., fusion polypeptide) 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 dimer (e.g., a homodimer). In some embodiments, the cancer is gastric cancer or gastroesophageal junction (GEJ) cancer. In some embodiments, the individual has had at least one prior treatment for gastric cancer or GEJ cancer. In some embodiments, the gastric cancer or GEJ cancer is HER2 overexpressing (e.g., HER2 +) gastric cancer or HER2-overexpressing GEJ cancer. In some embodiments, the individual has been prior treated with an anti-HER2 antibody, with an anti-HER2 antibody and a fluoropyrimidine, or with an anti-HER2 antibody and a platinum-based chemotherapy agent. In some embodiments, the individual has progressed (e.g., experienced disease progression) during or after prior treatment with an anti-HER2 antibody (e.g., trastuzumab) and a fluoropyrimidine-containing chemotherapy (e.g., fluorouracil). In some embodiments, the individual has progressed (e.g., experienced disease progression) during or after prior treatment with an anti-HER2 antibody (e.g., trastuzumab) and a platinum-containing chemotherapy. In some embodiments, the individual has progressed (e.g., experienced disease progression) during or after prior treatment with an anti-HER2 antibody (e.g., trastuzumab), a fluoropyrimidine-containing chemotherapy (e.g., fluorouracil), and a platinum-containing chemotherapy. In some embodiments, the individual has not been prior treated with an anti-CD47 agent or an anti-SIRPα agent.

[0219] In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 15 mg / kg once weekly in combination with trastuzumab at an initial dose of 8 mg / kg followed by 6 mg / kg once every three weeks (e.g., an initial dose of 8 mg / kg in week 1 followed by a 6 mg / kg dose in week 2 and 6 mg / kg doses once every three weeks thereafter after the first 6 mg / kg dose). In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 15 mg / kg once weekly in combination with trastuzumab at an initial dose of 8 mg / kg followed by 6 mg / kg once every three weeks and ramucirumab at an 8 mg / kg dose once every other week (e.g., on days 1 and 15 of each 28 day cycle). In some embodiments, a method of treating cancer in an individual includes administering a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant at a dose of 15 mg / kg once weekly, followed by an initial dose of 8 mg / kg, followed by trastuzumab at 6 mg / kg once every three weeks, ramucirumab at an 8 mg / kg dose once every other week (e.g., on days 1 and 15 of each 28 day cycle), and ramucirumab at 80 mg / kg once weekly for three weeks during each four week cycle (e.g., on days 1, 8, and 15 of each 28 day cycle). 2The method includes administering to an individual a dose of paclitaxel in combination with the compound. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant comprises a SIRPα variant comprising SEQ ID NO: 81 or SEQ ID NO: 85, wherein the Fc domain variant is selected from the group consisting of: (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations (wherein numbering is according to EU index of Kabat); or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations. In some embodiments, the polypeptide (e.g., fusion polypeptide) 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 dimer (e.g., a homodimer). In some embodiments, the cancer is gastric cancer or gastroesophageal junction (GEJ) cancer. In some embodiments, the individual has had at least one prior treatment for gastric cancer or GEJ cancer. In some embodiments, the gastric cancer or GEJ cancer is HER2 overexpressing (e.g., HER2 +) gastric cancer or HER2-overexpressing GEJ cancer. In some embodiments, the individual has been prior treated with an anti-HER2 antibody, with an anti-HER2 antibody and a fluoropyrimidine, or with an anti-HER2 antibody and a platinum-based chemotherapy agent. In some embodiments, the individual has progressed (e.g., experienced disease progression) during or after prior treatment with an anti-HER2 antibody (e.g., trastuzumab) and a fluoropyrimidine-containing chemotherapy (e.g., fluorouracil). In some embodiments, the individual has progressed (e.g., experienced disease progression) during or after prior treatment with an anti-HER2 antibody (e.g., trastuzumab) and a platinum-containing chemotherapy. In some embodiments, the individual has progressed (e.g., experienced disease progression) during or after prior treatment with an anti-HER2 antibody (e.g., trastuzumab), a fluoropyrimidine-containing chemotherapy (e.g., fluorouracil), and a platinum-containing chemotherapy. In some embodiments, the individual has not been prior treated with an anti-CD47 agent or an anti-SIRPα agent.

[0220] In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 30 mg / kg every two weeks in combination with trastuzumab at an initial dose of 6 mg / kg followed by 4 mg / kg every two weeks (e.g., an initial dose of 6 mg / kg followed by a 4 mg / kg dose two weeks after the first 6 mg / kg dose, followed by 4 mg / kg doses every two weeks thereafter). In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 30 mg / kg every two weeks in combination with trastuzumab at an initial dose of 6 mg / kg followed by 4 mg / kg every two weeks, and ramucirumab at an 8 mg / kg dose every two weeks (e.g., on days 1 and 15 of each 28 day cycle). In some embodiments, methods of treating cancer in an individual are provided comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 30 mg / kg once every two weeks in combination with an initial dose of 6 mg / kg (e.g., as described elsewhere herein), followed by trastuzumab at 4 mg / kg once every two weeks, ramucirumab at an 8 mg / kg dose once every two weeks (e.g., on days 1 and 15 of each 28 day cycle), and paclitaxel at an 80 mg / m2 dose once a week for three weeks of each four week cycle (e.g., on days 1, 8, and 15 of a 28 day cycle).In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant comprises a SIRPα variant comprising SEQ ID NO: 81 or SEQ ID NO: 85, wherein the Fc domain variant is selected from the group consisting of: (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations (wherein numbering is according to EU index of Kabat); (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations (wherein numbering is according to EU index of Kabat); (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations (wherein numbering is according to EU index of Kabat); or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations. In some embodiments, the polypeptide (e.g., fusion polypeptide) 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 dimer (e.g., a homodimer). In some embodiments, the cancer is gastric or gastroesophageal junction (GEJ) cancer. In some embodiments, the individual has had at least one prior treatment for gastric or GEJ cancer. In some embodiments, the gastric or GEJ cancer is HER2-overexpressing (e.g., HER2. +) gastric cancer or HER2-overexpressing GEJ cancer. In some embodiments, the individual has been prior treated with an anti-HER2 antibody, with an anti-HER2 antibody and a fluoropyrimidine, or with an anti-HER2 antibody and a platinum-based chemotherapy agent. In some embodiments, the individual has progressed (e.g., experienced disease progression) during or after prior treatment with an anti-HER2 antibody (e.g., trastuzumab) and a fluoropyrimidine-containing chemotherapy (e.g., fluorouracil). In some embodiments, the individual has progressed (e.g., experienced disease progression) during or after prior treatment with an anti-HER2 antibody (e.g., trastuzumab) and a platinum-containing chemotherapy. In some embodiments, the individual has progressed (e.g., experienced disease progression) during or after prior treatment with an anti-HER2 antibody (e.g., trastuzumab), a fluoropyrimidine-containing chemotherapy (e.g., fluorouracil), and a platinum-containing chemotherapy. In some embodiments, the individual has not been prior treated with an anti-CD47 agent or an anti-SIRPα agent.

[0221] Exemplary cancers In some embodiments, the cancer treated by the methods provided herein is breast cancer, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), head and neck cancer, mesothelioma, brain cancer, brain tumor, abdominal cancer, colon cancer, colorectal cancer, esophageal cancer, parapharyngeal cancer, gastrointestinal cancer, glioma, liver cancer, gastric cancer, oral cancer, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, renal cancer, bladder cancer, urinary tract cancer, pancreatic cancer, retinoblastoma, cervical cancer, uterine cancer, Wilms' tumor, multiple myeloma, skin cancer, lymphoma, leukemia, blood cancer, thyroid cancer, bone cancer, adenoid cystic tumor, chondrosarcoma, pancreatic islet cell tumor, neuroendocrine tumor, prostate cancer, glioblastoma, endometrial carcinoma, endometrial cancer, leiomyosarcoma, gallbladder cancer, hepatocellular carcinoma, melanoma, or a solid tumor.

[0222] In some embodiments, the cancer treated by the methods provided herein is gastric cancer. In some embodiments, the cancer treated by the methods provided herein is gastric adenocarcinoma. In some embodiments, the cancer treated by the methods provided herein is gastroesophageal junction adenocarcinoma. In some embodiments, the cancer treated by the methods provided herein is HER2-overexpressing gastroesophageal junction adenocarcinoma.

[0223] Treatment of solid tumors In some embodiments, provided are methods of treating a solid tumor in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), and (b) a platinum-based chemotherapeutic agent. In some embodiments, the solid tumor is colon cancer (e.g., colon cancer), lung cancer, head and neck cancer, esophageal cancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer, testicular cancer, brain cancer, mesothelioma, or neuroblastoma. In some embodiments, the platinum-based chemotherapeutic agent is carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and / or satraplatin. In some embodiments, the platinum-based chemotherapeutic agent is cisplatin. In some embodiments, the agent is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein). In some embodiments, the polypeptide (e.g., a fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat. In some embodiments, the polypeptide (eg, a fusion polypeptide) administered to the individual comprises the amino acid sequence of SEQ ID NO:136 or SEQ ID NO:135.In some embodiments, the polypeptide (e.g., a fusion polypeptide) forms a homodimer. In some embodiments, the polypeptide (e.g., a fusion polypeptide) and the platinum-based chemotherapeutic agent (e.g., cisplatin) are administered simultaneously, concurrently, or sequentially.

[0224] Platinum agents (such as carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin, etc.) are widely used antitumor drugs that cause crosslinking of DNA as monoadducts, interstrand crosslinks, intrastrand crosslinks, or DNA-protein crosslinks. Platinum agents typically act at adjacent N-7 positions of guanine, forming 1,2 intrastrand crosslinks (Poklar et al. (1996). Proc. Natl. Acad. Sci. USA 93(15):7606-11; Rudd et al. (1995). Cancer Chemother. Pharmacol. 35(4):323-6). The resulting crosslinks inhibit DNA repair and / or DNA synthesis in cancer cells.

[0225] Cisplatin is an exemplary platinum coordination compound for use in the methods described herein. The chemical name for cisplatin is dichloroplatinum diammoniate, and cisplatin has the following structural formula: [ka]

[0226] Cisplatin is an inorganic, water-soluble platinum complex with the molecular formula Pt(NH3)2Cl2 and a molecular weight of 300.046. After undergoing hydrolysis, cisplatin reacts with DNA to generate both intrastrand and interstrand crosslinks. These crosslinks are believed to impair DNA replication and transcription. The cytotoxicity of cisplatin correlates with cell arrest in the G2 phase of the cell cycle. Cisplatin, which has been assigned the CAS Registry Number 15663-27-1, is commercially available as PLATINOL®, PLATINOL®-AQ, CDDP, CISPLAN, CISPLAT, PLATIKEM, PLATIONCO, PRACTICIS, PLATICIS, BLASTOLEM, CISMAX, CISPLAN, CISPLATINUM, CISTEEN, DUPLAT, KEMOPLAT, ONCOPLATIN-AQ, PLATINEX, PLATIN, TEVAPLATIN, etc. Complete information regarding preparation, dispensing, dosage, and administration schedules of cisplatin may be found in the national package insert (in the United States, see, e.g., www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2011 / 018057s080lbl(dot)pdf and www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2015 / 018057s083lbl(dot)pdf). In some embodiments, cisplatin is administered according to the dosage and frequency recommended in the national package insert.

[0227] Carboplatin is another exemplary platinum coordination compound for use in the methods described herein. The chemical name of carboplatin is platinum, diamine[1,1 cyclobutane-dicarboxylato(2-)-0,0]-, (SP-4-2), and carboplatin has the following structural formula: [ka]

[0228] Carboplatin has the molecular formula CH 12Carboplatin is a water-soluble platinum complex with a molecular weight of 373.26 and is assigned the CAS Registry Number 41575-94-4. Its mechanism of action is similar to that of cisplatin. Carboplatin is typically more commonly prescribed than cisplatin. Carboplatin is commercially available as PARAPLATIN®, BLASTOCARB®, BLASTOPLATIN®, CARBOKEM®, CARBOMAX®, PARAPLATIN®, CARBOPA®, KARPLAT®, and others. Complete information regarding the preparation, dispensing, dosage, and administration schedule of carboplatin may be found in the national package insert (in the United States, see, for example, www(dot)accessdata(dot)fda(dot)gov / drugsatfda_docs / label / 2010 / 020452s005lbl(dot)pdf and www(dot)accessdata.fda(dot)gov / drugsatfda_docs / label / 2012 / 077139Orig1s016lbl(dot)pdf). In some embodiments, carboplatin is administered according to the dosage and frequency recommended in the national package insert.

[0229] In some embodiments, provided are methods of treating a solid tumor in an individual (e.g., a human individual), the method comprising administering to the individual effective amounts of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-HER2 antibody, and (c) an anti-PD-L1 antibody. In some embodiments, the anti-HER2 antibody is trastuzumab (CAS Registry Number: 180288-69-1). In some embodiments, the anti-PD-L1 antibody is atezolizumab (CAS Registry Number 1380723-44-3), avelumab (CAS Registry Number 1537032-82-8), or durvalumab (CAS Registry Number 1428935-60-7). In some embodiments, the agent is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein). In some embodiments, the polypeptide (e.g., a fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat. In some embodiments, the polypeptide (e.g., a fusion polypeptide) administered to the individual comprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., a fusion polypeptide) forms a homodimer.In some embodiments, the polypeptide (e.g., fusion polypeptide), anti-HER2 antibody, anti-PD-L1 antibody (e.g., anti-PD-L1 antagonist antibody) are administered simultaneously, concurrently, or sequentially. In some embodiments, the solid tumor is colon cancer, lung cancer, head and neck cancer, esophageal cancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer, testicular cancer, endometrial cancer, liver cancer, gastric cancer, gastroesophageal junction cancer, brain tumor, mesothelioma, or neuroblastoma. In some embodiments, the solid tumor is HER2. + In some embodiments, the solid tumor is a colon cancer (e.g., HER2 + colon cancer).

[0230] Treatment of Gastric Cancer (GC) or Gastroesophageal Junction (GEJ) Cancer In some embodiments, a method of treating gastric or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-HER2 antibody, (c) an anti-VEGFR2 antibody, and (d) paclitaxel. In some embodiments, the anti-HER2 antibody is trastuzumab (CAS Registry Number: 180288-69-1). In some embodiments, the anti-VEGFR2 antibody is ramucirumab (CAS Registry Number: 947687-13-0). In some embodiments, the agent is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein). In some embodiments, a polypeptide (eg, a fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat. In some embodiments, the polypeptide (e.g., a 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., a fusion polypeptide) forms a dimer (e.g., a homodimer).In some embodiments, the polypeptide (e.g., fusion polypeptide), anti-HER2 antibody, anti-VEGFR2 antibody and paclitaxel are administered simultaneously, in parallel or sequentially. In some embodiments, the polypeptide (e.g., fusion polypeptide) is administered to the individual at a dose of 10 mg / kg once a week or 15 mg / kg once a week or 30 mg / kg once every two weeks. In some embodiments, the individual being treated has gastric adenocarcinoma or GEJ adenocarcinoma. In some embodiments, the individual being treated has HER2. + Gastric cancer or HER2 + In some embodiments, the patient has a GEJ cancer (e.g., a HER2-overexpressing gastric cancer or a GEJ cancer). + Gastric cancer or HER2 +The GEJ cancer is progressive and / or metastatic. In some embodiments, the individual being treated has a gastric or GEJ cancer that has progressed during or after prior treatment(s) including an anti-HER2 antibody (e.g., trastuzumab). In some embodiments, the individual being treated has a gastric or GEJ cancer that has progressed during or after prior treatment(s) including an anti-HER2 antibody (e.g., trastuzumab) and a fluoropyrimidine (e.g., fluorouracil, also known as 5-fluorouracil). In some embodiments, the individual being treated has a gastric or GEJ cancer that has progressed during or after prior treatment(s) including a fluoropyrimidine (e.g., fluorouracil). In some embodiments, the individual being treated has a gastric or GEJ cancer that has progressed during or after prior treatment(s) including a platinum-based chemotherapy agent (e.g., carboplatin or cisplatin). In some embodiments, the individual being treated has gastric or GEJ cancer that has progressed during or after prior treatment(s) including an anti-HER2 antibody (e.g., trastuzumab) and a platinum-based chemotherapy agent (e.g., carboplatin or cisplatin). In some embodiments, the individual being treated has gastric or GEJ cancer (e.g., HER2 + In some embodiments, the individual undergoing treatment has gastric or GEJ cancer (e.g., HER2 cancer) that has progressed during or after prior treatment(s) that include an anti-HER2 antibody (e.g., trastuzumab) and / or a fluoropyrimidine (e.g., fluorouracil). + In some embodiments, the individual undergoing treatment has gastric or GEJ cancer (e.g., HER2 +In some embodiments, the individual has a gastric or GEJ cancer. In some embodiments, the individual has failed (e.g., relapsed after or did not respond to) prior treatment with an anti-HER2 antibody, an anti-HER2 antibody and a fluoropyrimidine (e.g., fluorouracil), an anti-HER2 antibody and a platinum-based chemotherapy agent (e.g., carboplatin or cisplatin), or a platinum-based chemotherapy agent (e.g., carboplatin or cisplatin). In some embodiments, the fluoropyrimidine was fluorouracil (also known as 5-fluorouracil). In some embodiments, the individual being treated has received a prior anti-VEGFR2 antibody-containing treatment for gastric or GEJ cancer. In some embodiments, the prior anti-VEGFR2 antibody-containing treatment was a treatment comprising ramucirumab. In some embodiments, the individual being treated has not received a prior anti-VEGFR2 antibody-containing treatment for gastric or GEJ cancer (e.g., a treatment comprising ramucirumab). In some embodiments, the individual being treated has had at least one, at least two, at least three, or at least four prior therapies for gastric or GEJ cancer. In some embodiments, treatment with the polypeptide, anti-HER2 antibody, anti-VEGFR2 antibody, and paclitaxel produces no adverse effects. In some embodiments, treatment with the polypeptide, anti-HER2 antibody, anti-VEGFR2 antibody, and paclitaxel produces only mild adverse effects.

[0231] In some embodiments, a method of treating gastric or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual) is provided, the method comprising administering to the individual effective amounts of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-PD-1 antibody (e.g., an anti-PD-1 antagonist antibody), (c) an anti-HER2 antibody, (d) 5-fluorouracil, and (e) a platinum-based chemotherapeutic agent. In some embodiments, a method of treating gastric or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-PD-1 antibody (e.g., an anti-PD-1 antagonist antibody), (c) an anti-HER2 antibody, (d) capecitabine, and (e) a platinum-based chemotherapeutic agent. In some embodiments, the anti-PD-1 antibody is pembrolizumab (CAS Registry Number: 1374853-91-4). In some embodiments, the anti-HER2 antibody is trastuzumab (CAS Registry Number: 180288-69-1). In some embodiments, the platinum-based chemotherapeutic agent is cisplatin. In some embodiments, the agent is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein). In some embodiments, the polypeptide (e.g., a fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85.In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat. In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to the individual comprises 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 dimer (e.g., homodimer). In some embodiments, the polypeptide (e.g., fusion polypeptide), anti-PD-1 antibody, anti-HER2 antibody, 5-fluorouracil, and platinum-based chemotherapeutic agent are administered simultaneously, in parallel, or sequentially. In some embodiments, the polypeptide (e.g., fusion polypeptide), anti-PD-1 antibody, anti-HER2 antibody, capecitabine, and platinum-based chemotherapeutic agent are administered simultaneously, in parallel, or sequentially. In some embodiments, the individual being treated has HER2-overexpressing gastric cancer or HER2-overexpressing GEJ cancer. In some embodiments, the gastric cancer or GEJ cancer is advanced and / or metastatic. In some embodiments, the individual has not been previously treated for gastric cancer or GEJ cancer.

[0232] In some embodiments, a method of treating gastric cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of about 10 to about 60 mg / kg once per week (qw). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of about 10 to about 60 mg / kg once every two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 10 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 15 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 20 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 30 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 40 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 45 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 50 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 60 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 10 mg / kg once every two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 15 mg / kg once every two weeks (q2w).In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 20 mg / kg once every two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 30 mg / kg once every two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 40 mg / kg once every two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 45 mg / kg once every two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 50 mg / kg once every two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 60 mg / kg once every two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered in combination with an initial dose of 6 mg / kg followed by 4 mg / kg trastuzumab once every two weeks, and ramucirumab at a dose of 8 mg / kg once every two weeks, and paclitaxel at a dose of 80 mg / m2 on days 1, 8, and 15 of a 28 day cycle, e.g., at any dosage and frequency of administration described herein (e.g., as described elsewhere herein).

[0233] In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 10 mg / kg once weekly in combination with trastuzumab at an initial dose of 8 mg / kg followed by 6 mg / kg once every three weeks (e.g., an initial dose of 8 mg / kg in week 1 followed by a 6 mg / kg dose in week 2 and 6 mg / kg doses once every three weeks thereafter after the first 6 mg / kg dose). In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 10 mg / kg once weekly in combination with trastuzumab at an initial dose of 8 mg / kg followed by 6 mg / kg once every three weeks and ramucirumab at 8 mg / kg doses on days 1 and 15 of every four weeks. In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 10 mg / kg once weekly in combination with an initial dose of 8 mg / kg followed by 6 mg / kg trastuzumab once every 3 weeks, ramucirumab at a dose of 8 mg / kg on days 1 and 15 every 4 weeks, and paclitaxel at a dose of 80 mg / m2 on days 1, 8, and 15 every 4 weeks.

[0234] In some embodiments, a method of treating gastric cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 15 mg / kg once weekly in combination with trastuzumab at an initial dose of 8 mg / kg followed by 6 mg / kg once every three weeks. In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 15 mg / kg once weekly in combination with trastuzumab at an initial dose of 8 mg / kg followed by 6 mg / kg once every three weeks, and ramucirumab at a dose of 8 mg / kg on days 1 and 15 of every four weeks. In some embodiments, a method of treating cancer in an individual is provided comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 15 mg / kg once weekly in combination with an initial dose of 8 mg / kg followed by 6 mg / kg trastuzumab once every 3 weeks, ramucirumab at a dose of 8 mg / kg on days 1 and 15 every 4 weeks, and paclitaxel at a dose of 80 mg / m2 on days 1, 8, and 15 every 4 weeks.

[0235] In some embodiments, a method of treating gastric cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 30 mg / kg once every two weeks, in combination with an initial dose of 6 mg / kg, followed by 4 mg / kg once every two weeks (e.g., an initial dose of 6 mg / kg, followed by a 4 mg / kg dose two weeks after the initial dose of 6 mg / kg, then 4 mg / kg doses every two weeks after the first 4 mg / kg dose), trastuzumab at an 8 mg / kg dose once every two weeks, and ramucirumab at an 80 mg / m2 dose on days 1, 8, and 15 of a 28 day cycle. In some embodiments, a method of treating gastric adenocarcinoma in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 30 mg / kg every two weeks in combination with an initial dose of 6 mg / kg followed by trastuzumab at 4 mg / kg every two weeks, and ramucirumab at a dose of 8 mg / kg every two weeks, and paclitaxel at a dose of 80 mg / m2 on days 1, 8, and 15 of a 28 day cycle. In some embodiments, a method of treating gastroesophageal junction adenocarcinoma in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 30 mg / kg every two weeks in combination with an initial dose of 6 mg / kg, followed by trastuzumab at 4 mg / kg every two weeks, ramucirumab at a dose of 8 mg / kg every two weeks, and paclitaxel at a dose of 80 mg / m2 on days 1, 8, and 15 of a 28 day cycle.

[0236] In some embodiments, a method of treating gastroesophageal junction cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of about 10 to about 60 mg / kg once per week (qw). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of about 10 to about 60 mg / kg once every two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 10 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 15 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 20 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 30 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 40 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 45 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 50 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 60 mg / kg once per week. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 10 mg / kg once every two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 15 mg / kg once every two weeks (q2w).In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 20 mg / kg once every two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 30 mg / kg once every two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 40 mg / kg once every two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 45 mg / kg once every two weeks. In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 50 mg / kg once every two weeks (q2w). In some embodiments, the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 60 mg / kg once every two weeks (q2w).

[0237] In some embodiments, a method of treating gastroesophageal junction cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 10 mg / kg once weekly in combination with trastuzumab at an initial dose of 8 mg / kg followed by 6 mg / kg once every three weeks. In some embodiments, a method of treating gastroesophageal junction cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 10 mg / kg once weekly in combination with trastuzumab at an initial dose of 8 mg / kg followed by 6 mg / kg once every three weeks, and ramucirumab at a dose of 8 mg / kg on days 1 and 15 every four weeks. In some embodiments, a method of treating gastroesophageal junction cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 10 mg / kg once weekly in combination with an initial dose of 8 mg / kg followed by 6 mg / kg trastuzumab once every 3 weeks, ramucirumab at a dose of 8 mg / kg on days 1 and 15 every 4 weeks, and paclitaxel at a dose of 80 mg / m2 on days 1, 8, and 15 every 4 weeks.

[0238] In some embodiments, a method of treating gastroesophageal junction cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 15 mg / kg once weekly in combination with trastuzumab at an initial dose of 8 mg / kg followed by 6 mg / kg once every three weeks. In some embodiments, a method of treating gastroesophageal junction cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 15 mg / kg once weekly in combination with trastuzumab at an initial dose of 8 mg / kg followed by 6 mg / kg once every three weeks, and ramucirumab at a dose of 8 mg / kg on days 1 and 15 of every four weeks. In some embodiments, a method of treating gastroesophageal junction cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 15 mg / kg once weekly in combination with an initial dose of 8 mg / kg followed by 6 mg / kg trastuzumab once every 3 weeks, ramucirumab at a dose of 8 mg / kg on days 1 and 15 every 4 weeks, and paclitaxel at a dose of 80 mg / m2 on days 1, 8, and 15 every 4 weeks.

[0239] In some embodiments, a method of treating gastroesophageal junction cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 30 mg / kg every two weeks in combination with an initial dose of 6 mg / kg followed by trastuzumab at 4 mg / kg every two weeks, and ramucirumab at a dose of 8 mg / kg every two weeks, and paclitaxel at a dose of 80 mg / m2 on days 1, 8, and 15 of a 28 day cycle. In some embodiments, a method of treating gastroesophageal junction cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 30 mg / kg every two weeks in combination with an initial dose of 6 mg / kg, followed by 4 mg / kg every two weeks (e.g., an initial dose of 6 mg / kg, followed by a 4 mg / kg dose two weeks after the initial dose of 6 mg / kg, then 4 mg / kg doses every two weeks after the first 4 mg / kg dose), trastuzumab at an 8 mg / kg dose every two weeks, and ramucirumab at an 80 mg / m2 dose on days 1, 8, and 15 of a 28 day cycle. In some embodiments, a method of treating gastroesophageal junction cancer in an individual is provided, comprising administering to the individual a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant administered at a dose of 30 mg / kg every two weeks in combination with an initial dose of 6 mg / kg, followed by 4 mg / kg every two weeks (e.g., an initial dose of 6 mg / kg, followed by a 4 mg / kg dose two weeks after the initial dose of 6 mg / kg, then 4 mg / kg doses every two weeks after the first 4 mg / kg dose), trastuzumab at an 8 mg / kg dose every two weeks, and ramucirumab at an 80 mg / m2 dose on days 1, 8, and 15 of a 28 day cycle.

[0240] In some embodiments, a method of treating gastric or GEJ cancer in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-HER2 antibody, (c) an anti-VEGFR2 antibody, and (d) paclitaxel, whereby across a patient population receiving the treatment, the overall response rate (ORR) of the population is greater than 50%. In some embodiments, a method of treating gastric or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual) is provided, the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-HER2 antibody, (c) an anti-VEGFR2 antibody, and (d) paclitaxel, whereby across a patient population receiving the treatment, the overall response rate (ORR) of the population is greater than 55%. In some embodiments, a method of treating gastric or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual) is provided, the method comprising administering to the individual effective amounts of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα); (b) an anti-HER2 antibody; (c) an anti-VEGFR2 antibody; and (d) paclitaxel, wherein across a population of patients receiving the treatment, the overall response rate (ORR) for the population is greater than 60%. In some embodiments, a method of treating gastric or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual) is provided, the method comprising administering to the individual effective amounts of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα); (b) an anti-HER2 antibody; (c) an anti-VEGFR2 antibody; and (d) paclitaxel, wherein across a population of patients receiving the treatment, the overall response rate (ORR) for the population is greater than 65%.In some embodiments, methods are provided for treating gastric or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual), comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-HER2 antibody, (c) an anti-VEGFR2 antibody, and (d) paclitaxel, whereby across a patient population receiving the treatment, the overall response rate (ORR) of the population is greater than 70%. In some embodiments, methods are provided for treating gastric or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual), comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-HER2 antibody, (c) an anti-VEGFR2 antibody, and (d) paclitaxel, whereby across a patient population receiving the treatment, the overall response rate (ORR) of the population is greater than 75%. In some embodiments, a method of treating gastric or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual) is provided, the method comprising administering to the individual effective amounts of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα); (b) an anti-HER2 antibody; (c) an anti-VEGFR2 antibody; and (d) paclitaxel, wherein across a population of patients receiving the treatment, the overall response rate (ORR) for the population is greater than 80%. In some embodiments, a method of treating gastric or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual) is provided, the method comprising administering to the individual effective amounts of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα); (b) an anti-HER2 antibody; (c) an anti-VEGFR2 antibody; and (d) paclitaxel, wherein across a population of patients receiving the treatment, the overall response rate (ORR) for the population is greater than 85%.In some embodiments, methods are provided for treating gastric or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual), comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-HER2 antibody, (c) an anti-VEGFR2 antibody, and (d) paclitaxel, whereby across a patient population receiving the treatment, the overall response rate (ORR) of the population is greater than 90%. In some embodiments, methods are provided for treating gastric or gastroesophageal junction (GEJ) cancer in an individual (e.g., a human individual), comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), (b) an anti-HER2 antibody, (c) an anti-VEGFR2 antibody, and (d) paclitaxel, whereby across a patient population receiving the treatment, the overall response rate (ORR) of the population is greater than 95%. In some embodiments, the overall response rate (ORR) is the proportion of a patient population that achieves a partial response (PR) or a complete response (CR) to a treatment. In some embodiments, PR and CR are determined according to RECIST criteria (Response Evaluation Criteria in Solid Tumors). Details regarding RECIST can be found, for example, at https: / / ctep(dot)cancer(dot)gov / protocolDevelopment / docs / recist_guideline.pdf or https: / / recist(dot)eortc(dot)org.

[0241] Cancer combination therapy including anti-TROP2 antibody In some embodiments, provided are methods of treating cancer in an individual (e.g., a human individual), the method comprising administering to the individual an effective amount of (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), and (b) an anti-TROP2 antibody. In some embodiments, the anti-TROP2 antibody is RS7, described in U.S. Pat. No. 10,179,171, the contents of which are incorporated herein in their entirety. In some embodiments, the anti-TROP2 antibody is conjugated to a drug (i.e., an antibody-drug conjugate or "ADC"). In some embodiments, the anti-TROP2 ADC is sacituzumab govitecan (also known as hRS7-SN38 or IMMU-132), which is described in US2017 / 0281791, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the agent is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein). In some embodiments, the polypeptide (e.g., a fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat.In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to the individual comprises 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 polypeptide (e.g., fusion polypeptide) and the anti-TROP2 antibody are administered simultaneously, in parallel, or sequentially. In some embodiments, the cancer is a solid tumor, gastric cancer, nasopharyngeal cancer, gallbladder cancer, cervical cancer, extranodal NK / T cell lymphoma, lung cancer, laryngeal squamous cell carcinoma, colon cancer, perihilar cholangiocarcinoma, pancreatic cancer, oral squamous cell carcinoma, endometrioid endometrial cancer, or ovarian cancer. In some embodiments, the cancer is characterized by overexpression of TROP2. In some embodiments, the cancer is not characterized by overexpression of TROP2.

[0242] Methods for increasing phagocytosis of target cells In some embodiments, provided are methods of increasing phagocytosis of a target cell (e.g., a cancer cell), the method comprising contacting the target cell with (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), and (b) an anti-TROP2 antibody. In some embodiments, the anti-TROP2 antibody is RS7, described in U.S. Pat. No. 10,179,171, the contents of which are incorporated herein in their entirety. In some embodiments, the anti-TROP2 antibody is conjugated to a drug (i.e., an antibody-drug conjugate or "ADC"). In some embodiments, the anti-TROP2 ADC is sacituzumab govitecan (also known as hRS7-SN38 or IMMU-132), which is described in US2017 / 0281791, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the agent is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein). In some embodiments, the polypeptide (e.g., a fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat.In some embodiments, the polypeptide (e.g., fusion polypeptide) administered to the individual comprises 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 target cell is a cancer cell. In some embodiments, the cancer cell is a solid tumor cell, a gastric cancer cell, a nasopharyngeal cancer cell, a gallbladder cancer cell, a cervical cancer cell, an extranodal NK / T cell lymphoma cell, a lung cancer cell, a laryngeal squamous cell carcinoma cell, a colon cancer cell, a hilar cholangiocarcinoma cell, a pancreatic cancer cell, an oral squamous cell carcinoma cell, an endometrioid endometrial cancer cell, or an ovarian cancer cell.

[0243] In some embodiments, a method of increasing phagocytosis of a target cell is provided, the method comprising contacting the target cell with (a) an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), and (b) a second agent capable of enhancing phagocytosis. In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein). In some embodiments, the polypeptide (e.g., a fusion polypeptide) comprises a SIRPα D1 domain variant comprising the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat. In some embodiments, the polypeptide (e.g., a fusion polypeptide) administered to the individual comprises the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 135. In some embodiments, the polypeptide (e.g., a fusion polypeptide) forms a homodimer. In some embodiments, the second agent enhances phagocytosis, for example, by blocking a "don't eat me" signal.Exemplary agents include, but are not limited to, for example, anti-LILRB2 antibodies, anti-LILRB1 antibodies, anti-SIGLEC-10 antibodies, anti-CD24 antibodies, anti-SIRPα antibodies, anti-PD1 antibodies (e.g., anti-PD1 antagonist antibodies), and anti-PD-L1 antibodies (e.g., anti-PD-L1 antagonist antibodies). In some embodiments, the second agent enhances phagocytosis, for example, by enhancing the "eat me" signal. Exemplary agents include, but are not limited to, for example, BTK activators, TLR agonists, agents that promote the interaction between Mac-1 and SLAMF7, agents that promote the interaction between calreticulin and LRP1. Additional exemplary agents that enhance phagocytosis include, but are not limited to, for example, agents that modulate podosome adhesion, agents that modulate the expression level of lamin A, activators of SHP-1 phosphatase activity, and activators of myosin IIa assembly. In some embodiments, the method comprises contacting the target cell with (a) a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein), and (b) an anti-LILBR2 antibody, an anti-CD24 antibody, or an anti-SIGLEC-10 antibody. In some embodiments, the method comprises contacting the target cell with (a) the fusion polypeptide and (b) a BTK activator. In some embodiments, the method comprises contacting the target cell with (a) the fusion polypeptide and (b) a TLR agonist.

[0244] In some embodiments, the method comprises contacting a target cell with (a) a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein), and (b) two or more agents capable of enhancing phagocytosis (such as, but not limited to, two or more agents described herein). In some embodiments, the method comprises contacting a target cell with (a) a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein), (b) and an anti-LILBR2 antibody, an anti-CD24 antibody, or an anti-SIGLEC-10 antibody, and (c) an anti-PD1 antibody (e.g., an anti-PD-1 antagonist antibody) or an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody). In some embodiments, the method comprises contacting a target cell with (a) a fusion polypeptide, (b) an anti-LILBR2 antibody, and (c) an anti-PD1 antibody (e.g., an anti-PD-1 antagonist antibody). In some embodiments, the method comprises contacting a target cell with (a) a fusion polypeptide, (b) an anti-LILBR2 antibody, and (c) an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody).

[0245] In some embodiments, the contacting occurs in vitro. In some embodiments, the contacting occurs in vivo. In some embodiments, the target cell is a cancer cell. In some embodiments, contacting a target cell with (a) a polypeptide comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein), and (b) one or more agents capable of enhancing phagocytosis increases phagocytosis of the target cell by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more than 99%, compared to contacting the target cell with one or more agents capable of enhancing phagocytosis (i.e., in the absence of a polypeptide comprising a SIRPα D1 domain variant (e.g., a SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein).

[0246] Kits and manufactured products In another embodiment of the invention, an article of manufacture or kit is provided that includes a polypeptide (e.g., a fusion polypeptide described herein) comprising a SIRPα D1 domain variant and an Fc domain variant, in some embodiments, the SIRPα D1 domain variant comprises an amino acid sequence selected from the group consisting of SEQ ID NO:81 and SEQ ID NO:85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, where numbering is according to EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, where numbering is according to EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, where numbering is according to EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, where numbering is according to EU index of Kabat. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO:91. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 135 or SEQ ID NO: 136. In some embodiments, the kit or article of manufacture is for use in accordance with the methods of treatment provided herein.

[0247] In some embodiments, the kit or article of manufacture further comprises an anti-HER2 antibody (e.g., trastuzumab), an anti-VEGFR2 antibody (e.g., ramucirumab), and paclitaxel. In some embodiments, the kit includes a package insert or label containing instructions for using the polypeptide (e.g., a fusion polypeptide) in combination with an anti-HER2 antibody (e.g., trastuzumab), an anti-VEGFR2 antibody (e.g., ramucirumab), and paclitaxel to treat or delay the progression of gastric cancer or gastroesophageal junction (GEJ) cancer in an individual (such as a human individual), e.g., according to the methods described herein.

[0248] In some embodiments, the polypeptide (e.g., fusion polypeptide) and one or more additional anti-cancer agents (e.g., as outlined in the embodiments above) are provided together in a kit. In some embodiments, the polypeptide (e.g., fusion polypeptide) and one or more additional anti-cancer agents are provided in the same container or in separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers can be formed from a variety of materials, such as glass, plastic (such as polyvinyl chloride or polyolefin), or alloys (such as stainless steel or Hastelloy). In some embodiments, the container holds the formulation, and a label on or associated with the container can indicate instructions for use. The article of manufacture or kit can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further includes one or more of another agent (e.g., chemotherapeutic and anti-tumor agents, therapeutic antibodies, etc.). Suitable containers for one or more agents include, for example, bottles, vials, bags, and syringes.

[0249] The present specification is deemed sufficient to enable one skilled in the art to practice the invention. In addition to those shown and described herein, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description and are included within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. EXAMPLES

[0250] The present disclosure will be more fully understood by referring to the following examples. However, the examples should not be interpreted as limiting the scope of the present disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes in consideration thereof will be suggested to those skilled in the art, and they are included within the spirit and scope of the present application and the scope of the appended claims.

[0251] Example 1: Antitumor activity of Drug A in combination with trastuzumab and anti-PD-L1 antibody in a colon cancer model. MC38 mouse / human HER2 expressing cells ("MC38 m / h HER2 cells") were generated by infecting MC38 mouse colon adenocarcinoma cells with a lentiviral vector encoding a chimera of mouse and human HER2 transmembrane and extracellular domains. MC38 m / h HER2 cells were maintained in DMEM (Thermo Fisher Scientific 11965092) supplemented with 10% FBS, 1% penicillin-streptomycin, 1% GlutaMAX, and 1 mM sodium pyruvate (Thermo Fisher Scientific 11360070) at 37°C in a 5% CO2 incubator. All tissue culture was performed under sterile conditions.

[0252] Prior to implantation, a master cell bank was generated for each cell line to ensure that cells used in subsequent experiments were at the same passage number. Cells were harvested and washed twice with 50 mL of cold PBS (Life Technologies 10010072). After the final wash, for the MC38 m / h HER2 cell line, cells were diluted to 5 × 10 6 The cells were resuspended in PBS or RPMI at 100 cells / mL. For MC38 m / h HER2, 100 μL of the cell suspension was injected subcutaneously into the right flank of C57BL / 6 mice. The tumor size of MC38 m / h HER2 tumors averaged 65–69 mm. 3 When the animals reached maturity, they were randomized into eight groups of 10 mice each. Each group was assigned to a treatment group as outlined in Table A. [Table 13]

[0253] Drug A is an exemplary fusion protein comprising a SIRPα variant that binds hCD47 with high affinity and an inactive Fc region (ie, an Fc region that does not exhibit ADCC effector function).

[0254] Tumor volume (mm 3 ) was assessed using a Mitutoyo Digital Caliper (Mitutoyo America, Aurora, Illinois). Tumor volume and body weight were recorded two or three times weekly. 3 Mice that exceeded 10 cm or lost 20% of their body weight were euthanized according to IACUC guidelines. Tumor volume was calculated as ([length × {width × width}] × 0.5 = volume (mm 3 Statistical analyses and p-values ​​were calculated using GraphPad Prism software.

[0255] Chimeric mouse / human HER2, which contains the extracellular domain of human HER2 and the intracellular domain of mouse HER2, was expressed on MC38 colon cells to allow the activity of trastuzumab against MC38 mouse tumors to be evaluated. As shown in Figure 1, monotherapy with trastuzumab had no effect on tumor growth, while drug A monotherapy and anti-PD-L1 antibody monotherapy each had a moderate effect on tumor growth. Treatment with the dual combination of drug A + anti-PD-L1 antibody or trastuzumab + anti-PD-L1 antibody showed improved tumor growth inhibition compared to trastuzumab monotherapy, drug A monotherapy, and anti-PD-L1 monotherapy. Treatment with the triple combination of drug A + anti-PD-L1 + trastuzumab showed improved tumor inhibition when compared to each dual treatment. Compared with the dual combination of drug A + anti-PD-L1 antibody or trastuzumab + anti-PD-L1 antibody, the effect of the triple combination on tumor growth inhibition was most evident on days 19 and 22 (3 to 6 days after the last dose). Days 19 and 22 are shown as “ *By day 26, the triple combination was minimally better at reducing tumor growth compared to the doublet of drug A plus anti-PD-L1 antibody or the doublet of trastuzumab plus anti-PD-L1 antibody. No adverse effects were observed in any of the treatment cohorts in the MC38 m / h HER2 colon tumor model.

[0256] Example 2A. Exemplary Clinical Trial to Evaluate Antitumor Activity of Drug A Combination Therapy in Human Patients Drug A, trastuzumab, ramucirumab, and paclitaxel in gastric or gastroesophageal junction (GEJ) adenocarcinoma HER2 disease that has progressed during or after prior treatment with trastuzumab and fluoropyrimidine-containing chemotherapy (e.g., fluorouracil); during or after prior treatment with trastuzumab and platinum-containing chemotherapy; or during or after prior treatment with trastuzumab, fluoropyrimidine-containing chemotherapy (e.g., fluorouracil), and platinum-containing chemotherapy. + A clinical trial was conducted to evaluate the safety, tolerability, and efficacy of the combination of Drug A, trastuzumab, ramucirumab, and paclitaxel in patients with overexpressing advanced or metastatic gastric or GEJ adenocarcinoma. Patients enrolled in the study were eligible for treatment with trastuzumab. Patients had not received prior treatment with anti-CD47 or anti-SIRPα agents.

[0257] Example 2B. Preliminary Safety Results from an Exemplary Clinical Trial One patient with previously untreated advanced head and neck squamous cell carcinoma (HNSCC) received drug A (10 mg / kg IV QW), pembrolizumab (200 mg IV Q3W), and 5-fluorouracil (1,000 mg / m 2 / day, days 1, 2, 3, and 4, Q3W × 6), and carboplatin (AUC = 5 mg / ml / min, day 1, Q3W × 6). (In the expansion study, cisplatin (100 mg / m 2, Q3W×6) or carboplatin (AUC=5mg / ml / min, day 1, Q3W×6) will be administered in combination with drug A, pembrolizumab, and fluorouracil. Patients who received carboplatin continued to receive carboplatin for the duration of the expansion study. Patients who received cisplatin continued to receive cisplatin for the duration of the expansion study.

[0258] Three patients with HER2-positive gastric / gastroesophageal cancer that had progressed on prior treatment(s) with trastuzumab, fluorouracil, and a platinum agent were treated with drug A (10 mg / kg IV QW), trastuzumab (initial dose 8 mg / kg IV, then 6 mg / kg Q3W), ramucirumab (8 mg / kg on days 1 and 15, Q4W), and paclitaxel (80 mg / m2 on days 1, 8, and 15, Q4W).

[0259] Three additional patients with HER2-positive gastric / gastroesophageal cancer who had progressed on prior treatment(s) with trastuzumab, fluorouracil, and a platinum agent were treated with drug A (15 mg / kg IV QW), trastuzumab (initial dose 8 mg / kg IV, then 6 mg / kg Q3W), ramucirumab (8 mg / kg on days 1 and 15, Q4W), and paclitaxel (80 mg / m2 on days 1, 8, and 15, Q4W).

[0260] Initial results suggest that drug A is well tolerated with no dose-limiting toxicities to date when administered at doses of 10 mg / kg or 15 mg / kg QW in the above combination regimens. Three patients (50%) receiving drug A + trastuzumab + ramucirumab + paclitaxel experienced treatment-related adverse events (TRAEs), and none (0%) receiving drug A + pembrolizumab + fluorouracil + carboplatin. There were no dose-limiting toxicities in patients receiving drug A + pembrolizumab + fluorouracil + carboplatin or drug A + trastuzumab + ramucirumab + paclitaxel. Additionally, there were no treatment-related adverse events (TRAEs) occurring in more than two patients in the following three cohorts: Drug A (10 mg / kg QW) + pembrolizumab + fluorouracil + carboplatin (N=1) Drug A (10 mg / kg QW) + trastuzumab + ramucirumab + paclitaxel (N=3) Drug A (15 mg / kg QW) + trastuzumab + ramucirumab + paclitaxel (N=3)

[0261] Finally, there were no treatment-related adverse events of grade 3 or higher (TRAEs ≥ grade 3) reported in patients treated with drug A + pembrolizumab + fluorouracil + carboplatin or drug A + trastuzumab + ramucirumab + paclitaxel.

[0262] Example 2C: Preliminary Efficacy Results from the Exemplary Clinical Trial Described in Example 2A Patients with previously untreated advanced head and neck squamous cell carcinoma (HNSCC) treated with Drug A, pembrolizumab, 5-fluorouracil, and a platinum agent at the dosages and administration schedule described in Example 2B achieved partial responses (PR) based on investigator-assessed response using RECIST v1.1 criteria.

[0263] Of three patients with HER2-positive gastric / gastroesophageal cancer treated with Drug A (10 mg / kg QW), trastuzumab, ramucirumab, and paclitaxel (see Example 2B), two remained evaluable. One patient achieved a PR based on investigator-assessed response using RECIST v1.1 criteria.

[0264] Of the three patients with HER2-positive gastric / gastroesophageal cancer treated with Drug A (15 mg / kg QW), trastuzumab, ramucirumab, and paclitaxel (see Example 2B), two remained evaluable. One patient achieved PR based on investigator-assessed response using RECIST v1.1 criteria. Low rates of cytopenias were observed.

[0265] Drug A in combination with pembrolizumab, 5-fluorouracil, and a platinum agent has demonstrated clinical activity in the treatment of advanced 1L HNSCC (i.e., as initial treatment for patients with advanced HNSCC who have not received prior HNSCC therapy). Drug A in combination with trastuzumab, ramucirumab, and paclitaxel has demonstrated clinical activity in the treatment of advanced >2L gastric / gastroesophageal cancer (i.e., as treatment for patients who have received at least one prior therapy for gastric or GEJ cancer).

[0266] Pharmacodynamic analysis results showed that when combined with chemotherapy-containing regimens, nearly complete CD47 target occupancy (also known as receptor occupancy) was maintained throughout the entire Drug A dosing interval.

[0267] Example 2D: Additional Results from the Exemplary Clinical Trial Described in Example 2A CD47 is a myeloid checkpoint that is upregulated by tumors to evade anti-cancer immune responses. Drug A is an exemplary high-affinity CD47-blocking fusion protein with an inactive Fc region designed to safely potentiate anti-cancer therapeutics (Kauder et al. (2018) PLoS ONE. 13(8): e0201832; Chow et al. (2020) Journal of Clinical Oncology. 38: 15_suppl, 3056-3056; and Lakhani et al. (2021) Lancet Oncology. 22(12): 1740-1751). Drug A in combination with standard chemotherapy and antibody regimens was evaluated in patients with advanced HER2-positive gastric cancer (GC) or head and neck squamous cell carcinoma (HNSCC).

[0268] method Patients with previously treated advanced HER2-positive GC received drug A (A) 10 mg / kg QW or 15 mg / kg QW in combination with trastuzumab (T) + ramucirumab (ram) + paclitaxel (pac) as second-line or subsequent treatment. GC patients had progressed during or after prior fluoropyrimidine treatment (or fluoropyrimidine-containing therapy). GC patients who had progressed during or after prior treatment with trastuzumab and / or platinum-based chemotherapy agents were included. Patients with previously untreated advanced HNSCC received drug A (A) 10 mg / kg QW or 15 mg / kg QW in combination with pembrolizumab (P) + 5FU + platinum (cisplatin or carboplatin) as first-line treatment. The primary endpoint was dose-limiting toxicity (DLT). Tumor response, pharmacokinetic (PK), and pharmacodynamic (PD) markers were evaluated in all patients.

[0269] result Fifty-five patients were enrolled in the study. Their baseline characteristics are shown in Table B. [Table 14]

[0270] One patient with GC ≥2L received A+T+ram+pac and safety was evaluated. No dose-limiting toxicities (DLTs) were reported, and the maximum dose of drug A was 15 mg / kg QW. Of the nine patients who experienced any adverse events, eight patients reported treatment-related adverse events (TRAEs). The most common TRAEs were mild diarrhea, fatigue, pruritus / urticaria, and rash (n=21% each). TRAEs of grade 3 or greater severity were infrequent. There were no treatment-related SAEs reported in GC patients treated with A+T+ram+pac. Of the 11 GC patients who received drug A + trastuzumab + ramucirumab + paclitaxel at 15 mg / kg qw, seven had partial responses, three had stable disease, and one had disease progression. Of the three patients who received drug A 10 mg / kg qw + trastuzumab + ramucirumab + paclitaxel, two had partial responses and one had stable disease.

[0271] As mentioned above, three patients with previously untreated HNSCC received A+P+5FU+platinum. No DLTs were reported. Three patients experienced any adverse events (AEs), none of which were treatment-related. The HNSCC patient who received drug A + pembrolizumab + 5-fluorouracil + platinum-based chemotherapy at 15 mg / kg qw was CPI-naïve and had a partial response. The three patients who received drug A + pembrolizumab + 5-fluorouracil + platinum-based chemotherapy at 10 mg / kg qw were all CPI-naïve. One patient had a complete response, one had a partial response, and one had disease progression.

[0272] The clinical activity of the drug A chemotherapy combinations in response-evaluable patients is summarized in Table C below. [Table 15]

[0273] Initial PK and CD47 target occupancy of the drug A combination are similar to those of the single agents. When combined with chemotherapy-containing regimens, near-complete (80%-100%) CD47 target occupancy is maintained throughout the drug A dosing interval. Circulating immune cell profiles (CD4 + T cells, CD8 + T cells, CD19 + B cells and CD16 + CD56 + NK cells) are generally unchanged after drug A in combination with chemotherapy-containing regimens. The PK of drug A after combination therapy with pembrolizumab or trastuzumab is similar with or without chemotherapy.

[0274] conclusion Preliminary data indicate that drug A is well tolerated and can be safely combined with the tested anticancer antibody plus multiagent chemotherapy regimens without reaching the maximum tolerated dose. The maximum combined dose of drug A was 15 mg / kg QW.

[0275] Drug A, in combination with trastuzumab and ramucirumab plus paclitaxel, demonstrated an initial ORR of 64% in patients with ≥2L HER2-positive GC, which is comparable to the clinical experience with ramucirumab plus paclitaxel in patients whose disease had progressed on a prior trastuzumab-containing regimen.

[0276] Drug A shows early anticancer activity, including objective complete and partial responses, in combination with pembrolizumab + 5FU + platinum in previously untreated patients with advanced HNSCC.

[0277] Preliminary pharmacokinetic and pharmacodynamic analyses indicate no effect of combination partners on the level of Drug A exposure at which CD47 receptors are fully occupied.

[0278] Example 2E: A Phase 1 Study of Drug A, an Agent That Blocks the Interaction Between CD47 and SIRPα, in Combination with Trastuzumab, Ramucirumab, and Paclitaxel in Patients with Secondary HER2-Positive Advanced Gastric or Gastroesophageal (GC) Cancer The data provided herein further support the use of Drug A as a single agent and in combination with established anti-cancer agents, namely, the anti-HER2 antibody trastuzumab, the anti-VEGFR2 antibody ramucirumab, and paclitaxel. Drug A was administered at doses of 10 mg / kg (mpk) QW or 15 mg / kg (mpk) QW in combination with trastuzumab, ramucirumab, and paclitaxel to patients with HER2-overexpressing advanced or metastatic gastric cancer (GC) or gastroesophageal junction (GEJ) adenocarcinoma that had progressed during or after treatment with a prior HER2-targeted agent and a fluoropyrimidine-containing therapy, or a prior platinum-containing chemotherapy. Patients had received one or two prior therapies for GC or GEJ adenocarcinoma. The maximum dose of combined Drug A was 15 mg / kg QW. No dose-limiting toxicities, on-study deaths, or drug A-related serious adverse events (SAEs) occurred in patients receiving the drug A + trastuzumab + ramucirumab + paclitaxel combination.

[0279] The results of a Phase 1 clinical trial of combination therapy of Drug A with (a) an anti-HER2 antibody (trastuzumab), (b) an anti-VEGFR antibody (ramucirumab), and (c) paclitaxel are briefly shown in Figures 2A and 2B. Figure 2A shows a graph of the best percent change in measurable lesions from baseline in each study participant. Three of 18 patients achieved stable disease (SD), 12 of 18 patients achieved a partial response (PR), and one patient achieved a complete response (CR). Figure 2B shows a graph of the percent change in measurable lesions from baseline as a function of time in each study participant.

[0280] Table D below shows further analysis of the clinical activity of the drug A combination in response-evaluable patients with ≥2L HER2-positive GC or GEJ adenocarcinoma. The table shows that the overall response rate (ORR) for patients treated with drug A + trastuzumab + ramucirumab + paclitaxel was approximately 72%. [Table 16]

[0281] Preliminary data suggest that Drug A can be safely combined with trastuzumab, ramucirumab, and paclitaxel without reaching the maximum tolerated dose.

[0282] Preliminary PK / PD analysis indicates no effect of combination partner on the drug A exposure level at which CD47 receptors are fully occupied.

[0283] Agent A in combination with trastuzumab, ramucirumab, and paclitaxel has demonstrated an initial ORR of 72.2% and an estimated overall survival (OS) of 75.8% at 12 months in patients with ≥2L HER2-positive GC or GEJ adenocarcinoma that had progressed after a prior trastuzumab-containing regimen, which is comparable to historical controls in both RAINBOW (see Wilke et al., Lancet October 2014) and DESTINY-01 (see Enhertu prescribing information and Shitara et al., NEJM June 18, 2020).

[0284] Recent data from patients who received drug A plus trastuzumab after tumor progression on prior trastuzumab treatment suggest clinical activity exceeds that expected from either trastuzumab or chemotherapy alone.

[0285] Example 3: Phase 2 / 3 study of drug A in patients with advanced HER2-overexpressing gastric / gastroesophageal junction adenocarcinoma The data described herein support a follow-up study to validate the efficacy of the combination therapy described herein. This is a randomized, Phase 2 (open-label) / Phase 3 (double-blind), international, multicenter study in patients with metastatic HER2-overexpressing gastric / GEJ adenocarcinoma who have progressed on or after prior HER2-targeted therapy and fluoropyrimidine- or platinum-containing chemotherapy and are eligible for chemotherapy (2nd or 3rd line). Approximately 450 adult patients are expected to be enrolled in the study across both phases.

[0286] Symptoms: Gastric cancer; Gastroesophageal junction adenocarcinoma; Gastric adenocarcinoma

[0287] Experimental Arms: Phase 2 - Arm A: Drug A-30 mg / kg Q2W IV, trastuzumab (initial dose 6 mg / kg then 4 mg / kg) Q2W IV, ramucirumab 8 mg / kg Q2W IV, and paclitaxel 80 mg / m2 IV on days 1, 8, and 15 of a 28 day cycle. Active Comparator Arms: Phase 2 - Arm B: Trastuzumab (initial dose 6 mg / kg then 4 mg / kg) Q2W IV, ramucirumab 8 mg / kg Q2W IV, and paclitaxel 80 mg / m2 IV on days 1, 8, and 15 of a 28 day cycle.

[0288] Experimental Arms: Phase 3 - Arm A: Drug A- 30 mg / kg Q2W IV, trastuzumab (initial dose 6 mg / kg, then 4 mg / kg) Q2W IV, ramucirumab 8 mg / kg Q2W IV, and paclitaxel 80 mg / m2 IV on days 1, 8, and 15 of a 28 day cycle. Active Comparator Arms: Phase 3 - Arm B: Ramucirumab 8 mg / kg Q2W IV, and paclitaxel 80 mg / m2 IV on days 1, 8, and 15 of a 28 day cycle.

[0289] Outcome Measures: Primary outcome measures include objective response rate by RECIST 1.1 for phase 2 and overall survival for phase 3.

[0290] The following are the eligibility criteria for the study: Minimum eligible age: 18 years; Maximum age: None; Gender: All; Allowance of healthy volunteers: Not allowed.

[0291] Selection criteria: HER2-overexpressing advanced or metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma that has progressed on or after previous HER2-targeted agents and fluoropyrimidine- or platinum-containing chemotherapy (second or third line); adequate bone marrow function; adequate renal and liver function; adequate performance status.

[0292] Exclusion criteria: patients with known symptomatic CNS metastases or leptomeningeal disease requiring steroids, prior treatment with anti-CD47 or anti-SIRPα agents, prior treatment with ramucirumab.

[0293] Example 4: HER2 + Efficacy of Drug B in Combination with Anti-Mouse VEGFR-2, Paclitaxel, and Trastuzumab in a Syngeneic Mouse Model of Colorectal Cancer overview Drug B, a murine surrogate for Drug A, contains a SIRPα variant that exhibits high affinity for mouse CD47 ("mCD47") and an inactive Fc domain (i.e., an Fc domain that does not exhibit effector function). Drug B was evaluated in a four-drug combination treatment with trastuzumab (i.e., an anti-human HER2 antibody), anti-mouse VEGFR-2 (i.e., a murine ramucirumab surrogate), and paclitaxel in chimeric mouse / human HER2 CT26 (CT26 m:h HER2), a murine colorectal cancer model expressing mouse and human HER2. Triple treatment with trastuzumab, paclitaxel, and anti-mouse VEGFR-2 resulted in modest tumor growth inhibition. However, the addition of Drug B to the three-drug regimen significantly enhanced tumor growth inhibition and prolonged survival.

[0294] The therapeutic effect of drug B in the absence of trastuzumab was also evaluated. Combination of drug B with paclitaxel and anti-mouse VEGFR-2 dual therapy in the CT26 mouse colorectal cancer model showed no enhanced antitumor activity compared to paclitaxel and anti-mouse VEGFR-2 therapy.

[0295] These data indicate that inhibition of the CD47-SIRPα axis with drug B enhances the antitumor efficacy of trastuzumab, anti-mouse VEGFR-2, and paclitaxel treatment in a HER2-expressing tumor model. In the absence of trastuzumab, drug B does not enhance the antitumor activity of the paclitaxel and anti-mouse VEGFR-2 dual therapy.

[0296] Drug A has been shown to bridge innate and adaptive immunity, relieving immunosuppression and activating adaptive immune responses in an antigen-specific manner. Treatment with Drug A, in combination with anti-PD-1 and anti-PD-L1 antibodies, resulted in increased anti-tumor responses, significant tumor growth inhibition, and prolonged survival in both checkpoint inhibitor-sensitive (i.e., using the murine CT26 colon cancer cell line and the murine MC38 colon adenocarcinoma cell line) and checkpoint inhibitor-resistant (i.e., using the murine 4T1 breast cancer cell line) syngeneic mouse cancer models. See Kauder et al. (2018) PLoS One. 2018;13(8):e0201832.

[0297] In HER2-positive gastric / gastroesophageal junction (G / GEJ) cancer, specific targeting of the HER2 antigen with trastuzumab and concomitant engagement of FcγRs on macrophages with the active Fc IgG1 domain provide the necessary pro-phagocytic signal. Combining this selective pro-phagocytic signal with blockade of anti-phagocytic myeloid checkpoint signals with drug A maximizes G / GEJ cancer-specific ADCP activity in macrophages. The drug A combination, by providing blockade of the anti-phagocytic CD47-SIRPα interaction in concert with the tumor targeting and macrophage FcγR association provided by trastuzumab, can also exert antitumor activity in clinical settings where trastuzumab activity has been shown to be negligible, such as the treatment of HER2-positive gastric cancer with trastuzumab and paclitaxel in the second-line treatment of disease that has progressed on trastuzumab (T-ACT clinical trial; Makiyama et al. (2020) Clin Oncol. 8(17):1919-1927).

[0298] Purpose of the test The experiments in this example were performed to characterize the in vivo antitumor activity of Drug B, a murine surrogate of Drug A, in combination with paclitaxel and anti-mouse VEGFR-2 in the presence or absence of trastuzumab in a syngeneic mouse colorectal cancer model.

[0299] Materials and Methods The reagents used in this example include those listed in Table E below. [Table 17]

[0300] Animals for in vivo studies BALB / c mice were purchased from Charles River Laboratories International (Hollister, CA). All animals were housed in a pathogen-free facility in accordance with IACUC guidelines. Animals used in all studies were 6–8 weeks of age.

[0301] cell line CT26 m:h HER2 was generated by transduction of CT26 (ATCC CRL-2638) with a lentivirus expressing murine HER2 engrafted with a trastuzumab epitope on its extracellular domain.

[0302] CT26 and CT26 m:h HER2 were cultured in complete RPMI-1640 medium consisting of RPMI-1640 (Thermo Fisher Scientific 11875119) medium supplemented with 10% fetal bovine serum (FBS) (Millipore TMS-013B), 1% penicillin-streptomycin (Thermo Fisher Scientific 15140163), and 1% GlutaMAX (Thermo Fisher Scientific 35050061) at 37°C in a 5% CO2 incubator.

[0303] Subcutaneous tumor model in mice All tissue cultures were performed under sterile conditions. CT26 and CT26 m:h HER2 cells were harvested and washed twice with 50 mL of cold PBS (Thermo Fisher Scientific 10010072). After the final wash, 20 × 10 6Cells were resuspended in serum-free RPMI-1640 at 100 cells / mL of CT26 or CT26 m:h HER2. 100 mL of cell suspension was injected subcutaneously into the right flank of BALB / c mice.

[0304] In the CT26 tumor model, tumors averaged 47-85 mm 3 When the IL-16 receptor agonist (IL-16) level was reached, animals were randomized into groups of 8 mice and treatment was initiated. All treatments were administered intraperitoneally (IP). Paclitaxel was administered at 20 mg / kg every 5 days for a total of 3 doses until 1 day before the regimen was stopped. Drug B was administered at 30 mg / kg, 5 days apart for 5 doses. Anti-mouse VEGFR-2 was administered at 40 mg / kg every 2–3 days for 5 doses.

[0305] In the CT26 m:h HER2 tumor model, tumors averaged 45-73 mm 3 When the sigma-positive mice reached 100 mg / kg, animals were randomized into groups of 10 mice and treatment was initiated. All treatments were administered IP. Paclitaxel was administered at 20 mg / kg every 3 days for a total of 3 doses until 1 day before the regimen was stopped. Drug B was administered at 30 mg / kg, 5 days apart for 4 doses. Anti-mouse VEGFR-2 was administered at 40 mg / kg, 4 days apart for 5 doses. Trastuzumab was administered at 30 mg / kg, 5 days apart for 4 doses.

[0306] Tumor volume (mm 3 ) was assessed using a Mitutoyo Digital Caliper (Mitutoyo America, Aurora, Illinois). Tumor volume and body weight were recorded 1 to 3 times per week. Tumor volume was calculated as ([length × {width × width}] × 0.5 = volume (mm 3 )). P values ​​were calculated using the Student's t test for tumor volume, and survival rates were calculated using the log-rank (Mantel-Cox) test using Prism 9 software (GraphPad). 3 Mice that exceeded 10 min and lost 20% body weight were euthanized according to IACUC guidelines.

[0307] Results and Discussion Drug B does not enhance antitumor activity in combination with anti-mouse VEGFR-2 and paclitaxel compared to the anti-mouse VEGFR-2 + paclitaxel dual agent in the CT26 syngeneic tumor model In the CT26 colon cancer tumor model, animals treated with a two-drug regimen of 20 mg / kg paclitaxel and 40 mg / kg anti-mouse VEGFR-2 showed suboptimal tumor growth inhibition. As shown in Figure 3, a three-drug combination containing 20 mg / kg paclitaxel, 40 mg / kg anti-mouse VEGFR-2, and 30 mg / kg Drug B did not inhibit tumor growth compared to the two-drug regimen of paclitaxel and anti-mouse VEGFR-2 alone.

[0308] Addition of drug B to the triplet of trastuzumab, anti-mouse VEGFR-2, and paclitaxel enhances antitumor responses compared to the triplet in the CT26 m:h HER2-expressing colon cancer tumor model In the CT26 m:h HER2-expressing colon cancer tumor model, animals treated with a triple regimen of 30 mg / kg trastuzumab, 20 mg / kg paclitaxel, and 40 mg / kg anti-mouse VEGFR-2 had modest tumor growth inhibition. The addition of 30 mg / kg drug B to the triple of trastuzumab+paclitaxel+anti-mouse VEGFR-2 significantly enhanced tumor growth inhibition compared to the triple without drug B (day 26, unpaired t-test, p<0.0022). In the four-drug cohort, 3 out of 10 animals achieved complete tumor eradication at day 26. No tumor eradication was observed in the three-drug or PBS cohorts. See Figure 4A, showing tumor growth ± SEM for n=10 mice per cohort. In addition, the combination of drug B with a triple-drug regimen of trastuzumab, anti-mouse VEGFR-2, and paclitaxel significantly increased survival compared to the triple-drug or PBS cohorts alone (log-rank (Mantel-Cox) test, p<0.0001). See Figure 4B.

[0309] These results indicate that in the CT26 m:h HER2-expressing colon cancer tumor model, the combination of trastuzumab, paclitaxel, and anti-mouse VEGFR-2 with drug B significantly increased the antitumor response and enhanced tumor growth inhibition, resulting in complete tumor eradication and prolonged survival in a subset of treated animals.

[0310] conclusion The antitumor efficacy of drug B in combination with paclitaxel and anti-mouse VEGFR2 (i.e., without trastuzumab) was evaluated in the CT26 syngeneic mouse tum...

Claims

1. 1. A method of treating cancer in an individual, comprising administering to the individual an effective amount of (a) a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant, (b) an anti-HER2 antibody, (c) an anti-VEGF2 antibody, and (d) paclitaxel; The SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; The Fc domain variant comprises: (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, the numbering of which is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, the numbering of which is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, the numbering of which is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, the numbering of which is according to the EU index of Kabat; The method, wherein the cancer is gastric cancer (GC) or gastroesophageal junction (GEJ) cancer, and the individual has undergone at least one prior treatment for gastric or GEJ cancer.

2. 2. The method of claim 1, wherein the individual has undergone prior treatment with an anti-HER2 antibody, with an anti-HER2 antibody and a fluoropyrimidine, or with an anti-HER2 antibody and a platinum-based chemotherapy agent.

3. The method of claim 1 or 2, wherein the anti-HER2 antibody is trastuzumab.

4. 2. The method of claim 1, wherein the anti-VEGF antibody is ramucirumab.

5. The gastric cancer or the GEJ cancer is HER2 ＋ Gastric cancer or HER2 ＋ The method of claim 1, which is a GEJ cancer.

6. 2. The method of claim 1, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 10 mg / kg once weekly.

7. 2. The method of claim 1, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 15 mg / kg once weekly.

8. 2. The method of claim 1, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 30 mg / kg once weekly.

9. 2. The method of claim 1, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 10 mg / kg once every two weeks.

10. 2. The method of claim 1, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 15 mg / kg once every two weeks.

11. 2. The method of claim 1, wherein the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant is administered at a dose of 30 mg / kg once every two weeks.

12. 4. The method of claim 3, wherein the trastuzumab is administered at 6 mg / kg once every three weeks after an initial dose of 8 mg / kg.

13. 4. The method of claim 3, wherein the trastuzumab is administered at 4 mg / kg every two weeks after an initial dose of 6 mg / kg.

14. The paclitaxel is administered at 80 mg / m once weekly for 3 weeks of each 4-week cycle. ２ The method of claim 1 , wherein the dose is

15. The paclitaxel is administered at 80 mg / m on days 1, 8, and 15 of each 28-day cycle. ２ The method of claim 1 , wherein the dose is

16. 10. The method of claim 1, wherein the overall response rate (ORR) of the treated patient population is greater than 65%.

17. A kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharma- tically acceptable carrier for use in combination with an anti-HER2 antibody, an anti-VEGFR2 antibody, and paclitaxel, The SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; The Fc domain variant comprises: (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations, the numbering of which is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations, the numbering of which is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations, the numbering of which is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, the numbering of which is according to the EU index of Kabat; The kit comprises instructions for administering the polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in combination with the anti-HER2 antibody, the anti-VEGFR2 antibody, and the paclitaxel to an individual with gastric or gastroesophageal (GEJ) cancer who has received at least one prior treatment for the gastric or GEJ cancer.

18. The gastric cancer or the GEJ cancer is HER2 ＋ Gastric cancer or HER2 ＋ 18. The kit of claim 17, wherein the cancer is GEJ cancer.

19. The kit of claim 17 or 18, wherein the anti-HER2 antibody is trastuzumab.

20. The kit of claim 17, wherein the anti-VEGFR2 antibody is ramucirumab.

21. 18. The kit of claim 17, wherein the individual has undergone prior treatment with an anti-HER2 antibody, with an anti-HER2 antibody and a fluoropyrimidine, or with an anti-HER2 antibody and a platinum-based chemotherapy agent.

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