Combination therapy for treating urothelial carcinoma

Abstract

A method for treating cancer (e.g., urothelial carcinoma), the method comprising administering a polypeptide (e.g., a fusion polypeptide) comprising an SIRPα-D1 domain variant and an Fc domain variant in combination with an antibody-drug conjugate (e.g., enfortumab vedotin). Related kits are also provided.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 347,939, filed on June 1, 2022, the disclosure of which is hereby incorporated by reference in its entirety.

[0002] Reference to a Sequence Listing in Electronic Form The content of the electronic sequence listing (757972001900SEQLIST.xml; size: 365,943 bytes; and creation date: May 25, 2023) is hereby incorporated by reference in its entirety.

[0003] The present invention relates to a method for treating cancer, the method comprising administering to a subject in need thereof, an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) in combination with an antibody - drug conjugate (e.g., enfortumab vedotin).

Background Art

[0004] Bladder cancer is the sixth most common cancer in the United States (US). According to the National Cancer Institute's estimates, more than 83,000 new cases of urothelial carcinoma were diagnosed in 2021, and more than 17,000 people died from this disease in the US (SEER Cancer Stat Facts: Bladder Cancer, 2021. National Cancer Institute. Bethesda, MD, https: / / seer.cancer.gov / statfacts / html / urinb.html. Accessed 09 March 2022). Bladder cancer mainly develops in people aged 55 and over, and the median age at diagnosis is 73 years. The male-to-female ratio of people developing this cancer is approximately 4:1. Whites are more likely to be diagnosed with bladder cancer than African American or Hispanic Americans (SEER Cancer Stat Facts: Bladder Cancer, 2021. National Cancer Institute. Bethesda, MD, https: / / seer.cancer.gov / statfacts / html / urinb.html. Accessed 09 March 2022). Approximately 90% of bladder cancers are urothelial carcinomas, and the prognosis is poor if the cancer is advanced at the time of diagnosis (Simeone JC, Nordstrom BL, Patel K, Mann H, Klein AB, Horne L. Treatment patterns and overall survival in metastatic urothelial carcinoma in a real-world, US setting. Cancer Epidemiol. 2019;60:121-7). Most urothelial carcinomas are diagnosed at the muscle-invasive stage. At this stage, disease management involves resection, with or without intravesical therapy. Despite such treatment, patients often develop more advanced incurable disease and ultimately die.Approximately 12% of patients have locally advanced or metastatic disease at the time of diagnosis (SEER Cancer Stat Facts: Bladder Cancer, 2021. National Cancer Institute. Bethesda, MD, https: / / seer.cancer.gov / statfacts / html / urinb.html. Accessed 09 March 2022).

[0005] The first-line treatment for locally advanced or metastatic urothelial carcinoma in patients with adequate renal function consists of a combination of methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) or a cisplatin-based combination therapy such as gemcitabine + cisplatin, showing an overall response rate of up to 50% including a complete response (CR) of approximately 10% - 15% (Bellmunt J, Orsola A, Wiegel T, Guix M, De Santis M, Kataja V; ESMO Guidelines Working Group. Bladder cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2011 Sep;v22 Suppl 6:vi45-9.doi:10(.)1093 / annonc / mdr376(.)PMID:21908503). Carboplatin and gemcitabine are commonly used in patients in whom cisplatin is ineligible, but the outcomes are generally not good. Despite initial chemosensitivity, patients are not cured, the outcomes of metastatic urothelial carcinoma after these regimens are poor, the median time to progression is only 7 months, and the median overall survival (OS) is 14 months. Approximately 15% of patients survive at least 5 years, and the prognosis is particularly poor in patients with visceral metastases, with a 5-year overall survival rate of 7% (von der Maase H, Sengelov L, Roberts JT, Ricci S, Dogliotti L, Oliver T, et al. Long-term survival results of a randomized trial comparing gemcitabine plus cisplatin, with methotrexate, vinblastine, doxorubicin, plus cisplatin in patients with bladder cancer J Clin Oncol. 2005;23(21):4602-8).Despite recent advances in the treatment of urothelial carcinoma, there remains a significant unmet need in the art for improved treatments for patients with locally advanced or metastatic urothelial carcinoma that has recurred after treatment with platinum-based regimens and immunotherapy.

[0006] All references cited herein are incorporated by reference in their entirety as if each individual reference were specifically and individually indicated to be incorporated by reference, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers.

Summary of the Invention

[0007] In some embodiments, provided herein is a method of treating urothelial carcinoma in an individual, the method comprising administering to the individual: (a) an effective amount of a fusion polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant; and (b) an effective amount of enfortumab vedotin, wherein the SIRPα D1 domain variant of the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85, and the Fc domain variant of the fusion polypeptide is: (i) a human IgG1 Fc region comprising the mutations L234A, L235A, G237A, and N297A (numbering according to the Kabat EU index); (ii) a human IgG2 Fc region comprising the mutations A330S, P331S, and N297A (numbering according to the Kabat EU index); (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, and delG236 (numbering according to the Kabat EU index); or (iv) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A (numbering according to the Kabat EU index).

[0008] In some embodiments, the individual is a human. In some embodiments, the urothelial cancer is locally advanced urothelial cancer or metastatic urothelial cancer. In some embodiments, the urothelial cancer is bladder cancer, renal pelvic cancer, ureteral cancer, or urethral cancer. In some embodiments, the individual has received prior treatment with an immune checkpoint inhibitor (CPI). In some embodiments, the CPI was a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the CPI was atezolizumab, pembrolizumab, durvalumab, avelumab, or nivolumab. In some embodiments, the individual has received prior treatment with platinum-containing chemotherapy. In some embodiments, the individual developed progression or recurrence of urothelial cancer during or after receiving the most recent prior treatment. In some embodiments, the individual has not received prior treatment with a monomethyl auristatin (MMAE)-based antibody-drug conjugate. In some embodiments, the individual has not received prior treatment with enfortumab vedotin. In some embodiments, the individual has not received prior treatment with a therapeutic agent that blocks the interaction between CD47 and SIRPα.

[0009] In some embodiments, enfortumab vedotin is administered to the individual in one or more 28-day cycles, and enfortumab vedotin is administered intravenously to the individual at a dose of 1.25 mg / kg on days 1, 8, and 15 of each 28-day cycle. In some embodiments, enfortumab vedotin is administered intravenously. In some embodiments, the fusion polypeptide is administered to the individual at a dose of up to about 60 mg / kg. In some embodiments, the fusion polypeptide is administered to the individual once every two weeks (q2w) at a dose of about 30 mg / kg. In some embodiments, the fusion polypeptide is administered at a dose of about 20 mg / kg once every two weeks (q2w). In some embodiments, the fusion polypeptide is administered at a dose of about 15 mg / kg once every two weeks (q2w). In some embodiments, the fusion polypeptide is administered intravenously.

[0010] In some embodiments, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, the Fc domain variant is a human IgG1 Fc region comprising the mutations L234A, L235A, G237A, and N297A, where the numbering follows the Kabat EU index. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 136. In some embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the fusion polypeptide forms a homodimer.

[0011] In some embodiments, a kit comprising a polypeptide comprising a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier for use in combination with enfortumab vedotin in an individual in need thereof for treating urothelial cancer, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85, and the Fc domain variant is (i) a human IgG1 Fc region comprising the mutations L234A, L235A, G237A, and N297A (numbering follows the Kabat EU index); (ii) a human IgG2 Fc region comprising the mutations A330S, P331S, and N297A (numbering follows the Kabat EU index); (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, and delG236 (numbering follows the Kabat EU index); or (iv) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A (numbering follows the Kabat EU index), and the kit comprises instructions for administering the polypeptide comprising the SIRPα D1 domain variant and the Fc domain variant in combination with enfortumab vedotin to an individual.

[0012] It should be understood that one, several, or all of the characteristics of the various embodiments described in this specification can be combined to form other embodiments of the present invention. These aspects and other aspects of the present invention will become apparent to those skilled in the art. These embodiments and other embodiments of the present invention are further described by the following detailed description.

Brief Description of the Drawings

[0013]

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Modes for Carrying Out the Invention

[0014] The following description sets forth exemplary methods, parameters, and the like. However, such description is not intended to limit the scope of the present disclosure, but rather is to be recognized as being provided as an illustration of exemplary embodiments.

[0015] The headings provided herein are not intended to limit the various aspects or embodiments that can be obtained by referring to the entire specification. Accordingly, the terms immediately defined below are more fully defined by reference to the entire specification.

[0016] Definitions 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 and depends in part on the method of measuring or determining the value, i.e., the limitations of the measuring system. For example, "about" can mean within one or greater than one standard deviation in accordance with the convention 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, this term can mean within a factor of 10, preferably within a factor of 5, more preferably within a factor of 2 of the value. Unless otherwise stated, when a particular value is recited in this application and the claims, the term "about" is considered to mean within an acceptable error range for the particular value.

[0017] The terms used herein are for the purpose of describing particular instances only and are not intended to be limiting. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Further, as long as the terms "including", "includes", "having", "has", "with" or variations thereof are used in either the detailed description or the claims, such terms are intended to be inclusive in the same manner as the term "comprising".

[0018] The terms "treat", "treating", or "treatment" with respect to a particular disease state in a mammal refer to causing a desirable or beneficial effect in a mammal having that disease state. Desirable or beneficial effects can include a reduction in the frequency or severity of one or more symptoms of the disease (i.e., tumor growth and / or metastasis, or other effects mediated by the number and / or activity of immune cells, etc.), or the prevention or suppression of further development of the disease, condition, or disorder. In the context of cancer treatment in a mammal, desirable or beneficial effects can include inhibition of further growth or spread of cancer cells, death of cancer cells, inhibition of cancer recurrence, reduction of cancer-related pain, or improvement of the survival rate of the mammal. The effect can be either subjective or objective. For example, if the mammal is a human, the human can indicate an improvement in strength or vitality or a reduction in pain as a subjective symptom of the response to the improvement or treatment. Alternatively, a clinician can indicate a decrease in tumor size or tumor tissue mass based on a physical examination, clinical test parameters, tumor markers, or x-ray findings. In addition, a clinician can observe a decrease in detectable tumor markers. Alternatively, other tests such as computed tomography (CT), magnetic resonance imaging (MRI), etc. can be used to evaluate objective improvement.

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

[0020] As used herein, the term "pharmaceutical composition" refers to a medical or pharmaceutical preparation that contains an active ingredient and an excipient or diluent (or both an excipient and a diluent) and enables administration of the active ingredient by a suitable method of administration. In some embodiments, the pharmaceutical compositions disclosed herein contain pharmaceutically acceptable components compatible with the polypeptide. In some embodiments, the pharmaceutical composition is in the form of a tablet or capsule for oral administration or, for example, an aqueous form for intravenous or subcutaneous administration by injection.

[0021] As used herein, the terms "subject", "individual", and "patient" are used interchangeably to refer to a vertebrate, e.g., a mammal. Mammals include, but are not limited to, mice, monkeys, humans, livestock, sport animals, and pets. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro. None of the terms implies the accompaniment of a medical professional.

[0022] As used herein, the terms "affinity" or "binding affinity" refer to the strength of the binding interaction between two molecules. In general, binding affinity refers to the total strength of non-covalent interactions between a molecule and its binding partner, e.g., between a SIRPα D1 domain variant and CD47. Unless otherwise indicated, binding affinity refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair. The binding affinity between two molecules is generally described by the dissociation constant (K D ) or the association constant (K A ). Two molecules with low binding affinity for each other generally bind slowly and tend to dissociate easily, showing a large K D . Two molecules with high binding affinity for each other generally bind easily and tend to maintain the binding for a long time, showing a small K D . In some embodiments, the KD of two interacting molecules is determined using known methods and techniques, such as surface plasmon resonance (SPR). K D can be calculated as the ratio of k off / k on .

[0023] As used herein, the term "K D less than" means that the K D value is numerically smaller and the binding affinity increases for the listed K D value. As used herein, the term "K D greater than" means that the K D value is numerically larger and the binding affinity decreases for the listed KD value.

[0024] "Effective amount" refers to at least an effective amount at the dosage and time necessary to achieve one or more desired or indicated effects, including therapeutic or prophylactic results. An effective amount can be provided in one or more administrations. For purposes of the present disclosure, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve a prophylactic or therapeutic treatment. As understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition (e.g., the effective amount when administered as monotherapy or combination therapy). Thus, an "effective amount" can be considered in relation to the administration of one or more therapeutic agents, and a single agent can be considered to have been administered in an effective amount if the desired effect can be achieved, or is achieved, in combination with one or more other agents.

[0025] The methods and techniques of the present disclosure are generally carried out according to methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification, unless otherwise indicated. Such references include, for example, Sambrook and Russell, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001), Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990). Enzyme reactions and purification techniques can be performed according to the manufacturer's specifications, as commonly practiced in the art, or as described herein. The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as their experimental procedures and methods, are well known and commonly used in the art. Standard methods are used for chemical synthesis, preparation of pharmaceuticals, formulation, and delivery, as well as treatment of patients.

[0026] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)).

[0027] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0028] Summary Provided herein is a method of treating cancer (e.g., urothelial cancer) in an individual (e.g., a human individual), the method comprising administering to the individual (a) an agent in an amount effective to block the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα), and (b) an effective amount of an antibody-drug conjugate.

[0029] 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.). Exemplary small molecule inhibitors of the CD47-SIRPα pathway include, for example, Miller et al. (2019) “Quantitative high-throughput screening assays for the discovery and development of SIRPα-CD47 interaction inhibitors.” PLoS ONE 14(7):e0218897 and Sasikumar et al. ACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; October 26-30, 2017; Philadelphia, PA; Abstract B007, but are not limited thereto.

[0030] In some embodiments, the 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 has a K of about 10 nM or better D(For example, any one of at least about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM, or less than 10 pM) binds to CD47 (e.g., hCD47). In some embodiments, the agent that binds to CD47 (e.g., hCD47) exhibits at least about 50% CD47 receptor occupancy (e.g., any one of at least about 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 CD47 (e.g., hCD47) 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 agent that binds to CD47 (e.g., hCD47) is a polypeptide. In some embodiments, the agent that binds to 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 of the anti-CD47 antibody is Fab, Fab’, Fab’-SH, F(ab’)2, Fv, scFv, a one-armed antibody, or a 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” includes, but is not limited to, antibody-based constructs (such as multispecific constructs) including triomab, DART (i.e., dual affinity retargeting antibody), T and Ab (i.e., tandem diabody), tandem scFv, CrossMab, DNL (i.e., dock-and-lock antibody), DVD-Ig (i.e., dual variable domain immunoglobulin), tetravalent bispecific IgG, nanobody, dual-targeting domain, and ART-Ig (i.e., asymmetric engineering technology immunoglobulin).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 a full-length antibody, e.g., Hu5F9-G4, B6H12.2, BRIC126, CC-90002, SRF231, or IBI188 (Innovent Biologics) (for further information on 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).

[0031] In some embodiments, an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) binds to SIRPα (e.g., hSIRPα). In some embodiments, the agent has a K of about 10 nM or better D(e.g., any one of at least about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM, or less than 10 pM) binds to SIRPα (e.g., hSIRPα). In some embodiments, the agent that binds to SIRPα (e.g., hSIRPα) exhibits at least about 50% SIRPα receptor occupancy (e.g., any one of at least about 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 a polypeptide. 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 of the anti-SIRPα antibody is a Fab, Fab’, Fab’-SH, F(ab’)2, Fv, scFv, one-arm 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 a full-length antibody, e.g., KWAR23, SE12C3, 040, or MY-1 (for further information on 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 hereby incorporated by reference in their entirety.

[0032] In some embodiments, an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is an anti-SIRPβ antibody or an anti-SIRPγ antibody (e.g., an anti-SIRPβ antibody or an anti-SIRPγ antibody capable of binding to SIRPα), or an antigen-binding fragment thereof. In some embodiments, the agent is an antibody (or an antigen-binding fragment thereof) capable of binding to two or more of SIRPα, SIRPβ, and SIRPγ. In some embodiments, such an antibody (or an antigen-binding fragment thereof) has a K of about 10 nM or better D(e.g., any one of at least about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM, or less than 10 pM) binds to SIRPα (e.g., hSIRPα). In some embodiments, the antibody (or antigen-binding fragment thereof) exhibits at least about 50% SIRPα receptor occupancy (e.g., any one of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%) in a human subject. In some embodiments, the antibody (or antigen-binding fragment thereof) 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 antigen-binding fragment is a Fab, Fab’, Fab’-SH, F(ab’)2, Fv, scFv, one-arm antibody, or diabody. In some embodiments, the antibody is a monospecific antibody or monospecific antibody construct (including, but not limited to, those described above). In some embodiments, the antibody is a multispecific (e.g., bispecific) antibody or multispecific antibody construct (including, but not limited to, those described above).

[0033] In some embodiments, an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is a fusion polypeptide comprising a portion that binds to CD47. In some embodiments, the fusion polypeptide comprises 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 of about 10 nM or better D(For example, any one of at least about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM or less than 10 pM) binds to CD47 (for example, hCD47). In some embodiments, the fusion polypeptide exhibits at least about 50% CD47 receptor occupancy (for example, any one of at least 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, 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 wild-type human antibody Fc region. In some embodiments, the fusion polypeptide comprises an Fc variant (for example, a variant of the wild-type human antibody Fc region) that includes one or more amino acid insertions, deletions, and / or substitutions relative to the amino acid sequence of the wild-type human antibody Fc region. In some embodiments, the Fc variant exhibits a decrease in effector function (for example, loss, etc.) compared to the WT Fc region. Exemplary Fc variants are described in WO2017 / 027422 and US2017 / 0107270, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the portion of the fusion protein that binds to CD47 (for example, hCD47) is WT SIRPα (for example, hSIRPα), or WT SIRPγ (for example, hSIRPγ). In some embodiments, the portion that binds to CD47 (for example, hCD47) is the CD47-binding fragment (for example, the D1 domain) of WT SIRPα (for example, hSIRPα) or WT SIRPγ (for example, hSIRPγ). In some embodiments, the portion that binds to CD47 (for example, hCD47) is a SIRPα variant, a SIRPγ variant, a SIRPβ variant, or a CD47-binding fragment thereof (for example, the D1 domain).In some embodiments, the SIRPα variant, SIRPγ variant, SIRPβ variant, or any of their CD47-binding fragments (e.g., the D1 domain) described above each contain one or more amino acid insertions, deletions, or substitutions relative to the amino acid sequence of wild-type SIRPα, SIRPγ, SIRPβ, or any of their CD47-binding fragments described above. Exemplary SIRPγ variants and SIRPβ variants are described, for example, in WO2013 / 109752; US2015 / 0071905; USP9,944,911; WO2016 / 023040; WO2017 / 027422; US2017 / 0107270; USP10,259,859; US9845345; WO2016187226; US20180155405; WO2017177333; WO2014094122; US2015329616; US20180312563; WO2018176132; WO2018081898; WO2018081897; PCT / US2019 / 048921; US20180141986A1; and EP3287470A1, the contents of which are hereby incorporated by reference in their entirety. Exemplary SIRPα variants are described in further detail elsewhere herein.

[0034] In some embodiments, an 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 an SIRPα variant. In some embodiments, the SIRPα variant has a K of about 10 nM or better D(For example, any one of at least about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 750 pM, 500 pM, 250 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 10 pM, or less than 10 pM) binds to CD47 (e.g., hCD47). In some embodiments, the fusion polypeptide exhibits at least about 50% CD47 receptor occupancy (e.g., any one of at least 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 the WT human antibody Fc region) that exhibits a decrease in effector function (e.g., loss, etc.) compared to the WT Fc region, e.g., those described in the references cited herein. In some embodiments, the fusion polypeptide comprises a SIRPα variant described in WO2013 / 109752; US2015 / 0071905; WO2016 / 023040; WO2017 / 027422; US2017 / 0107270; USP10,259,859; US9845345; WO2016187226; US20180155405; WO2017177333; WO2014094122; US2015329616; US20180312563; WO2018176132; WO2018081898; WO2018081897; US20180141986A1; and EP3287470A1, the contents of which are hereby incorporated by reference in their entirety.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).

[0035] In some embodiments, an 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). Further details regarding such fusion polypeptides are provided below.

[0036] Exemplary fusion polypeptides comprising a signal regulatory protein alpha (SIRPα) D1 domain variant and an Fc variant Signal regulatory protein alpha (SIRPα) D1 domain variant In some embodiments, the fusion polypeptide comprises a signal regulatory protein alpha (SIRPα) D1 domain or a variant thereof. In some embodiments, the SIRPα D1 domain variant comprises one or more amino acid insertions, deletions, and / or substitutions relative to the amino acid sequence of the wild-type SIRPα D1 domain. In some embodiments, the amino acid mutation at position 80 relative to the wild-type signal regulatory protein alpha (SIRP-α) D1 domain (e.g., the wild-type SIRPα D1 domain described in SEQ ID NO: 1 or 2); and at least one additional amino acid mutation at an amino acid position 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., the wild-type SIRPα D1 domain described in SEQ ID NO: 1 or 2), a polypeptide (e.g., a fusion polypeptide) comprising a SIRPα D1 domain or a SIRP-α D1 variant comprising a CD47-binding fragment thereof.

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

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

[0039] Native SIRPα contains three highly homologous immunoglobulin (Ig)-like extracellular domains D1, D2, and D3. The SIRPα D1 domain (the "D1 domain") refers to the membrane-distal extracellular domain of SIRPα and mediates the binding of SIRPα to CD47. As used herein, the term "SIRPα polypeptide" refers to any SIRPα polypeptide or fragment thereof capable of binding to CD47. There are at least 10 variants of wild-type human SIRPα. Table 1 shows the amino acid sequences of the D1 domains of naturally occurring wild-type human SIRPα D1 domain variants (SEQ ID NOs: 1 and 2). In some embodiments, the SIRPα polypeptide comprises the SIRPα D1 domain. In some embodiments, the SIRPα polypeptide comprises a wild-type D1 domain such as those set forth 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 the CD47-binding portion of a SIRPα polypeptide that has a higher affinity for CD47 than wild-type SIRPα. A SIRPα D1 domain variant comprises at least one amino acid substitution, deletion, or insertion (or a combination thereof) relative to the amino acid sequence of wild-type SIRPα.

[0041] In some embodiments, the fusion polypeptide comprises a SIRPα D1 domain variant that comprises one or more amino acid substitutions, insertions, additions, or deletions relative to the wild-type D1 domains 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 fusion polypeptide comprises a fragment of a SIRPα D1 domain variant (e.g., a CD47-binding fragment). In some, the fragment of the SIRPα D1 domain variant (e.g., a CD47-binding fragment) comprises an amino acid sequence of less than 10 amino acids in length, about 10 amino acids in length, about 20 amino acids in length, about 30 amino acids in length, about 40 amino acids in length, about 50 amino acids in length, about 60 amino acids in length, about 70 amino acids in length, about 80 amino acids in length, about 90 amino acids in length, about 100 amino acids in length, or more than about 100 amino acids in length.

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

[0043] As used herein, the terms "optimized affinity" or "optimized binding affinity" refer to the optimized strength of the binding interaction between a fusion polypeptide (e.g., a fusion polypeptide comprising a SIRPα D1 domain variant) disclosed herein and CD47. For example, in some embodiments, the fusion polypeptide binds primarily or with high affinity to CD47 on cancer cells and binds substantially or with low affinity to CD47 on non-cancer cells. In some embodiments, the binding affinity between the fusion polypeptide and CD47 is optimized such that the interaction does not cause clinically relevant toxicity or has lower toxicity compared to variants that bind with maximal affinity. In some embodiments, to achieve an optimized binding affinity between the fusion polypeptide and CD47, the fusion polypeptide comprising a SIRPα D1 domain variant is developed to have a binding affinity for CD47 that is lower than that which can be maximally achieved. In some embodiments, the fusion polypeptide comprises a SIRPα D1 domain variant that cross-reacts with rodent CD47 (e.g., mouse CD47 or rat CD47), non-human primate (NHP) CD47 (e.g., cynomolgus monkey CD47), and human CD47.

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

[0045] As used herein, the term "minimal immunogenicity" refers to, for example, a polypeptide (e.g., a therapeutic polypeptide) modified by amino acid substitution having an immunogenicity that is lower (e.g., at least 10%, 25%, 50%, or 100% lower) than the immunogenicity before the amino acid substitution was introduced (e.g., an unmodified protein). In some embodiments, a fusion polypeptide (e.g., a polypeptide comprising a SIRPα D1 domain variant and an Fc variant) is modified to have minimal immunogenicity and does not elicit an immune response, or elicits little to no immune response, in a subject (e.g., a human subject), even when recognized as a foreign antigen by the immune system of the subject.

[0046] In some embodiments, a fusion polypeptide comprising a SIRPα D1 domain variant exhibits minimal immunogenicity. In some embodiments, the fusion polypeptide administered to a subject comprises a SIRPα D1 domain variant having the same amino acid sequence as the endogenous SIRPα of the subject, except for amino acid changes that increase the affinity of the SIRPα D1 domain variant. In some embodiments, the fusion polypeptide comprises a SIRPα D1 domain variant that reduces the risk of side effects as compared to an anti-CD47 antibody or wild-type SIRPα. In some embodiments, the fusion polypeptide comprises a SIRPα D1 domain variant that reduces the risk of anemia as compared to an anti-CD47 antibody or wild-type SIRPα. In some embodiments, the fusion polypeptide comprises a SIRPα D1 domain variant that does not cause acute anemia in rodent or non-human primate (NHP) studies.

[0047] Table 2 lists the specific amino acid substitutions for each D1 domain sequence in the SIRPα D1 domain variants. In some embodiments, the SIRPα D1 domain variant of the fusion polypeptide comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more) of the substitutions listed in Table 2. In some embodiments, the SIRPα D1 domain variant of the fusion polypeptide comprises up to 15 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant of the fusion polypeptide comprises up to 10 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant of the fusion polypeptide comprises up to 7 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the fusion polypeptide comprises an SIRPα D1 domain variant having at least 90% (e.g., at least 92%, 95%, 97% or more than 97%) amino acid sequence identity relative to the sequence of the wild-type D1 domain.

[0048] In some embodiments, the fusion polypeptide comprises a chimeric SIRPα D1 domain variant, e.g., a variant comprising portions of two or more wild-type D1 domains or variants thereof (e.g., a portion of a first wild-type D1 domain (or a variant thereof) from a first species or and a portion of a second wild-type D1 domain (or a variant thereof) from a second species). In some embodiments, the chimeric SIRPα D1 domain variant comprises portions (e.g., 2, 3, 4, 5, or more portions) derived from at least two wild-type D1 domains (or variants thereof), where each of the portions is derived from a different wild-type D1 domain (e.g., each wild-type D1 domain is from a different species). In some embodiments, the fusion polypeptide further comprises a chimeric SIRPα D1 domain variant that further comprises one or more of the amino acid substitutions listed in Table 2.

Table 2

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

[0050] In some embodiments, the fusion 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, X11 is K or R. In some embodiments, X 12 is V or I. In some embodiments, X 13 is F, L, V. In some embodiments, X 14 is F or V. In some embodiments, the fusion polypeptide comprises a SIRPα D1 domain variant (or a CD47-binding fragment thereof) that contains 6 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO: 1.

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

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

[0053] In some embodiments, the fusion polypeptide comprises the sequence of SEQ ID NO: 14, wherein X1 is L, I, or V. In some embodiments, X2 is V, L, or I. In some embodiments, X3 is A or V. In some embodiments, X4 is V, I, or L. In some embodiments, X5 is I, T, S, or F. In some embodiments, X6 is E, V, or L. In some embodiments, X7 is K or R. In some embodiments, X8 is E or Q. In some embodiments, X9 is H, P, or R. In some embodiments, X 10 is S, T, or G. In some embodiments, X 11 is K or R. In some embodiments, X12 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 fusion polypeptide comprises a SIRPα D1 domain variant (or a CD47-binding fragment thereof) that contains six or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO: 2.

[0054] In some embodiments, the fusion polypeptide binds to CD47 with a binding affinity that is at least 10-fold greater than that of the wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO: 2. In some embodiments, the fusion polypeptide binds to CD47 with a binding affinity that is at least 100-fold greater than that of the wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO: 2. In some embodiments, the fusion polypeptide binds to CD47 with a binding affinity that is at least 1000-fold greater than that of the wild-type SIRPα D1 domain comprising the sequence of SEQ ID NO: 2. In some embodiments, the fusion polypeptide binds to CD47 with a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M or less than 1×10 D M and comprises a SIRPα D1 domain variant (or a CD47-binding fragment thereof) that binds to CD47. In some embodiments, the fusion polypeptide has a K D for binding to CD47 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 and comprises a SIRPα D1 domain variant (or a CD47-binding fragment thereof) that binds to CD47.

[0055] In some embodiments, the fusion polypeptide is EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSLX11 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 It contains a SIRPα D1 domain variant containing the sequence of KPS (SEQ ID NO: 23), where X1 is E or G, X2 is L, I, or V, X3 is V, L, or I, X4 is S or F, X5 is L or S, X6 is S or T, X7 is A or V, X8 is I or T, X9 is H or R, X 10 is A, V, I, or L, X 11 is I, T, S, or F, X 12 is A or G, X 13 is E, V, or L, X 14 is K or R, X 15 is E or Q, X 16 is H, P, or R, X 17 is D or E, X 18 is S, L, T, or G, X 19 is K or R, X 20 is E or D, X 21 is S or P, X 22 is S or R, X 23 is S or G, X 24 is V or I, X 25 is F, L, V, X 26 is D or absent, X 27 is T or V, X 28 is F or V, X29 is A or G, and the variant contains 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, in this aspect of the present disclosure, X2 is L, I, or V. In any of the foregoing embodiments, X3 is V, L, or I. In an embodiment, 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, X 18 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 25is F, L, V. In some embodiments, X 26 is D or absent. In some embodiments, X 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 fusion polypeptide comprises a SIRPα D1 domain variant (or a CD47-binding fragment thereof) that contains six or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2.

[0057] In some embodiments, the fusion polypeptide binds to CD47 with a binding affinity that is at least 10-fold greater than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the fusion polypeptide binds to CD47 with a binding affinity that is at least 100-fold greater than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the fusion polypeptide binds to CD47 with a binding affinity that is at least 1000-fold greater than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the fusion polypeptide binds to CD47 with a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M or less than 1×10 D M of a SIRPα D1 domain variant (or a CD47-binding fragment thereof). In some embodiments, the fusion polypeptide has a K D for binding to CD47 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 and comprises a SIRPα D1 domain variant (or a CD47-binding fragment thereof).

[0058] In some embodiments, the fusion polypeptide comprises a SIRPα D2 domain comprising the sequence of SEQ ID NO: 24 or a SIRPα D3 domain having the sequence of SEQ ID NO: 25. In some embodiments, the fusion polypeptide comprises a SIRPα D2 domain comprising SEQ ID NO: 24 and a D3 domain comprising SEQ ID NO: 25 (see 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 connected to the D2 or D3 domain by a linker. In some embodiments, the SIRPα D1 domain variant is connected to the D2 and D3 domains by a linker. [Table 3]

[0059] In some embodiments, the fusion polypeptide comprises a SIRPα D1 domain variant conjugated (e.g., fused, e.g., genetically fused) to an Fc domain or Fc domain variant. In some embodiments, the fusion polypeptide comprises a SIRPα D1 domain variant conjugated (e.g., fused, e.g., genetically fused) to an Fc domain variant that cannot dimerize. In some embodiments, a fusion polypeptide comprising a SIRPα D1 domain variant and an Fc domain or Fc domain variant exhibits improved pharmacokinetic properties, e.g., an increase in serum half-life, compared to a fusion polypeptide that does not comprise an Fc domain or Fc domain variant. In some embodiments, the fusion polypeptide comprising a SIRPα D1 domain variant does not comprise any one of the sequences of SEQ ID NOs: 26-36 shown in Table 4. [Table 4]

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

[0061] In some embodiments, the fusion polypeptides described herein are bound to various carriers and used to detect the presence of specific antigen-expressing cells. Examples of carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and denatured cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier may be 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 conjugating a label to the polypeptides disclosed herein.

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

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

[0065] Also, in some embodiments, a polypeptide comprising an Fc domain variant is disclosed herein, wherein the Fc domain variant dimer comprises two Fc domain variants, and each Fc domain variant is independently selected from (i) a human IgG1 Fc region consisting of the mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of the mutations A330S, P331S and N297A; or (iii) a human IgG4 Fc region consisting of the mutations S228P, E233P, F234V, L235A, delG236, and N297A.

[0066] In some embodiments, the polypeptide in the compositions 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 of the sequence N80ITP. Expression of the SIRPα D1 domain in Chinese hamster ovary (CHO) cells results in a major band at 16 kDa (non-glycosylated) and minor high molecular weight bands that are removed by Endo Hf. Endo Hf is a recombinant protein fusion of endoglycosidase H and maltose binding protein. Endo Hf cleaves within the high mannose chitobiose core and within some hybrid oligosaccharides of N-linked glycoproteins. This suggests that the proline at amino acid position 83 reduces the efficiency of glycosylation, resulting in proteins with different degrees of glycosylation and thus heterogeneity. In drug development, heterogeneity can be a challenge in process development. Thus, in some embodiments, amino acid N80 of the SIRPα D1 variant is mutated to Ala to investigate the possibility of generating a homogeneous non-glycosylated form of the SIRPα D1 variant. In some embodiments, to generate a non-glycosylated SIRPα D1 domain variant, amino acid N80 of the SIRPα D1 domain variant is replaced with any amino acid that occurs naturally or non-naturally, including, for example, N80A and N80Q. In some embodiments, the SIRPα D1 domain variant comprises an N80A mutation and at least one additional mutation (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more additional mutations). In some embodiments, the additional mutation is in the CD47 binding site. In some embodiments, the additional mutation is in the hydrophobic core of the D1 domain.

[0067] In some embodiments, the polypeptide in the compositions disclosed herein comprises a SIRPα D1 domain variant with increased glycosylation compared to the wild-type SIRPα D1 domain. Another option for enhancing the homogeneity of the final product is to increase the efficiency of glycosylation at amino acid N80 and 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 of the SIRPα D1 domain variant is replaced with any amino acid, including naturally and non-naturally occurring amino acids, such as P83V, P83A, P83I, and P83L. In some embodiments, the polypeptides of the present disclosure are expressed in cells optimized to not glycosylate proteins expressed by such cells, for example, by genetic manipulation of cell lines (e.g., genetically engineered yeast or mammalian hosts) or by changing cell culture conditions such as the addition of kifunensine, or by using hosts that are naturally non-glycosylated such as prokaryotes (e.g., E. coli).

[0068] Table 5 lists the specific amino acid substitutions for each D1 domain variant sequence in the SIRPα D1 domain variants. In some embodiments, the SIRPα D1 domain variant comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more) of the substitutions listed in Table 5. In some embodiments, the SIRPα D1 domain variant is not glycosylated or has minimal glycosylation. In some embodiments, the SIRPα D1 domain variant is fully glycosylated or nearly fully glycosylated. In some embodiments, the SIRPα D1 domain variant comprises up to 14 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant comprises up to 10 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant comprises up to 7 amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPα D1 domain variant of the present disclosure has at least 90% (e.g., at least 92%, 95%, 97% or 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 comprising two or more wild-type D1 domains or portions of variants thereof (e.g., a portion of a wild-type D1 domain or variant thereof and a portion of another wild-type D1 domain or variant thereof). In some embodiments, the chimeric SIRPα D1 domain variant comprises at least two portions (e.g., 3, 4, 5 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 is EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSPDDVEX 16 comprises a SIRPα D1 domain variant having the sequence of KSGAGTELSVRAKPS (SEQ ID NO: 37), wherein X1 is L, I, or V, X2 is V, L, or I, X3 is A or V, X4 is A, I, or L, X5 is I, T, S, or F, X6 is E, V, or L, X7 is K or R, X8 is E or Q, X9 is H, P, or R, X 10 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, and the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1.

[0071] In some embodiments, in this aspect of the present disclosure, the polypeptide comprises a SIRPα D1 domain variant having the sequence of SEQ ID NO: 37, 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 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, V. In some embodiments, X 16 is F or V.

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

[0073] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold greater than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold greater than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold greater than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain mutant polypeptide or a fragment thereof binds to CD47 with a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M or a K D less than 1×10 D M. In some embodiments, the SIRPα D1 domain mutant polypeptide or a fragment thereof binds to CD47 with a K

[0074] 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. 10 TX 11 RENMDFSISISX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSPDTEX 16Comprising an SIRPα D1 domain variant having the sequence of KSGAGTELSVRAKPS (SEQ ID NO: 38), wherein X1 is L, I, or V; X2 is V, L, or I; X3 is A or V; X4 is V, I, or L; X5 is I, T, S, or F; X6 is E, V, or L; X7 is K or R; X8 is E or Q; X9 is H, P, or R; X 10 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; X 16 is F or V, and the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2.

[0075] In some embodiments, in this aspect of the present disclosure, the polypeptide comprises an 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 12is 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.

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

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

[0078] In another aspect, the present disclosure provides 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 A polypeptide comprising an SIRPα D1 domain variant having the sequence of KPS (SEQ ID NO: 47), wherein X1 is E or G, X2 is L, I, or V, X3 is V, L, or I, X4 is S or F, X5 is L or S, X6 is S or T, X7 is A or V, X8 is I or T, X9 is H, R, or L, X 10 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, X16 is H, P, or R, and X 17 is D or E, and X 18 is S, L, T, or G, and X 19 is K or R, and X 20 is E or N, and X 21 is S or P, and X 22 is S or R, and X 23 is S or G, and X 24 is any amino acid, and X 25 is any amino acid, and X 26 is V or I, and X 27 is F, L, V, and X 28 is D or absent, and X 29 is T or V, and X 30 is F or V, and X 31 is A or G, and the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2.

[0079] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 47, where X1 is E or G. In any of the foregoing embodiments, in this aspect of the present disclosure, X2 is L, I, or V. In any of the foregoing embodiments, X3 is V, L, or I. In any of the foregoing embodiments, X4 is S or F. In any of the foregoing embodiments, X5 is L or S. In any of the foregoing embodiments, X6 is S or T. In any of the foregoing embodiments, X7 is A or V. In any of the foregoing embodiments, X8 is I or T. In any of the foregoing embodiments, X9 is H or R. In any of the foregoing embodiments, X 10 is A, V, I, or L. In any of the foregoing embodiments, X 11 is I, T, S, or F. In any of the foregoing embodiments, X 12 is A or G. In any of the foregoing embodiments, X 13is E, V, or L. In any of the foregoing embodiments, X 14 is K or R. In any of the foregoing embodiments, X 15 is E or Q. In any of the foregoing embodiments, X 16 is H, P, or R. In any of the foregoing embodiments, X 17 is D or E. In any of the foregoing embodiments, X 18 is S, L, T, or G. In any of the foregoing embodiments, X 19 is K or R. In any of the foregoing embodiments, X 20 is E or N. In any of the foregoing embodiments, X 21 is S or P. In any of the foregoing embodiments, X 22 is S or R. In any of the foregoing embodiments, X 23 is S or G. In any of the foregoing embodiments, X 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 foregoing embodiments, X 25 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. In any of the foregoing embodiments, X 26 is V or I. In any of the foregoing embodiments, X 27 is F, L, V. In any of the foregoing embodiments, X 28 is D or absent. In any of the foregoing embodiments, X 29 is T or V. In any of the foregoing embodiments, X 30 is F or V. In any of the foregoing embodiments, X 31 is A or G.

[0080] In some embodiments, the polypeptide of this aspect of the present disclosure contains 10 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptide of this aspect of the present disclosure contains 7 or fewer amino acid substitutions relative to the 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 a binding affinity that is at least 10-fold greater than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold greater than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold greater than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the SIRPα D1 domain mutant polypeptide or a fragment thereof has a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M or less than 1×10 D M and binds to CD47. In some embodiments, the SIRPα D1 domain mutant polypeptide or a fragment thereof has a K D of about 500 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 10 nM, about 10 nM to 5 nM, about 5 nM to 1 nM, about 1 nM to 500 pM, about 500 pM to 100 pM, about 100 pM to 50 pM, or about 50 pM to 10 pM and binds to CD47.

[0082] In some embodiments, the polypeptide is EEELQX1IQPDKSVX2VAAGEX3AX4LX5CTX6TSLX7PVGPIQWFRGAGPX8RX9LIYNQX 10 X 11 GX 12 FPRVTTVSX13 X 14 TKRX 15 NMDFSIX 16 IX 17 X 18 ITPADAGTYYCX 19 KFRKGX 20 X 21 X 22 DX 23 The sequence of EFKSGAGTELSVRAKPS (SEQ ID NO: 48) (X1 is V or I, X2 is L or S, X3 is T or S, X4 is T or I, X5 is R or H, X6 is A, V, or I, X7 is I, R, Y, K or F, X8 is G or A, X9 is E or V, X 10 is K or R, X 11 is E, D or Q, X 12 is H or P, X 13 is D or E, X 14 is S, L or T, X 15 is N or E, X 16 is R or S, X 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 absent, X 22 is D or P, X 23 is V or T) or a fragment thereof, and includes an SIRPα D1 domain variant.

[0083] In another aspect, the present disclosure provides EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9ITPADAGTYYCX 10A polypeptide comprising a SIRPα D1 domain variant having the sequence of KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 49), wherein X1 is V, L, or I, X2 is A, I, V, or L, X3 is I, F, S, or T, X4 is E, V, or L, X5 is K or R, X6 is E or Q, X7 is H, P, or R, X8 is L, T, S, or G, X9 is A, X 10 is V or I, and the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1.

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

[0085] 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: 49, wherein each of X1, X2, X3, X4, X5, X6, X7, X8, X9, and X 10 is not a wild-type amino acid.

[0086] In some embodiments, the polypeptide of this aspect of the present disclosure contains 10 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1. In some embodiments, the polypeptide of this aspect of the present disclosure contains 7 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having any one of the sequences of SEQ ID NO: 1.

[0087] In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 10-fold greater than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 100-fold greater than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity that is at least 1000-fold greater than that of the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the SIRPα D1 domain mutant polypeptide or a fragment thereof has a K -8 less than 1×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 5×10 -10 M, less than 1×10 -11 M or a K D less than 1×10 D M for binding to CD47. In some embodiments, the SIRPα D1 domain mutant polypeptide or a fragment thereof has a K

[0088] In another aspect, the present disclosure provides EEELQX1IQPDKSVSVAAGESAILHCTX2TSLX3PVGPIQWFRGAGPARX4LIYNQX5X6GX7FPRVTTVSEX8TKRENMDFSISISX9ITPADAGTYYCX 10A polypeptide comprising an SIRPα D1 domain variant having the sequence of KFRKGSPDTEFKSGAGTELSVRAKPS (SEQ ID NO: 50), wherein X1 is V or I, X2 is V or I, X3 is I or F, X4 is E or V, X5 is K or R, X6 is E or Q, X7 is H or P, X8 is S or T, X9 is N or A, and X 10 is V or I, and 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, wherein X1 is V or I. In any of the foregoing embodiments, in this aspect of the present disclosure, X2 is V or I. In any of the foregoing embodiments, X3 is I or F. In any of the foregoing embodiments, X4 is 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 an SIRPα D1 domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 50, wherein each of X1, X2, X3, X4, X5, X6, X7, X8, X9, and X 10 is not a wild-type amino acid.

[0091] In some embodiments, the polypeptide of this aspect of the present disclosure contains 10 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide of this aspect of the present disclosure contains 7 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2.

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

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

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

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

[0097] 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 NO: 51, wherein each of X1, X2, X3, X4, X5, X6, X7, X8, and X9 is not a wild-type amino acid.

[0098] In some embodiments, the polypeptide of this aspect of the disclosure comprises 10 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide of this aspect of the disclosure comprises 7 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1.

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

[0100] In another aspect, the present disclosure features a polypeptide comprising a SIRPα D1 domain variant having the sequence of EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRELIYNQX4EGX5FPRVTTVSDX6TKRNNMDFSIRIGX7ITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 222), where X1 is V, L, or I, X2 is A, I, or L, X3 is I, T, S, or F, X4 is K or R, X5 is H or P, X6 is L, T, or G, X7 is N or A, and the variant comprises at least one amino acid substitution relative to the wild-type SIRPα D1 domain having the sequence according to 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, in this aspect of the present disclosure, X2 is A, I, or L. In any of the foregoing embodiments, X3 is I, T, S, or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In 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, in this aspect of the present disclosure, X2 is A or I. In any of the foregoing embodiments, X3 is I or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In 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, in this aspect of the present disclosure, X7 is A and X1 is V or I. In any of the foregoing embodiments, in this aspect of the present disclosure, X7 is A and X2 is A or I. In any of the foregoing embodiments, X7 is A and X3 is I or F. In any of the foregoing embodiments, X7 is A and X4 is K or R. In any of the foregoing embodiments, X7 is A and X5 is H or P. In any of the foregoing embodiments, X7 is A and X6 is L or T.

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

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

[0106] In some embodiments, the polypeptide of this aspect of the disclosure comprises 10 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide of this aspect of the disclosure comprises 7 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 1.

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

[0108] In another aspect, the present disclosure features a polypeptide comprising a SIRPα D1 domain variant having the sequence of EEELQX1IQPDKSVSVAAGESAILHCTX2TSLX3PVGPIQWFRGAGPARELIYNQX4EGX5FPRVTTVSEX6TKRENMDFSISISX7ITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS (SEQ ID NO: 212), where X1 is V, L, or I, X2 is V, I, or L, X3 is I, T, S, or F, X4 is K or R, X5 is H, P, or R, X6 is S, T, or G, X7 is N or A, and 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, in this aspect of the present disclosure, X2 is V, I, or L. In any of the foregoing embodiments, X3 is I, T, S, or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In 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, in this aspect of the present disclosure, X2 is V or I. In any of the foregoing embodiments, X3 is I or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In 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, in this aspect of the present disclosure, X7 is A and X1 is V or I. In any of the foregoing embodiments, in this aspect of the present disclosure, X7 is A and X2 is V or I. In any of the foregoing embodiments, X7 is A and X3 is I or F. In any of the foregoing embodiments, X7 is A and X4 is K or R. In any of the foregoing embodiments, X7 is A and X5 is H or P. In any of the foregoing embodiments, X7 is A and X6 is S or T.

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

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

[0114] In some embodiments, the polypeptide of this aspect of the disclosure comprises 10 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide of this aspect of the disclosure comprises 7 or fewer amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence of SEQ ID NO: 2.

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

[0116] In some embodiments, EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9X 10 X 11 X 12 ADAGTYYCX 13 Polypeptides comprising an SIRPα D1 domain variant having a sequence according to KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 218) are described herein, where X1 is V, L, or I, X2 is A, V, L, or I, X3 is I, S, T, or F, X4 is E, L, or V, X5 is K or R, X6 is E or Q, X7 is H, R, or P, X8 is S, G, L, or T, X9 is any amino acid, X 10 is any amino acid, X 11 is any amino acid, X 12 is any amino acid, X 13 is V or I, and the SIRPα D1 domain variant comprises at least two amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence according to SEQ ID NO: 1.

[0117] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, where X1 and X9 are A. In any of the foregoing embodiments, in this aspect of the present disclosure, X9 is N. In any of the foregoing embodiments, in this aspect of the present disclosure, X 10 is I. In any of the foregoing embodiments, in this aspect of the present disclosure, X9 is N and X10 is P. In any of the foregoing embodiments, in this aspect of the present disclosure, X9 is N and X11 is any amino acid other than S, T, or C. In any of the foregoing embodiments, in this aspect of the present disclosure, X 11 is T. In any of the foregoing embodiments, in this aspect of the present disclosure, X 11 is an amino acid other than T. In any of the foregoing embodiments, in this aspect of the present disclosure, X 12 is P. In any of the foregoing embodiments, in this aspect of the present disclosure, X9 is N and X 12 is any amino acid other than P.

[0118] In some embodiments, a polypeptide comprising a SIRPα D1 domain variant having a sequence according to EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9ITX 10 ADAGTYYCX 11 KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 219) is described herein, where 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, and X 10 is any amino acid other than P, X 11 is V or I, and the SIRPα D1 domain variant comprises at least two amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence according to SEQ ID NO: 1.

[0119] In another aspect of the present disclosure, a composition comprising an SIRPα D1 domain mutant polypeptide having the amino acid sequence of SEQ ID NO: 48 or a fragment thereof is disclosed herein. In some embodiments, the SIRPα D1 domain mutant polypeptide or a fragment thereof binds to CD47 with a higher affinity compared to the affinity of the SIRPα polypeptide for binding to CD47. In some embodiments, the SIRPα D1 domain mutant polypeptide binds to CD47 with a K -8 less than 1×10 -9 M, less than 1×10 -10 M, less than 1×10 -11 M or less than 1×10 D M. In some embodiments, the above-described SIRPα D1 domain mutant polypeptide is bound or fused to a second polypeptide. In some embodiments, the second polypeptide includes, but is not limited to, an Fc polypeptide, an Fc mutant, or a fragment as described above.

[0120] Without being limited to the foregoing, in some embodiments, the SIRPα D1 domain mutant polypeptide is selected from any one of SEQ ID NOs: 53 to 87 and 213 shown in Table 6.

Table 6-1

Table 6-2

Table 6-3

Table 6-4

[0121] In some embodiments, the polypeptide comprises a SIRPα D1 variant domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to any of the variants provided 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 variant The fusion polypeptides disclosed herein comprise a signal regulatory protein alpha (SIRP-α) D1 variant (or a CD47-binding fragment thereof) and an Fc domain variant. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising the mutations L234A, L235A, G237A, and N297A (amino acid numbering according to the EU index); (ii) a human IgG2 Fc region comprising the mutations A330S, P331S and N297A (amino acid numbering according to the EU index); or (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A (amino acid numbering according to the EU index), or comprises the same.

[0124] Antibodies that target cell surface antigens can induce immune activation and effector functions associated with Fc receptor (FcR) binding on immune cells. There are numerous Fc receptors that are specific for particular antibody classes, including IgG (gamma receptor), IgE (epsilon receptor), IgA (alpha receptor), and IgM (mu receptor). Binding of the Fc region to Fc receptors on the cell surface can trigger a number of biological responses, including phagocytosis of antibody-coated particles (antibody-dependent cell-mediated phagocytosis or ADCP), clearance of immune complexes, lysis of antibody-coated cells by killer cells (antibody-dependent cell-mediated cytotoxicity or ADCC), and release of inflammatory mediators, placental passage, and regulation of immunoglobulin production. Furthermore, binding of the C1 component of complement to an antibody can activate the complement system. Activation of the complement can be important for lysis of cellular pathogens. However, activation of the complement can also sometimes stimulate an inflammatory response and be involved in autoimmune hypersensitivity or other immune disorders. Mutant Fc regions with reduced or abolished ability to bind to certain Fc receptors are useful for the development of therapeutic antibodies and Fc fusion polypeptide constructs that act by targeting, activating, or neutralizing ligand function without damaging or destroying local cells or tissues.

[0125] In some embodiments, the fusion protein comprises a SIRPα D1 domain variant (or a CD47-binding fragment thereof) linked (e.g., fused, e.g., genetically fused) to an Fc domain variant that forms an Fc domain with diminished or abolished effector function.

[0126] In some embodiments, the Fc domain variant refers to a polypeptide chain comprising the second and third antibody constant domains (e.g., CH2 and CH3). In some embodiments, the Fc domain variant also comprises a hinge domain. In some embodiments, the Fc domain variant is of any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, and IgD. Further, in some embodiments, the Fc domain variant is of any IgG subtype (e.g., IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, and IgG4). In some embodiments, the Fc domain variant comprises 10 amino acid modifications (e.g., insertions, deletions and / or substitutions) (e.g., substitution, addition or insertion, deletion of 1 to 10, 1 to 8, 1 to 6, 1 to 4 amino acids, or combinations thereof) that alter the interaction between the Fc domain and the Fc receptor relative to the wild-type Fc domain monomer sequence.

[0127] As used herein, the term "Fc domain dimer" refers to a dimer of two Fc domains or two Fc domain variants. In a wild-type Fc domain dimer, the two wild-type Fc domains are dimerized by the interaction between the two CH3 antibody constant domains and one or more disulfide bonds formed between the two dimerization Fc domain hinge domains.

[0128] As used herein, the term "Fc domain dimer variant" comprises two Fc domain variants. In some embodiments, the Fc domain dimer variant comprises Fc domain variants that are mutated to lack effector function, e.g., a "dead Fc domain dimer variant". In some embodiments, each of the Fc domains in the Fc domain dimer variant comprises an amino acid substitution in the CH2 and / or CH3 antibody constant domains that reduces the interaction or binding between the Fc domain dimer variant and an Fc receptor such as Fcγ receptor (FcγR), Fcα receptor (FcαR), or Fcε (FcεR).

[0129] In some embodiments, the fusion polypeptide comprises a SIRPα D1 domain variant (e.g., any of the variants described in Tables 2, 5, and 6) fused (e.g., genetically fused) to an Fc domain variant of an immunoglobulin or a fragment of such an Fc domain variant. In some embodiments, the fusion polypeptide comprises an Fc domain variant (or a fragment thereof) of an immunoglobulin that can form an Fc domain dimer with another Fc domain variant. In some embodiments, the fusion polypeptide comprises an Fc domain variant (or a fragment thereof) of an immunoglobulin that cannot form an Fc domain dimer with another Fc domain variant. In some embodiments, a fusion polypeptide comprising an Fc domain variant (or a fragment thereof) exhibits an increase in the serum half-life of the polypeptide as compared to a polypeptide that does not comprise the Fc domain variant (or a fragment thereof). In some embodiments, the fusion polypeptide comprises an Fc domain variant (or a fragment thereof) that dimerizes with a second Fc domain variant to form an Fc domain dimer variant that binds to an Fc receptor. In some embodiments, the fusion polypeptide comprises an Fc domain variant (or a fragment thereof) that dimerizes with a second Fc domain variant to form an Fc domain dimer variant that does not bind to an Fc receptor. In some embodiments, the fusion polypeptide comprises an Fc domain variant (or a fragment thereof) that does not induce any immune system-related response after administration to a subject (e.g., a human subject).

[0130] In some embodiments, the fusion polypeptide comprises a SIRPα D1 domain or a variant thereof connected to a first Fc domain variant and an antibody variable domain connected to a second Fc domain variant, and the first and second Fc domain variants bind to form an Fc domain dimer variant (e.g., a heterodimeric Fc domain dimer variant). In some embodiments, the fusion polypeptide comprises a SIRPα D1 domain variant connected to (e.g., fused to, e.g., genetically fused to) a first Fc domain variant and a second SIRPα D1 domain variant connected to (e.g., fused to, e.g., genetically fused to) a second Fc domain variant, and the first and second Fc domain variants bind to form an Fc domain dimer variant (e.g., a heterodimeric Fc domain dimer variant). In some embodiments, the fusion polypeptide herein comprises a homodimer comprising a first SIRPα D1 domain variant connected to (e.g., fused to, e.g., genetically fused to) a first Fc domain.

[0131] The Fc domain dimer is a protein structure found at the C-terminus of immunoglobulins. The Fc domain dimer comprises two Fc domains dimerized by the interaction between the CH3 antibody constant domains. The wild-type Fc domain dimer forms the minimal structure that binds to Fc receptors, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and FcγRIV.

[0132] The Fc domain dimer is not directly involved in the binding of the antibody to its target, but can be involved in various effector functions such as the involvement of the antibody in antibody-dependent cytotoxicity. In some embodiments, the fusion polypeptide has a decrease in effector function, for example, a decrease in antibody-dependent cell-mediated cytotoxicity (ADCC), a decrease in complement-dependent cytotoxicity (CDC), a decrease in antibody-dependent cell-mediated phagocytosis (ADCP), or any combination thereof, relative to the amino acid sequence of the corresponding wild-type Fc domain, including amino acid substitutions, additions or insertions, deletions, or any combination thereof, and includes an Fc domain variant. In some embodiments, the fusion polypeptide is characterized by a decrease in binding to human Fc receptors (e.g., minimal binding or no binding) and a decrease in binding to complement protein C1q (e.g., minimal binding or no binding). In some embodiments, the fusion polypeptide is characterized by a decrease in binding to human FcγRI, FcγRIIA, FcγRIIB, FcγRIIIB, or any combination thereof, and C1q (e.g., minimal binding or no binding). To alter or reduce antibody-dependent effector functions such as ADCC, CDC, ADCP, or any combination thereof, the fusion polypeptide, in some embodiments, includes a human IgG Fc domain variant that includes 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 Kabat's EU index (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991))).

[0133] In some embodiments, the fusion polypeptide, when dimerized to form an Fc domain dimer variant, comprises a non-native Fc domain (e.g., an Fc domain variant) that exhibits a decrease or loss of binding to at least one of the Fcγ receptors CD16a, CD32a, CD32b, CD32c, and CD64 as compared to a fusion polypeptide comprising a native Fc domain dimer. In some cases, the fusion polypeptide exhibits a decrease or loss of binding to the CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors when dimerized (e.g., homodimerized or heterodimerized).

[0134] CDC refers to a form of cell injury in which complement cascade activation occurs by the binding of complement component C1q to the antibody Fc domain. In some embodiments, the fusion polypeptide, when dimerized to form an Fc domain dimer variant, comprises an Fc domain variant that exhibits a decrease in C1q binding of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more as compared to a polypeptide construct comprising a wild-type Fc region. In some embodiments, the fusion polypeptide, when dimerized to form an Fc domain dimer variant, comprises an Fc domain variant that exhibits a decrease in CDC as compared to a polypeptide construct comprising a wild-type Fc domain. In some embodiments, the fusion polypeptide, when dimerized to form an Fc domain dimer variant, comprises an Fc domain variant that exhibits a decrease in CDC of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more as compared to a fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the fusion polypeptide, when dimerized to form an Fc domain dimer variant, comprises an Fc domain variant that exhibits negligible CDC as compared to a polypeptide construct comprising a wild-type Fc region.

[0135] In some embodiments, the fusion polypeptide comprises an Fc domain variant that has minimal glycosylation or reduced glycosylation relative to the wild-type Fc domain. In some embodiments, deglycosylation is achieved by a mutation of N297A (amino acid numbering follows the EU index), or by mutating N297 to any amino acid other than N. In some embodiments, deglycosylation is achieved by breaking the motif N-Xaa1-Xaa2-Xaa3, where N = asparagine; Xaa1 = any amino acid except P (proline); Xaa2 = T (threonine), S (serine) or C (cysteine); and Xaa3 = any amino acid except P (proline). In one embodiment, the N-Xaa1-Xaa2-Xaa3 motif refers to residues 297-300 as designated according to Kabat et al., 1991. In some embodiments, a mutation to any one or more of N, Xaa1, Xaa2, or Xaa3 results in deglycosylation of the Fc domain variant.

[0136] In some embodiments, the fusion polypeptide comprises an IgG Fc domain variant that exhibits a reduced ability to specifically bind to an Fcγ receptor or a reduced ability to induce phagocytosis when dimerized. For example, in some embodiments, the fusion polypeptide comprises an Fc domain variant (e.g., an IgG Fc domain variant) lacking a function that is typical of a "dead" Fc domain variant (e.g., a "dead" IgG Fc domain variant) when dimerized. For example, in some embodiments, the Fc domain variant (e.g., an IgG Fc domain variant) comprises amino acid substitutions known to minimize the interaction between the Fc domain dimer and the Fcγ receptor. In some embodiments, the fusion polypeptide comprises an Fc domain variant (e.g., an IgG Fc domain variant) comprising 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, the Fc domain variant comprises one or more additional mutations. Non-limiting examples of such additional mutations to the human IgG1 Fc domain variant include E318A and K322A (amino acid numbering follows the EU index). In some embodiments, the fusion polypeptide comprises an Fc domain variant (e.g., an IgG Fc domain variant) that contains a total of up to 12, 11, 10, 9, 8, 7, 6, 5, or 4 or fewer mutations compared to the amino acid sequence of the wild-type human IgG1 domain. In some embodiments, the Fc domain variant further comprises one or more additional deletions. For example, in some embodiments, for example, when the polypeptide is produced in bacterial or mammalian cells, the C-terminal lysine of the Fc domain IgG1 heavy chain constant region provided in SEQ ID NO: 88 in Table 7 is deleted to enhance the homogeneity of the polypeptide. In some embodiments, the human IgG1 Fc domain variant contains a total of up to 12, 11, 10, 9, 8, 7, 6, 5, or 4 or fewer deletions compared to the wild-type human IgG1 sequence (e.g., see SEQ ID NO: 161 below).In some embodiments, the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NO: 135, SEQ ID NO: 136, or SEQ ID NO: 137. DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 161)

[0137] In some embodiments, the Fc domain variant is a variant of the human IgG2 or human IgG4 antibody Fc domain. In some embodiments, the IgG2 variant or IgG4 variant comprises the amino acid substitutions A330S, P331S, or both A330S and P331S (amino acid numbering follows the EU index). In some embodiments, the IgG2 Fc domain variant comprises a human IgG2 Fc domain comprising one or more of the amino acid substitutions A330S, P331S and N297A (designated according to the EU numbering system by Kabat, et al. (1991)). In some embodiments, the IgG2 Fc domain variant comprises one or more additional mutations. Non-limiting examples of such additional mutations include, for example, V234A, G237A, P238S, V309L and H268A (designated according to the EU numbering system by Kabat et al. (1991)), but are not limited thereto. In some cases, the human IgG2 Fc domain variant comprises a total of up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or fewer mutations compared to the wild-type human IgG2 sequence. In some embodiments, the C-terminal lysine of the wild-type human IgG2 Fc domain (e.g., SEQ ID NO: 89 in Table 7) is deleted to generate an IgG2 Fc domain variant. In some embodiments, the IgG2 Fc domain variant comprises 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 (e.g., see SEQ ID NO: 162 below). ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 162).

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

[0139] In some embodiments, the Fc domain variant is a variant of a human IgG1 Fc domain monomer that comprises at least one mutation (such as 2, 3, or all 4 mutations) selected from the group consisting of L234A, L235A, G237A, and N297A. In some embodiments, the Fc domain variant is a variant of a human IgG2 Fc domain monomer that comprises at least one mutation (such as 2 or all 3 mutations) selected from the group consisting of A330S, P331S, and N297A.

[0140] In some embodiments, the Fc domain variant exhibits a decrease in binding to Fc receptors as compared to the wild-type human IgG Fc domain. In some embodiments, the Fc domain variant exhibits a loss of binding to the Fc receptors of interest as compared to the wild-type human IgG Fc domain. In some embodiments, the Fc domain variant exhibits a decrease in the ability to mediate phagocytosis as compared to the wild-type human IgG Fc domain. In some embodiments, the Fc domain variant exhibits a loss of phagocytosis as compared to the wild-type human IgG Fc domain.

[0141] SEQ ID NO: 88 and SEQ ID NO: 89 are the amino acid sequences of the IgG1 and IgG2 Fc domains, respectively. In some embodiments, the Fc domain variant comprises (or is) any one of SEQ ID NOs: 90-95 shown in Table 7. **Table 7**

[0142] Antibody-dependent cell-mediated cytotoxicity, also referred to herein as ADCC, refers to a form of cell injury in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells and neutrophils), thereby enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill their target cells. Antibody-dependent cell-mediated phagocytosis, also referred to herein as ADCP, refers to a form of cell injury in which secreted Ig binds to Fc receptors (FcRs) present on certain phagocytic cells (e.g., macrophages), thereby enabling phagocytic effector cells to specifically bind to antigen-bearing target cells and subsequently take up and digest their target cells. Ligand-specific high-affinity IgG antibodies against the surface of target cells can stimulate cytotoxic cells or phagocytic cells and can be used for such killing. In some embodiments, the polypeptides (e.g., fusion polypeptides) provided herein comprise Fc domain variants or Fc domain dimer variants that exhibit a decrease in ADCC or ADCP as compared to polypeptides (e.g., fusion polypeptides) comprising a wild-type Fc domain (e.g., wild-type Fc domain dimer). In some embodiments, the polypeptides (e.g., fusion polypeptides) provided herein comprise Fc domain variants or Fc domain dimer variants that exhibit a decrease in ADCC or ADCP of about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more as compared to polypeptides (e.g., fusion polypeptides) comprising a wild-type Fc domain. In some embodiments, the polypeptides (e.g., fusion polypeptides) provided herein comprise Fc domain variants or Fc domain dimer variants that exhibit a loss of ADCC or ADCP as compared to polypeptides (e.g., fusion polypeptides) comprising a wild-type Fc region.

[0143] Complement-dependent cytotoxicity, also referred to herein as CDC, refers to a form of cell injury in which the complement cascade is activated by the binding of complement component C1q to the Fc domain of an antibody. In some embodiments, the polypeptides (e.g., fusion polypeptides) provided herein include an Fc domain variant or Fc domain dimer variant that exhibits a reduction in C1q binding of at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more as compared to a polypeptide (e.g., fusion polypeptide) that includes a wild-type Fc region. In some embodiments, the polypeptides (e.g., fusion polypeptides) provided herein include an Fc domain variant or Fc domain dimer variant that exhibits a reduction in CDC as compared to a polypeptide (e.g., fusion polypeptide) that includes a wild-type Fc region. In some embodiments, the polypeptides (e.g., fusion polypeptides) provided herein include an Fc domain variant or Fc domain dimer variant that exhibits a reduction in CDC of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more as compared to a polypeptide (e.g., fusion polypeptide) that includes a wild-type Fc region. In some cases, the polypeptides (e.g., fusion polypeptides) provided herein include an Fc domain variant or Fc domain dimer variant that exhibits negligible CDC as compared to a polypeptide construct that includes a wild-type Fc region.

[0144] The Fc domain variants or Fc domain dimer variants described herein exhibit a decrease in binding to Fcγ receptors as compared to the wild-type human IgG Fc region. For example, in some embodiments, the Fc domain variants or Fc domain dimer variants have a lower affinity for Fcγ receptors than the affinity of the wild-type IgG Fc domain for Fcγ receptors, as described in the Examples. In some embodiments, the binding of the Fc domain variants or Fc domain dimer variants described herein to Fcγ receptors is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (complete loss of effector function) compared to the binding of the wild-type Fc domain to Fcγ receptors. In some embodiments, the decrease in binding is to one or more Fcγ receptors selected from the group consisting of CD16a, CD32a, CD32b, CD32c, and CD64.

[0145] In some embodiments, the Fc domain variants or Fc domain dimer variants disclosed herein exhibit a decrease in phagocytosis as compared to the wild-type human IgG Fc region. In some embodiments, the ability of the Fc domain variants or Fc domain dimer variants described herein to mediate phagocytosis is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% compared to the binding of the wild-type Fc domain. In some cases, the Fc domain variants or Fc domain dimer variants exhibit a loss of phagocytosis as compared to the wild-type human IgG Fc region.

[0146] In some embodiments, the SIRPα variant 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 amino acids or more. Optionally, in some embodiments, a cleavable linker is utilized.

[0147] In some embodiments, the polypeptides (e.g., fusion polypeptides) herein include targeting or signal sequences that direct the polypeptide to a desired cellular location or extracellular environment. In some embodiments, certain signaling sequences target either secretion of the polypeptide into the growth medium or secretion into the periplasmic space located between the inner and outer membranes of the cell. In some embodiments, the polypeptide (e.g., fusion polypeptide) includes an epitope or tag that enables purification or screening. Such epitopes or tags include polyhistidine tags (His-tags) (e.g., His6 (SEQ ID NO: 223) and His10 (SEQ ID NO: 224)) or other tags used in immobilized metal affinity chromatography (IMAC) systems (e.g., Ni+2 affinity columns), GST fusions, MBP fusions, Strep tags, the BSP biotinylation target sequence of bacterial enzyme BirA, and epitope tags that are targets for antibodies (e.g., c-myc tag, flag tag, etc.), but are not limited thereto. In some embodiments, such tags are useful for purification, screening, or both. For example, in some embodiments, the polypeptide (e.g., fusion polypeptide) is purified by immobilization on a Ni+2 affinity column using a His-tag, and then, after purification, the same His-tag is used to immobilize an antibody on a Ni+2-coated plate and an ELISA or other binding assay described elsewhere herein is performed.

[0148] In some embodiments, the use of a selection method for screening the Fc domain variants or Fc domain dimer variants described herein by a fusion partner becomes possible. A variety of fusion partners enabling a variety of selection methods are available. For example, phage display can be utilized by fusing members of a library of Fc domain variants or Fc domain dimer variants to the gene III protein. In some embodiments, the fusion partner Fc domain variant or Fc domain dimer variant is labeled. Alternatively, in some embodiments, the fusion partner binds to a specific sequence on an expression vector, thereby enabling the fusion partner and the associated Fc domain variant or Fc domain dimer variant to be linked covalently or non-covalently to the nucleic acid encoding them.

[0149] In some embodiments, when the fusion partner is a therapeutic moiety, the therapeutic moiety is, for example, a peptide, protein, antibody, siRNA, or small molecule. Non-limiting examples of therapeutic antibodies that bind to the Fc domain variants or Fc domain dimer variants of the present disclosure include, but are not limited to, antibodies that recognize CD47. Non-limiting examples of therapeutic polypeptides that bind to the Fc domain variants or Fc domain dimer variants of the present disclosure include CD47-binding polypeptides including the SIRPα polypeptide, but are not limited thereto. In such cases, the CD47-binding polypeptide is bound or fused to the Fc domain variant or Fc domain dimer variant of the present disclosure. Examples of CD47-binding polypeptides include, but are not limited to, anti-CD47 antibodies or fragments thereof, and ligands of CD47 such as SIRPα or fragments thereof. Further examples of CD47-binding polypeptides include, but are not limited to, the naturally occurring form of SIRPα and variants thereof.

[0150] In some embodiments, polypeptides comprising Fc domain dimer variants are disclosed herein, where the Fc domain dimer variant comprises two Fc domain variants, and each Fc domain variant is independently selected from (i) a human IgG1 Fc region consisting of the mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting of the mutations A330S, P331S and N297A; or (iii) a human IgG4 Fc region consisting of the mutations S228P, E233P, F234V, L235A, delG236, and N297A. In some embodiments, the Fc domain variants are identical (i.e., homodimer). In some embodiments, the Fc domain variants are different (i.e., heterodimer). In some embodiments, at least one of the Fc domain variants of 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 of 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 a loss or decrease in binding to Fcγ receptors as compared to the wild-type version of the human IgG Fc region. In some embodiments, the Fc domain dimer variant exhibits a loss or decrease in binding to the Fcγ receptors CD16a, CD32a, CD32b, CD32c, and CD64 as compared to the wild-type version of the human IgG Fc region. In some embodiments, the Fc domain dimer variant exhibits a loss or decrease in binding to C1q as compared to the wild-type version of the human IgG Fc fusion. In some embodiments, at least one of the Fc domain variants of 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 a loss or decrease in binding to Fcγ receptors as compared to the wild-type human IgG4 Fc region.In some embodiments, the Fc domain dimer variant exhibits a loss or reduction of binding to the Fcγ receptors CD16a and CD32b as compared to the wild-type version of its human IgG4 Fc region. In some embodiments, the Fc domain dimer variant has a K -6 greater than about 5×10 D and binds to the Fcγ receptor.

[0151] In some embodiments, the Fc domain dimer variant further comprises a CD47-binding polypeptide. In some embodiments, the Fc domain dimer variant exhibits a loss or reduction of binding to the Fcγ receptors as compared to the wild-type version of the human IgG Fc region. In some embodiments, the CD47-binding polypeptide does not cause acute anemia in rodents and non-human primates. In some embodiments, the CD47-binding polypeptide does not cause acute anemia in humans.

[0152] 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 amino acid sequence EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMDFSIRIGX8ITPADAGTYYCX9KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 221), where X1 is V or I, X2 is A or I, X3 is I or F, X4 is E or V, X5 is K or R, X6 is H or P, X7 is L or T, X8 is any amino acid other than N, and X9 is V or I. In some embodiments, the SIRPα polypeptide comprises a SIRPα D1 domain variant, where X1 is V or I, X2 is A or I, X3 is I or F, X4 is E, X5 is K or R, X6 is H or P, X7 is L or T, X8 is not N, and X9 is V.

[0153] In some embodiments, a SIRPα D1 domain variant, wherein the SIRPα D1 domain variant is a non-naturally occurring high-affinity SIRPα D1 domain, and the SIRPα D1 domain variant binds to human CD47 with an affinity that is at least 10-fold greater than the affinity of the naturally occurring D1 domain, and an Fc domain variant, wherein the Fc domain variant is linked to a second polypeptide comprising a second Fc domain variant to form an Fc domain dimer variant, and the Fc domain dimer variant has a loss or reduction of effector function, are disclosed herein. In some embodiments, the non-naturally occurring high-affinity SIRPα D1 domain comprises an amino acid mutation at residue 80.

[0154] In some embodiments, SIRPα D1 domain variants are disclosed herein, where the SIRPα D1 domain variant binds to CD47 derived from a first species with a KD of less than 250 nM, the SIRPα D1 domain variant binds to CD47 derived from a second species with a KD of less than 250 nM, the KD of CD47 derived from the first species and the KD of CD47 derived from the second species are within 100-fold of each other, and the first species and the second species are selected from the group consisting of humans, rodents, and non-human primates. In some embodiments, the SIRPα D1 domain variant binds to CD47 derived from at least three different species. In some embodiments, the non-human primate is a cynomolgus monkey.

[0155] In some embodiments, (a) K less than 250 nM DA polypeptide comprising (a) an SIRPα D1 domain that binds to human CD47 and (b) an Fc domain or a variant thereof linked to the N-terminus or C-terminus of the SIRPα D1 domain, which does not cause acute anemia in rodents and non-human primates, is disclosed herein. In some embodiments, the polypeptide is a non-naturally occurring variant of human SIRP-α. In some embodiments, in vivo administration of the polypeptide results in less than 50% decrease in hemoglobin during the first week after administration. In some embodiments, administration of the polypeptide to humans results in less than 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 is selected from an Fc domain variant comprising (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 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.

[0156] The SIRPα constructs of the present disclosure include an SIRPα domain or a variant thereof having a C-terminus connected to the N-terminus of an Fc domain or a variant thereof by a linker, using conventional genetic or chemical means, such as chemical conjugation. In some embodiments, a linker (e.g., a spacer) is inserted between the polypeptide and the Fc domain or a variant thereof. In some embodiments, the polypeptides of the present disclosure comprising an SIRPα D1 domain variant are fused to an Fc domain variant incapable of forming a dimer. In some embodiments, the polypeptides of the present disclosure are fused to an Fc domain or a variant thereof capable of forming a dimer, such as a heterodimer, with another Fc domain or a variant thereof. In some embodiments, the polypeptides of the present invention are fused to an Fc domain or a variant thereof, and this fusion protein forms a homodimer. In some embodiments, the polypeptides of the present disclosure are fused to a first Fc domain or a variant thereof, and a different protein or peptide (e.g., an antibody variable region) is fused to a second Fc domain or a variant thereof. In some embodiments, the SIRPα D1 domain or a variant thereof is connected to a first Fc domain or a variant thereof, and a therapeutic protein (e.g., a cytokine, an interleukin, an antigen, a steroid, an anti-inflammatory agent, or an immunomodulatory agent) is connected to a second Fc domain or a variant thereof. In some embodiments, the first and second Fc domains or variants thereof form a heterodimer.

[0157] Without limitation to the foregoing, in some embodiments, an SIRPα D1 domain variant polypeptide (e.g., any of the variants described in Tables 2, 5, and 6) is fused to an Fc polypeptide or an Fc variant polypeptide, such as an Fc domain or a variant thereof. Examples of polypeptides comprising an SIRPα D1 domain variant polypeptide and a fused Fc domain variant polypeptide include, but are not limited to, SEQ ID NOs: 96-137, 214, and 216 shown in Table 8. [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

[0158] In some embodiments, the polypeptide comprises an SIRPα D1 variant domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to any of the variants provided in Table 8.

[0159] In some embodiments, the polypeptide comprises an SIRPα D1 domain variant having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NOs: 98-104, 107-113, 116-122, or 135-137 in Table 8.

[0160] In some embodiments, the polypeptide is (a) a signal regulatory protein alpha (SIRP-α) D1 variant, wherein the SIRPα D1 domain variant has 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 comprises KPS (SEQ ID NO: 47), wherein X1 is E or G, X2 is L, I, or V, X3 is V, L, or I, X4 is S or F, X5 is L or S, X6 is S or T, X7 is A or V, X8 is I or T, X9 is H, R, or L, X 10 is A, V, I, or L, X 11 is I, T, S, or F, X 12 is A or G, X 13 is E, V, or L, X 14 is K or R, X 15 is E or Q, X 16 is H, P, or R, X 17 is D or E, X 18 is S, L, T, or G, X 19 is K or R, X 20 is E or N, X 21 is S or P, X 22 is S or R, X 23 is S or G, X24 is any amino acid, X 25 is any amino acid, X 26 is V, or I, X 27 is F, L, or V, X 28 is D or absent, X 29 is T, or V, X 30 is F, or V, X 31 is A, or G, and the SIRPα D1 domain variant comprises at least two amino acid substitutions relative to the wild-type SIRPα D1 domain having the sequence according to any one of SEQ ID NOs: 1-10, an SIRPα D1 variant, and (b) an Fc domain dimer variant having two Fc domain variants, wherein each Fc domain variant independently is (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; (vii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations; or (viii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, an Fc domain dimer variant.

[0161] In some embodiments, the polypeptide comprises an SIRPα D1 domain variant, wherein the SIRPα D1 domain variant comprises an amino acid sequence according to SEQ ID NO: 47, an Fc domain dimer having two Fc domains, wherein one of the Fc domains is an Fc domain variant comprising a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations.

[0162] Dimerization of the Fc domain In some embodiments, the SIRPα D1 domain variant polypeptide (e.g., any of the variants described in Tables 2, 5, and 6) is fused to a first Fc domain (e.g., an Fc domain variant) at either the N-terminus or the C-terminus. In some embodiments, the first Fc domain is a variant that cannot form a dimer. In some embodiments, the first Fc domain forms a dimer with a second Fc domain. In some embodiments, the first and second Fc domains include amino acid substitutions that promote heterodimerization between the first and second domain Fc domains.

[0163] In some embodiments, each of the two Fc domains of the Fc domain dimer includes amino acid substitutions that promote heterodimerization of the two monomers. In some embodiments, the SIRPα construct includes a first subunit that includes, for example, a SIRPα D1 domain variant polypeptide fused to a first Fc domain, and a second subunit that includes a second Fc domain (e.g., that 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 the Fc domain dimer (e.g., single-arm). In some embodiments, the construct has two SIRPα D1 domain variant polypeptides linked to the Fc domain dimer (e.g., double-arm). In some embodiments, a SIRPα D1 domain variant having a K D of about 500 nM is particularly useful for double-arm constructs. In some embodiments, a SIRPα D1 domain variant having a K D of about 50 nM is particularly useful for double-arm constructs. In some embodiments, a SIRPα D1 domain variant having a K D of about 5 nM is useful for both double-arm constructs and single-arm constructs. In some embodiments, a SIRPα D1 domain variant having a K DThe SIRPα D1 domain variant having [it] is useful for double-arm constructs and single-arm constructs. In some embodiments, a K of about 100 pM D The SIRPα D1 domain variant having [it] is useful for double-arm constructs and single-arm constructs. In some embodiments, a K of about 50 pM D The SIRPα D1 domain variant having [it] is useful for double-arm constructs and single-arm constructs. In some embodiments, a K of about 10 pM D The SIRPα D1 domain variant having [it] is useful for double-arm constructs and single-arm constructs.

[0164] In some embodiments, the heterodimerization of the Fc domain is facilitated by introducing different but compatible substitutions into the two Fc domains, such as "knob-into-hole" residue pairs and charged residue pairs. The knob-hole interaction is favorable for heterodimer formation, while the knob-knob and hole-hole interactions interfere with homodimer formation due to steric clashes and lack of favorable interactions. A hole refers to a cavity that occurs when the original amino acid in the protein is replaced with a different amino acid having a smaller side-chain volume. A knob refers to a protrusion that occurs when the original amino acid in the protein is replaced with a different amino acid having a larger side-chain volume. For example, in some embodiments, the amino acids to be replaced are in the CH3 antibody constant domain of the Fc domain and are involved in the dimerization of the two Fc domains. In some embodiments, a hole is formed in one CH3 antibody constant domain to accommodate the knob in the other CH3 antibody constant domain. Thereby, the knob and hole amino acids act to promote or support the heterodimerization of the two Fc domains. In some embodiments, the hole in one CH3 antibody constant domain is created to better accommodate the original amino acid in the other CH3 antibody constant domain. In some embodiments, the knob in one CH3 antibody constant domain is created to form additional interactions with the original amino acid in the other CH3 antibody constant domain.

[0165] In some embodiments, the hole is constructed by replacing an amino acid having a larger side chain, such as tyrosine or tryptophan, in the CH3 antibody constant domain with an amino acid having a smaller side chain, such as alanine, valine, or threonine (e.g., Y407V mutation). Similarly, in some embodiments, the knob is constructed by replacing an amino acid having a smaller side chain in the CH3 antibody constant domain with an amino acid having a larger side chain (e.g., T366W mutation). In some embodiments, one Fc domain contains the knob mutation T366W and the other Fc domain contains the hole mutations T366S, L358A, and Y407V. In some embodiments, the polypeptides of the present disclosure comprising the SIRPα D1 domain variant are fused to an Fc domain containing the knob mutation T366W to limit unwanted knob-knob homodimer formation. Examples of knob-into-hole amino acid pairs include, but are not limited to, those in Table 9, and knob-into-hole Fc domain variants and SIRPα-Fc fusions are described in Table 10.

Table 9

Table 10-1

Table 10-2

Table 10-3

[0166] In addition to the knob-into-hole strategy, in some embodiments, electrostatic steering is also used to control dimerization of the Fc domain. Electrostatic steering refers to using favorable electrostatic interactions between peptides, protein domains, and oppositely charged amino acids in a protein to control the formation of higher-order protein molecules. In particular, to control dimerization of the Fc domain using electrostatic steering, one or more amino acid residues that make up the CH3-CH3 interface are replaced with positively or negatively charged amino acid residues, such that depending on the particular charged amino acid introduced, the interaction is made electrostatically favorable or unfavorable. In some embodiments, positively charged amino acids such as lysine, arginine, or histidine on the interface are replaced with negatively charged amino acids such as aspartic acid or glutamic acid. In some embodiments, negatively charged amino acids on the interface are replaced with positively charged amino acids. In some embodiments, the charged amino acids are introduced into one or both of the interacting CH3 antibody constant domains. In some embodiments, introducing charged amino acids into the CH3 antibody constant domains of two interacting Fc domains is controlled by the electrostatic steering effect resulting from the interaction between the charged amino acids, promoting the selective formation of heterodimers of the Fc domain. Examples of electrostatic steering amino acid pairs include, but are not limited to, those in Table 11.

Table 11

[0167] In particular, in the context of constructing bispecific antibodies, other methods for controlling heterodimerization of the Fc domain are also available.

[0168] In some embodiments, the first Fc domain and the second Fc domain each comprise one or more of the following amino acid substitutions relative to the sequence of human IgG1: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, and K409I.

[0169] In some embodiments, the Fc domain comprises (a) with respect to wild-type human IgG1, the following amino acid substitutions: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, or K409I, or (b) (i) a mutation of N297A with respect to the human IgG1 Fc region; (ii) mutations of L234A, L235A, and G237A with respect to the human IgG1 Fc region; (iii) mutations of L234A, L235A, G237A, and N297A with respect to the human IgG1 Fc region; (iv) a mutation of N297A with respect to the human IgG2 Fc region; (v) mutations of A330S and P331S with respect to the human IgG2 Fc region; (vi) mutations of A330S, P331S, and N297A with respect to the human IgG2 Fc region; (vii) mutations of S228P, E233P, F234V, L235A, and delG236 with respect to the human IgG4 Fc region; or (viii) mutations of S228P, E233P, F234V, L235A, delG236, and N297A with respect to the human IgG4 Fc region.In some embodiments, the Fc domain variant comprises (a) with respect to wild-type human IgG1, the following amino acid substitutions: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, or K409I, and (b) (i) with respect to the human IgG1 Fc region, the N297A mutation; (ii) with respect to the human IgG1 Fc region, the L234A, L235A, and G237A mutations; (iii) with respect to the human IgG1 Fc region, the L234A, L235A, G237A, and N297A mutations; (iv) with respect to the human IgG2 Fc region, the N297A mutation; (v) with respect to the human IgG2 Fc region, the A330S and P331S mutations; (vi) with respect to the human IgG2 Fc region, the A330S, P331S, and N297A mutations; (vii) with respect to the human IgG4 Fc region, the S228P, E233P, F234V, L235A, and delG236 mutations; or (viii) with respect to the human IgG4 Fc region, the S228P, E233P, F234V, L235A, delG236, and N297A mutations.

[0170] In some embodiments, the first and second Fc domains comprise different amino acid substitutions. In some embodiments, the first Fc domain comprises T366W. In some embodiments, the second Fc domain comprises T366S, L368A, and Y407V. In some embodiments, the first Fc domain comprises D399K. In some embodiments, the second Fc domain comprises K409D.

[0171] Linkage of polypeptides or protein domains In some embodiments, an amino acid mutation at residue 80 relative to the wild-type signal regulatory protein alpha (SIRP-α) D1 domain; and at least one additional amino acid mutation at a residue selected from the group consisting of residues 6, 27, 31, 47, 53, 54, 56, 66, and 92 relative to the wild-type SIRPα D1 domain, a polypeptide comprising a SIRPα D1 variant comprising a SIRP-α D1 domain or a fragment thereof is disclosed herein.

[0172] Also, in some embodiments, a polypeptide comprising an Fc variant is disclosed herein, wherein the Fc variant comprises an Fc domain dimer comprising two Fc domain variants, and each Fc domain variant is independently selected from (i) the human IgG1 Fc region consisting of the mutations L234A, L235A, G237A, and N297A; (ii) the human IgG2 Fc region consisting of the mutations A330S, P331S and N297A; or (iii) the human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A.

[0173] In some embodiments, the signal regulatory protein alpha (SIRP-α) D1 variant and the Fc variant are connected. In some embodiments, the C-terminus of the SIRPα D1 domain variant is connected to the N-terminus of the Fc domain variant, whereby the two polypeptides are connected in tandem with each other.

[0174] In some embodiments, the signal regulatory protein alpha (SIRP-α) D1 variant and the Fc variant are connected via a covalent bond, such as a peptide bond, a synthetic polymer, or any type of bond generated from a chemical reaction, such as chemical conjugation. When connected via a peptide bond, in some embodiments, the carboxylic acid group at the C-terminus of one protein domain (e.g., the SIRPα D1 domain variant) is reacted with the amino group at the N-terminus of another protein domain (e.g., the Fc variant) in a condensation reaction to form a peptide bond. In some embodiments, the peptide bond is formed by synthetic means via conventional organic chemical reactions or by natural production from host cells, where the nucleic acid molecule encoding the DNA sequences of both tandem proteins (e.g., the Fc domain variant and the SIRPα D1 domain variant) can be directly transcribed in the host cell by the necessary molecular machinery (e.g., DNA polymerase and ribosome) and translated into a continuous polypeptide encoding both proteins.

[0175] When the signal regulatory protein alpha (SIRP-α) D1 variant and the Fc variant are connected by a synthetic polymer, in some embodiments, the polymer is functionalized with reactive chemical functional groups at both ends such that the connecting ends of the two proteins react with the terminal amino acids.

[0176] In some embodiments, the signal regulatory protein alpha (SIRP-α) D1 variant and the Fc variant are connected by a bond other than a peptide bond, such as a bond formed by a chemical reaction. In some embodiments, chemical functional groups (e.g., amine, carboxylic acid, ester, azide, or other functional groups) synthetically bond the C-terminus of one protein to the N-terminus of another protein, respectively. In some embodiments, a chemical bond is then formed by reacting the two functional groups via synthetic chemical means, thereby connecting the two proteins together.

[0177] Spacer In the present disclosure, in some embodiments, the linker between the Fc domain monomer of the present disclosure and the SIRPα D1 variant polypeptide is an amino acid spacer comprising from about 1 to 200 amino acids. Suitable peptide spacers include peptide linkers containing flexible amino acid residues such as glycine and serine. Examples of linker sequences are provided in Table 12. In some embodiments, the spacer contains a motif of GS, GG, GGS, GGG, GGGGS (SEQ ID NO: 163), GGSG (SEQ ID NO: 164), or SGGG (SEQ ID NO: 165), for example, a plurality or repeating motif. In some embodiments, the spacer contains from 2 to 12 amino acids and includes a motif of GS, for example, 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 contains from 3 to 12 amino acids and includes a motif of GGS, for example, GGS, GGSGGS (SEQ ID NO: 171), GGSGGSGGS (SEQ ID NO: 172), and GGSGGSGGSGGS (SEQ ID NO: 173). In some embodiments, the spacer contains from 4 to 12 amino acids and includes a motif of GGSG (SEQ ID NO: 164), for example, GGSG (SEQ ID NO: 164), GGSGGGSG (SEQ ID NO: 174), or GGSGGGSGGGSG (SEQ ID NO: 175). In some embodiments, the spacer contains a motif of GGGGS (SEQ ID NO: 163), for example, GGGGSGGGGSGGGGS (SEQ ID NO: 176).In some embodiments, the spacer contains amino acids other than glycine and serine, such as AAS (SEQ ID NO: 177), AAAL (SEQ ID NO: 178), AAAK (SEQ ID NO: 179), AAAR (SEQ ID NO: 180), EGKSSGSGSESKST (SEQ ID NO: 181), GSAGSAAGSGEF (SEQ ID NO: 182), AEAAAKEAAAKA (SEQ ID NO: 183), KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), GGGGAGGGG (SEQ ID NO: 185), GENLYFQSGG (SEQ ID NO: 186), SACYCELS (SEQ ID NO: 187), RSIAT (SEQ ID NO: 188), RPACKIPNDLKQKVMNH (SEQ ID NO: 189), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 190), AAANSSIDLISVPVDSR (SEQ ID NO: 191), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 192).

[0178] In some embodiments, the spacer contains a motif of EAAAK (SEQ ID NO: 193), such as a plurality or repeating motif. In some embodiments, the spacer contains a motif of a proline-rich sequence such as (XP)n (where X is any amino acid (e.g., A, K, or E) and n is 1 to 5) and PAPAP (SEQ ID NO: 194), such as a plurality or repeating motif. [Table 12]

[0179] In some embodiments, the length of the peptide spacer and amino acids used are adjusted according to the degree of flexibility desired for the two proteins involved and the final protein fusion polypeptide. In some embodiments, the length of the spacer is adjusted to ensure proper protein folding and avoid the formation of aggregates. In some embodiments, the spacer is A or AAAL (SEQ ID NO: 178).

[0180] Vectors, host cells, and protein production In some embodiments, the polypeptide comprises a 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 signal regulatory protein alpha (SIRP-α) D1 domain; and at least one additional amino acid mutation at a residue selected from the group consisting of residues 6, 27, 31, 47, 53, 54, 56, 66, and 92 relative to the wild-type SIRPα D1 domain, as disclosed herein.

[0181] Also, in some embodiments, a polypeptide comprising an Fc variant is disclosed herein, wherein the Fc variant comprises an Fc domain dimer comprising two Fc domain monomers, and each Fc domain monomer is independently selected from (i) a human IgG1 Fc region comprising the mutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc region comprising the mutations A330S, P331S, and N297A; or (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A.

[0182] In some embodiments, the polypeptide of the present disclosure is produced from a host cell. A host cell refers to a medium containing the necessary cellular components, such as organelles, required to express the polypeptides and fusion polypeptides described herein from the corresponding nucleic acids. In some embodiments, the nucleic acid is contained in a nucleic acid vector that is introduced into the host cell by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, or the like. In some embodiments, the selection of the nucleic acid vector depends on the host cell used. In some embodiments, the host cell is of either prokaryotic (e.g., bacterial) or eukaryotic (e.g., mammalian) origin.

[0183] In some embodiments, a polypeptide construct comprising a polypeptide, such as a SIRPα D1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and a fusion partner such as an Fc variant, is produced by culturing a host cell transformed with an expression vector containing a nucleic acid, preferably a nucleic acid encoding the polypeptide construct (e.g., an Fc variant, a linker, and a fusion partner), under conditions appropriate to induce or cause expression of the polypeptide construct. In some embodiments, the conditions suitable for expression vary depending on the selected expression vector and host cell. 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 found to be useful in the present disclosure are described in the ATCC® cell line catalog available from the American Type Culture Collection. In some embodiments, the Fc domain variants of the present disclosure are expressed in cells optimized to not glycosylate the proteins expressed by such cells, either by genetic manipulation of the cell line or by changing cell culture conditions such as the addition of kifunensine, or by using a host that is naturally non-glycosylated such as a prokaryote (e.g., E. coli). In some cases, modification of the glycosylation sequence in the Fc is not necessary.

[0184] Construction of Nucleic Acid Vectors and Host Cells The nucleic acid sequences encoding the amino acid sequences of the polypeptides of the present disclosure can be prepared by various methods. These methods include, but are not limited to, mutagenesis via oligonucleotides (or site-directed mutagenesis) and PCR mutagenesis. In some embodiments, the nucleic acid molecules encoding the polypeptides of the present disclosure are obtained by using standard techniques, such as gene synthesis. Alternatively, a nucleic acid molecule encoding the 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 molecules are synthesized using nucleotide synthesizers or PCR techniques.

[0185] In some embodiments, a nucleic acid encoding a polypeptide construct, such as a fusion partner including a SIRPα D1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and an Fc variant, is incorporated into an expression vector for protein expression. A variety of expression vectors can be utilized for protein expression. The expression vector can include an extrachromosomal vector that self-replicates or a vector that integrates into the host genome. The vector can also include various elements or elements. For example, in some embodiments, vector elements include transcriptional and translational regulatory sequences such as promoter sequences, ribosome binding sites, signal sequences, transcription start and stop sequences, translation start and stop sequences, 3' and 5' untranslated regions (UTRs), and enhancer or activation sequences; an origin of replication; a selectable marker gene; as well as a nucleic acid sequence encoding the polypeptide of interest and a transcription termination sequence, but are not limited thereto. 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 include transcriptional and translational regulatory nucleic acids operably linked to a nucleic acid encoding an Fc variant and are typically suitable for the host cells used for protein expression. Without limitation, a selection gene or marker such as an antibiotic resistance gene or a fluorescent protein gene can be used to select host cells containing the expression vector, for example, by the expression of an antibiotic or fluorescence. A variety of selection genes are available.

[0186] In some embodiments, the elements or elements of the vector are optimized such that the expression vector is compatible with the type of host cell. Expression vectors found useful in the present disclosure include, but are not limited to, those that enable protein expression in mammalian cells, bacteria, insect cells, yeast, and in vitro systems.

[0187] In some embodiments, mammalian cells are used as host cells for producing the polypeptides of the present disclosure. Examples of mammalian cell types include 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, but are not limited thereto. In some embodiments, E. coli cells are used as host cells for producing the polypeptides of the present disclosure. Examples of E. coli strains include E. coli 294 (ATCC® 31,446), E. coli λ 1776 (ATCC® 31,537), E. coli BL21(DE3) (ATCC® BAA-1025), and E. coli RV308 (ATCC® 31,608), but are not limited thereto.

[0188] Different host cells have characteristic and specific mechanisms for post-translational processing and modification (e.g., glycosylation) of protein products. In some embodiments, an appropriate cell line or host system is selected to ensure proper modification and processing of the expressed polypeptide. After introducing the vector into the host cell for protein production, the host cell is cultured in a conventional nutrient medium that has been appropriately modified for induction of the promoter, selection of transformants, or amplification of the gene encoding the desired sequence.

[0189] In some embodiments, a polypeptide construct, such as a polypeptide construct comprising a fusion partner such as an SIRPα D1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and an Fc variant, is expressed in a mammalian expression system, including systems in which an expression construct is introduced into mammalian cells using a virus such as a retrovirus or an adenovirus. In some embodiments, cells of human, mouse, rat, hamster, or primate origin are utilized. Suitable cells include, but are not limited to, known research cells such as Jurkat T cells, NIH3T3, CHO, COS, and 293 cells. Alternatively, in some embodiments, the protein is expressed in bacterial cells. Bacterial expression systems are well known in the art and include Escherichia coli (E. coli), Bacillus subtilis, Streptococcus cremoris, and Streptococcus lividans. In some cases, a polypeptide construct comprising an Fc domain variant is produced in insect cells such as Sf9 and Sf21 cells, or in yeast cells such as organisms of the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, and Yarrowia. In some cases, a polypeptide construct comprising an Fc domain variant is expressed in vitro using a cell-free translation system. In vitro translation systems derived from both prokaryotic (e.g., E. coli) and eukaryotic (e.g., wheat germ, rabbit reticulocyte) cells are available and are selected in some embodiments based on the expression level and functional properties of the protein of interest. For example, as recognized 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 variant is generated by chemical synthesis methods such as liquid-phase peptide synthesis and solid-phase peptide synthesis.In the case of in vitro transcription using a non-glycosylated system such as a bacterial extract, Fc is not glycosylated even if a natural glycosylation site is present, so inactivation of Fc can be obtained in the same manner.

[0190] In some embodiments, the polypeptide construct includes unnatural amino acids, amino acid analogs, amino acid mimics, or any combination thereof that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids generally refer to the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrrolysine and selenocysteine. Amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids, e.g., compounds having a carbon bonded to hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. In some embodiments, such analogs have a modified R group (such as norleucine) or a modified peptide backbone, but generally retain the same basic chemical structure as naturally occurring amino acids.

[0191] 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. Examples of media suitable for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Expi293™ Expression Medium, DMEM containing fetal bovine serum (FBS), and RPMI-1640. Examples of media suitable for bacterial host cells include Luria broth (LB) supplemented with necessary additives such as a selective agent, for example, ampicillin. In some embodiments, the host cells are cultured at a suitable temperature, such as about 20°C to about 39°C, for example, about 25°C to about 37°C, preferably 37°C, and a CO2 level of about 5% to 10%. In some embodiments, the pH of the medium is from about pH 6.8 to pH 7.4, for example, pH 7.0, and mainly depends on the host organism. When an inducible promoter is used in the expression vector, the expression of the protein can be induced under conditions suitable for the activation of the promoter.

[0192] In some embodiments, protein recovery involves disrupting the host cells, for example, by osmotic shock, sonication, or lysis. Once the cells are disrupted, the cell debris is removed by centrifugation or filtration. The protein can then be further purified. In some embodiments, the polypeptides of the present disclosure are purified by various protein purification methods, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. For example, in some embodiments, the protein is isolated and purified by appropriately selecting and combining an affinity column, such as a Protein A column (e.g., POROS Protein A chromatography), and a chromatography column (e.g., POROS HS-50 cation exchange chromatography), filtration, ultrafiltration, desalting, and dialysis procedures. In some embodiments, the polypeptide is conjugated to a marker sequence, such as a peptide, to facilitate purification. An example of a marker amino acid sequence is the hexahistidine peptide (His6 tag), which can bind to a nickel-functionalized agarose affinity column with micromolar affinity. Alternatively, a hemagglutinin "HA" tag corresponding to an epitope derived from the influenza hemagglutinin protein can be used.

[0193] In some embodiments, polypeptide constructs of the disclosure, such as polypeptides comprising a fusion partner such as an SIRPα D1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and an Fc variant, are produced by a cell of a subject (e.g., a human) by administering a viral vector (e.g., a retroviral vector, an adenoviral vector, a poxviral vector (e.g., a vaccinia viral vector such as modified vaccinia Ankara (MVA)), an adeno-associated viral vector, and an alphavirus vector) containing a nucleic acid molecule encoding the polypeptide of the disclosure, for example, in the context of gene therapy. The vector can be used for the expression of the polypeptides disclosed herein by entering into the interior of the cells of the subject (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.). In some cases, the polypeptide is secreted from the cell. In some embodiments, if the treatment of a disease or disorder is the desired outcome, no further treatment is necessary. In some embodiments, if protein recovery is desired, blood is collected from the subject and the protein is purified from the blood by various methods.

[0194] Method for treating urothelial carcinoma In some embodiments, a method of treating cancer (e.g., urothelial cancer, e.g., urothelial carcinoma) in an individual (e.g., a human individual) is provided, the method comprising administering to the individual: (a) an effective amount of an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα); and (b) an effective amount of an antibody-drug conjugate (ADC). In some embodiments, the urothelial cancer is a histologically confirmed unresectable locally advanced or metastatic urothelial carcinoma. Additionally or alternatively, the urothelial carcinoma is a cancer of the bladder, renal pelvis, ureter, or urethra. In some embodiments, the individual has a transitional cell carcinoma with squamous differentiation or a mixed cell type, and the urothelial carcinoma is the predominant histotype. In some embodiments, the individual does not have a small cell carcinoma or a neuroendocrine histotype.

[0195] In some embodiments, the ADC comprises an antibody that specifically binds to nectin-4 (e.g., human nectin-4) conjugated to a cytotoxic drug. In some embodiments, the anti-nectin-4 antibody is enfortumab, also known as AGS-22C3 (CAS Registry Number 1448664-46-7). In some embodiments, the cytotoxic drug is monomethyl auristatin E (MMAE), a small molecule microtubule disrupting agent also known as vedotin or SGD-1006 (CAS Registry Number 474645-27-7). In some embodiments, the ADC is enfortumab vedotin (also known as PADCEV®; CAS Registry Number 1346452-25-2). Enfortumab vedotin is a nectin-4-directed antibody-drug conjugate (ADC) comprising a full-length human anti-nectin-4 IgG1 kappa monoclonal antibody conjugated to MMAE via a protease-cleavable maleimidocaproyl valine-citrulline (vc) linker. Conjugation occurs at a cysteine residue on the heavy chain of the antibody, resulting in a product with a drug-to-antibody ratio (DAR) of approximately 3.8:1. The molecular weight is approximately 152 kDa. In some embodiments, the ADC (e.g., enfortumab vedotin) is administered to an individual in one or more 28-day cycles. In some embodiments, the ADC (e.g., enfortumab vedotin) is administered at a dose of 1.25 mg / kg on days 1, 8, and 15 of one or more 28-day cycles. In some embodiments, the ADC (e.g., enfortumab vedotin) is administered to an individual in one or more 28-day cycles. In some embodiments, the ADC (e.g., enfortumab vedotin) is administered at a dose of 1.25 mg / kg every three weeks (Q3W). In some embodiments, the ADC (e.g., enfortumab vedotin) is administered by intravenous infusion. In some embodiments, the ADC (e.g., enfortumab vedotin) is administered by intravenous infusion over 30 minutes on days 1, 8, and 15 of one or more 28-day cycles.In some embodiments, the maximum dose of the ADC (e.g., enfortumab vedotin) is administered to the individual up to a maximum dose of 125 mg on each of days 1, 8, and 15 of one or more 28-day cycles. In some embodiments, the ADC (e.g., enfortumab vedotin) is administered according to the regimen provided in the local prescribing information (for the United States, see, for example, https: / / astellas.us / docs / PADCEV_label.pdf). Details regarding the mechanism of action of the ADC can also be found in the prescribing information.

[0196] In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is an agent described elsewhere herein (e.g., any agent). In some embodiments, the agent that blocks the interaction between CD47 and SIRPα is a polypeptide (e.g., a fusion polypeptide) comprising an SIRPα D1 domain variant (e.g., an SIRPα D1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein). In some embodiments, the C-terminus of the SIRPα D1 domain variant of the fusion polypeptide (e.g., an SIRPα D1 domain variant described herein) is fused to the N-terminus of the Fc domain variant. In some embodiments, the polypeptide (e.g., a fusion polypeptide) comprises an SIRPα 1 domain variant comprising the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising the L234A, L235A, G237A, and N297A mutations (numbering according to the Kabat EU index); (ii) a human IgG2 Fc region comprising the A330S, P331S, and N297A mutations (numbering according to the Kabat EU index); (iii) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, and delG236 mutations (numbering according to the Kabat EU index); or (iv) a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations (numbering according to the Kabat EU index) (e.g., the C-terminus of the SIRPα D1 domain variant is fused to the N-terminus of the Fc domain variant). 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 is administered to an individual (e.g., a human individual) at a dose of up to about 60 mg / kg (e.g., any one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 mg / kg; including any range between these values). In some embodiments, the polypeptide is administered to the individual by intravenous infusion. In some embodiments, the polypeptide is administered to the individual at a dose of about 15 mg / kg. In some embodiments, the polypeptide is administered to the individual at a dose of about 15 mg / kg q2w (i.e., once every two weeks or once every 14 days). In some embodiments, the polypeptide is administered to the individual at a dose of about 20 mg / kg. In some embodiments, the polypeptide is administered to the individual at a dose of about 20 mg / kg q2w (i.e., once every two weeks or once every 14 days). In some embodiments, the polypeptide is administered to the individual at a dose of about 30 mg / kg. In some embodiments, the polypeptide is administered to the individual at a dose of about 30 mg / kg q2w (i.e., once every two weeks or once every 14 days). In some embodiments, the polypeptide is administered by intravenous infusion at 15, 20, or 30 mg / kg over 60 hours q2w (i.e., once every two weeks or once every 14 days). In some embodiments, the fusion polypeptide is supplied in a 1000 mg / 50 ml type I clear glass vial sealed with a 20 mm Teflon-coated rubber septum stopper and a tamper-evident aluminum seal for use (e.g., intravenous administration). In some embodiments, the fusion polypeptide is stored in its original container at 2 - 8°C (36 - 46°F) until use (e.g., preparation for intravenous administration).

[0197] In some embodiments, on days when the administration of the polypeptide (e.g., fusion polypeptide) overlaps with the administration of the ADC (e.g., enfortumab vedotin), the polypeptide is administered before the ADC. In some embodiments, the ADC (e.g., enfortumab vedotin) is administered approximately 30 minutes (e.g., about 20 to about 40 minutes, 25 to about 45 minutes, or about 30 to 50 minutes) after the administration of the polypeptide is completed.

[0198] In some embodiments, the subject has been previously treated with an immune checkpoint inhibitor (CPI) for locally advanced or metastatic urothelial carcinoma. In some embodiments, the subject has received CPI for urothelial carcinoma with neoadjuvant or adjuvant therapy and had recurrent or progressive disease either during CPI therapy or within 12 months after completion of CPI therapy. In some embodiments, the CPI therapy included or was a programmed cell death protein 1 (PD-1) inhibitor or a programmed cell death ligand 1 (PD-L1) inhibitor. In some embodiments, the PD-1 inhibitor or PD-L1 inhibitor was a therapeutic antibody. In some embodiments, the therapeutic anti-PD-1 antibody or therapeutic anti-PD-L1 antibody was or included one or more of atezolizumab, pembrolizumab, durvalumab, avelumab, and nivolumab. In some embodiments, the subject has been previously treated with platinum-containing chemotherapy for urothelial carcinoma. In some embodiments, the subject has received platinum-containing chemotherapy for urothelial carcinoma with adjuvant or neoadjuvant therapy and had recurrent or progressive disease within 12 months after completion. In some embodiments, the subject has received platinum-containing chemotherapy for metastatic urothelial carcinoma or unresectable locally advanced urothelial carcinoma. In some embodiments, the platinum-containing chemotherapy was or included one or more of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin.

[0199] In some embodiments, the subject has progressed during or after receiving a most recent prior treatment for urothelial cancer (e.g., the subject's urothelial cancer has shown progression of the disease). In some embodiments, the subject's cancer has recurred during or after receiving a most recent treatment method (e.g., has shown recurrence). In some embodiments, the subject has not received prior treatment with enfortumab vedotin. In some embodiments, the subject has not received treatment with a monomethyl auristatin (MMAE)-based agent (e.g., a vedotin-based antibody-drug conjugate (ADC)). In some embodiments, the subject has not received prior treatment with an agent that disrupts the interaction between hCD47 and hSIRPα, such as an anti-CD47 agent and / or an anti-SIRPα agent. In some embodiments, the subject does not have an allergy to enfortumab vedotin or any excipients (including histidine, trehalose dihydrate, and polysorbate 20) contained in a pharmaceutical formulation of enfortumab vedotin. In some embodiments, the subject is not allergic to biopharmaceuticals produced in Chinese hamster ovary (CHO) cells. In some embodiments, the subject is not intolerant to, or does not have a severe allergic or anaphylactic reaction to, an antibody or a therapeutic protein to be injected. In some embodiments, the subject is not intolerant to, or does not have a severe allergic reaction or anaphylactic reaction to, any of the substances contained in a polypeptide formulation.

[0200] In some embodiments, the cancer treated by the methods provided herein is urothelial cancer, head and neck cancer, gastric cancer, non-small cell lung cancer (NSCLC), hormone receptor-positive breast cancer that does not overexpress (or express) HER2, e.g., HR + HER2 - breast cancer.

[0201] Kits and Manufactured Articles In another embodiment of the invention, provided are a manufactured article or a kit comprising a polypeptide (e.g., a fusion polypeptide described herein) comprising an SIRPα D1 domain variant and an Fc domain variant. In some embodiments, the SIRPα D1 domain variant is for use in combination with an antibody-drug conjugate (e.g., enfortumab vedotin) for the treatment of urothelial carcinoma in an individual (e.g., a human individual). In some embodiments, the SIRPα D1 domain variant is for use in combination with an antibody-drug conjugate (e.g., enfortumab vedotin) for the treatment of urothelium in an individual (e.g., a human individual). In some embodiments, the SIRPα D1 domain variant comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 81 and SEQ ID NO: 85. In some embodiments, the Fc domain variant is: (i) a human IgG1 Fc region comprising the mutations L234A, L235A, G237A, and N297A (numbering according to the Kabat EU index); (ii) a human IgG2 Fc region comprising the mutations A330S, P331S, and N297A (numbering according to the Kabat EU index); (iii) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, and delG236 (numbering according to the Kabat EU index); or (iv) a human IgG4 Fc region comprising the mutations S228P, E233P, F234V, L235A, delG236, and N297A (numbering according to the Kabat EU index). In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 135 or SEQ ID NO: 136. In some embodiments, the polypeptide comprising an SIRPα D1 domain variant and an Fc domain variant forms a homodimer. In some embodiments, the kit or manufactured article is for use according to the treatment methods provided herein.

[0202] In some embodiments, the kit or article of manufacture further comprises an antibody-drug conjugate (ADC). In some embodiments, the ADC comprises an anti-nectin-4 antibody (e.g., enfortumab). In some embodiments, the ADC comprises an antibody that specifically binds to nectin-4 (e.g., human nectin-4) conjugated to a cytotoxic drug. In some embodiments, the cytotoxic drug is monomethyl auristatin-E (MMAE), a small molecule microtubule-disrupting agent also known as vedotin. In some embodiments, the ADC is enfortumab vedotin. In some embodiments, the polypeptide (e.g., fusion polypeptide) and the ADC are provided in the same container or in separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The container can be formed from a variety of materials, such as glass, plastic (such as polyvinyl chloride or polyolefin), or alloy (such as stainless steel or hastelloy). In some embodiments, the container holds the formulation, and a label on the container or a label associated with the container can indicate instructions for use.

[0203] The manufactured product or kit may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and accompanying documents including instructions for use. In some embodiments, the kit includes an accompanying document or label containing instructions for using a polypeptide (e.g., a fusion polypeptide) in combination with an antibody-drug conjugate (e.g., enfortumab vedotin) for the treatment or delay of progression of cancer (e.g., urothelial cancer, such as urothelial cancer further detailed elsewhere herein) in an individual (such as a human individual). In some embodiments, the accompanying document or label provides instructions for administering the polypeptide (e.g., a fusion polypeptide) to an individual in need thereof at a dose of up to 60 mg / kg. In some embodiments, the accompanying document or label provides instructions for administering the polypeptide (e.g., a fusion polypeptide) to an individual once every two weeks (q2w) or once every 14 days at a dose of 20 mg / kg. In some embodiments, the accompanying document or label provides instructions for administering the polypeptide (e.g., a fusion polypeptide) to an individual once every two weeks (q2w) or once every 14 days at a dose of 30 mg / kg. In some embodiments, the accompanying document or label provides instructions for administering the polypeptide (e.g., a fusion polypeptide) to an individual once every two weeks (q2w) or once every 14 days at a dose of 15 mg / kg.

[0204] Suitable containers include, for example, bottles, vials, bags, and syringes. The container can be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or an alloy (such as stainless steel or Hastelloy). In some embodiments, the container holds the formulation, and a label on the container or a label associated with the container can indicate usage instructions. The manufactured product or kit can further include other buffers, diluents, filters, needles, syringes, and accompanying documents including usage instructions, as well as other materials desirable from a commercial and user perspective. In some embodiments, the manufactured product further includes one or more of another agent (such as a chemotherapeutic agent, an anti-neoplastic agent, a therapeutic antibody, etc.). Suitable containers for one or more agents include, for example, bottles, vials, bags, and syringes.

[0205] This specification is considered to be sufficient to enable those skilled in the art to practice the invention. Various modifications of the invention other than those shown and described herein will be apparent to those skilled in the art from the above description and are 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

[0206] The present disclosure will be more fully understood by reference to the following examples. However, the examples should not be construed as limiting the scope of the present disclosure. The examples and embodiments described herein are for illustrative purposes only, and it should be understood that various modifications or changes in view of them will occur to those skilled in the art and are within the spirit and scope of the present application and the scope of the appended claims.

[0207] Example 1: Evaluation of the Binding of Drug B and Drug C to Human Fcγ Receptors Overview IgG antibodies mediate the phagocytosis of target tumor cells by inducing antibody-dependent cell phagocytosis (ADCP) or antibody-dependent cell-mediated cytotoxicity (ADCC) through binding to Fc gamma receptors (FcγR) on effector cells (e.g., macrophages). Most effector cells express multiple FcγRs. The Fc gamma receptors for the IgG1 class of antibodies are FcγRI / CD64, FcγRII / CD32, and FcγRIII / CD16. FcγRI / CD64 is a high-affinity receptor, while FcγRII and FcγRIII are low-affinity receptors. In humans, FcγRIIA has an H / R131 single nucleotide polymorphism, and one well-characterized FcγRIII single nucleotide polymorphism is V / F158. The IgG Fc domain binds to multiple FcγRs with varying affinities and contributes to the clearance of target cells even in the case of low-affinity interactions for immune complexes with high avidity (Armour et al. (2003) Mol. Immunol. 40:585-593; Nagelkerke et al. (2019) Front Immunol. 10:2237; Kang et al. (2019) Front Immunol. doi.org / 10.3389 / fimmu.2019.00562). The human IgG1 isotope binds to human FcRn with a published K of 760 + / - 60 nM at 25°C, pH 5.8 (Abdiche et al. (2015) MAbs. 7(2):331-43). D at (Abdiche et al. (2015) MAbs. 7(2):331-43).

[0208] Enfortumab vedotin, an antibody-drug conjugate (ADC), contains an mc-vc-PAB-MMAE linker payload conjugated to an interchain cysteine. Such conjugation may limit the binding of the Fc region of enfortumab to Fc receptors and may affect the ability of the ADC to mediate antibody-dependent cell phagocytosis (ADCP). To determine whether the conjugation of the linker payload affects the ability of the Fc region of enfortumab to retain its effector functions (e.g., those mediated through binding to human Fc gamma receptors (FcγR) and the neonatal Fc receptor (FcRn)), the affinities of hFcγIa, hFcγIIa-H131, hFcγIIa-R131, hFcγIIIa, FcγIIIaV158F, and hFcRn for drug B (i.e., an enfortumab analog) and drug C (i.e., an enfortumab vedotin analog) were evaluated by surface plasmon resonance (SPR).

[0209] Materials and Methods Gene Synthesis, Antibody Expression, and Purification of Drug B Monoclonal Antibody The amino acid sequence of drug B (i.e., an enfortumab analog that specifically binds to human nectin-4) was based on the published amino acid sequence of enfortumab (see KEGG database entry D1154; CAS: 1448664-46-7; PubChem database entry 384585500). The sequences of the antibody heavy chain and antibody light chain of drug B (see below) were generated by gene synthesis and codon-optimized for expression in mammalian cells (ATUM). The heavy chain and light chain genes were cloned into separate mammalian expression vectors and co-transfected transiently into Expi293F cells (ThermoFisher). Antibody expression was carried out in Expi293 expression medium, and the cell culture supernatant was harvested 5 days after transfection. Drug B was purified using MABSELECT PrismA Resin (Cytiva) and buffer-exchanged into 1x phosphate-buffered saline (pH 7.4). Data from analytical size-exclusion chromatography (Cytiva, Superdex 200 10 / 300) indicated that drug B was approximately 99% monomeric.

[0210] The amino acid sequences of the light chain of Drug B and the heavy chain of Drug B are provided below. The light chain variable domain and the heavy chain variable domain are underlined. Light chain of Drug B:

Chemical formula

Chemical formula

[0211] The amino acid sequences of the extracellular domain (ECD) of human nectin-4 and the ECD of cynomolgus nectin-4 are provided below. His6 (HHHHHH SEQ ID NO: 223) is fused to the C-terminal domain of the ECD of human and cynomolgus nectin-4 (shown in bold below). Human nectin-4-ECD

Chemical formula

Chemical formula

[0212] Determination of the affinity of Drug B for nectin-4 All experiments were performed at 25 °C and 37 °C using a Biacore 8K high-throughput high-sensitivity SPR system (Cytiva, Global Life Sciences Solutions USA LLC, Marlborough, MA) equipped with an S-type sensor chip. All kinetic data analyses were performed using Biacore Insight Evaluation Software Version 3.0.12.15655.

[0213] The running buffer was 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% (v / v) Surfactant P20 (HBS-EP+). All analytes (human and cynomolgus nectin-4-ECD) were used at the nominal concentrations determined using the absorbance at A280 and the molar extinction coefficient calculated. The kinetic injection method used was "single cycle kinetics" (also known as "kinetic titration") (Karlsson et al. (2006) Analytical Biochemistry. 349(1):136-147).

[0214] The interaction of Drug B, that is, the anti-nectin-4 monoclonal antibody with the extracellular domain (ECD) of human nectin-4, was analyzed by flowing nectin-4-ECD over Drug B (200 - 400 RU) captured on a Biacore Series S Sensor Chip ProteinA. Drug B was captured at a concentration of approximately 1 μg / mL in flow cell 2 of each channel at a flow rate of 10 μL / min for a contact time of 60 seconds (s), and buffer was used in flow cell 1. The human nectin-4-ECD analyte was prepared as a 5-step 3-fold dilution series at a nominal maximum concentration of 300 nM, and each analyte series was injected in ascending order of concentration using the single cycle kinetics method. The association time and dissociation time were monitored for 120 seconds and 1800 seconds, respectively, at a flow rate of 30 μL / min. The surface was regenerated using two 15-second pulses at a flow rate of 30 μL / min with 75 mM phosphoric acid (pH 1.6).

[0215] To analyze the data, the following processing steps were applied. The reference reaction of flow cell 1 (reference surface) was subtracted from the active response of flow cell 2 (reaction surface) to obtain subtracted data (2 - 1). Then, the reaction of the closest buffer blank injection (temporally) was subtracted from the reference - subtracted data (2 - 1) to obtain "double - reference" data (Myszka, D.G. (1999). J. Mol. Recognit. 12, 279 - 284). These double - reference data were globally fitted to a simple 1:1 Langmuir binding model with mass transport to determine the apparent association (k a ) and dissociation (k d ) rate constants. Then, from their ratio, the apparent equilibrium dissociation constant or "affinity" constant (K D ) was estimated as K D = k d / k a . Drug B bound to human nectin - 4 - ECD with a K D of approximately 36.3 ± 3.3 nM at 37°C and approximately 14.5 ± 0.7 nM at 25°C. Drug B bound to cynomolgus monkey nectin - 4 - ECD with a K D of approximately 50.7 ± 1.6 nM at 37°C and approximately 22.6 ± 1.7 nM at 25°C. The results suggest that emfortumab and drug B have comparable apparent affinity binding to human nectin - 4 - ECD (in SPR, the K D of emfortumab vedotin at 25°C is about 16 nM (Satpayev D, Morrison RK, Morrison KJM, Gudas J, Jakobovits A, Torgov M, An Z. 2018. Antibody drug conjugates (ADC) that bind to 191P4D12 proteins. US9962454B2), while the K D of drug B was determined to be about 14.5 nM).

[0216] Preparation of Drug C To prepare an antibody-drug conjugate drug C similar to enfortumab vedotin (CAS number 1346452-25-2), drug B of an enfortumab-like antibody was conjugated to maleimidocaproyl-valyl-citrullinyl-p-aminobenzyloxycarbonyl-monomethyl auristatin E linker-payload (MC-Val-Cit-PAB-MMAE, CAS 646502-53-6; obtained from BroadPharm) via the inter-chain cysteine of the antibody. Briefly, a 2.2 mg / ml drug B monoclonal antibody in 1x PBS (pH 7.4), 10% sucrose, 5 mM EDTA, 30 mM Tris-HCL (pH 7.5) was partially reduced at 20 °C for 20 minutes by adding 20 molar equivalents of TCEP (ThermoFisher) to the monoclonal antibody. MC-Val-Cit-PAB-MMAE was dissolved in 100% DMSO and added to the reaction mixture as a 10 molar equivalent 5% v / v DMSO solution to the monoclonal antibody, and the reaction solution was stirred at 20 °C for 2 hours. Thereafter, N-acetylcysteine (SigmaAldrich) was added at 1 molar equivalent to the linker-payload, and the reaction mixture was incubated at 20 °C for 20 minutes. The quenched excess MC-Val-Cit-PAB-MMAE was separated from the antibody-drug conjugate by cation exchange chromatography (Cytiva, HiTrap SP HP resin), and the antibody-drug conjugate of drug C was eluted with 10% sucrose, 150 mM NaCl, 12.5 mM sodium acetate buffer (pH 5.0). From analytical size exclusion chromatography (Cytiva, Superdex 200 10 / 300), drug C was shown to be approximately 99% monomeric.

[0217] Determination of the drug-to-antibody ratio (DAR) of drug C Enfortumab vedotin (CAS number 1346452-25-2) has an average drug-antibody ratio (DAR) of approximately 3.8:1 (see the 2019 US prescribing information for PADCEV®). The DAR of Drug C, an ADC analog of enfortumab vedotin, was determined at CRO Novatia, LLC (USA) using liquid chromatography mass spectrometry (LC-MS). Briefly, samples of Drug C were deglycosylated and reduced using PNGase F treatment (New England Biolabs Rapid PNGase F), and then analyzed by reversed-phase liquid chromatography coupled to mass spectrometry (RPLC-MS). HPLC was performed on an Acquity I-Class UPLC coupled to a halogenated phenyl column 2.1×50 mm, 2.7 mm. Phase A was 0.05% TFA / water and Phase B was 0.05% trifluoroacetic acid / acetonitrile. The gradient was 10–20% solution B for 1 minute, 20–50% solution B for 9 minutes, 0.5 mL / min, 80 °C. The mass spectrometer was a Waters Xevo G2-XS Q-Tof. Data were processed using MassLynx software via Novatia ProMass HR. The results showed that the average drug-antibody ratio of Drug C was 3.85:1, similar to the DAR value (3.8:1) of enfortumab vedotin.

[0218] Binding of Drug B and Drug C to human FcγR All SPR experiments were performed at 25 °C using a Biacore 8K equipped with an S-type sensor chip, and kinetic data analysis was performed using Biacore Insight Evaluation Software.

[0219] The running buffer was HBS-EP+ (10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.05% (v / v) Surfactant P20) for all hFcγR interactions. All hFcγR analytes were used at their nominal concentrations determined using the absorbance at A280 and the molar extinction coefficient calculated.

[0220] The interaction of Drug B and Drug C with human FcγR was analyzed by flowing the extracellular domain (ECD) of hFcγR over Drug B or Drug C captured on a CM5 chip coated with nectin-4. Up to 2400 RU of human nectin-4 ECD was immobilized on both flow cells (1 and 2) of the CM5 chip using amine chemistry according to the instructions of the Cytiva amine coupling kit. Drug B and Drug C were captured in flow cell 2 of each channel at 2 μg / mL in HBS-EP+ at a flow rate of 10 μL / min for 120 seconds (100 - 200 RU). The analyte was injected in single-cycle kinetics mode at a nominal maximum concentration of 30 nM for hFcγRI (CD64) in a 3-fold dilution series, or 3000 nM for hFcγRIIa (CD32a) or hFcγRIIIa (CD16a) in a 3-fold dilution series. The association time was monitored for 120 seconds and the dissociation time for 600 seconds (except for hFcγRI where the dissociation time was 1800 seconds). The surface was regenerated using 75 mM phosphoric acid (pH 1.6) with two 15-second pulses at a flow rate of 30 μL / min.

[0221] The data were processed and analyzed using Biacore 8K Evaluation Software Version 3.0.12.15655 (Cytiva, Global Life Sciences Solutions USA LLC, Marlborough, MA). The reference reaction in flow cell 1 was subtracted from the active response in flow cell 2 to obtain subtracted data (2 - 1). Then, the reaction of the closest buffer blank injection was subtracted from the reference-subtracted data (2 - 1) to obtain double-reference data. For hFcγRI, these double-reference data were fitted to a simple 1:1 Langmuir binding model with mass transport to determine the apparent association (ka) and dissociation rate constants (kd). Then, based on their ratio, the apparent equilibrium dissociation constant or affinity constant (K Dwas calculated as (kd / ka). The binding affinity K for the interaction of all other hFcγRs (hFcγIIa-H131, hFcγIIa-R131, hFcγIIIa, and FcγIIIaV158F) D values were analyzed using the "steady state" (or "equilibrium binding") method due to the fast on-rate and fast off-rate.

[0222] Binding of Drug B and Drug C to Human FcRn All experiments were performed at 25 °C using a Biacore 8K equipped with an S-type sensor chip, and kinetic data analysis was performed using Biacore Insight Evaluation Software. The running buffer for the hFcRn interaction was PBS (pH 5.8) containing 0.01% Tween-20, and the running buffer for capture was HBS-EP+. The hFcRn ECD protein of the analyte was used at its nominal concentration. The "single cycle kinetics" injection mode was used.

[0223] The interaction of Drug B and Drug C with hFcRn was analyzed by flowing the hFcRn ECD protein over Drug B or Drug C captured on a CM5 chip coated with nectin-4. Up to 2400 RU of human nectin-4 ECD was immobilized on both flow cells (1 and 2) of the CM5 chip using amine chemistry according to the instructions of the Cytiva amine coupling kit. Drug B or Drug C was captured in flow cell 2 of each channel at 2 μg / mL in HBS-EP+ at 10 μL / min for 120 s (100 - 200 RU) by surface treatment. The hFcRn experiments were performed in PBS (pH 5.8) containing 0.01% Tween-20. The hFcRn analyte was prepared as a 5-step 3-fold dilution series with a nominal highest concentration of 3000 nM, and these samples were injected in ascending order of concentration using the single cycle mode. The association time and dissociation time were monitored for 120 s and 600 s, respectively. The surface was regenerated using 2 pulses of 30 s at a flow rate of 30 μL / min with PBS (pH 7.4). The hFcRn was efficiently removed while maintaining the capture antibody on the chip with a neutral pH buffer.

[0224] The data was processed and analyzed using Biacore 8K Evaluation Software Version 3.0.12.15655 (Cytiva, Global Life Sciences Solutions USA LLC, Marlborough, MA). The reference reaction of flow cell 1 was subtracted from the active response of flow cell 2 to obtain subtraction data (2-1). Then, the reaction of the closest buffer blank injection was subtracted from the reference subtraction data (2-1) to obtain double reference data. The binding affinity K D values were analyzed using the "steady state" (or "equilibrium binding") method due to the fast on-rate and fast off-rate.

[0225] Results Binding of Drug B and Drug C to Human FcγR Drug C (i.e., an emfortumab vedotin analog) and Drug B (i.e., an emfortumab analog without a conjugate) were found to have comparable k a k d and apparent affinity K D bindings. See Table A below.

[0226] Binding of Drug B and Drug C to Human FcRn Drug C (i.e., an emfortumab vedotin analog) and Drug B (i.e., an emfortumab analog without a conjugate) were found to have comparable k a k d and apparent affinity K D bindings. See Table A below. [Table 13]

[0227] Conclusion The results of SPR kinetics comparing the binding of Drug B (enfortumab-like mAb) and Drug C (enfortumab vedotin-like ADC) to human hFcγR and hFcRn indicate that Drug B (i.e., enfortumab analog) and Drug C (i.e., enfortumab vedotin analog) bind to human hFcγR and hFcRn with similar affinities. Therefore, the presence of the linker-payload mc-vc-PAB-MMAE conjugated to the interchain cysteine of enfortumab vedotin at an average drug-to-antibody ratio of 3.85:1 does not appear to affect the ability of Drug C to bind to human FcγR and human FcRn receptors.

[0228] Example 2: Evaluation of the effect of Drug A on antibody-dependent cell phagocytosis (ADCP) in combination with Drug B or Drug C Overview Enfortumab vedotin-ejfv is a nectin-4-directed antibody-drug conjugate (ADC) consisting of a full-length human anti-nectin-4 IgG1 kappa monoclonal antibody conjugated to the small molecule microtubule-disrupting agent monomethyl auristatin E via a protease-cleavable maleimidocaproyl valine-citrulline linker (referred to herein as mc-vc-PAB-MMAE). The enfortumab vedotin linker-payload is conjugated to the interchain cysteine residue contained in the interchain disulfide bond of the antibody, resulting in a product with a drug-to-antibody ratio of approximately 3.8:1 (see PADCEV® package insert, United States). In the human IgG1 antibody structure, the interchain cysteine between the heavy chains is located within the hinge region, and the interchain cysteine between the heavy and light chains is located at the interface of the human IgG1 heavy chain domain CH1 and the human IgG1 light chain kappa constant domain (CK). Therefore, conjugation of the linker-payload mc-vc-PAB-MMAE to the interchain cysteine of enfortumab vedotin may introduce steric hindrance to the binding of the Fc region to FcγR and FcRn receptors.

[0229] The purpose of this experiment was to evaluate whether the presence of the linker payload in the Fc and Fc-proximal regions of Drug C affects the Fc effector function mediated by ADCP. Therefore, in combination with Drug B (i.e., an enfortumab analog) and Drug C (i.e., an enfortumab vedotin analog), the effect of Drug A on antibody-dependent cell phagocytosis (ADCP) was evaluated in three human cancer cell lines expressing human nectin-4 at different levels. Drug A is an exemplary SIRPα variant-Fc variant fusion polypeptide that has high affinity for human CD47 and lacks Fc effector function.

[0230] Materials and Methods Cell Lines OE19 (Sigma 96071721-1VL) and T47D (ATCC HTB-133) cells were maintained in growth medium consisting of RPMI-1640 (Thermo Fisher Scientific 11875119) supplemented with 10% FBS (Thermo Fisher Scientific 26140079), 1% penicillin / streptomycin (Thermo Fisher Scientific 15140163), and 1% GlutaMAX (Thermo Fisher Scientific 35050061). OE19 is a human esophageal adenocarcinoma cell line. T47D is a human breast cancer (invasive ductal carcinoma) cell line. Information regarding the nectin-4 expression levels of OE19 and T47D is provided in Table B.

[0231] HT-1376 (ATCC CRL-1472) cells were maintained in growth medium consisting of DMEM (Thermo Fisher Scientific 11965092) supplemented with 10% FBS (Millipore TMS-013-B), 1% penicillin / streptomycin (Thermo Fisher Scientific 15140163), and 1% GlutaMAX (Thermo Fisher Scientific 35050061). HT-1376 is a human bladder carcinoma cell line. Information regarding the nectin-4 expression level of HT-1376 is provided in Table B.

[0232] Quantification of Receptor The cell line was recovered with TryPLE Select (Thermo Fisher Scientific 12563029), counted, and 2×10 5 cells were seeded into a U-bottom 96-well plate (Falcon 353227). After centrifugation, the cells were washed with ice-cold FACS buffer composed of PBS containing 0.5% BSA (Thermo Fisher Scientific 15260-037). The cells were incubated with 10 μg / mL of nectin-4-AF647 conjugate antibody (clone 337516, R&D Systems FAB2659R) at 4°C. After 1 hour of incubation, the cell suspension was washed twice with ice-cold FACS buffer and centrifuged at 400×g for 5 minutes. The sample was resuspended in 100 μL of FACS buffer and the cells were analyzed with an Attune NxT cytometer (Thermo Fisher Scientific). The effective fluorescence dye-to-protein ratio (F / P) was determined by using SIMPLE CELLULAR® anti-human IgG beads (Bangs Laboratories 816A). One drop of SIMPLE CELLULAR® anti-human IgG beads was added to 100 μL of 10 μg / ml nectin-4-AF647 solution. The mixture was incubated on ice in the dark for 30 minutes. Then, the sample was washed twice with 2 mL of ice-cold FACS buffer and centrifuged at 400×g for 5 minutes. 500 μL of FACS buffer was added to the sample and then analyzed with an Attune NxT cytometer on the same day as the cells. A total of 10,000 events were recorded and analyzed with FlowJo (BD).

[0233] Induction and Culture of Human Monocyte-Derived Macrophages Human leukocyte-depleted whole blood (Vitalant Blood Center) was diluted 1:3 with PBS (Thermo Scientific 10010072). The diluted blood was overlaid with 10 mL of Ficoll-Paque Premium (Cytiva 17-5442-02). The tube was centrifuged at 400×g for 30 minutes. PBMCs were collected from the interface, washed twice by adding 40 mL of PBS, centrifuged at 400×g for 10 minutes, and resuspended in magnetic-activated cell sorting (MACS) buffer (PBS containing 0.5% BSA (Thermo Fisher Scientific 15260-037) and 2 mM EDTA (Teknova E0307-06)). CD14 + Monocytes were purified by negative selection using the Monocyte Isolation Kit II (Miltenyi Biotec 130-091-153) and LS column (Miltenyi Biotec 130-042-401) according to the manufacturer's protocol. CD14 + Monocytes were seeded at 10 million cells / dish in 150 mm tissue culture dishes (Falcon 353025) containing 25 mL of medium composed of RPMI-1640 supplemented with 10% FBS (Thermo Fisher Scientific 26140079), 1% penicillin / streptomycin (Thermo Fisher Scientific 15140163), 1% GlutaMAX (Thermo Fisher Scientific 35050061), and 50 ng / mL of M-CSF (Miltenyi 130-096-492). The cells were cultured for 7 days.

[0234] In vitro phagocytosis assay HT-1376, T47-D, and OE19 cells were detached from the culture plates by washing once with 10 mL of PBS and incubated in 5 mL of TrypLE Select at 37 °C for 10 minutes. The cells were washed twice with PBS and resuspended in PBS. HT-1376, T-47D, and OE19 cells were labeled by suspending with 150 nM of CFSE according to the manufacturer's instructions using the Celltrace CFSE Cell Proliferation Kit (Thermo Fisher Scientific C34554) and resuspended in RPMI-1640. Macrophages were detached from the culture plates by washing once with 10 mL of PBS and incubated in 5 mL of TrypLE Select at 37 °C for 20 minutes. The cells were removed with a cell scraper (Corning 3008), washed with PBS, and resuspended in RPMI-1640.

[0235] CFSE-labeled T47-D and OE19 target cells were added to ultra-low attachment U-bottom 96-well plates at 100,000 cells / well. Drug B or Drug C was added at a concentration of 40 ng / mL or 8 ng / mL, and Drug A was added at a concentration of 6.25 nM, 0.40 nM, or 90 pM. EC 50To determine, HT-1376 and OE19 target cells labeled with CFSE were added to an ultra-low attachment U-bottom 96-well plate (Corning 7007) at 100,000 cells / well. Drug B or Drug C was added at a concentration of 200 ng / ml. Next, a 10-fold dilution series of Drug A from 100 nM to 0.1 pM was added. The plates containing the target cells, Drug A, and Drug B or Drug C were incubated at 37 °C for 20 minutes in a humidified incubator with 5% carbon dioxide before adding 50,000 macrophages. After 20 minutes, the cultured macrophages were added, and the plates were incubated for an additional 2 hours at 37 °C in a humidified incubator with 5% carbon dioxide. The cells were pelleted by centrifugation at 400 g for 5 minutes and stained for 30 minutes at 4 °C in 100 μL of Fixable Viability Dye eFluor 780 (ebioscience 65-0865-14) diluted 1:5000 in PBS. The cells were washed with 200 μL of FACS buffer (PBS + 2% FBS) and stained on ice for 60 minutes in 50 μL of FACS buffer containing 2 μL of human FcR blocking reagent (Miltenyi Biotec 130-059-901), 0.5 μL of BV421 anti-CD163 (clone GHI / 61, Biolegend 333612), 0.5 μL of PE-Cyanine7 CD11b (clone ICRF44, Thermo Scientific 25-0118-42), and 0.5 μL of PECD326 (clone 9C4, Biolegend 324206). The cells were washed twice with 250 μL of FACS buffer and fixed overnight at 4 °C in 100 μL of 0.5% paraformaldehyde (Electron Microscopy Sciences 15710) PBS solution. The cells were analyzed on a FACS Canto II (BD Biosciences), and the data were subsequently analyzed by Flowjo 10.8 (BD). Dead cells were excluded by gating on the e780-negative population. Macrophages were identified as cells positive for the lineage markers CD11b and CD163. Among this population, macrophages that had phagocytosed tumor cells were identified as CFSE-positive cells.To remove non-phagocytosed CFSE-labeled tumor cells from the analysis, cells positive for the epithelial cell marker CD326 were excluded. The phagocytosis percentage was calculated as the percentage of viable CD11b - ) that were CFSE-positive stained and CD163 + negative (CD326 + ) and was calculated as the percentage of human monocyte-derived macrophages.

[0236] The data was plotted to identify the maximum phagocytosis value, and the EC 50 value was calculated using Prism 9 software (Graphpad). The average phagocytosis level and average EC 50 value were calculated using Excel (Microsoft). Error bars represent the standard deviation from the mean.

[0237] Results Table B provides a summary of the nectin-4 receptor numbers of the tested cell lines. In Table B, the number of nectin-4 receptors expressed on the cell surface of the tested cell lines (e.g., the average number) ranged from a maximum of 110,312 to a minimum of 43,784. [Table 14]

[0238] Table C provides a summary of the effects of Drug B, Drug C, Drug A + Drug B, and Drug A + Drug C on the phagocytosis of TROP2-expressing cell lines by macrophages derived from monocytes obtained from human donors. [Table 15]

[0239] In the absence of Drug A, Drugs B and C stimulated ADCP by 1.37-fold and 1.37-fold, respectively, compared to medium alone. The combination of Drug A with Drug B or Drug C enhanced ADCP for all cell lines by 2.39-fold on average compared to medium alone. Drug A enhanced the ADCP of Drugs B and C in OE19 and HT-1376, and the EC 50The overall averages were 24.54 pM and 6.19 pM, respectively.

[0240] The results of drug A, which increased the in vitro phagocytosis of drugs B and C using T-47D and OE19 from two different donors, are shown in FIGS. 1A and 1B. FIG. 1A shows the enhancing effect of drug A on the drug B or drug C-induced phagocytosis of OE19 cells and T47D cells by human monocyte-derived macrophages obtained from the first donor. The percent phagocytosis, defined as the percentage of viable macrophages that phagocytosed CFSE-labeled OE19 cells or T47D cells, is shown on the y-axis. The single-agent or combination parameters are shown on the x-axis. FIG. 1B shows the enhancing effect of drug A on the drug B or drug C-induced phagocytosis of OE19 cells and T47D cells by human monocyte-derived macrophages obtained from the second donor. The percent phagocytosis, defined as the percentage of viable macrophages that phagocytosed CFSE-labeled OE19 cells or T47D cells, is shown on the y-axis. The single-agent or combination parameters are shown on the x-axis. The EC 50 of the in vitro phagocytosis assay using OE19 cells is shown in FIG. 2. In FIG. 2, the percent phagocytosis, defined as the percentage of viable macrophages that phagocytosed CFSE-labeled OE19 cells, is shown on the y-axis. The concentration of drug A (nM) is shown on the x-axis. The percentage of phagocytosis of cells treated with drug B alone, drug C alone, or medium alone is shown at 0 nM of drug A and indicated by an arrow. The percentage of phagocytosis of cells treated with drug A + drug B (open circles), drug A + drug C (filled circles), and drug A alone (open squares) is shown. Error bars represent the standard deviation of three technical replicates. The EC 50 was calculated for each curve by sigmoid dose-response variable slope fitting. The EC of the in vitro phagocytosis assay using HT-1376 50The results are shown in Figure 3. In Figure 3, the y-axis shows the percentage of phagocytosis defined as the percentage of viable macrophages that phagocytosed CFSE-labeled tumor cells. The x-axis shows the concentration of Drug A (nM). The phagocytosis percentages of cells treated with Drug B alone, Drug C alone, or medium alone are shown at 0 nM of Drug A and indicated by arrows. The phagocytosis percentages of cells treated with Drug A + Drug B (open circles), Drug A + Drug C (filled circles), or Drug A alone (open squares) are shown. Error bars represent the standard deviation of three technical replicates. EC 50 was calculated for each curve by sigmoid dose-response variable slope fitting.

[0241] Conclusion The effect of Drug A on the ADCP of Drugs B and C was evaluated in an in vitro phagocytosis assay based on flow cytometry. In multiple tumor cell lines expressing nectin-4 receptor over a range, Drug A enhanced the ADCP of Drugs B and C, and the overall mean of EC 50 was 24.54 pM and 6.19 pM, respectively. As single agents, Drugs B and C stimulated ADCP by an average of 1.41-fold and 1.32-fold, respectively, across the cell lines compared to the background levels observed with medium alone. Combinations of Drug A with Drug B and Drug A with Drug C enhanced ADCP by an average of 2.42-fold and 2.36-fold, respectively, compared to the medium-alone control. In conclusion, the presence of mc-vc-PAB-MMAE of linker-payload conjugated to the interchain cysteine of Drug C at an average drug-to-antibody ratio of 3.85:1 does not appear to affect the ability of Drug C to mediate ADCP, either alone or in combination with Drug A.

[0242] Example 3: Phase 1 Safety, Pharmacokinetics, and Pharmacodynamics Study of the Combination of Drug A and Enfortumab Vedotin in Subjects with Urothelial Carcinoma This example describes a Phase 1 clinical trial regarding the combination of Drug A and enfortumab vedotin in subjects with locally advanced or metastatic urothelial carcinoma.

[0243] (A) Test Design This trial includes a dose escalation part (Phase 1a) and a dose expansion part (Phase 1b). The test design is shown in Figure 4. Approximately 30 adult subjects (i.e., 18 years old and above) are enrolled. This trial is designed to establish the safety and tolerability, maximum tolerated dose (MTD), Phase 2 recommended dose (RP2D), PK profiles for single and multiple administrations, and PD markers (including but not limited to target occupancy) of Drug A and Enfortumab Vedotin, and to characterize the preliminary activity (e.g., therapeutic activity) of the combination of Drug A and Enfortumab Vedotin.

[0244] Drug A, in combination with Enfortumab Vedotin, is administered intravenously (IV) once every 2 weeks (Q2W) at a dose escalation level cohort starting from an initial dose of 20 mg / kg, and Enfortumab Vedotin is administered at a standard dose and schedule of 1.25 mg / kg IV on days 1, 8, and 15 of each 28-day cycle.

[0245] The dose of Drug A is escalated, and the occurrence of dose-limiting toxicity (DLT) is evaluated using the Bayesian optimal interval (BOIN) design (see Liu et al. (2015) Journal of the Royal Statistical Society. Series C: Applied Statistics. 64(3):507-523 and Yuan et al. (2016) Clin Cancer Res. 22(17):4291-4301). Drug A is evaluated at two dose levels: 20 mg / kg Q2W and 30 mg / kg Q2W. If 20 mg / kg Q2W is not tolerated, the low dose level of Drug A (i.e., 15 mg / kg Q2W) is evaluated. Other dose levels and / or schedules below the maximum tolerated dose (MTD) may also be evaluated.

[0246] In the BOIN design with increasing dose levels, the target dose-limiting toxicity (DLT) rate for the maximum tolerated dose (MTD) is set to 0.25. A cohort of three subjects is enrolled and evaluated for DLT. DLT is evaluated for each cohort and is described in more detail below. DLT is evaluated with a 28-day evaluation period in cycle 1. The BOIN design uses the following rules with overdose control to guide dose escalation / de-escalation: · If the DLT rate observed at the current dose is 0.197 or less, increase the dose to the next higher dose level; · If the observed DLT is greater than 0.298, decrease the dose to the next lower dose level; · Otherwise, maintain the current dose level.

[0247] The selection of the MTD is based on isotonic regression as described in Liu et al. (2015) Journal of the Royal Statistical Society. Series C: Applied Statistics. 64(3):507 - 523. Specifically, the MTD is selected as the dose at which the isotonic estimate of the DLT rate is closest to the target DLT rate. In case of ties, the higher dose level is selected if the isotonic estimate is lower than the target DLT rate, and the lower dose level is selected if the isotonic estimate is greater than or equal to the target DLT rate.

[0248] Once the dose levels have been reviewed and approved by the Safety Review Committee (SRC), additional subjects will be registered in the backfill cohort at the same dose level, and for the purpose of dose optimization, further characterization of the safety, PK, PD, and preliminary anti-tumor activity of Drug A and enfortumab vedotin will be performed. Subjects registered in the backfill cohort will not be evaluated for DLT. In the Phase 1a portion, which includes both the dose escalation cohort and the backfill cohort, approximately 15 subjects will be treated per dose level. For the selection of the RP2D, the sponsor will review with the SRC all available safety, PK, PD, and preliminary anti-cancer activity data obtained from the Phase 1a portion, which includes both the dose escalation cohort and the backfill cohort, and make recommendations regarding the dose of Drug A in combination with enfortumab vedotin to be used in the Phase 2 trial.

[0249] At the discretion of the SRC, dose expansion will be initiated in a selected subject population to further evaluate the safety and tolerability of Drug A and enfortumab vedotin and to characterize the preliminary anti-cancer activity (see Figure 4). In the dose expansion portion, the safety and tolerability of other anti-cancer agents, such as checkpoint inhibitors in combination with Drug A and enfortumab vedotin, will be characterized.

[0250] To evaluate the pharmacodynamic endpoints within the tumor, fresh biopsies before and during treatment are required for the backfill and expansion cohorts. For subjects enrolled in the dose-escalation cohort, these biopsies are optional. Subjects have up to 28 days until they complete the screening assessment and receive combination therapy with Drug A and enfortumab vedotin until (a) disease progression, (b) a decision to discontinue treatment by the subject or the physician, (c) the occurrence of unacceptable toxicity, (d) withdrawal of consent, or (e) the end of the trial. Tumor evaluations are performed at baseline and approximately every 8 weeks during the treatment period of the trial. Patients may continue treatment after progression on imaging if, in the judgment of the treating investigator, the subject (i) is deriving a clinical benefit from the trial treatment and shows no clinical signs or symptoms of clinically significant disease progression, (ii) has not had a decline in performance status (PS), (iii) shows no evidence of rapid disease progression or threat to an organ or critical anatomical site essential for life support requiring urgent alternative medical intervention, and (iv) shows no serious, unacceptable, or irreversible toxicity related to the trial treatment. The visit at the end of treatment (EOT) is conducted approximately 4 weeks (at least 28 and within 35 days) after the last dose of Drug A or prior to the initiation of the next cancer therapy to confirm / collect concomitant medications, vital signs, adverse events (AE), and serious adverse events (SAE) and to assess the recovery of any treatment-related toxicity. Subsequent follow-up consists of overall survival data and is collected by telephone every 3 months for 24 months.

[0251] This trial is conducted in accordance with the protocol, the standards for the conduct of clinical trials (GCP), and the applicable regulatory requirements (s).

[0252] (B) Study Objectives and Endpoints The primary objectives of this trial are (1) to evaluate the safety and tolerability of Drug A in combination with enfortumab vedotin in subjects with treatment-experienced locally advanced or metastatic urothelial carcinoma and (2) to determine the maximum tolerated dose (MTD) and the phase 2 recommended phase 2 dose (RP2D) of Drug A in combination with enfortumab vedotin.

[0253] The secondary objectives of this study are: (1) to evaluate the overall safety profile of Drug A in combination with Enfortumab vedotin; (2) to characterize the pharmacokinetics (PK) of single and multiple doses of Drug A in combination with Enfortumab vedotin; (3) to evaluate the immunogenicity of Drug A; and (4) to evaluate the evidence of antitumor activity of Drug A in combination with Enfortumab vedotin.

[0254] The exploratory objectives of this study are: (1) to explore the pharmacodynamic (PD) effects of Drug A in combination with Enfortumab vedotin; and (2) to evaluate methods to mitigate the interference of Drug A in serological tests used for blood product transfusions.

[0255] The primary endpoints of this study are: (1) the first cycle dose-limiting toxicity (DLT) (described in more detail below), and (2) adverse events (AEs) characterized by type, frequency, severity (evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE v.5.0, see, for example, https: / / ctep.cancer.gov / protocoldevelopment / electronic_applications / docs / CTCAE_v5_Quick_Reference_5x7.pdf)), timing, severity, and relationship to the study treatment.

[0256] The secondary endpoints of this study are: (1) clinical laboratory abnormalities characterized by type, frequency, severity (evaluated according to NCI CTCAE v.5.0) and timing; (2) pharmacokinetic parameters of Drug A to the extent data permit, such as maximum serum concentration (C max ), time to reach maximum plasma concentration (T max ), drug exposure over time (area under the curve or AUC), clearance (CL), and half-life (t1 / 2 ) (3) Response Evaluation Criteria in Solid Tumors (RECIST 1.1, see, for example, Eisenhauer et al. (2009) Eur J Cancer 45:228-247), (4) disease control rate (DCR), best overall response rate (BOR), duration of response (DOR), time to progression (TTP), progression-free survival (PFS), and overall survival (OS).

[0257] The exploratory endpoints of this study are: (1) pharmacodynamic effects, such as (a) CD47 target occupancy in peripheral blood before and after drug A dose level, (b) immunophenotyping of circulating leukocyte populations, (c) tumor marker expression, infiltrating leukocyte populations, and immunomodulatory molecules in tumor biopsy tissues before and after test treatment, (d) exploratory molecular analysis (including but not limited to tumor markers and immune markers) in peripheral blood and / or tumor biopsy samples before and after treatment, and (2) characterization of methods for alleviating drug A interference in indirect antiglobulin test (IAT) and direct antiglobulin test (DAT) during drug A treatment.

[0258] (C) Study population Inclusion criteria Subjects must meet the following inclusion criteria to be eligible for enrollment in this study: · Subjects must have histologically confirmed unresectable locally advanced or metastatic urothelial carcinoma (i.e., cancer of the bladder, renal pelvis, ureter, or urethra). Subjects with urothelial carcinoma (transitional cell) with squamous differentiation or mixed cell type are eligible provided that the urothelial carcinoma is the dominant histotype. Subjects with elements of small cell or neuroendocrine histotype are excluded. · The subject has received prior treatment with an immune checkpoint inhibitor (CPI) under the condition of locally advanced or metastatic urothelial carcinoma. Eligible subjects are those who have received CPI therapy in neoadjuvant / adjuvant therapy and have had recurrent or progressive disease either during the therapy or within 12 months after the completion of the therapy. CPI is defined as a programmed cell death protein 1 (PD-1) inhibitor or a programmed cell death ligand 1 (PD-L1) inhibitor (including but not limited to atezolizumab, pembrolizumab, durvalumab, avelumab, and nivolumab). · The subject has received prior treatment with platinum-containing chemotherapy defined as those who have been administered platinum in adjuvant / neoadjuvant therapy and have had recurrent or progressive disease within 12 months after completion, or those who have received treatment with platinum for metastatic or unresectable locally advanced disease. · The subject has progressive or recurrent urothelial carcinoma during or after recent treatment. · The subject must have measurable disease according to RECIST (version 1.1). To be considered measurable, lesions in the previous radiation field must have progressed after radiotherapy. · Have adequate bone marrow function according to the following clinical laboratory values, depending on the disease under test: · Absolute neutrophil count (ANC) ≥ 1,500 / mm 3 (≥ 1.5 × 10 9 / L); · Platelets ≥ 100,000 / mm 3 (≥ 100 × 10 9 / L); · Hemoglobin ≥ 9 g / dL (≥ 90 g / L) · Renal function is appropriate as indicated by a creatinine clearance estimated by the Cockcroft-Gault formula or other medically acceptable formulas such as the Modification of Diet in Renal Disease (MDRD) or Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) of ≧ 30 mL / min. · Adequate liver function according to the following clinical test values, corresponding to the disease under test: · Serum total bilirubin ≦ 1.5 × upper limit of normal value (ULN) (≦ 3.0 × ULN if the subject is confirmed to have Gilbert syndrome); · Aspartate aminotransferase and alanine aminotransferase (AST and ALT) ≦ 3.0 × ULN or ≦ 5.0 × ULN in subjects with liver metastases; · Alkaline phosphatase ≦ 2.5 × ULN or ≦ 5.0 × ULN in subjects with bone metastases or liver metastases. · The interval of the Fridericia formula corrected QT interval (QTcF) ≦ 480 msec (based on the average of three electrocardiograms [ECG]). · 18 years of age or older. · Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1. · Subjects in the dose-escalation cohort shall provide a stored (or fresh) biopsy sample available before study registration. Subjects in the backfill cohort and expansion cohort have a tumor amenable to sequential biopsy and are willing to provide fresh tumor tissue biopsies (core needle biopsy or resection required) before treatment and during the study. · Serum pregnancy test (for women of childbearing potential) is negative at screening. · Male and female subjects with reproductive potential shall consent to use an effective contraceptive method during the study period and for at least 4 months after the last dose of study treatment.

[0259] Exclusion Criteria Subjects with any of the following characteristics / conditions will not be included in the study: · Having sensory or motor neuropathy of grade 2 or higher. · Having symptomatic or uncontrolled central nervous system (CNS) metastases. Subjects who have received treatment for CNS metastases will be permitted to participate in the study if all of the following apply: · The CNS metastases have been clinically stable for at least 6 weeks prior to screening; · If steroid treatment is required for CNS metastases, the subject has been taking a constant dose of prednisone or equivalent at 20 mg / day or less for at least 2 weeks; · No new or enlarging brain metastases are detected on the baseline scan; and · The subject does not have leptomeningeal disease. · Prior treatment with enfortumab vedotin or other monomethyl auristatin (MMAE)-based antibody-drug conjugate (ADC) · Prior treatment with any anti-CD47 or anti-signal regulatory protein alpha (SIRPα) agent · Having a known hypersensitivity to enfortumab vedotin or any excipient (including histidine, trehalose dihydrate, and polysorbate 20) contained in the pharmaceutical formulation of enfortumab vedotin; or having a known hypersensitivity to biopharmaceuticals produced in Chinese hamster ovary (CHO) cells. · Subjects who are intolerant to antibodies or injected therapeutic proteins, or who have had a severe allergic reaction or anaphylactic reaction, or who have had a severe allergic reaction or anaphylactic reaction to any of the substances contained in Drug A. · Clinically significant toxicity (excluding alopecia grade 2 or higher) associated with prior treatment (including systemic therapy, radiotherapy, or surgery) persists. Subjects with hypothyroidism or panhypopituitarism related to immunotherapy grade 2 or lower are enrolled if they are well maintained / controlled with a certain dose of hormone replacement therapy (if necessary). Subjects with hypothyroidism or panhypopituitarism related to immunotherapy grade 3 or higher that persists are excluded. Subjects with ongoing colitis, uveitis, myocarditis, or pneumonia related to immunotherapy, or other immunotherapy-related AEs requiring high-dose steroids (prednisone or equivalent >20 mg / day) are excluded. · Currently undergoing treatment with systemic antibacterial agents for active infectious diseases (viral, bacterial, or fungal). Regular antibacterial prophylaxis is acceptable. · Have active, uncontrolled hepatitis B (HBV), hepatitis C (HCV), and human immunodeficiency virus (HIV) infections. · Have met any of the following in the past 6 months: myocardial infarction, severe / unstable angina, coronary / peripheral artery bypass surgery, congestive heart failure New York Heart Association (NYHA) class II or higher, uncontrolled hypertension, cerebrovascular attack, transient ischemic attack, deep vein thrombosis (excluding device-related thrombi not considered clinically problematic), arterial thrombosis, symptomatic pulmonary embolism, or any other major thromboembolic event. · Currently receiving active drug treatment in any other interventional treatment clinical trial. · Radiation therapy or major surgery within 14 days before the first dose of the study drug. · Antitumor treatment with chemotherapy, biological agents, investigational drugs, and / or immunotherapy that has not been completed within 28 days or 5 half-lives (whichever is shorter) before the first dose of the study drug. · Any experimental antibody or live vaccine within 28 days before the first administration of the investigational drug. Examples of live vaccines include, but are not limited to: measles, mumps, rubella, varicella / zoster, yellow fever, rabies, Bacillus Calmette-Guérin (BCG), and typhoid vaccine. Injectable seasonal influenza vaccine is generally an inactivated virus vaccine and is acceptable. However, intranasal influenza vaccine (e.g., FluMist®) is a live attenuated vaccine and is not permitted. · Subjects with a history of another malignancy within 3 years before the first administration of the investigational drug, or any evidence of residual lesions of a previously diagnosed malignancy. Subjects with non-melanoma skin cancer, localized prostate cancer without evidence of progression treated with intent to cure, low-risk or very low-risk (according to standard guidelines) localized prostate cancer under surveillance / observation therapy without intent to treat, or any type of intraepithelial neoplasia (if complete resection has been performed) are permitted. · Subjects with an active autoimmune disease that required systemic treatment in the past 1 year (i.e., use of disease-modifying agents, corticosteroids, or immunosuppressive drugs). Replacement therapy (e.g., thyroxine, insulin, or physiological corticosteroid replacement therapy for adrenal or pituitary insufficiency) is not considered a form of systemic treatment and is permitted. · Other severe and acute or chronic medical or psychiatric conditions, including recent (within the past 1 year) or ongoing suicidal ideation or suicidal behavior, or clinical laboratory abnormalities that may increase the risk associated with participation in the trial or administration of the investigational drug, or may interfere with the interpretation of the trial results, and for which the subject's participation in this trial is considered inappropriate. · A history of autoimmune hemolytic anemia, autoimmune thrombocytopenia, or hemolytic transfusion reaction. · Having active keratitis or corneal ulcer. Subjects with punctate superficial keratitis are permitted if the disease is appropriately treated. · A history of uncontrolled type 2 diabetes within 3 months of the first administration of the investigational drug. Uncontrolled diabetes is defined as hemoglobin A1C (HbA1c) ≥ 8% or HbA1c < 7-8% with associated diabetes symptoms (e.g., polyuria or polydipsia) without additional explanation. · Currently pregnant or breastfeeding.

[0260] (D) Investigational drug, dosage, and administration In the dose escalation part of the study, the initial starting dose of Drug A is 20 mg / kg IV Q2W. If it is considered safe, the dose of Drug A will be escalated to the maximum dose of 30 mg / kg IV Q2W as defined in the protocol. If 20 mg / kg Q2W is not tolerated, a lower dose level of Drug A (i.e., 15 mg / kg IV Q2W) may be evaluated. Other dose levels and / or schedules below the MTD may also be evaluated. Dose escalation and backfill cohorts will determine the optimal dose and schedule of the Phase 2 recommended Phase 2 dose (RP2D) of Drug A in combination with enfortumab vedotin. In the dose expansion part of the study, Drug A will be administered at a dose below the MTD in combination with enfortumab vedotin determined in the dose escalation part of the study. Enfortumab vedotin will be administered IV at a standard dose of 1.25 mg / kg on days 1, 8, and 15 of each 28-day cycle. See Table D below.

Table 16

[0261] As described elsewhere in this specification, the maximum tolerated dose (MTD) is determined using the Bayesian optimal interval (BOIN) design (Liu et al. (2015) Journal of the Royal Statistical Society. Series C: Applied Statistics. 64(3):507-523 and Yuan et al. (2016) Clin Cancer Res. 22(17):4291-4301). The BOIN design is implemented in a simple manner similar to the conventional 3+3 design, but is more flexible and has excellent operating characteristics comparable to complex model-based designs such as the continual reassessment method (CRM) (see Zhou et al. (2018) Clin Cancer Res. 24(18):4357-4364).

[0262] Each single-use vial of Drug A is supplied in a 1000 mg / 50 mL Type I clear glass vial sealed with a 20 mm Teflon-coated rubber serum stopper and a tamper-evident aluminum seal, delivering 1000 mg of Drug A (50 mL) and intended for intravenous (IV) administration.

[0263] Complete information regarding the dosage form and packaging of enfortumab vedotin can be found in the U.S. or local prescribing information. For the U.S., see, for example, https: / / astellas(dot)us / docs / PADCEV(underscore)label(dot)pdf.

[0264] Drug A is administered as an outpatient intravenous infusion once every two weeks. The dose is infused over approximately 60 minutes. To ensure accurate delivery of the investigational drug, use of an infusion pump is the preferred method of administration, but gravity infusion is also acceptable.

[0265] One cycle is defined as the time from the first dosing day to the next first dosing day. If there is no delay in treatment, one cycle is 28 days with dosing once every two weeks. All investigational treatments are conducted on an outpatient basis. Subjects are observed at the clinic for at least 2 hours after the infusion on the first day of cycle 1 and then clinically as needed thereafter.

[0266] Pre - dosing of Drug A is not required. The guidelines for the infusion of Enfortumab Vedotin follow those of the United States or the local prescribing information for Enfortumab Vedotin. The recommended dose of Enfortumab Vedotin is 1.25 mg / kg (maximum 125 mg for subjects weighing 100 kg or more) and is administered as an intravenous infusion over 30 minutes on days 1, 8, and 15 of a 28 - day cycle until disease progression or unacceptable toxicity.

[0267] On dosing days when the dosing schedules overlap, Enfortumab Vedotin is initiated approximately 30 minutes after the completion of Drug A therapy.

[0268] Interruptions and changes in dose are permitted for toxicity. Dose changes of Drug A can occur in one of the following three cases: · Within a cycle: During a given treatment cycle, dosing is interrupted until sufficient recovery (only for once - every - two - weeks dosing of Drug A), and the dose is reduced if necessary; · Between cycles: The start of the next cycle of dosing may be delayed due to persistent toxicity at the planned start of a new cycle; · Next cycle: Based on the toxicity experienced in the previous cycle, a dose reduction may be required in subsequent cycles.

[0269] If Drug A is permanently discontinued, the subject may continue receiving Enfortumab Vedotin if the principal investigator of the study determines that there is a clinical benefit to the subject. If Enfortumab Vedotin is permanently discontinued, the subject may continue receiving Drug A if the principal investigator of the study determines that there is a clinical benefit of Drug A to the subject.

[0270] While the dosage is gradually increasing, the subjects are sequentially assigned to the next available treatment frame at the dosage level and schedule determined after the safety evaluation of the previous cohort.

[0271] (E) Statistical Methods Approximately 30 adult subjects will be enrolled in the study in total. Enrollment depends on the observed safety profile, thereby determining the number of subjects at each dosage level and the number of dosage levels to be explored. The sample size of Phase 1 depends on the underlying dosage toxicity profile and the variability of actual data realization. In the dosage escalation part, the number of subjects treated at each dosage (i.e., 3 - 15) is based on the BOIN (Bayesian Optimal Interval) design (see, for example, Yuan et al. (2016) Clin Cancer Res. 22(17):4291 - 4301).

[0272] Once the Safety Review Committee (SRC) determines that the dosage level is safe and tolerable, additional subjects will be enrolled in the backfill cohort at the same dosage level to further evaluate the safety, pharmacokinetics, pharmacodynamics, and preliminary anti - tumor activity of Drug A administered in combination with enfortumab vedotin in subjects with locally advanced or metastatic urothelial carcinoma with a treatment history. In these backfill cohorts, the evaluation of DLT will not be performed. In the dosage escalation part including the backfill cohorts, approximately 15 subjects will be treated at each dosage level.

[0273] For the selection of the RP2D, the sponsor, together with the SRC, will review all available safety, pharmacokinetic, pharmacodynamic, and preliminary anti - cancer activity data obtained from Phase 1a including both the dosage escalation cohort and the backfill cohort, and make recommendations regarding the dosage of Drug A to be used in the Phase 2 trial.

[0274] (F) Safety Adverse events (AEs) are recorded regardless of causality, based on the judgment of the principal investigator of the clinical trial. The timing of all AEs, the overall frequency of toxicity, and the severity were classified according to toxicity grades 1-5 and recorded. The severity of adverse events is evaluated according to CTCAE version 5.0 (for example, see https: / / ctep.cancer.gov / protocoldevelopment / electronic_applications / docs / CTCAE_v5_Quick_Reference_5x7.pdf). For AEs observed at a high frequency or AEs considered to be serious adverse events (e.g., cases of Hy's law), additional summaries are provided.

[0275] Adverse events, electrocardiogram (ECG), blood pressure (BP), pulse rate, cardiac monitoring, and safety test data are continuously reviewed and summarized during the trial to assess the safety of the subjects. Safety data are presented in tabular and / or graphical form and summarized descriptively as necessary.

[0276] This trial uses a trial SRC composed of the principal investigator of the trial and representatives of the trial sponsor to provide continuous monitoring of AEs. AEs, serious adverse events (SAEs), and safety test values that occur during the trial and are considered related to the investigational drug are regularly evaluated to determine whether continued administration will compromise the safety of future subjects.

[0277] (G) Dose-limiting toxicity (DLT) The dose-limiting toxicity (DLT) evaluation period is the first 28 days of treatment (i.e., cycle 1). All AEs in cycle 1 that meet the following definitions are considered DLTs, except when they are clearly and unambiguously unrelated to drug A. · Hematology: · Grade 4 neutropenia persists for more than 7 days. · Febrile neutropenia (defined as neutropenia greater than grade 3 with a body temperature exceeding 38.3°C once or a body temperature exceeding 38°C for more than 1 hour). · Grade 3 or higher neutropenia accompanied by an infection. · Grade 3 thrombocytopenia accompanied by clinically significant bleeding. · Grade 4 thrombocytopenia. · Cases meeting the criteria of Hy's Law · Grade 3 or higher non-hematological toxicity. However, the following are excluded: · Grade 3 nausea, vomiting, or diarrhea that recovers to Grade 1 or lower by the next infusion (Grade 3 nausea, vomiting, or diarrhea that persists for more than 72 hours despite adequate antiemetic and other supportive therapies shall be considered DLT). · Grade 3 fatigue that recovers to Grade 2 or lower within 7 days. · Grade 3 or higher clinical laboratory abnormalities that recover to Grade 1 or lower within 24 hours, or those considered not clinically significant by the principal investigator of the clinical trial. · Grade 3 infusion reactions that are successfully managed and resolve within 72 hours (Grade 3 infusion reactions that occur regardless of recurrence or previous administration shall be considered DLT). · If the administration of the dose of Drug A on Day 1 of Cycle 2 is delayed by more than 2 weeks due to the persistence of toxicity attributable to Drug A.

[0278] In addition, Grade 2 toxicity that is clinically significant or persistent may be considered DLT.

[0279] (H) Efficacy In this study, preliminary antitumor activity is a secondary objective. The overall response rate (ORR), best overall response (BOR), disease control rate (DCR), duration of response (DOR), time to progression (TTP), progression-free survival (PFS), and overall survival (OS) will be analyzed in the full analysis set (FAS) and evaluable population to the extent possible based on the data.

[0280] Tumor evaluation includes all disease sites that are confirmed or suspected. Computed tomography (CT) is the preferred imaging method, although magnetic resonance imaging (MRI) is also used. Imaging includes the chest, abdomen, and pelvis (head and neck are optional). For subjects with confirmed or suspected brain metastases, a brain CT or MRI scan will be performed. The same imaging technique used to characterize each lesion identified and reported at baseline will be employed for subsequent tumor evaluations.

[0281] Antitumor activity is evaluated by radiation oncology examinations performed at baseline, during treatment (if disease progression is suspected, e.g., worsening symptoms), and at the end of treatment. Assessment of response for relevant secondary endpoints is done using RECIST version 1.1 (see, e.g., Eisenhauer et al. (2009) Eur J Cancer 45:228-247), evaluated by the treating investigator. Changes in tumor size are classified as complete response (CR), partial response (PR), stable disease (SD), or progression, the latter including the appearance of new lesions.

[0282] (I) Tumor biopsy markers Analysis of biopsy tissue is performed at a central reference laboratory. Analysis includes additional immunohistochemical (IHC) evaluations such as nectin-4 expression, PD-L1 status, as well as CD47 expression, and the prevalence and location of infiltrating immune cells such as T cells and tumor-associated macrophages (TAMs). Additional multiplex immunofluorescence assays and exploratory molecular assays for tumor, immune, and checkpoint markers will be performed if sufficient biopsy material is available.

[0283] (J) Pharmacokinetics / pharmacodynamics To provide serum for PK analysis, blood samples are collected. All efforts are made to obtain pharmacokinetic samples at the exact times planned for administration. The drug concentration of Drug A is measured using a validated method. Pharmacokinetic (PK) parameters are determined from the respective concentration-time data using standard non-compartmental methods. The sample collection times are used for parameter calculation. For Drug A, pharmacokinetic parameters are calculated including maximum concentration (Cmax), time to reach the highest concentration (T max ), area under the concentration-time curve from time 0 to the final measurement (AUC last ), AUC from time 0 to infinity (AUC inf ), and / or area under the plasma concentration-time curve during the dosing interval (τ) (AUCτ). If necessary, additional PK parameters are calculated including clearance (CL), volume of distribution (V z ), terminal phase elimination half-life (t 1 / 2 ), and accumulation ratio (R ac ). The drug concentration of Drug A is tabulated by graph and descriptive statistics for each dose, cycle, and planned PK sampling time. Non-compartmental PK parameters are tabulated descriptively for each dose and cycle. Pharmacodynamic data are tabulated by graph and descriptive statistics for each time and dose. To better understand the exposure-response relationship, PK / PD analysis using appropriate model-based methods may be explored and the results reported separately.

[0284] Collect whole blood samples for CD47 target occupancy, immunophenotyping of circulating white blood cells, peripheral blood circulating tumor DNA (ctDNA), and exploratory molecular analysis.

[0285] Blood samples collected at baseline visit and any remaining blood collected at other visits are stored for potential pharmacogenomic analysis related to drug response. For example, SIRPα gene polymorphisms, putative safety biomarkers, drug-metabolizing enzyme genes, drug transporter protein genes, or genes thought to be related to the mechanism of action of the drug can be examined.

[0286] (K) Evaluation of Neutralizing Reagents for Pre-Transfusion Crossmatch Testing Since red blood cells (RBCs) express CD47, drug A can not only exist unbound in the serum or plasma of a subject but also bind to the patient's circulating RBCs. After initiation of treatment with drug A, antibody screening including indirect antiglobulin test (IAT) and direct antiglobulin test (DAT) may report false positives as anti-human globulin (AHG) binds to the Fc portion of drug A, which may affect the interpretation of pre-transfusion crossmatch. Evaluate potential mitigation methods to neutralize this interference. For this purpose, a study neutralizing reagent may be provided to the blood bank of each facility, or at the time of performing ABO Rh typing, antibody screening, and crossmatch for RBC transfusion, blood samples may be sent to a designated reference laboratory and tested by an exploratory neutralization assay.

[0287] The foregoing examples are provided for illustrative purposes only and are in no way intended to limit the scope of the present invention. Various modifications of the present invention other than those shown and described herein will be apparent to those skilled in the art from the above description and are included within the scope of the appended claims.

Claims

1. A pharmaceutical agent for treating urothelial carcinoma in an individual by combination therapy, comprising a fusion polypeptide containing a SIRPα D1 domain variant and an Fc domain variant, wherein the pharmaceutical agent is administered in combination with enfortumab vedotin. The SIRPα D1 domain variant of the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO:

85. The Fc domain variant of the fusion polypeptide is (i) Human IgG1 Fc regions containing mutations L234A, L235A, G237A, and N297A (numbering follows Kabat's EU index); (ii) Human IgG2 Fc regions containing the A330S, P331S, and N297A mutations (numbering follows Kabat's EU index); (iii) Human IgG4 Fc region containing mutations in S228P, E233P, F234V, L235A, and delG236 (numbering follows Kabat's EU index); or (iv) Human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (numbering follows Kabat's EU index), a pharmaceutical drug.

2. A pharmaceutical agent for treating urothelial carcinoma in an individual, comprising a fusion polypeptide containing a SIRPα D1 domain variant and an Fc domain variant, and enfortumab vedotin, The SIRPα D1 domain variant of the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO:

85. The Fc domain variant of the fusion polypeptide is (i) Human IgG1 Fc regions containing mutations L234A, L235A, G237A, and N297A (numbering follows Kabat's EU index); (ii) Human IgG2 Fc regions containing the A330S, P331S, and N297A mutations (numbering follows Kabat's EU index); (iii) Human IgG4 Fc region containing mutations in S228P, E233P, F234V, L235A, and delG236 (numbering follows Kabat's EU index); or (iv) Human IgG4 Fc region containing S228P, E233P, F234V, L235A, delG236, and N297A mutations (numbering follows Kabat's EU index), a pharmaceutical drug.

3. The pharmaceutical product according to claim 1 or claim 2, wherein the urothelial carcinoma is locally advanced urothelial carcinoma or metastatic urothelial carcinoma.

4. The pharmaceutical product according to claim 1 or claim 2, wherein the urothelial carcinoma is bladder cancer, renal pelvis cancer, ureteral cancer, or urethral cancer.

5. The pharmaceutical product according to claim 1 or 2, wherein the individual has received prior treatment with an immune checkpoint inhibitor (CPI).

6. The pharmaceutical product according to claim 5, wherein the CPI is a PD-1 inhibitor or a PD-L1 inhibitor.

7. The pharmaceutical product according to claim 6, wherein the CPI is atezolizumab, pembrolizumab, durvalumab, avelumab, or nivolumab.

8. The pharmaceutical product according to claim 1 or claim 2, wherein the individual has received prior treatment with platinum-containing chemotherapy.

9. The pharmaceutical product according to claim 1 or 2, wherein the individual has experienced progression or recurrence of urothelial carcinoma during or after receiving the most recent prior treatment.

10. The pharmaceutical product according to claim 1 or 2, wherein the individual has not received prior treatment with a monomethyl auristatin (MMAE) antibody-drug conjugate.

11. The pharmaceutical product according to claim 10, wherein the individual has not received prior treatment with enfortumab vedotin.

12. The pharmaceutical product according to claim 1 or 2, wherein the individual has not received prior treatment with a therapeutic agent that blocks the interaction between CD47 and SIRPα.

13. The pharmaceutical product according to claim 1 or 2, wherein the enfortumab vedotin is administered to the individual in one or more 28-day cycles, and the enfortumab vedotin is administered intravenously to the individual at a dose of 1.25 mg / kg on days 1, 8 and 15 of each 28-day cycle.

14. The pharmaceutical product according to claim 1 or claim 2, wherein the enfortumab vedotin is to be administered intravenously.

15. The pharmaceutical product according to claim 1 or claim 2, wherein the fusion polypeptide is administered to the individual at a maximum dose of approximately 60 mg / kg.

16. The pharmaceutical product according to claim 15, wherein the fusion polypeptide is administered to the individual once every two weeks (q2w) at a dose of approximately 30 mg / kg.

17. The pharmaceutical product according to claim 15, wherein the fusion polypeptide is administered at a dose of approximately 20 mg / kg once every two weeks (q2w).

18. The pharmaceutical product according to claim 15, wherein the fusion polypeptide is administered at a dose of approximately 15 mg / kg once every two weeks (q2w).

19. The pharmaceutical product according to claim 1 or claim 2, wherein the fusion polypeptide is to be administered intravenously.

20. The pharmaceutical product according to claim 1 or claim 2, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO:

85.

21. The pharmaceutical product according to claim 1 or claim 2, wherein the SIRPα D1 domain variant comprises the amino acid sequence of SEQ ID NO:

81.

22. The pharmaceutical product according to claim 1 or claim 2, wherein the Fc domain variant is a human IgG1 Fc region containing the L234A, L235A, G237A, and N297A mutations, and the numbering follows the EU index of Kabat.

23. The pharmaceutical product according to claim 22, wherein the Fc domain variant comprises the amino acid sequence of SEQ ID NO:

91.

24. The pharmaceutical product according to claim 1 or claim 2, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:

136.

25. The pharmaceutical product according to claim 1 or claim 2, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:

135.

26. The pharmaceutical product according to claim 1 or claim 2, wherein the fusion polypeptide forms a homodimer.

27. The pharmaceutical product according to claim 1 or claim 2, wherein the individual is a human.

28. A kit for treating urothelial carcinoma in an individual requiring treatment, comprising a polypeptide containing a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, for use in combination with enfortumab vedotin, The SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO:

85. The Fc domain variant is (i) Human IgG1 Fc regions containing mutations L234A, L235A, G237A, and N297A (numbering follows Kabat's EU index); (ii) Human IgG2 Fc regions containing the A330S, P331S, and N297A mutations (numbering follows Kabat's EU index); (iii) Human IgG4 Fc region containing mutations in S228P, E233P, F234V, L235A, and delG236 (numbering follows Kabat's EU index); or (iv) Human IgG4 Fc region containing mutations in S228P, E233P, F234V, L235A, delG236, and N297A (numbering follows Kabat's EU index), The kit includes instructions for administering the polypeptide, which contains the SIRPα D1 domain variant and the Fc domain variant, to the organism in combination with enfortumab vedotin.

29. A kit for treating urothelial carcinoma in an individual requiring treatment, comprising a polypeptide containing a SIRPα D1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, and enfortumab vedotin, The SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO:

85. The Fc domain variant is (i) Human IgG1 Fc regions containing mutations L234A, L235A, G237A, and N297A (numbering follows Kabat's EU index); (ii) Human IgG2 Fc regions containing the A330S, P331S, and N297A mutations (numbering follows Kabat's EU index); (iii) Human IgG4 Fc region containing mutations in S228P, E233P, F234V, L235A, and delG236 (numbering follows Kabat's EU index); or (iv) Human IgG4 Fc region containing mutations in S228P, E233P, F234V, L235A, delG236, and N297A (numbering follows Kabat's EU index), The kit includes instructions for administering the polypeptide, which contains the SIRPα D1 domain variant and the Fc domain variant, to the organism in combination with enfortumab vedotin.

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