Combination therapy for treating urothelial cancer

Abstract

The present invention provides methods of treating cancer (e.g., urothelial cancer) comprising administering a polypeptide (e.g., a fusion polypeptide) comprising a SIRP [alpha]-D1 domain variant and an Fc domain variant in combination with an antibody-drug conjugate (e.g., Wientumeumab). Related kits are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to 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.

[0003] Reference to an electronic sequence listing

[0004] The contents of the electronic Sequence Listing (757972001900SEQLIST.xml; Size: 365,943 bytes; Creation Date: May 25, 2023) are incorporated herein by reference in its entirety. Technical Field

[0005] The present invention relates to a method of treating cancer, comprising administering to an individual in need thereof a combination of an agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) and an antibody drug conjugate (e.g., enfortumab vedotin). Background Art

[0006] Bladder cancer is the sixth most common cancer in the United States (US). According to estimates by the National Cancer Institute, more than 83,000 new cases of urothelial cancer were diagnosed in 2021, and more than 17,000 people died from the disease in the United States (SEER Cancer Stat Facts: Bladder Cancer, 2021. National Cancer Institute. Bethesda, MD, https: / / seer(dot)ca ncer(dot)gov / statfacts / html / urinb(dot)html. Accessed on March 09, 2022). Bladder cancer occurs mainly in people over the age of 55, with a median age of 73 at diagnosis. The male:female ratio of developing this cancer is approximately 4:1. Caucasians are more likely to be diagnosed with bladder cancer than African Americans or Hispanics (SEER Cancer Stat Facts: Bladder Cancer, 2021. National Cancer Institute. Bethesda, MD, https: / / seer(dot)cancer(dot)gov / statfacts / html / urinb(dot)html. Accessed on March 09, 2022). Approximately 90% of bladder cancers are urothelial carcinomas; if the cancer is advanced at the time of diagnosis, the prognosis is poor (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 non-muscle invasive stage. At this stage, disease management includes resection with or without intracystic therapy. Despite this treatment, patients often progress to more advanced disease that is incurable, ultimately leading to death. Approximately 12% of patients have locally advanced or metastatic disease at diagnosis (SEER Cancer Stat Facts: Bladder Cancer, 2021. National Cancer Institute. Bethesda, MD, https: / / seer(dot)cancer(dot)gov / statfacts / html / urinb(dot)html. Accessed on March 09, 2022).

[0007] First-line therapy for locally advanced or metastatic urothelial carcinoma in patients with adequate renal function consists of cisplatin-based combinations, such as with methotrexate, vinblastine, doxorubicin and cisplatin (MVAC) or gemcitabine plus cisplatin, which have shown overall response rates up to 50%, including approximately 10% to 15% complete responses (CR) (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(dot)1093 / annonc / mdr376(dot)PMID:21908503). Carboplatin and gemcitabine are often used in patients who are not suitable for cisplatin, but the results are generally poor. Despite the initial chemotherapy sensitivity, patients are not cured, and the results of metastatic urothelial carcinoma after these regimens are poor: the median progression time is only 7 months, and the median overall survival (OS) is 14 months. About 15% of patients survive at least 5 years, and the prognosis is particularly poor in patients with visceral metastasis and a 5-year OS rate of 7% (von der Maase H, Sengelov L, Roberts JT, Ricci S, Dogliotti L, Oliver T, et al. Long-term survival results of arandomized 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 progress in the treatment of urothelial carcinoma, there is still an urgent need in the art for patients with locally advanced or metastatic urothelial carcinoma and relapse after treatment with platinum-based regimens and immunotherapy.

[0008] All references cited herein (including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers) are hereby incorporated by reference in their entirety to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the invention

[0009] In some embodiments, provided herein is a method of treating urothelial cancer in an individual, comprising administering to the individual (a) an effective amount of a fusion polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) an effective amount of ventozumab, wherein the SIRPαD1 domain variant of the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; and wherein the Fc domain variant of the fusion polypeptide is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations. or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236 and N297A mutations, wherein the numbering is according to the EU index as of Kabat.

[0010] In some embodiments, the individual is a human. In some embodiments, urothelial carcinoma is locally advanced urothelial carcinoma or metastatic urothelial carcinoma. In some embodiments, urothelial carcinoma 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 is a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the CPI is atezolizumab, pembrolizumab, durvalumab, avelumab or nivolumab. In some embodiments, the individual has received prior treatment with platinum-containing chemotherapy. In some embodiments, the individual suffers from progression or recurrence of urothelial carcinoma during or after receiving the most recent prior treatment. In some embodiments, the individual has not received prior treatment with an antibody-drug conjugate based on monomethylauristatin (MMAE). In some embodiments, the individual has not received prior treatment with ventozumab. In some embodiments, the individual has not received prior treatment with a therapeutic agent that blocks the interaction between CD47 and SIRPα.

[0011] In some embodiments, venetumab is administered to an individual in one or more 28-day cycles, and wherein venetumab is administered to an individual IV at a dose of 1.25 mg / kg on the 1st, 8th, and 15th days of each 28-day cycle. In some embodiments, venetumab is administered intravenously. In some embodiments, the fusion polypeptide is administered to an individual at a dose of up to about 60 mg / kg. In some embodiments, the fusion polypeptide is administered to an individual once every two weeks (q2w) at a dose of about 30 mg / kg. In some embodiments, the fusion polypeptide is administered once every two weeks (q2w) at a dose of about 20 mg / kg. In some embodiments, the fusion polypeptide is administered once every two weeks (q2w) at a dose of about 15 mg / kg. In some embodiments, the fusion polypeptide is administered intravenously.

[0012] In some embodiments, the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:85. In some embodiments, the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81. In some embodiments, the Fc domain variant is a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein the numbering is according to the EU index of Kabat. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO:91. In some embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:136. In some embodiments, the fusion polypeptide comprises the amino acid sequence of SEQID NO:135. In some embodiments, the fusion polypeptide forms a homodimer.

[0013] In some embodiments, a kit is provided, comprising a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, the polypeptide being used in combination with ventuumab to treat urothelial carcinoma in an individual in need thereof, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein the numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein the numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A and delG236 mutations. Fc region, wherein the numbering is according to the EU index as in Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index as in Kabat, and wherein the kit comprises instructions for administering a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant in combination with ventuumab to an individual.

[0014] It should be understood that one, some or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will become apparent to those skilled in the art. These and other embodiments of the present invention are further described by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1A Shown are the results of an experiment performed to determine whether drug A enhances the antibody-dependent cellular phagocytosis (ADCP) activity of drug B and drug C using macrophages derived from monocytes of a first human donor as well as T47D ductal carcinoma cells and OE19 esophageal adenocarcinoma cells as target cells.

[0016] Figure 1B Shown are the results of an experiment conducted using macrophages derived from monocytes obtained from a second human donor, as well as T47D ductal carcinoma cells and OE19 esophageal adenocarcinoma cells as target cells to determine whether drug A enhances the antibody-dependent cellular phagocytosis (ADCP) activity of drug B and drug C.

[0017] Figure 2Shown are the results of experiments performed to determine the effect of drug A on drug B-dependent (left panel) or drug C-dependent (right panel) ADCP of macrophages derived from monocytes obtained from a third human donor against OE19 esophageal adenocarcinoma cells.

[0018] Figure 3 Shown are the results of assays performed to determine the effect of drug A on drug B-dependent (left panel) or drug C-dependent (right panel) ADCP of macrophages derived from monocytes obtained from a third human donor against HT-1376 bladder cancer cells.

[0019] Figure 4 The study design for the Phase 1 clinical trial described in Example 3 is provided. DETAILED DESCRIPTION

[0020] The following description sets forth exemplary methods, parameters, etc. However, it should be appreciated that such description is not intended to limit the scope of the present disclosure, but is provided as a description of exemplary embodiments.

[0021] The headings provided herein are not limitations of the various aspects or embodiments, which can be had by reference to the specification as a whole.Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0022] definition

[0023] The term "about" or "approximately" means that a particular value is within an acceptable error range as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5 times, and more preferably within 2 times of a value. When describing specific values ​​in the application and claims, unless otherwise indicated, the term "about" should be considered to mean that the specific value is within an acceptable error range.

[0024] The terms used herein are for the purpose of describing a particular situation only and are not intended to be limiting. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, to the extent the terms "including", "having", "has", or variations thereof are used in the detailed description or claims, these terms are intended to be included in a manner similar to the term "comprising".

[0025] The term "treat / treating / treatment" refers to producing a desired or beneficial effect in a mammal suffering from a disease condition when it relates to a certain disease condition in a mammal. The desired or beneficial effect may include reducing 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 preventing or inhibiting the further development of a disease, condition or illness. In the case of treating cancer in a mammal, the desired or beneficial effect may include inhibiting the further growth or spread of cancer cells, cancer cell death, inhibiting cancer recurrence, alleviating pain associated with cancer, or increasing the survival rate of the mammal. This effect may be subjective or objective. For example, if the mammal is a human, the person may notice an improvement in vitality or vitality or a reduction in pain as a subjective symptom of improvement or response to treatment. Alternatively, a clinician may notice a reduction in tumor size or tumor burden based on physical examination, laboratory parameters, tumor markers, or radiographic findings. In addition, a clinician may observe a reduction in detectable tumor markers. Alternatively, other tests may be used to assess objective improvement, such as computed tomography (CT), magnetic resonance imaging (MRI), etc.

[0026] As used herein, the term "joint" refers to the connection between two elements (e.g., protein domains). In some embodiments, the joint can be a covalent bond or a spacer. The term "spacer" refers to a portion (e.g., a polyethylene glycol (PEG) polymer) or an amino acid sequence (e.g., a 1-200 amino acid sequence) that appears between two polypeptides or polypeptide domains to provide space or flexibility (or both space and flexibility) between two polypeptides or polypeptide domains. In some embodiments, the amino acid spacer is part of the primary sequence of the polypeptide (e.g., connected to the polypeptide or polypeptide domain of the interval via the polypeptide backbone).

[0027] As used herein, the term "pharmaceutical composition" refers to a medical or pharmaceutical formulation comprising an active ingredient and an excipient or diluent (or both an excipient and a diluent) and enabling the active ingredient to be administered by a suitable administration method. In some embodiments, the pharmaceutical composition disclosed herein comprises a pharmaceutically acceptable component that is compatible with the polypeptide. In some embodiments, the pharmaceutical composition is in the form of a tablet or capsule for oral administration, or in an aqueous form for intravenous or subcutaneous administration (e.g., by injection).

[0028] As used herein, the terms "subject," "individual," and "patient" are used interchangeably and refer to a vertebrate, such as a mammal. Mammals include, but are not limited to, mice, monkeys, humans, farm animals, sports animals, and pets. Tissues, cells, and progeny of biological entities obtained in vivo or cultured in vitro are also encompassed. None of these terms include supervision of a medical professional.

[0029] As used herein, the term "affinity" or "binding affinity" refers to the strength of the binding interaction between two molecules. Typically, binding affinity refers to the total strength of non-covalent interactions between a molecule and its binding partner (such as SIRPαD1 domain variants and CD47). Unless otherwise specified, binding affinity refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair. The binding affinity between two molecules is usually expressed in terms of the dissociation constant (K D ) or association constant (K A Two molecules that have low binding affinity for each other typically associate slowly, dissociate easily, and exhibit a large K D Two molecules that have a high affinity for each other generally bind easily, tend to remain bound longer, and exhibit a small K D In some embodiments, the KD of two interacting molecules is determined using known methods and techniques (e.g., surface plasmon resonance (SPR)). D It can be calculated as k off / k on ratio.

[0030] As used herein, the term "K D "Less than" refers to the smaller K in value D value and relative to the K D As used herein, the term "K D "Greater than" refers to a numerically larger K D Values ​​and reduced binding affinity relative to the KD value.

[0031] "Effective amount" refers to at least an effective amount that achieves one or more desired or indicated effects (including therapeutic or preventive results) at the necessary dosage within the necessary time period. The effective amount can be provided in one or more administrations. For the purposes of this disclosure, an effective amount of an antibody, drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve a preventive or therapeutic treatment. As understood in a 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., an effective amount administered as a monotherapy or combination therapy). Therefore, an "effective amount" can be considered in the context of administering one or more therapeutic agents, and if a desirable result can be achieved or achieved in combination with one or more other agents, then a single agent can be considered to be given in an effective amount.

[0032] Unless otherwise indicated, the methods and techniques disclosed herein are generally performed according to methods well known in the art, and as described in various general and more specific references cited and discussed throughout this specification. These references include, for example, Sambrook and Russell, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY (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, NY (1990). Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as generally accomplished in the art or as described herein. The terms and laboratory procedures and techniques used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry as described herein are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, formulation and administration, and patient treatment.

[0033] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd edition, ES Golub and DR Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)).

[0034] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0035] Overview

[0036] Provided herein is a method of treating cancer (e.g., urothelial carcinoma) in an individual (e.g., a human individual), 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.

[0037] 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 and others). Exemplary small molecule inhibitors of the CD47-SIRPα pathway include, but are not limited to, for example, Miller et al. (2019) "Quantitative high-throughput screening assays for the discovery and development of SIRPα-CD47interaction 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.

[0038] 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 (such as at least about any of 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). DBinding 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 in a human subject (e.g., at least about any of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%). In some embodiments, the agent that binds to CD47 (e.g., hCD47) has an EC50 of about 80 ng / ml or less, such as about any of 75 ng / ml, 70 ng / ml, 65 ng / ml, 60 ng / ml, 55 ng / ml, 50 ng / ml, 45 ng / ml, 40 ng / ml, 35 ng / ml, 30 ng / ml, 25 ng / ml, 20 ng / ml, 15 ng / ml, 10 ng / ml, 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 bivalent antibody (diabody). In some embodiments, the anti-CD47 antibody is a monospecific antibody. In some embodiments, the anti-CD47 antibody is a multispecific (e.g., bispecific) antibody. In some embodiments, the term "anti-CD47 antibody" encompasses antibody-based constructs (such as multispecific constructs), including but not limited to triomabs, DARTs (i.e., dual affinity retargeting antibodies), TandAbs (i.e., tandem bivalent antibodies), tandem scFvs, CrossMabs, DNLs (i.e., docking and locking antibodies), DVD-Igs (i.e., dual variable domain immunoglobulins), tetravalent bispecific IgGs, nanobodies, dual targeting domains, and ART-Igs (i.e., asymmetric remodeling technology-immunoglobulins). Additional details on 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, such as Hu5F9-G4, B6H12.2, BRIC126, CC-90002, SRF231, or IBI188 (from Innovent Biologics) (for additional 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).

[0039] In some embodiments, the agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) binds to SIRPα (e.g., hSIRPα). In some embodiments, the agent has a K of about 10 nM or better (such as 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). DBinding 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 in human subjects (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%). In some embodiments, the agent that binds to SIRPα (e.g., hSIRPα) has an EC50 of about 80ng / ml or less, such as about 75ng / ml, 70ng / ml, 65ng / ml, 60ng / ml, 55ng / ml, 50ng / ml, 45ng / ml, 40ng / ml, 35ng / ml, 30ng / ml, 25ng / ml, 20ng / ml, 15ng / ml, 10ng / ml, or any one of 5ng / 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 Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv, a one-armed antibody, or a bivalent antibody (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, such as KWAR23, SE12C3,040, or MY-1 (for additional 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 WO 2018 / 057669, US-2018-0105600-A1, US20180312587, WO2018107058, WO2019023347, US20180037652, WO2018210795, WO2017178653, WO2018149938, WO2017068164, and WO2016063233, the contents of which are incorporated herein by reference in their entireties.

[0040] In some embodiments, the 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 antibodies (or antigen-binding fragments thereof) have a K of about 10 nM or better (such as 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). D Binding to SIRPα (e.g., hSIRPα). In some embodiments, the antibody (or its antigen-binding fragment) exhibits at least about 50% SIRPα receptor occupancy in human subjects (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or about 100%). In some embodiments, the antibody (or its antigen-binding fragment) has an EC50 of about 80 ng / ml or less, for example, 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 Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv, one-armed antibody or bivalent antibody (diabody). In some embodiments, the antibody is a monospecific antibody or a monospecific antibody construct (including but not limited to those described above). In some embodiments, the antibody is a multispecific (eg, bispecific) antibody or a multispecific antibody construct (including but not limited to those described above).

[0041] In some embodiments, the agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is a fusion polypeptide comprising a portion that binds CD47. In some embodiments, the fusion polypeptide comprises an antibody Fc region and a portion that binds CD47. In some embodiments, the fusion polypeptide portion that binds CD47 (e.g., hCD47) binds to CD47 with a KD of about 10 nM or better (such as 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)例如, hCD47). In some embodiments, the fusion polypeptide exhibits at least about 50% CD47 receptor occupancy in a human subject (e.g., at least about any of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%). In some embodiments, the fusion polypeptide has an EC50 of about 80 ng / ml or less, such as about any of 75 ng / ml, 70 ng / ml, 65 ng / ml, 60 ng / ml, 55 ng / ml, 50 ng / ml, 45 ng / ml, 40 ng / ml, 35 ng / ml, 30 ng / ml, 25 ng / ml, 20 ng / ml, 15 ng / ml, 10 ng / ml, 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 (e.g., a variant of a wild-type human antibody Fc region), and the variant comprises 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 reduced (e.g., such as eliminated) effector functions compared to the WT Fc region. Exemplary Fc variants are described in WO 2017 / 027422 and US2017 / 0107270, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the portion of the fusion protein that binds CD47 (e.g., hCD47) is WT SIRPα (e.g., hSIRPα) or WT SIRPγ (e.g., hSIRPγ). In some embodiments, the portion that binds CD47 (e.g., hCD47) is a CD47 binding fragment (e.g., D1 domain) of WT SIRPα (e.g., hSIRPα) or WT SIRPγ (e.g., hSIRPγ). In some embodiments, the portion that binds to CD47 (e.g., hCD47) is a SIRPα variant, a SIRPγ variant, a SIRPβ variant, or a CD47 binding fragment thereof (e.g., a D1 domain). In some embodiments, any of the aforementioned SIRPα variants, SIRPγ variants, SIRPβ variants, or CD47 binding fragments thereof (e.g., a D1 domain) comprises one or more amino acid insertions, deletions, or substitutions relative to the amino acid sequences of any of the aforementioned wild-type SIRPα, SIRPγ, SIRPβ, or CD47 binding fragments thereof, respectively.Exemplary SIRPγ variants and SIRPβ variants are described in, for example, WO 2013 / 109752, US 2015 / 0071905, USP 9,944,911, WO 2016 / 023040, WO 2017 / 027422, US 2017 / 0107270, USP 10,259,859, US9845345, WO2016187226, US20180155405, WO2017177333, WO2014094122, US2015329616, US20180312563, WO2018176132, WO2018081898, WO2018081897, PCT / US2019 / 048921, US20180141986A1 and EP3287470A1, the contents of which are incorporated herein by reference in their entirety. Exemplary SIRPα variants are described in more detail elsewhere herein.

[0042] In some embodiments, the agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is a fusion polypeptide comprising an antibody Fc region and a SIRPα variant. In some embodiments, the SIRPα variant has a K of about 10 nM or better (such as 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). DBinding to CD47 (e.g., hCD47). In some embodiments, the fusion polypeptide exhibits at least about 50% CD47 receptor occupancy in a human subject (e.g., at least about any of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%). In some embodiments, the fusion polypeptide has an EC50 of about 80 ng / ml or less, such as about any of 75 ng / ml, 70 ng / ml, 65 ng / ml, 60 ng / ml, 55 ng / ml, 50 ng / ml, 45 ng / ml, 40 ng / ml, 35 ng / ml, 30 ng / ml, 25 ng / ml, 20 ng / ml, 15 ng / ml, 10 ng / ml, or 5 ng / ml. In some embodiments, the fusion polypeptide comprises a WT human antibody Fc region. In some embodiments, the fusion polypeptide comprises an Fc variant (e.g., a variant of a WT human antibody Fc region) that exhibits reduced (e.g., such as abolished) effector function compared to a WT Fc region, such as those described in the references cited herein. In some embodiments, the fusion polypeptide comprises a variant described in WO 2013 / 109752, US2015 / 0071905, WO 2016 / 023040, WO 2017 / 027422, US2017 / 0107270, USP 10,259,859, US9845345, WO2016187226, US20180155405, WO2017177333, WO2014094122, US2015329616, US20180312563, WO2018176132, WO2018081898, WO2018081897, US20180141986A1 and EP3287470A1, the contents of which are incorporated herein 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).

[0043] In some embodiments, the agent that blocks the interaction between CD47 (e.g., hCD47) and SIRPα (e.g., hSIRPα) is a fusion polypeptide comprising a SIRPαD1 domain variant (e.g., a SIRPαD1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein). Further details about such fusion polypeptides are provided below.

[0044] Exemplary fusion polypeptides comprising a signal regulatory protein alpha (SIRPα) D1 domain variant and an Fc variant

[0045] Signal regulatory protein α (SIRPα) D1 domain variant

[0046] In some embodiments, the fusion polypeptide comprises a signal regulatory protein alpha (SIRPα) D1 domain or a variant thereof. In some embodiments, the SIRPαD1 domain variant comprises one or more amino acid insertions, deletions and / or substitutions relative to the amino acid sequence of the wild-type SIRPαD1 domain. In some embodiments, disclosed herein is a polypeptide (e.g., a fusion polypeptide) comprising a signal regulatory protein alpha (SIRP-α) D1 variant, the variant comprising a SIRPαD1 domain or a CD47 binding fragment thereof, comprising an amino acid mutation at position 80 relative to a wild-type SIRPαD1 domain (e.g., a wild-type SIRPαD1 domain shown in SEQ ID NO: 1 or 2); and at least one additional amino acid mutation at an amino acid position selected from the group consisting of: residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92 relative to a wild-type SIRPαD1 domain (e.g., a wild-type SIRPαD1 domain shown in SEQ ID NO: 1 or 2).

[0047] In some embodiments, the present invention also discloses a fusion polypeptide comprising an Fc domain variant, wherein the Fc domain variant dimer comprises two Fc domain variants, wherein each Fc domain variant is independently selected from (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236 and N297A.

[0048] Signal regulatory protein alpha ("SIRP-α" or "SIRP-α") is a transmembrane glycoprotein belonging to the Ig superfamily that is widely expressed on the membranes of myeloid cells. SIRPα interacts with CD47, a protein widely expressed on many cell types in the body. The interaction of SIRPα with CD47 prevents the phagocytosis of "self" cells that would otherwise be recognized by the immune system. It has been observed that in acute myeloid leukemia and several solid tumor cancers, high expression of CD47 on tumor cells can serve as a negative prognostic factor for survival.

[0049] Natural SIRPα contains three highly homologous immunoglobulin (Ig)-like extracellular domains: D1, D2 and D3. The SIRPαD1 domain ("D1 domain") refers to the membrane-distal extracellular domain of SIRPα and mediates the binding of SIRPα to CD47. As used herein, the term "SIRPα polypeptide" refers to any SIRPα polypeptide or fragment thereof that is capable of binding to CD47. There are at least ten variants of wild-type human SIRPα. Table 1 shows the amino acid sequence of the D1 domain of naturally occurring wild-type human SIRPαD1 domain variants (SEQ ID NOs: 1 and 2). In some embodiments, the SIRPα polypeptide comprises the SIRPαD1 domain. In some embodiments, the SIRPα polypeptide comprises a wild-type D1 domain, such as those provided in SEQ ID NOs: 1 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).

[0050] Table 1. Sequences of wild-type SIRPαD1 domains

[0051]

[0052]

[0053] As used herein, the term "SIRPαD1 domain variant" refers to a polypeptide comprising a SIRPαD1 domain or a CD47 binding portion of a SIRPα polypeptide, which has a higher affinity for CD47 than wild-type SIRPα. The SIRPαD1 domain variant comprises at least one amino acid substitution, deletion or insertion (or a combination thereof) relative to the amino acid sequence of wild-type SIRPα.

[0054] In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant, which comprises one or more amino acid substitutions, insertions, additions or deletions relative to the wild-type D1 domain shown in SEQ ID NO: 1 and 2. Table 2 lists exemplary amino acid substitutions in each SIRPαD1 domain variant (SEQ ID NO: 13-14). In some embodiments, the fusion polypeptide comprises a fragment of a SIRPαD1 domain variant (e.g., a CD47 binding fragment). In some embodiments, a fragment of a SIRPαD1 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.

[0055] In some embodiments, the binding affinity of the fusion polypeptide comprising a SIRPαD1 domain variant to CD47 is higher than that of the wild-type human SIRPαD1 domain. In some embodiments, the SIRPαD1 domain variant binds to human CD47 with an affinity at least 1 times (e.g., at least 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 5 times, or more than 5 times) than the affinity of the naturally occurring D1 domain. In some embodiments, the SIRPαD1 domain variant binds to human CD47 with an affinity at least 1 times (e.g., at least 10 times, 100 times, 1000 times, or more than 1000 times) than the affinity of the naturally occurring D1 domain.

[0056] As used herein, the term "optimized affinity" or "optimized binding affinity" refers to the optimized strength of the binding interaction between a fusion polypeptide disclosed herein (e.g., a fusion polypeptide comprising a SIRPαD1 domain variant) and CD47. For example, in some embodiments, the fusion polypeptide binds primarily or with a higher affinity to CD47 on cancer cells, and does not substantially bind or binds with a lower affinity to CD47 on non-cancer cells. In some embodiments, the binding affinity between the fusion polypeptide and CD47 is optimized so that the interaction does not cause clinically relevant toxicity or reduces toxicity compared to variants that bind with maximum affinity. In some embodiments, in order to achieve optimized binding affinity between the fusion polypeptide and CD47, a fusion polypeptide comprising a SIRPαD1 domain variant is developed to have a binding affinity to CD47 that is lower than the maximum achievable binding affinity. In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant that cross-reacts with rodent CD47 (eg, mouse CD47 or rat CD47), non-human primate (NHP) CD47 (eg, cynomolgus monkey CD47), and human CD47.

[0057] As used herein, the term "immunogenicity" refers to the property of a protein (e.g., a therapeutic protein) that causes an immune response in a host as if it were a foreign antigen. The immunogenicity of a protein can be determined in vitro in a variety of different ways, such as by an in vitro T cell proliferation assay.

[0058] As used herein, the term "minimal immunogenicity" refers to the immunogenicity of a polypeptide (e.g., a therapeutic polypeptide) that has been modified, for example, by amino acid substitutions, that is lower (e.g., at least 10%, 25%, 50% or 100%) than the immunogenicity before the introduction of the amino acid substitutions (e.g., unmodified protein). In some embodiments, the fusion polypeptide (e.g., a polypeptide comprising a SIRPαD1 domain variant and an Fc variant) is modified to have minimal immunogenicity and does not cause or causes a very small immune response in a subject (e.g., a human subject), even though it may be recognized as a foreign antigen by the subject's immune system.

[0059] In some embodiments, fusion polypeptides comprising SIRPαD1 domain variants exhibit minimal immunogenicity. In some embodiments, the fusion polypeptide administered to a subject comprises a SIRPαD1 domain variant having the same amino acid sequence as the subject's endogenous SIRPα, except for amino acid changes that increase the affinity of the SIRPαD1 domain variant. In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant, which reduces the risk of side effects compared to anti-CD47 antibodies or wild-type SIRPα. In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant, which reduces the risk of anemia compared to anti-CD47 antibodies or wild-type SIRPα. In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant that does not cause acute anemia in rodent or non-human primate (NHP) studies.

[0060] Table 2 lists specific amino acid substitutions in SIRPαD1 domain variants relative to each D1 domain sequence. In some embodiments, the SIRPαD1 domain variant of the fusion polypeptide comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more) substitutions listed in Table 2. In some embodiments, the SIRPαD1 domain variant of the fusion polypeptide comprises up to fifteen amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPαD1 domain variant of the fusion polypeptide comprises up to ten amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the SIRPαD1 domain variant of the fusion polypeptide comprises up to seven amino acid substitutions relative to the wild-type D1 domain. In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant having at least 90% (e.g., at least 92%, 95%, 97% or greater than 97%) amino acid sequence identity with the sequence of the wild-type D1 domain.

[0061] In some embodiments, the fusion polypeptide comprises a chimeric SIRPαD1 domain variant, such as a variant comprising a portion of two or more wild-type D1 domains or variants thereof (e.g., a portion of a first wild-type D1 domain (or variant thereof) from a first species, or a portion of a second wild-type D1 domain (or variant thereof) from a second species). In some embodiments, the chimeric SIRPαD1 domain variant includes portions from at least two (e.g., two, three, four, five or more portions) of wild-type D1 domains (or variants thereof), wherein each portion is 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 comprises a chimeric SIRPαD1 domain variant, which further comprises one or more amino acid substitutions listed in Table 2.

[0062] Table 2. Amino Acid Substitutions in SIRPαD1 Domain Variants

[0063]

[0064]

[0065] In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant comprising the sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGNITPADAGTYYCX 12 KX 13 RKGSPDDVEX 14 KSGAGTELSVRAKPS (SEQ ID NO: 13), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is A, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10 is L, T or G; X 11 is K or R; X 12 is V or I; X 13 is F, L or V; and X 14 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain comprising the sequence of SEQ ID NO: 1.

[0066] In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant comprising the sequence of SEQ ID NO: 13, wherein X1 is L, I or V. In any of the foregoing embodiments, X2 is V, L or I. In some embodiments, X3 is A or V. In some embodiments, X4 is A, I or L. In some embodiments, X5 is I, T, S or F. In some embodiments, X6 is E, V or L. In some embodiments, X7 is K or R. In some embodiments, X8 is E or Q. In some embodiments, X9 is H, P or R. In some embodiments, X 10 is L, T or G. In some embodiments, X 11 is K or R. In some embodiments, X 12 is V or I. In some embodiments, X 13 is F, L, V. In some embodiments, X 14is F or V. In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant (or a CD47-binding fragment thereof) comprising no more than six amino acid substitutions relative to a wild-type SIRPαD1 domain comprising the sequence of SEQ ID NO:1.

[0067] In some embodiments, the fusion polypeptide binds to CD47 with a binding affinity that is at least 10 times higher than the wild-type SIRPαD1 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 times higher than the wild-type SIRPαD1 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 times higher than the wild-type SIRPαD1 domain comprising the sequence of SEQ ID NO: 1. In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant or a CD47 binding fragment thereof that binds to the K of CD47. D Less than 1x 10 -8 M, less than 5x 10 -9 M, less than 1x 10 -9 M, less than 5x 10 -10 M, less than 1x 10 -10 M or less than 1x 10 - 11 M. In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant or a CD47 binding fragment thereof, which binds to the K domain of CD47. D Between about 500nM and 100nM, between about 100nM and 50nM, between about 50nM and 10nM, between about 10nM and 5nM, between about 5nM and 1nM, between about 1nM and 500pM, between about 500pM and 100pM, between about 100pM and 50pM, or between about 50pM and 10pM.

[0068] In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant comprising the sequence: EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISNITPADAGTYYCX 12 KX 13 RKGSPDTEX 14KSGAGTELSVRAKPS (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; and X 14 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain comprising the sequence of SEQ ID NO:2.

[0069] 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, X 12 is V or I. In some embodiments, X 13 is F, L or V. In some embodiments, X 14 is F or V. In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant (or a CD47-binding fragment thereof) comprising no more than six amino acid substitutions relative to a wild-type SIRPαD1 domain comprising the sequence of SEQ ID NO:2.

[0070] In some embodiments, the fusion polypeptide binds to CD47 with a binding affinity that is at least 10 times higher than the wild-type SIRPαD1 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 times higher than the wild-type SIRPαD1 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 times higher than the wild-type SIRPαD1 domain comprising the sequence of SEQ ID NO: 2. In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant or a CD47 binding fragment thereof that binds to the K of CD47. D Less than 1x 10-8 M, less than 5x 10 -9 M, less than 1x 10 -9 M, less than 5x 10 -10 M, less than 1x 10 -10 M or less than 1x 10 -11 M. In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant or a CD47 binding fragment thereof, which binds to the K domain of CD47. D Between about 500nM and 100nM, between about 100nM and 50nM, between about 50nM and 10nM, between about 10nM and 5nM, between about 5nM and 1nM, between about 1nM and 500pM, between about 500pM and 100pM, between about 100pM and 50pM, or between about 50pM and 10pM.

[0071] In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant comprising the sequence: EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSX 11 PVGPIQWFRGAGPX 12 RX 13 SXWh 14 X 15 GX 16 FPRVTTVSX17X 18 TX 19 RX 20 NMDFX 21 IX 22 IX 23 NITPADAGTYYCX 24 KX 25 RKGSPDX 26 X 27 EX 28 KSGAGTELSVRX 29 KPS (SEQ ID NO: 23), wherein X1 is E or G; X2 is L, I or V; X3 is V, L or I; X4 is S or F; X5 is L or S; X6 is S or T; X7 is A or V; X8 is I or T; X9 is H or R; X 10 is A, V, I or L; X 11 is I, T, S or F; X 12 A or G; X 13 is E, V or L; X 14 is K or R; X 15 is E or Q; X 16 is H, P or R; X 17 Is D or E; X18 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 It is F, L, V; X 26 Is D or does not exist; X 27 is T or V; X 28 is F or V; and X 29 is A or G; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1 or 2.

[0072] In any of the foregoing embodiments of this aspect of the disclosure, X2 is L, I, or V. In any of the foregoing embodiments, X3 is V, L, or I. In embodiments, X4 is S or F. In some embodiments, X5 is L or S. In some embodiments, X6 is S or T. In some embodiments, X7 is A or V. In some embodiments, X8 is I or T. In some embodiments, X9 is H or R. In some embodiments, X 10 is A, V, I or L. In some embodiments, X 11 is I, T, S or F. In some embodiments, X 12 is A or G. In some embodiments, X 13 is E, V or L. In some embodiments, X 14 is K or R. In some embodiments, X 15 is E or Q. In some embodiments, X 16 is H, P or R. In some embodiments, X 17 is D or E. In some embodiments, 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 25 is F, L, V. In some embodiments, X 26 is D or absent. In some embodiments, X 27 is T or V. In some embodiments, X28 is F or V. In some embodiments, X 29 is A or G. In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant (or a CD47-binding fragment thereof) having no more than six amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of SEQ ID NO: 1 or 2.

[0073] In some embodiments, the fusion polypeptide binds to CD47 with a binding affinity that is at least 10 times higher than the wild-type SIRPαD1 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 times higher than the wild-type SIRPαD1 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 times higher than the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant or a CD47 binding fragment thereof that binds to the K of CD47. D Less than 1x 10 -8 M, less than 5x 10 -9 M, less than 1x 10 -9 M, less than 5x 10 -10 M, less than 1x10 -10 M or less than 1x 10 -11 M. In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant or a CD47 binding fragment thereof, which binds to the K domain of CD47. D Between about 500nM and 100nM, between about 100nM and 50nM, between about 50nM and 10nM, between about 10nM and 5nM, between about 5nM and 1nM, between about 1nM and 500pM, between about 500pM and 100pM, between about 100pM and 50pM, or between about 50pM and 10pM.

[0074] In some embodiments, the fusion polypeptide comprises a SIRPαD2 domain containing a sequence of SEQ ID NO: 24 or a SIRPαD3 domain having a sequence of SEQ ID NO: 25. In some embodiments, the fusion polypeptide comprises a SIRPαD2 domain containing SEQ ID NO: 24 and a D3 domain containing SEQ ID NO: 25 (see Table 3). In some embodiments, the SIRPαD1 domain variant also comprises a fragment or variant of the D2 domain or a fragment or variant of the D3 domain. In some embodiments, the SIRPαD1 domain variant also comprises a fragment or variant of the D2 domain and a fragment or variant of the D3 domain. In some embodiments, the SIRPαD1 domain variant is joined to the D2 or D3 domain via a linker. In some embodiments, the SIRPαD1 domain variant is joined to the D2 and D3 domains via a linker.

[0075] Table 3. Amino acid sequences of SIRPα D2 and D3 domains

[0076]

[0077]

[0078] In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant, which is attached to an Fc domain or an Fc domain variant (e.g., fused, such as a genetic fusion). In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant, which is attached to an Fc domain variant that cannot dimerize (e.g., fused, such as a genetic fusion). In some embodiments, compared to a fusion polypeptide that does not comprise an Fc domain or an Fc domain variant, a fusion polypeptide comprising a SIRPαD1 domain variant and an Fc domain or an Fc domain variant exhibits improved pharmacokinetic properties, such as an increased serum half-life. In some embodiments, the fusion polypeptide comprising a SIRPαD1 domain variant does not comprise the sequence of any one of SEQ ID NOs: 26-36 shown in Table 4.

[0079] Table 4. Exemplary SIRPαD1 domain variants

[0080]

[0081]

[0082] In some embodiments, the fusion polypeptides described herein are used in vitro for binding assays, such as immunoassays. For example, in some embodiments, the fusion polypeptides described herein are used in liquid phase or bound to a solid support. In some embodiments, the fusion polypeptides used in immunoassays are detectably labeled in various ways.

[0083] In some embodiments, the fusion polypeptides described herein are combined with various carriers and used to detect the presence of specific antigen-expressing cells. Examples of carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier can be soluble or insoluble.

[0084] Various different labels and labeling methods are known. Examples of labels include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds. Various techniques for binding labels to the polypeptides disclosed herein are available.

[0085] In some embodiments, the fusion polypeptide is coupled to a low molecular weight hapten. These haptens are then specifically detected by a second reaction. For example, in some embodiments, the hapten biotin is used together with avidin, or the hapten dinitrophenol, pyridoxal, or fluorescein is detected with a specific anti-hapten antibody (e.g., anti-dinitrophenol antibody, anti-pyridoxal antibody, and anti-fluorescein antibody, respectively).

[0086] SIRPαD1 domain variants with altered glycosylation patterns

[0087] In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein α (SIRP-α) D1 variant, the variant comprising a SIRPαD1 domain or a fragment thereof having an amino acid mutation at residue 80 relative to a wild-type SIRPαD1 domain (e.g., the wild-type SIRPαD1 domain shown in SEQ ID NO: 1 or 2); and at least one additional amino acid mutation relative to the wild-type SIRPαD1 domain (e.g., the wild-type SIRPαD1 domain shown in SEQ ID NO: 1 or 2) at a residue selected from the group consisting of: residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92.

[0088] In some embodiments, the present invention also discloses a polypeptide comprising an Fc domain variant, wherein the Fc domain variant dimer comprises two Fc domain variants, wherein each Fc domain variant is independently selected from (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236 and N297A.

[0089] In some embodiments, the polypeptides in the compositions disclosed herein comprise SIRPαD1 domain variants having reduced or minimal glycosylation. The D1 domains of SEQ ID NO: 1 and 2 in Table 1 each contain a single potential N-linked glycosylation site at amino acid N80 in the sequence N80ITP. Expression of the SIRPαD1 domain in Chinese hamster ovary (CHO) cells results in a major band of 16 kDa (non-glycosylated) and a secondary band of higher molecular weight, which is removed by Endo Hf. Endo Hf is a recombinant protein fusion of endoglycosidase H and maltose binding protein. Endo Hf cleaves within a high mannose chitobiose core and some hybrid oligosaccharides from N-linked glycoproteins. This means that the proline at amino acid position 83 can reduce the efficiency of glycosylation, resulting in proteins with varying degrees of glycosylation and therefore heterogeneity. For drug development, heterogeneity can pose challenges to process development. Therefore, in order to study the possibility of producing a homologous, non-glycosylated form of SIRPαD1 domain variants, in some embodiments, the amino acid N80 of the SIRPαD1 variant is mutated to Ala. In some embodiments, in order to prepare a non-glycosylated SIRPαD1 domain variant, the amino acid N80 in the SIRPαD1 domain variant is substituted by any amino acid, including any naturally and non-naturally occurring amino acids, such as N80A and N80Q. In some embodiments, the SIRPαD1 domain variant comprises an N80A mutation and at least one additional mutation (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional mutations or more 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.

[0090] In some embodiments, the polypeptides in the compositions disclosed herein comprise SIRPαD1 domain variants with increased glycosylation relative to the wild-type SIRPαD1 domain. Another option to increase the homogeneity of the final product is to increase the glycosylation efficiency at amino acid N80 and produce SIRPαD1 domain variants with increased glycosylation relative to the wild type. In some embodiments, the amino acid P83 in the sequence NITP83 affects the degree of glycosylation at amino acid N80. In some embodiments, changing P83 to any amino acid increases the glycosylation efficiency at N80. In some embodiments, the amino acid P83 in the SIRPαD1 domain variant is substituted with any amino acid, including natural and non-natural amino acids, such as P83V, P83A, P83I and P83L. In some embodiments, the polypeptides of the present disclosure are expressed in cells that are optimized not to glycosylate proteins expressed by such cells, for example, by genetically engineering cell lines (e.g., genetically engineering yeast or mammalian hosts) or by altering cell culture conditions (such as adding kifunensine) or by using naturally non-glycosylating hosts (e.g., prokaryotes (E. coli, etc.)).

[0091] Table 5 lists specific amino acid substitutions in SIRPαD1 domain variants relative to each D1 domain variant sequence. In some embodiments, the SIRPαD1 domain variant comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more) substitutions listed in Table 5. In some embodiments, the SIRPαD1 domain variant is not glycosylated or is minimally glycosylated. In some embodiments, the SIRPαD1 domain variant is fully glycosylated or almost fully glycosylated. In some embodiments, relative to the wild-type D1 domain, the SIRPαD1 domain variant comprises up to fourteen amino acid substitutions. In some embodiments, relative to the wild-type D1 domain, the SIRPαD1 domain variant comprises up to ten amino acid substitutions. In some embodiments, relative to the wild-type D1 domain, the SIRPαD1 domain variant comprises up to seven amino acid substitutions. In some embodiments, the SIRPα D1 domain variants disclosed herein have at least 90% (eg, at least 92%, 95%, 97%, or greater than 97%) amino acid sequence identity to the sequence of the wild-type D1 domain.

[0092] In some embodiments, the SIRPαD1 domain variant is a chimeric SIRPαD1 domain variant, which includes two or more wild-type D1 domains or a portion of a variant thereof (e.g., a portion of a wild-type D1 domain or a variant thereof and a portion of another wild-type D1 domain or a variant thereof). In some embodiments, the chimeric SIRPαD1 domain variant includes at least two portions (e.g., three, four, five or more portions) of a wild-type D1 domain or a variant thereof, wherein each portion is from a different wild-type D1 domain. In some embodiments, the chimeric SIRPαD1 domain variant also includes one or more amino acid substitutions listed in Table 5.

[0093] Table 5. Amino Acid Substitutions in SIRPαD1 Domain Variants

[0094]

[0095]

[0096]

[0097]

[0098] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having the following sequence: EEEX1QX2IQPDKSVLVAAGETX3TLRCTX4TSLX5PVGPIQWFRGAGPGRX6LIYNQX7X8GX9FPRVTTVSDX 10 TX 11 RNNMDFSIRIGX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSPDDVEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 37), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is A, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; 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 15is F, L or V; and X 16 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1.

[0099] In some embodiments of this aspect of the disclosure, the polypeptide comprises a SIRPαD1 domain variant having a sequence of SEQ ID NO: 37, wherein X1 is L, I or V. In some embodiments, X2 is V, L or I. In some embodiments, X3 is A or V. In some embodiments, X4 is A, I or L. In some embodiments, X5 is I, T, S or F. In some embodiments, X6 is E, V or L. In some embodiments, X7 is K or R. In some embodiments, X8 is E or Q. In some embodiments, X9 is H, P or R. In some embodiments, X 10 is L, T or G. In some embodiments, X 11 is K or R. In some embodiments, X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. In some embodiments, X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W or Y. In some embodiments, X 14 is V or I. In some embodiments, X 15 is F, L, V. In some embodiments, X 16 It is F or V.

[0100] In some embodiments, the polypeptides provided herein comprise no more than ten amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptides provided herein comprise no more than seven amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1.

[0101] In some embodiments, the polypeptide binds to CD47 with at least 10 times higher binding affinity than the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 100 times higher binding affinity than the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 1000 times higher binding affinity than the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof binds to CD47 with less than 1 x 10 -8M, less than 5x 10 -9 M, less than 1x10 -9 M, less than 5x 10 -10 M, less than 1x 10 -10 M or less than 1x 10 -11 M of K D Binds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

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

[0103] EEEX1QX2IQPDKSVSVAAGESX3ILHCTX4TSLX5PVGPIQWFRGAGPARX6LIYNQX7X8GX9FPRVTTVSEX 10 TX 11 RENMDFSISISX 12 ITX 13 ADAGTYYCX 14 KX 15 RKGSPDTEX 16 KSGAGTELSVRAKPS (SEQ ID NO: 38), wherein X1 is L, I or V; X2 is V, L or I; X3 is A or V; X4 is V, I or L; X5 is I, T, S or F; X6 is E, V or L; X7 is K or R; X8 is E or Q; X9 is H, P or R; X 10 is S, T or G; X 11 is K or R; X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; X 13 is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; X 14 is V or I; X 15 is F, L or V; and X 16 is F or V; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2.

[0104] In some embodiments of this aspect of the disclosure, the polypeptide comprises a SIRPαD1 domain variant having a sequence of SEQ ID NO: 38, wherein X1 is L, I, or V. In some embodiments, X2 is V, L, or I. In some embodiments, X3 is A or V. In some embodiments, X4 is V, I, or L. In some embodiments, X5 is I, T, S, or F. In some embodiments, X6 is E, V, or L. In some embodiments, X7 is K or R. In some embodiments, X8 is E or Q. In some embodiments, X9 is H, P, or R. In some embodiments, X 10 is S, T or G. In some embodiments, X 11 is K or R. In some embodiments, X 12 is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. In some embodiments, X 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 It is F or V.

[0105] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having no more than ten amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of SEQ ID NO: 2. In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having no more than seven amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of SEQ ID NO: 2.

[0106] In some embodiments, the polypeptide binds to CD47 with at least 10 times higher binding affinity than the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with at least 100 times higher binding affinity than the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with at least 1000 times higher binding affinity than the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof binds to CD47 with less than 1 x 10 -8 M, less than 5x 10 -9 M, less than 1x10 -9 M, less than 5x 10 -10 M, less than 1x 10 -10M or less than 1x 10 -11 M of K D Binds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

[0107] In another aspect, the disclosure features a polypeptide comprising a SIRPαD1 domain variant having the sequence:

[0108] EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSL 11 PVGPIQWFRGAGPX 12 RX 13 SXWh 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 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 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; X19 is K or R; X 20 is E or N; X 21 is S or P; X 22 is S or R; X 23 is S or G; X 24 is any amino acid; X 25 is any amino acid; X 26 is V or I; X 27 It is F, L, V; X 28 Is D or does not exist; X 29 is T or V; X 30 is F or V; and X 31 is A or G; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1 or 2.

[0109] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:47, wherein X1 is E or G. In any of the foregoing embodiments in this aspect of the disclosure, X2 is L, I or V. In any of the foregoing embodiments, X3 is V, L or I. In any of the foregoing embodiments, X4 is S or F. In any of the foregoing embodiments, X5 is L or S. In any of the foregoing embodiments, X6 is S or T. In any of the foregoing embodiments, X7 is A or V. In any of the foregoing embodiments, X8 is I or T. In any of the foregoing embodiments, X9 is H or R. In any of the foregoing embodiments, X 10 is A, V, I or L. In any of the foregoing embodiments, X 11 is I, T, S or F. In any of the foregoing embodiments, X 12 is A or G. In any of the foregoing embodiments, X 13 is E, V or L. In any of the foregoing embodiments, X 14 is K or R. In any of the foregoing embodiments, X 15 is E or Q. In any of the foregoing embodiments, X 16 is H, P or R. In any of the foregoing embodiments, X 17 is D or E. In any of the foregoing embodiments, X 18 is S, L, T or G. In any of the foregoing embodiments, X 19 is K or R. In any of the foregoing embodiments, X 20 is E or N. In any of the foregoing embodiments, X 21 is S or P. In any of the foregoing embodiments, X 22is 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 It is A or G.

[0110] In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than ten amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than seven amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of SEQ ID NO: 1 or 2.

[0111] In some embodiments, the polypeptide binds to CD47 with at least 10 times higher binding affinity than the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptide binds to CD47 with at least 100 times higher binding affinity than the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptide binds to CD47 with at least 1000 times higher binding affinity than the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1 or 2. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof binds to CD47 with less than 1x10 -8 M, less than 5x 10 -9 M, less than 1x 10 -9 M, less than 5x 10 -10 M, less than 1x 10 -10 M or less than 1x 10 -11 M of K DBinds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

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

[0113] EEELQX1IQPDKSVX2VAAGEX3AX4LX5CTX6TSLX7PVGPIQWFRGAGPX8RX9LIYNQX 10 X 11 GX 12 FPRVTTVSX 13 X 14 TKRX 15 NMDFSIX 16 IX 17 X 18 ITPADAGTYYCX 19 KFRKGX 20 X 21 X 22 DX 23 EFKSGAGTELSVRAKPS (SEQ ID NO: 48), wherein X1 is V or I; X2 is L or S; X3 is T or S; X4 is T or I; X5 is R or H; X6 is A, V or I; X7 is I, R, Y, K or F; X8 is G or A; X9 is E or V; X 10 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 N or A; X 19 is V or I; X 20 is S, I or M; X 21 Is P or does not exist; X 22 is D or P; and X 23 is V or T, or a fragment thereof.

[0114] In another aspect, the disclosure features a polypeptide comprising a SIRPαD1 domain variant having the sequence:

[0115] EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9ITPADAGTYYCX 10 KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 49), wherein X1 is V, L or I; X2 is A, I, V or L; X3 is I, F, S or T; X4 is E, V or L; X5 is K or R; X6 is E or Q; X7 is H, P or R; X8 is L, T, S or G; X9 is A; and X 10 is V or I; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1.

[0116] 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 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 It is V or I.

[0117] In some embodiments, the polypeptide comprises a SIRPαD1 domain comprising at least 85% sequence identity to SEQ ID NO: 49 (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity), wherein X 1、 X 2、 X 3、 X 4、 X 5、 X 6、 X 7、 X 8、 X9 and X 10 Each of these is not a wild-type amino acid.

[0118] In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than ten amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 1. In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than seven amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence of any one of SEQ ID NO: 1.

[0119] In some embodiments, the polypeptide binds to CD47 with at least 10 times higher binding affinity than the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 100 times higher binding affinity than the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 1000 times higher binding affinity than the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof binds to CD47 with less than 1 x 10 -8 M, less than 5x 10 -9 M, less than 1x10 -9 M, less than 5x 10 -10 M, less than 1x 10 -10 M or less than 1x 10 -11 M of K D Binds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

[0120] In another aspect, the disclosure features a polypeptide comprising a SIRPαD1 domain variant having the sequence:

[0121] EEELQX1IQPDKSVSVAAGESAILHCTX2TSLX3PVGPIQWFRGAGPARX4LIYNQX5X6GX7FPRVTTVSEX8TKRENMDFSISISX9ITPADAGTYYCX 10KFRKGSPDTEFKSGAGTELSVRAKPS, (SEQ ID NO: 50), wherein X1 is V or I; X2 is V or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is E or Q; X7 is H or P; X8 is S or T; X9 is N or A; and X 10 is V or I; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2.

[0122] 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 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 It is V or I.

[0123] In some embodiments, the polypeptide comprises a SIRPαD1 domain comprising at least 85% sequence identity to SEQ ID NO:50 (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity), wherein X 1、 X 2、 X 3、 X 4、 X 5、 X 6、 X 7、 X 8、 X9 and X 10 Each of these is not a wild-type amino acid.

[0124] In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than ten amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than seven amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 2.

[0125] In some embodiments, the polypeptide binds to CD47 with at least 10 times higher binding affinity than the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with at least 100 times higher binding affinity than the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with at least 1000 times higher binding affinity than the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof binds to CD47 with less than 1 x 10 -8 M, less than 5x 10 -9 M, less than 1x10 -9 M, less than 5x 10 -10 M, less than 1x 10 -10 M or less than 1x 10 -11 M of K D Binds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

[0126] In another aspect, the disclosure features a polypeptide comprising a SIRPαD1 domain variant having the sequence:

[0127] EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMDFSIRIGX8ITPADAGTYYCX9KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO:51), wherein X1 is V or I; X2 is A or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is H or P; X7 is L or T; X8 is N or A; and X9 is V or I; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:1.

[0128] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:51, wherein X1 is V or I. In any of the foregoing embodiments in this aspect of the disclosure, X2 is A or I. In any of the foregoing embodiments, X3 is I or F. In any of the foregoing embodiments, X4 is E or V. In any of the foregoing embodiments, X5 is K or R. In any of the foregoing embodiments, X6 is H or P. In any of the foregoing embodiments, X7 is L or T. In any of the foregoing embodiments, X8 is N or A. In any of the foregoing embodiments, X9 is V or I. In one embodiment, X4 is not V.

[0129] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:51, wherein X8 is A. In any of the foregoing embodiments in this aspect of the disclosure, X8 is A and X1 is V or I. In any of the foregoing embodiments in this aspect of the disclosure, X8 is A and X2 is A or I. In any of the foregoing embodiments, X8 is A and X3 is I or F. In any of the foregoing embodiments, X8 is A and X4 is E or V. In one embodiment, 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.

[0130] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO:51, wherein X8 is A. In any of the foregoing embodiments in this aspect of the disclosure, X8 is A and X1 is I. In any of the foregoing embodiments in this aspect of the 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.

[0131] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant comprising at least 85% sequence identity to SEQ ID NO: 51 (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity), wherein X 1、 X2、 X 3、 X 4、 X 5、 X 6、 X 7、 Each of X8 and X9 is not a wild-type amino acid.

[0132] In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than ten amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than seven amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1.

[0133] In some embodiments, the polypeptide binds to CD47 with at least 10 times higher binding affinity than the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 100 times higher binding affinity than the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with at least 1000 times higher binding affinity than the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof binds to CD47 with less than 1 x 10 -8 M, less than 5x10 -9 M, less than 1x 10 -9 M, less than 5x 10 -10 M, less than 1x 10 -10 M or less than 1x 10 -11 M of K D Binds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

[0134] In another aspect, the disclosure features a polypeptide comprising a SIRPαD1 domain variant having the sequence:

[0135] EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRELIYNQX4EGX5FPRVTTVSDX6TKRNNMDFSIRIGX7ITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO:222), wherein X1 is V, L or I; X2 is A, I or L; X3 is I, T, S or F; X4 is K or R; X5 is H or P; X6 is L, T or G; X7 is N or A; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having a sequence according to SEQ ID NO:1.

[0136] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 222, wherein X1 is V, L or I. In any of the preceding embodiments in this aspect of the disclosure, X2 is A, I or L. In any of the preceding embodiments, X3 is I, T, S or F. In any of the preceding embodiments, X4 is K or R. In any of the preceding embodiments, X5 is H or P. In any of the preceding embodiments, X6 is L, T or G. In any of the preceding embodiments, X7 is N or A.

[0137] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 222, wherein X1 is V or I. In any of the preceding embodiments in this aspect of the disclosure, X2 is A or I. In any of the preceding embodiments, X3 is I or F. In any of the preceding embodiments, X4 is K or R. In any of the preceding embodiments, X5 is H or P. In any of the preceding embodiments, X6 is L or T. In any of the preceding embodiments, X7 is N or A.

[0138] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 222, wherein X7 is A. In any of the foregoing embodiments in this aspect of the disclosure, X7 is A and X1 is V or I. In any of the foregoing embodiments in this aspect of the disclosure, X7 is A and X2 is A or I. In any of the foregoing embodiments, X7 is A and X3 is I or F. In any of the foregoing embodiments, X7 is A and X4 is K or R. In any of the foregoing embodiments, X7 is A and X5 is H or P. In any of the foregoing embodiments, X7 is A and X6 is L or T.

[0139] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 222, wherein X7 is A. In any of the foregoing embodiments in this aspect of the disclosure, X7 is A and X1 is I. In any of the foregoing embodiments in this aspect of the disclosure, X7 is A and X2 is I. In any of the foregoing embodiments, X7 is A and X3 is F. In any of the foregoing embodiments, X7 is A and X4 is R. In any of the foregoing embodiments, X7 is A and X5 is P. In any of the foregoing embodiments, X7 is A and X6 is T.

[0140] In some embodiments, the polypeptide comprises a SIRPαD1 domain comprising at least 85% sequence identity to SEQ ID NO: 222 (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity), wherein X 1、 X 2、 X 3、 X 4、 X 5、 Each of X6 and X7 is not a wild-type amino acid.

[0141] In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than ten amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than seven amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 1.

[0142] In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 10 times higher than that of a wild-type SIRPαD1 domain having a sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 100 times higher than that of a wild-type SIRPαD1 domain having a sequence of SEQ ID NO: 1. In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 1000 times higher than that of a wild-type SIRPαD1 domain having a sequence of SEQ ID NO: 1. In some embodiments, the fragment comprises a polypeptide having a length of less than 10 amino acids, a length of about 10 amino acids, a length of about 20 amino acids, a length of about 30 amino acids, a length of about 40 amino acids, a length of about 50 amino acids, a length of about 60 amino acids, a length of about 70 amino acids, a length of about 80 amino acids, a length of about 90 amino acids, a length of about 100 amino acids, or a length of more than about 100 amino acids. The fragment retains the ability to bind to CD47. Preferably, the SIRPαD1 domain variant polypeptide and its fragment binds to CD47 with a higher affinity than the SIRPα polypeptide binds to CD47. For example, in some embodiments, the SIRPαD1 domain variant polypeptide or its fragment binds to CD47 with an affinity of less than 1 x 10 -8 M, less than 5x 10 -9 M, less than 1x 10 -9 M, less than 5x 10 -10 M, less than 1x10 -10 M or less than 1x 10 -11 M of K D Binds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

[0143] In another aspect, the disclosure features a polypeptide comprising a SIRPαD1 domain variant having the sequence:

[0144] EEELQX1IQPDKSVSVAAGESAILHCTX2TSLX3PVGPIQWFRGAGPARELIYNQX4EGX5FPRVTTVSEX6TKRENMDFSISISX7ITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS (SEQ ID NO:212), wherein X1 is V, L or I; X2 is V, I or L; X3 is I, T, S or F; X4 is K or R; X5 is H, P or R; X6 is S, T or G; X7 is N or A; and wherein the variant comprises at least one amino acid substitution relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO:2.

[0145] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, wherein X1 is V, L or I. In any of the foregoing embodiments in this aspect of the disclosure, X2 is V, I or L. In any of the foregoing embodiments, X3 is I, T, S or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In any of the foregoing embodiments, X6 is S, T or G. In any of the foregoing embodiments, X7 is N or A.

[0146] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, wherein X1 is V or I. In any of the foregoing embodiments in this aspect of the disclosure, X2 is V or I. In any of the foregoing embodiments, X3 is I or F. In any of the foregoing embodiments, X4 is K or R. In any of the foregoing embodiments, X5 is H or P. In any of the foregoing embodiments, X6 is S or T. In any of the foregoing embodiments, X7 is N or A.

[0147] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, wherein X7 is A. In any of the foregoing embodiments in this aspect of the disclosure, X7 is A and X1 is V or I. In any of the foregoing embodiments in this aspect of the disclosure, X7 is A and X2 is V or I. In any of the foregoing embodiments, X7 is A and X3 is I or F. In any of the foregoing embodiments, X7 is A and X4 is K or R. In any of the foregoing embodiments, X7 is A and X5 is H or P. In any of the foregoing embodiments, X7 is A and X6 is S or T.

[0148] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, wherein X7 is A. In any of the foregoing embodiments in this aspect of the disclosure, X7 is A and X1 is I. In any of the foregoing embodiments in this aspect of the disclosure, X7 is A and X2 is I. In any of the foregoing embodiments, X7 is A and X3 is F. In any of the foregoing embodiments, X7 is A and X4 is R. In any of the foregoing embodiments, X7 is A and X5 is P. In any of the foregoing embodiments, X7 is A and X6 is T.

[0149] In some embodiments, the polypeptide comprises a SIRPαD1 domain having at least 85% sequence identity to SEQ ID NO: 212 (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity), wherein X 1、 X 2、 X 3、 X 4、 X 5、 X 6、 Each of X7 and X8 is not a wild-type amino acid.

[0150] In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than ten amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide of this aspect of the disclosure comprises no more than seven amino acid substitutions relative to the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 2.

[0151] In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 10 times higher than that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 100 times higher than that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds to CD47 with a binding affinity at least 1000 times higher than that of the wild-type SIRPαD1 domain having the sequence of SEQ ID NO: 2. In some embodiments, the fragment comprises a polypeptide having a length of less than 10 amino acids, a length of about 10 amino acids, a length of about 20 amino acids, a length of about 30 amino acids, a length of about 40 amino acids, a length of about 50 amino acids, a length of about 60 amino acids, a length of about 70 amino acids, a length of about 80 amino acids, a length of about 90 amino acids, a length of about 100 amino acids, or a length of more than about 100 amino acids. The fragment retains the ability to bind to CD47. Preferably, the SIRPαD1 domain variant polypeptide and its fragment binds to CD47 with a higher affinity than the SIRPα polypeptide binds to CD47. For example, in some embodiments, the SIRPαD1 domain variant polypeptide or its fragment binds to CD47 with an affinity of less than 1 x 10 -8 M, less than 5x 10 -9 M, less than 1x 10 -9 M, less than 5x 10 -10 M, less than 1x10 -10 M or less than 1x 10 -11 M of K D Binds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide or fragment thereof has a K between about 500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pM and 50 pM, or between about 50 pM and 10 pM. D Binds to CD47.

[0152] In some embodiments, described herein are polypeptides comprising a SIRPαD1 domain variant having a sequence according to:

[0153] EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9X 10 X 11 X 12 ADAGTYYCX 13KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 218), wherein X1 is V, L or I; X2 is A, V, L or I; X3 is I, S, T or F; X4 is E, L or V; X5 is K or R; X6 is E or Q; X7 is H, R or P; X8 is S, G, L or T; X9 is any amino acid; X 10 is any amino acid; X 11 is any amino acid; X 12 is any amino acid; and X 13 is V or I; and wherein the SIRPαD1 domain variant comprises at least two amino acid substitutions relative to the wild-type SIRPαD1 domain having a sequence according to SEQ ID NO:1.

[0154] In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 212, wherein X1, wherein X9 is A. In any of the foregoing embodiments in this aspect of the disclosure, X9 is N. In any of the foregoing embodiments in this aspect of the disclosure, X 10 is I. In any of the foregoing embodiments in this aspect of the disclosure, X9 is N and X10 is P. In any of the foregoing embodiments in this aspect of the disclosure, X9 is N and X11 is any amino acid except S, T or C. In any of the foregoing embodiments in this aspect of the disclosure, X 11 is T. In any of the foregoing embodiments in this aspect of the disclosure, X 11 is an amino acid other than T. In any of the foregoing embodiments in this aspect of the disclosure, X 12 is P. In any of the foregoing embodiments in this aspect of the disclosure, X9 is N and X 12 Any amino acid except P.

[0155] In some embodiments, described herein are polypeptides comprising a SIRPαD1 domain variant having a sequence according to:

[0156] EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5X6GX7FPRVTTVSDX8TKRNNMDFSIRIGX9ITX 10 ADAGTYYCX 11KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 219), wherein X1 is V, L or I; X2 is A, V, L or I; X3 is I, S, T or F; X4 is E, L or V; X5 is K or R; X6 is E or Q; X7 is H, R or P; X8 is S, G, L or T; X9 is N; X 10 is any amino acid except P; and X 11 is V or I; and wherein the SIRPαD1 domain variant comprises at least two amino acid substitutions relative to the wild-type SIRPαD1 domain having a sequence according to SEQ ID NO:1.

[0157] In another aspect of the present disclosure, disclosed herein is a composition comprising a SIRPαD1 domain variant polypeptide or a fragment thereof having an amino acid sequence of SEQ ID NO: 48. In some embodiments, the SIRPαD1 domain variant polypeptide or a fragment thereof binds to CD47 with an affinity higher than that of the SIRPα polypeptide to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide binds to CD47 with an affinity less than 1 x 10 -8 M, less than 1x 10 -9 M, less than 1x 10 -10 M or less than 1x 10 -11 K D Binds to CD47. In some embodiments, the SIRPαD1 domain variant polypeptide is linked or fused to a second polypeptide. In some embodiments, the second polypeptide includes but is not limited to an Fc polypeptide, an Fc variant, or a fragment thereof.

[0158] Without limiting the foregoing, in some embodiments, the SIRPαD1 domain variant polypeptide is selected from any one of SEQ ID NOs: 53-87 and 213 shown in Table 6.

[0159] Table 6. Exemplary SIRPαD1 domain variant polypeptides

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to any variant provided in Table 6.

[0167] In some embodiments, the polypeptide comprises a SIRPαD1 domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to SEQ ID NO: 80, 81 or 85 in Table 6.

[0168] Fc domain variants

[0169] The fusion polypeptide disclosed herein comprises a signal regulatory protein α (SIRP-α) D1 variant (or a CD47 binding fragment thereof) and an Fc domain variant. In some embodiments, the Fc domain variant is or comprises (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations (wherein the amino acid numbering is according to the EU index); (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations (wherein the amino acid numbering is according to the EU index); or (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236 and N297A mutations (wherein the amino acid numbering is according to the EU index).

[0170] Antibodies targeting cell surface antigens can trigger immunostimulation and effector functions associated with Fc receptors (FcR) on immune cells. There are many Fc receptors that are specific to antibodies of a particular class, including IgG (gamma receptors), IgE (η receptors), IgA (alpha receptors) and IgM (μ receptors). The combination of Fc regions with Fc receptors on the cell surface can trigger many biological responses, including phagocytosis of antibody-coated particles (antibody-dependent cell-mediated phagocytosis, or ADCP), the removal of immune complexes, killer cells to the lysis of antibody-coated cells (antibody-dependent cell-mediated cytotoxicity, or ADCC) and the release of inflammatory mediators, placental transfer and control of immunoglobulin production. In addition, the combination of the C1 component of complement with antibodies can activate the complement system. The activation of complement may be important for the lysis of cell pathogens. However, the activation of complement can also stimulate inflammatory responses, and can also participate in autoimmune hypersensitivity or other immune disorders. Variant Fc regions with reduced or eliminated ability to bind certain Fc receptors can be used to develop therapeutic antibodies and Fc fusion polypeptide constructs that act by targeting, activating or neutralizing ligand function without damaging or destroying local cells or tissues.

[0171] In some embodiments, the fusion protein comprises a SIRPαD1 domain variant (or a CD47-binding fragment thereof) linked (eg, fused, such as a genetic fusion) to an Fc domain variant, thereby forming an Fc domain with abolished or reduced effector function.

[0172] In some embodiments, Fc domain variant refers to a polypeptide chain comprising a second and third antibody constant domain (e.g., CH2 and CH3). In some embodiments, Fc domain variants also include a hinge domain. In some embodiments, Fc domain variants belong to any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA and IgD. In addition, in some embodiments, Fc domain variants belong to any IgG subtype (e.g., IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3 and IgG4). In some embodiments, Fc domain variants include up to ten amino acid modifications (e.g., insertions, deletions and / or substitutions) relative to wild-type Fc domain monomer sequences (e.g., 1-10, 1-8, 1-6, 1-4 amino acid substitutions, additions or insertions, deletions or combinations thereof), which change the interaction between the Fc domain and the Fc receptor.

[0173] 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, two wild-type Fc domains are dimerized by the interaction between two CH3 antibody constant domains and one or more disulfide bonds formed between the hinge domains of the two dimerized Fc domains.

[0174] As used herein, the term "Fc domain dimer variant" comprises two Fc domain variants. In some embodiments, the Fc domain dimer variant comprises an Fc domain variant that lacks effector function by mutation, such as a "dead Fc domain dimer variant". In some embodiments, each Fc domain in the Fc domain dimer variant comprises an amino acid substitution in the CH2 and / or CH3 antibody constant domain to reduce the interaction or binding between the Fc domain dimer variant and an Fc receptor, such as an Fcγ receptor (FcγR), an Fcα receptor (FcαR) or an Fcε (FcεR).

[0175] In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant (e.g., any variant described in Tables 2, 5, and 6), which is fused to an Fc domain variant of an immunoglobulin or a fragment of such an Fc domain variant (e.g., genetic fusion). In some embodiments, the fusion polypeptide comprises an Fc domain variant of an immunoglobulin (or a fragment thereof), which is capable of forming an Fc domain dimer with another Fc domain variant. In some embodiments, the fusion polypeptide comprises an Fc domain variant of an immunoglobulin (or a fragment thereof), which is unable to form an Fc domain dimer with another Fc domain variant. In some embodiments, the fusion polypeptide comprising an Fc domain variant (or a fragment thereof) shows an increase in the serum half-life of the polypeptide compared to a polypeptide not comprising an Fc domain variant (or a fragment thereof). In some embodiments, the fusion polypeptide comprises an Fc domain variant (or a fragment thereof), which 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).

[0176] In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain or a variant thereof joined to a first Fc domain variant and an antibody variable domain joined to a second Fc domain variant, wherein the first and second Fc domain variants combine to form an Fc domain dimer variant (e.g., a heterodimeric Fc domain dimer variant). In some embodiments, the fusion polypeptide comprises a SIRPαD1 domain variant connected to a first Fc domain variant (e.g., fused, such as genetic fusion) and a second SIRPαD1 domain variant connected to a second Fc domain variant (e.g., fused, such as genetic fusion), wherein the first Fc domain variant and the second Fc domain variant are combined 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 SIRPαD1 domain variant connected to a first Fc domain (e.g., fused, such as genetic fusion).

[0177] Fc domain dimer is a protein structure found at the C-terminus of immunoglobulins. Fc domain dimer includes two Fc domains dimerized by the interaction between CH3 antibody constant domains. Wild-type Fc domain dimer forms the minimum structure that binds to Fc receptors, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb and FcγRIV.

[0178] Fc domain dimers are not directly involved in the binding of antibodies to their targets, but may be involved in various effector functions, such as antibody involvement in antibody-dependent cellular toxicity. In some embodiments, the fusion polypeptide comprises an Fc domain variant, which comprises an amino acid substitution, addition or insertion, deletion or any combination thereof relative to the amino acid sequence of the corresponding wild-type Fc domain, resulting in a reduction in effector function, such as a reduction in antibody-dependent cell-mediated cytotoxicity (ADCC), a reduction in complement-dependent cytolysis (CDC), a reduction in antibody-dependent cell-mediated phagocytosis (ADCP) or any combination thereof. In some embodiments, the fusion polypeptide is characterized by reduced binding to human Fc receptors (e.g., minimal binding or absence of binding), and reduced binding to complement protein C1q (e.g., minimal binding or absence of binding). In some embodiments, the fusion polypeptide is characterized by reduced binding to human FcγRI, FcγRIIA, FcγRIIB, FcγRIIIB or any combination thereof and C1q (e.g., minimal binding or absence of binding). To alter or reduce antibody-dependent effector functions, such as ADCC, CDC, ADCP, or any combination thereof, in some embodiments, the fusion polypeptide comprises a human IgG Fc domain variant comprising one or more amino acid substitutions at E233, L234, L235, G236, G237, D265, D270, N297, E318, K320, K322, A327, A330, P331, or P329 (numbering according to the EU index of Kabat (Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD. (1991))).

[0179] In some embodiments, the fusion polypeptide comprises a non-natural Fc domain (e.g., an Fc domain variant), which, when dimerized to form an Fc domain dimer variant, exhibits reduced or eliminated binding to at least one of the Fcγ receptors CD16a, CD32a, CD32b, CD32c, and CD64 compared to a fusion polypeptide comprising a native Fc domain dimer. In some cases, when dimerized (e.g., homodimerization or heterodimerization), the fusion polypeptide exhibits reduced or eliminated binding to CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors.

[0180] CDC refers to a form of cytotoxicity in which the complement cascade is activated by complement component C1q bound to the Fc domain of an antibody. In some embodiments, the fusion polypeptide comprises an Fc domain variant that, when dimerized to form an Fc domain dimer variant, exhibits at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in C1q binding compared to a polypeptide construct comprising a wild-type Fc region. In some embodiments, the fusion polypeptide comprises an Fc domain variant that, when dimerized to form an Fc domain dimer variant, exhibits reduced CDC compared to a polypeptide construct comprising a wild-type Fc domain. In some embodiments, the fusion polypeptide comprises an Fc domain variant that, when dimerized to form an Fc domain dimer variant, exhibits at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in CDC compared to a fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the fusion polypeptide comprises an Fc domain variant that, when dimerized to form an Fc domain dimer variant, exhibits negligible CDC compared to a polypeptide construct comprising a wild-type Fc region.

[0181] In some embodiments, the fusion polypeptide comprises an Fc domain variant that is minimally glycosylated or has reduced glycosylation relative to a wild-type Fc domain. In some embodiments, deglycosylation is accomplished by mutation of N297A (wherein the amino acid numbering is according to the EU index) or by mutating N297 to any non-N amino acid. In some embodiments, deglycosylation is accomplished by destroying the motif N-Xaa1-Xaa2-Xaa3, wherein N=asparagine; Xaa1=any amino acid except P (proline); Xaa2=T (threonine), S (serine) or C (cysteine); Xaa3=any amino acid except P (proline). In one embodiment, the N-Xaa1-Xaa2-Xaa3 motif refers to residues 297-300, as specified according to Kabat et al., 1991. In some embodiments, mutation of any one or more of N, Xaa1, Xaa2 or Xaa3 results in deglycosylation of the Fc domain variant.

[0182] In some embodiments, fusion polypeptide includes IgG Fc domain variants, which show the ability of specific binding Fcγ receptors reduced or the ability of inducing phagocytosis reduced when dimerization.For example, in some embodiments, fusion polypeptide includes Fc domain variants (for example, IgG Fc domain variants), which lack the typical functions of "dead" Fc domain variants (for example, "dead" IgG Fc domain variants) when dimerization.For example, in some embodiments, Fc domain variants (for example, IgG Fc domain variants) include amino acid substitutions that are known to minimize the interaction between Fc domain dimers and Fcγ receptors.In some embodiments, fusion polypeptide includes Fc domain variants (for example, IgG Fc domain variants), which include one or more of amino acid substitutions L234A, L235A, G237A and N297A (as specified by the EU numbering system according to Kabat et al., 1991).In some embodiments, Fc domain variants include one or more additional mutations. Non-limiting examples of such additional mutations of human IgG1 Fc domain variants include E318A and K322A (wherein amino acid numbering is according to EU index). In some embodiments, the fusion polypeptide comprises an Fc domain variant (e.g., an IgG Fc domain variant), and the variant comprises up to 12, 11, 10, 9, 8, 7, 6, 5 or 4 or fewer mutations in total compared to the amino acid sequence of the wild-type human IgG1 domain. In some embodiments, the Fc domain variant also comprises one or more additional deletions. For example, in some embodiments, the C-terminal lysine of the Fc domain IgG1 heavy chain constant region provided in SEQ ID NO:88 in Table 7 is missing, for example, to increase the homogeneity of the polypeptide when the polypeptide is produced in bacteria or mammalian cells. In some embodiments, compared to the wild-type human IgG1 sequence, the human IgG1 Fc domain variant comprises up to 12, 11, 10, 9, 8, 7, 6, 5 or 4 or fewer deletions in total (see, e.g., SEQ ID NO:161, as follows). 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.

[0183] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:161)

[0184] In some embodiments, Fc domain variants are variants of human IgG2 or human IgG4 antibody Fc domains. In some embodiments, IgG2 variants or IgG4 variants include amino acid substitutions A330S, P331S or both A330S and P331S (wherein amino acid numbering is according to EU index). In some embodiments, IgG2 Fc domain variants include human IgG2 Fc domains, and the domain includes one or more of A330S, P331S and N297A amino acid substitutions (as specified by the EU numbering system according to Kabat et al. (1991)). In some embodiments, IgG2 Fc domain variants include one or more additional mutations. The non-limiting examples of such additional mutations include but are not limited to, for example, V234A, G237A, P238S, V309L and H268A (as specified by the EU numbering system according to Kabat et al. (1991)). In some cases, the human IgG2 Fc domain variant comprises up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or fewer mutations in total 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 up to 12, 11, 10, 9, 8, 7, 6, 5 or 4 or fewer deletions in total compared to the wild-type human IgG2 sequence (see, e.g., SEQ ID NO: 162, below).

[0185] ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK TISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:162).

[0186] In some embodiments, Fc domain variants are IgG4 Fc domain variants. In some embodiments, IgG4 Fc domain variants include S228P mutations (wherein amino acid numbering is according to EU index). In some embodiments, compared with the amino acid sequence of wild-type human IgG4 Fc domains, IgG4 Fc domain variants include up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations in total. In some embodiments, Fc domain variants include one or more of S228P, E233P, F234V, L235A and delG236 (wherein amino acid numbering is specified according to the EU numbering system of Kabat et al. (1991)). In some embodiments, Fc domain variants include one or more of S228P, E233P, F234V, L235A, delG236 and N297A amino acid substitutions (as specified according to the EU numbering system of Kabat et al. (1991)).

[0187] In some embodiments, the Fc domain variant is a variant of a human IgG1 Fc domain monomer comprising at least one mutation (e.g., two, three, or all four 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 comprising at least one mutation (e.g., two or all three mutations) selected from the group consisting of A330S, P331S, and N297A.

[0188] In some embodiments, the Fc domain variants exhibit reduced binding to Fc receptors compared to wild-type human IgG Fc domains. In some embodiments, the Fc domain variants exhibit eliminated binding to the subject's Fc receptors compared to wild-type human IgG Fc domains. In some embodiments, the Fc domain variants exhibit reduced ability to mediate phagocytosis compared to wild-type human IgG Fc domains. In some embodiments, the Fc domain variants exhibit eliminated phagocytosis compared to wild-type human IgG Fc domains.

[0189] SEQ ID NO:88 and SEQ ID NO:89 are the amino acid sequences of IgG1 and IgG2 Fc domains, respectively. In some embodiments, the Fc domain variant comprises (or is) any one of SEQ ID NO:90-95 as shown in Table 7.

[0190] Table 7. Amino acid sequences of Fc domain variants

[0191]

[0192]

[0193] Antibody-dependent cell-mediated cytotoxicity (also referred to herein as ADCC) refers to a form of cytotoxicity, wherein secreted Ig is bound to Fc receptors (FCR) present in certain cytotoxic cells (e.g., natural killer (NK) cells and neutrophils), enabling these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill target cells. Antibody-dependent cell-mediated phagocytosis (also referred to herein as ADCP) refers to a form of cytotoxicity, wherein secreted Ig is bound to Fc receptors (FCR) present in certain phagocytic cells (e.g., macrophages), enabling these phagocytic effector cells to specifically bind to target cells carrying antigens and subsequently engulf and digest the target cells. Ligand-specific high-affinity IgG antibodies for the surface of target cells can stimulate cytotoxicity or phagocytic cells and can be used for such killing. In some embodiments, compared with a polypeptide (e.g., fusion polypeptide) comprising a wild-type Fc domain (e.g., a wild-type Fc domain dimer), a polypeptide provided herein (e.g., a fusion polypeptide) includes an Fc domain variant or an Fc domain dimer variant showing reduced ADCC or ADCP. In some embodiments, a polypeptide (e.g., a fusion polypeptide) provided herein includes an Fc domain variant or an Fc domain dimer variant, which, compared with a polypeptide (e.g., a fusion polypeptide) comprising a wild-type Fc domain, shows about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more ADCC or ADCP reduction in any one. In some embodiments, compared with a polypeptide (e.g., a fusion polypeptide) comprising a wild-type Fc region, a polypeptide (e.g., a fusion polypeptide) provided herein includes an Fc domain variant or an Fc domain dimer variant, and the variant shows eliminated ADCC or ADCP.

[0194] Complement-mediated cytotoxicity (also referred to herein as CDC) refers to a form of cytotoxicity in which the complement cascade is activated by complement component C1q bound to the Fc domain of an antibody. In some embodiments, the polypeptides provided herein (e.g., fusion polypeptides) comprise Fc domain variants or Fc domain dimer variants, which exhibit at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater C1q binding reduction compared to polypeptides (e.g., fusion polypeptides) comprising wild-type Fc regions. Any one of the reduced. In some embodiments, the polypeptides provided herein (e.g., fusion polypeptides) comprise Fc domain variants or Fc domain dimer variants, which exhibit reduced CDC compared to polypeptides (e.g., fusion polypeptides) comprising wild-type Fc regions. In some embodiments, provided herein is a polypeptide (e.g., fusion polypeptide) comprising an Fc domain variant or an Fc domain dimer variant, which exhibits at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater CDC reduction compared to a polypeptide (e.g., fusion polypeptide) comprising a wild-type Fc region. In some cases, provided herein is a polypeptide (e.g., fusion polypeptide) comprising an Fc domain variant or an Fc domain dimer variant, which exhibits negligible CDC compared to a polypeptide construct comprising a wild-type Fc region.

[0195] The Fc domain variants or Fc domain dimer variants herein show reduced binding to Fcγ receptors compared to wild-type human IgG Fc regions. For example, in some embodiments, the affinity of Fc domain variants or Fc domain dimer variants to Fcγ receptors is lower than the affinity of wild-type IgG Fc domains to Fcγ receptors, as described in the Examples. In some embodiments, compared with the binding of wild-type Fc domains to Fcγ receptors, the binding of 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% (completely eliminated effector functions). In some embodiments, the reduced binding is for any one or more Fcγ receptors selected from the group consisting of CD16a, CD32a, CD32b, CD32c and CD64.

[0196] In some embodiments, compared with wild-type human IgG Fc region, Fc domain variants or Fc domain dimer variants disclosed herein show reduced phagocytosis. In some embodiments, compared with the combination of wild-type Fc domains, the ability of Fc domain variants or Fc domain dimer variants mediated phagocytosis described herein is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% in any one. In some cases, Fc domain variants or Fc domain dimer variants show eliminated phagocytosis compared with wild-type human IgG Fc region.

[0197] In some embodiments, the SIRPα variant is connected 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 amount of amino acids, such as less than ten amino acids, although longer linkers are also used. In some cases, the length of the linker is less than 10, 9, 8, 7, 6 or 5 amino acids or shorter. 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 longer. Optionally, in some embodiments, a cleavable linker is used.

[0198] In some embodiments, the polypeptide (e.g., fusion polypeptide) herein comprises a targeting or signal sequence that directs the polypeptide to a desired cell location or extracellular environment. In some embodiments, certain signal transduction sequences target polypeptides to be secreted into a growth medium or located in the periplasmic space between the inner and outer membranes of a cell. In some embodiments, a polypeptide (e.g., fusion polypeptide) comprises an epitope or tag that can be purified or screened. Such epitopes or tags include, but are not limited to, polyhistidine tags (His tags) (e.g., His6 (HHHHHH SEQ ID NO: 223) and His10 (HHHHHHHHHHH SEQID NO: 224)) or other tags used with an immobilized metal affinity chromatography (IMAC) system (e.g., Ni+2 affinity column), GST fusions, MBP fusions, Strep tags, BSP biotinylated target sequences of bacterial enzyme BirA, and epitope tags (e.g., c-myc tags, flag tags, etc.) targeted by antibodies. In some embodiments, such tags can be used for purification, screening, or both. For example, in some embodiments, a His tag is used to purify a polypeptide (e.g., a fusion polypeptide) by immobilizing it to a Ni+2 affinity column, and then after purification, the same His tag is used to immobilize the antibody to a Ni+2 coated plate for ELISA or other binding assays as described elsewhere herein.

[0199] In some embodiments, fusion partner enables to use selection method to screen Fc domain variants or Fc domain dimer variants as described herein. Various fusion partners realizing multiple selection methods can be obtained. For example, by merging the member of Fc domain variant or Fc domain dimer variant library with gene III protein, phage display can be adopted. In some embodiments, fusion partner Fc domain variant or Fc domain dimer variant will be marked. Alternatively, in some embodiments, fusion partner is attached to the specific sequence on expression vector, so that fusion partner and relevant Fc domain variant or Fc domain dimer variant can be covalently or non-covalently connected with the nucleic acid encoding it.

[0200] In some embodiments, when the fusion partner is a therapeutic moiety, the therapeutic moiety is, for example, a peptide, protein, antibody, siRNA or small molecule. Non-limiting examples of therapeutic antibodies coupled to the Fc domain variants or Fc domain dimer variants of the present disclosure include, but are not limited to, antibodies that recognize CD47. Non-limiting examples of therapeutic polypeptides coupled to the Fc domain variants or Fc domain dimer variants of the present disclosure include, but are not limited to, CD47 binding polypeptides, including SIRPα polypeptides. In such cases, the CD47 binding polypeptide is connected or fused to the Fc domain variants or Fc domain dimer variants of the present disclosure. Examples of CD47 binding polypeptides include, but are not limited to, anti-CD47 antibodies or fragments thereof, and ligands for CD47, such as SIRPα or fragments thereof. Additional examples of CD47 binding polypeptides include, but are not limited to, naturally occurring forms of SIRPα and mutants thereof.

[0201] In some embodiments, disclosed herein are polypeptides comprising Fc domain dimer variants, wherein the Fc domain dimer variant comprises two Fc domain variants, wherein each Fc domain variant is independently selected from (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236 and N297A. In some embodiments, the Fc domain variants are identical (i.e., homodimers). In some embodiments, the Fc domain variants are different (i.e., heterodimers). In some embodiments, at least one Fc domain variant in the Fc domain dimer is a human IgG1 Fc region consisting of mutations L234A, L235A, G237A, and N297A. In some embodiments, at least one Fc domain variant in the Fc domain dimer is a human IgG2 Fc region consisting of mutations A330S, P331S, and N297A. In some embodiments, compared with the wild-type form of the human IgG Fc region, the Fc domain dimer variant exhibits eliminated or reduced binding to Fcγ receptors. In some embodiments, compared with the wild-type form of the human IgG Fc region, the Fc domain dimer variant exhibits eliminated or reduced binding to CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors. In some embodiments, compared with the wild-type form of the human IgG Fc fusion, the Fc domain dimer variant exhibits eliminated or reduced binding to C1q. In some embodiments, at least one of the Fc domain dimer variants is a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236, and N297A. In some embodiments, the Fc domain dimer variant exhibits eliminated or reduced binding to Fcγ receptors compared to the wild-type human IgG4 Fc region. In some embodiments, the Fc domain dimer variant exhibits eliminated or reduced binding to CD16a and CD32b Fcγ receptors compared to the wild-type form of its human IgG4 Fc region. In some embodiments, the Fc domain dimer variant is greater than about 5x 10 -6 M of K D Binds to Fcγ receptors.

[0202] In some embodiments, the Fc domain dimer variant further comprises a CD47 binding polypeptide. In some embodiments, the Fc domain dimer variant exhibits eliminated or reduced binding to Fcγ receptors compared to the wild-type form 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.

[0203] In some embodiments, the CD47 binding polypeptide is a signal regulatory protein alpha (SIRP-α) polypeptide or a fragment thereof. In some embodiments, the SIRPα polypeptide comprises a SIRPα D1 domain variant comprising the following amino acid sequence: EEELQX1IQPDKSVLVAAGETATLRCTX2TSLX3PVGPIQWFRGAGPGRX4LIYNQX5EGX6FPRVTTVSDX7TKRNNMDFSIRIGX8ITPADAGTYYCX9KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 221), wherein X1 is V or I; X2 is A or I; X3 is I or F; X4 is E or V; X5 is K or R; X6 is H or P; X7 is L or T; X8 is any amino acid except N; and X9 is V or I. In some embodiments, the SIRPα polypeptide comprises a SIRPα D1 domain variant, wherein X1 is V or I; X2 is A or I; X3 is I or F; X4 is E; X5 is K or R; X6 is H or P; X7 is L or T; X8 is not N; and X9 is V.

[0204] In some embodiments, disclosed herein is a polypeptide comprising: a SIRPαD1 domain variant, wherein the SIRPαD1 domain variant is a non-naturally occurring high-affinity SIRPαD1 domain, wherein the SIRPαD1 domain variant binds to human CD47 with an affinity at least 10 times greater than that of 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, wherein the Fc domain dimer variant has an effector function that is eliminated or reduced. In some embodiments, the non-naturally occurring high-affinity SIRPαD1 domain comprises an amino acid mutation at residue 80.

[0205] In some embodiments, a SIRPαD1 domain variant is disclosed herein, wherein the SIRPαD1 domain variant binds CD47 from a first species with a KD of less than 250nM; and wherein the SIRPαD1 domain variant binds CD47 from a second species with a KD of less than 250nM; and the KD of CD47 from the first species and the KD of CD47 from the second species are within 100 times of each other; wherein the first species and the second species are selected from the group consisting of: humans, rodents, and non-human primates. In some embodiments, the SIRPαD1 domain variant binds CD47 from at least 3 different species. In some embodiments, the non-human primate is a cynomolgus monkey.

[0206] In some embodiments, disclosed herein is a polypeptide comprising (a) a polypeptide having a K of less than 250 nM. D A SIRPαD1 domain that binds to human CD47; and (b) an Fc domain or a variant thereof connected to the N-terminus or C-terminus of the SIRPαD1 domain, wherein the polypeptide does not cause acute anemia in rodents and non-human primates. In some embodiments, the polypeptide is a non-naturally occurring variant of human SIRP-α. In some embodiments, in vivo administration of the polypeptide results in a decrease in hemoglobin of less than 50% during the first week after administration. In some embodiments, administration of the polypeptide in humans results in a decrease in hemoglobin of less than 50% during the first week after administration. In some embodiments, the polypeptide further comprises at least one Fc domain dimer variant, wherein the Fc domain dimer variant comprises an Fc domain variant selected from the following: (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236 and N297A. In some embodiments, the Fc domain variant is a human IgG1 Fc region consisting of mutations L234A, L235A, G237A and N297A. In some embodiments, the Fc domain variant is a human IgG2 Fc region consisting of mutations A330S, P331S and N297A.

[0207] The SIRPα construct of the present disclosure includes a SIRPα domain or a variant thereof, the C-terminus of which is joined to the N-terminus of an Fc domain or a variant thereof by a linker using conventional genetic or chemical means (e.g., chemical conjugation). In some embodiments, a linker (e.g., a spacer) is inserted between a polypeptide and an Fc domain or a variant thereof. In some embodiments, a polypeptide of the present disclosure including a SIRPαD1 domain variant is fused with an Fc domain variant that cannot form a dimer. In some embodiments, the polypeptide of the present disclosure is fused with an Fc domain or a variant thereof that can form a dimer (e.g., a heterodimer) with another Fc domain or a variant thereof. In some embodiments, the polypeptide of the present disclosure is fused with an Fc domain or a variant thereof, and this fusion protein forms a homodimer. In some embodiments, the polypeptide of the present disclosure is fused with a first Fc domain or a variant thereof, and a different protein or peptide (e.g., an antibody variable region) is fused with a second Fc domain or a variant thereof. In some embodiments, the SIRPαD1 domain or a variant thereof is joined to a first Fc domain or a variant thereof, and a therapeutic protein (e.g., a cytokine, an interleukin, an antigen, a steroid, an anti-inflammatory agent, or an immunomodulator) is joined to a second Fc domain or a variant thereof. In some embodiments, the first and second Fc domains or variants thereof form a heterodimer.

[0208] Without limiting the foregoing, in some embodiments, a SIRPαD1 domain variant polypeptide (e.g., any variant described in Tables 2, 5, and 6) is fused to an Fc polypeptide or Fc variant polypeptide (such as an Fc domain or a variant thereof). Examples of polypeptides comprising a SIRPαD1 domain variant polypeptide and a fused Fc domain variant polypeptide include, but are not limited to, SEQ ID NOs: 96-137, 214, and 216 shown in Table 8.

[0209] Table 8. Polypeptides comprising SIRPαD1 domain variants fused to Fc domain variants

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223] In some embodiments, the polypeptide comprises a SIRPαD1 variant domain having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to any variant provided in Table 8.

[0224] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to SEQ ID NO: 98-104, 107-113, 116-122 or 135-137 in Table 8.

[0225] In some embodiments, the polypeptide comprises (a) a signal regulatory protein alpha (SIRP-α) D1 variant, wherein the SIRPα D1 domain variant comprises an amino acid sequence,

[0226] EEX1X2QX3IQPDKX4VX5VAAGEX6X7X8LX9CTX 10 TSL 11 PVGPIQWFRGAGPX 12 RX 13 SXWh 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 X29 EX 30 KSGAGTELSVRX 31 KPS (SEQ ID NO: 47), wherein X1 is E or G; X2 is L, I or V; X3 is V, L or I; X4 is S or F; X5 is L or S; X6 is S or T; X7 is A or V; X8 is I or T; X9 is H, R or L; X 10 is A, V, I or L; X 11 is I, T, S or F; X 12 A or G; X 13 is E, V or L; X 14 is K or R; X 15 is E or Q; X 16 is H, P or R; X 17 Is D or E; X 18 is S, L, T or G; X 19 is K or R; X 20 is E or N; X 21 is S or P; X 22 is S or R; X 23 is S or G; X 24 is any amino acid; X 25 is any amino acid; X 26 is V or I; X 27 is F, L or V; X 28 Is D or does not exist; X 29 is T or V; X 30 is F or V; and X 31 is A or G; and wherein the SIRPαD1 domain variant comprises at least two amino acid substitutions relative to a wild-type SIRPαD1 domain having a sequence according to any one of SEQ ID NOs: 1 to 10; and (b) an Fc domain dimer variant having two Fc domain variants, wherein each Fc domain variant is independently (i) a human IgG1 Fc region comprising an N297A mutation; (ii) a human IgG1 Fc region comprising L234A, L235A, and G237A mutations; (iii) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations; (iv) a human IgG2 Fc region comprising an N297A mutation; (v) a human IgG2 Fc region comprising A330S and P331S mutations; (vi) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations. Fc region; (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.

[0227] In some embodiments, the polypeptide comprises a SIRPαD1 domain variant, wherein the SIRPαD1 domain variant comprises an amino acid sequence according to SEQ ID NO: 47; an Fc domain dimer having two Fc domains, wherein one of the Fc domains is an Fc domain variant comprising a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations.

[0228] Dimerization of the Fc domain

[0229] In some embodiments, the SIRPαD1 domain variant polypeptide (e.g., any variant described in Tables 2, 5, and 6) is fused to the first Fc domain (e.g., Fc domain variant) at the N-terminus or C-terminus. In some embodiments, the first Fc domain is a variant that cannot form a dimer. In some embodiments, the first Fc domain forms a dimer with the second Fc domain. In some embodiments, the first and second Fc domains comprise amino acid substitutions that promote heterodimerization between the first and second domain Fc domains.

[0230] In some embodiments, each of the two Fc domains in the Fc domain dimer includes an amino acid substitution that promotes heterodimerization of the two monomers. In some embodiments, the SIRPα construct is formed by, for example, a first subunit comprising a SIRPαD1 domain variant polypeptide fused to a first Fc domain and a second subunit comprising a second Fc domain (e.g., without a SIRPαD1 domain variant polypeptide or any other polypeptide). In some embodiments, the construct has a single SIRPαD1 domain variant polypeptide connected to an Fc domain dimer (e.g., a single arm). In some embodiments, the construct has two SIRPαD1 domain variant polypeptides connected to an Fc domain dimer (e.g., two arms). In some embodiments, K D SIRPαD1 domain variants with a K of about 500 nM are particularly useful in two-arm constructs. D SIRPαD1 domain variants with a K of about 50 nM are particularly useful in two-arm constructs. D SIRPαD1 domain variants with a K of about 5 nM are useful in both two-arm and one-arm constructs. D SIRPαD1 domain variants with a K of about 500 pM are useful in both two-arm and one-arm constructs. D SIRPαD1 domain variants with a K of about 100 pM are useful in both two-arm and one-arm constructs. DSIRPαD1 domain variants with a K of about 50 pM are useful in both two-arm and one-arm constructs. D SIRPαD1 domain variants at about 10 pM are useful in both two-armed and one-armed constructs.

[0231] In some embodiments, heterodimerization of Fc domains is promoted by introducing different but compatible substitutions in two Fc domains, such as "knob-into-hole" residue pairs and charge residue pairs. Knob and hole interactions favor heterodimer formation, while knob-knob and hole-hole interactions hinder homodimer formation due to steric conflicts and lack of favorable interactions. Hole refers to the gap created when the original amino acid in a protein is replaced by a different amino acid with a smaller side chain volume. Knob refers to the bump created when the original amino acid in a protein is replaced by a different amino acid with a larger side chain volume. For example, in some embodiments, the replaced amino acid is located in the CH3 antibody constant domain of the Fc domain and participates in the dimerization of the two Fc domains. In some embodiments, the hole in one CH3 antibody constant domain is created to accommodate the knob in another CH3 antibody constant domain, so that the knob and hole amino acids play a role in promoting or favoring the heterodimerization of the two Fc domains. In some embodiments, the hole in one CH3 antibody constant domain is generated to better accommodate the original amino acid in another CH3 antibody constant domain. In some embodiments, the knob in one CH3 antibody constant domain is generated to form additional interactions with the original amino acid in another CH3 antibody constant domain.

[0232] In some embodiments, the hole is constructed by replacing an amino acid with a larger side chain (such as tyrosine or tryptophan) with an amino acid with a smaller side chain (such as alanine, valine, or threonine), such as the Y407V mutation in the CH3 antibody constant domain. Similarly, in some embodiments, the knob is constructed by replacing an amino acid with a smaller side chain with an amino acid with a larger side chain, such as the T366W mutation in the CH3 antibody constant domain. In some embodiments, one Fc domain includes the knob mutation T366W, and the other Fc domain includes the hole mutations T366S, L358A, and Y407V. In some embodiments, a polypeptide of the present disclosure comprising a SIRPαD1 domain variant is fused to an Fc domain comprising the knob mutation T366W to limit unwanted knob-knob homodimer formation. Examples of knob-hole amino acid pairs are included, but are not limited to, in Table 9, and examples of knob-hole Fc domain variants and SIRPα–Fc fusions are provided in Table 10.

[0233] Table 9. Knob-hole mutations

[0234]

[0235] Table 10. Exemplary Fc domain variants and SIRPαD1 domain variant-Fc domain variant fusion polypeptides

[0236]

[0237]

[0238]

[0239]

[0240] In addition to the knob-and-hole strategy, in some embodiments, electrostatic manipulation is also used to control the dimerization of the Fc domain. Electrostatic manipulation refers to the use of favorable electrostatic interactions between oppositely charged amino acids in peptides, protein domains, and proteins to control the formation of higher-order protein molecules. Specifically, in order to use electrostatic manipulation to control the dimerization of the Fc domain, one or more amino acid residues that make up the CH3-CH3 interface are replaced by positively or negatively charged amino acid residues, so that the interaction becomes electrostatically favorable or unfavorable according to the specific charged amino acids introduced. In some embodiments, positively charged amino acids (such as lysine, arginine, or histidine) in the interface are replaced by negatively charged amino acids (such as aspartic acid or glutamic acid). In some embodiments, negatively charged amino acids in the interface are replaced by positively charged amino acids. In some embodiments, charged amino acids are introduced into one or two of the interacting CH3 antibody constant domains. In some embodiments, charged amino acids are introduced into the interacting CH3 antibody constant domains of two Fc domains to promote the selective formation of heterodimers of the Fc domain, as controlled by the electrostatic manipulation effect generated by the interaction between the charged amino acids. Table 11 includes, but is not limited to, examples of electrostatically manipulated amino acid pairs.

[0241] Table 11. Electrostatically manipulated amino acid mutations

[0242]

[0243] Other methods are available for controlling heterodimerization of Fc domains, particularly in the context of constructing bispecific antibodies.

[0244] In some embodiments, the first and second Fc domains 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, 71T, Y407H, Y407I, K409E, K409D, K409T, and K409I.

[0245] In some embodiments, the Fc domain comprises: (a) one of the following amino acid substitutions relative to wild-type human IgG1: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T or K409I; or (b)(i) relative to human IgG1 N297A mutation in the Fc region; (ii) L234A, L235A and G237A mutations relative to human IgG1 Fc region; (iii) L234A, L235A, G237A and N297A mutations relative to human IgG1 Fc region; (iv) N297A mutation relative to human IgG2 Fc region; (v) A330S and P331S mutations relative to human IgG2 Fc region; (vi) A330S, P331S and N297A mutations relative to human IgG2 Fc region; (vii) S228P, E233P, F234V, L235A and delG236 mutations relative to human IgG4 Fc region; or (viii) S228P, E233P, F234V, L235A, delG236 and N297A mutations relative to human IgG4 Fc region.In some embodiments, the Fc domain variant comprises: (a) one of the following amino acid substitutions relative to wild-type human IgG1: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L 368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T or K409I; and (b) further comprising (i) an N297A mutation relative to the Fc region of human IgG1; (ii) an N297A mutation relative to the Fc region of human IgG1 (iii) L234A, L235A and G237A mutations relative to human IgG1 Fc region; (iv) N297A mutation relative to human IgG2 Fc region; (v) A330S and P331S mutations relative to human IgG2 Fc region; (vi) A330S, P331S and N297A mutations relative to human IgG2 Fc region; (vii) S228P, E233P, F234V, L235A and delG236 mutations relative to human IgG4 Fc region; or (viii) S228P, E233P, F234V, L235A, delG236 and N297A mutations relative to human IgG4 Fc region.

[0246] 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.

[0247] Connecting peptides or protein domains

[0248] In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein α (SIRP-α) D1 variant, wherein the variant comprises a SIRPαD1 domain or a fragment thereof having an amino acid mutation at residue 80 relative to the wild-type SIRPαD1 domain; and at least one additional amino acid mutation relative to the wild-type SIRPαD1 domain at a residue selected from the group consisting of: residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92.

[0249] In some embodiments, the present invention also discloses a polypeptide comprising an Fc variant, wherein the Fc variant comprises an Fc domain dimer comprising two Fc domain variants, wherein each Fc domain variant is independently selected from (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236 and N297A.

[0250] In some embodiments, the signal regulatory protein alpha (SIRP-α) D1 variant and the Fc variant are linked. In some embodiments, the C-terminus of the SIRPα D1 domain variant is linked to the N-terminus of the Fc domain variant, such that the two polypeptides are joined to each other in tandem.

[0251] In some embodiments, the signal regulatory protein α (SIRP-α) D1 variant and the Fc variant are connected by a covalent bond, such as a peptide bond, a synthetic polymer, or any kind of bond produced by a chemical reaction (e.g., chemical conjugation). When connected via a peptide bond, in some embodiments, the carboxylic acid group at the C-terminus of one protein domain (e.g., SIRPαD1 domain variant) reacts with the amino group at the N-terminus of another protein domain (e.g., Fc variant) in a condensation reaction to form a peptide bond. In some embodiments, the peptide bond is formed by synthetic means through conventional organic chemical reactions, or by natural production of host cells, wherein the nucleic acid molecule encoding the DNA sequence of the two proteins in series (e.g., Fc domain variant and SIRPαD1 domain variant) can be directly transcribed and translated into a continuous polypeptide encoding the two proteins by the necessary molecular machinery (e.g., DNA polymerase and ribosome) in the host cell.

[0252] When the SIRP-α D1 variant and the Fc variant are linked by a synthetic polymer, in some embodiments, the polymer is functionalized at each end with a reactive chemical functional group to react with the terminal amino acid at the linking end of the two proteins.

[0253] 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, a chemical functional group (such as an amine, carboxylic acid, ester, azide or other functional group) is synthetically connected to the C-terminus of one protein and the N-terminus of another protein, respectively. In some embodiments, the two functional groups are then reacted by synthetic chemical means to form a chemical bond, thereby connecting the two proteins together.

[0254] Spacer

[0255] In the present disclosure, in some embodiments, the linker between the Fc domain monomer and the SIRPαD1 variant polypeptide of the present disclosure is an amino acid spacer comprising about 1-200 amino acids. Suitable peptide spacers include peptide linkers containing flexible amino acid residues such as glycine and serine. Examples of linker sequences are provided in Table 12. In some embodiments, the spacer contains a motif of GS, GG, GGS, GGG, GGGGS (SEQ ID NO: 163), GGSG (SEQ ID NO: 164) or SGGG (SEQ ID NO: 165), for example, multiple or repeated motifs. In some embodiments, the spacer contains 2 to 12 amino acids, including GS, for example, a motif of 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 3 to 12 amino acids, including GGS, for example, GGS, GGSGGS (SEQ ID NO: 171), GGSGGSGGS (SEQ ID NO: 172), and GGSGGSGGSGGS (SEQ ID NO: 173) motifs. In some embodiments, the spacer contains 4 to 12 amino acids, including GGSG (SEQ ID NO: 164), for example, a motif of 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 comprises 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), NO:191) or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO:192).

[0256] In some embodiments, the spacer contains a motif, e.g., multiple or repeated, of EAAAK (SEQ ID NO: 193). In some embodiments, the spacer contains a motif, e.g., multiple or repeated, of a proline-rich sequence (such as (XP)n, where X is any amino acid (e.g., A, K, or E), and n is 1-5) and PAPAP (SEQ ID NO: 194), e.g., multiple or repeated.

[0257] Table 12. Linker sequences

[0258]

[0259]

[0260] In some embodiments, the length of the peptide spacer and amino acid used is adjusted according to the flexibility required in the two proteins involved and the final protein fusion polypeptide. In some embodiments, the length of the spacer is adjusted to ensure that the protein is correctly folded and to avoid aggregate formation. In some embodiments, the spacer is A or AAAL (SEQ ID NO: 178).

[0261] Vectors, host cells and protein production

[0262] In some embodiments, disclosed herein is a polypeptide comprising a signal regulatory protein α (SIRP-α) D1 variant, wherein the variant comprises a SIRPαD1 domain or a fragment thereof having an amino acid mutation at residue 80 relative to the wild-type SIRPαD1 domain; and at least one additional amino acid mutation relative to the wild-type SIRPαD1 domain at a residue selected from the group consisting of: residue 6, residue 27, residue 31, residue 47, residue 53, residue 54, residue 56, residue 66, and residue 92.

[0263] In some embodiments, the present invention also discloses a polypeptide comprising an Fc variant, wherein the Fc variant comprises an Fc domain dimer having two Fc domain monomers, wherein each Fc domain monomer is independently selected from (i) a human IgG1 Fc region consisting of mutations L234A, L235A, G237A and N297A; (ii) a human IgG2 Fc region consisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A, delG236 and N297A.

[0264] In some embodiments, polypeptides of the present disclosure are produced from host cells.Host cells refer to vehicles including necessary cellular components (e.g., organelles) required for expressing polypeptides and fusion polypeptides described herein from their corresponding nucleic acids.In some embodiments, nucleic acids are included in nucleic acid vectors introduced into host cells by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc. In some embodiments, the selection of nucleic acid vectors depends on the host cell to be used.In some embodiments, host cells belong to prokaryotic (e.g., bacteria) or eukaryotic (e.g., mammalian) sources.

[0265] In some embodiments, a polypeptide (e.g., a polypeptide construct comprising a SIRPαD1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and a fusion partner (such as an Fc variant)) is produced by culturing a host cell transformed with a nucleic acid under suitable conditions, wherein the nucleic acid is preferably an expression vector containing a nucleic acid encoding a polypeptide construct (e.g., an Fc variant, a linker, and a fusion partner) to induce or cause expression of the polypeptide construct. In some embodiments, conditions suitable for expression vary with the expression vector and host cell selected. In some embodiments, a variety of suitable host cells are used, including but not limited to mammalian cells, bacteria, insect cells, and yeast. For example, the nucleic acid available from the American Type Culture Collection is A variety of cell lines useful for the present disclosure are described in the cell line catalog. In some embodiments, the Fc domain variants of the present disclosure are expressed in cells that are optimized to not glycosylate proteins expressed by such cells by genetic engineering of the cell line or modification of cell culture conditions (such as the addition of kissin) or by using a naturally non-glycosylating host (such as a prokaryote) (E. coli, etc.), and in some cases, there is no need to modify the glycosylation sequence in the Fc.

[0266] Nucleic acid vector construction and host cells

[0267] Nucleic acid sequences encoding the amino acid sequences of the polypeptides of the present disclosure can be prepared by a variety of methods. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. In some embodiments, nucleic acid molecules encoding the polypeptides of the present disclosure are obtained using standard techniques (e.g., gene synthesis). Alternatively, standard techniques (e.g., QuikChange TM Mutagenesis) Mutates a nucleic acid molecule encoding a wild-type SIRPαD1 domain to include a specific amino acid substitution. In some cases, the nucleic acid molecule is synthesized using a nucleotide synthesizer or PCR technology.

[0268] In some embodiments, a nucleic acid encoding a polypeptide construct (e.g., a polypeptide construct comprising a SIRPαD1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and a fusion partner (such as an Fc variant)) is incorporated into an expression vector to express the protein. A variety of expression vectors can be used for protein expression. The expression vector may comprise a self-replicating extrachromosomal vector or a vector that integrates into the host genome. The vector may also include various components or elements. For example, in some embodiments, vector components include, but are not limited to, transcriptional and translational regulatory sequences, such as promoter sequences, ribosome binding sites, signal sequences, transcriptional start and stop sequences, translational start and stop sequences, 3' and 5' untranslated regions (UTRs), and enhancer or activator sequences; origin of replication; selection marker genes; and nucleic acid sequences encoding a polypeptide of interest, and transcriptional termination sequences. In some embodiments, the expression vector comprises a protein operably linked to a control or regulatory sequence, a selective marker, any fusion partner, additional elements, or any combination thereof. The term "operably linked" refers to a nucleic acid being in a functional relationship with another nucleic acid sequence. Typically, these expression vectors include transcription and translation regulatory nucleic acids operably linked to the nucleic acid encoding the Fc variants, and are generally suitable for host cells expressing the protein. Selection genes or markers (such as, but not limited to, antibiotic resistance genes or fluorescent protein genes) can be used to select host cells containing expression vectors, for example, by antibiotic or fluorescent expression. Various selection genes are available.

[0269] In some embodiments, components or elements of the vector are optimized so that the expression vector is compatible with the host cell type.Expression vectors that find use in the present disclosure include, but are not limited to, those capable of protein expression in mammalian cells, bacteria, insect cells, yeast, and in vitro systems.

[0270] In some embodiments, mammalian cells are used as host cells to produce polypeptides of the present disclosure. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK 293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NS0, Sp2 / 0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030, and HsS78Bst cells. In some embodiments, E. coli cells are used as host cells to produce polypeptides of the present disclosure. Examples of E. coli strains include, but are not limited to, E. coli 294 ( 31,446), Escherichia coli λ1776( 31,537), Escherichia coli BL21(DE3)( BAA-1025) and Escherichia coli RV308 ( 31,608).

[0271] Different host cells have characteristic and specific mechanisms for post-translational processing and modification of protein products (e.g., glycosylation). In some embodiments, a suitable cell line or host system is selected to ensure the correct modification and processing of the expressed polypeptide. Once the vector is introduced into the host cell for protein production, the host cell is cultured in a conventional nutrient medium modified to be suitable for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequence.

[0272] In some embodiments, a polypeptide construct (e.g., a polypeptide construct comprising a SIRPαD1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and a fusion partner (such as an Fc variant)) is expressed in a mammalian expression system, including systems in which a virus (such as a retrovirus or an adenovirus) is used to introduce the expression construct into a mammalian cell. In some embodiments, human, mouse, rat, hamster, or primate cells are utilized. Suitable cells also include known research cells, including, but not limited to, Jurkat T cells, NIH3T3, CHO, COS, and 293 cells. Alternatively, in some embodiments, 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, polypeptide constructs comprising Fc domain variants are produced in insect cells (such as, but not limited to, Sf9 and Sf21 cells) or yeast cells (such as, but not limited to, organisms from the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, and Yarrowia). In some cases, polypeptide constructs comprising Fc domain variants are expressed in vitro using a cell-free translation system. In vitro translation systems derived from prokaryotic (e.g., E. coli) and eukaryotic (e.g., wheat germ, rabbit reticulocyte) cells are available, and in some embodiments, are selected based on the expression level and functional properties of the protein of interest. For example, as will be appreciated by those skilled in the art, some display technologies (e.g., ribosome display) require in vitro translation. In addition, in some embodiments, Fc domain variants are produced by chemical synthesis methods, such as, but not limited to, liquid phase peptide synthesis and solid phase peptide synthesis. In the case of in vitro transcription using a non-glycosylation system such as a bacterial extract, Fc will not be glycosylated even if native glycosylation sites are present, and thus inactivation of Fc will be equivalently obtained.

[0273] In some embodiments, the polypeptide construct includes non-natural amino acids, amino acid analogs, amino acid mimetics, or any combination thereof, which function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids generally refer to 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) as well as pyrrolysine and selenocysteine. Amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids, for example, carbon bonded to hydrogen, carboxyl, amino, and R groups, such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. In some embodiments, such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but generally retain the same basic chemical structure as naturally occurring amino acids.

[0274] Protein production, recovery and purification

[0275] In some embodiments, host cells used to produce the polypeptides of the present disclosure are grown in a medium suitable for culturing the selected host cells. Examples of suitable culture media for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Expi293 TM Expression medium, DMEM and RPMI-1640 supplemented with fetal bovine serum (FBS). Examples of suitable culture media for bacterial host cells include Luria broth (LB) plus necessary supplements, such as selection agents, such as ampicillin (ampicillin). In some embodiments, host cells are cultured at a suitable temperature (such as about 20°C to about 39°C, for example, about 25°C to about 37°C, preferably 37°C) and CO2 levels (such as about 5% to 10%). In some embodiments, the pH of the culture medium is about pH 6.8 to pH 7.4, for example pH 7.0, depending primarily on the host organism. If an inducible promoter is used in the expression vector, protein expression can be induced under conditions suitable for activating the promoter.

[0276] In some embodiments, protein recovery includes destroying host cells, such as by osmotic shock, ultrasonic treatment or lysis. Once the cells are destroyed, cell debris is removed by centrifugation or filtration. The protein can then be further purified. In some embodiments, the polypeptide of the present disclosure is purified by various protein purification methods, such as by chromatography (e.g., ion exchange chromatography, affinity chromatography and size exclusion column chromatography), centrifugation, differential dissolution or by any other standard technique for protein purification. For example, in some embodiments, by appropriately selecting and combining affinity columns, such as protein A columns (e.g., POROS protein A chromatography) and chromatographic columns (e.g., POROS HS-50 cation exchange chromatography), filtering, ultrafiltration, desalting and dialysis procedures to separate and purify proteins. In some embodiments, polypeptides are conjugated to marker sequences (such as peptides) for purification. An example of a marker amino acid sequence is a six-histidine peptide (His6-tag), which can be bound to a nickel-functionalized agarose affinity column with a micromolar affinity. As an alternative, a hemagglutinin "HA" tag can be used, which corresponds to an epitope derived from an influenza hemagglutinin protein.

[0277] In some embodiments, for example, in the context of gene therapy, polypeptides of the present disclosure (e.g., polypeptide constructs comprising a SIRPαD1 domain variant (e.g., any variant provided in Tables 2, 5, and 6) and a fusion partner (such as an Fc variant)) are produced by cells of a subject (e.g., a human) by administering a vector containing a nucleic acid molecule encoding a polypeptide of the present disclosure, such as a viral vector (e.g., a retroviral vector, an adenoviral vector, a poxvirus vector (e.g., a vaccinia virus vector, such as Modified Vaccinia Ankara (MVA)), an adeno-associated virus vector, and an alphavirus vector). Once the vector enters the cells of the subject (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.), it can be used to express the polypeptides disclosed herein. In some cases, the polypeptide is secreted from the cell. In some embodiments, if treatment of the disease or condition is the desired outcome, no further action is required. In some embodiments, if protein collection is desired, blood is collected from the subject and the protein is purified from the blood by various methods.

[0278] Treatment options for urothelial cancer

[0279] In some embodiments, a method of treating cancer (e.g., urothelial carcinoma, such as 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, urothelial carcinoma is histologically confirmed, unresectable locally advanced or metastatic urothelial carcinoma. Additionally or alternatively, urothelial carcinoma is bladder cancer, renal pelvic cancer, ureteral cancer, or urethral cancer. In some embodiments, the individual suffers from transitional cell carcinoma of squamous differentiation or mixed cell types, in which urothelial carcinoma is the predominant histology. In some embodiments, the individual does not suffer from small cell carcinoma or neuroendocrine histology.

[0280] In some embodiments, the ADC comprises an antibody that specifically binds to fibronectin-4 (e.g., human fibronectin-4) linked to a cytotoxic drug. In some embodiments, the anti-fibronectin-4 antibody is enfortumab, which is also known as AGS-22C3 (CAS Reg. No. 1448664-46-7). In some embodiments, the cytotoxic drug is the small molecule microtubule disruptor monomethyl auristatin-E (MMAE), also known as vedotin or SGD-1006 (CAS Reg. No. 474645-27-7). In some embodiments, the ADC is ventozumab (also known as CAS Registry No. 1346452-25-2). Vientoumab is a fibronectin-4 directed antibody-drug conjugate (ADC) comprising a fully human anti-fibronectin-4 IgG1κ monoclonal antibody conjugated to MMAE via a protease-cleavable maleimidoalanylvaline-citrulline (vc) linker. Conjugation occurs at cysteine ​​residues on the heavy chain of the antibody, resulting in a product with a drug-antibody ratio (DAR) of about 3.8:1. The molecular weight is approximately 152 kDa. In some embodiments, the ADC (e.g., Vientoumab) is administered to an individual for one or more 28-day cycles. In some embodiments, the ADC (e.g., Vientoumab) is administered at a dose of 1.25 mg / kg on each of days 1, 8, and 15 of one or more 28-day cycles. In some embodiments, the ADC (e.g., Vientoumab) is administered to an individual for one or more 28-day cycles. In some embodiments, ADC (e.g., Vientoumab) is administered at a dose of 1.25 mg / kg every 3 weeks (Q3W). In some embodiments, ADC (e.g., Vientoumab) is administered by intravenous infusion. In some embodiments, ADC (e.g., Vientoumab) is administered by intravenous infusion within a 30-minute time period on each day of the 1st, 8th, and 15th days of one or more 28-day cycles. In some embodiments, the maximum dose of ADC (e.g., Vientoumab) is administered to an individual on each day of the 1st, 8th, and 15th days of one or more 28-day cycles, with a maximum dose of up to 125 mg. In some embodiments, ADC (e.g., Vientoumab) is administered according to the regimen provided on the local package insert (for the United States, see, e.g., https: / / astellas(dot)us / docs / PADCEV(underscore)label(dot)pdf). Details about the mechanism of action of ADC can also be found on the package insert.

[0281] 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., fusion polypeptide) comprising a SIRPαD1 domain variant (e.g., a SIRPαD1 domain variant described herein) and an Fc domain variant (e.g., an Fc domain variant described herein). In some embodiments, the C-terminus of the SIRPαD1 domain variant of the fusion polypeptide (e.g., a SIRPαD1 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 a SIRPαD1 domain variant, which comprises an amino acid sequence of SEQ ID NO:81 or SEQ ID NO:85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein the numbering is according to the EU index as in Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein the numbering is according to the EU index as in Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, wherein the numbering is according to the EU index as in Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein the numbering is according to the EU index as in Kabat (e.g., wherein the C-terminus of the SIRPαD1 domain variant is fused to the N-terminus of the Fc domain variant). In some embodiments, the polypeptide (eg, 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 (eg, fusion polypeptide) forms a dimer, such as 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., such as about 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 an individual by intravenous infusion. In some embodiments, the polypeptide is administered to an individual at a dose of about 15 mg / kg.In some embodiments, the polypeptide is applied to an individual at a dosage of about 15 mg / kg q2w (i.e., once every two weeks or once every 14 days). In some embodiments, the polypeptide is applied to an individual at a dosage of about 20 mg / kg. In some embodiments, the polypeptide is applied to an individual at a dosage of about 20 mg / kgq2w (i.e., once every two weeks or once every 14 days). In some embodiments, the polypeptide is applied to an individual at a dosage of about 30 mg / kg. In some embodiments, the polypeptide is applied to an individual at a dosage of about 30 mg / kg q2w (i.e., once every two weeks or once every 14 days). In some embodiments, the polypeptide is applied by intravenous infusion at 15, 20 or 30 mg / kgq2w (i.e., once every two weeks or once every 14 days) within a time period of 60 hours. In some embodiments, the fusion polypeptide is provided for use (e.g., intravenous administration) in a 1000 mg / 50 ml type I transparent glass vial, and the glass vial is sealed with a 20 mm Teflon-coated rubber septum plug and a tamper-proof aluminum seal. In some embodiments, the fusion polypeptide is stored in the original container at 2-8°C (36-46°F) prior to use (eg, preparation for intravenous administration).

[0282] In some embodiments, when the administration of a polypeptide (e.g., a fusion polypeptide) and an ADC (e.g., veentumab) overlaps, the polypeptide is administered before the ADC. In some embodiments, the ADC (e.g., veentumab) is administered approximately 30 minutes (e.g., about 20 minutes to about 40 minutes, 25 minutes to about 45 minutes, or about 30 minutes to 50 minutes) after the polypeptide administration is completed.

[0283] In some embodiments, the subject has received prior treatment with an immune checkpoint inhibitor (CPI) for locally advanced urothelial carcinoma or metastatic urothelial carcinoma. In some embodiments, the subject has received a CPI for urothelial carcinoma in a neoadjuvant setting or an adjuvant setting, and has recurrent or progressive disease during CPI treatment or within 12 months after completion of CPI treatment. In some embodiments, CPI therapy includes or is a programmed cell death protein 1 (PD-1) inhibitor or a programmed cell death ligand 1 (PD-L1) inhibitor. In some embodiments, a PD-1 inhibitor or a PD-L1 inhibitor is a therapeutic antibody. In some embodiments, a therapeutic anti-PD-1 antibody or a therapeutic anti-PD-L1 antibody is or comprises one or more of atezolizumab, pembrolizumab, durvalumab, avelumab, and nivolumab. In some embodiments, the subject has received prior treatment for urothelial carcinoma with platinum-containing chemotherapy. In some embodiments, the subject receives platinum-containing chemotherapy for urothelial carcinoma in an adjuvant setting or a neoadjuvant setting, and has 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 is or comprises one or more of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin.

[0284] In some embodiments, the subject has developed during or after receiving the most recent prior treatment for urothelial carcinoma (e.g., the subject's urothelial carcinoma has shown disease progression). In some embodiments, the subject's cancer relapses during or after receiving the most recent treatment (e.g., confirmed relapse). In some embodiments, the subject has not received prior treatment with venetumab. In some embodiments, the subject has not received treatment with an antibody-drug conjugate (ADC) based on monomethyl auristatin (MMAE) (e.g., based on bedotin). In some embodiments, the subject has not received prior treatment with an agent that destroys the interaction between hCD47 and hSIRPα, such as an anti-CD47 agent and / or an anti-SIRPα agent. In some embodiments, the subject has no hypersensitivity to any excipients (including histidine, trehalose dihydrate, and polysorbate 20) contained in the pharmaceutical formulation of venetumab or venetumab. In some embodiments, the subject has no hypersensitivity to biological drugs produced in Chinese hamster ovary (CHO) cells. In some embodiments, the subject has no intolerance or severe allergic or anaphylactic reaction to the antibody or infused therapeutic protein. In some embodiments, the subject has no intolerance or severe allergic or anaphylactic reaction to any substance contained in the polypeptide formulation.

[0285] In some embodiments, the cancer treated by the methods provided herein is urothelial carcinoma, head and neck cancer, gastric cancer, non-small cell lung cancer (NSCLC), hormone receptor positive breast cancer that does not overexpress (or expresses) HER2 (e.g., HR + HER2 - breast cancer).

[0286] Kits and Products

[0287] In another embodiment of the present invention, a product or kit comprising a polypeptide (e.g., a fusion polypeptide described herein) is provided, the polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant. In some embodiments, the SIRPαD1 domain variant is used in combination with an antibody-drug conjugate (e.g., Vientoumab) for treating urothelial carcinoma in an individual (e.g., a human individual). In some embodiments, the SIRPαD1 domain variant is used in combination with an antibody-drug conjugate (e.g., Vientoumab) for treating urothelium in an individual (e.g., a human individual). In some embodiments, the SIRPαD1 domain variant comprises an amino acid sequence selected from SEQ ID NO: 81 and SEQ ID NO: 85. In some embodiments, the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A, and N297A mutations, wherein numbering is according to the EU index of Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S, and N297A mutations, wherein numbering is according to the EU index of Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, and delG236 mutations, wherein numbering is according to the EU index of Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering is according to the EU index of Kabat. In some embodiments, the Fc domain variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 135 or SEQ ID NO: 136. In some embodiments, the polypeptide comprising the SIRPαD1 domain variant and the Fc domain variant forms a homodimer. In some embodiments, the kit or article of manufacture is used according to the treatment methods provided herein.

[0288] In some embodiments, the kit or article also includes an antibody-drug conjugate (ADC). In some embodiments, ADC includes an anti-adhesion protein-4 antibody (e.g., ennomonab). In some embodiments, ADC includes an antibody that specifically binds to adhesion protein-4 (e.g., human adhesion protein-4) connected to a cytotoxic drug. In some embodiments, the cytotoxic drug is a small molecule microtubule disruptor monomethyl auristatin-E (MMAE), also known as bedotin. In some embodiments, ADC is venetumab. In some embodiments, polypeptides (e.g., fusion polypeptides) and ADC are provided in the same container or a separate container. Suitable containers include, for example, bottles, vials, bags, and syringes. The container can be formed of a variety of materials such as glass, plastics (such as polyvinyl chloride or polyolefins) or metal alloys (such as stainless steel or Hastelloy). In some embodiments, the formulation and label on or associated with the container may indicate directions for use.

[0289] The article or kit may also include other materials required from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the kit includes a package insert or label with instructions for using the combination of the polypeptide (e.g., fusion polypeptide) and the antibody-drug conjugate (e.g., venetumab) to treat or delay cancer (e.g., urothelial carcinoma, such as urothelial carcinoma described in further detail elsewhere herein) in an individual (e.g., a human individual). In some embodiments, the package insert or label provides instructions for administering the polypeptide (e.g., fusion polypeptide) to an individual in need at a dose of up to 60 mg / kg. In some embodiments, the package insert or label provides instructions for administering the polypeptide (e.g., fusion polypeptide) to an individual at a dose of 20 mg / kg once every 2 weeks (q2w) or once every 14 days. In some embodiments, the package insert or label provides instructions for administering a polypeptide (e.g., a fusion polypeptide) to an individual in need thereof at a dosage of 30 mg / kg once every 2 weeks (q2w) or once every 14 days. In some embodiments, the package insert or label provides instructions for administering a polypeptide (e.g., a fusion polypeptide) to an individual in need thereof at a dosage of 15 mg / kg once every 2 weeks (q2w) or once every 14 days.

[0290] Suitable containers include, for example, bottles, vials, bags, and syringes. The container can be formed of a variety of materials such as glass, plastics (such as polyvinyl chloride or polyolefins) or metal alloys (such as stainless steel or Hastelloy). In some embodiments, the container or the formulation and label associated therewith can indicate the direction of use. The article or test kit may also include other materials required from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article also includes one or more other medicaments (for example, chemotherapeutic agents, antitumor agents, therapeutic antibodies, etc.). Suitable containers for one or more medicaments include, for example, bottles, vials, bags, and syringes.

[0291] This specification is considered sufficient to enable those skilled in the art to practice the invention. Various modifications of the invention, except those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. For all purposes, all publications, patents and patent applications cited herein are hereby incorporated herein by reference in their entirety.

[0292] Example

[0293] By reference to the following examples, the present disclosure will be more fully understood. However, these examples should not be construed as limiting the scope of the present disclosure. It should be understood that the embodiments and embodiments described herein are only for illustrative purposes, and it will be suggested that those skilled in the art make various modifications or changes based on these, and various modifications or changes will be included in the spirit and scope of the application and the scope of the appended claims.

[0294] Example 1: Evaluation of the binding of Drug B and Drug C to human Fcγ receptors.

[0295] Overview

[0296] IgG antibodies mediate phagocytosis of target tumor cells via the association of Fcγ receptors (FcγRs) on effector cells (e.g., macrophages) by inducing antibody-dependent cellular phagocytosis (ADCP) or antibody-dependent cellular toxicity (ADCC). Most effector cells express multiple FcγRs. The Fcγ receptors of 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 a H / R131 single nucleotide polymorphism, and a well-characterized FcγRIII single nucleotide polymorphism is V / F158. IgG Fc domains bind to multiple FcγRs with varying affinities, and even participate in low-affinity interactions with high-affinity immune complexes that contribute to target cell clearance (Armour et al. (2003) Mol. Immunol. 40:585-593; Nagelkerke et al. (2019) Front Immunol. 10:2237; Kang et al. (2019) Front Immunol. doi(dot)org / 10(dot)3389 / fimmu(dot)2019(dot)00562). The published K of human IgG1 isotype binding to human FcRn at 25°C, pH 5.8 is D is 760+ / -60nM (Abdiche et al. (2015) MAbs. 7(2):331-43).

[0297] The antibody drug conjugate (ADC) venotumab contains a mc-vc-PAB-MMAE linker payload conjugated to an interchain cysteine. This conjugation may limit the binding of the Fc region of enroku to Fc receptors and affect the ability of the ADC to mediate antibody-dependent cellular phagocytosis (ADCP). To determine whether the conjugation of the linker payload affects the ability of the Fc region of enroku to retain its effector function (e.g., by binding to human Fc-γ receptors (FcγRs) and neonatal Fc receptors (FcRn)), the affinity of hFcγIa, hFcγIIa-H131, hFcγIIa-R131, hFcγIIIa, FcγIIIaV158F and hFcRn to drug B (i.e., enroku analogs) and drug C (i.e., venotumab analogs) was evaluated by surface plasmon resonance (SPR).

[0298] Materials and methods

[0299] Drug B monoclonal antibody gene synthesis, antibody expression and purification

[0300] The amino acid sequence of drug B (i.e., an ennomonab-like antibody that specifically binds to human adhesion protein-4) is based on the publicly available ennomonab amino acid sequence (see KEGG database entry D1154; CAS: 1448664-46-7; PubChem database entry 384585500). The antibody heavy chain and antibody light chain sequences of drug B were generated by gene synthesis (see below) and codon optimized for expression in mammalian cells (ATUM). The heavy chain and light chain genes were cloned into separate mammalian expression vectors and transiently co-transfected into Expi293F cells (ThermoFisher). Antibody expression was performed in Expi293 expression medium, and cell culture supernatant was harvested 5 days after transfection. Drug B was purified using MABSELECT PrismA resin (Cytiva), and the buffer was exchanged for 1x phosphate buffered saline (pH 7.4). Analytical size exclusion chromatography (Cytiva, Superdex 200 10 / 300) data indicated that Drug B was approximately 99% monomeric.

[0301] The amino acid sequences of the Drug B light chain and the Drug B heavy chain are provided below. The light chain variable domain and the heavy chain variable domain are underlined.

[0302] Drug B light chain:

[0303] DIQMTQSPSSSVSASVGDRVTITCRASQGISGWLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQANSFPPTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:225)

[0304] Drug B Heavy Chain:

[0305] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRF TISRDNAKNSLSLQMNSLRDEDTAVYYCARAYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:226)

[0306] The amino acid sequences of the human nectin-4 extracellular domain (ECD) and the cynomolgus monkey nectin-4 ECD are provided below. His6 (HHHHHH SEQ ID NO: 223) is fused to the C-terminal domain of the human and cynomolgus monkey nectin-4 ECD (indicated in bold below).

[0307] Human fibronectin-4-ECD

[0308] GELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRS FKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASHHHHHH(SEQ ID NO:227)

[0309] Cynomolgus monkey adhesion protein-4-ECD

[0310] GELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARADAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRS FKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHVGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASHHHHHH(SEQ ID NO:228)

[0311] Determination of the affinity of drug B for adhesion protein-4

[0312] 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 analysis was performed using Biacore Insight evaluation software version 3.0.12.15655.

[0313] 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 monkey adhesion protein-4-ECD) were used at their nominal concentrations, as determined by A280 absorbance and using their molar extinction coefficients. The kinetic injection method used was "single cycle kinetics" (also called "kinetic titration") (Karlsson et al. (2006) Analytical Biochemistry. 349 (1): 136-147).

[0314] The interaction of drug B (i.e., anti-nectin-4 monoclonal antibody) with the extracellular domain (ECD) of human nectin-4 was analyzed by flowing nectin-4-ECD through drug B (200-400RU) captured on Biacore series S sensor chip protein A. Respectively, drug B was captured in the flow cell 2 of each channel at a concentration of about 1 μg / mL at a flow rate of 10 μL / min for a contact time of 60 seconds, and buffer was used for flow cell 1. Human nectin-4-ECD analyte was prepared into a 5-yuan 3-fold dilution series, with a maximum nominal concentration of 300nM, and each analyte series was injected in the order of increasing concentration using a single-cycle kinetic method. At a flow rate of 30 μL / min, the association and dissociation times of 120s and 1800s were monitored respectively. Surface regeneration was performed using two 15s pulses with 75mM phosphoric acid at pH 1.6 at a flow rate of 30 μL / min.

[0315] To analyze the data, the following processing steps were applied. The reference response from flow cell 1 (reference surface) was subtracted from the active response from flow cell 2 (reactive surface) to obtain subtraction data (2-1). The response of the most recent (in time) buffer blank injection was then subtracted from the reference subtraction data (2-1) to obtain "double reference" data (Myszka, DG (1999). J. Mol. Recognit. 12, 279-284). These double reference data were overall fit to a simple 1:1 Langmuir binding model with mass transfer to determine the apparent association rate constant (k a ) and the dissociation rate constant (k d ). The apparent equilibrium dissociation or "affinity" constant (K) is then derived from their ratio. D ), K D =k d / k a Drug B binds to human adhesion protein-4-ECD, K D At 37°C, it is approximately 36.3±3.3 nM, and at 25°C, it is approximately 14.5±0.7 nM. Drug B binds to cynomolgus monkey adhesion protein-4-ECD, K D The results showed that enroku and drug B have similar apparent affinity for binding to human adhesion protein-4-ECD (K for venetosumab SPR assay at 25°C is about 50.7±1.6 nM at 37°C and about 22.6±1.7 nM at 25°C). Dis about 16 nM (Satpayev D, Morrison RK, Morrison KJM, Gudas J, JakobovitsA, Torgov M, An Z. 2018. Antibody drug conjugates (ADC) that bind to 191P4D12 proteins. US9962454B2) compared to the K determined for drug B D is about 14.5 nM).

[0316] Production of Drug C

[0317] To generate the antibody drug conjugate drug C similar to ventoumab (CAS Number 1346452-25-2), the enroku analog antibody drug B was conjugated to maleimidopropionyl-valyl-citrullyl-p-aminobenzyloxycarbonyl-monomethyl auristatin E linker-payload (MC-Val-Cit-PAB-MMAE, CAS 646502-53-6; obtained from BroadPharm) via the interchain cysteines of the antibody. Briefly, 2.2 mg / ml drug B monoclonal antibody in 1×PBS pH 7.4, 10% sucrose, 5 mM EDTA, 30 mM Tris-HCL pH 7.5 was partially reduced by adding 20 molar equivalents of TCEP (ThermoFisher) relative to the monoclonal antibody at 20°C for 20 minutes. MC-Val-Cit-PAB-MMAE was dissolved in 100% DMSO and added to the reaction mixture as a 5% v / v DMSO solution in an amount of 10 molar equivalents relative to the monoclonal antibody, and the reaction solution was swung at 20°C for 2 hours. Afterwards, 1 molar equivalent of N-acetylcysteine ​​(SigmaAldrich) relative to the linker-payload was added, and the reaction mixture was incubated at 20°C for 20 minutes. Excess quenched MC-Val-Cit-PAB-MMAE was separated from the antibody-drug conjugate by cation exchange chromatography (Cytiva, HiTrap SP HP resin), and the drug C antibody-drug conjugate was eluted in 10% sucrose, 150mM NaCl, 12.5mM sodium acetate pH 5.0 buffer. Analytical size exclusion chromatography (Cytiva, Superdex 200 10 / 300) showed that drug C was about 99% monomer.

[0318] Determination of drug-antibody ratio (DAR) of drug C

[0319] The average drug-to-antibody ratio (DAR) of ventozumab (CAS No. 1346452-25-2) is approximately 3.8:1 (see US package insert, 2019). CRO Novatia, LLC (USA) used liquid chromatography mass spectrometry (LC-MS) to determine the DAR of drug C (i.e., venetolumab ADC analog). Briefly, samples of drug C were deglycosylated and reduced using PNGase F treatment (New England Biolabs Rapid PNGase F) and then analyzed by reverse phase liquid chromatography coupled to mass spectrometry (RPLC-MS). HPLC was an AcquityI-Class UPLC coupled with a Halo Diphenyl column 2.1x50 mm, 2.7 mm. Phase A was 0.05% TFA in water and phase B was 0.05% trifluoroacetic acid in acetonitrile. The gradient was 10-20% solution B in 1 minute, 20-50% solution B in 9 minutes, 0.5 mL / min, 80°C. The mass spectrometer was a Waters XevoG2-XS Q-Tof. Novatia ProMass HR processed the data using MassLyxn software. The results showed that the average drug-to-antibody ratio of drug C was 3.85:1, similar to the DAR value of ventuinumab (3.8:1).

[0320] Human FcγR binding to drug B and drug C

[0321] All SPR experiments were performed at 25 °C using a Biacore8K equipped with an S-type sensor chip, and kinetic data analysis was performed using Biacore Insight evaluation software.

[0322] For all hFcγR interactions, the running buffer was HBS-EP+ (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% (v / v) surfactant P20). All hFcγR analytes were used at their nominal concentrations determined by A280 absorbance and their molar calculated extinction coefficients were used.

[0323] The interaction of drug B and drug C with human FcγR was analyzed by flowing the extracellular domain (ECD) of hFcγR through drug B or drug C captured on a CM5 chip coated with adhesion protein-4. According to the Cytiva amine coupling kit instructions, up to 2400RU of human adhesion protein-4 ECD was immobilized on two flow cells (1 and 2) of the CM5 chip using amine chemistry. Drug B and drug C were captured in HBS-EP+ (100-200RU) at 10μL / min, 2μg / mL on flow cell 2 of each channel for 120 seconds. The analytes were injected in the capture method using a single cycle kinetic mode, with a nominal maximum concentration of 30nM and 3-fold serial dilutions for hFcγRI (CD64), or a nominal maximum concentration of 3000nM and 3-fold serial dilutions for hFcγRIIa (CD32a) or hFcγRIIIa (CD16a). Association times were monitored for 120 s, and dissociation times were monitored for 600 s (except for hFcγRI which had a dissociation time of 1800 s).The surface was regenerated using two 15 s pulses with 75 mM phosphoric acid, pH 1.6 at a flow rate of 30 μL / min.

[0324] 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 response from flow cell 1 was subtracted from the active response from flow cell 2 to obtain subtraction data (2-1). The response of the latest buffer blank injection was then subtracted from the reference subtraction data (2-1) to obtain dual reference data. For hFcγRI, these dual reference data were fit to a simple 1:1 Langmuir binding model with mass transfer to determine the apparent association rate constant (ka) and dissociation rate constant (kd). The apparent equilibrium dissociation constant or affinity constant, (Kd) was then calculated based on their ratio. D = kd / ka). Due to their fast association and dissociation rates, the “steady-state” (or “equilibrium binding”) approach was used to analyze the binding affinity K of all other hFcγR interactions (hFcγIIa-H131, hFcγIIa-R131, hFcγIIIa, and FcγIIIaV158F). D value.

[0325] Human FcRn binding to drug B and drug C

[0326] 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 BiacoreInsight evaluation software. The running buffer for hFcRn interaction was PBS pH 5.8 with 0.01% Tween-20, and the running buffer for capture was HBS-EP+. The analyte hFcRn ECD protein was used at its nominal concentration. The "single cycle kinetics" injection mode was used.

[0327] The interaction of drug B and drug C with hFcRn was analyzed by flowing hFcRn ECD protein through drug B or drug C captured on a CM5 chip coated with adhesion protein-4. According to the instructions of the Cytiva amine coupling kit, up to 2400RU of human adhesion protein-4 ECD was immobilized on two flow cells (1 and 2) of the CM5 chip using amine chemistry. In the surface preparation method, drug B or drug C was captured in HBS-EP+ (100-200RU) at 10μL / min, 2μg / mL on flow cell 2 of each channel for 120 seconds. The hFcRn run was performed in PBS pH 5.8 containing 0.01% Tween-20. The hFcRn analyte was prepared into 5-yuan 3-fold serial dilutions with a maximum nominal concentration of 3000nM, and these samples were injected in the order of increasing concentration using a single cycle mode. The association time and dissociation time were monitored for 120s and 600s, respectively. The surface was regenerated using two 30 s pulses with PBS pH 7.4 at a flow rate of 30 μL / min. The neutral pH buffer effectively removed hFcRn while retaining the captured antibody on the chip.

[0328] 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 response from flow cell 1 was subtracted from the active response from flow cell 2 to obtain subtraction data (2-1). The response of the most recent buffer blank injection was then subtracted from the reference subtraction data (2-1) to obtain double reference data. Due to its fast association rate and fast dissociation rate, the "steady-state" (or "equilibrium binding") method was used to analyze the binding affinity K. D value.

[0329] result

[0330] Human FcγR binding to drug B and drug C

[0331] Drug C (i.e., venetosumab analog) and drug B (i.e., unconjugated enrokus analog) were found to have similar k values ​​for binding to hFcγR. a, k d and apparent affinity K D . See Table A below.

[0332] Human FcRn binding to drug B and drug C

[0333] Drug C (i.e., venetosumab analog) and Drug B (i.e., unconjugated enrokus analog) were found to have similar k values ​​for binding to hFcR. a , k d and apparent affinity K D . See Table A below.

[0334] Table A. Affinity of FcγR and FcRn for Drug B and Drug C (K D )

[0335]

[0336] in conclusion

[0337] Comparison of the SPR kinetic results of the binding of drug B (ennolumab analog mAb) and drug C (veentumab analog ADC) to human hFcγR and hFcRn showed that drug B (i.e., ennolumab analog) and drug C (i.e., veentumab analog) bind to human hFcγR and hFcRn with similar affinity. Therefore, the presence of the interchain cysteine-conjugated linker-payload mc-vc-PAB-MMAE with an average drug-antibody ratio of 3.85:1 to veentumab does not appear to affect the ability of drug C to bind to human FcγR and human FcRn receptors.

[0338] Example 2: Evaluation of the effect of the combination of drug A with drug B or drug C on antibody-dependent cellular phagocytosis (ADCP)

[0339] Overview

[0340] Veintoumab-ejfv is a fibronectin-4 directed antibody-drug conjugate (ADC) comprising a fully human anti-fibronectin-4 IgG1κ monoclonal antibody (referred to herein as mc-vc-PAB-MMAE) conjugated to the small molecule microtubule disruptor monomethyl auristatin E via a protease-cleavable maleimidoalanylvaline-citrulline linker. Veintoumab linker-payload is conjugated to an interchain cysteine ​​residue containing an antibody interchain disulfide bond to produce a product with a drug-antibody ratio of approximately 3.8:1 (see U.S. package insert). In the human IgG1 antibody structure, the heavy-heavy chain intercysteine ​​is located in the hinge region, and the heavy-light chain intercysteine ​​is located at the interface of the human IgG1 antibody heavy chain domain CH1 and the human IgG1 light chain kappa constant domain (CK). Therefore, the conjugation of the linker-payload mc-vc-PAB-MMAE to the interchain cysteine ​​in ventoumab may cause steric hindrance to the binding of the Fc region to the FcγR and FcRn receptors.

[0341] The purpose of this experiment was to evaluate whether the presence of a linker payload in the Fc and Fc-proximal regions of drug C affects Fc effector functions that mediate ADCP. Therefore, the effects of drug A in combination with drug B (i.e., enroku analogs) and drug C (i.e., veentumab analogs) on antibody-dependent cellular phagocytosis (ADCP) were 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 but lacks Fc effector functions.

[0342] Materials and methods

[0343] Cell lines

[0344] OE19 (Sigma 96071721-1VL) and T47D (ATCC HTB-133) cells were maintained in growth medium containing 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 on the expression levels of adhesion protein-4 by OE19 and T47D is provided in Table B.

[0345] HT-1376 (ATCC CRL-1472) cells were maintained in a growth medium containing DMEM (Therm o 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 cancer cell line. Information on the expression level of adhesion protein-4 of HT-1376 is provided in Table B.

[0346] Receptor quantification

[0347] The cell lines were harvested using TryPLE select (Thermo Fisher Scientific 12563029), counted, and 2×10 5 Cells were seeded into U-bottom 96-well plates (Falcon 353227). After centrifugation, the cells were washed with ice-cold FACS buffer containing PBS with 0.5% BSA (Thermo Fisher Scientific 15260-037). 10 μg / mL of adhesion protein-4-AF647 conjugated antibody (clone 337516, R&D Systems FAB2659R) was incubated with the cells at 4°C. After incubation for 1 hour, the cell suspension was washed twice with ice-cold FACS buffer and centrifuged at 400×g for 5 minutes. The samples were resuspended in 100 μL FACs buffer and the cells were analyzed on an Attune NxT cell counter (Thermo Fisher Scientific). By using SIMPLE Anti-human IgG beads (Bangs Laboratories 816A) were used to determine the effective fluorophore to protein ratio (F / P). Anti-human IgG beads were added to 100 μL of 10 μg / ml nectin-4-AF647 solution. The mixture was incubated on ice in the dark for 30 minutes. The samples were then 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 samples and then analyzed on the Attune NxT Cytometer on the same day as the cell analysis. A total of 10,000 events were recorded and analyzed using FlowJo (BD).

[0348] Derivation and culture of human monocyte-derived macrophages

[0349] Human leukocyte-depleted whole blood (Vitalant Blood Center) was diluted 1:3 with PBS (Thermo Scientific 10010072). 10 mL Ficoll-Paque Premium (Cytiva 17-5442-02) was placed under the diluted blood. The tube was centrifuged at 400 x g for 30 minutes. PBMCs were collected from the interface, washed twice by adding 40 mL PBS, centrifuged at 400 x g for 10 minutes, and resuspended in magnetic activated cell sorting (MACS) buffer (PBS containing 0.5% BSA (Thermo Fisher Scientific 15260-037), 2 mM EDTA (Teknova E0307-06)). CD14 was purified by negative selection using a monocyte isolation kit II (Miltenyi Biotec 130-091-153) and LS columns (Miltenyi Biotec 130-042-401) according to the manufacturer's protocol. + Monocytes. CD14 + Monocytes were seeded at 10 million cells per dish in 150 mm tissue culture dishes (Falcon 353025) in 25 mL of culture medium containing 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 M-CSF (Miltenyi 130-096-492). The cells were cultured for seven days.

[0350] In vitro phagocytosis assay

[0351] HT-1376, T47-D and OE19 cells were isolated from culture plates by washing once with 10 mL PBS and incubating in 5 mL TrypLE Select for 10 minutes at 37 ° C. Cells were washed twice in PBS and resuspended in PBS. HT-1376, T-47D and OE19 cells were labeled in a suspension containing 150 nM CFSE with Celltrace CFSE cell proliferation kit (Thermo Fisher Scientific C34554) according to the manufacturer's instructions and resuspended in RPMI-1640. Macrophages were isolated from culture plates by washing once with 10 mL PBS and incubating in 5 mL TrypLE Select for 20 minutes at 37 ° C. Cell scrapers (Corning 3008) were removed, washed in PBS, and resuspended in RPMI-1640.

[0352] CFSE-labeled T47-D and OE19 target cells were added to ultra-low attachment U-bottom 96-well plates at 100,000 cells per 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. To determine the EC 50CFSE-labeled HT-1376 and OE19 target cells were added to ultra-low attachment U-bottom 96-well plates (Corning 7007) at 100,000 cells per well. Drug B or Drug C was added at a concentration of 200 ng / ml. Next, a ten-fold serial dilution of Drug A between 100 nM and 0.1 pM was added. The plates containing target cells, Drug A, and Drug B or Drug C were incubated at 37°C in a humidified incubator containing 5% carbon dioxide for 20 minutes before adding 50,000 macrophages. After 20 minutes, cultured macrophages were added and the plates were incubated at 37°C in a humidified incubator containing 5% carbon dioxide for another two hours. Cells were pelleted by centrifugation at 400 g for five minutes and stained for 30 minutes at 4° C. in 100 μL of fixable vital dye eFluor 780 (ebioscience 65-0865-14) diluted 1:5000 in PBS. Cells were washed in 200 μL FACS buffer (PBS + 2% FBS) and stained on ice for 60 minutes in 50 μL FACS buffer containing 2 μL human FcR blocking reagent (Miltenyi Biotec 130-059-901), 0.5 μL BV421 anti-CD163 (Clone GHI / 61, Biolegend 333612), 0.5 uL PE-Cyanine7 CD11b (clone ICRF44, Thermo Scientific 25-0118-42) and 0.5 μL PE CD326 (Clone 9C4, Biolegend 324206). The cells were washed twice in 250 μL FACS buffer and fixed overnight at 4 ° C in 100 μL 0.5% paraformaldehyde (Electron Microscopy Sciences 15710) PBS solution. Cells were analyzed on FACS Canto II (BD Biosciences), followed by data analysis by Flowjo10.8 (BD). Dead cells were excluded by gating the e780 negative population. Macrophages were identified as cells that were positive for lineage markers CD11b and CD163. In this population, macrophages that engulfed tumor cells were identified as cells that were positive for CFSE. In order to exclude unengulfed CFSE-labeled tumor cells from the analysis, cells that were positive for the epithelial cell marker CD326 were not included. The percentage of engulfment was calculated as live CD11b + CD163 + Percentage of human monocyte-derived macrophages expressing target cell markers (CD326 - ) stained negatively and stained positively for CFSE.

[0353] Data were plotted, maximal phagocytosis values ​​were determined, and EC was calculated using Prism 9 software (Graphpad) 50 The average phagocytic level and average EC were calculated using Excel (Microsoft). 50 Error bars represent standard deviation from the mean.

[0354] result

[0355] A summary of the number of fibronectin-4 receptors for the cell lines tested is provided in Table B. The number (eg, average number) of fibronectin-4 receptors expressed on the cell surface of the cell lines tested in Table B ranged from a high of 110,312 to a low of 43,784.

[0356] Table B: Number of fibronectin-4 receptors in human tumor cell lines

[0357] Cell lines Adhesion protein 4 receptor HT-1376 110,312 T47D 86,139 OE19 43,784

[0358] Table C provides a summary of the effects of Drug B, Drug C, Drug A + Drug B, and Drug A + Drug C on phagocytosis of TROP2-expressing cell lines by macrophages derived from monocytes obtained from human donors.

[0359] Table C. Summary of ADCP activity of drug B, drug C, drug A + drug B, and drug A + drug C against ADCP of human cell lines expressing TROP2

[0360]

[0361] a The percentage of macrophages phagocytosing target cells in response to drug B or drug C was normalized to the percentage of macrophages phagocytosing target cells treated with medium alone.

[0362] b The maximum percentage of macrophages phagocytosing target cells in response to Drug A plus Drug B or Drug C was normalized to the percentage of macrophages phagocytosing target cells treated with medium alone.

[0363] c Calculate the EC for cells receiving drug A plus drug B or drug C 50 value.

[0364] nd = not determined

[0365] Note: Each row represents data from a different donor.

[0366] In the absence of Drug A, Drug B, and Drug C, each stimulated ADCP by an average of 1.37-fold relative to medium alone. The combination of Drug A with either Drug B or Drug C enhanced ADCP by an average of 2.39-fold across all cell lines relative to medium alone. Drug A enhanced ADCP of Drug B and Drug C in OE19 and HT-1376, with an overall average EC 50 24.54pM and 6.19pM respectively.

[0367] The results of drug A enhancing the in vitro phagocytosis of drug B and drug C using T-47D and OE19 and two different donors are shown in Figure 1A and Figure 1B middle. Figure 1A It is shown that drug A enhances drug B or drug C-induced phagocytosis of OE19 and T47D cells by human monocyte-derived macrophages obtained from the first donor. Percent phagocytosis, defined as the percentage of viable macrophages engulfing CFSE-labeled OE19 cells or T47D cells, is shown on the y-axis. Single agent or combination parameters are shown on the x-axis. Figure 1B Drug A is shown to enhance drug B or drug C-induced phagocytosis of OE19 and T47D cells by human monocyte-derived macrophages obtained from a second donor. Percent phagocytosis, defined as the percentage of viable macrophages engulfing CFSE-labeled OE19 cells or T47D cells, is shown on the y-axis. Single agent or combination parameters are shown on the x-axis. Figure 2 The EC in an in vitro phagocytosis assay using OE19 cells is shown in 50 Results. Figure 2 In the figure, the percentage of phagocytosis, defined as the percentage of live macrophages that engulfed 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 drug A and is 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. The error bars represent the standard deviation of three technical replicates. The EC of each curve was calculated based on a sigmoidal dose response, variable slope fit 50 . Figure 3 EC of in vitro phagocytosis assay using HT-1376 is shown. 50 Results. Figure 3In the figure, the percentage of phagocytosis, defined as the percentage of live macrophages that engulf CFSE-labeled tumor 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 drug A and indicated by arrows. The percentage of phagocytosis of cells treated with drug A + drug B (open circles) or drug A + drug C (filled circles) and drug A alone (open squares) is shown. The error bars represent the standard deviation of three technical replicates. The EC of each curve was calculated based on a sigmoidal dose response, variable slope fit 50 .

[0368] in conclusion

[0369] The effect of drug A on the ADCP of drug B and drug C was evaluated by flow cytometry-based in vitro phagocytosis assay. Drug A enhanced the ADCP of drug B and drug C in multiple tumor cell lines expressing multiple fibronectin-4 receptors, with an overall mean EC 50 Respectively, 24.54 and 6.19 pM. Relative to the background level observed with the medium control alone, as a single agent, drug B and drug C stimulated ADCP across cell lines by an average of 1.41 times and 1.32 times, respectively. Relative to the medium control alone, the combination of drug A with drug B and the combination of drug A with drug C enhanced ADCP by an average of 2.42 times and 2.36 times, respectively. In summary, the presence of a linker-payload mc-vc-PAB-MMAE conjugated to the interchain cysteine ​​of drug C with an average drug-antibody ratio of 3.85:1 does not seem to affect the ability of drug C to mediate ADCP alone or in combination with drug A.

[0370] Example 3: Phase 1 Safety, Pharmacokinetics, and Pharmacodynamics Study of the Combination of Drug A and Veentumumab in Subjects with Urothelial Carcinoma

[0371] This example describes a Phase 1 clinical study of the combination of Drug A and Viagra in subjects with locally advanced or metastatic urothelial carcinoma

[0372] (A) Research design

[0373] This study includes a dose escalation part (Phase 1a) and a dose expansion part (Phase 1b). The study design is presented in Figure 4 Approximately 30 adult subjects (i.e., 18 years of age or older) will be enrolled. This study is designed to determine the safety and tolerability, maximum tolerated dose (MTD), recommended phase 2 dose (RP2D), single-dose and multiple-dose PK profiles, and PD markers (including but not limited to target occupancy) of Drug A combined with Vicente, and to determine the preliminary activity (e.g., therapeutic activity) of the combination of Drug A and Vicente.

[0374] Drug A was administered intravenously (IV) in escalating dose level cohorts at a starting dose of 20 mg / kg, administered once every 2 weeks (Q2W), in combination with ventuumab, given at the standard dose and schedule of ventuumab of 1.25 mg / kg IV on Days 1, 8, and 15 of each 28-day cycle.

[0375] The dose of drug A was escalated stepwise using a Bayesian optimal interval (BOIN) design, and the incidence of dose-limiting toxicity (DLT) was evaluated (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). Two dose levels of drug A were evaluated: 20 mg / kg Q2W and 30 mg / kg Q2W. If 20 mg / kg Q2W was not tolerated, a lower dose level of drug A (i.e., 15 mg / kg Q2W) was evaluated. Other dose levels and / or schedules at or below the maximum tolerated dose (MTD) may be evaluated.

[0376] During dose escalation, a BOIN design was used with a target dose-limiting toxicity (DLT) rate of 0.25 at the MTD. Cohorts of 3 subjects were enrolled and assessed for DLT. DLTs were assessed for each cohort and are described in further detail below. DLTs were assessed during the 28-day assessment window in Cycle 1. The BOIN design used the following overdose control rule to guide dose increases / decreases:

[0377] If the observed DLT rate at the current dose is ≤ 0.197, dose escalation to the next higher dose level;

[0378] If DLT > 0.298 is observed, dose escalation to the next lower dose level;

[0379] Otherwise, maintain current dose level.

[0380] The selection of MTD is based on the ordinal regression specified in Liu et al. (2015) Journal of the Royal Statistical Society. Series C: Applied Statistics. 64 (3): 507-523. Specifically, MTD is selected as the dose whose ordinal estimate of DLT rate is closest to the target DLT rate. If there is a tie, when the ordinal estimate is lower than the target DLT rate, a higher dose level is selected, and when the ordinal estimate is greater than or equal to the target DLT rate, a lower dose level is selected.

[0381] Once the Safety Review Committee (SRC) reviews and approves the dose level, additional subjects are enrolled at the same dose level in the backfill cohort to further characterize the safety, PK, PD, and preliminary anti-tumor activity of Drug A and Vicente to optimize the dose. DLTs were not evaluated for patients enrolled in the backfill cohort. In the Phase 1a portion, including dose escalation and backfill cohorts, approximately 15 subjects were treated at each dose level. To select the RP2D, the sponsor reviewed with the SRC all available safety, PK, PD, and preliminary anti-cancer activity data from the Phase 1a portion, including dose escalation and backfill cohorts, to recommend the dose of the combination of Drug A and Vicente in the Phase 2 setting.

[0382] Based on the SRC's decision, dose expansion was conducted to further evaluate the safety, tolerability and characterize the preliminary anticancer activity of Agent A and ventozumab in the selected subject population (see Figure 4 ). In the dose expansion part, the safety and tolerability of other anticancer drugs such as checkpoint inhibitors in combination with drug A and ventuumab were characterized.

[0383] New pre-treatment and on-treatment biopsies were required for the backfill and expansion cohorts for the assessment of intratumoral pharmacodynamic endpoints. These biopsies were optional for subjects enrolled in the dose escalation cohorts. Subjects had up to 28 days to complete the screening assessment and were treated with the combination of Drug A and ventozumab until (a) disease progression, (b) subject or physician decision to discontinue treatment, (c) unacceptable toxicity, (d) withdrawal of consent, or (e) study termination. Tumor assessments were performed at baseline and approximately every 8 weeks during the treatment phase of the study. Patients could continue treatment after radiographic progression if the investigator determined that the subject (i) had a clinical benefit from study treatment and demonstrated the absence of clinical symptoms or signs indicative of clinically significant disease progression; (ii) had no decline in performance status (PS); (iii) demonstrated the absence of rapid disease progression or threat to vital organs or critical anatomical sites requiring urgent alternative medical intervention; and (iv) had not demonstrated significant, unacceptable, or irreversible toxicity related to study treatment. The end-of-treatment (EOT) visit occurred approximately 4 weeks (at least 28 days and no more than 35 days), or before starting the next cancer treatment, after the last dose of Drug A, to review / collect concomitant medications, vital signs, adverse events (AEs) and serious adverse events (SAEs), and to assess resolution of any treatment-related toxicities. Thereafter, follow-up consisted of overall survival data collected by telephone every 3 months for 24 months.

[0384] This study was conducted in accordance with the protocol, good clinical practice (GCP), and applicable regulatory requirements.

[0385] (B) Study objectives and endpoints

[0386] This study Main Purpose The objectives of this study are to: (1) evaluate the safety and tolerability of the combination of Drug A and Vicentinatumomab in subjects who have previously received treatment for locally advanced or metastatic urothelial carcinoma; and (2) determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of the combination of Drug A and Vicentinatumomab.

[0387] This study Secondary Purpose The objectives were to: (1) evaluate the overall safety profile of the combination of Drug A and veentumab; (2) characterize the single-dose and multiple-dose pharmacokinetics (PK) of the combination of Drug A and veentumab; (3) evaluate the immunogenicity of Drug A; and (4) evaluate the evidence of anti-tumor activity of the combination of Drug A and veentumab.

[0388] This study Exploratory purpose The objectives were: (1) to explore the pharmacodynamic (PD) effects of the combination of Drug A and ventuinumab; and (2) to evaluate methods to reduce interference from Drug A in serological tests used for blood product transfusions.

[0389] This study Primary End Point are (1) first cycle dose-limiting toxicity (DLT), as described in further detail below; and (2) adverse events (AEs), as characterized by type, frequency, severity (graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE v.5.0, see, e.g., https: / / ctep(dot)cancer(dot)gov / protocoldevelopment / electronic(underscore)applications / docs / CTCAE(underscore)v5(underscore)Quick(underscore)Reference(underscore)5x7(dot)pdf)), timing, severity, and relationship to study therapy.

[0390] This study Secondary End Points The following are (1) laboratory abnormalities characterized by type, frequency, severity (according to NCICTCAE v.5.0 classification), and timing; (2) pharmacokinetic parameters of drug A, such as maximum serum concentration (C max ), time to reach maximum serum concentration (T max ), drug exposure over time (area under the curve or AUC), clearance (CL), and half-life (t 1 / 2 ), (3) evaluated using the Response Evaluation Criteria in Solid Tumors (RECIST 1.1, see, e.g., Eisenhauer et al. (2009) Eur J Cancer 45:228-247); (4) disease control rate (DCR), best overall response (BOR), duration of response (DOR), time to tumor progression (TTP), progression-free survival (PFS), and overall survival (OS).

[0391] This study Exploratory End PointsThe invention relates to (1) pharmacodynamic effects, including (a) pre-drug A dose levels and post-drug A dose levels of CD47 target occupancy in peripheral blood; (b) immunophenotype of circulating leukocyte populations; (c) investigation of tumor marker expression, infiltrating leukocyte populations, and immunomodulatory molecules in tumor biopsies before and after treatment; (d) exploratory molecular analysis (including, but not limited to, tumor and immune markers) in peripheral blood and / or tumor biopsy samples before and after treatment; and (2) characterization of methods for mitigating drug A interference in the indirect antiglobulin test (IAT) and direct antiglobulin test (DAT) during drug A treatment.

[0392] (C) Research Group

[0393] Inclusion criteria

[0394] Subjects must meet the following inclusion criteria to be eligible for this study:

[0395] Subjects have histologically confirmed, unresectable locally advanced or metastatic urothelial carcinoma (i.e., bladder, renal pelvis, ureter, or urethra). Subjects with urothelial carcinoma (transitional cell) with squamous differentiation or mixed cell types are eligible, provided that urothelial carcinoma is the predominant histology. Subjects with any small cell or neuroendocrine histological component are excluded.

[0396] Subjects who have received prior treatment with an immune checkpoint inhibitor (CPI) in the setting of locally advanced or metastatic urothelial carcinoma. Subjects who received a CPI in the neoadjuvant / adjuvant setting and developed recurrent or progressive disease during treatment or within 12 months after completion of treatment are eligible. CPIs are defined as programmed cell death protein 1 (PD-1) inhibitors or programmed cell death ligand 1 (PD-L1) inhibitors (including but not limited to: atezolizumab, pembrolizumab, durvalumab, avelumab, and nivolumab).

[0397] Subjects have received prior treatment with platinum-containing chemotherapy, defined as receiving platinum in the adjuvant / neoadjuvant setting with recurrent or progressive disease within 12 months of completing treatment, or receiving platinum in the metastatic setting or for unresectable locally advanced disease.

[0398] The subject has urothelial carcinoma that has progressed or recurred during or after the last treatment.

[0399] Subjects must have measurable disease according to RECIST (version 1.1). Lesions in the previous radiation field must have developed after radiation therapy to be considered measurable.

[0400] Adequate bone marrow function, as demonstrated by the following laboratory values, appropriate for the disease being studied:

[0401] Absolute neutrophil count (ANC) ≥ 1,500 / mm 3 (≥1.5x 10 9 / L);

[0402] Platelet count ≥ 100,000 / mm 3 (≥100x 10 9 / L);

[0403] Hemoglobin ≥9 g / dL (≥90 g / L)

[0404] Adequate renal function as demonstrated by creatinine clearance ≥ 30 mL / min estimated by the Cockcroft-Gault equation or other medically accepted formulas such as Modification of Diet in Renal Disease (MDRD) or Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI).

[0405] Adequate liver function, as demonstrated by the following laboratory values, appropriate for the disease being studied:

[0406] Serum total bilirubin ≤1.5x upper limit of normal (ULN) (≤3.0x ULN if the subject has documented Gilbert syndrome);

[0407] For subjects with liver metastases, aspartate and alanine aminotransferase (AST and ALT) ≤ 3.0 x ULN or ≤5.0xULN;

[0408] For subjects with bone or liver metastases, alkaline phosphatase ≤ 2.5x ULN or ≤5.0x ULN.

[0409] QT interval corrected for heart rate Fridericia equation (QTcF) interval ≤ 480 ms (based on the average of three electrocardiograms [ECGs]).

[0410] Age ≥ 18 years.

[0411] Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.

[0412] Subjects in the dose escalation cohort provided available archived (or fresh) biopsy samples prior to study entry. Subjects in the backfill and expansion cohorts had tumors available for follow-up biopsies and were willing to provide fresh pre-treatment and on-study tumor tissue biopsies (needing core biopsy or resection).

[0413] Negative serum pregnancy test (for females of childbearing potential) at screening.

[0414] Male and female subjects of reproductive potential agree to use highly effective contraceptive methods throughout the study and for at least 4 months after the last dose of study treatment.

[0415] Exclusion criteria

[0416] Subjects with any of the following characteristics / conditions were not included in this study:

[0417] Pre-existing sensory or motor neuropathy grade ≥ 2;

[0418] Presence of symptomatic or uncontrolled central nervous system (CNS) metastases. Subjects receiving treatment for CNS metastases are allowed to participate in the study if all of the following conditions are met:

[0419] CNS metastases have been clinically stable for at least 6 weeks prior to screening;

[0420] If CNS metastases require steroid therapy, the subject has received a stable dose of ≤20 mg / day of prednisone or equivalent for at least 2 weeks;

[0421] Baseline scans show no evidence of new or enlarging brain metastases; and

[0422] The subject does not have leptomeningeal disease

[0423] Prior treatment with ventuinumab or other monomethyl auristatin (MMAE)-based antibody-drug conjugates (ADCs)

[0424] Prior treatment with any anti-CD47 or anti-signal regulatory protein-α (SIRPα) agents.

[0425] Known hypersensitivity to ventocumab or any excipients contained in the pharmaceutical formulation of ventocumab (including histidine, trehalose dihydrate, and polysorbate 20); or the subject is known to have hypersensitivity to biologic drugs produced in Chinese hamster ovary (CHO) cells.

[0426] Subjects who are intolerant to or have severe allergic or anaphylactic reactions to the antibody or infused therapeutic protein, or to any substance contained in Drug A.

[0427] · Ongoing clinically significant toxicity (Grade 2 or higher, excluding alopecia) related to prior treatment (including systemic therapy, radiation therapy, or surgery). Subjects with ≤ Grade 2 immunotherapy-related hypothyroidism or panhypopituitarism were enrolled when they were well maintained / controlled on stable doses of hormone replacement therapy (if indicated). Subjects with ongoing Grade ≥ 3 immunotherapy-related hypothyroidism or panhypopituitarism were excluded. Subjects with ongoing immunotherapy-related colitis, uveitis, myocarditis, or pneumonitis, or with other immunotherapy-related AEs requiring high-dose steroids (>20 mg / day of prednisone or equivalent) were excluded.

[0428] Current systemic antimicrobial therapy for active infection (viral, bacterial, or fungal). Routine antimicrobial prophylaxis is permitted.

[0429] Known active uncontrolled hepatitis B (HBV), hepatitis C (HCV), and human immunodeficiency virus (HIV) infection.

[0430] Any of the following within the past 6 months: myocardial infarction, severe / unstable angina, coronary artery / peripheral artery bypass graft, New York Heart Association (NYHA) class II or higher congestive heart failure, uncontrolled hypertension, cerebrovascular accident, transient ischemic attack, deep vein thrombosis (except those considered device-related and not clinically significant), arterial thrombosis, symptomatic pulmonary embolism, or any other major thromboembolism.

[0431] Any other currently active treatment in clinical investigational interventions.

[0432] Radiation therapy or major surgery within 14 days before the first dose of study drug.

[0433] Uncompleted anti-tumor treatment with chemotherapy, biologics, investigational drugs, and / or immunotherapy within 28 days or 5 half-lives (whichever is shorter) prior to the first dose of study drug.

[0434] Any experimental antibody or live vaccine within the last 28 days before the first dose of study drug. Examples of live vaccines include, but are not limited to, the following: measles, mumps, rubella, varicella / zoster, yellow fever, rabies, Bacillus Calmette–Guérin (BCG), and typhoid vaccines. Injectable seasonal influenza vaccines are usually inactivated virus vaccines and are permitted; however, intranasal influenza vaccines (e.g. ) is a live attenuated vaccine and is not permitted for use.

[0435] Subjects with a history of other malignancies within 3 years prior to the first dose of study drug, or any evidence of residual disease from a previously diagnosed malignancy. Subjects with non-melanoma skin cancer, localized prostate cancer treated with curative intent without evidence of progression, low-risk or very low-risk (according to standard guidelines) localized prostate cancer on active surveillance / watchful waiting without treatment intent, or any type of carcinoma in situ (if completely resected) are permitted.

[0436] Subjects with active autoimmune disease who have required systemic treatment (i.e., use of disease-modifying agents, corticosteroids, or immunosuppressive drugs) in the past year. Replacement therapy (e.g., thyroxine, insulin, or physiologic corticosteroid replacement for adrenal or pituitary insufficiency) is not considered a form of systemic treatment and is permitted.

[0437] Other serious acute or chronic medical or psychiatric disorders, including recent (within the past year) or active suicidal ideation or behavior, or laboratory abnormalities that may increase the risks associated with participation in the study or administration of study drugs or that may interfere with the interpretation of study results and make the subject unsuitable for participation in this study.

[0438] History of autoimmune hemolytic anemia, autoimmune thrombocytopenia, or hemolytic transfusion reaction.

[0439] Known active keratitis or corneal ulcer. Subjects with superficial punctate keratitis are permitted if the condition is adequately treated.

[0440] History of uncontrolled diabetes within 3 months after the first dose of study drug. Uncontrolled diabetes is defined as hemoglobin A1C (HbA1c) ≥8% or HbA1c between 7% and <8% with associated diabetic symptoms not otherwise explained (e.g., polyuria or polydipsia).

[0441] Currently pregnant or breastfeeding.

[0442] (D) Study of pharmaceutical products, dosages and routes of administration

[0443] In the dose escalation part of this study, the initial starting dose of drug A is 20mg / kg IV Q2W, and if it is considered safe, the dose of drug A is increased to the maximum dose of 30mg / kg IV Q2W specified in the scheme. If 20mg / kgQ2W cannot be tolerated, drug A (i.e. 15mg / kg IV Q2W) at a lower dose level can be evaluated. Other dose levels and / or schedules below MTD or MTD can be evaluated. The dose escalation group and the backfill group determine the optimal dose and schedule of the combination of drug A recommended 2-phase dose (RP2D) and Vientoumab. In the dose expansion part of this study, drug A is administered in combination with Vientoumab determined in the dose escalation part equal to or lower than MTD and this study. Vientoumab is administered at the standard dose of 1.25mg / kg IV on the 1st day, the 8th day and the 15th day of each 28-day cycle. See Table D below.

[0444] Table D. Dose escalation doses and administration schedule

[0445]

[0446] As described elsewhere herein, 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) is used to find the maximum tolerated dose (MTD). The BOIN design is implemented in a simple manner similar to the traditional 3+3 design, but is more flexible and has excellent operating characteristics comparable to more complex model-based designs such as the continuous reassessment method (CRM) (see Zhou et al. (2018) Clin Cancer Res. 24(18): 4357-4364).

[0447] Drug A is packaged in 1000 mg / 50 mL Type 1 clear glass vials, sealed with 20 mm polytetrafluoroethylene-coated rubber serum stoppers and tamper-evident aluminum seals. Each disposable vial can deliver 1000 mg of Drug A (50 mL) and is intended for intravenous (IV) administration.

[0448] Complete information on ventuumab dosage forms and packaging can be found in the U.S. or local package insert. For the U.S., see, e.g., https: / / astellas(dot)us / docs / PADCEV(underscore)label(dot)pdf.

[0449] Drug A is administered every 2 weeks as an IV infusion on an outpatient basis. The dose is infused over approximately 60 minutes. Use of an infusion pump is the preferred method of administration to ensure accurate delivery of study drug, but gravity infusion is also permitted.

[0450] A cycle was defined as the time from the Day 1 dose to the next Day 1 dose. If there were no treatment delays, a cycle was 28 days with dosing every 2 weeks. All trial treatments were administered on an outpatient basis. Following the Day 1 infusion of Cycle 1, subjects were observed in the clinic for at least 2 hours and thereafter as clinically indicated.

[0451] No premedication with Drug A is required. Follow the veentumab infusion guidelines according to the veentumab U.S. or local package insert. The recommended dose of veentumab is 1.25 mg / kg (maximum dose of 125 mg for subjects ≥100 kg) 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.

[0452] On the day of administration consistent with the dosing schedule, ventuumab was administered beginning approximately 30 minutes after the end of Drug A therapy.

[0453] For toxicity, interruptions and dose adjustments are allowed. Dose adjustments of drug A can occur in one of three ways:

[0454] Within a cycle: During a given treatment cycle, (for every 2-week dosing of Drug A only) interrupt dosing until adequate recovery and dose reduction (if necessary);

[0455] Between cycles: At the start of a new cycle, administration of the next cycle may be delayed due to continued toxicity;

[0456] In the next cycle: Based on toxicity experienced in the previous cycle, a dose reduction may be necessary in the next cycle.

[0457] If drug A is permanently discontinued, the subject may continue to receive veentumab if the investigator believes the subject has achieved clinical benefit. If veentumab is permanently discontinued prematurely, the subject may continue to receive drug A if the investigator believes the subject has achieved clinical benefit from drug A.

[0458] During dose escalation, subjects were consecutively assigned to the next available treatment period, with the dose level and schedule determined after safety assessment of the previous cohort.

[0459] (E) Statistical methods

[0460] A total of approximately 30 adult subjects were enrolled in this study. Enrollment depended on the observed safety profile, which determined the number of subjects at each dose level and the number of dose levels explored. The sample size for Phase 1 depended on the potential dose-toxicity profile and the variability of actual data realization. The number of subjects treated at each dose (i.e., between 3 and 15) in the dose-escalation portion was based on a BOIN (Bayesian Optimal Interval) design (see, e.g., Yuan et al. (2016) Clin Cancer Res. 22(17):4291–4301).

[0461] Once the Safety Review Committee (SRC) determines that the dose level is safe and tolerable, additional subjects are recruited at the same dose level in the backfill cohort to further evaluate the safety, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of the combination of Drug A administered with ventozumab in subjects previously treated for locally advanced or metastatic urothelial carcinoma. No DLT assessments were performed in these backfill cohorts. Approximately 15 subjects were treated at each dose level in the dose escalation portion, including the backfill cohort.

[0462] To select the RP2D, the sponsor, together with the SRC, reviewed all available safety, pharmacokinetic, pharmacodynamic, and preliminary anticancer activity data from Phase 1a, including dose escalation and backfill cohorts, to recommend the dose of Agent A to be used in the Phase 2 setting.

[0463] (F) Security

[0464] Adverse events (AEs) were described as causally related or not, as judged by the investigator. The timing, overall toxicity frequency, and severity of all AEs were described, classified on a toxicity scale of 1 to 5. The severity of AEs was graded according to CTCAE version 5.0 (see, e.g., https: / / ctep(dot)cancer(dot)gov / protocoldevelopment / electronic(underscore)applications / docs / CTCAE(underscore)v5(underscore)Quick(underscore)Reference(underscore)5x7(dot)pdf). Additional summaries are provided for AEs that were observed at a higher frequency or were considered serious adverse events (e.g., Hy's Law cases).

[0465] During the study, adverse events, electrocardiogram (ECG), blood pressure (BP), pulse rate, cardiac monitoring, and safety laboratory data were continuously reviewed and summarized to assess the safety of the subjects. Safety data were presented in tables and / or graphs and summarized descriptively where appropriate.

[0466] This study uses a trial SRC comprised of study investigators and sponsor representatives to monitor AEs on an ongoing basis. AEs, serious adverse events (SAEs), and safety laboratory values ​​that occur during the study and are considered related to study drug are regularly evaluated to determine if continued dosing would jeopardize the safety of future subjects.

[0467] (G) Dose-limiting toxicity (DLT)

[0468] Dose-limiting toxicity (DLT) - The assessment period is the first 28 days of treatment (i.e., Cycle 1). All Cycle 1 AEs that meet the following definition are considered DLTs unless clearly and unequivocally unrelated to Drug A.

[0469] ·hematology:

[0470] Grade 4 neutropenia lasting > 7 days.

[0471] Febrile neutropenia (defined as neutropenia ≥ Grade 3 and a single temperature > 38.3°C or a temperature ≥ 38°C for more than one hour).

[0472] ≥Grade 3 neutropenia associated with infection.

[0473] Grade 3 thrombocytopenia associated with clinically significant bleeding.

[0474] Grade 4 thrombocytopenia.

[0475] Cases that meet the criteria of Hy's Law

[0476] Non-hematologic toxicity of grade 3 or higher, except for the following:

[0477] Grade 3 nausea, vomiting, or diarrhea that resolves to ≤ Grade 1 before the next infusion (Grade 3 nausea, vomiting, or diarrhea that persists for >72 hours with adequate antiemetics and other supportive care should be considered a DLT).

[0478] Grade 3 fatigue, which resolves to ≤ Grade 2 within 7 days.

[0479] Laboratory abnormalities of grade 3 or higher that resolve to grade 1 or lower within 24 hours or are considered by the investigator to be clinically insignificant.

[0480] Grade 3 infusion reactions, if managed successfully and resolved within 72 hours (Grade 3 infusion reactions that recur or occur despite premedication should be considered a DLT).

[0481] Delay of more than 2 weeks in receiving the scheduled Cycle 2 Day 1 dose of Drug A due to persistent toxicity attributable to Drug A.

[0482] In addition, clinically important or persistent grade 2 toxicities could be considered DLTs.

[0483] (H) Efficacy

[0484] In this study, preliminary antitumor activity was a secondary objective. If data permitted, overall response rate (ORR), best overall response (BOR), disease control rate (DCR), duration of response (DOR), time to tumor progression (TTP), progression-free survival (PFS), and overall survival (OS) were analyzed in the full analysis set (FAS) population and the evaluable population.

[0485] Tumor assessments include all known or suspected sites of disease. Computed tomography (CT) is the preferred imaging modality, but magnetic resonance imaging (MRI) is also used. Imaging includes the chest, abdomen, and pelvis (head and neck are optional). For subjects with known or suspected brain metastases, a brain CT or MRI scan should be performed. In the following tumor assessments, the same imaging techniques used to characterize each identified and reported lesion at baseline are used.

[0486] Antitumor activity was assessed by radioactive tumor assessments performed at baseline, during treatment, when suspected disease progression (e.g., worsening symptoms) and when treatment was stopped. According to the investigator's assessment, the response to the relevant secondary endpoints was assessed using RECIST version 1.1 (see, e.g., Eisenhauer et al. (2009) Eur J Cancer 45: 228-247). The change in tumor size was classified as complete remission (CR), partial remission (PR), stable disease (SD) or disease progression, the latter including the appearance of new lesions.

[0487] (I) Tumor biopsy markers

[0488] Analysis of biopsy tissue was performed at a central reference laboratory. Analysis included fibronectin-4 expression, PD-L1 status, and additional immunohistochemistry (IHC) assessments, such as CD47 expression, and the frequency and location of infiltrating immune cells such as T cells and tumor-associated macrophages (TAMs). If biopsy material was adequate, additional multiplex immunofluorescence assays and exploratory molecular assays of tumor, immune, and checkpoint markers were performed.

[0489] (J) Pharmacokinetics / pharmacodynamics

[0490] Blood samples were collected to provide serum for PK analysis. Every effort was made to obtain pharmacokinetic samples at the exact nominal time relative to dosing. Drug concentrations of Drug A were measured using validated methods. Pharmacokinetic (PK) parameters were determined from the corresponding concentration-time data using standard non-compartmental methods. Sample collection times were used for parameter calculations. For Drug A, pharmacokinetic parameters were calculated, including maximum concentration (Cmax), time to maximum concentration (Tmax), and time to maximum concentration (Tmax). max ), the area under the concentration-time curve (AUC last ), AUC from time 0 to infinity (AUC inf ) and / or the area under the plasma concentration-time curve (AUCτ) during the dosing interval (τ). Other PK parameters were calculated as appropriate, including clearance (CL), volume of distribution (V z ), terminal elimination half-life (t 1 / 2 ) and cumulative ratio (R ac ). Drug concentrations for Drug A are summarized graphically with descriptive statistics by dose, cycle, and nominal PK sampling time. Non-compartmental PK parameters are summarized descriptively by dose and cycle. Pharmacodynamic data are summarized graphically with descriptive statistics by time and dose. PK / PD analyses are performed using appropriate model-based approaches to better understand exposure-response relationships, and the results may be reported separately.

[0491] Whole blood samples were collected for CD47 target occupancy, circulating leukocyte immunophenotype, peripheral blood circulating tumor DNA (ctDNA), and exploratory molecular analyses.

[0492] The blood sample collected at the baseline visit and any remaining blood collected at other visits is retained for potential pharmacogenomic analyses related to drug response. For example, SIRPα gene polymorphisms, putative safety biomarkers, drug-metabolizing enzyme genes, drug transporter genes, or genes thought to be related to the drug's mechanism of action can be examined.

[0493] (K) Evaluation of neutralizing agents for use in pre-transfusion cross-matching testing

[0494] Because red blood cells (RBCs) express CD47, Drug A can bind to a patient's circulating RBCs in addition to being present in the subject's serum or plasma in an unbound form. After initiating treatment with Drug A, antibody screens, including the indirect antiglobulin test (IAT) and direct antiglobulin test (DAT), can report false positives due to antihuman globulin (AHG) binding to the Fc portion of Drug A, potentially affecting the interpretation of pretransfusion crossmatches. Potential mitigation approaches for neutralizing this interference were evaluated. Therefore, when performing ABO Rh typing, antibody screening, and crossmatching for RBC transfusions, research neutralizing reagents are available to local blood banks, or blood samples can be sent to designated reference laboratories for testing by exploratory neutralization assays.

[0495] The foregoing embodiments are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Various modifications of the present invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Claims

1. A method for treating urothelial carcinoma in an individual, comprising administering to the individual (a) an effective amount of a fusion polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant, and (b) an effective amount of ventozumab, wherein the SIRPαD1 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 L234A, L235A, G237A and N297A mutations, wherein numbering is according to the EU index as in Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein numbering is according to the EU index as in Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A and delG236 mutations, wherein numbering is according to the EU index as in Kabat; or (iv) human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236 and N297A mutations, wherein the numbering is according to the EU index as in Kabat.

2. The method of claim 1, wherein the urothelial carcinoma is locally advanced urothelial carcinoma or metastatic urothelial carcinoma.

3. The method of claim 1 or claim 2, wherein the urothelial cancer is bladder cancer, renal pelvis cancer, ureter cancer, or urethral cancer.

4. The method of any one of claims 1-3, wherein the individual has received prior treatment with an immune checkpoint inhibitor (CPI).

5. The method of claim 4, wherein the CPI is a PD-1 inhibitor or a PD-L1 inhibitor.

6. The method of claim 4 or 5, wherein the CPI is atezolizumab, pembrolizumab, durvalumab, avelumab, or nivolumab.

7. The method of any one of claims 1-6, wherein the individual has received prior treatment with platinum-containing chemotherapy.

8. The method of any one of claims 1-7, wherein the individual has progression or recurrence of urothelial carcinoma during or after receiving the most recent prior therapy.

9. The method of any one of claims 1-8, wherein the individual has not received prior treatment with a monomethyl auristatin (MMAE)-based antibody-drug conjugate.

10. The method of claim 9, wherein the individual has not received prior treatment with ventuumab.

11. The method of any one of claims 1-10, wherein the individual has not received prior treatment with a therapeutic agent that blocks the interaction between CD47 and SIRPα.

12. The method of any one of claims 1-11, wherein the veentumab is administered to the individual in one or more 28-day cycles, and wherein the veentumab is administered to the individual at a dose of 1.25 mg / kg IV on Day 1, Day 8, and Day 15 of each 28-day cycle.

13. The method of any one of claims 1-12, wherein the ventuumab is administered intravenously.

14. The method of any one of claims 1-13, wherein the fusion polypeptide is administered to the individual at a dose of up to about 60 mg / kg.

15. The method of claim 14, wherein the fusion polypeptide is administered to the individual once every two weeks (q2w) at a dose of about 30 mg / kg.

16. The method of claim 14, wherein the fusion polypeptide is administered once every two weeks (q2w) at a dose of about 20 mg / kg.

17. The method of claim 14, wherein the fusion polypeptide is administered once every two weeks (q2w) at a dose of about 15 mg / kg.

18. The method of any one of claims 1-17, wherein the fusion polypeptide is administered intravenously.

19. The method of any one of claims 1-18, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:

85.

20. The method of any one of claims 1-18, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO:

81.

21. The method of any one of claims 1-20, wherein the Fc domain variant is a human IgGl Fc region comprising L234A, L235A, G237A and N297A mutations, wherein numbering is according to the EU index as in Kabat.

22. The method of claim 21, wherein the Fc domain variant comprises the amino acid sequence of SEQ ID NO:

91.

23. The method of any one of claims 1-19 and 21-22, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:

136.

24. The method of any one of claims 1-18 and 20-22, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:

135.

25. The method of any one of claims 1-24, wherein the fusion polypeptide forms a homodimer.

26. The method of any one of claims 1-25, wherein the individual is a human.

27. A kit comprising a polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant in a pharmaceutically acceptable carrier, the polypeptide for use in combination with ventoumab for treating urothelial carcinoma in an individual in need thereof, wherein the SIRPαD1 domain variant comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 85; wherein the Fc domain variant is (i) a human IgG1 Fc region comprising L234A, L235A, G237A and N297A mutations, wherein numbering is according to the EU index as in Kabat; (ii) a human IgG2 Fc region comprising A330S, P331S and N297A mutations, wherein numbering is according to the EU index as in Kabat; (iii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A and delG236 mutations, wherein numbering is according to the EU index as in Kabat; or (iv) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A, delG236 and N297A mutations, wherein the numbering is according to the EU index as in Kabat, And wherein the kit comprises instructions for administering to the individual a combination of the polypeptide comprising a SIRPαD1 domain variant and an Fc domain variant and ventozumab.

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