SIRP-ALPHA FUSION POLYPEPTIDES HAVING MODIFIED Fc DOMAINS

Abstract

Provided herein are signal regulatory protein alpha (SIRPα) fusion polypeptides that contain a modified Fc domain, and methods of making and using the same. The inclusion of a modified Fc domain results in improved pharmacokinetics and / or pharmacodynamics of the SIRPα fusion polypeptide. The compositions and methods described herein can be used to treat a variety of diseases, such as cardiovascular diseases.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 323,417, filed March 24, 2022, the contents of which are incorporated by reference herein in their entirety.

[0002] Incorporation by reference of sequence listing The contents of the electronic sequence listing (BTRT_010_01WO_SeqList_ST26.xml, size: 13,201 bytes, created on: March 21, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] Optimization of IgG Fc domain-containing therapies remains a challenge because these polypeptides may exhibit variable half-life in the bloodstream. One example is a signal-regulatory protein alpha (SIRPα) fusion polypeptide with an IgG Fc domain designed to bind to CD47 protein, which is applicable to a wide range of diseases that benefit from optimal pharmacological inhibition of CD47. Thus, there is a need for signal-regulatory protein alpha (SIRPα)-IgG Fc therapy with improved pharmacokinetics and / or pharmacodynamics. Provided herein are compositions and methods that address this need. Summary of the Invention

[0004] The present disclosure provides signal regulatory protein alpha (SIRPα) fusion polypeptide therapeutics that contain a modified Fc domain to improve the pharmacokinetics and / or pharmacodynamics of the fusion polypeptide.

[0005] In some embodiments of the present disclosure, a fusion polypeptide is provided comprising a signal regulatory protein alpha (SIRPα) domain and a modified Fc domain, the modified Fc domain comprising one or more amino acid modifications relative to the wild-type Fc domain, wherein inclusion of the modified Fc domain increases binding affinity to FcRn.

[0006] In some embodiments of the present disclosure, the inclusion of a modified Fc domain increases the half-life of the fusion polypeptide. In some embodiments of the present disclosure, the inclusion of a modified Fc domain increases the blood clearance time of the fusion polypeptide.

[0007] In some embodiments of the present disclosure, the inclusion of a modified Fc domain increases the binding affinity of the fusion polypeptide to CD47. In some embodiments of the present disclosure, the inclusion of a modified Fc domain increases the EC 50 Decreases.

[0008] In some embodiments, the inclusion of a modified Fc domain reduces the effective dose of the fusion polypeptide required to achieve a therapeutic effect in a subject, hi some embodiments, the inclusion of a modified Fc domain reduces the frequency of administration of the fusion polypeptide in a subject.

[0009] In some embodiments, the inclusion of a modified Fc domain reduces the toxicity of the fusion polypeptide, hi some embodiments, the inclusion of a modified Fc domain reduces antibody-dependent cellular cytotoxicity (ADCC).

[0010] In some embodiments, the inclusion of a modified Fc domain increases the effector function of the fusion polypeptide. In some embodiments, the inclusion of a modified Fc domain increases phagocytosis by macrophages. In some embodiments, the inclusion of a modified Fc domain increases the interaction of the fusion polypeptide with cells expressing the CD47 protein. In some embodiments, the inclusion of a modified Fc domain increases endocytosis of the CD47 protein.

[0011] In some embodiments, the inclusion of a modified Fc domain increases degradation of the CD47 protein, hi some embodiments, the CD47 protein is a human or mouse CD47 protein.

[0012] In some embodiments, the inclusion of a modified Fc domain reduces aggregation of the fusion polypeptide, hi some embodiments, the inclusion of a modified Fc domain improves purification of the polypeptide.

[0013] In some embodiments, the modified Fc domain comprises an IgG4 Fc domain amino acid sequence of SEQ ID NO: 7 or 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0014] In some embodiments, the modified Fc domain comprises one or more substitutions selected from the group consisting of T250Q, M252Y, S254T, T256E, S267E, N325S, L328F, N343S, M428L, N434F, and H443K relative to SEQ ID NO: 7 or 8 according to the EU numbering scheme. In some embodiments, the modified Fc domain comprises one or more substitutions selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F relative to SEQ ID NO: 7 or 8 according to the EU numbering scheme. In some embodiments, the modified Fc domain comprises SEQ ID NO:9, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0015] In some embodiments, the modified Fc domain comprises an IgG1 Fc domain amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the modified Fc domain comprises one or more substitutions selected from the group consisting of V215A, G236A, S239D, and I332E relative to SEQ ID NO: 6 according to the EU numbering scheme. In some embodiments, the modified Fc domain comprises one or more substitutions selected from the group consisting of T250Q, M252Y, S254T, T256E, S267E, N325S, L328F, N343S, M428L, H433K, and N434F relative to SEQ ID NO: 6 according to the EU numbering scheme. In some embodiments, the modified Fc domain comprises one or more substitutions selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F relative to SEQ ID NO:6, according to the EU numbering scheme.

[0016] In some embodiments, the modified Fc domain is a modified human IgG1, IgG2, IgG3, or IgG4 domain.

[0017] In some embodiments, the SIRPα domain comprises an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the SIRPα domain comprises one or more of the following mutations relative to SEQ ID NO: 1: V6I, S14L, S20T, I22T, H24R, V27I, I31F, A45G, E47V, K53R, E54Q, H56P, S66T, E70N, S77R, V92I, and / or a duplication of the D100 residue. In some embodiments, the SIRPα domain comprises the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0018] In some embodiments, the modified Fc domain comprises any of SEQ ID NOs: 6-9, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, and the SIRPα domain comprises an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the modified Fc domain comprises SEQ ID NO:9, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, and the SIRPα domain comprises SEQ ID NO:2, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0019] In some embodiments, provided herein is a method for treating a disease in a subject in need thereof, the method comprising administering a SIRPα fusion polypeptide to the subject. In some embodiments, the disease is a cardiovascular disease. In some embodiments, the fusion polypeptide is administered subcutaneously.

[0020] In some embodiments, provided herein is a method of increasing phagocytosis by macrophages comprising contacting a population of macrophages with a SIRPα fusion polypeptide, in some embodiments, the macrophages are human macrophages.

[0021] In some embodiments, provided herein are nucleotides encoding SIRPα fusion polypeptides. [Brief description of the drawings]

[0022] [Figure 1] 1 is a chart showing the binding affinity to different CD47 proteins for an exemplary SIRPα fusion polypeptide with a YTE Fc domain mutation (construct 67) compared to a SIRPα fusion polypeptide with a wild-type Fc domain and the MIAP410 anti-CD47 monoclonal antibody. [Figure 2A] Examples of the performance of individual CD47 binding assays for SIRPα fusion polypeptide construct 50 (FIG. 2A) and construct 67 (FIG. 2B) are shown. [Figure 2B] Examples of the performance of individual CD47 binding assays for SIRPα fusion polypeptide construct 50 (FIG. 2A) and construct 67 (FIG. 2B) are shown. [Diagram 3] 1 shows the EC50 effect of SIRPα fusion polypeptide construct 50 and construct 67 on the amount of construct bound to human CD45-negative erythrocytes in vitro. [Figure 4A] 4A and 4B show the levels of phagocytosis induced by SIRPα fusion polypeptide construct 67, and construct 50, and an anti-CD47 antibody. Figures 4A and 4B each show the results for a different human donor. [Figure 4B] 4A and 4B show the levels of phagocytosis induced by SIRPα fusion polypeptide construct 67, and construct 50, and an anti-CD47 antibody. Figures 4A and 4B each show the results for a different human donor. [Figure 5A] Figure 5 shows the effect of SIRPα fusion polypeptide constructs 67 and 50 on antibody-dependent cellular cytotoxicity (ADCC) compared to an antibody against CD20. Figure 5A shows that constructs 67 and 50 showed minimal ADCC compared to an anti-CD20 antibody. [Figure 5B]Figure 5B shows the effect of SIRPα fusion polypeptide constructs 67 and 50 on antibody-dependent cellular cytotoxicity (ADCC) compared to an antibody against CD20. Figure 5B is an inset to Figure 5A and has been rescaled to show the difference between construct 67, which contains the YTE mutation in the Fc domain, and construct 50, which contains the wild-type Fc domain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Provided herein are signal-regulatory protein alpha (SIRPα) fusion polypeptide therapeutics that include a modified Fc domain, where the inclusion of the modified Fc domain improves the pharmacokinetics and / or pharmacodynamics of the fusion polypeptide. The signal-regulatory protein alpha (SIRPα) fusion polypeptides of the present disclosure can be used, for example, to treat cardiovascular disease, fibrosis, cancer, infectious diseases, hematological diseases, and neurological diseases.

[0024] I. Definition Unless otherwise defined herein, scientific and technical terms used herein shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the nomenclature and techniques utilized in connection with chemistry, molecular biology, cell biology, immunology, pharmacology, and protein chemistry described herein are those well known and commonly used in the art.

[0025] As used in this specification and the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" can refer to one candidate agent or a mixture of such candidate agents, and reference to "a method" includes references to equivalent steps and methods known to those skilled in the art, and so forth.

[0026] As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value that is of a similar order of magnitude and / or within a similar range to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that is included within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (above or below) of the stated reference value, unless otherwise stated or otherwise evident from the context (except where such number may exceed 100% of the possible values).

[0027] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, as well as any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also encompassed within the disclosure, subject to any specific excluded limits in the stated range. Where a stated range includes one or both of its upper and lower limits, ranges excluding either or both of those included upper and lower limits are also encompassed within the disclosure.

[0028] As used herein, the terms "polypeptide," "peptide," and "protein" refer to polymers of amino acids of any length. The terms also encompass amino acid polymers that have been modified (e.g., to include disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling moiety).

[0029] As used herein, the term "nucleic acid sequence" or "nucleotide sequence" refers to a molecule that comprises either a sequence of DNA or RNA nucleotides presented 5' to 3'.

[0030] As used herein, "antibody" includes reference to full-length immunoglobulin molecules immunologically reactive with a particular antigen, including both polyclonal and monoclonal antibodies. The term also includes humanized antibodies, chimeric antibodies, e.g., murine variable regions with human constant regions, and conjugated antibodies.

[0031] The term "Fc domain" (also referred to herein interchangeably as "Fc sequence", "Fc region", or simply "Fc"), as used herein, refers to a fragment crystallizable region monomer comprising a constant heavy chain 2 domain (CH2) and a constant heavy chain 3 domain (CH3). In some embodiments, the "Fc domain" sequence comprises an IgG hinge region sequence. In some embodiments, the Fc domain dimerizes or forms other multimers. Exemplary human Fc domains include IgG1, IgG2, IgG3, and IgG4 Fc domains.

[0032] Unless otherwise noted, modifications in the Fc domain are presented according to the EU numbering scheme, however, multiple numbering schemes exist that can be readily cross-referenced by one of skill in the art (www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html#refs).

[0033] The terms "treatment", "treating", and the like, as used herein, generally refer to obtaining a pharmacological and / or physiological effect by a therapeutic agent. The effect may be prophylactic in terms of completely or partially preventing the disease or condition, e.g., reducing the likelihood of the disease or its symptoms occurring in a subject, and / or may be therapeutic in terms of completely or partially reducing the symptoms, or partially or completely curing the disease and / or side effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, including (a) preventing onset in a subject susceptible to, but not yet diagnosed with, the disease, (b) arresting or delaying the onset or manifestation of the disease, and (c) relieving the disease, e.g., causing regression of the disease or symptoms associated with the disease. The therapeutic agent may be administered before, during, or after the onset of the disease. Treatment of ongoing diseases, where the treatment stabilizes or reduces undesirable clinical symptoms in the patient, may be of particular interest. In some embodiments, treatment is administered before complete loss of function in the affected tissue. In some embodiments, the subject treatment is administered during the symptomatic stage of the disease, and in some embodiments after the symptomatic stage of the disease.

[0034] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to any subject for whom treatment is desired. The subject may be a mammalian subject. Mammalian subjects include, for example, humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), invertebrates (e.g., cows, sheep, pigs, horses, goats, etc.), and the like. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate, e.g., a cynomolgus monkey. In some embodiments, the subject is a companion animal (e.g., cat, dog).

[0035] II. Signal Regulatory Protein Alpha (SIRPα) Fusion Polypeptides Blocking the binding of endogenous signal-regulatory protein alpha (SIRPα) to CD47 protein on cells, e.g., on human cells, allows for phagocytic engulfment of CD47-expressing cells. Provided herein are fusion polypeptides comprising a SIRPα domain and a modified Fc domain, useful for blocking endogenous SIRPα binding to CD47 protein, where the inclusion of the modified Fc domain improves the pharmacokinetics and / or pharmacodynamics of the fusion polypeptide. In some embodiments, the inclusion of the modified Fc domain increases the binding affinity of the fusion polypeptide to fetal Fc receptors, among other effects, thereby extending the half-life of the fusion polypeptide in a subject, and the fusion polypeptide provides improved blocking of endogenous SIRPα binding to CD47 by the fusion polypeptide. In some embodiments, the inclusion of the modified Fc domain increases the binding affinity of the fusion polypeptide to CD47 protein, among other effects. In some embodiments, such increased binding affinity allows for a reduction in the dose and / or frequency of administration of the fusion polypeptide in a subject, which may lead to improved blocking of endogenous SIRPα binding to CD47 by the fusion polypeptide. In some embodiments, the inclusion of a modified Fc domain can enhance the safety of a SIRPα fusion polypeptide, among other effects.

[0036] In some embodiments, SIRPα fusion polypeptides of the disclosure form monomers, dimers, trimers, tetramers, pentamers, or other multimers. In exemplary embodiments, SIRPα fusion polypeptides comprising modified Fc domains of the disclosure form dimers, in some embodiments the dimers are homodimers, and in other embodiments the dimers are heterodimers.

[0037] SIRPα Polypeptide Sequence The SIRPα fusion polypeptides provided herein comprise a SIRPα domain and a modified Fc domain. In this section, aspects of the SIRPα domain of the fusion polypeptide are described in more detail.

[0038] The SIRPα domains provided herein comprise the membrane distal (D1) domain of SIRPα that binds to the CD47 protein (either wild-type or modified forms thereof). In some embodiments, the SIRPα fusion polypeptides of the present disclosure comprise a wild-type human SIRPα D1 sequence comprising SEQ ID NO:1, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. SEQ ID NO:1 EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS

[0039] In some embodiments, a SIRPα fusion polypeptide of the present disclosure comprises a SIRPα D1 domain having one or more amino acid modifications compared to the wild-type sequence of the D1 domain, e.g., the D1 domain of SEQ ID NO: 1. Modifications include amino acid substitutions, amino acid deletions, and amino acid additions. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least one amino acid modification compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least two amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least three amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least four amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least five amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least six amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least seven amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least eight amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least nine amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least ten amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least eleven amino acid modifications compared to the wild-type sequence of the D1 domain.In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least 12 amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least 13 amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least 14 amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least 15 amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least 16 amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least 17 amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least 18 amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least 19 amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least 20 amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least 21 amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least 22 amino acid modifications compared to the wild-type sequence of the D1 domain. In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least 23 amino acid modifications compared to the wild-type sequence of the D1 domain.In some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least 24 amino acid modifications compared to the wild-type sequence of the D1 domain, hi some embodiments, a SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least 25 amino acid modifications compared to the wild-type sequence of the D1 domain.

[0040] In some embodiments, the SIRPα fusion polypeptide has at least 5-fold, 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold or more higher binding affinity for CD47 (i.e., a lower K D The present invention includes a SIRPα D1 polypeptide exhibiting a specific IL-1 binding domain (IL-2 binding domain) and a specific IL-2 binding domain (IL-3 binding domain).

[0041] In some embodiments, SIRPα fusion polypeptides of the disclosure include modifications relative to the wild-type SIRPα D1 domain sequence of SEQ ID NO:1 at one or more of the following residues: V6, S14, S20, I22, H24, V27, I31, A45, E47, K53, E54, H56, S66, E70, S77, V92, and / or a overlap of the D100 residue.

[0042] In some embodiments, SIRPα fusion polypeptides of the disclosure include modifications relative to the wild-type SIRPα D1 domain sequence of SEQ ID NO:1 at one or more of the following residues: V6I, S14L, S20T, I22T, H24R, V27I, I31F, A45G, E47V, K53R, E54Q, H56P, S66T, E70N, S77R, V92I, and / or a duplication of the D100 residue.

[0043] In some embodiments, the SIRPα fusion polypeptides of the present disclosure comprise the SIRPα D1 sequence of SEQ ID NO: 2 (referred to herein as CV1, which is an exemplary SIRPα D1 domain that exhibits higher binding affinity for CD47), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. SEQ ID NO:2 EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPS

[0044] In some embodiments, a SIRPα fusion polypeptide of the present disclosure comprises the SIRPα D1 sequence of SEQ ID NO: 3, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. SEQ ID NO:3 XXELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVR

[0045] In some embodiments, a SIRPα fusion polypeptide of the present disclosure comprises the SIRPα D1 sequence of SEQ ID NO: 4, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. SEQ ID NO:4 XXELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVR

[0046] In some embodiments, a SIRPα fusion polypeptide of the present disclosure comprises the SIRPα D1 sequence of SEQ ID NO:5, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. SEQ ID NO:5 EEXLQVIQPDKXVXVAAGEXAXLXCTXTSLIPVGPIQWFRGAGPXRELIYNQKEGHFPRVTTVSXXDLTKRXNMDFXIXIXNITPADAGTYYCVKFRKGSPDDXEFKSGAGTELSVR

[0047] In some embodiments, SIRPα fusion polypeptides of the disclosure comprise any of the following sequences relative to the SIRPα D1 sequence of SEQ ID NOs: 1-5: E3G, L4V, L4I, V6I, V6L, S12F, S14L, S20T, A21V, I22T, H24L, H24R, V27A, V27I, V27L, I31F, I31S, I31T, Q37H, A45G, E47V, E47L, K53R, E54Q, E54P, H56P, H56R, V63I, E65D, S66T, S66G, S66L, K68R, E and one or more of the following substitutions: 70N, M72R, S75P, R77S, S79G, N80A, N80X, I81N, T82N, P83N, P83X, V92I, F94L, F94V, a duplication of D100, E102V, E102T, E102F, F103E, F103V, K104F, K104V, A115G, K116A, and K116G (where X=any amino acid).

[0048] In some embodiments, a SIRPα fusion polypeptide sequence of the present disclosure may include any of the SIRPα D1 sequences described in WO2013109752, WO2014094122A1, WO2017027422, WO2016023040, and WO2016024021A1, which are incorporated herein in their entireties.

[0049] III. Modified Fc Domains for Improved Pharmacokinetics of SIRPα Fusion Polypeptides The present disclosure provides signal regulatory protein alpha (SIRPα) fusion polypeptides comprising modified Fc domains useful for improving pharmacokinetics and / or pharmacodynamics, for example, by increasing binding affinity to FcRn and / or CD47 protein. As provided herein, one or more modifications (e.g., substitutions) can be introduced into a wild-type IgG Fc domain sequence, e.g., a human IgG1, IgG2, IgG3, or IgG4 domain, to increase binding to fetal Fc receptor (FcRn) and extend the half-life of the fusion polypeptide. One or more modifications introduced into the wild-type IgG Fc domain sequence can also improve efficacy and / or safety of the fusion polypeptide.

[0050] Modified Fc domain sequences The SIRPα fusion polypeptides provided herein comprise modified Fc domains that are useful for improving the pharmacokinetics and / or pharmacodynamics of the fusion polypeptide. The Fc domains provided herein can be modified domains of any species, e.g., human or mouse, or can be engineered non-naturally occurring Fc domains, e.g., human or mouse IgG domains that contain one or more modifications. In some embodiments, the Fc domain is a modified human IgG1 or IgG4 Fc domain. These standard wild-type sequences are provided herein.

[0051] In some embodiments, the Fc domain is a modified human IgG2 or IgG3 domain, or a murine IgG1, IgG2a, IgG2b, or IgG3 domain.

[0052] In some embodiments, a SIRPα fusion polypeptide of the disclosure comprises the human IgG1 Fc amino sequence of SEQ ID NO: 6, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. SEQ ID NO:6 ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0053] In some embodiments, a SIRPα fusion polypeptide of the disclosure comprises a human IgG4 Fc amino sequence of SEQ ID NO: 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. SEQ ID NO:7 PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0054] In some embodiments, the SIRPα fusion polypeptides of the present disclosure comprise an Fc domain of an IgG4 human Fc domain, where the polypeptide is susceptible to the dynamic process of Fab-arm exchange. Thus, in some embodiments, the IgG4 Fc domain may comprise a S228P substitution relative to SEQ ID NO: 7 according to the EU numbering scheme, resulting in a reduction of this process. In some embodiments, the SIRPα fusion polypeptides of the present disclosure comprise an IgG4 Fc amino sequence of SEQ ID NO: 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. SEQ ID NO:8 PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0055] In some embodiments, the IgG4Fc amino acid sequence comprises the substitution L445P relative to SEQ ID NO:8, according to the EU numbering scheme.

[0056] In some embodiments, a SIRPα fusion polypeptide of the disclosure comprises a human IgG1Fc sequence of SEQ ID NO: 6 that includes one or more modifications (e.g., substitutions) to enhance effector function. In some embodiments, the substitutions include one or more substitutions selected from the group consisting of V215A, G236A, S239D, I332E, T250Q, M252Y, S254T, T256E, S267E, N325S, L328F, N343S, M428L, H433K, and N434F relative to SEQ ID NO: 6 according to the EU numbering scheme. Exemplary combinations include, according to the EU numbering scheme, G236A-S239D, G236A-I332E, S239D-I332E, V215A-G236A-S239D-I332E, G236A-S239D-I332E, K326W-E333S, S267E-H268F-S324T, and E345R-E430G-S440Y, F243L-R292P-Y300L-V305I-P396L, S239D-I332E, S298A-E333A-K334A, L234Y-L235Q-G236W-S239M-H268D-D270E-S298A, and D270E-K326D-A330M-K334E.

[0057] In some embodiments, a SIRPα fusion polypeptide of the disclosure comprises a human IgG1 Fc sequence of SEQ ID NO: 6 that includes one or more modifications (e.g., substitutions) to reduce effector function. In some embodiments, the substitutions are selected from the group consisting of N297A, N297Q, N297G, L235E, L234A, L235A, K214R, P329G, D356E, and L358M.

[0058] In some embodiments, a SIRPα fusion polypeptide of the disclosure comprises a human IgG4 Fc sequence of SEQ ID NO: 7 or 8 that includes one or more modifications (e.g., substitutions) to reduce effector function. In some embodiments, the substitutions are selected from the group consisting of L235A, L235E, S228P, and F234A. Exemplary combinations include L235E-S228P, S228P-F234A, and S228P-F234A-L235A.

[0059] In other embodiments, the SIRPα fusion polypeptides of the disclosure comprise an IgG1 Fc or IgG4 Fc domain in which a modification to increase serum half-life is present, in some embodiments the mutation is selected from the group consisting of T250Q, M252Y, S254T, T256E, S267E, N325S, L328F, N343S, M428L, N434F, and H443K, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In some embodiments, the mutations are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8, according to the EU numbering scheme.

[0060] In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 that comprises the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E.

[0061] In some embodiments, a SIRPα fusion polypeptide of the disclosure comprises a human IgG4 Fc amino sequence of SEQ ID NO: 9, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. SEQ ID NO:9 PPCPPCPAPEFLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0062] In some embodiments, the SIRPα fusion polypeptides of the disclosure include a peptide linker connecting the SIRPα domain and the Fc domain. In some embodiments, the SIRPα fusion polypeptides include a peptide linker that is about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, or about 16 amino acids in length. In some embodiments, the peptide linker includes alanine (A), glycine (G), and / or serine (S) amino acids. In some embodiments, the peptide linker is eight amino acids of G and S amino acids. In some embodiments, the linker is AAA. In some embodiments, the linker is GGGSGGGS (SEQ ID NO: 11). In some embodiments, the linker includes a human IgG sequence, e.g., ASTKGPSVFPLAP (SEQ ID NO: 12).

[0063] In exemplary embodiments, the SIRPα fusion polypeptides provided herein comprise a SIRPα domain sequence of any of SEQ ID NOs: 1-5, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, and a modified Fc domain sequence of any of SEQ ID NOs: 6-9, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0064] In exemplary embodiments, the SIRPα fusion polypeptides provided herein comprise an Fc domain sequence of SEQ ID NO: 9, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, and a SIRPα domain sequence of SEQ ID NO: 2, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0065] Exemplary SIRPα fusion polypeptides comprising a SIRPα domain and a modified Fc domain include those comprising the sequence of SEQ ID NO: 10, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85% thereto. At least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. SEQ ID NO:10 EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSAAAPPCPPCPAPEFLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0066] Effect of modified Fc domains In some embodiments, one or more modifications can be introduced into the Fc domain of a SIRPα fusion polypeptide of the present disclosure to increase the binding affinity of the fusion polypeptide to FcRn and / or to increase the binding affinity to a CD47 protein. In some embodiments, containing a modified Fc domain increases the binding affinity to FcRn by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 1000-fold, or about 10,000-fold compared to that of the wild-type Fc domain. In some embodiments, the inclusion of the modified Fc domain increases the binding affinity for CD47 by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold compared to that of the wild-type Fc domain. In some embodiments, the CD47 protein is a human, mouse, non-human primate, or rat CD47 protein. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0067] In some embodiments, the inclusion of a modified Fc domain increases the half-life of a SIRPα fusion polypeptide in vivo or in vitro.

[0068] In some embodiments, a SIRPα fusion polypeptide comprising a modified Fc domain exhibits about a 1-fold, about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold increase in half-life in vivo compared to the half-life of a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0069] In some embodiments, the inclusion of a modified Fc domain increases the in vitro (e.g., cell culture) half-life of a SIRPα fusion polypeptide by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold compared to the half-life of a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0070] In some embodiments, the inclusion of a modified Fc domain delays blood clearance of a SIRPα fusion polypeptide in a subject, hi some embodiments, the inclusion of a modified Fc domain delays blood clearance of a SIRPα fusion polypeptide by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, or about 1000-fold, as compared to a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0071] In some embodiments, the inclusion of a modified Fc domain increases the binding affinity of the SIRPα fusion polypeptide to CD47 protein in vivo or in vitro. In some embodiments, a SIRPα fusion polypeptide comprising a modified Fc domain is effective at a lower dose in a subject compared to a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S,, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8, comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0072] In some embodiments, the inclusion of a modified Fc domain enhances the EC 50is reduced by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold, as compared to a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, relative to the IgG1 Fc domain sequence SEQ ID NO: 6, or the IgG4 Fc domain sequence SEQ ID NO: 7 or 8, according to the EU numbering scheme. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8 comprising the following substitutions relative to SEQ ID NO: 8: M252Y, S254T, and T256E. An exemplary SIRPα fusion polypeptide of the disclosure comprises an amino acid sequence of SEQ ID NO: 10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0073] In some embodiments, the inclusion of a modified Fc domain reduces the dosage of a SIRPα fusion polypeptide required to achieve a desired therapeutic effect by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold, as compared to a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0074] In some embodiments, SIRPα fusion polypeptides comprising modified Fc domains are effective in subjects at lower dosing frequency (necessary to achieve a desired therapeutic effect) compared to SIRPα fusion polypeptides comprising wild-type Fc domains. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S,, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8, comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0075] In some embodiments, the inclusion of a modified Fc domain reduces the frequency of administration of a SIRPα fusion polypeptide required to achieve a desired therapeutic effect by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold, as compared to a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Another exemplary SIRPα fusion polypeptide of the present disclosure comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0076] In some embodiments, the inclusion of a modified Fc domain enhances the effector function of the fusion polypeptide in a SIRPα fusion polypeptide. In some embodiments, the inclusion of a modified Fc domain reduces complement-dependent cytotoxicity. In some embodiments, the inclusion of a modified Fc domain increases the complement-dependent cytotoxicity of a SIRPα fusion polypeptide by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold compared to that of a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0077] In some embodiments, the inclusion of a modified Fc domain increases the interaction of a SIRPα fusion polypeptide with cells expressing a CD47 polypeptide. In some embodiments, the inclusion of a modified Fc domain increases binding of a SIRPα fusion polypeptide to CD47-expressing cells by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold compared to that of a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0078] In some embodiments, the inclusion of a modified Fc domain increases SIRPα fusion polypeptide induction of endocytosis of CD47 protein in cells expressing a CD47 polypeptide. In some embodiments, the inclusion of a modified Fc domain increases SIRPα fusion polypeptide induction of endocytosis of CD47 protein in CD47 expressing cells by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0079] In some embodiments, the inclusion of a modified Fc domain increases SIRPα fusion polypeptide induction of phagocytosis by macrophages, hi some embodiments, the inclusion of a modified Fc domain increases SIRPα fusion polypeptide induction of phagocytosis by macrophages by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0080] In some embodiments, SIRPα fusion polypeptides comprising a modified Fc domain are associated with increased degradation of CD47 protein compared to SIRPα fusion polypeptides comprising a wild-type Fc domain, in some embodiments, the inclusion of a modified Fc domain increases SIRPα fusion polypeptide-associated degradation of CD47 protein by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold compared to that of a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0081] In some embodiments, the inclusion of a modified Fc domain reduces SIRPα fusion polypeptide toxicity, hi some embodiments, the inclusion of a modified Fc domain reduces SIRPα fusion polypeptide toxicity by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold compared to that of a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0082] In some embodiments, the inclusion of a modified Fc domain reduces SIRPα fusion polypeptide antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the inclusion of a modified Fc domain reduces SIRPα fusion polypeptide antibody-dependent cellular cytotoxicity (ADCC) by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold compared to that of a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0083] In some embodiments, the inclusion of a modified Fc domain improves the stability of hemoglobin levels associated with a SIRPα fusion polypeptide, hi some embodiments, the inclusion of a modified Fc domain stabilizes the stability of hemoglobin levels associated with a SIRPα fusion polypeptide by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold, relative to that of a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0084] In some embodiments, the inclusion of a modified Fc domain reduces SIRPα fusion polypeptide-associated anemia. In some embodiments, the inclusion of a modified Fc domain reduces SIRPα fusion polypeptide-associated anemia by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold compared to that of a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0085] In some embodiments, the inclusion of a modified Fc domain reduces aggregation of a SIRPα fusion polypeptide, hi some embodiments, the inclusion of a modified Fc domain reduces aggregation of a SIRPα fusion polypeptide by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold compared to that of a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0086] In some embodiments, the inclusion of a modified Fc domain improves purification of a SIRPα fusion polypeptide, hi some embodiments, the inclusion of a modified Fc domain improves purification of a SIRPα fusion polypeptide by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold, compared to that of a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0087] In some embodiments, the inclusion of a modified Fc domain reduces the formation of anti-drug antibodies (ADA) by a SIRPα fusion polypeptide. In some embodiments, the inclusion of a modified Fc domain reduces the formation of anti-drug antibodies (ADA) by a SIRPα fusion polypeptide by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold compared to that of a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0088] In some embodiments, the inclusion of a modified Fc domain reduces red blood cell (RBC) aggregation associated with a SIRPα fusion polypeptide. In some embodiments, the inclusion of a modified Fc domain reduces red blood cell (RBC) aggregation associated with a SIRPα fusion polypeptide by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold compared to that of a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0089] In some embodiments, the inclusion of a modified Fc domain reduces the formation of antibodies against red blood cells associated with the SIRPα fusion polypeptide as measured by a Coombs assay. In some embodiments, the inclusion of a modified Fc domain reduces the formation of antibodies against red blood cells associated with the SIRPα fusion polypeptide by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, about 1000-fold, about 5000-fold, or about 10,000-fold relative to that of a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the one or more modifications in the Fc domain are selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F, according to the EU numbering scheme, to the IgG1 Fc domain sequence SEQ ID NO:6, or the IgG4 Fc domain sequence SEQ ID NO:7 or 8. In embodiments, an exemplary SIRPα fusion polypeptide of the disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8 comprising the following substitutions relative to SEQ ID NO:8: M252Y, S254T, and T256E. Exemplary SIRPα fusion polypeptides of the present disclosure include an amino acid sequence having the sequence of SEQ ID NO:10, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0090] III. Methods for Producing SIRPα Fusion Polypeptides Containing Modified Fc Domains Also provided herein is a polynucleotide encoding a SIRPα fusion polypeptide comprising a modified Fc domain for improved pharmacokinetics of the present disclosure. In some embodiments, the polynucleotide encodes any of the aforementioned SIRPα fusion polypeptides, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0091] In some embodiments, a polynucleotide encoding an exemplary SIRPα fusion polypeptide of the present disclosure is introduced (e.g., transfected or transformed) and expressed in a human cell line or a bacterial cell line. Exemplary cell lines available for production include, but are not limited to, Expi293 and CHO cell lines.

[0092] IV. METHODS OF USING SIRPα FUSION POLYPEPTIDES CONTAINING MODIFIED Fc DOMAINS In some embodiments, a SIRPα fusion polypeptide comprising a modified Fc domain is administered as a therapeutic to a subject in need thereof. In some embodiments, a SIRPα fusion polypeptide of the present disclosure is administered to treat, for example, a cardiovascular disease, cancer, fibrosis, infectious disease, hematological disease, or neurological disease. In some embodiments, the inclusion of a modified Fc domain improves the efficacy and / or safety of the SIRPα fusion polypeptide as a therapeutic.

[0093] In some embodiments, subjects selected for treatment with a SIRPα fusion polypeptide have cardiovascular disease and are determined to have or be at risk of having one or more of atherosclerosis, heart failure, myocardial infarction, cardiomyopathy, acute coronary syndrome, myocarditis, cardiac remodeling, hypertension, angina, restenosis, stroke, aneurysm, thrombosis, phlebitis, peripheral vascular disease, pulmonary arterial hypertension, and autoimmune vasculitis.

[0094] In some embodiments, the subject selected for treatment with a SIRPα fusion polypeptide has cancer, hi some embodiments, the SIRPα fusion polypeptides of the present disclosure may treat tumor growth and / or tumor metastasis, e.g., lymphoma, leukemia, carcinoma, melanoma, glioblastoma, sarcoma, or myeloma.

[0095] In some embodiments, the subject selected for treatment with a SIRPα fusion polypeptide of the present disclosure has fibrosis or a fibrotic disease, such as a liver or lung fibrotic disease. In some embodiments, the subject has or is at risk of having end-stage liver disease, kidney disease, idiopathic pulmonary fibrosis (IPF), retinal fibrosis, chronic transplant rejection from progressive myopathy, or heart failure from cardiac fibrosis.

[0096] In some embodiments, the subject selected for treatment with a SIRPα fusion polypeptide has an infection, an infection associated with a viral, bacterial, or fungal pathogen. In some embodiments, the subject has a viral infection, such as an infection associated with one of retroviruses, lentiviruses, hepadnaviruses, herpesviruses, poxviruses, or human papillomaviruses. In some embodiments, the subject has an intracellular bacterial infection, such as an infection associated with one of Mycobacterium, Chlamydophila, Ehrlichia, Rickettsia, Brucella, Legionella, Francisella, Listeria, Coxiella, Neisseria, Salmonella, or Yersinia species. In some embodiments, the subject has an intracellular protozoan pathogen infection, such as an infection associated with one of Plasmodium species, Trypanosoma species, Giardia species, Toxoplasma species, or Leishmania species.

[0097] In some embodiments, a subject has a blood disease or disorder, e.g., an inherited blood disorder or severe combined immunodeficiency, and is selected for treatment with a SIRPα fusion polypeptide of the present disclosure. In some embodiments, a SIRPα fusion polypeptide of the present disclosure may be used alone or in combination with other agents to promote engraftment of endogenous stem cells prior to hematopoietic stem cell transplantation.

[0098] In some embodiments, the subject selected for treatment with a SIRPα fusion polypeptide has a neurological disorder.

[0099] In some embodiments, SIRPα fusion polypeptides can be delivered to a subject in need thereof subcutaneously, intravenously, intravitreally, orally, intranasally, transdermally, intraperitoneally, intramuscularly, intrathecally, intrapulmonary, vaginally, or rectally, hi some embodiments, SIRPα fusion polypeptides are administered subcutaneously.

[0100] In some embodiments, the SIRPα fusion polypeptide is conjugated to a fluorophore, radionucleotide, or other imaging or diagnostic moiety. In some embodiments, the SIRPα fusion polypeptide is administered to a cell or organism to image the location or concentration of CD47 protein. In some embodiments, the SIRPα fusion polypeptide is administered to a cell or organism to diagnose disease.

[0101] In some embodiments, the SIRPα fusion polypeptide comprises a conjugated toxin for delivery of the toxin to cells expressing CD47.

[0102] In some embodiments, SIRPα fusion polypeptides are administered in combination with a CD20 antibody and / or a CD47 antibody, or fragments thereof. In some embodiments, multivalent SIRPα fusion polypeptides are administered in combination with an antibody or antibody fragment to a protein selected from the group consisting of TNFalpha, TNF-alphaR, IL6, IL6R, IL1beta, IL1-betaR, IL17A, CD117, EGFR, HER2, CD20, PD1 / PDL1, CD137, CTLA4, LAG3, CD3, CD2, CD4, CD19, CD38, GD2, VEGF, VEGF-R, P-selectin, CCR4, CD52, IL2, and IL2 R.

[0103] VI. Pharmaceutical Compositions In some embodiments, the SIRPα fusion polypeptide comprising a modified Fc domain is present in a pharmaceutical composition. In certain embodiments, the pharmaceutical composition may be in a water-soluble form, such as a pharma- ceutically acceptable salt, which is meant to include both acid and base addition salts. Pharmaceutically acceptable acid addition salts include, but are not limited to, organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid, and salicylic acid. Pharmaceutically acceptable acid addition salts include those derived from inorganic bases, such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Salts derived from pharma- ceutically acceptable non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.

[0104] The pharmaceutical compositions described herein may also include one or more of the following: a carrier protein, such as serum albumin; buffers; excipients, such as microcrystalline cellulose, lactose, corn and other starches; binders; and polyethylene glycol.

[0105] The composition for administration generally comprises the polypeptide dissolved in a pharma- ceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline. The composition can include pharma- ceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH, and buffers, toxicity counteractants, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentration of the active agent in the formulation can vary and is selected based on fluid volume, viscosity, and body weight, according to the particular mode of administration selected and the needs of the patient (e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0106] VII. Kit The SIRPα fusion polypeptides comprising modified Fc domains described herein may also be included in therapeutic or diagnostic kits for administration by a medical professional or a subject in need thereof. The kits may include, for example, a container, a dose of the SIRPα fusion polypeptide, a syringe and / or a vial, and instructions for their use. In some embodiments, the kits include the SIRPα fusion polypeptide and instructions for administering the polypeptide to treat a disease.

[0107] In some embodiments, the SIRPα fusion polypeptide of the kit is conjugated to a fluorophore, radionucleotide, or other diagnostic moiety. EXAMPLES

[0108] Example 1: Effect of YTE mutations in SIRPα fusion polypeptides on CD47 binding affinity. To determine the effect of the YTE Fc domain mutation on CD47 binding affinity, CD47 binding experiments were performed on a SIRPα fusion polypeptide of SEQ ID NO: 10, which contains a SIRPα domain of SEQ ID NO: 2 and an IgG4 Fc domain with a YTE mutation (construct 67). The CD47 binding affinity of the SIRPα fusion polypeptide SEQ ID NO: 10 construct without the YTE mutation, i.e., with a wild-type IgG4 Fc domain (construct 50), was also measured, as was the binding affinity of MIAP410, a commercially available CD47 monoclonal antibody. MIAP410 is a mouse anti-human CD47 antibody that reacts with human, mouse, and rat CD47.

[0109] A Sartorius Biosensor Octet® R8 was used to measure the affinity octets of the above constructs and antibodies to AMC (anti-mouse Fc capture) Cat. No. 18-5088 or AHC (anti-human Fc capture) Cat. No. 18-5060.

[0110] Binding affinity assays were performed at 30°C using the following method: running buffer was PBS, 1% BSA, and 0.05% Tween-20. SIRPα fusion polypeptides and CD47 antibodies were immobilized on AMC or AHC for 3 min, followed by rhCD47-his association and dissociation for 15 min. Data were analyzed with Sartorius software Octet Analysis Studio using reference subtracted values. Dissociation constants (KD) were determined by applying a global (full kinetics) 1:1 fitting model (N=1 per sample for SIRPα fusion polypeptides and N=5 for anti-CD47 antibodies). Data from the best kinetic curve fitting experiment are presented in Figure 1, and examples of individual runs are shown in Figure 2A (construct 50) and Figure 2B (construct 67).

[0111] These data demonstrate that both Construct 67, a SIRPα fusion polypeptide of SEQ ID NO: 10, and Construct 50, the same construct as Construct 67 but without the YTE mutation, have significantly higher binding affinity to all CD47 proteins tested (cynomolgus monkey, human, mouse, and rat) compared to the binding affinity of the MIAP410 anti-CD47 antibody. Notably, Construct 67, which contains the YTE mutation in the IgG4 Fc domain, showed binding affinity comparable to or better than that of Construct 50 SIRPα fusion polypeptide without the YTE mutation.

[0112] Example 2: Effect of YTE mutations in SIRPα fusion polypeptides on human erythrocyte binding in vitro. To determine the effect of the YTE mutations on erythrocyte binding of the SIRPα fusion polypeptide of SEQ ID NO: 10 (construct 67), the following experiment was performed with construct 67 and construct 50 as a comparison.

[0113] Normal human whole blood in EDTA from two different donors was purchased from Stanford Blood Center and received the next day at room temperature. Cells were incubated with 0.18-400 nM of construct 50 or construct 67 for 20 min at 4°C. Cells were washed three times with PBS and subsequently incubated with AF488-conjugated anti-human CD45 and 30 μg / mL of AF647-labeled anti-human IgG4 antibody for 20 min at 4°C. After washing, binding was measured using CD45 gating (N=2 donors) using an Agilent Quanteon to select for effects in red blood cells.

[0114] EC measured for the two constructs 50 Values ​​are the amount of construct bound to erythrocytes in vitro. As shown in Figure 3, the half-maximal binding of construct 67 containing the YTE mutation was comparable to or lower than that of construct 50.

[0115] Example 3: Effect of YTE mutations in SIRPα fusion polypeptides on phagocytosis in vitro. To determine the effect of YTE mutations in SIRPα fusion polypeptides on the induction of phagocytosis in vitro, the following experiments were performed using construct 67 containing the YTE Fc domain mutation, construct 50 containing the wild-type Fc domain, and a biosimilar anti-CD47 magrolimab-like antibody.

[0116] Healthy human buffy coats from LRS chambers were purchased from Stanford Blood Center 8 days before performing the assay and received the next day. PBMC cells were isolated from the buffy coats by applying a Ficoll-Paque gradient and monocytes were isolated using an easySep human monocyte isolation kit (catalog number 19359) purchased from Stemcell. The isolated monocytes were seeded and differentiated for 7 days in IMDM, 10% human serum (from Innovative Research), and 1% penicillin and streptomycin. On the day of the experiment, Raji cells were first labeled with calcein using the protocol below. Calcein-AM dye was reconstituted in 20 μl of DMSO and incubated with Raji cells (5×10 6 1 μl calcein-DMSO solution per cell was added and incubated at 37° C. for 10 min, followed by incubation on ice for 10 min. The Raji cells were then centrifuged at 1250 rpm for 5 min at 4° C. The cells were washed once with HBSS and resuspended in IMDM. 4×10 6 25 μl / well of calcein-stained Raji (target) cells suspended in IMDM at a density of 1000 cells / ml were pipetted into a Costar ultra-low attachment 96-well plate. 25 μl / well of biosimilar magrolimab-like antibodies, construct 50 and construct 67 at concentrations of 0.67 nM and 6.67 nM, and IgG4 isotype control at 6.67 nM in IMDM were added to the Raji cells in triplicate. Raji cells were pre-incubated with the test article for 30 min at 37°C. 1×10 650μl / well of in vitro differentiated macrophages from human donors in IMDM at 100 cells / ml were added to the target cells in triplicate. Cells were resuspended and incubated at 37°C for 2 hours. At the end of the incubation, plates were centrifuged and the supernatant was discarded. Cells were then resuspended in 50μl PBS+2%FBS+5ug / ml Alexa Fluor® 647 anti-human CD206 and incubated on ice in the dark for 30 minutes. Cells were washed once with 200μl FACS buffer and resuspended in 100μl FACS buffer+DAPI. Phagocytosis index was detected using a Flow cytometer and analyzed with Flowjo software. Percent macrophages is the percent macrophages with phagocytic calcein-AM labeled Raji.

[0117] As shown in Figures 4A and 4B, construct 50 and construct 67 induced increased levels of phagocytosis compared to the biosimilar magrolimab-like antibody. Furthermore, construct 67, which contains a YTE Fc domain, induced equal or better levels of phagocytosis than construct 50, which lacks the YTE mutation in the IgG4 Fc domain.

[0118] Example 4: Effect of YTE mutations in SIRPα fusion polypeptides on in vitro antibody-dependent cellular cytotoxicity (ADCC). To determine the effect of the YTE mutations in the SIRPα fusion polypeptide on the induction of antibody-dependent cellular cytotoxicity (ADCC), the following experiments were performed using construct 67 containing the YTE Fc domain mutation and construct 50 containing the wild-type Fc domain, as well as an anti-CD20 antibody as a positive control.

[0119] The ADCC assay was performed according to the instructions of the ADCC Reporter Bioassay Complete Kit (catalog number G7015) purchased from Promega. Briefly, 25 μl / well of Raji cells in RPMI 1640 in low IgG serum medium, prepared according to the kit's instructions, were pipetted into a white flat-bottom 96-well Corning 3610 microplate. Serial dilutions of anti-CD20 (14 pM to 10 nM), construct 50 and construct 67 (457 pM to 1 μM), and IgG4 at 1 μM were prepared in RPMI 1640 in low IgG serum. 25 μl / well of antibody / molecule was pipetted into wells containing target cells, and no antibody / molecule was added to one set of wells. IgG4 and anti-CD20 were used as negative and positive controls, respectively. 25 μl / well of effector cells prepared in RPMI 1640 in low IgG serum were pipetted into wells containing target Raji cells and drugs / molecules. The microplates were incubated at 37° C. for 6 hours, followed by luminescence detection using reagents provided in the kit.

[0120] As shown in Figure 5A, construct 67 and construct 50 exhibited minimal ADCC compared to the anti-CD20 antibody. The inset of Figure 5A is shown in Figure 5B and has been rescaled to show the difference between construct 67, which contains the YTE mutation in the Fc domain, and construct 50, which contains the wild-type Fc domain. These data demonstrate that the YTE containing construct 67 exhibited lower antibody-dependent cellular cytotoxicity than did construct 50.

[0121] Example 5: Effect of YTE mutations on in vivo receptor occupancy, hemoglobin levels, and pharmacokinetics of SIRPα fusion polypeptides in non-human primates. To determine the effect of YTE mutations in SIRPα fusion polypeptides on in vivo receptor occupancy, hemoglobin levels, and pharmacokinetics (PK), the following experiments were performed using constructs of the present disclosure (e.g., construct 67 with YTE Fc domain substitutions) and wild-type constructs (e.g., construct 50 with wild-type Fc domain). Biologically naive male cynomolgus non-human primates were dosed by subcutaneous and / or intravenous administration. Receptor occupancy, hemoglobin, and / or PK data were collected and analyzed before, during, and after dosing, as well as over the observation period. Analysis may be gated on CD45 negative cells.

[0122] It is expected that receptor occupancy of erythrocytes treated with certain constructs of the present disclosure containing YTE Fc domain substitutions will be greater than or equivalent to receptor occupancy of erythrocytes treated with wild-type constructs throughout the observation period.

[0123] It is expected that serum concentrations in subjects using certain constructs of the present disclosure containing YTE Fc domain substitutions will be higher than or equivalent to serum concentrations in subjects treated with wild-type constructs throughout the observation period.

[0124] Hemoglobin levels in subjects using certain constructs of the present disclosure containing YTE Fc domain substitutions are expected to be higher than or equivalent to the hemoglobin levels of subjects treated with wild-type constructs throughout the observation period.

Claims

1. A fusion polypeptide comprising a signal regulatory protein α (SIRPα) domain and a modified Fc domain, wherein the modified Fc domain comprises one or more amino acid modifications relative to the wild-type Fc domain, and the inclusion of the modified Fc domain increases the binding affinity to FcRn.

2. The fusion polypeptide according to claim 1, wherein the inclusion of the modified Fc domain increases the half-life of the fusion polypeptide.

3. The fusion polypeptide according to claim 1, wherein the inclusion of the modified Fc domain increases the blood clearance time of the fusion polypeptide.

4. The fusion polypeptide according to claim 1, wherein the inclusion of the modified Fc domain improves the binding affinity of the fusion polypeptide to the CD47 protein.

5. The fusion polypeptide according to claim 1, wherein the inclusion of the modified Fc domain reduces the Ec50 of the fusion polypeptide.

6. The fusion polypeptide according to claim 1, wherein the inclusion of the modified Fc domain reduces the effective dose of the fusion polypeptide required to achieve a therapeutic effect in the target.

7. The fusion polypeptide according to claim 1, wherein the inclusion of the modified Fc domain reduces the frequency of administration of the fusion polypeptide to the subject.

8. The fusion polypeptide according to claim 1, wherein the inclusion of the modified Fc domain reduces the toxicity of the fusion polypeptide.

9. The fusion polypeptide according to claim 8, wherein the inclusion of the modified Fc domain reduces antibody-dependent cell-mediated cytotoxicity (ADCC).

10. The fusion polypeptide according to claim 1, wherein the inclusion of the modified Fc domain enhances the effector function of the fusion polypeptide.

11. The fusion polypeptide according to claim 1, wherein the inclusion of the modified Fc domain increases phagocytosis by macrophages.

12. The inclusion of the modified Fc domain is (i) Increase the interaction of the fusion polypeptide with cells expressing the CD47 protein; (ii) Increase the endocytosis of the CD47 protein; and / or (iii) Increases the degradation of CD47 protein. The fusion polypeptide according to claim 1.

13. The fusion polypeptide according to claim 12, wherein the CD47 protein is human or mouse CD47 protein.

14. The fusion polypeptide according to claim 1, wherein the inclusion of the modified Fc domain reduces aggregation of the fusion polypeptide.

15. The fusion polypeptide according to claim 1, wherein the inclusion of the modified Fc domain improves the purification of the polypeptide.

16. The fusion polypeptide according to any one of claims 1 to 15, wherein the modified Fc domain comprises the IgG4 Fc domain amino acid sequence of SEQ ID NO: 7 or 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

17. The fusion polypeptide according to claim 16, wherein the modified Fc domain comprises one or more substitutions selected from the group consisting of T250Q, M252Y, S254T, T256E, S267E, N325S, L328F, N343S, M428L, N434F, and H443K for SEQ ID NO: 7 or 8, according to the EU numbering scheme.

18. The fusion polypeptide according to claim 17, wherein the modified Fc domain comprises one or more substitutions selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F for SEQ ID NO: 7 or 8, according to the EU numbering scheme.

19. The fusion polypeptide according to any one of claims 1 to 15, wherein the modified Fc domain comprises SEQ ID NO: 9, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

20. The fusion polypeptide according to any one of claims 1 to 15, wherein the modified Fc domain comprises the IgG1Fc domain amino acid sequence of Sequence ID No. 6, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

21. The fusion polypeptide according to claim 20, wherein the modified Fc domain comprises one or more substitutions selected from the group consisting of V215A, G236A, S239D, and I332E for SEQ ID NO: 6, in accordance with the EU numbering scheme.

22. The fusion polypeptide according to claim 20, wherein the modified Fc domain comprises one or more substitutions selected from the group consisting of T250Q, M252Y, S254T, T256E, S267E, N325S, L328F, N343S, M428L, H433K, and N434F with respect to SEQ ID NO: 6, according to the EU numbering scheme.

23. The fusion polypeptide according to claim 20, wherein the modified Fc domain comprises one or more substitutions selected from the group consisting of T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F with respect to Sequence ID No. 6, according to the EU numbering scheme.

24. The fusion polypeptide according to any one of claims 1 to 15, wherein the modified Fc domain is a modified human IgG1, IgG2, IgG3, or IgG4 domain.

25. The fusion polypeptide according to any one of claims 1 to 15, wherein the SIRPα domain comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

26. The fusion polypeptide according to claim 25, wherein the SIRPα domain contains one or more mutations from among V6I, S14L, S20T, I22T, H24R, V27I, I31F, A45G, E47V, K53R, E54Q, H56P, S66T, E70N, S77R, V92I, and / or D100 residue duplication relative to SEQ ID NO:

1.

27. The fusion polypeptide according to any one of claims 1 to 15, wherein the SIRPα domain comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

28. The fusion polypeptide according to any one of claims 1 to 15, wherein the modified Fc domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 6 to 9, and the SIRPα domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 1 or 2, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 1 or 2.

29. The fusion polypeptide according to any one of claims 1 to 15, wherein the modified Fc domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 9, and the SIRPα domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:

2.

30. A pharmaceutical composition for treating a disease in a subject requiring treatment of that disease, comprising the fusion polypeptide described in any one of claims 1 to 15.

31. The pharmaceutical composition according to claim 30, wherein the disease is a cardiovascular disease.

32. The pharmaceutical composition according to claim 30, wherein the pharmaceutical composition is administered subcutaneously.

33. A method for increasing phagocytosis by macrophages, comprising contacting a group of macrophages with a fusion polypeptide according to any one of claims 1 to 15.

34. The method according to claim 33, wherein the macrophage is a human macrophage.

35. A nucleotide encoding a fusion polypeptide according to any one of claims 1 to 15.

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