SIRP-alpha fusion polypeptides with modified FC domains

Abstract

Provided herein are signal regulatory protein alpha (SIRP alpha) fusion polypeptides comprising a modified Fc domain, methods of making and uses thereof. Comprising the modified Fc domain results in an improvement in the pharmacokinetics and / or pharmacokinetics of the SIRP [alpha] fusion polypeptide. The compositions and methods described herein are useful for the treatment of various diseases, such as cardiovascular diseases.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 323,417 filed on March 24, 2022, the contents of which are incorporated by reference in their entirety.

[0003] Incorporation by Reference into the Sequence Listing

[0004] The contents of the electronic sequence listing (BTRT_010_01WO_SeqList_ST26.xml; size: 13,201 bytes; and creation date: March 21, 2023) are incorporated herein by reference in their entirety. Background Art

[0005] Optimization of therapies containing IgG Fc domains remains a challenge because these polypeptides can exhibit variable half-lives in the bloodstream. An example is a signal regulatory protein alpha (SIRPα) fusion polypeptide with an IgG Fc domain, which is designed to engage the CD47 protein, which is suitable for a wide range of diseases that benefit from optimal pharmacological inhibition of CD47. Therefore, there is a need for signal regulatory protein alpha (SIRPα)-IgG Fc therapy with improved pharmacokinetics and / or pharmacodynamics; compositions and methods for addressing this need are provided herein. Summary of the invention

[0006] The present disclosure provides signal regulatory protein alpha (SIRPα) fusion polypeptide therapeutics comprising a modified Fc domain for improving the pharmacokinetics and / or pharmacodynamics of the fusion polypeptide.

[0007] In some embodiments of the present disclosure, a fusion polypeptide is provided, which comprises 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 a wild-type Fc domain, and wherein the modified Fc domain increases binding affinity to FcRn.

[0008] In some embodiments of the present disclosure, the Fc domain comprising the modification increases the half-life of the fusion polypeptide. In some embodiments of the present disclosure, the Fc domain comprising the modification increases the blood clearance time of the fusion polypeptide.

[0009] In some embodiments of the present disclosure, the Fc domain comprising the modification increases the binding affinity of the fusion polypeptide to the CD47 protein. In some embodiments of the present disclosure, the Fc domain comprising the modification decreases the EC of the fusion polypeptide. 50 .

[0010] In some embodiments, the Fc domain comprising the modification reduces the effective dose of the fusion polypeptide necessary to achieve a therapeutic effect in a subject. In some embodiments, the Fc domain comprising the modification reduces the frequency of administration of the fusion polypeptide in a subject.

[0011] In some embodiments, the Fc domain comprising the modification reduces the toxicity of the fusion polypeptide. In some embodiments, the Fc domain comprising the modification reduces antibody-dependent cellular cytotoxicity (ADCC).

[0012] In some embodiments, the Fc domain comprising the modification increases the effector function of the fusion polypeptide. In some embodiments, the Fc domain comprising the modification increases the phagocytosis of macrophages. In some embodiments, the Fc domain comprising the modification increases the interaction of the fusion polypeptide with cells expressing the CD47 protein. In some embodiments, the Fc domain comprising the modification increases the endocytosis of the CD47 protein.

[0013] In some embodiments, the Fc domain comprising the modification increases the degradation of the CD47 protein. In some embodiments, the CD47 protein is a human or mouse CD47 protein. In some embodiments, the Fc domain comprising the modification reduces the aggregation of the fusion polypeptide. In some embodiments, the Fc domain comprising the modification improves the purification of the polypeptide.

[0014] In some embodiments, 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.

[0015] In some embodiments, according to the EU numbering scheme, relative to SEQ ID NO: 7 or 8, 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. In some embodiments, according to the EU numbering scheme, relative to SEQ ID NO: 7 or 8, 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. 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.

[0016] In some embodiments, the modified Fc domain comprises the 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, according to the EU numbering scheme, relative to SEQ ID NO: 6, the modified Fc domain comprises one or more substitutions selected from the group consisting of V215A, G236A, S239D and I332E. In some embodiments, according to the EU numbering scheme, relative to SEQ ID NO: 6, 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. 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.

[0017] In some embodiments, the modified Fc domain is a modified human IgG1, IgG2, IgG3, or IgG4 domain.

[0018] In some embodiments, 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. 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 residue D100. 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.

[0019] In some embodiments, the modified Fc domain comprises any one 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 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.

[0020] In some embodiments, the present invention provides a method of treating a disease in a subject in need thereof, 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.

[0021] In some embodiments, provided herein is a method of increasing phagocytosis of macrophages, comprising contacting a population of macrophages with a SIRPα fusion polypeptide. In some embodiments, the macrophages are human macrophages.

[0022] In some embodiments, provided herein is a nucleotide encoding a SIRPα fusion polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a graph showing the binding affinity of an exemplary SIRPα fusion polypeptide having a YTE Fc domain mutation (Construct 67) to various CD47 proteins relative to a SIRPα fusion polypeptide having a wild-type Fc domain and the MIAP410 anti-CD47 monoclonal antibody.

[0024] Figure 2A and Figure 2B The results show that the SIRPα fusion polypeptide construct 50 ( Figure 2A ) and construct 67 ( Figure 2B ) Example of a single CD47 binding assay run performed.

[0025] Figure 3 The EC values ​​of SIRPα fusion polypeptide construct 50 and construct 67 are shown for the amount of constructs binding to human CD45-negative erythrocytes in vitro. 50 effect.

[0026] Figure 4A and Figure 4B Shown are the levels of phagocytosis induced by SIRPα fusion polypeptide constructs 67 and 50 and anti-CD47 antibodies. Figure 4A and Figure 4B Each shows different results for human donors.

[0027] Figure 5A and Figure 5B The effects of SIRPα fusion polypeptide construct 67 and construct 50 on antibody-dependent cellular cytotoxicity (ADCC) relative to CD-20 antibody are shown. Figure 5A It was shown that construct 67 and construct 50 displayed minimal ADCC compared to the anti-CD20 antibody. Figure 5B yes Figure 5A Inset, rescaled to show the difference between construct 67 containing a YTE mutation in the Fc domain and construct 50 containing a wild-type Fc domain. DETAILED DESCRIPTION

[0028] Provided herein are signal regulatory protein alpha (SIRPα) fusion polypeptide treatments comprising a modified Fc domain, wherein the modified Fc domain improves the pharmacokinetics and / or pharmacodynamics of the fusion polypeptide. The signal regulatory protein alpha (SIRPα) fusion polypeptides disclosed herein can be used to treat, for example, cardiovascular disease, fibrosis, cancer, infectious diseases, blood diseases, and nervous system diseases.

[0029] I. Definitions

[0030] 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 used in connection with the chemistry, molecular biology, cell biology, immunology, pharmacology and protein chemistry described herein are those well known and commonly used in the art.

[0031] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" refers to one of such agents or a mixture thereof, and reference to "a method" includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

[0032] As used herein, the term "about" or "approximately" applied to one or more target values ​​refers to a value similar in magnitude and / or in a similar range to a specified reference value. In certain embodiments, unless otherwise specified or in addition apparent from context (unless such numerals exceed 100% of possible values), the term "about" or "approximately" refers to a series of values ​​falling within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of a range of values ​​in either direction of a specified reference value (greater than or less than).

[0033] When providing a range of values, it should be understood that unless the context clearly indicates otherwise, each intermediate value (to one tenth of the lower limit unit) between the upper and lower limits of the range and any other specified value or intermediate value in the specified range are included in the scope of the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also included in the present disclosure, subject to any explicitly excluded limits in the specified range. When the stated range includes one or two limits, the range excluding any one or both of those included limits is also included in the present disclosure.

[0034] As used herein, the terms "polypeptide", "peptide" and "protein" refer to amino acid polymers of any length. These terms also encompass amino acid polymers that have been modified; for example, modifications include disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.

[0035] As used herein, the term "nucleic acid sequence" or "nucleotide sequence" refers to a molecule comprising a DNA or RNA nucleotide sequence presented from 5' to 3'.

[0036] As used herein, "antibody" includes reference to full-length immunoglobulin molecules that are immunoreactive with a specific antigen, including polyclonal antibodies and monoclonal antibodies. The term includes humanized antibodies, chimeric antibodies (e.g., mouse variable regions with human constant regions) and conjugated antibodies.

[0037] As used herein, the term "Fc domain" (also interchangeably referred to herein as "Fc sequence", "Fc region" or simply "Fc") refers to a fragment crystallizable region monomer of an antibody domain 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.

[0038] Unless otherwise indicated, modifications in the Fc domain are presented according to the EU numbering scheme. However, one skilled in the art can easily cross-reference multiple numbering schemes (www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html#refs).

[0039] The term "treatment" and the like are generally used herein to mean obtaining the desired pharmacological and / or physiological effect with a therapeutic agent. The effect may be preventive in terms of preventing a disease or its symptoms completely or partially, such as reducing the likelihood of the disease or its symptoms occurring in a subject, and / or in terms of reducing symptoms completely or partially, the effect may be therapeutic, or partially or completely cure the disease and / or the adverse effects attributable to the disease. "Treatment" as used herein includes any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting or slowing the onset or development of the disease; or (c) alleviating the disease, such as causing the disease or regression of symptoms associated with the disease. The therapeutic agent may be administered before, during, or after the onset of the disease. In the case of treatment stabilizing or reducing adverse clinical symptoms in patients, treatment of persistent diseases may be particularly interesting. In some embodiments, treatment is performed before complete loss of function in the affected tissue. In some embodiments, the treatment of the subject will be administered during the symptomatic phase of the disease, and in some cases after the symptomatic phase of the disease.

[0040] The terms "individual", "subject" and "patient" are used interchangeably herein and refer to any subject in need of treatment. The subject can be a mammalian subject. Mammalian subjects include, for example, humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cattle, sheep, pigs, horses, goats, etc.), etc. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate, such as a cynomolgus monkey. In some embodiments, the subject is a companion animal (e.g., a cat, a dog).

[0041] II. Signal regulatory protein α (SIRPα) fusion peptide

[0042] Blocking the binding of endogenous signal regulatory protein alpha (SIRPα) to CD47 protein on cells (e.g., human cells) allows phagocytic engulfment of CD47 expressing cells. Provided herein are fusion polypeptides comprising a SIRPα domain and a modified Fc domain, which can be used to block the binding of endogenous SIRPα to CD47 protein, wherein the modified Fc domain improves the pharmacokinetics and / or pharmacodynamics of the fusion polypeptide. In some embodiments, among other effects, the modified Fc domain may increase the binding affinity of the fusion polypeptide to the neonatal Fc receptor, thereby increasing the half-life of the fusion polypeptide in the subject, and causing the fusion polypeptide to block the binding of endogenous SIRPα to CD47. In some embodiments, among other effects, the modified Fc domain may increase the binding affinity of the fusion polypeptide to the CD47 protein. In some embodiments, such an increase in binding affinity allows for a reduction in the dose and / or frequency of administration of the fusion polypeptide in a subject, and can result in improved blocking of the binding of endogenous SIRPα to CD47 by the fusion polypeptide. In some embodiments, among other effects, the inclusion of the modified Fc domain can increase the safety of the SIRPα fusion polypeptide.

[0043] In some embodiments, the SIRPα fusion polypeptides of the present disclosure form monomers, dimers, trimers, tetramers, pentamers or other multimers. In an exemplary embodiment, the SIRPα fusion polypeptides comprising the modified Fc domains of the present disclosure form dimers, in some embodiments, the dimers are homodimers, and in other embodiments, the dimers are heterodimers.

[0044] SIRPα polypeptide sequence

[0045] The SIRPα fusion polypeptide as provided herein comprises a SIRPα domain and a modified Fc domain. In this section, the SIRPα domain aspect of the fusion polypeptide is described in more detail.

[0046] The SIRPα domain provided herein comprises a membrane distal (D1) domain of SIRPα that binds to a CD47 protein (wild type or modified form thereof). In some embodiments, the SIRPα fusion polypeptide of the present disclosure comprises a wild type human SIRPαD1 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.

[0047] SEQ ID NO:1

[0048] EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS

[0049] In some embodiments, the SIRPα fusion polypeptide of the present disclosure comprises a SIRPαD1 domain having one or more amino acid modifications relative to the wild-type sequence of the D1 domain, such as the D1 domain of SEQ ID NO: 1. Modifications include amino acid substitutions, amino acid deletions, and amino acid additions. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain having at least one amino acid modification relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain having at least two amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain having at least three amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain having at least four amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain having at least five amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain having at least six amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain having at least seven amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain having at least eight amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain having at least nine amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain having at least ten amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain having at least eleven amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain having at least twelve amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least thirteen amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least fourteen amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least fifteen amino acid modifications relative to the wild-type sequence of the D1 domain.In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain with at least sixteen amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain with at least seventeen amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain with at least eighteen amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain with at least nineteen amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain with at least twenty amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain with at least twenty amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain with at least twenty-one amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPαD1 domain with at least twenty-two amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least twenty-three amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least twenty-four amino acid modifications relative to the wild-type sequence of the D1 domain. In some embodiments, the SIRPα fusion polypeptide comprises a SIRPα D1 domain having at least twenty-five amino acid modifications relative to the wild-type sequence of the D1 domain.

[0050] In some embodiments, the SIRPα fusion polypeptide comprises a wild-type SIRPα D1 domain that exhibits 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 greater binding affinity for CD47 (i.e., lower K D value) of SIRPαD1 polypeptide.

[0051] In some embodiments, the SIRPα fusion polypeptides of the present disclosure comprise modifications at one or more of the following residues relative to the wild-type SIRPα D1 domain sequence of SEQ ID NO: 1: V6, S14, S20, I22, H24, V27, I31, A45, E47, K53, E54, H56, S66, E70, S77, V92 and / or a repeat of the D100 residue.

[0052] In some embodiments, the SIRPα fusion polypeptide of the present disclosure comprises one or more of the following modifications relative to the wild-type SIRPα D1 domain sequence of 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.

[0053] In some embodiments, the SIRPα fusion polypeptide of the present disclosure comprises the SIRPαD1 sequence of SEQ ID NO:2 (referred to herein as CV1, an exemplary SIRPαD1 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.

[0054] SEQ ID NO:2

[0055] EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPS

[0056] In some embodiments, the SIRPα fusion polypeptide of the present disclosure comprises the SIRPαD1 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.

[0057] SEQ ID NO:3

[0058] XXELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVR

[0059] In some embodiments, the SIRPα fusion polypeptide of the present disclosure comprises the SIRPαD1 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.

[0060] SEQ ID NO:4

[0061] XXELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVR

[0062] In some embodiments, the SIRPα fusion polypeptide of the present disclosure comprises the SIRPαD1 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.

[0063] SEQ ID NO:5

[0064] EEXLQVIQPDKXVXVAAGEXAXLXCTXTSLIPVGPIQWFRGAGPXRELIYNQKEGHFPRVTTVSXXDLTKRXNMDFXIXIXNITPADAGTYYCVKFRKGSPDDXEFKSGAGTELSVR

[0065] In some embodiments, the SIRPα fusion polypeptides of the present disclosure comprise one or more of the following substitutions relative to the SIRPαD1 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, E70N, M72R, S75P, R77S, S79G, N80A, N80X, I81N, T82N, P83N, P83X, V92I, F94L, F94V, repeat of D100, E102V, E102T, E102F, F103E, F103V, K104F, K104V, A115G, K116A, and K116G, wherein X = any amino acid.

[0066] In some embodiments, the SIRPα fusion polypeptide sequence of the present disclosure may comprise any one of the SIRPα D1 sequences described in WO2013109752, WO2014094122A1, WO2017027422, WO2016023040, and WO2016024021A1, which are incorporated herein in their entireties.

[0067] III. Modified Fc Domains for Improving the Pharmacokinetics of SIRPα Fusion Polypeptides

[0068] The present disclosure provides a signal regulatory protein alpha (SIRPα) fusion polypeptide comprising a modified Fc domain, and the modified Fc domain can be used to improve pharmacokinetics and / or pharmacodynamics, for example, by increasing binding affinity with FcRn and / or with CD47 protein. As provided herein, one or more modifications (e.g., substitutions) can be introduced into a wild-type IgG Fc domain sequence, such as a human IgG1, IgG2, IgG3, or IgG4 domain, to increase engagement with a neonatal Fc receptor (FcRn) and extend the half-life of the fusion polypeptide. The one or more modifications introduced into a wild-type IgG Fc domain sequence can also improve the effectiveness and / or safety of the fusion polypeptide.

[0069] Modified Fc domain sequence

[0070] The SIRPα fusion polypeptide provided herein comprises a modified Fc domain, which can be used to improve the pharmacokinetics and / or pharmacodynamics of the fusion polypeptide. The Fc domain provided herein can be a modified domain of any species (e.g., human or mouse), or can be an engineered non-naturally occurring Fc domain, such as a human or mouse IgG domain comprising one or more modifications. In some embodiments, the Fc domain is a modified human IgG1 or IgG4 Fc domain. The canonical wild-type sequences of these are presented herein.

[0071] In some embodiments, the Fc domain is a modified human IgG2 or IgG3 domain, or a mouse IgG1, IgG2a, IgG2b, or IgG3 domain.

[0072] In some embodiments, the SIRPα fusion polypeptide of the present disclosure comprises the human IgG1 Fc 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.

[0073] SEQ ID NO:6

[0074] ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0075] In some embodiments, the SIRPα fusion polypeptide of the present disclosure comprises the human IgG4 Fc amino acid 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.

[0076] SEQ ID NO:7

[0077] PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0078] In some embodiments, the SIRPα fusion polypeptide of the present disclosure comprises an Fc domain of an IgG4 human Fc domain, and the polypeptide is susceptible to the dynamic process of Fab-arm exchange. Therefore, in some embodiments, according to the EU numbering scheme, the IgG4 Fc domain may comprise an S228P substitution relative to SEQ ID NO: 7, causing the process to be reduced. In some embodiments, the SIRPα fusion polypeptide of the present disclosure comprises an IgG4 Fc amino acid 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.

[0079] SEQ ID NO:8

[0080] PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0081] In some embodiments, the IgG4 Fc amino acid sequence comprises the substitution L445P relative to SEQ ID NO: 8 according to the EU numbering scheme.

[0082] In some embodiments, the SIRPα fusion polypeptide of the present disclosure comprises a human IgG1 Fc sequence of SEQ ID NO: 6, which comprises one or more modifications (e.g., substitutions) that increase effector function. In some embodiments, according to the EU numbering scheme, relative to SE Q ID NO: 6, the substitution is selected from V215A, G236A, S239D, I332E, T250Q, M252Y, S254T, T256E, S267E, N325S, L328F, N343S, M428L, H433K, and N434F. According to the EU numbering scheme, relative to SEQ ID NO: 6, IDNO: 6, exemplary combinations include: G236A-S239D, G236A-I332E, S239D-I332E, V215A-G236A-S239D-I332E, G236A-S239D-I332E, K326W-E333S, S267E-H268F-S324T and E345R-E430 G-S440Y, F243L-R292P-Y300L-V305I-P396L, S239D-I332E, S298A-E333A-K334A , L234Y-L235Q-G236W-S239M-H268D-D270E-S298A and D270E-K326D-A330M-K334E.

[0083] In some embodiments, the SIRPα fusion polypeptide of the present disclosure comprises a human IgG1 Fc sequence of SEQ ID NO: 6, which comprises one or more modifications (e.g., substitutions) that reduce effector function. In some embodiments, the substitutions are selected from: N297A, N297Q, N297G, L235E, L234A, L235A, K214R, P329G, D356E, and L358M.

[0084] In some embodiments, the SIRPα fusion polypeptide of the present disclosure comprises a human IgG4 Fc sequence of SEQ ID NO: 7 or 8, which comprises one or more modifications (e.g., substitutions) that reduce effector function. In some embodiments, the substitution is selected from: L235A, L235E, S228P, and F234A. Exemplary combinations include L235E-S228P, S228P-F234A, and S228P-F234A-L235A.

[0085] In other embodiments, the SIRPα fusion polypeptide of the present disclosure comprises an IgG1 Fc or IgG4 Fc domain, wherein there is a modification to increase serum half-life. In some embodiments, according to the EU numbering scheme, relative to the IgG1 Fc domain sequence SEQ ID NO: 6 or the IgG4 Fc domain sequence SEQ ID NO: 7 or 8, the mutation is selected from T250Q, M252Y, S254T, T256E, S267E, N325S, L328F, N343S, M428L, N434F and H443K. In some embodiments, the mutation is selected from 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.

[0086] In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO:8, which comprises substitutions M252Y, S254T, and T256E relative to SEQ ID NO:8.

[0087] In some embodiments, the SIRPα fusion polypeptide of the present disclosure comprises the human IgG4 Fc amino acid 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.

[0088] SEQ ID NO:9

[0089] PPCPPCPAPEFLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0090] In some embodiments, the SIRPα fusion polypeptide of the present disclosure comprises a peptide linker connecting the SIRPα domain and the Fc domain. In some embodiments, the SIRPα fusion polypeptide comprises a peptide linker of 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 comprises alanine (A), glycine (G), and / or serine (S) amino acids. In some embodiments, the peptide linker is 8 amino acids of G and S amino acids. In some embodiments, the linker is AAA. In some embodiments, the linker is GGGSGGGS (SEQID NO: 11). In some embodiments, the linker comprises a human IgG sequence, such as ASTKGPSVFPLAP (SEQ ID NO: 12).

[0091] In an exemplary embodiment, a SIRPα fusion polypeptide as provided herein comprises a SIRPα domain sequence of any one 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 one 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.

[0092] In an exemplary embodiment, a SIRPα fusion polypeptide as provided herein comprises 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.

[0093] An exemplary SIRPα fusion polypeptide comprising a SIRPα domain and a modified Fc domain comprises 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0094] SEQ ID NO:10

[0095] EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSAAAPPPCPCPAPEFLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0096] Effects of modified Fc domains

[0097] In some embodiments, one or more modifications may be introduced into the Fc domain of the SIRPα fusion polypeptide of the present disclosure to increase the binding affinity of the fusion polypeptide to FcRn and / or increase the binding affinity to the CD47 protein. In some embodiments, the Fc domain comprising the modification causes the binding affinity to FcRn to increase by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 1000x or about 10,000x relative to the wild-type Fc domain. In some embodiments, the Fc domain comprising the modification causes an increase in binding affinity for CD47 relative to a wild-type Fc domain by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x, or about 10,000x. In some embodiments, the CD47 protein is a human, mouse, non-human primate, or rat CD47 protein. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from 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. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T, and T256E relative to SEQ ID NO: 8. Exemplary SIRPα fusion polypeptides of the present disclosure comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0098] In some embodiments, the modified Fc domain increases the in vivo or in vitro half-life of the SIRPα fusion polypeptide.

[0099] In some embodiments, the SIRPα fusion polypeptide comprising the modified Fc domain exhibits an increase in in vivo half-life of about 1x, about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x, or about 10,000x relative to the half-life of a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from 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. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T, and T256E relative to SEQ ID NO: 8. Exemplary SIRPα fusion polypeptides of the present disclosure comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0100] In some embodiments, the modified Fc domain increases the in vitro (e.g., cell culture) half-life of the SIRPα fusion polypeptide by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x, or about 10,000x relative to the half-life of a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from 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. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T, and T256E relative to SEQ ID NO: 8. Exemplary SIRPα fusion polypeptides of the present disclosure comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0101] In some embodiments, the modified Fc domain slows down the blood clearance rate of the SIRPα fusion polypeptide in a subject. In some embodiments, the modified Fc domain slows down the blood clearance rate of the SIRPα fusion polypeptide relative to the SIRPα fusion polypeptide comprising a wild-type Fc domain by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, or about 1000x. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from 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. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T, and T256E relative to SEQ ID NO: 8. Exemplary SIRPα fusion polypeptides of the present disclosure comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0102] In some embodiments, the Fc domain comprising the modification increases the in vivo or in vitro binding affinity of the SIRPα fusion polypeptide to the CD47 protein. In some embodiments, the SIRPα fusion polypeptide comprising the modified Fc domain is effective at a lower dose in a subject relative to a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, according to the EU numbering scheme, relative to the IgG1 Fc domain sequence SEQ ID NO: 6 or the IgG4 Fc domain sequence SEQ ID NO: 7 or 8, the one or more modifications in the Fc domain are selected from T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F and H433K-N434F. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T, and T256E relative to SEQ ID NO: 8. An exemplary SIRPα fusion polypeptide of the present disclosure comprises 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0103] In some embodiments, the SIRPα fusion polypeptide comprising a modified Fc domain has an EC of 1. ... 50Reduced by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x, or about 10,000x. In some embodiments, according to the EU numbering scheme, relative to the IgG1 Fc domain sequence SEQ ID NO: 6 or the IgG4 Fc domain sequence SEQ ID NO: 7 or 8, the one or more modifications in the Fc domain are selected from T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F and H433K-N434F. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T, and T256E relative to SEQ ID NO: 8. An exemplary SIRPα fusion polypeptide of the present disclosure comprises 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0104] In some embodiments, the modified Fc domain reduces the effective dose of the SIRPα fusion polypeptide necessary to achieve the desired therapeutic effect by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x, or about 10,000x, relative to a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from 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. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T, and T256E relative to SEQ ID NO: 8. Exemplary SIRPα fusion polypeptides of the present disclosure comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0105] In some embodiments, the SIRPα fusion polypeptide comprising the modified Fc domain is effective in a subject at a lower dosing frequency (the dosing frequency necessary to achieve the desired therapeutic effect) relative to a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, according to the EU numbering scheme, relative to the IgG1 Fc domain sequence SEQ ID NO: 6 or the IgG4 Fc domain sequence SEQ ID NO: 7 or 8, the one or more modifications in the Fc domain are selected from T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F and H433K-N434F. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T and T256E relative to SEQ ID NO: 8. An exemplary SIRPα fusion polypeptide of the present disclosure comprises 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0106] In some embodiments, the SIRPα fusion polypeptide comprising a modified Fc domain reduces the frequency of dosing of the SIRPα fusion polypeptide necessary to achieve a desired therapeutic effect by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x, or about 10,000x, relative to a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from T250Q-M428L, M252Y-S254T-T256E, M428L-N434S, S267E-L328F, N325S-L328F, and H433K-N434F relative to the IgG1 Fc domain sequence of SEQ ID NO: 6 or the IgG4 Fc domain sequence of SEQ ID NO: 7 or 8. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T, and T256E relative to SEQ ID NO: 8. Other exemplary SIRPα fusion polypeptides disclosed herein comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0107] In some embodiments, the modified Fc domain increases the effector function of the Fc domain in the SIRPα fusion polypeptide. In some embodiments, the modified Fc domain increases complement-dependent cytotoxicity. In some embodiments, relative to the complement-dependent cytotoxicity of a SIRPα fusion polypeptide comprising a wild-type Fc domain, the modified Fc domain increases the complement-dependent cytotoxicity of the SIRPα fusion polypeptide by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x, or about 10,000x. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from 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. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T and T256E relative to SEQ ID NO: 8. Exemplary SIRPα fusion polypeptides of the present disclosure comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0108] In some embodiments, the Fc domain comprising the modification increases the interaction of the SIRPα fusion polypeptide with cells expressing the CD47 polypeptide. In some embodiments, the Fc domain comprising the modification increases the binding of the SIRPα fusion polypeptide to CD47 expressing cells by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x, or about 10,000x relative to the binding of the SIRPα fusion polypeptide comprising the wild-type Fc domain. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from 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. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T, and T256E relative to SEQ ID NO: 8. Exemplary SIRPα fusion polypeptides of the present disclosure comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0109] In some embodiments, the modified Fc domain increases the induction of endocytosis of CD47 protein by SIRPα fusion polypeptide in cells expressing CD47 polypeptide. In some embodiments, the modified Fc domain increases the induction of endocytosis of CD47 protein by SIRPα fusion polypeptide in CD47 expressing cells by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x, or about 10,000x relative to the induction of SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from 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. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T, and T256E relative to SEQ ID NO: 8. Exemplary SIRPα fusion polypeptides of the present disclosure comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0110] In some embodiments, the modified Fc domain increases the induction of phagocytosis of macrophages by SIRPα fusion polypeptides. In some embodiments, the modified Fc domain increases the induction of phagocytosis of macrophages by SIRPα fusion polypeptides by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x, or about 10,000x relative to the induction of SIRPα fusion polypeptides comprising wild-type Fc domains. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from 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. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T, and T256E relative to SEQ ID NO: 8. Exemplary SIRPα fusion polypeptides of the present disclosure comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0111] In some embodiments, the SIRPα fusion polypeptide comprising a modified Fc domain is associated with increased degradation of the CD47 protein relative to a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, the inclusion of the modified Fc domain increases the degradation of the CD47 protein associated with the SIRPα fusion polypeptide by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x, or about 10,000x relative to a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from 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. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T and T256E relative to SEQ ID NO: 8. Exemplary SIRPα fusion polypeptides of the present disclosure comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0112] In some embodiments, the modified Fc domain reduces SIRPα fusion polypeptide toxicity. In some embodiments, the modified Fc domain reduces SIRPα fusion polypeptide toxicity by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x, or about 10,000x relative to the toxicity of a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from 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. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T and T256E relative to SEQ ID NO: 8. Exemplary SIRPα fusion polypeptides of the present disclosure comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0113] In some embodiments, the modified Fc domain reduces the antibody-dependent cellular toxicity (ADCC) of the SIRPα fusion polypeptide. In some embodiments, relative to the antibody-dependent cellular toxicity (ADCC) of the SIRPα fusion polypeptide comprising a wild-type Fc domain, the modified Fc domain reduces the antibody-dependent cellular toxicity of the SIRPα fusion polypeptide by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x or about 10,000x. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from 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. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T, and T256E relative to SEQ ID NO: 8. Exemplary SIRPα fusion polypeptides of the present disclosure comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0114] In some embodiments, the modified Fc domain increases the stability of hemoglobin levels associated with the SIRPα fusion polypeptide. In some embodiments, the modified Fc domain increases the stability of hemoglobin levels associated with the SIRPα fusion polypeptide by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x, or about 10,000x relative to the stability of hemoglobin levels associated with a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from 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. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T, and T256E relative to SEQ ID NO: 8. Exemplary SIRPα fusion polypeptides of the present disclosure comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0115] In some embodiments, the modified Fc domain reduces anemia associated with SIRPα fusion polypeptide. In some embodiments, the modified Fc domain reduces anemia associated with SIRPα fusion polypeptide by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x, or about 10,000x relative to anemia associated with SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from 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. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T, and T256E relative to SEQ ID NO: 8. Exemplary SIRPα fusion polypeptides of the present disclosure comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0116] In some embodiments, the modified Fc domain reduces aggregation of SIRPα fusion polypeptides. In some embodiments, the modified Fc domain reduces aggregation of SIRPα fusion polypeptides by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x, or about 10,000x relative to the aggregation of SIRPα fusion polypeptides comprising a wild-type Fc domain. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from 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. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T, and T256E relative to SEQ ID NO: 8. Exemplary SIRPα fusion polypeptides of the present disclosure comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0117] In some embodiments, comprising a modified Fc domain improves purification of SIRPα fusion polypeptides. In some embodiments, comprising the modified Fc domain improves purification of SIRPα fusion polypeptides by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x, or about 10,000x relative to purification of SIRPα fusion polypeptides comprising a wild-type Fc domain. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from 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. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T, and T256E relative to SEQ ID NO: 8. Exemplary SIRPα fusion polypeptides of the present disclosure comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0118] In some embodiments, the modified Fc domain reduces the formation of anti-drug antibodies (ADA) by SIRPα fusion polypeptides. In some embodiments, the modified Fc domain reduces the formation of ADA by SIRPα fusion polypeptides by about 1, about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x, or about 10,000x relative to the formation of ADA by SIRPα fusion polypeptides comprising wild-type Fc domains. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from 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. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T and T256E relative to SEQ ID NO: 8. Exemplary SIRPα fusion polypeptides of the present disclosure comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0119] In some embodiments, the modified Fc domain reduces red blood cell (RBC) agglutination associated with SIRPα fusion polypeptide. In some embodiments, the modified Fc domain reduces RBC agglutination associated with SIRPα fusion polypeptide by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x, or about 10,000x relative to RBC agglutination associated with SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from 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. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T and T256E relative to SEQ ID NO: 8. Exemplary SIRPα fusion polypeptides of the present disclosure comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0120] In some embodiments, the Fc domain comprising the modification reduces antibody formation against erythrocytes associated with the SIRPα fusion polypeptide as measured by a Coombs assay. In some embodiments, the Fc domain comprising the modification reduces antibody formation against erythrocytes associated with the SIRPα fusion polypeptide by about 1.5x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 20x, about 50x, about 100x, about 250x, about 500x, about 750x, about 1000x, about 5000x, or about 10,000x relative to antibody formation against a SIRPα fusion polypeptide comprising a wild-type Fc domain. In some embodiments, according to the EU numbering scheme, the one or more modifications in the Fc domain are selected from 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. In an embodiment, an exemplary SIRPα fusion polypeptide of the present disclosure comprises an IgG4 human Fc domain of SEQ ID NO: 8, which comprises substitutions M252Y, S254T and T256E relative to SEQ ID NO: 8. Exemplary SIRPα fusion polypeptides of the present disclosure comprise 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%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0121] III. Methods for preparing SIRPα fusion polypeptides comprising a modified Fc domain

[0122] Also provided herein are polynucleotides encoding SIRPα fusion polypeptides comprising modified Fc domains for improving the 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.

[0123] In some embodiments, polynucleotides encoding exemplary SIRPα fusion polypeptides of the present disclosure are introduced (e.g., transfected or transformed) into human cell lines or bacterial cell lines and expressed in human cell lines or bacterial cell lines. Exemplary cell lines that can be used for production include, but are not limited to, Expi 293 and CHO cell lines.

[0124] IV. Methods of Using SIRPα Fusion Polypeptides Comprising a Modified Fc Domain

[0125] In some embodiments, a SIRPα fusion polypeptide comprising a modified Fc domain is administered as a therapeutic agent to a subject in need thereof. In some embodiments, the SIRPα fusion polypeptide of the present disclosure is administered to treat, for example, cardiovascular disease, cancer, fibrosis, infectious disease, blood disease, or nervous system disease. In some embodiments, comprising a modified Fc domain improves the efficacy and / or safety of the SIRPα fusion polypeptide as a therapeutic agent.

[0126] In some embodiments, the subject selected for treatment with a SIRPα fusion polypeptide has cardiovascular disease and suffers from or is determined to be at risk of suffering from one or more of the following diseases: atherosclerosis, heart failure, myocardial infarction, cardiomyopathy, acute coronary syndrome, myocarditis, cardiac remodeling, hypertension, angina pectoris, restenosis, stroke, aneurysm, thrombosis, phlebitis, peripheral vascular disease, pulmonary hypertension, and autoimmune vasculitis.

[0127] In some embodiments, the subject selected for treatment with a SIRPα fusion polypeptide has cancer. In some embodiments, the SIRPα fusion polypeptides of the present disclosure can treat tumor growth and / or tumor metastasis, such as lymphoma, leukemia, carcinoma, melanoma, glioblastoma, sarcoma, or myeloma.

[0128] In some embodiments, the subject selected for treatment with the SIRPα fusion polypeptide of the present disclosure suffers from fibrosis or a fibrotic disease, such as a liver or lung fibrotic disease. In some embodiments, the subject suffers from or is at risk of suffering from the following diseases: advanced liver disease, kidney disease, idiopathic pulmonary fibrosis (IPF), retinal fibrosis, chronic transplant rejection caused by progressive myopathy, or heart failure caused by cardiac fibrosis.

[0129] In some embodiments, the subject selected for treatment with SIRPα fusion polypeptide suffers from an infectious disease, an infectious disease associated with a viral, bacterial or fungal pathogen. In some embodiments, the subject suffers from a viral infection, such as an infection associated with one of a retrovirus, a lentivirus, a hepadnavirus, a herpes virus, a poxvirus or a human papillomavirus. In some embodiments, the subject has an intracellular bacterial infection, such as an infection associated with one of the species of Mycobacterium, Chlamydophila, Ehrlichia, Rickettsia, Brucella, Legionella, Francisella, Listeria, Coxiella, Neisseria, Salmonella or Yersinia. In some embodiments, the subject has an intracellular protozoan pathogen infection, such as an infection associated with one of a Plasmodium species, a Trypanosoma species, a Giardia species, a Toxoplasma species, or a Leishmania species.

[0130] In some embodiments, a subject is selected to be treated with a SIRPα fusion polypeptide of the present disclosure for a blood disease or disorder, such as an inherited blood disorder or severe combined immunodeficiency. In some embodiments, the 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.

[0131] In some embodiments, the subject selected for treatment with a SIRPα fusion polypeptide suffers from a neurological disease.

[0132] In some embodiments, the SIRPα fusion polypeptide can be delivered to a subject in need thereof subcutaneously, intravenously, intravitreally, orally, intranasally, transdermally, intraperitoneally, intramuscularly, intrathecally, intrapulmonaryly, vaginally, or rectally. In some embodiments, the SIRPα fusion polypeptide is administered subcutaneously.

[0133] In some embodiments, the SIRPα fusion polypeptide is conjugated to a fluorophore, a 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 the CD47 protein. In some embodiments, the SIRPα fusion polypeptide is administered to a cell or organism to diagnose a disease.

[0134] In some embodiments, the SIRPα fusion polypeptide comprises a conjugated toxin to deliver the toxin to cells expressing CD47.

[0135] In some embodiments, the SIRPα fusion polypeptide is administered in combination with a CD20 antibody and / or a CD47 antibody or fragment thereof. In some embodiments, a multivalent SIRPα fusion polypeptide is administered in combination with an antibody or antibody fragment to a protein selected from the group consisting of TNFα, TNF-αR, IL6, IL6R, IL1β, IL1-βR, 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 IL2R.

[0136] VI. Pharmaceutical Compositions

[0137] In some embodiments, a SIRPα fusion polypeptide comprising a modified Fc domain is present in a pharmaceutical composition. In certain embodiments, these pharmaceutical compositions may be in a water-soluble form, such as in a pharmaceutically acceptable salt, which is intended to include acid addition salts and base addition salts. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, organic acids such as 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-toluenesulfonic acid and salicylic acid acid addition salts. Pharmaceutically acceptable base addition salts include those derived from inorganic bases, such as sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Salts derived from pharmaceutically acceptable organic non-toxic bases include 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.

[0138] Pharmaceutical compositions as described herein may also include one or more of the following: carrier proteins, such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn starch and other starches; binders; and polyethylene glycol.

[0139] The composition for administration will generally comprise a polypeptide dissolved in a pharmaceutically acceptable carrier (preferably an aqueous carrier). Various aqueous carriers may be used, for example, buffered saline. As required, the composition may contain pharmaceutically acceptable auxiliary substances to approach physiological conditions, such as pH agents and buffers, antitoxic agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate. The concentration of the active agent in these preparations may vary and may be selected based on fluid volume, viscosity and body weight according to the selected specific mode of administration and the needs of the patient (e.g., Remington's Pharmaceutical Science (1980, 15th edition) and Goodman and Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., ed., 1996)).

[0140] VII. Kit

[0141] The SIRPα fusion polypeptides comprising a modified Fc domain as described herein can also constitute a therapeutic or diagnostic kit for administration by a medical professional or a subject in need thereof. The kit may comprise, for example, a container, a dose of SIRPα fusion polypeptide, a syringe and / or a vial and instructions for use thereof. In some embodiments, the kit comprises a SIRPα fusion polypeptide and instructions for administering the polypeptide to treat a disease.

[0142] In some embodiments, the SIRPα fusion polypeptide of the kit is conjugated to a fluorophore, radionucleotide, or other diagnostic moiety.

[0143] Example

[0144] Example 1: Effect of YTE mutation in SIRPα fusion polypeptide on CD47 binding affinity.

[0145] To determine the effect of the YTE Fc domain mutation on CD47 binding affinity, a CD47 binding experiment was performed on a SIRPα fusion polypeptide of SEQ ID NO: 10 (construct 67) comprising a SIRPα domain of SEQ ID NO: 2 and an IgG4 Fc domain with a YTE mutation. The CD47 binding affinity of a SIRPα fusion polypeptide SEQ ID NO: 10 construct (construct 50) without a YTE mutation, i.e., with a wild-type IgG4 Fc domain, was also measured; as well as the binding affinity of the commercially available CD47 monoclonal antibody MIAP410. MIAP410 is a mouse anti-human CD47 antibody that reacts with human, mouse, and rat CD47.

[0146] Using Sartorius Biosensor R8 measures the affinity octet of the aforementioned constructs and antibodies to AMC (anti-mouse Fc-capture) catalog #18-5088 or AHC (anti-human Fc-capture) catalog #18-5060.

[0147] Binding affinity determinations were performed at 30°C using the following method. The running buffer was PBS, 1% BSA, and 0.05% Tween-20. SIRPα fusion polypeptides and CD47 antibodies were immobilized to AMC or AHC for 3 minutes, followed by rhCD47-his association and dissociation for 15 minutes. Data were analyzed using the Sartorius software Octet Analysis Studio using reference-subtracted values. The dissociation constant (KD) was determined by applying a global (full kinetic) 1:1 fit model (N=1 for each sample for SIRPα fusion polypeptides and N=5 for anti-CD47 antibodies); Figure 1 The data from the best kinetic curve fitting experiments are presented in Figure 2A (Construct 50) and Figure 2B An example of the results of a single run is shown in (Construct 67).

[0148] The data demonstrate that construct 67 (which is a SIRPα fusion polypeptide of SEQ ID NO: 10) and construct 50 (which is 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) relative to the binding affinity of the MIAP410 anti-CD47 antibody. Notably, construct 67 containing the YTE mutation in the IgG4 Fc domain exhibited binding affinity equal to or better than the construct 50 SIRPα fusion polypeptide without the YTE mutation.

[0149] Example 2: Effect of YTE mutation in SIRPα fusion polypeptide on human erythrocyte binding in vitro.

[0150] To determine the effect of the YTE mutation on erythrocyte binding of the SIRPα fusion polypeptide of SEQ ID NO: 10 (Construct 67), the following experiment was performed using Construct 67 and Construct 50 as a comparison.

[0151] Normal human whole blood in EDTA from two different donors was purchased from Stanford Blood Center and shipped overnight at room temperature. Cells were incubated with 0.18 to 400 nM construct 50 or construct 67 at 4°C for 20 minutes. Cells were washed three times with PBS, followed by incubation with AF488-conjugated anti-human CD45 and 30 μg / mL AF647-labeled anti-human IgG4 antibodies at 4°C for 20 minutes. After washing, binding was measured using Agilent Quanteon using CD45 gating (N=2 donors) to select for effects in erythrocytes.

[0152] EC values ​​measured for both constructs 50 The value is the amount of construct bound to erythrocytes in vitro. Figure 3 As shown, the half-maximal binding of construct 67 containing the YTE mutation is equal to or lower than the half-maximal binding of construct 50.

[0153] Example 3: Effect of YTE mutation in SIRPα fusion polypeptide on phagocytosis in vitro.

[0154] To determine the effect of the YTE mutation in the SIRPα fusion polypeptide 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.

[0155] Eight days before running the assay, healthy human buffy coats from LRS chambers were purchased from Stanford Blood Center and shipped overnight. PBMC cells were isolated from buffy coats by applying a ficoll-paque gradient, and monocytes were isolated using the EasySep Human Monocyte Isolation Kit (Catalog No. 19359) purchased from Stemcell. The isolated monocytes were plated 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 following protocol. Calcein-AM dye was reconstituted in 20 ml DMSO and added to Raji cells (5×10 6 The cells were incubated at 37°C for 10 minutes, followed by 10 minutes on ice. The Raji cells were then centrifuged at 1250 rpm for 5 minutes at 4°C. The cells were washed once with HBSS and resuspended in IMDM. 25 ml / well was added at 4 × 10 6Calcein-stained Raji (target) cells suspended in IMDM at a density of 10 cells / ml were pipetted into Costar ultra-low attachment 96-well plates. 25 ml / well of biosimilar mololimab-like antibodies, constructs 50 and 67 at concentrations of 0.67 nM and 6.67 nM in IMDM, and 6.67 nM IgG4 isotype control were added to the Raji cells in triplicate. Raji cells were pre-incubated with the test articles at 37°C for 30 minutes. 50 ml / well of 1×10 6 In vitro differentiated macrophages from human donors at 100 cells / ml in IMDM were added to target cells in triplicate. The cells were resuspended and incubated at 37°C for 2 hours. At the end of the incubation, the plate was centrifuged and the supernatant was discarded. The cells were then resuspended in 50 ml of PBS + 2% FBS + 5ug / ml Alexa Fluor 647 anti-human CD206 and incubated on ice in the dark for 30 minutes. The cells were washed once with 200 μl FACS buffer and resuspended in 100 μl FACS buffer + DAPI. The phagocytosis index was detected using a flow cytometer and analyzed by Flowjo software. The percentage of macrophages is the percentage of macrophages with phagocytic calcein-AM-labeled Raji.

[0156] like Figure 4A and Figure 4B As shown, constructs 50 and 67 induced increased levels of phagocytosis relative to a biosimilar mololimab-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.

[0157] Example 4: Effect of YTE mutation in SIRPα fusion polypeptide on antibody-dependent cellular cytotoxicity (ADCC) in vitro.

[0158] To determine the effect of the YTE mutation in the SIRPα fusion polypeptide on the induction of antibody-dependent cellular cytotoxicity (ADCC), the following experiment was performed using construct 67 with the YTE Fc domain mutation and construct 50 with the wild-type Fc domain and anti-CD20 antibody as a positive control.

[0159] ADCC assay is carried out according to the instructions of ADCC reporter bioassay complete kit (catalog number G7015) purchased from Promega. In brief, the Raji cells in RPMI1640 in low IgG serum culture medium prepared in 25ml / well according to the kit instructions are pipetted into white flat-bottom 96-well Corning 3610 microplates. Serial dilutions of anti-CD20 (14pM to 10nM), construct 50 and construct 67 (457pM to 1μM) and 1μM IgG4 are prepared in RPMI1640 in low IgG serum. The antibody / molecule of 25ml / well is pipetted into the hole containing target cells, and one group of holes does not accept antibody / molecule. IgG4 and anti-CD20 are used as negative control and positive control respectively. In the hole containing target Raji cells and drug / molecule, the effector cells prepared in RPMI 1640 in low IgG serum of 25ml / well are pipetted. The microplate was incubated at 37°C for 6 hours, followed by luminescence detection using the reagents provided with the kit.

[0160] like Figure 5A As shown, construct 67 and construct 50 displayed minimal ADCC compared to the anti-CD20 antibody. Figure 5A The illustration is shown in Figure 5B middle, Figure 5B Rescaled to show the difference between construct 67 containing a YTE mutation in the Fc domain and construct 50 containing a wild-type Fc domain. The data demonstrate that construct 67 containing YTE exhibits lower antibody-dependent cellular cytotoxicity than construct 50.

[0161] Example 5: Effects of YTE mutation on in vivo receptor occupancy, hemoglobin levels and pharmacokinetics of SIRPα fusion polypeptides in non-human primates.

[0162] To determine the effect of the YTE mutation in the SIRPα fusion polypeptide 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 a YTE Fc domain substitution) and wild-type constructs (e.g., construct 50 with a wild-type Fc domain). Administration was performed to biologically naive male cynomolgus monkey non-human primates by subcutaneous and / or intravenous administration. Receptor occupancy, hemoglobin and / or PK data were collected and analyzed before, during and after administration and during observation. Gating analysis can be performed on CD45-negative cells.

[0163] It is predicted that the receptor occupancy of erythrocytes treated with certain constructs of the present disclosure comprising a YTE Fc domain substitution will be greater than or equal to the receptor occupancy of erythrocytes treated with the wild-type construct over the course of the observation period.

[0164] The serum concentrations of subjects treated with certain constructs of the disclosure comprising a YTE Fc domain substitution are predicted to be higher than or equal to the serum concentrations of subjects treated with the wild-type construct during the observation period.

[0165] The hemoglobin levels of subjects treated with certain constructs of the disclosure comprising a YTE Fc domain substitution are predicted to be higher than or equal to the hemoglobin levels of subjects treated with the wild-type construct during 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 a wild-type Fc domain, and wherein the modified Fc domain increases binding affinity to FcRn.

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

3. The fusion polypeptide of claim 1 or claim 2, wherein the modified Fc domain increases the blood clearance time of the fusion polypeptide.

4. The fusion polypeptide of any one of claims 1 to 3, wherein the modified Fc domain improves the binding affinity of the fusion polypeptide to the CD47 protein.

5. The fusion polypeptide of any one of claims 1-4, wherein the modified Fc domain reduces the Ec50 of the fusion polypeptide.

6. The fusion polypeptide of any one of claims 1-5, wherein the Fc domain comprising the modification reduces the effective dose of the fusion polypeptide necessary to achieve a therapeutic effect in a subject.

7. The fusion polypeptide of any one of claims 1-6, wherein comprising the modified Fc domain reduces the dosing frequency of the fusion polypeptide in a subject.

8. The fusion polypeptide of any one of claims 1-7, wherein the modified Fc domain reduces the toxicity of the fusion polypeptide.

9. The fusion polypeptide of claim 8, wherein the modified Fc domain reduces antibody-dependent cellular cytotoxicity (ADCC).

10. The fusion polypeptide of any one of claims 1 to 9, wherein the modified Fc domain increases the effector function of the fusion polypeptide.

11. The fusion polypeptide of any one of claims 1-10, wherein the modified Fc domain increases phagocytosis of macrophages.

12. The fusion polypeptide of any one of claims 1 to 11, wherein the modified Fc domain increases the interaction of the fusion polypeptide with cells expressing CD47 protein.

13. The fusion polypeptide of any one of claims 1 to 12, wherein the modified Fc domain increases endocytosis of the CD47 protein.

14. The fusion polypeptide of any one of claims 1 to 13, wherein the modified Fc domain increases the degradation of CD47 protein.

15. The fusion polypeptide of any one of claims 12-14, wherein the CD47 protein is a human or mouse CD47 protein.

16. The fusion polypeptide of any one of claims 1-15, wherein the Fc domain comprising the modification reduces aggregation of the fusion polypeptide.

17. The fusion polypeptide of any one of claims 1-16, wherein the inclusion of the modified Fc domain improves purification of the polypeptide.

18. A fusion polypeptide as described in any one of claims 1-17, 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.

19. The fusion polypeptide of claim 18, 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 relative to SEQ ID NO: 7 or 8 according to the EU numbering scheme.

20. The fusion polypeptide of claim 18, 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 relative to SEQ ID NO: 7 or 8 according to the EU numbering scheme.

21. The fusion polypeptide of any one of claims 1-20, 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.

22. A fusion polypeptide as described in any one of claims 1-17, wherein the modified Fc domain comprises the 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.

23. The fusion polypeptide of claim 22, wherein 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.

24. The fusion polypeptide of claim 22, 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 relative to SEQ ID NO:6 according to the EU numbering scheme.

25. The fusion polypeptide of claim 22, 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 relative to SEQ ID NO:6 according to the EU numbering scheme.

26. The fusion polypeptide of any one of claims 1-17, wherein the modified Fc domain is a modified human IgG1, IgG2, IgG3 or IgG4 domain.

27. A fusion polypeptide as described in any one of claims 1-26, 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.

28. A fusion polypeptide as described in any one of claims 1-26, wherein 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.

29. A fusion polypeptide as described in any one of claims 1-26, 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.

30. A fusion polypeptide as described in any one of claims 1-17, wherein the modified Fc domain comprises any one 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 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.

31. A fusion polypeptide as described in any one of claims 1-17, 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; 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.

32. A method of treating a disease in a subject in need thereof, comprising administering to the subject the fusion polypeptide of any one of claims 1-31.

33. The method of claim 32, wherein the disease is cardiovascular disease.

34. The method of any one of claims 32-33, wherein the fusion polypeptide is administered subcutaneously.

35. A method for increasing phagocytosis of macrophages, comprising contacting a population of macrophages with the fusion polypeptide of any one of claims 1-31.

36. The method of claim 35, wherein the macrophages are human macrophages.

37. A polynucleotide encoding the fusion polypeptide of any one of claims 1-31.

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