Human signal regulatory protein alpha mutant having ph-dependent binding activity and use thereof

Abstract

Provided is a mutant of an immunoglobulin-like variable domain of a signal regulatory protein alpha (SIRPαV), which mutant, relative to wild-type SIRPαV, contains histidine (H) at at least one position corresponding to amino acid positions 29, 31, 36, 37, 51, 52, 53, 68, 69, 95 and 99 as set forth in SEQ ID NO: 1.

Description

Human signal regulatory protein α mutants with pH-dependent binding activity and their applications

[0001] This application claims priority to three Chinese patent applications filed on May 30, 2024 (application number: CN202410695510.9, title: Human signal regulatory protein α mutant with pH-dependent binding activity and its application), filed on August 1, 2024 (application number: CN202411055675.6, title: Human signal regulatory protein α mutant with pH-dependent binding activity and its application), and filed on January 23, 2025 (application number: CN202510112310.0, title: Human signal regulatory protein α mutant with pH-dependent binding activity and its application), the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of tumor treatment, and in particular to mutants of human signal regulatory protein α (SIRPα) with pH-dependent binding activity. Background Technology

[0003] CD47, also known as integrin-associated protein (IAP), binds to integrins, thromboretin-1 (TSP-1), and SIRPα. CD47 is widely expressed on the surface of normal cells, acting as a "self-marker" to prevent macrophages from phagocytosing normal cells. In oncology research, CD47 was first discovered to be expressed on the surface of human ovarian cancer cells. Subsequently, it was found that CD47 expression levels were significantly increased on the surface of various lymphoma and hematologic malignancies, which helps tumor cells evade immune clearance.

[0004] Signal regulatory protein alpha (SIRPα) is a class of transmembrane glycoproteins expressed on the surface of myeloid cells, including monocytes, macrophages, and neutrophils. The signal regulatory protein family comprises five members: SIRPα, SIRPβ1, SIRPγ, SIRPβ2, and SIRPδ. The extracellular domain of SIRPα contains three immunoglobulin superfamily domains, while the intracellular domain contains the ITIM motif (immunoreceptor tyrosine-based inhibition motif). Upon binding to CD47, intracellular ITIM phosphorylation recruits tyrosine phosphatases such as SHP-1 and SHP-2, activating the "don't eat me" signal, thereby inhibiting the phagocytic activity of macrophages.

[0005] Studies have shown that blocking the CD47-SIRPα signaling pathway can relieve immunosuppression caused by the "don't eat me" signal and enhance the phagocytic capacity of macrophages against tumors. However, given the widespread expression of CD47 across multiple cell types, particularly its high expression levels on the surface of erythrocytes and platelets, CD47-targeted therapeutic strategies may carry the risk of off-target effects. In particular, the interaction between CD47-targeting IgG1 and erythrocytes may lead to adverse reactions such as hemolytic anemia; furthermore, CD47 expression on T cells means that targeted drugs may induce T cell apoptosis, thereby affecting the function of the immune system. Considering the antigen deposition effect, higher doses or more frequent dosing regimens may be required to block the CD47 / SIRPα pathway, which may affect the efficacy or safety of the drug. Therefore, when developing CD47-targeted therapeutic strategies, their specificity and potential systemic side effects must be carefully evaluated to ensure an optimal balance between efficacy and safety. Summary of the Invention

[0006] This invention relates to a series of engineered human SIRPα immunoglobulin-like variable domain (SIRPαV) mutants. In some embodiments, the SIRPαV mutants of this invention possess pH-dependent binding to human CD47. At acidic pH levels (e.g., in acidic tumor microenvironments), the affinity between the SIRPαV mutants and their ligand CD47 is significantly enhanced. In some embodiments, the SIRPαV mutants of this invention, utilizing their pH-dependent binding properties, can more specifically target CD47 in the tumor microenvironment while reducing binding to CD47 on the surface of erythrocytes in normal tissues, thereby significantly reducing potential adverse reactions such as coagulation. This specific affinity modulation provides a novel strategy for cancer therapy, potentially improving treatment outcomes and reducing treatment-related side effects.

[0007] In a first aspect, the present invention provides a mutant of the immunoglobulin (Ig)-like variable domain (SIRPαV) of signal regulatory protein α (SIRPα), which, relative to wild-type SIRPαV, contains histidine (H) at at least one position corresponding to amino acid positions 29, 31, 36, 37, 51, 52, 53, 68, 69, 95 and 99 shown in SEQ ID NO:1.

[0008] In a second aspect, the present invention provides a fusion protein comprising the SIRPαV mutant of the first aspect and an immunoglobulin moiety.

[0009] In a third aspect, the present invention provides a nucleic acid encoding a SIRPαV variant of the first aspect or a fusion protein of the second aspect, or a nucleic acid construct comprising the thereof.

[0010] In a fourth aspect, the present invention provides a host cell comprising the nucleic acid of the third aspect.

[0011] In a fifth aspect, the present invention provides a pharmaceutical composition comprising, in the first aspect, a SIRPαV mutant, in the second aspect, a fusion protein, in the third aspect, a nucleic acid, or in the fourth aspect, a host cell.

[0012] In a sixth aspect, the present invention provides the use of the SIRPαV variant of the first aspect, the fusion protein of the second aspect, the nucleic acid of the third aspect, the cell of the fourth aspect, or the pharmaceutical composition of the fifth aspect in the preparation of a medicament for treating cancer.

[0013] In a seventh aspect, the present invention provides a method for preparing a SIRPαV variant of the first aspect or a fusion protein of the second aspect, comprising culturing a host cell of the sixth aspect and expressing nucleic acid under suitable conditions.

[0014] In an eighth aspect, the present invention provides a method for causing a protein containing SIRPαV to generate pH-dependent binding to CD47, comprising mutating an amino acid residue at at least one position corresponding to amino acid positions 36, 37, 51, 52, 53, 68, 69, 95 and 99 of the immunoglobulin-like variable domain of SIRPα to histidine (H).

[0015] In a ninth aspect, the present invention provides a method for enhancing the binding of SIRPαV to human CD47 protein, the method comprising mutating amino acid residues at positions 29 and / or 31 of SIRPαV corresponding to the amino acid position shown in SEQ ID NO:1 to histidine. Attached Figure Description

[0016] Figure 1: SIRPα mutation site selection and structural diagram. (A): SIRPα mutation site selected based on calculation results; (B): Schematic diagram of the structure and selection of the SIRPα-CD47 complex.

[0017] Figure 2: Schematic diagram of SIRPαV-Fc fusion protein.

[0018] Figure 3: pH-dependent binding of SIRPαV-Fc to CD47 protein as detected by ELISA. (A): Binding of S008-S017 mutant to CD47 protein; (B) Binding of S018-S027 mutant to CD47 protein; (C) Binding of S028-S037 mutant to CD47 protein; (D) Binding of S038-S047 mutant to CD47 protein; (E): Binding of single-site SIRPαV-Fc mutant to CD47 protein. Control: Negative control VISIG4-Fc.

[0019] Figure 4: pH-dependent cell binding assays. (A): Binding of S008-S022 mutants to Jurkat cells; (B) Binding of S023-S031 mutants to Jurkat cells; (C) Binding of S032-S040 mutants to Jurkat cells; (D) Binding of S041-S047 mutants to Jurkat cells. Control: Negative control VISIG4-Fc.

[0020] Figure 5: Target blocking assay. (A): Blocking WT SIRPα binding in Raji cells at pH 6.4; (B): Blocking WT SIRPα binding in Raji cells at pH 7.4. Control: Negative control VISIG4-Fc.

[0021] Figure 6: Binding ability of SIRPαV-Fc fusion protein to erythrocytes. (A): Binding of S009 to erythrocytes; (B) Binding of S017 to erythrocytes; (C) Binding of S019 to erythrocytes.

[0022] Figure 7: Binding ability of SIRPαV-Fc fusion protein to platelets. (A): Binding of WT to platelets; (B): Binding of S009 to platelets; (C) Binding of S017 to platelets; (D) Binding of S019 to platelets.

[0023] Figure 8: Detection of hemagglutination reactivity of SIRPαV-Fc fusion protein. (A): Hemagglutination reaction at pH 7.4; (B): Hemagglutination reaction at pH 6.8; (C): Hemagglutination reaction at pH 6.0.

[0024] Figure 9: (A): Sequence alignment of SIRPαV1, V2 and V8; (B): ELISA detection of pH-dependent binding of SIRPαV1-Fc mutant; and (C): ELISA detection of pH-dependent binding of SIRPαV8-Fc mutant.

[0025] Figure 10: (A): Results of macrophage phagocytosis induced by S34 / S41 fusion protein, (B): Results of macrophage phagocytosis induced by S31 / S45 fusion protein.

[0026] Figure 11: Results of in vivo pharmacodynamic experiments in the Raji subcutaneous xenograft model of human lymphoma cells. Detailed Implementation

[0027] Unless otherwise specified, the scientific and technical terms used herein shall have meanings commonly known to those skilled in the art. Furthermore, unless specifically required, singular terms shall include plural terms, and plural terms shall include singular terms. The foregoing techniques and methods are generally performed according to conventional methods well known in the art and as described in the references cited herein, such as those incorporated herein by reference: Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)); Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). All references cited in this article, including but not limited to patents, patent applications, articles, textbooks, etc., and all references cited therein, are incorporated herein by reference in their entirety.

[0028] Unless otherwise stated, when referring to nucleic acid sequences in this article, the direction is from left to right 5′ to 3′; when referring to amino acid sequences, the direction is from left (upstream) to right (downstream) amino (N) to carboxyl (C).

[0029] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and are defined as biomolecules composed of amino acid residues linked together by peptide bonds.

[0030] As used in this article, "pH-dependent binding" refers to the ability of a protein (e.g., SIRPα) to bind to its ligand (e.g., CD47) with greater binding force at acidic pH than at neutral pH.

[0031] As used herein, “acidic pH” means a pH of about 6.6 or lower (e.g., pH 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5 or lower, or any value in between); “neutral pH” means a pH of about 7.0 to about 7.4 (e.g., 7.0, 7.1, 7.2, 7.3, 7.4 or any value in between).

[0032] The specific binding force between two proteins can be determined using methods known in the art. For example, the receptor-ligand binding activity can be determined by measuring the rate of formation and dissociation of the protein receptor-ligand complex. Both the "binding rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation. The ratio of kdis / kon is equal to the dissociation constant KD. KD, kon, and kdis values ​​can be measured using any effective method. The smaller the equilibrium dissociation constant KD, the tighter the receptor-ligand binding, the higher the affinity between the receptor and ligand, and the stronger the binding ability.

[0033] In some embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In some embodiments, the binding force between a protein and its ligand is determined by detecting the EC50 and / or Emax (also known as the TOP) values ​​of the protein (e.g., SIRPα) binding to its ligand (e.g., CD47). The “EC50” value refers to the half-maximum effective concentration of the substance; the “Emax” value refers to the maximum effect achievable by the substance. In some embodiments, the EC50 value is determined by measuring the half-maximum binding concentration using the fluorescence intensity of the medium, and then nonlinearly fitting the sigmoid dose response within GraphPadPrism software; the Emax value is determined by nonlinear regression analysis of a four-parameter logarithmic equation (DeLean, A., A.A. Hancock, and R.J. Lefkowitz, Mol. Pharmacol. 21:5-16 (1981)).

[0034] This invention, based on extensive research, develops a pH-dependent binding mutant of SIRPαV, which, compared to the parental wild-type SIRPαV protein, possesses one or more histidine (His, H) mutations. Histidine is a positively charged basic amino acid, serving as both a proton donor and acceptor. Its side-chain imidazole group has a pKa of approximately 6.0. Under acidic conditions, histidine undergoes protonation. When key histidine residues at the protein-protein interaction interface are protonated, it can potentially affect the direct binding of the protein receptor to its ligand. If the histidine residue is a backbone amino acid maintaining protein conformation, protonation of this backbone amino acid residue can cause conformational changes and affect the binding of the protein receptor to its ligand. By introducing an appropriate number of histidine residues at suitable positions in the protein, a protein mutant with pH-dependent binding characteristics can be constructed. The positions of the histidine-mutated amino acid residues were determined using computational methods. Based on the crystal structure of the CD47 and SIRPα complex (PDB ID: 2JJS), structural and binding analyses were performed using computational methods such as molecular dynamics simulations (e.g., Amber, see https: / / ambermd.org / index.php) supplemented by 3D visualization software (e.g., PyMol, see https: / / pymol.org). Key amino acids affecting binding were identified from multiple perspectives, including residue energy contribution and spatial relationship. Potential mutation sites that may produce pH-dependent binding were selected, and random combinations of mutations were performed on the selected sites. The fusion protein SIRPαV-Fc (a fusion protein constructed from SIRPαV and Fc) containing the SIRPαV mutant was prepared using Expi293 cells.

[0035] In some implementation schemes, the CD47 binding of the prepared fusion proteins was verified by ELISA. Experiments showed that the screened SIRPαV-Fc fusion proteins maintained a strong binding to CD47 protein under acidic conditions, while their binding ability to CD47 decreased significantly at pH 7.4; furthermore, the binding ability significantly increased with decreasing pH (from pH 7.4 to pH 6.0). FACS experiments confirmed that the SIRPαV-Fc fusion proteins exhibited the same pH-dependent binding in CD47-highly expressed cell lines. Simultaneously, target blocking experiments demonstrated that this series of SIRPαV-Fc fusion proteins showed weak blocking activity against the binding of wild-type SIRPα and CD47 under physiological pH conditions, but exhibited a certain blocking ability at pH 6.0.

[0036] In some embodiments, the SIRPαV mutant proteins of the present invention exhibit a lower affinity for their ligand CD47 under physiological pH conditions (i.e., pH = 7.4) than under acidic conditions (e.g., pH = 6.0), reducing non-specific binding to blood cells and thus lowering the hematologic cytotoxicity of the drug. Notably, as the ambient pH decreases from neutral to acidic, the binding affinity of these SIRPαV mutant proteins to CD47 significantly increases, a characteristic that makes them more selective in the acidic tumor microenvironment. Therefore, these SIRPαV mutant proteins have potential therapeutic advantages in targeting tumor cells while minimizing the impact on normal cells.

[0037] In a first aspect, the present invention provides an immunoglobulin-like variable domain (SIRPαV) mutant of signal regulatory protein α (SIRPα), which, relative to the wild-type SIRPαV domain, contains histidine (H) at at least one position corresponding to amino acid positions 29, 31, 36, 37, 51, 52, 53, 68, 69, 95 and 99 as shown in SEQ ID NO:1.

[0038] In one embodiment, the SIRPαV contains histidine (H) at at least one position corresponding to amino acid positions 37, 51, 95 and 99 shown in SEQ ID NO:1.

[0039] As used in this article, "SIRPα" refers to signal regulatory protein α. SIRPα is a transmembrane protein widely expressed on the surface of myeloid cells such as macrophages and dendritic cells. It belongs to the immunoglobulin superfamily. The complete SIRPα consists of an extracellular region, a transmembrane region, and an intracellular region. The extracellular region of SIRPα is composed of a distal Ig variable region-like domain (the immunoglobulin-like variable domain of signal regulatory protein α, SIRPαV) and two proximal Ig constant region-like domains (SIRPαC). The SIRPαV domain of the extracellular region of SIRPα can bind to its ligand CD47. In humans, 10 human SIRPα alleles have been identified (Polymorphismin Sirpα modulates engraftment of human hematopoietic stem cells, NATURE IMMUNOLOGY VOLUME 8NUMBER 12DECEMBER 2007), which encode 10 human SIRPα subtypes (SIRPα1-10). Among them, the most common subtypes are SIRPα1 (whose extracellular SIRPαV domain includes the amino acid sequence SEQ ID NO:41), SIRPα2 (whose extracellular SIRPαV domain includes the amino acid sequence SEQ ID NO:1), and SIRPα8 (whose extracellular SIRPαV domain includes the amino acid sequence SEQ ID NO:51).

[0040] As used herein, “wild-type SIRPα” means wild-type signal regulatory protein α, or a recombinant or non-recombinant polypeptide containing the amino acid sequence of wild-type signal regulatory protein α, or a naturally occurring allele variant of SIRPα. In some embodiments, SIRPα may be derived from humans. In some embodiments, SIRPα may be derived from other animals, including but not limited to immunoglobulin-like variable domains and homologs of the native SIRPα protein derived from humans and non-human mammals (e.g., cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer). As used herein, “SIRPα mutant” means a SIRPα amino acid sequence with one or more amino acid alterations compared to wild-type SIRPα, provided that it still retains the desired binding function with its ligand CD47.

[0041] As used herein, "immunoglobulin-like variable domain of signal regulatory protein α" and "SIRPαV" refer to the Ig-like variable domain at the distal membrane of the extracellular region of SIRPα, which can effectively bind to the ligand CD47. The amino acid sequences of SIRPαV in the most common wild-type SIRPα subtypes, SIRPα1, 2, and 8, are SEQ ID NO:41, SEQ ID NO:1, and SEQ ID NO:51, respectively. As used herein, "SIRPαV mutant" refers to a protein with one or more amino acid alterations compared to wild-type SIRPαV, provided that it still retains the desired binding function to its ligand CD47.

[0042] As used herein, the terms “variant” and “mutant” are used interchangeably and, in the context of a peptide, refer to a peptide containing altered amino acid residues (through amino acid residue substitution, deletion, and / or addition). Amino acid residue substitution can be a conserved substitution that generally does not adversely affect or alter the intended properties of a protein containing the amino acid sequence. For example, conserved substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conserved amino acid substitutions include replacing amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0043] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between a receptor and its ligand. The specific binding properties between two molecules can be determined using methods known in the art. For example, binding activity can be determined by measuring the rates of formation and dissociation of the receptor-ligand complex. Both the "binding rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation. The ratio of kdis / kon is equal to the dissociation constant KD. KD, kon, and kdis values ​​can be measured using any effective method. In some embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In some embodiments, the strength of the specific binding of SIRPαV to human CD47 protein can be represented by the equilibrium dissociation constant (KD) of the receptor-ligand interaction. In some implementations, the protein containing SIRPαV binds to human CD47 with an equilibrium dissociation constant (KD) of about 1000 nM or less (e.g., less than 500 nM, 300 nM, 200 nM, 100 nM, 50 nM, 20 nM, 10 nM, 5 nM, 3 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM or less).

[0044] In this article, "weak binding" refers to a weak binding activity between two protein molecules. For example, the receptor protein SIRPαV binds with a binding activity greater than 1 × 10⁻⁶. -7 M's KD value is weakly bound to the ligand protein CD47, and the KD value can be determined by... The surface plasmon resonance assay was used to measure the intermolecular binding activity. The intermolecular binding activity can also be reflected by detecting the EC50 value, which can be determined by an ELISA method; for example, in some embodiments, the ligand binds weakly to its receptor with an EC50 value greater than 40 nM (e.g., greater than 40 nM, greater than 50 nM, greater than 100 nM, greater than 500 nM, greater than 1000 nM or greater); when the EC50 value is greater than 10000 nM, it is generally considered that there is almost no intermolecular binding.

[0045] The term "blocking the binding of A to B" refers to inhibiting the binding ability between A and B. In some embodiments, the peptide that blocks the binding between A and B inhibits the interaction between A and B by more than 50% (e.g., inhibition of 50%, 60%, 70%, 80%, 90%, 95%, or 100%, or any value between them). Methods for determining the ability of a peptide to block the binding of A to B are well known in the art, for example, by using a biolayer interference assay. Unless otherwise specified herein, when describing amino acid positions, the amino acid sites refer to the corresponding positions in the reference sequence SEQ ID NO:1. The positions in the sequence of interest corresponding to the positions in the reference sequence can be determined by methods known in the art.

[0046] The amino acid position corresponding to the reference sequence (e.g., SEQ ID NO:1) refers to the amino acid position identified when comparing the sequence with the reference sequence using a standard alignment algorithm such as the GAP algorithm to maximize similarity. By aligning the sequences, those skilled in the art can identify the corresponding residues, for example, using conserved / identical amino acid residues as guidance. Typically, to identify the corresponding position, the amino acid sequence is arranged to obtain the highest-order match (see, for example, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carrillo et al. (1988) SIAM J Applied Math 48:1073).

[0047] As used herein, sequence alignment refers to the comparison of two or more amino acid sequences using homology. Typically, two or more sequences with 50% or higher identity are aligned. Related or variant polypeptide molecules can be aligned using any method known to those skilled in the art. Such methods generally require maximizing the match and involve the use of a variety of available algorithms (e.g., BLASTP) and other methods known to those skilled in the art or manual alignment. Through alignment, those skilled in the art can identify corresponding amino acid residues between a sequence and a reference sequence. Corresponding positions can also be based on structural alignment, such as alignment using computer simulations of molecular structures.

[0048] As used herein, the term "identity" or "identity" of a sequence refers to the degree (percentage) to which the amino acids (amino acid sequence identity) or nucleic acids (nucleic acid sequence identity) of two sequences are identical at equivalent positions when two sequences are optimally aligned. To obtain optimal alignment of two sequences, gaps may be introduced if necessary to achieve the maximum percentage of sequence identity. For example, when two sequences are optimally aligned, if a position in the first sequence is occupied by the corresponding amino acid residue or nucleotide in the second sequence, the sequences are identical or identical at that position. In some embodiments, the percentage identity between two sequences is determined by the number of identical positions shared by the sequences: Percentage identity = Number of identical positions / Total number of positions × 100%. The percentage of amino acid sequence identity can be determined using various methods known in the art, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA.

[0049] A sequence having at least 70% (e.g., 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) identity with the parent polypeptide sequence includes polypeptide variants with altered (through amino acid residue substitution, deletion, and / or addition) amino acid sequences (e.g., polypeptide variants obtained through conserved substitution), and also includes modified (e.g., by covalently linking the molecule to the polypeptide) polypeptide variants (e.g., through glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins, etc.). Polypeptide variants can be generated by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. In some embodiments, the peptide variant has a function similar to, identical to, or improved upon that of the peptide from which it is derived (the parent peptide before mutation). As used herein, corresponding to SEQ ID NO:1 means determining the amino acid position of the sequence of interest by performing a maximum match alignment between the sequence of interest (e.g., a wild-type SIRPαV mutant) and the sequence of SEQ ID NO:1.

[0050] In some embodiments, the SIRPαV mutant of the present invention, relative to the wild-type SIRPαV, contains histidine (H) at at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11) position corresponding to amino acid positions 29, 31, 36, 37, 51, 52, 53, 68, 69, 95 and 99 shown in SEQ ID NO:1. In some embodiments, the wild-type SIRPαV comprises: SEQ ID NO:1 or an amino acid sequence having at least 70% (e.g., at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity; SEQ ID NO:41 or an amino acid sequence having at least 70% (e.g., at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity; or SEQ ID NO:1. NO:51 or an amino acid sequence that has at least 70% (e.g., at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with it.

[0051] In some embodiments, the wild-type SIRPαV comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the wild-type SIRPαV comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the wild-type SIRPαV comprises the amino acid sequence of SEQ ID NO:51.

[0052] In some embodiments, the SIRPαV mutant of the present invention, relative to the wild-type SIRPαV, also contains histidine (H) at at least one (e.g., 1, 2, or 3) positions corresponding to amino acid positions 35, 54, and 67 shown in SEQ ID NO:1.

[0053] In some embodiments, the SIRPαV mutant of the present invention, relative to the wild-type SIRPαV, contains histidine (H) at at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) position corresponding to amino acid positions 36, 37, 51, 52, 53, 68, 69, 95 and 99 shown in SEQ ID NO:1.

[0054] In some embodiments, the SIRPαV mutant of the present invention, relative to the wild-type SIRPαV, contains histidine (H) at at least one (e.g., 1, 2, 3, 4, 5 or 6) position corresponding to amino acid positions 51, 52, 53, 68, 95 and 99 shown in SEQ ID NO:1.

[0055] In some embodiments, the SIRPαV mutant of the present invention, relative to wild-type SIRPαV, contains histidine (H) at at least one (e.g., 1, 2, 3, 4 or 5) position corresponding to amino acid positions 51, 52, 68, 95 and 99 shown in SEQ ID NO:1.

[0056] In some embodiments, the SIRPαV mutant of the present invention contains histidine (H) at at least one (e.g., 1, 2, 3 or 4) position corresponding to amino acid positions 37, 51, 95 and 99 shown in SEQ ID NO:1, relative to wild-type SIRPαV.

[0057] In some embodiments, the SIRPαV mutant of the present invention contains histidine (H) at at least one (e.g., 1, 2, or 3) positions corresponding to amino acid positions 51, 95, and 99 shown in SEQ ID NO:1, relative to wild-type SIRPαV.

[0058] In some embodiments, the SIRPαV mutant of the present invention contains histidine (H) at at least one (e.g., one or two) positions corresponding to amino acid positions 95 and 99 shown in SEQ ID NO:1, relative to wild-type SIRPαV.

[0059] In some embodiments, the SIRPαV mutant of the present invention contains histidine (H) at position 95 corresponding to the amino acid shown in SEQ ID NO:1, relative to the wild-type SIRPαV.

[0060] In one embodiment, the SIRPαV variant of the present invention contains histidine (H) at at least one of the amino acid positions 29, 31, 35, 36, 52, 53, 54, 67, 68, and 69 shown in SEQ ID NO:1, for example, histidine (H) is contained at any 1, 2, 3, 4, 5, 6, 7, 8, 9, or all 10 positions. In a further embodiment, the SIRPαV variant contains histidine (H) at at least one of the amino acid positions 29, 31, 52, 53, 54, 67, 68, and 69 shown in SEQ ID NO:1, for example, histidine (H) is contained at any 1, 2, 3, 4, 5, 6, 7, or all 8 positions.

[0061] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at at least one position corresponding to amino acid positions 37, 51, and 95 shown in SEQ ID NO:1, and at at least one position corresponding to amino acid positions 29, 31, 52, 54, 67, and 68 shown in SEQ ID NO:1, for example, at any 1, 2, 3, 4, 5, or 6 positions, preferably at 1, 2, 3, or 4 positions.

[0062] In some embodiments, the SIRPαV variant contains histidine at position 95 corresponding to amino acid position 95 shown in SEQ ID NO:1, and contains histidine at at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) of amino acid positions 35, 36, 37, 51, 52, 53, 54, 67, 68, 69, and 99 shown in SEQ ID NO:1.

[0063] In some embodiments, the SIRPαV variant contains histidine at position 95 corresponding to amino acid position 95 shown in SEQ ID NO:1, and at at least one (e.g., 1, 2, 3, 4, or 5) of positions corresponding to amino acid positions 37, 51, 52, 68, and 99 shown in SEQ ID NO:1.

[0064] In some embodiments, the SIRPαV variant contains histidine at position 95 corresponding to amino acid position 95 shown in SEQ ID NO:1, and at at least one (e.g., 1, 2, 3, or 4) of positions corresponding to amino acid positions 37, 51, 52, and 68 shown in SEQ ID NO:1.

[0065] In some embodiments, the SIRPαV variant contains histidine at position 95 corresponding to amino acid position 95 shown in SEQ ID NO:1, and at at least one (e.g., 1, 2, or 3) of positions corresponding to amino acid positions 51, 52, and 68 shown in SEQ ID NO:1.

[0066] In some embodiments, the SIRPαV variant contains histidine at at least one position corresponding to amino acid positions 29 and 31 shown in SEQ ID NO:1.

[0067] In some embodiments, the SIRPαV variant contains histidine at position 29 corresponding to the amino acid shown in SEQ ID NO:1.

[0068] In some embodiments, the SIRPαV variant contains histidine at position 31 corresponding to the amino acid shown in SEQ ID NO:1.

[0069] In some embodiments, the SIRPαV variant contains histidine at positions 29 and 31 corresponding to the amino acid positions shown in SEQ ID NO:1.

[0070] In some embodiments, the SIRPαV mutant: A) contains histidine at at least one position corresponding to amino acid positions 29 and 31 shown in SEQ ID NO:1, and B) contains histidine at at least one position corresponding to amino acid positions 35, 36, 37, 51, 52, 53, 54, 67, 68, 69, 95, and 99 shown in SEQ ID NO:1.

[0071] In some embodiments, the SIRPαV mutant contains histidine at position 95 corresponding to amino acid position SEQ ID NO:1, contains histidine at at least one of positions corresponding to amino acid positions 36, 37, 51, 52, 53, 68, 69 and 99 corresponding to amino acid positions SEQ ID NO:1, and contains histidine at at least one of positions corresponding to amino acid positions 29 and 31 corresponding to amino acid positions SEQ ID NO:1.

[0072] In some embodiments, the SIRPαV mutant: A) contains histidine at position 95 corresponding to amino acid position SEQ ID NO:1, B) contains histidine at at least one position corresponding to amino acid positions 37, 51, 52 and 68 corresponding to amino acid positions SEQ ID NO:1, and C) contains histidine substitution at at least one position corresponding to amino acid positions 29 and 31 corresponding to amino acid positions SEQ ID NO:1.

[0073] In some embodiments, the SIRPαV mutant contains histidine at position 95 corresponding to amino acid position SEQ ID NO:1, contains histidine at two positions corresponding to amino acid positions 51, 52 and 68 corresponding to amino acid positions SEQ ID NO:1, and contains histidine at at least one position corresponding to amino acid positions 29 and 31 corresponding to amino acid positions SEQ ID NO:1.

[0074] In some embodiments, the amino acid residue at position 100 of SEQ ID NO:1 in the SIRPαV mutant is not histidine (H). In some embodiments, the amino acid residue at position 100 of SEQ ID NO:1 in the SIRPαV mutant is alanine (D).

[0075] In some embodiments, the SIRPαV mutant has histidine at the following position corresponding to the amino acid position shown in SEQ ID NO:1:

[0076] A1) 29, 37, 52, 53 and 68;

[0077] A2) 52 and 95;

[0078] A3) 31, 37, 51 and 95;

[0079] A4) 31, 52, 53 and 95;

[0080] A5) 31, 37, 51, 54 and 95;

[0081] A6) 29, 31, 51, 67, 68 and 95;

[0082] A7) 37, 52, 68, 69 and 95;

[0083] A8) 31, 37, 51, 52, 54 and 95;

[0084] A9)29, 52 and 95;

[0085] A10) 31, 52 and 95;

[0086] A11) 37, 52 and 95;

[0087] A12)51, 52 and 95;

[0088] A13) 52, 67 and 95;

[0089] A14) 52, 68 and 95;

[0090] A15) 29, 31, 52 and 95;

[0091] A16) 29, 37, 52 and 95;

[0092] A17)29, 51, 52 and 95;

[0093] A18)29, 52, 67 and 95;

[0094] A19)29, 52, 68 and 95;

[0095] A20) 31, 37, 52 and 95;

[0096] A21)31, 51, 52 and 95;

[0097] A22) 31, 52, 67 and 95;

[0098] A23) 31, 52, 68 and 95;

[0099] A24) 37, 51, 52 and 95;

[0100] A25) 37, 52, 67 and 95;

[0101] A26) 37, 52, 68 and 95;

[0102] A27)51, 52, 67 and 95;

[0103] A28) 51, 52, 68 and 95;

[0104] A29) 52, 67, 68 and 95;

[0105] A30) 31, 51, 68 and 95;

[0106] A31) 31, 51, 52 and 68;

[0107] A32) 29, 51, 68 and 95;

[0108] A33)29, 51, 52 and 68;

[0109] A34) 29, 31, 68 and 95;

[0110] A35) 29, 31, 51 and 95;

[0111] A36) 29, 31, 51 and 52;

[0112] A37)29;

[0113] A38)31;

[0114] A39)36;

[0115] A40)37;

[0116] A41)51;

[0117] A42)52;

[0118] A43)53;

[0119] A44)68;

[0120] A45)69;

[0121] A46)95; or

[0122] A47)99.

[0123] In one embodiment, the SIRPαV mutant contains histidine residues at positions 31, 52, 68, and 95 corresponding to the amino acid positions shown in SEQ ID NO:1, relative to the wild-type SIRPαV domain.

[0124] In one embodiment, the SIRPαV mutant contains histidine residues at positions 29, 31, 68, and 95 corresponding to the amino acid positions shown in SEQ ID NO:1, relative to the wild-type SIRPαV domain.

[0125] In one embodiment, the SIRPαV mutant contains histidine residues at positions 31, 51, 68, and 95 corresponding to the amino acid positions shown in SEQ ID NO:1, relative to the wild-type SIRPαV domain.

[0126] In one embodiment, the SIRPαV mutant contains histidine residues at positions 31, 37, 52, and 95 corresponding to the amino acid positions shown in SEQ ID NO:1, relative to the wild-type SIRPαV domain.

[0127] In one embodiment, the SIRPαV mutant contains histidine (H) at the amino acid position corresponding to the amino acid shown in SEQ ID NO:1, relative to the wild-type SIRPαV domain:

[0128] i)52, 95;

[0129] ii)29, 31, 51, 67, 68, 95;

[0130] iii)31, 37, 51, 52, 54, 95;

[0131] iv)29, 52, 68, 95;

[0132] v)31, 37, 52, 95;

[0133] vi) 31, 52, 68, 95;

[0134] vii)51, 52, 68, 95;

[0135] viii) 29, 31, 68, 95; or

[0136] ix)52, 68, 95.

[0137] In one embodiment, the SIRPαV variant contains histidine (H) at position 37 corresponding to amino acid position shown in SEQ ID NO:1, relative to the wild-type SIRPαV domain.

[0138] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at position 37 corresponding to amino acid position 37 of SEQ ID NO:1, and contains histidine (H) at at least one of the following positions corresponding to amino acid positions 29, 31, 35, 36, 51, 52, 53, 54, 67, 68, 69, 95, 99 of SEQ ID NO:1, for example, at any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all 13 positions, preferably at 2, 3, 4 or 5 positions.

[0139] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at position 37 corresponding to amino acid position 37 shown in SEQ ID NO:1, and contains histidine (H) at at least one of the following positions corresponding to amino acid positions 29, 31, 51, 52, 53, 54, 67, 68, 69, 95 shown in SEQ ID NO:1, for example, at any 2, 3, 4, 5, 6, 7, 8, 9 or all 10 positions, preferably at 2, 3, 4 or 5 positions.

[0140] In one embodiment, the SIRPαV variant contains histidine (H) at position 37 corresponding to amino acid position 37 of SEQ ID NO:1, relative to the wild-type SIRPαV domain, and also contains histidine (H) at positions corresponding to amino acid positions shown in SEQ ID NO:1 as follows:

[0141] i)29, 52, 53 and 68;

[0142] ii) 31, 51 and 95;

[0143] iii) 31, 51, 54 and 95;

[0144] iv) 52, 68, 69 and 95;

[0145] v)31, 51, 52, 54 and 95;

[0146] vi) 52 and 95;

[0147] vii)29, 52 and 95;

[0148] viii)31, 52 and 95;

[0149] ix)51, 52 and 95;

[0150] x)52, 67 and 95; or

[0151] xi)52, 68 and 95.

[0152] In one embodiment, the SIRPαV variant contains histidine (H) at position 51 corresponding to amino acid position 51 shown in SEQ ID NO:1, relative to the wild-type SIRPαV domain.

[0153] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at position 51 corresponding to amino acid position 51 shown in SEQ ID NO:1, and contains histidine (H) at at least one of the following positions corresponding to amino acid positions 29, 31, 35, 36, 37, 52, 53, 54, 67, 68, 69, 95, 99 shown in SEQ ID NO:1, for example, at any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all 13 positions, preferably at 2, 3, 4 or 5 positions.

[0154] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at position 51 corresponding to amino acid position 51 shown in SEQ ID NO:1, and contains histidine (H) at at least one of positions corresponding to amino acid positions 29, 31, 37, 52, 54, 67, 68, and 95 shown in SEQ ID NO:1, for example, at any 2, 3, 4, 5, 6, 7, or all 8 positions, preferably at 2, 3, 4, or 5 positions.

[0155] In one embodiment, the SIRPαV variant contains histidine (H) at position 51 corresponding to amino acid position 51 shown in SEQ ID NO:1, relative to the wild-type SIRPαV domain, and also contains histidine (H) at positions corresponding to amino acid positions shown in SEQ ID NO:1 as follows:

[0156] i)31, 37 and 95;

[0157] ii) 31, 37, 54 and 95;

[0158] iii) 29, 31, 67, 68 and 95;

[0159] iv) 31, 37, 52, 54 and 95;

[0160] v)52 and 95;

[0161] vi) 29, 52 and 95;

[0162] vii)31, 52 and 95;

[0163] viii) 37, 52 and 95;

[0164] ix)52, 67 and 95;

[0165] x)52, 68 and 95;

[0166] xi)31, 68 and 95;

[0167] xii)31, 52 and 68;

[0168] xiii) 29, 68 and 95;

[0169] xiv)29, 52 and 68;

[0170] xv)29, 31 and 95; or

[0171] xvi)29, 31 and 52.

[0172] In one embodiment, the SIRPαV variant contains histidine (H) at position 95 corresponding to the amino acid position shown in SEQ ID NO:1, relative to the wild-type SIRPαV domain.

[0173] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at position 95 corresponding to amino acid position 95 of SEQ ID NO:1, and contains histidine (H) at at least one of the following positions corresponding to amino acid positions 29, 31, 35, 36, 37, 51, 52, 53, 54, 67, 68, 69, and 99 of SEQ ID NO:1, for example, at any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all 13 positions, preferably at 1, 2, 3, 4, or 5 positions.

[0174] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at position 95 corresponding to amino acid position 95 of SEQ ID NO:1, and contains histidine (H) at at least one of positions corresponding to amino acid positions 29, 31, 37, 51, 52, 54, 67, 68, and 69 of SEQ ID NO:1, for example, at any 2, 3, 4, 5, 6, 7, 8, or all 9 positions, preferably at 1, 2, 3, 4, or 5 positions.

[0175] In one embodiment, the SIRPαV variant contains histidine (H) at position 95 corresponding to amino acid position shown in SEQ ID NO:1, relative to the wild-type SIRPαV domain, and also contains histidine (H) at positions corresponding to amino acid positions shown in SEQ ID NO:1 as follows:

[0176] i)52;

[0177] ii) 31, 37 and 51;

[0178] iii) 31, 52 and 53;

[0179] iv) 31, 37, 51 and 54;

[0180] v)29, 31, 51, 67 and 68;

[0181] vi) 37, 52, 68 and 69;

[0182] vii)31, 37, 51, 52 and 54;

[0183] viii)29 and 52;

[0184] ix)31 and 52;

[0185] x)37 and 52;

[0186] xi)51 and 52;

[0187] xii)52 and 67;

[0188] xiii) 52 and 68;

[0189] xiv)29, 31 and 52;

[0190] xv)29, 37 and 52;

[0191] xvi)29, 51 and 52;

[0192] xvii)29, 52 and 67;

[0193] xviii)29, 52 and 68;

[0194] xix)31, 37 and 52;

[0195] xx)31, 51 and 52;

[0196] xxi)31, 52 and 67;

[0197] xxii)31, 52 and 68;

[0198] xxiii)37, 51 and 52;

[0199] xxiv)37, 52 and 67;

[0200] xxv)37, 52 and 68;

[0201] xxvi)51, 52 and 67;

[0202] xxvii)51, 52 and 68;

[0203] xxviii)52, 67 and 68;

[0204] xxix)31, 51 and 68;

[0205] xxx)29, 51 and 68;

[0206] xxxi)29, 31 and 68; or

[0207] xxxii)29, 31 and 51.

[0208] In one embodiment, the SIRPαV variant contains histidine (H) at position 99 corresponding to amino acid position shown in SEQ ID NO:1, relative to the wild-type SIRPαV domain.

[0209] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at position 99 corresponding to amino acid position 99 of SEQ ID NO:1, and contains histidine (H) at at least one of the following positions corresponding to amino acid positions 29, 31, 35, 36, 37, 51, 52, 53, 54, 67, 68, 69, 95 of SEQ ID NO:1, for example, at any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, preferably at 1, 2, 3, 4, or 5.

[0210] In one embodiment, the SIRPαV variant contains histidine (H) at positions 37 and 51 corresponding to the amino acid positions shown in SEQ ID NO:1, relative to the wild-type SIRPαV domain.

[0211] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at positions corresponding to amino acid positions 37 and 51 shown in SEQ ID NO:1, and contains histidine (H) at at least one of the amino acid positions 29, 31, 35, 36, 52, 53, 54, 67, 68, 69, 95, 99 shown in SEQ ID NO:1, for example, at any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 positions, preferably 2, 3, 4 or 5 positions.

[0212] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at positions corresponding to amino acid positions 37 and 51 shown in SEQ ID NO:1, and contains histidine (H) at at least one of positions corresponding to amino acid positions 31, 52, 54, 68, and 95 shown in SEQ ID NO:1, for example, at any 2, 3, 4, or 5 positions, preferably at 2, 3, or 4 positions.

[0213] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at positions corresponding to amino acid positions 37 and 51 as shown in SEQ ID NO:1, and also contains histidine (H) at positions corresponding to the amino acid positions shown in SEQ ID NO:1 as follows:

[0214] i)31 and 95;

[0215] ii) 31, 54 and 95;

[0216] iii) 31, 52, 54 and 95; or

[0217] iv)52 and 95.

[0218] In one embodiment, the SIRPαV variant contains histidine (H) at positions corresponding to amino acid positions 37 and 95 as shown in SEQ ID NO:1, relative to the wild-type SIRPαV domain.

[0219] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at positions corresponding to amino acid positions 37 and 95 shown in SEQ ID NO:1, and contains histidine (H) at at least one of the amino acid positions 29, 31, 35, 36, 51, 52, 53, 54, 67, 68, 69, and 99 shown in SEQ ID NO:1, for example, at any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all 12 positions, preferably at 2, 3, 4, or 5 positions.

[0220] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at positions corresponding to amino acid positions 37 and 95 shown in SEQ ID NO:1, and at at least one position corresponding to amino acid positions 29, 31, 51, 52, 54, 67, 68, and 69 shown in SEQ ID NO:1, for example, at any 2, 3, 4, 5, 6, 7, or 8 positions, preferably at 1, 2, 3, or 4 positions.

[0221] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at positions corresponding to amino acid positions 37 and 95 as shown in SEQ ID NO:1, and also contains histidine (H) at positions corresponding to the amino acid positions shown in SEQ ID NO:1 as follows:

[0222] i)31 and 51;

[0223] ii) 31, 51 and 54;

[0224] iii) 52, 68, and 69;

[0225] iv)31, 51, 52 and 54;

[0226] v)52;

[0227] vi)29 and 52;

[0228] vii)31 and 52;

[0229] viii)51 and 52;

[0230] ix)52 and 67; or

[0231] x)52 and 68.

[0232] In one embodiment, the SIRPαV variant contains histidine (H) at positions 51 and 95 corresponding to the amino acid positions shown in SEQ ID NO:1, relative to the wild-type SIRPαV domain.

[0233] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at positions corresponding to amino acid positions 51 and 95 shown in SEQ ID NO:1, and contains histidine (H) at at least one of the amino acid positions 29, 31, 35, 36, 37, 52, 53, 54, 67, 68, 69, and 99 shown in SEQ ID NO:1, for example, at any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all 12 positions, preferably at 2, 3, 4, or 5 positions.

[0234] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at positions corresponding to amino acid positions 51 and 95 shown in SEQ ID NO:1, and contains histidine (H) at at least one of the positions corresponding to amino acid positions 29, 31, 37, 52, 54, 67, and 68 shown in SEQ ID NO:1, for example, at any 2, 3, 4, 5, 6, or 7 positions, preferably at 1, 2, 3, or 4 positions.

[0235] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at positions corresponding to amino acid positions 51 and 95 as shown in SEQ ID NO:1, and also contains histidine (H) at positions corresponding to the amino acid positions shown in SEQ ID NO:1 as follows:

[0236] i)31 and 37;

[0237] ii) 31, 37, and 54;

[0238] iii) 29, 31, 67 and 68;

[0239] iv) 31, 37, 52 and 54;

[0240] v)52;

[0241] vi)29 and 52;

[0242] vii)31 and 52;

[0243] viii)37 and 52;

[0244] ix)52 and 67;

[0245] x)52 and 68;

[0246] xi)31 and 68; or

[0247] xii)29 and 31.

[0248] In one embodiment, the SIRPαV variant contains histidine (H) at positions corresponding to amino acid positions 37, 51, and 95 as shown in SEQ ID NO:1, relative to the wild-type SIRPαV domain.

[0249] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at positions corresponding to amino acid positions 37, 51, and 95 shown in SEQ ID NO:1, and at at least one position corresponding to amino acid positions 29, 31, 35, 36, 52, 53, 54, 67, 68, 69, and 99 shown in SEQ ID NO:1, for example, at any 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 positions, preferably at 2, 3, 4, or 5 positions.

[0250] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at positions corresponding to amino acid positions 37, 51, and 95 shown in SEQ ID NO:1, and contains histidine (H) at at least one position corresponding to amino acid positions 31, 52, and 54 shown in SEQ ID NO:1, for example, containing histidine (H) at any one, two, or three of these positions.

[0251] In one embodiment, the SIRPαV variant, relative to the wild-type SIRPαV domain, contains histidine (H) at positions corresponding to amino acid positions 37, 51, and 95 as shown in SEQ ID NO:1, and also contains histidine (H) at positions corresponding to the amino acid positions shown in SEQ ID NO:1 as follows:

[0252] i)31;

[0253] ii) 31 and 54;

[0254] iii) 31, 52 and 54; or

[0255] iv)52.

[0256] In one embodiment, the SIRPαV mutant contains histidine at at least one position corresponding to amino acid positions 29 and 31 shown in SEQ ID NO:1, relative to the wild-type SIRPαV domain.

[0257] In one embodiment, the SIRPαV variant: A) contains histidine at at least one position corresponding to amino acid positions 29 and 31 shown in SEQ ID NO:1, and B) contains histidine at at least one position corresponding to amino acid positions 35, 36, 37, 51, 52, 53, 54, 67, 68, 69, 95 and 99 shown in SEQ ID NO:1.

[0258] In one embodiment, the SIRPαV variant: A) contains histidine at position 95 corresponding to amino acid position SEQ ID NO:1, B) contains histidine at at least one of positions corresponding to amino acid positions 36, 37, 51, 52, 53, 68, 69 and 99 corresponding to amino acid positions SEQ ID NO:1, and C) contains histidine at at least one of positions corresponding to amino acid positions 29 and 31 corresponding to amino acid positions SEQ ID NO:1;

[0259] In one embodiment, the SIRPαV variant contains histidine at position 95 corresponding to amino acid position SEQ ID NO:1, contains histidine at at least one of positions corresponding to amino acid positions 37, 51, 52 and 68 corresponding to amino acid positions SEQ ID NO:1, and contains histidine at at least one of positions corresponding to amino acid positions 29 and 31 corresponding to amino acid positions SEQ ID NO:1.

[0260] In one embodiment, the SIRPαV variant contains histidine at position 95 corresponding to amino acid position SEQ ID NO:1, contains histidine at two positions corresponding to amino acid positions 51, 52 and 68 corresponding to amino acid positions SEQ ID NO:1, and contains histidine at at least one position corresponding to amino acid positions 29 and 31 corresponding to amino acid positions SEQ ID NO:1.

[0261] In one embodiment, the SIRPαV mutant has an alanine (D) amino acid residue at position 100 corresponding to the amino acid shown in SEQ ID NO:1.

[0262] As used herein, "wild-type SIRPαV domain" or "wild-type SIRPαV" refers to the immunoglobulin-like variable domain of a naturally occurring SIRPα protein or its recombinant polypeptide, including but not limited to the immunoglobulin-like variable domain of a natural SIRPα protein derived from humans and non-human mammals (e.g., cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer) and their homologs. Homologs are two or more protein sequences with at least about 70% sequence identity (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher). Preferably, the homologs have the same or similar functions or activities, i.e., the SIRPαV domain homologs of the present invention have the function of binding CD47, and particularly preferably, the homologs have the same or similar spatial structure as the wild-type SIRPαV (SEQ ID NO: 1) of SIRPα2 described in the present invention. In one embodiment, the wild-type SIRPαV domain is selected from SIRPαV of subtypes SIRPα1, SIRPα8, and SIRPα2. In one embodiment, among the wild-type SIRPα subtypes, the SIRPαV domain of subtype SIRPα1 includes the amino acid sequence SEQ ID NO:41; the SIRPαV domain of SIRPα2 includes the amino acid sequence SEQ ID NO:1; and the SIRPαV domain of SIRPα8 includes the amino acid sequence SEQ ID NO:51.

[0263] In one embodiment, the wild-type SIRPαV domain comprises, or is composed of, the amino acid sequence shown in any of SEQ ID NO:1, 41, or 51. In one embodiment, the wild-type SIRPαV domain comprises, or is composed of, an amino acid sequence having at least about 70% (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher) sequence identity with, the amino acid sequence shown in any of SEQ ID NO:1. In one embodiment, the amino acid sequence of the wild-type SIRPαV domain is as shown in SEQ ID NO:1.

[0264] In one embodiment, the SIRPαV variant contains histidine (H) at a position corresponding to the amino acid position shown in SEQ ID NO:1 below:

[0265] i)29, 37, 52, 53 and 68;

[0266] ii) 52 and 95;

[0267] iii) 31, 37, 51 and 95;

[0268] iv)31, 52, 53 and 95;

[0269] v)31, 37, 51, 54 and 95;

[0270] vi) 29, 31, 51, 67, 68 and 95;

[0271] vii)37, 52, 68, 69 and 95;

[0272] viii) 31, 37, 51, 52, 54 and 95;

[0273] ix)29, 52 and 95;

[0274] x)31, 52 and 95;

[0275] xi)37, 52 and 95;

[0276] xii)51, 52 and 95;

[0277] xiii) 52, 67 and 95;

[0278] xiv)52, 68 and 95;

[0279] xv)29, 31, 52 and 95;

[0280] xvi)29, 37, 52 and 95;

[0281] xvii)29, 51, 52 and 95;

[0282] xviii)29, 52, 67 and 95;

[0283] xix)29, 52, 68 and 95;

[0284] xx)31, 37, 52 and 95;

[0285] xxi)31, 51, 52 and 95;

[0286] xxii)31, 52, 67 and 95;

[0287] xxiii)31, 52, 68 and 95;

[0288] xxiv)37, 51, 52 and 95;

[0289] xxv)37, 52, 67 and 95;

[0290] xxvi)37, 52, 68 and 95;

[0291] xxvii)51, 52, 67 and 95;

[0292] xxviii)51, 52, 68 and 95;

[0293] xxix)52, 67, 68 and 95;

[0294] xxx)31, 51, 68 and 95;

[0295] xxxi)31, 51, 52 and 68;

[0296] xxxii)29, 51, 68 and 95;

[0297] xxxiii)29, 51, 52 and 68;

[0298] xxxiv)29, 31, 68 and 95;

[0299] xxxv)29, 51, 68 and 95; or

[0300] xxxvi)29, 31, 51 and 52.

[0301] In some embodiments, the SIRPαV variant has amino acids identical to or with conserved substitutions (i.e., no deletions, additions, or non-conserved mutations) at all positions except those described herein.

[0302] In one embodiment, the SIRPαV variant has at least about 70% (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or higher) sequence identity with the wild-type SIRPαV domain. Specifically, the amino acid residues in the SIRPαV variant that differ from the wild-type SIRPαV domain are substituted with other amino acids at at least one position corresponding to amino acid positions 29, 31, 36, 37, 51, 52, 53, 68, 69, 95, and 99 shown in SEQ ID NO:1 (e.g., at least one position in 36, 37, 51, 52, 53, 68, 69, 95, and 99, optionally also at least one position in 35, 54, and 67; at least one position in 51, 52, 53, 68, 95, and 99; at least one position in 37, 51, 95, and 99; at least one position in 51, 95, and 99; at least one position in 95 and 99; at position 95; or at least one position in 29 or 31). In some embodiments, the amino acid substitution is a histidine substitution.

[0303] In one embodiment, the SIRPαV variant differs from the wild-type SIRPαV domain in having one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35). Specifically, the amino acid residues that distinguish the SIRPαV variant from the wild-type SIRPαV domain are at least one histidine residue among positions 29, 31, 36, 37, 51, 52, 53, 68, 69, 95, and 99, optionally including at least one histidine residue among positions 35, 54, and 67.

[0304] In one embodiment, the SIRPαV mutant described in any of the preceding claims comprises an amino acid sequence such as SEQ ID NO:61 or having at least 85% (e.g., approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with it, wherein the amino acid sequence of SEQ ID NO:61 is: EEELQVIQPDKSVSVAAGESAILHCTVTX1LX2PVGX3X4X5WFRGAGPARELIYX6X7X8X9GHFPRVTTVSESX 10 X 11 X 12 ENMDFSISISNITPADAGTYYCVKFX 13KGSX 14 DTEFKSGAGTELSVRAKPS, where X1 is S or H; X2 is I or H; X3 is P or H; X4 is I or H; X5 is Q or H; X6 is N or H; X7 is Q or H; X8 is K or H; X9 is E or H; X 10 For T or H; X 11 For K or H; X 12 For R or H; X 13 For R or H; X 14 For P or H, and X1-X 14 At least one amino acid residue is H.

[0305] In one implementation, X1, X2, X4-X8 and X 11 -X 14 At least one amino acid residue is H. In one embodiment, X1, X2, X4-X8, and X... 11 -X 14 The amino acid residues at positions 1, 2, 3, 4, 5, or 6 are H. In one embodiment, X1, X2, X4-X8, and X... 11 -X 14 The amino acid residues at the four positions in it are H.

[0306] In one embodiment, at least one amino acid residue at positions X1 and X2 is H.

[0307] In one implementation scheme, X4-X8, X 11 -X 14 At least one amino acid residue at the position is H.

[0308] In one implementation scheme, X6, X7, X8, X 11 X 13 and X 14 At least one amino acid residue at the position is H.

[0309] In one implementation scheme, X5, X6, X 13 and X 14 At least one amino acid residue at the position is H. In one embodiment, X6, X 13 X 14 At least one amino acid residue at position X is H. In one embodiment, X 13 The amino acid residue at that position is H.

[0310] In one implementation, A)X3-X 14 At least one amino acid residue at position X4-X8 is H, and at least one amino acid residue at positions X1 and X2 is H. In one embodiment, X4-X8, X 11-X 12 and X 14 At least one amino acid residue at the position is H.

[0311] In one implementation scheme, A)X4-X8, X 11 -X 12 and X 14 At least one amino acid residue at position X5 is H, preferably X5, X6, X7, or X6. 11 A) At least one amino acid residue at position H; B) At least one amino acid residue in X1 and X2 is H; and C) X 13 The amino acid residue is H.

[0312] In one embodiment, the amino acid sequence of the SIRPαV mutant is shown as any one of the amino acid sequences in SEQ ID NO:3-5, 7, 9-40.

[0313] In one embodiment, the SIRPαV variant comprises, or consists of, the amino acid sequence shown in any of SEQ ID NO:3-5, 7, 9-40, 42-50 or 52-60; or has at least 85% (e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with the amino acid sequence of one of SEQ ID NO:3-5, 7, 9-40, 42-50 or 52-60.

[0314] The present invention also provides an immunoglobulin-like variable domain (SIRPαV) variant of signal regulatory protein α (SIRPα), which, relative to the wild-type SIRPαV domain, contains histidine (H) at positions corresponding to amino acid positions 29, 52, 53 and 68 shown in SEQ ID NO:1.

[0315] The present invention also provides an immunoglobulin-like variable domain (SIRPαV) variant of signal regulatory protein α (SIRPα), which, relative to the wild-type SIRPαV domain, contains histidine (H) at positions corresponding to amino acid positions 29, 31, 52 and 67 shown in SEQ ID NO:1.

[0316] In one embodiment, the SIRPαV variant comprises, or consists of, the amino acid sequence shown in either SEQ ID NO:6 or 8.

[0317] In a second aspect, the present invention provides a fusion protein comprising the SIRPαV variant described in any of the preceding claims and an immunoglobulin portion.

[0318] As used herein, the term "immunoglobulin" refers to a protein composed of polypeptides encoded by one or more immunoglobulin genes. Antibodies are the most common immunoglobulins. Common human immunoglobulin genes include κ, λ, α (IgA1 and IgA2), γ (IgG1, IgG2, IgG3, IgG4), δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. The full-length immunoglobulin "light chain" (approximately 25 Dd) is encoded by a variable region gene at the NH2 end (approximately 110 amino acids) and by a κ or λ constant region gene at the COOH end; the full-length immunoglobulin "heavy chain" (approximately 50 Kd) is encoded by a variable region gene at the NH2 end (approximately 116 amino acids) and by a constant region gene (e.g., α, γ, δ, ε, or μ) at the COOH end.

[0319] As used herein, the term "antibody" refers to a protein molecule capable of specifically binding to an antigen. Unless the context clearly indicates otherwise, the term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, full-length antibodies, or their antigen-binding fragments, provided they exhibit the desired antigen-binding activity. A "full-length antibody" typically consists of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). The antibody light chain can be divided into κ (kappa) and λ (lambda) light chains. The heavy chain can be divided into μ, δ, γ, α, or ε, corresponding to the isotype antibodies IgM, IgD, IgG, IgA, and IgE, respectively. The full-length heavy chain consists of a variable region (VH) and a constant region (CH), with the typical IgG heavy chain constant region comprising three domains (CH1, CH2, and CH3). The full-length light chain consists of a variable region (VL) and a constant region (CL). Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions include highly degenerated domains (called hypervariable regions or complementarity-determining regions (CDRs)). More conserved framework regions (FRs) are interspersed between the CDRs. Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from the amino terminus to the carboxyl terminus). The allocation of antibody heavy / light chain amino acids in different regions or domains can follow the definitions of Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.

[0320] As used herein, the term "Fc region" refers to a region of the antibody heavy chain consisting of the CH2 and CH3 domains, or a region of the antibody heavy chain consisting of a hinge or portion thereof and the CH2 and CH3 domains. A typical IgG antibody's Fc region is the region of the heavy chain from cysteine ​​226 (EU system number) to the C-terminus, or from proline 230 (EU system number) to the C-terminus. Fc regions include native Fc regions and modified Fc regions. Native Fc regions contain an amino acid sequence identical to the amino acid sequence of Fc regions found in nature, such as the native human IgG1 Fc region, the native human IgG2 Fc region, the native human IgG3 Fc region, or the native human IgG4 Fc region. Modified Fc regions contain an amino acid sequence that differs from the amino acid sequence of native Fc regions due to at least one amino acid modification. In some implementations, the variant Fc region may have altered functions compared to the native Fc region (e.g., altered Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, complement function, half-life, etc.). For example, substitution of amino acid residues at positions 238, 265, 269, 270, 297, 327, and 329 (EU system number) in the IgG1 Fc region may reduce effector function.

[0321] Effector cell function refers to the biological activity attributable to the Fc region of an antibody and varying with antibody isotypes; examples of effector cell function include, but are not limited to, C1q binding and complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent phagocytosis (ADCP). ADCC is a mechanism that induces cell death by relying on the interaction between antibody-coated target cells and lytic effector cells (such as natural killer (NK) cells, monocytes, macrophages, and neutrophils) via Fcγ receptors expressed on the effector cells (e.g., FcγRIIIa expressed on NK cells, and FcγRI, FcγRII, and FcγRIIIa expressed on monocytes). ADCP is a mechanism by which antibody-coated target cells are eliminated through internalization by phagocytes (such as macrophages or dendritic cells); CDC refers to a mechanism that induces cell death in which the target cell binds to the Fc effector domain of the antibody and activates the complement component C1q, which in turn activates the complement cascade, leading to the death of the target cell. Effector cell function can be detected by methods known in the art. For example, ADCC activity can be determined by known in vitro methods, such as using cells expressing antigens as target cells and NK cells as effector cells, and detecting cell lysis rate based on markers (e.g., radioactive substrates, fluorescent dyes, or native intracellular proteins) released from lysed cells. Other methods for determining ADCC can be found in, for example, WO2006082515, WO2012130831, etc.

[0322] In some embodiments, the Fc region is an Fc region mutant with enhanced effector cell function; in some embodiments, the Fc mutant has enhanced ADCC / ADCP / CDC and / or affinity for the Fcγ receptor compared to wild-type Fc; for example, the Fc mutant can be obtained by amino acid substitutions at one or more of the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 28 9, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439, or see WO200042072 for a description. Furthermore, binding sites for FcγR1, FcγRII, FcγRIII, and FcRn on human IgG1 have been located, and variants with enhanced binding have been described (see Shields et al., 2001 J. Biol. Chem. 276: 6591-6604). Additionally, specific mutations at positions 256, 290, 298, 333, 334, and 339 enhance binding to FcγRIII, for example: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A mutation combinations. In some embodiments, the Fc mutant is the DLE mutation (S239D / A330L / I332E), which enhances binding to FcγRIIIa and enhances the ADCC effect. In some implementations, the Fc region is an IgG1 Fc mutant as shown in SEQ ID NO:66. The mutation sites of the aforementioned Fc mutants are numbered according to the EU index by Kabat et al.

[0323] In one embodiment, the immunoglobulin portion is an antibody or the Fc region of an antibody. In one embodiment, the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4. In one embodiment, the amino acid sequence of the Fc region is as shown in SEQ ID NO:2. In one embodiment, the amino acid sequence of the Fc region is as shown in SEQ ID NO:66. In one embodiment, the SIRPαV variant is directly or via a linker linked to the immunoglobulin portion. In one embodiment, in the fusion protein, the C-terminus of the SIRPαV variant is directly or via a linker linked to the N-terminus of the Fc region. Specifically, the fusion protein comprises, from the N-terminus to the C-terminus, a SIRPαV variant-Fc region or a SIRPαV variant-linker-Fc region. In one embodiment, in the fusion protein, the C-terminus of the SIRPαV variant is directly linked to the N-terminus of the Fc region.

[0324] As used in this article, direct linkage refers to linkage via peptide bonds.

[0325] As used in this article, linker linking refers to linking via peptide linkers.

[0326] As used herein, "linker" or "connector" refers to a peptide composed of amino acids of a certain length (typically amino acids with low hydrophobicity and low charge effect). Linkers, when used in fusion proteins, often allow the linked parts to fully unfold and fold into their respective native conformations without interference. Commonly used spacers in the art include, for example, flexible GS-type linkers rich in glycine (G) and serine (S); and rigid PT-type linkers rich in proline (P) and threonine (T). GS-type linkers are used in some embodiments of the present invention because they possess a suitable amino acid length, exhibit both hydrophobicity and flexibility, and enable functional proteins to have good stability and biological activity.

[0327] Those skilled in the art can determine suitable linkers for use in this invention. The linker preferably does not form an antigenic epitope. Typical amino acid residues used for the linker are glycine, serine, tyrosine, cysteine, lysine, glutamic acid, and aspartic acid, etc. Examples of such known linker portions include, but are not limited to, (GmS)n, where m and n are each independently an integer from 1 to 10, preferably, m and n are each independently 1, 2, 3, 4, 5, or 6.

[0328] In this article, the Fc region includes, but is not limited to, the Fc regions of immunoglobulin molecules IgG1, IgG2, IgG3, and IgG4.

[0329] The Fc region can be derived from any suitable source, such as humans or non-human mammals. In one embodiment, the Fc region is a human IgG Fc region, preferably an IgG1 Fc region. In particular, the Fc contains, or consists of, the amino acid sequence shown in SEQ ID NO:2.

[0330] In some embodiments, the amino acid sequence of the fusion protein has at least 85% (e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity with the amino acid sequence shown in any of SEQ ID NO:62-65 or 67;

[0331] In some embodiments, the fusion protein comprises any of the amino acid sequences shown in SEQ ID NO:62-65 or 67.

[0332] In some embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO:62.

[0333] In some embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO:63.

[0334] In some embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO:64.

[0335] In some embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO:65.

[0336] In some embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO:67.

[0337] In some embodiments, the fusion protein according to the invention may include a signal sequence. The signal sequence is present at the N-terminus of most newly synthesized proteins and is typically cleaved by a signal peptidase to produce a free signal peptide and the mature protein.

[0338] In some embodiments, the fusion protein according to the invention does not contain a signal sequence.

[0339] In some embodiments, the SIRPαV variant or fusion protein described in any of the preceding embodiments has at least one of the following functions:

[0340] A) Possesses the function of binding to human CD47 protein; in some embodiments, the SIRPαV mutant or fusion protein has pH-dependent binding to human CD47 protein; in some embodiments, the SIRPαV mutant or fusion protein has enhanced CD47 binding activity compared to wild-type SIRPαV; in some embodiments, the binding function of the SIRPαV mutant or fusion protein to human CD47 protein at pH=6.0 is the same as its binding function at pH=7.4. The synergistic effect is 1 times or more (e.g., 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 150 times, 200 times or more); in some embodiments, the SIRPαV mutant or fusion Proteins can be expressed at pH 6.0 at concentrations less than 350 nM (e.g., less than 350 nM, less than 300 nM, less than 250 nM, less than 200 nM, less than 100 nM, less than 50 nM, less than 45 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 1 nM, less than 0.8 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM). EC50 values ​​(less than 0.1 nM, less than 0.08 nM, less than 0.06 nM, less than 0.05 nM, less than 0.04 nM, less than 0.02 nM, less than 0.01 nM or smaller) bind to human CD47 protein; in some embodiments, the binding function of the SIRPαV mutant or fusion protein to human CD47 protein is determined by detecting EC50 and / or Emax values ​​using surface plasmon resonance; in some specific embodiments, the EC50 value is determined by the method of Example 3 of this invention;

[0341] B) Possesses the ability to bind to cells expressing human CD47 protein; in some embodiments, the SIRPαV mutant or fusion protein binds to cells expressing human CD47 protein in a pH-dependent manner; in some embodiments, the SIRPαV mutant or fusion protein exhibits pH-dependent binding activity to cells expressing human CD47 protein at pH 6.0 that is more than 1 times greater than that at pH 7.4 (e.g., 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 150 times, 200 times or higher). In some embodiments, the SIRPαV mutant or fusion protein can bind to cells expressing human CD47 protein at an EC50 value of less than 200 nM (e.g., less than 200 nM, less than 150 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.3 nM or less) at pH 6.0; in some embodiments, the EC50 value is determined by flow cytometry fluorescence sorting; in some specific embodiments, the EC50 value is determined by the method of Example 4 of this invention;

[0342] C) Possesses the function of blocking the binding of SIRPα to target cell Raji; in some embodiments, the SIRPαV mutant or fusion protein pH-dependently blocks the binding of SIRPα to target cell Raji; in some embodiments, the IC50 value of the SIRPαV mutant or fusion protein blocking the binding of SIRPα to target cell Raji at pH=7.4 is more than 1 times (e.g., 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or higher) of the IC50 value blocking the binding of SIRPα to target cell Raji at pH=6.0; in some embodiments, the IC50 value is detected by flow cytometry fluorescence sorting; in some specific embodiments, the IC50 value is determined by the method of Example 5 of this invention;

[0343] D) It does not bind or binds weakly to platelets at pH 7.4; in some embodiments, the platelet binding activity is determined by flow cytometry fluorescence sorting; in some specific embodiments, the platelet binding activity is determined by the method of Example 7 of this invention;

[0344] E) It does not bind or binds weakly to erythrocytes at pH 7.4; in some embodiments, the erythrocyte binding activity is determined by flow cytometry fluorescence sorting; in some specific embodiments, the erythrocyte binding activity is determined by the method of Example 6 of this invention; and / or

[0345] F) It mediates macrophage phagocytosis; in some embodiments, the macrophage phagocytosis mediated by the fusion protein at pH 6.0 is stronger than that mediated at pH 7.4; in some specific embodiments, the erythrocyte binding activity is determined by the method of Example 9 of this invention.

[0346] In a third aspect, the present invention provides isolated nucleic acids encoding SIRPαV variants of the first aspect or fusion proteins of the second aspect.

[0347] As used herein, the terms "nucleotide sequence," "polynucleotide," "nucleic acid," and "nucleic acid sequence" are used interchangeably and refer to a molecule composed of multiple nucleotides linked by 3'-5'-phosphodiester bonds, wherein the nucleotides include ribonucleotides and deoxyribonucleotides. The sequences of the polynucleotides of the present invention can be codon-optimized for different host cells (such as *E. coli*) to improve the expression of fusion proteins. Methods for codon optimization are known in the art.

[0348] In one embodiment, the nucleic acid is contained in a nucleic acid construct, such as an expression construct. In the expression nucleic acid construct of the present invention, a sequence of a multinucleotide encoding the SIRPαV variant or fusion protein is operatively linked to an expression control sequence to perform desired transcription and ultimately produce the SIRPαV variant or fusion protein in a host cell. Suitable expression control sequences include, but are not limited to, promoters, enhancers, ribosome-acting sites such as ribosome binding sites, polyadenylation sites, transcription splicing sequences, transcription termination sequences, and sequences stabilizing mRNA, etc.

[0349] Vectors used to construct the expression constructs of the present invention include those that replicate autonomously in host cells, such as plasmid vectors; and also include vectors capable of integrating into and replicating with the host cell DNA. Many commercially available vectors suitable for the present invention are readily available. In one embodiment, the plasmid is suitable for prokaryotic or eukaryotic expression systems. In a specific embodiment, the plasmid is or is derived from pcDNA3.4.

[0350] In some embodiments, the nucleic acid molecule encoding the protein according to the invention is codon-optimized for expression in mammalian cells (e.g., human cells). Methods for codon optimization are known in the art, for example, as described in WO 96 / 09378. A sequence is considered codon-optimized if at least one non-preferred codon is replaced by a more preferred codon compared to a wild-type sequence. Codon usage frequencies for a particular organism can be found in codon frequency tables, such as http: / / www.kazusa.or.jp / codon. In some embodiments, preferred codon replacements result in higher expression.

[0351] Those skilled in the art will understand that, due to the degeneracy of the genetic code, many different polynucleotide and nucleic acid molecules can encode the same protein. Therefore, unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate to each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may or may not include introns.

[0352] Nucleic acid sequences can be generated using conventional molecular biology techniques or regenerated through DNA synthesis.

[0353] The present invention also provides vectors comprising nucleic acid molecules as described above. In some embodiments, the nucleic acid molecule according to the invention is therefore part of the vector. The vector can be readily manipulated by methods well known to those skilled in the art and can, for example, be designed to replicate in prokaryotic and / or eukaryotic cells. Furthermore, many vectors can be used for the transformation of eukaryotic cells and to integrate wholly or partially into the genome of these cells, producing stable host cells whose genomes contain the desired nucleic acid. The vector used can be any vector suitable for cloning DNA and can be used for transcription of nucleic acids for a specific purpose. Suitable vectors according to the invention are, for example, adenovirus vectors, alphaviruses, paramyxoviruses, vaccinia virus, herpesviruses, retroviral vectors, etc. Those skilled in the art can select suitable expression vectors and insert them functionally into the nucleic acid sequences of the invention.

[0354] In a fourth aspect, the present invention provides a host cell comprising the nucleic acid of the third aspect. Specifically, the host cell is capable of expressing the nucleic acid to produce the SIRPαV variant or fusion protein.

[0355] Host cells used to express the fusion protein of the present invention include prokaryotes, yeast, and higher eukaryotic cells. Exemplary prokaryotic hosts include bacteria of the genera *Escherichia*, *Bacillus*, *Salmonella*, *Pseudomonas*, and *Streptomyces*. In a preferred embodiment, the host cell is *Escherichia* cells, mammalian cells (e.g., Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines (e.g., HEK293 cells, PER.C6 cells), or insect cells. In a more preferred embodiment, the host cell is *Escherichia coli*, *Bacillus subtilis*, or *Bacillus megaterium*. In a specific embodiment of the present invention, the host cell used is *Escherichia coli* DH5α strain cells.

[0356] The nucleic acids of the present invention can be introduced into host cells to express the encoded amino acid sequence using one of many well-known techniques, including but not limited to: heat shock conversion, electroporation, DEAE-glucan transfection, microinjection, liposome-mediated transfection, calcium phosphate precipitation, protoplasmic fusion, microparticle bombardment, viral transformation, and similar techniques.

[0357] In one embodiment, the nucleic acid can be integrated into the genome of a host cell, which can express the encoded variant or fusion protein under appropriate conditions or constitutively express the encoded variant or fusion protein.

[0358] In one implementation, the nucleic acid encoding the variant or fusion protein exists in the host cell in an extrachromosomal form (e.g., a plasmid or construct such as an expression vector).

[0359] In a fifth aspect, the present invention provides a pharmaceutical composition comprising a SIRPαV variant of the first aspect, a fusion protein of the second aspect, a nucleic acid of the third aspect, or a host cell of the fourth aspect, optionally comprising a pharmaceutically acceptable carrier. In particular, the pharmaceutical composition is intended for the treatment of cancer.

[0360] "Pharmaceutically acceptable carrier" refers to a substance that facilitates the delivery and absorption of an active substance by a subject, and may be included in the compositions of the present invention without causing significant toxic side effects to the patient. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, physiological saline, sucrose, glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions, alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and colorants, etc. Those skilled in the art will understand that other pharmaceutical carriers may be used in the present invention.

[0361] If desired, the pharmaceutical composition may be contained in a kit, vial, or dispenser, which may, for example, contain one or more unit doses of the SIRPαV variant, fusion protein, nucleic acid, or host cell described in this invention. The kit, vial, or dispenser may be accompanied by instructions for use.

[0362] In a sixth aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need a therapeutically effective amount of the SIRPαV variant, fusion protein, pharmaceutical composition, nucleic acid, or host cell described herein; or providing the use of the SIRPαV variant of the first aspect, the fusion protein of the second aspect, or the nucleic acid of the third aspect, the host cell of the fourth aspect, and the pharmaceutical composition of the fifth aspect in the preparation of a medicament for treating cancer; or providing the SIRPαV variant of the first aspect, the fusion protein of the second aspect, the nucleic acid of the third aspect, the host cell of the fourth aspect, and the pharmaceutical composition of the fifth aspect as a medicament for treating cancer.

[0363] In one embodiment, the cancer is a cancer that highly expresses CD47 on the cell surface, including, but not limited to, solid tumors or metastatic cancer. In some embodiments, the cancer includes, but is not limited to, cancers of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testis, cervix, or liver. In one embodiment, the cancer described herein is a solid tumor, such as lung and bronchial cancer, breast cancer, colon and rectal cancer, kidney cancer, stomach cancer, esophageal cancer, liver and intrahepatic bile duct cancer, bladder cancer, brain and other nervous system cancers, head and neck cancer, oral and pharyngeal cancer, cervical cancer, endometrial cancer, thyroid cancer, ovarian cancer, testicular cancer, prostate cancer, melanoma, bile duct cancer, thymic cancer, skin cancer, and hematologic malignancies, such as leukemias like T-cell lymphoblastic leukemia and lymphoma. In one embodiment, the cancer is selected from melanoma, lung cancer, lymphomas such as B-cell lymphoma, pancreatic cancer, ovarian cancer, hematologic malignancies, colon cancer, prostate cancer, cervical cancer, head and neck cancer, and breast cancer. In some embodiments, the cancer is selected from melanoma, lung cancer, and lymphomas such as B-cell lymphoma. In one embodiment, the cancer is selected from lymphoma and leukemias such as T-cell leukemia.

[0364] As used herein, the term "treatment" refers to the relief of at least one symptom. This term includes administering medication to a subject and / or applying one or more of the SIRPαV variants, fusion proteins or nucleic acids, or host cells and / or pharmaceutical compositions comprising them to provide treatment for a disease. As used herein, "treatment" of a subject with cancer means that the subject's cancer is partially or completely eliminated, or remains stable and no longer progresses after treatment. Treatment includes prevention, treatment, and / or cure. Prevention refers to preventing the occurrence of potential cancer and / or preventing the progression or worsening of cancer; prevention of cancer includes mitigating or eliminating one or more risk factors that contribute to cancer development; because it is generally not possible to determine whether cancer has never occurred, prevention also includes reducing the risk of developing or having cancer. When used herein to address harmful proliferating cells (including cancer), "treatment" includes the partial or complete destruction of said harmful proliferating cells, but with minimal impact on normal cells.

[0365] As used herein, "patient" or "object of need" means an organism that suffers from or is susceptible to a disease or condition that can be treated by administration of the SIRPαV variant, fusion protein, nucleic acid, cell, or pharmaceutical composition provided herein. Non-limiting examples include humans, other mammals such as cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammals. In some embodiments, the patient or object is a human.

[0366] As used herein, “therapeutic effective amount” or “therapeutic effective dose” means an amount of a pharmaceutical agent, compound, or material in a preparation that is at least sufficient to produce a therapeutic effect in a subject. The exact amount depends on the therapeutic purpose and can be determined by a person skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0367] As used herein, the term "administration" means administration by any suitable route, including but not limited to parenteral administration, such as intradermal, intramuscular, subcutaneous, transdermal, or mucosal administration, such as intranasal or oral administration. In one embodiment, the composition is administered by intramuscular injection.

[0368] In a seventh aspect, the present invention provides a method for preparing a SIRPαV variant of the first aspect or a fusion protein of the second aspect, comprising culturing a host cell of the sixth aspect under conditions suitable for the expression of the nucleic acid, and optionally recovering the expressed variant or fusion protein.

[0369] In one embodiment, the method includes: transforming host cells with a nucleic acid from the third aspect of the present invention, culturing the transformed host cells under conditions suitable for the expression of the nucleic acid, and recovering the variant or fusion protein.

[0370] Conditions for protein expression, such as temperature, pH, and culture medium, are known in the art. Any suitable methods for protein recovery are known in the art, including but not limited to chromatography, centrifugation, dialysis, etc.

[0371] As described herein, the recovery of variants or fusion proteins can be performed using any suitable method known in the art. For example, after protein expression, host cells can be collected, cells can be lysed, supernatants can be collected (e.g., by centrifugation to remove cell debris), and the protein can optionally be separated (e.g., by a specific tag or specifically binding molecule such as an antibody).

[0372] In an eighth aspect, the present invention provides a method for causing a protein containing SIRPαV to produce pH-dependent binding to CD47, comprising mutating an amino acid residue in SIRPαV at at least one (e.g., at positions 1, 2, 3, 4, 5, 6, 7, 8 or all nine positions) corresponding to amino acid positions 36, 37, 51, 52, 53, 68, 69, 95 and 99 shown in SEQ ID NO:1 to histidine (H).

[0373] In one embodiment, the amino acid sequence of the SIRPαV comprises, or is composed of, an amino acid sequence having at least about 70% (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher) sequence identity with the amino acid sequence shown in any of SEQ ID NO:1, 41, or 51. In some embodiments, the amino acid sequence of the SIRPαV is as shown in SEQ ID NO:1.

[0374] In one embodiment, the method includes mutating an amino acid residue at at least one position corresponding to amino acid positions 37, 51, 95, and 99 of the immunoglobulin-like variable domain of SIRPα to histidine (H). In one embodiment, the method includes mutating an amino acid residue at at least one position corresponding to amino acid positions 51, 95, and 99 of the immunoglobulin-like variable domain of SIRPα to histidine (H). In one embodiment, the method includes mutating an amino acid residue at at least one position corresponding to amino acid positions 95 and 99 of the immunoglobulin-like variable domain of SIRPα to histidine (H). In one embodiment, the method includes mutating an amino acid residue at position 95 of the immunoglobulin-like variable domain of SIRPα to histidine (H).

[0375] In one embodiment, the method further includes containing histidine (H) at at least one position in the SIRPαV domain corresponding to amino acid positions 29, 31, 35, 54 and 67 shown in SEQ ID NO:1, for example, by mutating any 1, 2, 3, 4 or 5 of the amino acids to histidine (H).

[0376] In a further embodiment, the method includes mutating an amino acid at at least one of the positions corresponding to amino acid positions 29, 31, 52, 53, 54, 67, 68 and 69 of the SIRPαV domain to histidine (H), for example, mutating any 1, 2, 3, 4, 5, 6, 7 or all 8 of these positions to histidine (H).

[0377] In one embodiment, the method includes mutating an amino acid at at least one position in the SIRPαV domain corresponding to amino acid positions 37, 51, and 95 as shown in SEQ ID NO:1 to histidine (H), and mutating an amino acid at at least one position corresponding to amino acid positions 29, 31, 52, 54, 67, and 68 as shown in SEQ ID NO:1 to histidine (H), for example, mutating an amino acid at any 1, 2, 3, 4, 5, or 6 positions, preferably 1, 2, 3, or 4 positions, to histidine (H).

[0378] In one embodiment, the method includes mutating an amino acid in the SIRPαV domain corresponding to the amino acid position shown in SEQ ID NO:1 to histidine (H):

[0379] A1) 29, 37, 52, 53 and 68;

[0380] A2) 52 and 95;

[0381] A3) 31, 37, 51 and 95;

[0382] A4) 31, 52, 53 and 95;

[0383] A5) 31, 37, 51, 54 and 95;

[0384] A6) 29, 31, 51, 67, 68 and 95;

[0385] A7) 37, 52, 68, 69 and 95;

[0386] A8) 31, 37, 51, 52, 54 and 95;

[0387] A9)29, 52 and 95;

[0388] A10) 31, 52 and 95;

[0389] A11) 37, 52 and 95;

[0390] A12)51, 52 and 95;

[0391] A13) 52, 67 and 95;

[0392] A14) 52, 68 and 95;

[0393] A15) 29, 31, 52 and 95;

[0394] A16) 29, 37, 52 and 95;

[0395] A17)29, 51, 52 and 95;

[0396] A18)29, 52, 67 and 95;

[0397] A19)29, 52, 68 and 95;

[0398] A20) 31, 37, 52 and 95;

[0399] A21)31, 51, 52 and 95;

[0400] A22) 31, 52, 67 and 95;

[0401] A23) 31, 52, 68 and 95;

[0402] A24) 37, 51, 52 and 95;

[0403] A25) 37, 52, 67 and 95;

[0404] A26) 37, 52, 68 and 95;

[0405] A27)51, 52, 67 and 95;

[0406] A28) 51, 52, 68 and 95;

[0407] A29) 52, 67, 68 and 95;

[0408] A30) 31, 51, 68 and 95;

[0409] A31) 31, 51, 52 and 68;

[0410] A32) 29, 51, 68 and 95;

[0411] A33)29, 51, 52 and 68;

[0412] A34) 29, 31, 68 and 95;

[0413] A35) 29, 31, 51 and 95;

[0414] A36) 29, 31, 51 and 52;

[0415] A37)36;

[0416] A38)37;

[0417] A39)51;

[0418] A40)52;

[0419] A41)53;

[0420] A42)68;

[0421] A43)69;

[0422] A44)95; or

[0423] A45)99.

[0424] In one embodiment, the method includes mutating an amino acid in the SIRPαV domain corresponding to the amino acid position shown in SEQ ID NO:1 to histidine (H):

[0425] i)52, 95;

[0426] ii)29, 31, 51, 67, 68, 95;

[0427] iii)31, 37, 51, 52, 54, 95;

[0428] iv)29, 52, 68, 95;

[0429] v)31, 37, 52, 95;

[0430] vi) 31, 52, 68, 95;

[0431] vii)51, 52, 68, 95;

[0432] viii) 29, 31, 68, 95; or

[0433] ix)52, 68, 95.

[0434] In one embodiment, the method includes mutating an amino acid at position 37 of the SIRPαV domain corresponding to amino acid position 37 shown in SEQ ID NO:1 to histidine (H).

[0435] In one embodiment, the method includes mutating an amino acid at position 37 of the SIRPαV domain corresponding to amino acid position 37 of SEQ ID NO:1 to histidine (H), and mutating an amino acid at at least one of positions 29, 31, 35, 36, 51, 52, 53, 54, 67, 68, 69, 95, 99 of SEQ ID NO:1 to histidine (H), for example, mutating any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all 13 positions, preferably 2, 3, 4 or 5 positions, to histidine (H).

[0436] In one embodiment, the method includes mutating an amino acid at position 37 of the SIRPαV domain corresponding to amino acid position 37 of SEQ ID NO:1 to histidine (H), and mutating an amino acid at at least one of positions 29, 31, 51, 52, 53, 54, 67, 68, 69, 95 of the amino acid domain shown in SEQ ID NO:1 to histidine (H), for example, mutating any 2, 3, 4, 5, 6, 7, 8, 9 or all 10 positions, preferably 2, 3, 4 or 5 positions, to histidine (H).

[0437] In one embodiment, the method includes mutating an amino acid at position 37 of the SIRPαV domain corresponding to amino acid position 37 as shown in SEQ ID NO:1 to histidine (H), and mutating an amino acid at position corresponding to amino acid position 37 as shown in SEQ ID NO:1 to histidine (H):

[0438] i)29, 52, 53 and 68;

[0439] ii) 31, 51 and 95;

[0440] iii) 31, 51, 54 and 95;

[0441] iv) 52, 68, 69 and 95;

[0442] v)31, 51, 52, 54 and 95;

[0443] vi) 52 and 95;

[0444] vii)29, 52 and 95;

[0445] viii)31, 52 and 95;

[0446] ix)51, 52 and 95;

[0447] x)52, 67 and 95; or

[0448] xi)52, 68 and 95.

[0449] In one embodiment, the method includes mutating an amino acid at position 51 of the SIRPαV domain corresponding to amino acid position 51 shown in SEQ ID NO:1 to histidine (H).

[0450] In one embodiment, the method includes mutating an amino acid at position 51 of the SIRPαV domain corresponding to amino acid position 51 shown in SEQ ID NO:1 to histidine (H), and mutating an amino acid at at least one of the positions 29, 31, 35, 36, 37, 52, 53, 54, 67, 68, 69, 95, 99 shown in SEQ ID NO:1 to histidine (H), for example, mutating any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all 13 positions, preferably 2, 3, 4 or 5 positions, to histidine (H).

[0451] In one embodiment, the method includes mutating an amino acid at position 51 of the SIRPαV domain corresponding to amino acid position 51 shown in SEQ ID NO:1 to histidine (H), and mutating an amino acid at at least one of positions 29, 31, 37, 52, 54, 67, 68 and 95 shown in SEQ ID NO:1 to histidine (H), for example, mutating any 2, 3, 4, 5, 6, 7 or all 8 positions, preferably 2, 3, 4 or 5 positions, to histidine (H).

[0452] In one embodiment, the method includes mutating an amino acid at position 51 of the SIRPαV domain corresponding to amino acid position 51 as shown in SEQ ID NO:1 to histidine (H), and mutating an amino acid at position corresponding to amino acid position 51 as shown in SEQ ID NO:1 to histidine (H):

[0453] i)31, 37 and 95;

[0454] ii) 31, 37, 54 and 95;

[0455] iii) 29, 31, 67, 68 and 95;

[0456] iv) 31, 37, 52, 54 and 95;

[0457] v)52 and 95;

[0458] vi) 29, 52 and 95;

[0459] vii)31, 52 and 95;

[0460] viii) 37, 52 and 95;

[0461] ix)52, 67 and 95;

[0462] x)52, 68 and 95;

[0463] xi)31, 68 and 95;

[0464] xii)31, 52 and 68;

[0465] xiii) 29, 68 and 95;

[0466] xiv)29, 52 and 68;

[0467] xv)29, 68 and 95; or

[0468] xvi)29, 31 and 52.

[0469] In one embodiment, the method includes mutating an amino acid at position 95 of the SIRPαV domain corresponding to amino acid position 95 shown in SEQ ID NO:1 to histidine (H).

[0470] In one embodiment, the method includes mutating an amino acid at position 95 of the SIRPαV domain corresponding to amino acid position 95 of SEQ ID NO:1 to histidine (H), and mutating an amino acid at at least one of positions 29, 31, 35, 36, 37, 51, 52, 53, 54, 67, 68, 69, 99 of SEQ ID NO:1 to histidine (H), for example, mutating any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all 13 positions, preferably 1, 2, 3, 4 or 5 positions, to histidine (H).

[0471] In one embodiment, the method includes mutating an amino acid at position 95 of the SIRPαV domain corresponding to amino acid position 95 of SEQ ID NO:1 to histidine (H), and mutating an amino acid at at least one of positions 29, 31, 37, 51, 52, 54, 67, 68, and 69 of SEQ ID NO:1 to histidine (H), for example, mutating any 2, 3, 4, 5, 6, 7, 8, or all 9 positions, preferably 1, 2, 3, 4, or 5 positions, to histidine (H).

[0472] In one embodiment, the method includes mutating an amino acid at position 95 of the SIRPαV domain corresponding to amino acid position 95 as shown in SEQ ID NO:1 to histidine (H), and mutating an amino acid at position corresponding to amino acid position 95 as shown in SEQ ID NO:1 to histidine (H):

[0473] i)52;

[0474] ii) 31, 37 and 51;

[0475] iii) 31, 52 and 53;

[0476] iv) 31, 37, 51 and 54;

[0477] v)29, 31, 51, 67 and 68;

[0478] vi) 37, 52, 68 and 69;

[0479] vii)31, 37, 51, 52 and 54;

[0480] viii)29 and 52;

[0481] ix)31 and 52;

[0482] x)37 and 52;

[0483] xi)51 and 52;

[0484] xii)52 and 67;

[0485] xiii) 52 and 68;

[0486] xiv)29, 31 and 52;

[0487] xv)29, 37 and 52;

[0488] xvi)29, 51 and 52;

[0489] xvii)29, 52 and 67;

[0490] xviii)29, 52 and 68;

[0491] xix)31, 37 and 52;

[0492] xx)31, 51 and 52;

[0493] xxi)31, 52 and 67;

[0494] xxii)31, 52 and 68;

[0495] xxiii)37, 51 and 52;

[0496] xxiv)37, 52 and 67;

[0497] xxv)37, 52 and 68;

[0498] xxvi)51, 52 and 67;

[0499] xxvii)51, 52 and 68;

[0500] xxviii)52, 67 and 68;

[0501] xxix)31, 51 and 68;

[0502] xxx)29, 51 and 68;

[0503] xxxi)29, 31 and 68; or

[0504] xxxii)29, 51 and 68.

[0505] In one embodiment, the method includes mutating an amino acid at position 99 of the SIRPαV domain corresponding to amino acid position 99 shown in SEQ ID NO:1 to histidine (H).

[0506] In one embodiment, the method includes mutating an amino acid at position 99 of the SIRPαV domain corresponding to amino acid position 99 of SEQ ID NO:1 to histidine (H), and mutating an amino acid at at least one of positions 29, 31, 35, 36, 37, 51, 52, 53, 54, 67, 68, 69, 95 of the amino acid domain shown in SEQ ID NO:1 to histidine (H), for example, mutating an amino acid at any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of these positions, preferably at 1, 2, 3, 4, or 5 of these positions, to histidine (H).

[0507] In one embodiment, the method includes mutating an amino acid at position 37 and 51 of the SIRPαV domain corresponding to amino acid positions shown in SEQ ID NO:1 to histidine (H).

[0508] In one embodiment, the method includes mutating amino acids at positions 37 and 51 of the SIRPαV domain corresponding to amino acid positions shown in SEQ ID NO:1 to histidine (H), and mutating amino acids at at least one of the positions 29, 31, 35, 36, 52, 53, 54, 67, 68, 69, 95, 99 of the amino acid domain shown in SEQ ID NO:1 to histidine (H), for example, mutating amino acids at any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of these positions, preferably 2, 3, 4, or 5 of these positions, to histidine (H).

[0509] In one embodiment, the method includes mutating amino acids at positions 37 and 51 of the SIRPαV domain corresponding to amino acid positions 37 and 51 of SEQ ID NO:1 to histidine (H), and mutating amino acids at at least one of positions 31, 52, 54, 68 and 95 of SEQ ID NO:1 to histidine (H), for example, mutating amino acids at any two, three, four or five positions, preferably two, three or four positions, to histidine (H).

[0510] In one embodiment, the method includes mutating amino acids at positions 37 and 51 of the SIRPαV domain corresponding to amino acid positions shown in SEQ ID NO:1 to histidine (H), and mutating amino acids at positions corresponding to amino acid positions shown in SEQ ID NO:1 to histidine (H):

[0511] i)31 and 95;

[0512] ii) 31, 54 and 95;

[0513] iii) 31, 52, 54 and 95; or

[0514] iv)52 and 95.

[0515] In one embodiment, the method includes mutating an amino acid at positions 37 and 95 in the SIRPαV domain corresponding to amino acid positions shown in SEQ ID NO:1 to histidine (H).

[0516] In one embodiment, the method includes mutating amino acids at positions 37 and 95 of the SIRPαV domain corresponding to amino acid positions shown in SEQ ID NO:1 to histidine (H), and mutating amino acids at at least one of the amino acid positions 29, 31, 35, 36, 51, 52, 53, 54, 67, 68, 69, and 99 of SEQ ID NO:1 to histidine (H), for example, mutating amino acids at any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all 12 positions, preferably 2, 3, 4, or 5 positions, to histidine (H).

[0517] In one embodiment, the method includes mutating amino acids at positions 37 and 95 of the SIRPαV domain corresponding to amino acid positions shown in SEQ ID NO:1 to histidine (H), and mutating amino acids at at least one of positions 29, 31, 51, 52, 54, 67, 68, and 69 of the amino acid domain shown in SEQ ID NO:1 to histidine (H), for example, mutating amino acids at any 2, 3, 4, 5, 6, 7, or 8 positions, preferably 1, 2, 3, or 4 positions, to histidine (H).

[0518] In one embodiment, the method includes mutating amino acids at positions 37 and 95 of the SIRPαV domain corresponding to amino acid positions shown in SEQ ID NO:1 to histidine (H), and mutating amino acids at positions corresponding to amino acid positions shown in SEQ ID NO:1 to histidine (H):

[0519] i)31 and 51;

[0520] ii) 31, 51 and 54;

[0521] iii) 52, 68, and 69;

[0522] iv)31, 51, 52 and 54;

[0523] v)52;

[0524] vi)29 and 52;

[0525] vii)31 and 52;

[0526] viii)51 and 52;

[0527] ix)52 and 67; or

[0528] x)52 and 68.

[0529] In one embodiment, the method includes mutating an amino acid in the SIRPαV domain corresponding to positions 51 and 95 of the amino acids shown in SEQ ID NO:1 to histidine (H).

[0530] In one embodiment, the method includes mutating amino acids at positions 51 and 95 of the SIRPαV domain corresponding to amino acid positions shown in SEQ ID NO:1 to histidine (H), and mutating amino acids at at least one of the amino acid positions 29, 31, 35, 36, 37, 52, 53, 54, 67, 68, 69, and 99 of the amino acid domain shown in SEQ ID NO:1 to histidine (H), for example, mutating amino acids at any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all 12 positions, preferably 2, 3, 4, or 5 positions, to histidine (H).

[0531] In one embodiment, the method includes mutating amino acids at positions 51 and 95 of the SIRPαV domain corresponding to amino acid positions shown in SEQ ID NO:1 to histidine (H), and mutating amino acids at at least one of positions 29, 31, 37, 52, 54, 67, 68 of the amino acid domain shown in SEQ ID NO:1 to histidine (H), for example, mutating amino acids at any 2, 3, 4, 5, 6 or 7 positions, preferably 1, 2, 3 or 4 positions, to histidine (H).

[0532] In one embodiment, the method includes mutating amino acids at positions 51 and 95 of the SIRPαV domain corresponding to amino acid positions shown in SEQ ID NO:1 to histidine (H), and mutating amino acids at positions corresponding to amino acid positions shown in SEQ ID NO:1 to histidine (H):

[0533] i)31 and 37;

[0534] ii) 31, 37, and 54;

[0535] iii) 29, 31, 67 and 68;

[0536] iv) 31, 37, 52 and 54;

[0537] v)52;

[0538] vi)29 and 52;

[0539] vii)31 and 52;

[0540] viii)37 and 52;

[0541] ix)52 and 67;

[0542] x)52 and 68;

[0543] xi)31 and 68; or

[0544] xii)29 and 68.

[0545] In one embodiment, the method includes mutating amino acids at positions 37, 51, and 95 in the SIRPαV domain corresponding to amino acid positions shown in SEQ ID NO:1 to histidine (H).

[0546] In one embodiment, the method includes mutating amino acids at positions 37, 51, and 95 of the SIRPαV domain corresponding to amino acid positions shown in SEQ ID NO:1 to histidine (H), and mutating amino acids at at least one of positions 29, 31, 35, 36, 52, 53, 54, 67, 68, 69, and 99 of the amino acid domain shown in SEQ ID NO:1 to histidine (H), for example, mutating amino acids at any 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 positions, preferably 2, 3, 4, or 5 positions, to histidine (H).

[0547] In one embodiment, the method includes mutating amino acids at positions 37, 51, and 95 of the SIRPαV domain corresponding to amino acid positions 37, 51, and 95 of SEQ ID NO:1 to histidine (H), and mutating amino acids at at least one position corresponding to amino acid positions 31, 52, and 54 of SEQ ID NO:1 to histidine (H), for example, mutating amino acids at any one, two, or three of these positions to histidine (H).

[0548] In one embodiment, the method includes mutating amino acids at positions 51 and 95 of the SIRPαV domain corresponding to amino acid positions shown in SEQ ID NO:1 to histidine (H), and mutating amino acids at positions corresponding to amino acid positions shown in SEQ ID NO:1 to histidine (H):

[0549] i)31;

[0550] ii) 31 and 54;

[0551] iii) 31, 52 and 54; or

[0552] iv)52.

[0553] This invention provides a method for inducing pH-dependent binding of SIRPα to CD47, comprising mutating an amino acid residue in the immunoglobulin-like variable domain of SIRPα at the position corresponding to the amino acid position shown in SEQ ID NO:1 to histidine (H):

[0554] i)31, 52, 68 and 95;

[0555] ii) 29, 31, 68, and 95;

[0556] iii) 31, 51, 68, and 95; or

[0557] iv)31, 37, 52 and 95.

[0558] In a ninth aspect, the present invention provides a method for enhancing the binding of SIRPαV to human CD47 protein, the method comprising mutating amino acid residues at positions 29 and / or 31 of SIRPαV corresponding to the amino acid position shown in SEQ ID NO:1 to histidine. In some embodiments, the method comprises mutating amino acid residues at position 29 of SIRPαV corresponding to the amino acid position shown in SEQ ID NO:1 to histidine. In some embodiments, the method comprises mutating amino acid residues at position 31 of SIRPαV corresponding to the amino acid position shown in SEQ ID NO:1 to histidine.

[0559] In one embodiment, prior to the mutation at amino acid residues 29 and / or 31, the SIRPαV protein comprises, or is composed of, an amino acid sequence having at least about 70% (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher) sequence identity with the amino acid sequence shown in any of SEQ ID NO:1, 41, or 51. In some embodiments, prior to the mutation at amino acid residues 29 and / or 31, the SIRPαV protein comprises, the amino acid sequence shown in any of SEQ ID NO:1, 41, or 51. In some embodiments, prior to the mutation at amino acid residues 29 and / or 31, the SIRPαV protein has the amino acid sequence shown in SEQ ID NO:1.

[0560] In some embodiments, the method for enhancing SIRPαV binding to human CD47 protein enhances the binding of SIRPαV to human CD47 protein at pH 6.0.

[0561] In some embodiments, the method further includes mutating at least one amino acid residue in SIRPαV corresponding to the amino acid position shown in SEQ ID NO:1 at positions 35, 36, 37, 51, 52, 53, 54, 67, 68, 69, 95, and 99 to histidine.

[0562] In some embodiments, the method further includes mutating at least one amino acid residue in SIRPαV corresponding to the amino acid position shown in SEQ ID NO:1 at positions 36, 37, 51, 52, 53, 68, 69, 95, and 99 to histidine.

[0563] In some embodiments, the method further includes mutating at least one amino acid residue in SIRPαV corresponding to the amino acid positions shown in SEQ ID NO:1 at positions 37, 51, 52, 68, 95, and 99 to histidine.

[0564] In some embodiments, the method further includes mutating at least one amino acid residue in SIRPαV corresponding to amino acid positions shown in SEQ ID NO:1 at positions 37, 51, 95, and 99 to histidine.

[0565] In some embodiments, the method further includes mutating at least one amino acid residue in SIRPαV corresponding to amino acid positions 95 and 99 shown in SEQ ID NO:1 to histidine.

[0566] In some embodiments, the method further includes mutating amino acid residue 95 in SIRPαV, corresponding to the amino acid position shown in SEQ ID NO:1, to histidine.

[0567] In some embodiments, the method further includes mutating amino acid residue 95 corresponding to the amino acid position shown in SEQ ID NO:1 to histidine, and mutating at least one amino acid residue corresponding to amino acid positions 35, 36, 37, 51, 52, 53, 54, 67, 68, 69 and 99 corresponding to the amino acid positions shown in SEQ ID NO:1 to histidine.

[0568] In some embodiments, the method further includes mutating amino acid residue 95 corresponding to the amino acid position shown in SEQ ID NO:1 to histidine, and mutating at least one amino acid residue corresponding to amino acid positions 37, 51, 52 and 68 shown in SEQ ID NO:1 to histidine.

[0569] Unless the context otherwise indicates, the term "and / or" means both "and" and "or". For example, the phrase "A, B and / or C" is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0570] As used herein, "optional" or "optionally" means that the event or situation subsequently described occurs or does not occur, including both the occurrence and non-occurrence of said event or situation. For example, an optional step means that the step is present or absent.

[0571] As used herein, the term "about" refers to a range of values ​​that includes a specific value and that a person skilled in the art would reasonably consider similar to that specific value. In some embodiments, the term "about" refers to within a standard error using measurements generally accepted in the art. For example, in some embodiments, "about" refers to + / - 10% or 5% of a specific value.

[0572] The scope disclosed herein should be considered to specifically disclose all possible subranges and the individual values ​​within those ranges. For example, a description of the range from 1 to 4 should be considered as explicitly disclosing subranges from 1 to 2, from 1 to 3, from 1 to 4, from 2 to 3, from 2 to 4, from 3 to 4, etc., and the individual numbers within those ranges, such as 1, 2, 3, or 4. This applies regardless of the breadth of the range.

[0573] Example

[0574] The present invention is further illustrated by the following embodiments, but any embodiment or combination thereof should not be construed as limiting the scope or implementation of the invention. The scope of the invention is defined by the appended claims, and those skilled in the art can clearly understand the scope defined by the claims in conjunction with this specification and common knowledge in the art. Without departing from the spirit and scope of the invention, those skilled in the art can make any modifications or changes to the technical solutions of the invention, and such modifications and changes are also included within the scope of the invention. Unless otherwise specified, the methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used can be readily obtained from commercial companies.

[0575] Example 1: Design of pH-dependent SIRPαV mutation sites

[0576] A pH-dependent binding mutant of SIRPαV protein was designed, possessing one or more histidine mutations compared to the wild-type SIRPαV protein. The amino acid residue positions of the histidine mutations were determined using computational methods. Based on the crystal structure of the CD47 and SIRPα2 complex (PDB ID: 2JJS), structural and binding analyses were performed using computational methods such as molecular dynamics simulations (e.g., Amber et al.) supplemented by three-dimensional visualization software (e.g., PyMol et al.). Key amino acids affecting binding were identified from multiple perspectives, including residue energy contribution and spatial relationship. Histidine residue mutations were preferentially considered to be located at the SIRPα2 interface interacting with CD47. Table 1-1 lists the key SIRPαV amino acids that can be obtained using the histidine-mutated SIRPα2 (amino acid residue numbers are the natural sequence numbers relative to the SIRPαV sequence (SEQ ID NO: 1) of the extracellular region of wild-type SIRPα2).

[0577] Table 1-1: Key amino acids at the CD47-SIRPα2 interface

[0578] The key amino acids for identification were calculated using the Free Energy Perturbation (FEP) method to determine histidine residue mutations. This method assesses the binding strength between proteins based on a physical model, thereby calculating the energy difference between the binding of SIRPαV of SIRPα2 with histidine mutations to CD47 under acidic and physiological conditions. Based on the pH-dependent binding design goal described in this invention, the selected mutation sites are shown in bold underlined in Figure 1A. Computational modeling methods and empirical observations can also be used to confirm the changes in the three-dimensional structure of SIRPαV and its binding with CD47 after histidine mutation. A schematic diagram of the CD47 / SIRPαV structure and selected sites is shown in Figure 1B.

[0579] Table 1-2. Amino acid sequences of SIRPαV mutants

[0580] Note: In Table 1-2, the general formula for SIRPαV mutants is that at least one of the following sites is a histidine mutation at position 29, 31, 35, 36, 37, 51, 52, 53, 54, 67, 68, 69, 95 or 99 of SIRPαV (SEQ ID NO:1) of wild-type human SIRPα2.

[0581] Example 2: Expression and purification of SIRPαV-Fc fusion protein

[0582] The pcDNA3.4 vector was used as the dedicated vector for expressing the fusion protein. The pcDNA3.4 vector contains the CMV promoter used by the fusion protein, the WPRE signal which can significantly increase mRNA expression levels and translation efficiency, the HSV TK polyadenylation signal, the eukaryotic selection tag G418 tag, and the prokaryotic selection tag ampicillin resistance gene (AmpR).

[0583] Various fusion proteins were synthesized using gene synthesis. (The fusion protein formed by directly linking the SIRPαV mutant of SIRPα2 designed in Example 1 with the Fc region shown in SEQ ID NO:2 or the Fc mutant shown in SEQ ID NO:66 (Fc-DLE) is named as follows: the fusion protein formed by linking the SIRPαV mutant with the Fc region shown in SEQ ID NO:2 is named after the SIRPαV mutant without the suffix "-V". For example, the SIRPαV-Fc fusion protein constructed by directly linking the C-terminus of the SIRPαV mutant "S034-V" with the N-terminus of the Fc region (amino acid sequence as shown in SEQ ID NO:2) is named "S034", and the amino acid sequence of S034 is shown in SEQ ID NO:63, and so on.) The fusion protein formed by linking the mutant (Fc-DLE) shown in NO:66 is named after replacing the suffix "-V" with "-DLE" in the name of the SIRPαV mutant. For example, the fusion protein constructed by directly linking the C-terminus of the SIRPαV mutant "S034-V" to the N-terminus of Fc-DLE is "S034-DLE". The amino acid sequence of S034-DLE is shown in SEQ ID NO:67, and so on. The vector was double-digested with Hind III and Xho I, recovered, and seamlessly cloned using DNA homologous recombinase. It was then transformed into E. coli competent cells DH5α. Positive clones were selected, and plasmids were extracted and sequenced for verification. Plasmids were then extracted and transfected. One day before transfection, Expi293F cells that could be cultured in suspension were cultured in serum-free Expi293F expression medium (Gibco). On the day of transfection, the cell density was adjusted to 3 × 10⁶ cells / mL with fresh Expi293F expression medium. 6The concentration of each cell was determined by mixing 1 μg of plasmid DNA and 3.2 μL of ExpiFectamine (Gibco) with 116 μL of Opti-MEM I medium (Thermo Fisher) per mL of culture medium, and then adding the mixture to Expi293F cells. 18-22 h post-transfection, 6 μL of Enhancer1 and 60 μL of Enhancer2 feed (Gbico) were added, and the cells were cultured for 5-7 days. The supernatant was then collected for further purification. Purification was performed on an AKTA pure protein purifier. The first step of purification was performed using a MabSelect PrismA affinity purification column, and the purity of the purified protein was assessed by SEC-HPLC. The second step of purification was performed using a HiLoad 16 / 600 Superdex 200 pg molecular sieve purification column. The final purified product was used... Protein concentration was determined by N60 (IMPLEN) at 280 nm absorbance, and protein purity was assessed by SDS-PAGE and SEC-HPLC. Endotoxin levels were detected using the recombinant factor C endotoxin assay kit (Adamas Life).

[0584] The amino acid sequence of hIgG1-Fc (SEQ ID NO:2):

[0585] The amino acid sequence of hIgG1-Fc-DLE (SEQ ID NO:66)

[0586] (Note: DLE mutation sites are marked in bold underline)

[0587] For example, the amino acid sequences of the fusion proteins S031, S034, S041, S045, and S034-DLE are as follows:

[0588] The amino acid sequence of S031 (SEQ ID NO:62):

[0589] The amino acid sequence of S034 (SEQ ID NO:63):

[0590] The amino acid sequence of S041 (SEQ ID NO:64):

[0591] The amino acid sequence of S045 (SEQ ID NO:65):

[0592] The amino acid sequence of S034-DLE (SEQ ID NO:67):

[0593] In the above fusion protein sequence, the underlined part is the SIRPαV part, and the italicized part is the Fc part.

[0594] The molecular weight of the obtained fusion protein was confirmed to be correct by SDS-PAGE and SEC-HPLC. Endotoxin testing confirmed that the endotoxin level was below 1 EU mg⁻¹, meeting the standards for activity and physicochemical analysis. A schematic diagram of the SIRPαV-Fc fusion protein is shown in Figure 2. The yield (amount of protein obtained per liter of cell culture medium after purification) and purity are shown in Table 2. Experimental results showed that the purity of the fusion proteins all exceeded 96%.

[0595] Table 2: Yield and purity of SIRPαV-Fc fusion protein

[0596] Example 3: ELISA detection of pH-dependent binding of SIRPαV mutant

[0597] The plate was coated with 1 μg / mL recombinant human CD47 protein (Acro Biosystems, Cat#:CD7-H5227), 100 μL per well, and incubated overnight at 4°C. The following day, after washing the plate three times with washing buffer (PBS containing 0.05% Tween-20), 150 μL of blocking buffer (5% skim milk powder, 0.05% Tween-20, PBS) was added, and the plate was blocked at room temperature for one hour. After washing, each well was added with SIRPαV-Fc protein prepared in Example 2 (starting concentration 200 nM, serially diluted 5 times) diluted with different pH (pH=7.4, pH=6.0) buffer (1% skim milk powder, 0.05% Tween-20, phosphate buffer corresponding to the pH), and incubated at room temperature for one hour. After washing the plate five times, 100 μL of horseradish peroxidase (HRP)-labeled goat antibody against human IgG Fc fragment (Jackson ImmunoResearch Inc., Cat#:109-035-098) was added, and the plate was incubated at room temperature for one hour. After washing the plate five times, 100 μL of... The TMB single-component chromogenic reagent (Solarbio, Cat#: PR1200) was used for color development in the dark for 3 minutes. Then, 100 μL of ELISA stop solution (Solarbio, Cat#: C1058) was added to terminate the reaction. The absorbance (OD) values ​​were read at 450 nm using a Multiskan FC microplate reader (Thermo Scientific). A four-parameter logistic model was used to fit the dose-response data and absorbance data using GraphPad Prism. The EC50 results of the ELISA detection are shown in Table 3.1.

[0598] Table 3.1: EC50 of SIRPαV-Fc fusion protein binding to CD47 protein at different pH values. (Table 3.1a)

[0599] Table 3.1b

[0600] Table 3.1c

[0601] Table 3.1d

[0602] Note: In Table 3.1, at the highest detection concentration, an OD value <1 for binding with CD47 is defined as minimal binding; at the highest detection concentration, an OD value greater than 1 for binding with CD47 but not reaching the binding plateau, and an EC50 > 50 nM fitted by the software, is defined as weak binding.

[0603] In addition, the present invention also tested the binding activity of SIRPαV mutants obtained by single histidine substitution at positions 29, 31, 35, 36, 37, 51, 52, 53, 54, 67, 68, 69, 95, 99, and 100 of wild-type SIRPαV (amino acid sequence as shown in SEQ ID NO:1) with the Fc region (amino acid sequence as shown in SEQ ID NO:2) and the binding activity of the SIRPαV-Fc fusion protein (construction method as in Example 2) with CD47 protein at different pH values ​​using the same method. The experimental results are shown in Table 3.2 and Figure 3.

[0604] Table 3.2 EC50 and TOP values ​​of SIRPαV-Fc single-mutation site fusion protein binding to CD47 protein at different pH values

[0605] Note: The top value is the highest signal value of the combination (also known as the Emax value).

[0606] The experimental results are shown in Figure 3 and Tables 3.1 and 3.2. The binding activity of the SIRPαV-Fc mutant fusion protein at pH 6.0 was stronger than its binding activity to CD47 protein at pH 7.4. Furthermore, the binding activity of these mutants exhibited a strong pH dependence; compared to pH 7.4, their binding activity to CD47 protein was significantly enhanced when the pH decreased to 6.0. Specifically, single-point mutations of I36H, Q37H, N51H, Q52H, K53H, K68H, R69H, R95H, and P99H produced pH-dependent binding, especially the single-point mutations of I36H, N51H, Q52H, K53H, K68H, R95H, and P99H, which produced significant pH-dependent binding. However, the fusion protein with the single-point mutation of D100H did not bind to CD47 protein at either pH 6.0 or pH 7.4.

[0607] Example 4: pH-dependent cell binding assay

[0608] The binding activity assay for CD47 was performed using Jurkat cells, which are human T-lymphoblastic leukemia cells. After centrifugation and washing, the cells were adjusted to a density of 1 × 10⁻⁶. 6 Cells / mL. Cells were transferred to 96-well V-type cell culture plates, with 100 μL of cell suspension added to each well, and centrifuged. The SIRPαV-Fc fusion protein prepared in Example 2 (starting concentration 1000 nM, 4-fold serial dilutions) was diluted with FACS buffer (1% FBS, corresponding to different pH values) at different pH levels (pH = 6.0, pH = 7.4), and incubated at 4°C for one hour. After washing, 100 μL of diluted Alexa was added. Cells were incubated with goat anti-human IgG-Fc antibody (Jackson ImmunoResearch Inc.) conjugated with 647 cells at 4°C for 30 minutes. After washing, the cells were resuspended in phosphate-buffered saline at the appropriate pH and detected using flow cytometry (Intelligent iQue 3, Sartorius). Dose-response data and fluorescence signals were fitted using a four-parameter logistic model with GraphPad Prism. The cell binding EC50 is shown in Table 4.

[0609] Table 4: EC50 values ​​of SIRPαV-Fc mutant fusion protein binding to Jurkat cells at different pH levels

[0610] Table 4a

[0611] Table 4b

[0612] Table 4c

[0613] Table 4d

[0614] Note: In Table 4, MFI 最高检测浓度 / MFI 最低检测浓度 <7 is defined as minimum binding; "NA" indicates that the software cannot fit EC50.

[0615] For the EC50 mutant that the software could not fit at pH=6.0, the MFI (mean fluorescence intensity) values ​​of cell binding at 1 μM concentration under pH=6.0 and pH=7.4 environments are shown in Table 5.

[0616] Table 5: MFI of SIRPαV-Fc mutant fusion protein with Jurkat cells at different pH levels

[0617] As shown in Figure 4, FACS analysis showed that the binding activity of the SIRPαV-Fc mutant fusion protein to Jurkat cells was stronger at pH 6.0 than at pH 7.4.

[0618] Example 5: Target Blockade Experiment

[0619] Raji cells were used in the target blockade assay. After centrifugation and washing, the cells were adjusted to a density of 1×10⁶. 6Cells / mL. Cells were transferred to 96-well V-type cell culture plates, with 100 μL of cell suspension added to each well, and centrifuged. 100 nM biotin-labeled wild-type SIRPαV-Fc fusion protein was added to the cells, followed by SIRPαV-Fc mutant fusion protein prepared in Example 2 (starting concentration 1000 nM, 3-fold serial dilutions) diluted at different pH levels (pH = 6.4, pH = 7.4), and incubated at 4°C for one hour. After washing, 100 μL of APC-conjugated streptavidin (Invitrogen, Cat#: SA1005) was added, and incubated at 4°C for 30 minutes. After washing, the cells were resuspended in phosphate-buffered saline at the appropriate pH, and analyzed using a flow cytometer (Intelligent iQue 3, Sartorius). Dose-response data and fluorescence signals were fitted using a four-parameter logistic model using GraphPad Prism, and the target blocking IC50 is shown in Table 6.

[0620] Table 6: Target blocking activity of SIRPαV-Fc fusion protein at different pH levels

[0621] Note: In Table 6, NA indicates that the software cannot fit EC50.

[0622] As shown in Figure 5, the WT SIRPαV-Fc fusion protein could block the binding of SIRPα to target cells Raji in a dose-dependent manner at both pH 6.4 and pH 7.4; the S017, S034, and S041 fusion proteins only showed some blocking activity at pH 6.4, and had no blocking function at pH 7.4; the blocking activity of the S019, S031, and S045 fusion proteins at pH 6.4 was much higher than their activity at pH 7.4.

[0623] Example 6: Detection of the binding ability of SIRPαV-Fc fusion protein to erythrocytes

[0624] Place anticoagulant tubes containing whole blood cells (Shanghai Aoneng Biotechnology, Cat#:FTS-WB-10) into a centrifuge and centrifuge at 170×g for 10 min, with both the centrifuge speed and acceleration set to the lowest possible level. Discard the plasma and human peripheral blood leukocytes in the upper layer to obtain the red blood cells in the lower layer. Transfer the red blood cells to 15 mL centrifuge tubes, wash twice with PBS, and count using a flow cytometer (FACS Celesta™, BD). Resuspend the red blood cells in NaH2PO4 / Na2HPO4 buffer solutions at pH 6.0, 6.4, 6.6, 6.8, 7, and 7.4 to a final volume of 6×10⁻⁶. 7Cells / mL were added at 50 μL per well to a 96-well V plate. An equal volume of SIRPαV-Fc fusion protein prepared in Example 2 (S009 and S017 starting at 1000 nM, S019 starting at 100 nM, 3-fold dilution) prepared with serially diluted buffers of the same pH (pH=6.0, pH=6.4, pH=6.6, pH=6.8, pH=7.0, pH=7.4) was added. Cells were incubated on ice for 1 hour, washed twice by centrifugation with buffers of the appropriate pH, and 100 μL of 1:1000 diluted goat anti-human-IgG-AF647 secondary antibody (Invitrogen, Cat#:109-605-098) was added to each well. Cells were incubated on ice for 1 hour. After washing twice, cells were resuspended in 150 μL of buffer of the appropriate pH. Results were detected and analyzed using flow cytometry (FACS Celesta™, BD). The dose response data and fluorescence signals were fitted using a four-parameter logical model via GraphPad Prism.

[0625] The results are shown in Figure 6, which represents the average fluorescence intensity of the cell population as measured by MFI. The experimental results indicate that the S009 and S017 fusion proteins do not bind to erythrocytes at all, while the S019 fusion protein binds to erythrocytes only at pH 6.0, pH 6.4, and pH 6.6, with weaker binding at pH 6.4 and pH 6.6.

[0626] Example 7: Detection of the binding ability of SIRPαV-Fc fusion protein to platelets

[0627] Human platelet cells (purchased from Shanghai Aoneng Biotechnology, FTS-PLT-5) were washed twice with PBS buffer and counted using a flow cytometer (FACS Celesta™, BD). Platelets were resuspended in NaH2PO4 / Na2HPO4 buffer at pH 6.0 and pH 7.4 to a final volume of 6 × 10⁻⁶. 7 Cells / mL were added at 50 μL per well to a 96-well V plate, along with an equal volume of serially diluted SIRPαV-Fc fusion protein (starting concentration 100 nM, 4-fold dilution) prepared in Example 2. The cells were incubated on ice for 1 hour, washed twice by centrifugation with buffer at the appropriate pH, and then 100 μL of a 1:1000 dilution of goat anti-human-IgG-AF647 secondary antibody (Invitrogen, Cat#: 109-605-098) was added to each well. The cells were incubated on ice for 1 hour. After washing twice, the cells were resuspended in 150 μL of buffer at the appropriate pH, and the results were detected and analyzed using flow cytometry (FACS Celesta™, BD). A four-parameter logistic model was used to fit the dose-response data and fluorescence signals using GraphPad Prism.

[0628] As shown in Figure 7, WT SIRPα showed comparable platelet binding capacity at pH 6.0 and pH 7.4. The S009, S017, and S019 fusion proteins only showed platelet binding at pH 6.0, and their platelet binding capacity was significantly weaker than that of WT at pH 7.4.

[0629] Example 8: Detection of hemagglutination reactivity of SIRPαV-Fc fusion protein

[0630] The anticoagulant tubes containing whole blood cells (Shanghai Aoneng Biotechnology, Cat#:FTS-WB-10) were placed in a centrifuge and centrifuged at 170×g for 10 min, with the centrifuge speed set to the lowest setting. The plasma and human peripheral blood leukocytes in the upper layer were discarded to obtain the bottom layer of red blood cells. These were washed twice with PBS and then diluted to 10% (v / v) with NaH2PO4 / Na2HPO4 buffers at pH 6.0, pH 6.8, and pH 7.4, respectively. 50 μL of each buffer was added to each well of a 96-well round-bottom plate. The SIRPαV-Fc fusion protein prepared in Example 2 (starting concentration 1000 nM, 3-fold dilution) was diluted with buffer of the appropriate pH and added to the red blood cells in an equal volume. The mixture was mixed, incubated at 37°C for 4 hours, and photographed. The area of ​​the red spots in the images was quantified using Photoshop software. The hemagglutination index was determined by standardizing the area of ​​spots containing SIRPαV-Fc fusion protein relative to the data measured without the protein. The positive control, 5F9 analogue, was purchased from Baiying Biotechnology (Cat#:B3048). As shown in Figure 8, red blood cells that did not undergo hemagglutination settled completely at the bottom of the well with clear, dot-like boundaries, while those that did undergo hemagglutination appeared blurred, forming large circles. The results indicate that 5F9-analogue induces hemagglutination under all pH conditions, while the SIRPαV-Fc fusion protein showed no hemagglutination activity under all pH conditions.

[0631] Example 9: Macrophage phagocytosis induced by SIRPαV-Fc fusion protein

[0632] Collect Raji cells and wash twice with PBS. Adjust the cell density to 1×10⁶. 6 Cells / mL, add 0.1 μM CFSE (Sigma, 21888) and label at room temperature for 8 min, then add an equal volume of bovine serum and stop the reaction at 37°C for 10 min. Digest Raw264.7 cells with trypsin and wash twice with PBS. Resuspend cells in 1640 medium + 10% FBS at pH 6.4 and pH 7.4 respectively, and then add at 5 × 10⁶ cells / mL. 4 and 1×10 5Raw264.7 cells and labeled Raji cells were added to 96-well ultra-low adsorption cell culture plates (Liwo Biotechnology, Cat#LV-ULA002-96UW) at a density of cells per well. Different concentrations of the SIRPαV-Fc fusion protein prepared in Example 2 were then added, and the plates were incubated at 37°C for 2 hours. The supernatant was then discarded, and unphased Raji cells were washed away. Raw264.7 cells were then labeled with APC-conjugated mouse CD11b antibody (Biolegend, 101212). After incubation at 4°C for 1 hour, the cells were washed twice with PBS, and flow cytometry was used to detect cell scattering and fluorescence. In the four-quadrant scatter plot, cells double-positive for mouse CD11b and CFSE were considered to have phagocytosed Raji cells, while cells positive for mouse CD11b and negative for CFSE were considered to have not phagocytosed. The proportion of phagocytosed cells was calculated by dividing the number of cells in the former category by the sum of the former and CFSE, expressed as a percentage.

[0633] The experimental results are shown in Figures 10A-B. The results show that the fusion proteins constructed from SIRPα and Fc can induce phagocytosis of Raji cells by Raw264.7 cells. However, the phagocytic effect induced by the wild-type SIRPα and Fc fusion protein (WT) showed no significant pH difference. In contrast, the phagocytic effect induced by the SIRPαV and Fc fusion proteins of this invention, such as S034, S041, S031 and S045, was stronger at pH 6.4 than at pH 7.4.

[0634] Experimental results show that the ADCP effect of the fusion protein of the present invention is stronger at pH=6.4 than its killing effect at pH=7.4.

[0635] Example 10: ELISA detection of pH-dependent binding of SIRPα1 and SIRPα8 SIRPαV mutants

[0636] All of the above Examples 1-9 were modified using SIRPαV of SIRPα2 as the parent material. The SIRPαV of human SIRPα1 and SIRPα8 subtypes has a high sequence similarity to SIRPαV of SIRPα2, as shown in Figure 9A. The SIRPαV mutation sites of SIRPα2 in Examples 1-9 were transplanted into SIRPαV of SIRPα1 and SIRPα8 respectively to obtain SIRPαV mutants of SIRPα1 and SIRPα8 (SIRPαV mutant sequences are shown in Table 8). Then, the SIRPαV mutants of SIRPα1 and SIRPα8 were directly linked to the Fc region (amino acid sequence as shown in SEQ ID NO:2) to construct the SIRPαV-Fc fusion protein (method is the same as in Example 2 of this invention). For example, the V1-09 fusion protein is constructed by directly linking the SIRPαV mutant V1-09-V of SIRPα1 with the Fc region (amino acid sequence as shown in SEQ ID NO:2). The SIRPαV mutant V1-09-V of SIRPα1 is constructed by transplanting a histidine mutation from the SIRPαV mutant S009-V of SIRPα2 to the corresponding site of SIRPαV in SIRPα1, and so on. The constructed SIRPα1 and SIRPα8 SIRPαV-Fc proteins were detected by pH-dependent ELISA binding assays, using the same method as in Example 3 of this invention. The experimental results are shown in Figure 9 and Table 7.

[0637] Table 7: EC50 of SIRPαV-Fc fusion protein binding to CD47 protein at different pH values

[0638] Table 7a

[0639] Table 7b

[0640] Note: In Table 7, at the highest detection concentration, an OD value <1 for binding with CD47 is defined as minimal binding; at the highest detection concentration, an OD value for binding with CD47 that does not reach the binding plateau, and an EC50 > 50 nM fitted by the software, is defined as weak binding.

[0641] As shown in Figure 9, the SIRPαV-Fc fusion protein constructed from wild-type SIRPα1 and SIRPα8 SIRPαV and Fc exhibits comparable binding activity to CD47 protein at pH 6.0 and pH 7.4. After introducing the corresponding mutation sites, all SIRPαV-Fc mutant fusion proteins showed significant pH-dependent binding activity, with binding under acidic conditions (pH 6.0) being significantly higher than under neutral conditions (pH 7.4).

[0642] Table 8. Amino acid sequences of SIRPαV mutants of SIRPα1 and SIRPα8 subtypes

[0643] Note: In Table 8, V1-WT-V is the wild-type SIRPαV of the human SIRPα1 subtype, V8-WT-V is the wild-type SIRPαV of the human SIRPα8 subtype, and the other amino acid sequences are its mutants; the single underlined part in the amino acid sequence is the amino acid residue with histidine mutation relative to its wild-type SIRPαV. The histidine mutation site is the same as the mutation site of the corresponding SIRPα2 subtype SIRPαV mutant in Tables 1-2 of Example 1 of this invention. For example, V1-09-V and V8-09-V both have histidine mutations at positions 52 and 95 relative to SEQ ID NO:1, which is the same as the mutation site of S009-V.

[0644] Example 11: In vivo pharmacodynamic experiments in a Raji subcutaneous xenograft model of human lymphoma cells

[0645] Experimental animals: Female CB-17SCID mice, 6-8 weeks old, provided by Shanghai Lingchang Biotechnology Co., Ltd.

[0646] Experimental methods: All experimental animals were housed in a specific pathogen-free animal facility with a barrier. Experiments began after 3 days of acclimatization. Human lymphoma Raji tumor cells were cultured in vitro in RPMI-1640 complete medium (supplemented with 10% fetal bovine serum and 1% penicillin & streptomycin) at 37°C in air containing 5% CO2. Tumor cells were routinely passaged 2-3 times per week. Cells in the exponential growth phase were collected and counted for tumor cell seeding. 6 x 10⁶ cells were then cultured in a single layer in air containing 5% CO2. 6 Raji tumor cells were resuspended in 0.1 ml PBS and subcutaneously injected into the right side of each mouse to form tumors; once the average tumor volume of the tumor-bearing mice reached 100 mm, the tumors were induced to grow. 3 Around 1000 mg / kg, the tumor volume of all mice was measured. Suitable tumor-bearing mice were randomly divided into the following groups: blank control group (dose: 10 ml / kg DPBS (Dubor's phosphate buffer) per administration), fusion protein of the present invention 0.5 mg / kg group (dose: 0.5 mg / kg of the fusion protein of the present invention per administration), and fusion protein of the present invention 2 mg / kg group (dose: 2 mg / kg of the fusion protein of the present invention per administration). The blank control group was injected intraperitoneally with the solvent (DPBS), and the other groups were injected intraperitoneally with the corresponding test product. The drugs were administered twice a week for 4 consecutive weeks. During the experiment, the tumor volume and animal weight of each group were monitored twice a week.

[0647] The formula for calculating TGI (tumor growth inhibition rate) is as follows:

[0648] TGI=[1-(Ti-T0) / (Vi-V0)]×100%,

[0649] Wherein, Ti is the average tumor volume measured in the treatment group (fusion protein of the present invention - 0.5 mg / kg group or fusion protein of the present invention - 2 mg / kg group), T0 is the average tumor volume measured in the treatment group, Vi is the average tumor volume measured in the blank control group, and V0 is the average tumor volume measured in the blank control group.

[0650] Experimental results show that the fusion protein of this invention can effectively slow down tumor growth. The experimental results for the test sample S045 of this invention are shown in Figure 11 and Table 9. The results show that, compared with the blank control group, the p-values ​​of the S045_0.5mg / kg group and the S045_2mg / kg group of this invention were 0.053 and 0.0020, respectively, indicating significant differences.

[0651] Table 9: In vivo efficacy results in the Raji subcutaneous xenograft model of human lymphoma cells

[0652] Notes: S045 0.5 mg / kg group (dosage per administration: 0.5 mg / kg of the present invention's S045; administered twice weekly for 4 weeks); S045 2 mg / kg group (dosage per administration: 2 mg / kg of the present invention's S045; administered twice weekly for 4 weeks); blank control group (dosage per administration: 10 ml / kg of DPBS, administered twice weekly for 4 weeks)

[0653] Example 12: Pharmacokinetic Experiment

[0654] In this study, hSIRPA&hCD47 transgenic mice (6-9 weeks old, Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd.) were used as test animals. The concentration of the test product in plasma at different time points after intravenous injection of the test product (SIRPαV-Fc fusion protein prepared in Example 2 of this invention) was determined by ELISA to evaluate the pharmacokinetic characteristics of the test product in the animals.

[0655] The test solution was injected into animals via the tail vein (no fasting, solvent: DPBS) at a dose of 10 mg / kg. Whole blood samples were collected submandibularly at 0 hours before and 0.25, 8, 24, 48, 96, 168, 240, and 336 hours after intravenous administration. The samples were placed in EDTA-K2 anticoagulant tubes, mixed, and centrifuged at 8000 rpm for 7 minutes at 2–8°C to obtain plasma. Plasma concentration was determined using ELISA. Drug molecules in serum were captured by Anti-SIRPA mouse mAb (Sino Biologics, 11612-MM03) and detected using anti-human IgG (Fc specific)-peroxidase antibody (Sigma, A0170). The standard concentration range was 0.781–100 ng / ml. Phoenix WinNonlin was used. TM Version 8.4 (Pharsight, USA) pharmacokinetic software calculates relevant pharmacokinetic parameters using a non-compartmental model method.

[0656] Experimental results showed that the pH-modified fusion protein of this invention had a better in vivo half-life, indicating that the drug remained in the body for a longer period, which could improve patient compliance by reducing the frequency of administration in clinical practice. Specifically, the experimental results for S045 of this invention are shown in Table 10. The results showed that in transgenic mice, the plasma half-life of WT was 24.27 hours, while the plasma half-life of pH-modified S045 was extended to 50.27 hours. The initial plasma drug concentrations (C0) of both were comparable, but the AUC of S045 increased by 2.16 times compared to WT.

[0657] Table 10. Pharmacokinetic parameters of the test product in transgenic mice

[0658] Note: In Table 10, C0 refers to the initial concentration of the drug in the body, AUC represents the total exposure of the drug in the body, and T... 1 / 2 The time required for the drug concentration in the body to decrease by half; WT and S045 are the SIRPαV-Fc fusion proteins prepared in Example 2 of this invention: WT and S045, respectively.

[0659] Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the present invention.

Claims

1. A mutant of an immunoglobulin-like variable domain of signal-regulatory protein alpha (SIRPaV) comprising, relative to a wild-type SIRPaV, a histidine (H) at at least one position corresponding to amino acid positions 29, 31, 36, 37, 51, 52, 53, 68, 69, 95, and 99 of SEQ ID NO: 1; Optionally, the SIRPaV mutant further comprises a histidine at at least one position corresponding to amino acid positions 35, 54, and 67 of SEQ ID NO: 1; Optionally, the wild-type SIRPaV comprises: the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70% sequence identity thereto, the amino acid sequence of SEQ ID NO: 41 or an amino acid sequence having at least 70% sequence identity thereto, or the amino acid sequence of SEQ ID NO: 51 or an amino acid sequence having at least 70% sequence identity thereto; preferably, the wild-type SIRPaV comprises the amino acid sequence of SEQ ID NO:

1.

2. The SIRPaV mutant of claim 1, wherein the SIRPaV mutant; (i) comprises a histidine at at least one position corresponding to amino acid positions 36, 37, 51, 52, 53, 68, 69, 95, and 99 of SEQ ID NO: 1; (ii) comprises a histidine at at least one position corresponding to amino acid positions 51, 52, 53, 68, 95, and 99 of SEQ ID NO: 1; (iii) comprises a histidine at at least one position corresponding to amino acid positions 37, 51, 95, and 99 of SEQ ID NO: 1; (iv) comprises a histidine at at least one position corresponding to amino acid positions 51, 95, and 99 of SEQ ID NO: 1; (v) comprises a histidine at at least one position corresponding to amino acid positions 95 or 99 of SEQ ID NO: 1; (vi) comprises a histidine at a position corresponding to amino acid position 95 of SEQ ID NO: 1; (vii) comprises a histidine at a position corresponding to amino acid position 95 of SEQ ID NO: 1 and a histidine at at least one (e.g., 1, 2, 3, 4, or 5) position corresponding to amino acid positions 35, 36, 37, 51, 52, 53, 54, 67, 68, 69, and 99 of SEQ ID NO: 1; (viii) comprises a histidine at a position corresponding to amino acid position 95 of SEQ ID NO: 1 and a histidine at at least one position corresponding to amino acid positions 37, 51, 52, 68, and 99 of SEQ ID NO: 1; (ix) comprises a histidine at a position corresponding to amino acid position 95 of SEQ ID NO: 1 and a histidine at at least 1 position corresponding to amino acid positions 37, 51, 52, and 68 of SEQ ID NO: 1; or (x) comprises a histidine at a position corresponding to amino acid position 95 of SEQ ID NO: 1 and comprises a histidine at 2 positions corresponding to amino acid positions 51, 52 and 68 of SEQ ID NO: 1 ; Optionally, the SIRPaV mutant comprises an alanine at the amino acid residue corresponding to amino acid position 100 of SEQ ID NO: 1 (D).

3. The SIRPaV mutant of claim 1 or 2, which comprises a histidine at at least one position corresponding to amino acid positions 29 and 31 of SEQ ID NO: 1 ; Optionally, i) the SIRPaV mutant further comprises a histidine at at least one position corresponding to amino acid positions 35, 36, 37, 51, 52, 53, 54, 67, 68, 69, 95 and 99 of SEQ ID NO: 1 ; ii) the SIRPaV mutant further comprises a histidine at a position corresponding to amino acid position 95 of SEQ ID NO: 1 and a histidine at at least 1 position corresponding to amino acid positions 36, 37, 51, 52, 53, 68, 69 and 99 of SEQ ID NO: 1 ; iii) the SIRPaV mutant further comprises a histidine at a position corresponding to amino acid position 95 of SEQ ID NO: 1 and a histidine at at least 1 position corresponding to amino acid positions 37, 51, 52 and 68 of SEQ ID NO: 1 ; or iv) the SIRPaV mutant further comprises a histidine at a position corresponding to amino acid position 95 of SEQ ID NO: 1 and a histidine at 2 positions corresponding to amino acid positions 51, 52 and 68 of SEQ ID NO: 1 ; Optionally, the SIRPaV mutant comprises an alanine at the amino acid residue corresponding to amino acid position 100 of SEQ ID NO: 1 (D).

4. The SIRPaV mutant of any one of claims 1 to 3, which comprises a histidine at the amino acid corresponding to the following positions of SEQ ID NO: 1 : A1 ) 29, 37, 52, 53 and 68; A2) 52 and 95; A3) 31, 37, 51 and 95; A4) 31, 52, 53 and 95; A5) 31, 37, 51, 54 and 95; A6) 29, 31, 51, 67, 68 and 95; A7) 37, 52, 68, 69 and 95; A8) 31, 37, 51, 52, 54 and 95; A9) 29, 52 and 95; A10) 31, 52 and 95; A1 1 ) 37, 52 and 95; A12) 51, 52 and 95; A13) 52, 67 and 95; A14) 52, 68 and 95; A15) 29, 31, 52 and 95; A16) 29, 37, 52 and 95; A17) 29, 51, 52 and 95; A18) 29, 52, 67 and 95; A19) 29, 52, 68 and 95; A20) 31, 37, 52 and 95; A21) 31, 51, 52 and 95; A22) 31, 52, 67 and 95; A23) 31, 52, 68 and 95; A24) 37, 51, 52 and 95; A25) 37, 52, 67 and 95; A26) 37, 52, 68 and 95; A27) 51, 52, 67 and 95; A28) 51, 52, 68 and 95; A29) 52, 67, 68 and 95; A30) 31, 51, 68 and 95; A31) 31, 51, 52 and 68; A32) 29, 51, 68 and 95; A33) 29, 51, 52 and 68; A34) 29, 31, 68 and 95; A35) 29, 31, 51 and 95; A36) 29, 31, 51 and 52; A37)29； A38)31； A39)36； A40)37； A41)51； A42)52； A43)53； A44)68； A45)69； A46) 95; or A47)99； Preferably, the SIRPaV mutant comprises histidine substitutions at positions 31, 52, 68 and 95; Preferably, the SIRPaV mutant comprises histidine substitutions at positions 29, 31, 68 and 95; Preferably, the SIRPaV mutant comprises histidine substitutions at positions 31, 51, 68 and 95; Preferably, the SIRPaV mutant comprises histidine substitutions at positions 31, 37, 52 and 95; Preferably, the SIRPaV mutant comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 3-5, 7, 9-40, 42-50 or 52-60; or an amino acid sequence having at least 85% sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 3-5, 7, 9-40, 42-50 and 52-60.

5. A SIRPαV mutant comprising an amino acid sequence as shown in SEQ ID NO:61 or having at least 85% sequence identity with it, wherein the amino acid sequence of SEQ ID NO:61 is: EEELQVIQPDKSVSVAAGESAILHCTVTX1LX2PVGX3X4X5WFRGAGPARELIYX6X7X8X9GHFPRVTTVSESX 10 X 11 X 12 ENMDFSISISNITPADAGTYYCVKFX 13 KGSX 14 DTEFKSGAGTELSVRAKPS, where: X1is S or H; X2is I or H; X3is P or H; X4is I or H; X5is Q or H; X6is N or H; X7is Q or H; X8is K or H; X9is E or H; X 10 is T or H; X 11 is K or H; X 12 is R or H; X 13 is R or H; X 14 is P or H; and X1-X 14 at least one amino acid residue is H; Preferably, at least one of the amino acid residues at positions X1, X2, X4-X8 and X 11 -X 14 is H; more preferably, at least two of the amino acid residues at positions X1, X2, X4-X8 and X 11 -X 14 are H; more preferably, at least three of the amino acid residues at positions X1, X2, X4-X8 and X 11 -X 14 are H; more preferably, at least four of the amino acid residues at positions X1, X2, X4-X8 and X Preferably, at least one of the amino acid residues at positions X1 and X2 is H; Preferably, X4-X8, X 11 -X 14 at least one amino acid residue at position X is H; Preferably, at least one of the amino acid residues at positions X6, X7, X8, X 11 , X 13 , and X 14 is H. Preferably, at least one of the amino acid residues at positions X5, X6, X 13 and X 14 is H; preferably, at least one of the amino acid residues at positions X6, X 13 , X 14 is H; more preferably, the amino acid residue at position X 13 is H. Preferably, i) at least one amino acid residue at position X3-X 14 position X4-X8, X 11 -X 12 and X 14 position X4-X8, X Preferably, i) at least one of the amino acid residues X4-X8, X 11 -X 12 and X 14 at position X5, X6, X7 and X 11 at position X5, X6, X7 and X 13 at position X5, X6, X7 and X Preferably, the SIRPaV mutant comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 3-5, 7, 9-40.

6. A SIRPaV mutant comprising an amino acid mutation at a position corresponding to amino acid position 95 and / or 99 of SEQ ID NO: 1 relative to wild-type SIRPaV. Preferably, the SIRPaV mutant comprises a histidine residue at a position corresponding to amino acid position 95 and / or 99 of SEQ ID NO:

1. Preferably, the wild-type SIRPaV comprises: the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70% sequence identity thereto, the amino acid sequence of SEQ ID NO: 41 or an amino acid sequence having at least 70% sequence identity thereto, or the amino acid sequence of SEQ ID NO: 51 or an amino acid sequence having at least 70% sequence identity thereto; preferably, the wild-type SIRPaV comprises the amino acid sequence of SEQ ID NO:

1.

7. A fusion protein comprising the SIRPaV mutant of any one of claims 1 to 6, and an immunoglobulin moiety. Preferably, the immunoglobulin moiety is an antibody or an Fc region of an antibody; preferably, the Fc region is an Fc region of human IgGl, IgG2, IgG3 or IgG4; preferably, the amino acid sequence of the Fc region is set forth in SEQ ID NO: 2; Optionally, the Fc region is an Fc region mutant with enhanced effector cell function; optionally, the Fc mutant has enhanced ADCC and / or ADCP function compared to wild type Fc; optionally, the amino acid sequence of the Fc region is set forth in SEQ ID NO: 66; Optionally, the SIRPaV variant is C-terminally linked to the N-terminus of the Fc region directly or via a linker; preferably, the linker is (GmS)n, wherein each of m, n is independently an integer from 1 to 10, preferably, each of the m, n is independently 1, 2, 3, 4, 5 or 6; Optionally, the fusion protein has at least 85% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 62-65 or 67; preferably, the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 62-65 or 67.

8. The SIRPaV mutant of any one of claims 1 to 6 or the fusion protein of claim 7, wherein, The SIRPaV mutant or fusion protein has at least one of the following A)-F) functions: A) has the function of binding to human CD47 protein; optionally, the SIRPaV mutant or fusion protein has the function of pH-dependent binding to human CD47 protein; optionally, the SIRPaV mutant or fusion protein has enhanced CD47 binding activity compared to wild type SIRPaV; optionally, the SIRPaV mutant or fusion protein has more than 1-fold binding activity to human CD47 protein at pH = 6.0 than at pH = 7.4; optionally, the SIRPaV mutant or fusion protein can bind to human CD47 protein with an EC50 value of less than 350 nM at pH = 6.0; optionally, the binding function of the SIRPaV mutant or fusion protein to human CD47 protein is determined by surface plasmon resonance method by detecting EC50 value and / or Emax value; B) has the function of binding to cells expressing human CD47 protein; optionally, the SIRPaV mutant or fusion protein has the function of pH-dependent binding to cells expressing human CD47 protein; optionally, the SIRPaV mutant or fusion protein has more than 1-fold binding activity to cells expressing human CD47 protein at pH = 6.0 than at pH = 7.4; optionally, the SIRPaV mutant or fusion protein can bind to cells expressing human CD47 protein with an EC50 value of less than 200 nM at pH = 6.0; optionally, the EC50 value is determined by flow cytometry sorting technology method; C) has the function of blocking the binding of SIRPa to target cell Raji; optionally, the SIRPaV mutant or fusion protein pH-dependently blocks the binding of SIRPa to target cell Raji; optionally, the IC50 value of the SIRPaV mutant or fusion protein for blocking the binding of SIRPa to target cell Raji at pH = 7.4 environment is more than 1-fold of the IC50 value for blocking the binding of SIRPa to target cell Raji at pH = 6.0 environment; optionally, the IC50 value is detected by flow cytometry sorting technique method; D) does not bind or weakly binds to platelets at pH = 7.4 environment; E) does not bind or weakly binds to red blood cells at pH = 7.4 environment; and / or F) has the function of mediating macrophage phagocytosis; optionally, the fusion protein mediates macrophage phagocytosis at pH = 6.4 environment is stronger than that at pH = 7.4 environment.

9. A nucleic acid encoding the SIRPaV mutant of any one of claims 1 to 6 and 8 or the fusion protein of claim 7 or 8.

10. A host cell comprising the nucleic acid of claim 9 or a nucleic acid construct comprising the same; preferably, the host cell is a prokaryotic cell or a eukaryotic cell; more preferably, the host cell is selected from the group consisting of a bacterium, a yeast cell or a mammalian cell; more preferably, the host cell is selected from the group consisting of E. coli, human embryonic kidney cell, African green monkey kidney cell or Chinese hamster ovary cell.

11. A method of producing the SIRPaV mutant of any one of claims 1 to 6 and 8 or the fusion protein of claim 7 or 8, comprising culturing the host cell of claim 10 under conditions suitable for nucleic acid expression, and recovering the expressed SIRPaV mutant or fusion protein.

12. A pharmaceutical composition comprising the SIRPaV mutant of any one of claims 1 to 6 and 8, the fusion protein of claim 7 or 8, the nucleic acid of claim 9, or the host cell of claim 10; optionally, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

13. Use of the SIRPaV mutant of any one of claims 1 to 8, the fusion protein of claim 7 or 8, the nucleic acid of claim 9, the host cell of claim 10 or the pharmaceutical composition of claim 12 in the manufacture of a medicament for treating cancer; optionally, the cancer is a cancer that highly expresses CD47.

14. A method of making a protein comprising SIRPaV to have pH-dependent binding to CD47, the method comprising mutating an amino acid residue in SIRPaV at at least one position corresponding to amino acid positions 36, 37, 51, 52, 53, 68, 69, 95 and 99 as set forth in SEQ ID NO: 1 to histidine; optionally, the method comprises mutating an amino acid residue in SIRPaV at (i) at least one position corresponding to amino acid positions 37, 51, 95 and 99 as set forth in SEQ ID NO: 1 to histidine; (ii) the amino acid residue at at least one position corresponding to amino acid positions 51, 95 and 99 of SEQ ID NO: 1 is mutated to histidine; (iii) the amino acid residue at at least one position corresponding to amino acid positions 95 and 99 of SEQ ID NO: 1 is mutated to histidine; or (iv) the amino acid residue at position corresponding to amino acid position 95 of SEQ ID NO: 1 is mutated to histidine; Optionally, the method further comprises mutating the amino acid residue in SIRPaV at at least one position corresponding to amino acid positions 29, 31, 35, 54 and 67 of SEQ ID NO: 1 to histidine; preferably mutating the amino acid residue at at least one position corresponding to amino acid positions 29 and 31 of SEQ ID NO: 1 to histidine; Preferably, the SIRPaV comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70% sequence identity thereto, the amino acid sequence of SEQ ID NO: 41 or an amino acid sequence having at least 70% sequence identity thereto, or the amino acid sequence of SEQ ID NO: 51 or an amino acid sequence having at least 70% sequence identity thereto; more preferably, the SIRPaV comprises the amino acid sequence of any one of SEQ ID NO: 1, 41 or 51 ; Preferably, the method comprises mutating the amino acid in SIRPaV at at least one position corresponding to amino acid positions of SEQ ID NO: 1 to histidine: A1 ) 29, 37, 52, 53 and 68; A2) 52 and 95; A3) 31, 37, 51 and 95; A4) 31, 52, 53 and 95; A5) 31, 37, 51, 54 and 95; A6) 29, 31, 51, 67, 68 and 95; A7) 37, 52, 68, 69 and 95; A8) 31, 37, 51, 52, 54 and 95; A9) 29, 52 and 95; A10) 31, 52 and 95; A1 1 ) 37, 52 and 95; A12) 51, 52 and 95; A13) 52, 67 and 95; A14) 52, 68 and 95; A15) 29, 31, 52 and 95; A16) 29, 37, 52 and 95; A17) 29, 51, 52 and 95; A18) 29, 52, 67 and 95; A19) 29, 52, 68 and 95; A20) 31, 37, 52 and 95; A21 ) 31, 51, 52 and 95; A22) 31, 52, 67 and 95; A23) 31, 52, 68 and 95; A24) 37, 51, 52 and 95; A25) 37, 52, 67 and 95; A26) 37, 52, 68 and 95; A27) 51, 52, 67 and 95; A28) 51, 52, 68 and 95; A29) 52, 67, 68 and 95; A30) 31, 51, 68 and 95; A31 ) 31, 51, 52 and 68; A32) 29, 51, 68 and 95; A33) 29, 51, 52 and 68; A34) 29, 31, 68 and 95; A35) 29, 31, 51 and 95; A36) 29, 31, 51 and 52; A39)36； A40)37； A41)51； A42)52； A43)53； A44)68； A45)69； A46) 95; or A47)99。 15. A method of enhancing SIRPav binding to a human CD47 protein, the method comprising mutating an amino acid residue in SIRPav at a position corresponding to 29 and / or 31 of the amino acid positions set forth in SEQ ID NO: 1 to histidine; Preferably, the method comprises mutating an amino acid residue in SIRPav at a position corresponding to 29 of the amino acid positions set forth in SEQ ID NO: 1 to histidine; Preferably, the method comprises mutating an amino acid residue in SIRPav at a position corresponding to 31 of the amino acid positions set forth in SEQ ID NO: 1 to histidine; Optionally, the method enhances SIRPav binding to a human CD47 protein at a pH = 6.0 environment; Optionally, the method further comprises mutating an amino acid residue in SIRPav at a position corresponding to (i) at least one of 35, 36, 37, 51, 52, 53, 54, 67, 68, 69, 95 and 99 of the amino acid positions set forth in SEQ ID NO: 1 to histidine; (ii) at least one of 36, 37, 51, 52, 53, 68, 69, 95 and 99 of the amino acid positions set forth in SEQ ID NO: 1 to histidine; (iii) at least one of 37, 51, 52, 68, 95 and 99 of the amino acid positions set forth in SEQ ID NO: 1 to histidine; (iv) at least one of 37, 51, 95 and 99 of the amino acid positions set forth in SEQ ID NO: 1 to histidine; (v) at least one of 95 and 99 of the amino acid positions set forth in SEQ ID NO: 1 to histidine; (vi) the amino acid residue at a position corresponding to 95 of the amino acid positions set forth in SEQ ID NO: 1 to histidine; (vii) the amino acid residue at a position corresponding to 95 of the amino acid positions set forth in SEQ ID NO: 1 to histidine, and at least one of 35, 36, 37, 51, 52, 53, 54, 67, 68, 69 and 99 of the amino acid positions set forth in SEQ ID NO: 1 to histidine; or (viii) the amino acid residue at a position corresponding to 95 of the amino acid positions set forth in SEQ ID NO: 1 to histidine, and at least one of 37, 51, 52 and 68 of the amino acid positions set forth in SEQ ID NO: 1 to histidine; Preferably, the SIRPaV comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70% sequence identity thereto, the amino acid sequence of SEQ ID NO: 41 or an amino acid sequence having at least 70% sequence identity thereto, or the amino acid sequence of SEQ ID NO: 51 or an amino acid sequence having at least 70% sequence identity thereto; more preferably, the SIRPaV comprises the amino acid sequence set forth in any one of SEQ ID NO: 1, 41 or 51. Preferably, the SIRPaV comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70% sequence identity thereto, the amino acid sequence of SEQ ID NO: 41 or an amino acid sequence having at least 70% sequence identity thereto, or the amino acid sequence of SEQ ID NO: 51 or an amino acid sequence having at least 70% sequence identity thereto; more preferably, the SIRPaV comprises the amino acid sequence set forth in any one

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