TWI859619B - SIRPa MUTANT AND APPLICATION THEREOF - Google Patents

Abstract

The application discloses a high affinity SIRPa polypeptide, which comprises at least one amino acid mutations compared with a wild type protein, the polypeptide also has an increased affinity for CD47 compared with the wild-type protein. The application also discloses the nucleic acid molecule which encoding the SIRPa polypeptide and the vector expressing the polypeptide. And the drug compositions and methods are also provided, for the treatment of CD47-expressing tumors.

Description

SIRPa mutants and their applications

The present invention relates to the field of biomedicine, and more particularly, to SIRPα mutants and applications thereof.

CD47 is a glycosylated transmembrane protein that can mediate a series of processes such as cell proliferation, migration, phagocytosis, cell apoptosis, and immune homeostasis after activation. The ligands of CD47 include signal regulatory protein α (SIRPα), thrombospondin-1 (TSP-1), and integrins. CD47 is generally upregulated in a variety of malignant tumors, and high expression of CD47 is associated with the treatment response and prognosis of cancer deterioration. The CD47/SIRPa "don't eat me" mechanism is considered to be a potential new mechanism in tumor immunology. The high expression of CD47 in tumor cells can inhibit the activation of macrophages and avoid subsequent phagocytosis of tumor cells. Currently, some immunotherapies targeting CD47, including monoclonal antibodies, fusion proteins, CAR-T cells, and ADC, have entered the clinical research stage. The mechanisms of action, safety, and efficacy of these products vary.

Drugs currently in clinical research include a variety of CD47 monoclonal antibodies, bispecific antibodies, and SIRPa proteins. Among them, magrolimab, the CD47 antibody that was developed the earliest and has the fastest clinical progress, was developed by Forty Seven. It is currently advancing Phase II/III clinical trials for hematological tumors and solid tumors. However, it causes certain safety issues, and grade III or higher side effects include anemia events in about 14% and neutropenia events in about 16%. The SIRPa protein drugs with the fastest clinical progress include TTI621 and TTI622 developed by Trillium. TTI621 is a natural SIRPa IgV domain coupled to the Fc segment of human IgG1. Trillium is developing TTI621 for Phase I clinical trials for hematological tumors, and has a certain objective response rate for single-drug treatment of NHL. Grade III and above side effects include anemia events of about 9%, thrombocytopenia of 20%, and neutropenia of about 9%. TTI622 is a natural SIRPa IgV domain coupled to the Fc segment of human IgG4, and its current efficacy and safety data are better than TTI621. In addition, the combination or bi-antibody of multiple immune checkpoints and CD47 antibodies, the combination or bi-antibody of tumor-related antigen antibodies and CD47 antibodies, etc. have also gradually entered the clinic.

In addition to the above-mentioned dual-antibody combination mechanism, increasing the affinity of SIRPα to CD47, improving the efficiency of blocking the interaction between CD47 and SIRPα, and promoting the release of the inhibition of macrophage phagocytosis may provide new research and development ideas and innovative significance for the field of CD47 targeted drugs. In view of this, the present invention is proposed.

The present invention provides a SIRPα variant and a fusion protein thereof having higher target binding activity, which are used to detect the presence or extent of CD47, block the binding of SIRPα to CD47, and achieve tumor immunotherapy.

The first aspect of the present invention provides a polypeptide for inhibiting the growth and/or proliferation of CD47+ disease cells, comprising a signal regulatory protein alpha (SIRPα) mutant or a fragment thereof, wherein the mutant has at least one amino acid modification selected from residue 31, residue 37, residue 51, residue 66, residue 68 or residue 82 relative to wild-type SIRPα; preferably, the amino acid modification is a substitution; preferably, the wild-type SIRPα has a sequence according to any one of SEQ ID NOs: 1-2.

In some embodiments, the SIRPα mutant comprises at least one amino acid modification selected from I31, Q37, N51, L66, K68, or T82.

In some embodiments, the amino acid modification is selected from at least one of the following amino acid modifications: (1) I31M; I31Y; (2) Q37T; (3) N51G; N51R; N51A; N51E; N51F; N51K; N51L; N51M; N51Q; N51T; N51W; (4) L66A; L66V; L66P; L66M; L66 N; L66E; L66K; L66H; (5) K68D; K68M; K68Q; K68L; K68F; K68I; or (6) T82F; T82D; T82N; T82E; T82Q; T82G; T82H; T82L; ;T82W;T82Y.

In some embodiments, the polypeptide comprises an amino acid mutation selected from the following group relative to the wild-type SIRPα having SEQ ID NO: 1: (1) N51G; (2) N51R; (3) I31M; (4) I31Y; (5) I31M; N51G; (6) I31M; N51R; (7) I31Y; N51G; (8) I31Y; N51R; (9) Q37T; N51G; (10) Q37T; N51R; (11) I31Y; N51R; L66A; (12) I31Y; N51R; L66V; (13) I31 1Y; N51R; L66P; (14) I31Y; N51R; L66M; (15) I31Y; N51R; L66N; (16) I31Y; N51R; L66E; (17) I31Y; N51R; L66K; (18) I31Y; N51R; L66H; (19) N51A; (20) N51E; (21) N51F; (22) N51K; (23) N51L; (24 )N51M; (25) N51Q; (26) N51T; (27) N51W; (28) I31M; N51R; L66N; K68D; (29) I31M; N51R; L66M; K68M; (30) I31Y; N51R; L66N; K68D; (31) I31M; 6N; K68Q; (32) I31M; N51R; L66N; K68L; (3 3) I31Y; N51R; L66N; K68F; (34) I31M; N51R; L66N; K68I; (35) I31Y; N51R; L66N; T82F; (36) I31Y; N51R; L66N; T82D; (37) I31Y; )I31Y; N51R; L66N; T82E; (39) I31Y; N51R; L66N; T82Q; (40) I31Y; N51R; L66N; T82G; (41) I31Y; N51R; L66N; T82H; (42) I31Y; N51R; L66N; N; T82K; (45) I31Y; N51R; L66N; T82M; ( 46) I31Y; N51R; L66N; T82P; (47) I31Y; N51R; L66N; T82R; (48) I31Y; N51R; L66N; T82V; (49) I31Y; )I31M; N51R; L66N; K68Q; T82G; (52) I31M ; N51R; L66N; K68Q; T82H; (53) I31M; N51R; L66N; K68Q; T82L; (54) I31M; N51R; L66N; K68Q; T82V; (55) I31M; N51R; ;L66N; K68L; T82H; (57) I31M; N51R; L66N; K68L; T82L; (58) I31M; N51R; L66N; K68L; T82V; (59) I31M; N51R; L66N; K68I; T82G; (60) I31M; 2L; (62) I31M; N51R; L66N; K68I; T82V.

Or, relative to the wild-type SIRPα having SEQ ID NO: 2, comprising N51R; N51M or N51W mutations.

In some embodiments, the polypeptide is fused to an immunoglobulin Fc region at its N-terminus or C-terminus; preferably, the immunoglobulin Fc region is selected from the Fc region of human IgG1, IgG2 or IgG4 or a mutant thereof; more preferably, the immunoglobulin Fc region is selected from the Fc region of human IgG1 or a mutant thereof.

In some embodiments, the Fc mutant comprises at least one of the following amino acid modifications relative to wild-type human IgG1: L234A, L235A, G237A and N297A; preferably, the amino acid modifications comprise (1) N297A mutation; (2) L234A, L235A and G237A mutations; or (3) L234A, L235A, G237A and N297A mutations.

In some embodiments, the Fc mutant exhibits abolished or reduced effector function, abolished or reduced C1q binding and Fcγ receptor binding compared to the wild-type human IgG Fc region.

In some embodiments, the polypeptide has an amino acid sequence of any one of SEQ ID NOs: 4-14, 17-51, 55-110.

In some embodiments, the polypeptide has a KD for CD47 of at least 6×10 ⁻⁹ M.

In some embodiments, the polypeptide is further linked to other functional molecules, which can be selected from one or more of the following: signal peptides, protein tags, other antigen binding molecules, interleukins, cytokines, steroids, anti-inflammatory agents, immunomodulators or cytotoxins.

In some embodiments, the polypeptide is monomeric, or polymeric.

The second aspect of the present invention provides a chimeric antigen receptor (CAR), which comprises at least an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises any one of the above-mentioned polypeptides.

The third aspect of the present invention provides an immune cell, which expresses the above-mentioned chimeric antigen receptor, or contains a nucleic acid fragment encoding the chimeric antigen receptor described in claim 12; preferably, the immune cell is selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), DNT cells (double negative T cells), monocytes, macrophages, dendritic cells or mast cells, and the T cells are preferably selected from cytotoxic T cells, regulatory T cells or helper T cells; preferably, the immune cell is an autologous immune cell or an allogeneic immune cell.

A fourth aspect of the present invention provides a nucleic acid molecule encoding any of the above polypeptides.

A fifth aspect of the present invention provides an expression vector comprising the above-mentioned nucleic acid fragment.

The sixth aspect of the present invention provides a host cell comprising the above-mentioned vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (E. coli), fungi (yeast), insect cells or mammalian cells (CHO cell strain or 293T cell strain).

The seventh aspect of the present invention provides a method for preparing any of the above polypeptides, comprising culturing the host cells and isolating the polypeptide expressed by the host cells.

An eighth aspect of the present invention provides a method for preparing the above-mentioned immune cell, which comprises introducing a nucleic acid fragment encoding the above-mentioned CAR into the immune cell, and optionally, further comprises activating the immune cell to express the above-mentioned CAR.

The ninth aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and any of the above-mentioned polypeptides, immune cells, nucleic acid molecules, expression vectors, or products prepared by any of the above-mentioned methods in an amount that can effectively inhibit the growth or proliferation of CD47+ disease cells.

The tenth aspect of the present invention provides a kit comprising any of the above-mentioned polypeptides, immune cells, nucleic acid molecules, expression vectors, host cells, products prepared by the method, or pharmaceutical compositions.

The eleventh aspect of the present invention provides the use of any of the above-mentioned polypeptides, immune cells, nucleic acid molecules, expression vectors, host cells, products prepared by the method, or pharmaceutical compositions for preparing a drug for treating a tumor expressing CD47; preferably, the tumor is a CD47-positive hematological tumor or a CD47-positive solid tumor, and more preferably, the CD47-positive hematological tumor includes leukemia, lymphoma and myeloma, and the CD47-positive solid tumor includes sarcoma.

The twelfth aspect of the present invention provides a method for treating a tumor expressing CD47, the method comprising administering to a subject an effective amount of the polypeptide, immune cells, nucleic acid molecules, expression vectors, host cells, the product prepared by the method, and a pharmaceutical composition; preferably, the tumor is a CD47-positive hematological tumor or a CD47-positive solid tumor, and more preferably, the CD47-positive hematological tumor includes leukemia, lymphoma and myeloma, and the CD47-positive solid tumor includes sarcoma.

The thirteenth aspect of the present invention provides the polypeptide, immune cell, nucleic acid molecule, expression vector, host cell, product prepared by the method, or pharmaceutical composition for treating a tumor expressing CD47; preferably, the tumor is a CD47-positive hematological tumor or a CD47-positive solid tumor, and more preferably, the CD47-positive hematological tumor includes leukemia, lymphoma and myeloma, and the CD47-positive solid tumor includes sarcoma.

A fourteenth aspect of the present invention provides a method for detecting the expression of CD47 in a biological sample, characterized in that the method comprises contacting the biological sample with the polypeptide under conditions where a complex can be formed between the polypeptide and CD47; preferably, the method further comprises detecting the formation of the complex, indicating the presence or expression level of CD47 in the sample.

Cross-reference to related applications: This application claims the rights and priority of the following two Chinese invention patent applications, the entire contents of which are hereby incorporated by reference into this article: Patent Application No. 202111283381.5 filed with the National Intellectual Property Administration of China on October 31, 2021; and Patent Application No. 202210800901.3 filed with the National Intellectual Property Administration of China on July 7, 2022.

Unless otherwise defined in the present invention, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art to which the present invention belongs.

Furthermore, unless otherwise indicated herein, terms in the singular shall include the plural and terms in the plural shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly stated otherwise.

The terms "include", "comprising", and "having" are used interchangeably herein and are intended to indicate the inclusiveness of the solution, meaning that the solution may contain other elements in addition to the listed elements. At the same time, it should be understood that the descriptions using "include", "comprising", and "having" herein also provide "consisting of" solutions. Exemplarily, "a composition comprising A and B" should be understood as the following technical solutions: a composition consisting of A and B, and a composition containing other components in addition to A and B, all fall within the scope of the aforementioned "a composition".

The term "and/or" as used herein includes the meanings of "and", "or" and "all or any other combinations of elements linked by the term".

The term "SIRPa" herein refers to a cell surface type I transmembrane protein expressed on macrophages that is a member of the SIRP/SHPS (CD172) family of the Ig superfamily. The term "SIRPa" may be used interchangeably with "signal regulatory protein alpha," "SIRPα," or "SIRP-alpha." SIRPa is a receptor for CD47. In humans, the SIRPa protein is found in two main forms. One form (variant 1 or V1 form) has an amino acid sequence listed as NCBI RefSeq NP_542970.1 (residues 27-504 constitute the mature form). The other form (variant 2 or V2 form) differs by 13 amino acids and has an amino acid sequence listed in GenBank as CAA71403.1 (residues 30-504 constitute the mature form). These two forms of SIRPα make up approximately 80% of the various types of SIRPα present in humans.

The terms "CD47+" and "CD47 positive" generally refer to the property of expressing CD47 protein, fragments thereof, or mutants thereof with one or more amino acid substitutions on the surface of an organism or a cell. CD47 positive cells may be cells that overexpress CD47. The CD47 positive cells may generally be used as an indicator of a disease. For example, in the case of a disease, the density of CD47 protein on the surface of the CD47 positive cells may exceed the density of CD47 protein that such cells have under normal conditions. In certain embodiments, the tumor or tumor cell may be CD47 positive. For example, the tumor may be selected from the following groups: CD47 positive hematological tumors and/or CD47 positive solid tumors.

The term "CD47 protein" herein generally refers to integrin-associated protein (IAP), which is a multi-transmembrane receptor belonging to the immunoglobulin superfamily. For example, CD47 protein can bind to membrane integrins and to its ligands thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRPα). CD47 protein is widely expressed on the surface of cell membranes. In the present application, the CD47 protein may include any variants, isoforms and species homologs of human CD47.

The term "mutant" herein generally refers to a protein molecule having sequence homology with a protein without any mutation/modification, which retains at least a portion of the therapeutic and/or biological activity of the biologically active protein. For example, the mutant protein may share at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the reference biologically active protein. In some embodiments, a "mutant" may include a protein that has been intentionally modified (e.g., by site-directed mutagenesis, synthesis of an encoding gene, insertion, or by accident through mutation).

In the present invention, an amino acid residue in an amino acid sequence "relative to" an amino acid residue in another amino acid sequence generally refers to the amino acid residue correspondence relationship obtained when the amino acid sequences are aligned under optimized conditions. The sequence alignment can be performed in a manner known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithm required to achieve optimal alignment in the full-length sequences being compared.

The amino acid substitutions described in this application may be non-conservative substitutions. The non-conservative substitutions may include changing the amino acid residues in the target protein or polypeptide in a non-conservative manner, such as changing an amino acid residue with a certain side chain size or a certain property (e.g., hydrophilicity) to an amino acid residue with a different side chain size or different property (e.g., hydrophobicity).

The amino acid substitution may also be a conservative substitution. The conservative substitution may include changing the amino acid residues in the target protein or polypeptide in a conservative form, such as changing an amino acid residue with a certain side chain size or a certain property (e.g., hydrophilicity) to an amino acid residue with the same or similar side chain size or the same or similar property (e.g., still hydrophilicity). Such conservative substitutions usually do not have a significant impact on the structure or function of the resulting protein. In the present application, the amino acid sequence variants of the fusion protein, its fragment, or its mutants in which one or more amino acids are substituted may include conservative amino acid substitutions that do not significantly change the protein structure or its function (e.g., blocking the ability of CD47 to bind to its ligand).

As an example, mutual substitutions between amino acids in each of the following groups can be considered conservative substitutions in this application: 1) Alanine (A), serine (S), threonine (T); 2) Aspartic acid (D), glutamine (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), lysine (K), histidine (H); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

The term "fusion protein" herein generally refers to a protein obtained by fusing two or more proteins or polypeptides. Fusion proteins can be artificially prepared by recombinant DNA technology. For example, genes or nucleic acid molecules encoding the two or more proteins or polypeptides can be linked to each other to form a fusion gene or a fused nucleic acid molecule, which can encode the fusion protein. Translation of the fusion gene can produce a single polypeptide, which can have the properties of at least one, or even each, of the two or more proteins or polypeptides before fusion.

In the present invention, the fusion protein includes a human SIRPα domain capable of specifically binding to the CD47 protein and an immunoglobulin Fc region, wherein the human SIRPα domain can be directly or indirectly connected to the immunoglobulin Fc region. For example, the human SIRPα domain can be located at the N-terminus or C-terminus of the immunoglobulin Fc region. For example, the C-terminus of the human SIRPα domain can be directly or indirectly connected to the N-terminus of the immunoglobulin Fc, or the C-terminus of the immunoglobulin Fc is directly or indirectly connected to the N-terminus of the human SIRPα domain. For example, the human SIRPα domain can be connected to the immunoglobulin Fc through a linker.

The term "immunoglobulin Fc region" herein generally refers to the base region of the Y-shaped structure of the antibody structure, also known as the fragment crystallizable region (Fc region). In IgG, IgA and IgD antibody isotypes, the Fc region may be composed of two identical protein fragments, which come from the second and third constant domains of the two heavy chains of the antibody; the Fc region of IgM and IgE may contain three heavy chain constant domains in each polypeptide chain. The Fc region of IgG has highly conserved N-glycosylation sites. In certain embodiments, the immunoglobulin Fc region may include the Fc region of IgG. In certain embodiments, the immunoglobulin Fc region may include the CH2 and CH3 regions of the heavy chain constant region. In certain embodiments, the immunoglobulin Fc region may include a hinge region. For example, the immunoglobulin Fc region may comprise an amino acid sequence selected from any one of the following: SEQ ID NO: 3.

As an alternative embodiment, one or more alterations, usually no more than about 5 such alterations, including amino acid substitutions that affect certain Fc properties are introduced into the Fc region.

The term "IgG" herein generally refers to immunoglobulin G. IgG is one of the human immunoglobulins. According to the difference in the antigenicity of the γ chain in the IgG molecule, human IgG has four subtypes: IgG1, IgG2, IgG3 and IgG4. In this application, the term "IgG1" generally refers to the subtype with the highest proportion in IgG, which has a higher affinity with Fc receptors. For example, the IgG may be human IgG. For another example, the IgG may be selected from the following groups: IgG1 and/or IgG4.

The term "KD" herein generally refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, and the unit is M (mol/L), wherein a lower KD indicates a higher affinity. KD is calculated as follows: KD = Kd/Ka, wherein Kd represents the dissociation rate and Ka represents the association rate. The equilibrium dissociation constant KD can be measured by methods known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis. For example, the method for obtaining the KD value shown in Example 4 herein can be referred to.

The term "antigen binding molecule" is used in the broadest sense herein to refer to a molecule that specifically binds to an antigen. Exemplarily, antigen binding molecules include, but are not limited to, antibodies or antibody mimetics. "Antibody mimetics" refer to organic compounds or binding domains that are able to specifically bind to an antigen but are unrelated to the structure of an antibody, and exemplary antibody mimetics include, but are not limited to, affibodies, affitins, affilins, designed anchor protein repeat proteins (DARPins), nucleic acid aptamers, or Kunitz-type domain peptides.

The term "chimeric antigen receptor (CAR)" herein refers to an artificial cell surface receptor that is engineered to be expressed on immune cells and specifically binds to an antigen, and comprises at least (1) an extracellular antigen binding domain, such as a variable heavy chain or light chain of an antibody, (2) a transmembrane domain that anchors the CAR into immune cells, and (3) an intracellular signaling domain. CAR is able to redirect T cells and other immune cells to selected targets, such as cancer cells, in a non-MHC-restricted manner using the extracellular antigen binding domain.

The term "nucleic acid molecule" herein includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide is composed of an alkali group, in particular a purine or pyrimidine alkali group (i.e., cytosine (C), guanosine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by a sequence of alkali groups, whereby the alkali groups represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of alkali groups is usually expressed as 5' to 3'. In this article, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules that are suitable as vectors for direct expression of the antibodies of the present invention in vitro and/or in vivo, for example in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038/nm.4356 or EP 2 101 823 B1). An "isolated" nucleic acid herein refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present outside the chromosome or at a chromosomal location that is different from its natural chromosomal location.

The term "expression vector" herein refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

The term "host cell" herein refers to a cell into which an exogenous nucleic acid is introduced, including the offspring of such a cell. Host cells include "transformants" and "transformed cells", which include the first generation of transformed cells and offspring derived therefrom, without considering the number of generations. Offspring may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant offspring having the same function or biological activity as screened or selected in the initial transformed cell are included herein.

The term "pharmaceutical composition" herein refers to a preparation which is in a form which permits the biological activity of the active ingredient contained therein to be effective, and which contains no additional ingredients which are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

The term "treatment" herein refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathological changes in the treated subject, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete), whether detectable or undetectable. Subjects in need of treatment include those who already have a disorder or disease, as well as those who are susceptible to a disorder or disease or those for whom a disorder or disease is to be prevented. When we use terms like reduce, lessen, lessen, alleviate, relieve, etc., they also mean to eliminate, disappear, or not occur.

The term "subject" herein refers to an organism that is treated for a particular disease or condition as described herein. Examples of subjects and patients include mammals, such as humans, primates (e.g., monkeys), or non-primate mammals that are treated for a disease or condition.

As used herein, the term "effective amount" refers to an amount of a therapeutic agent that is effective in preventing or alleviating a disease symptom or the progression of a disease when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject. "Effective amount" also refers to an amount of a compound sufficient to relieve symptoms, such as to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate at which such conditions are treated, cured, prevented, or alleviated. When an active ingredient is administered to an individual alone, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

The term "cancer" as used herein refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are both benign and malignant cancers. The terms "tumor" or "neoplasm" as used herein refer to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" as used herein are not mutually exclusive.

In this application, the term "CD47-positive blood tumor" generally refers to blood tumors that overexpress CD47, which may include various types of leukemias, lymphomas, and myelomas. The term "leukemia" generally refers to a cancer of the blood in which too many white blood cells that are not effective in fighting infection are produced, thereby crowding out other parts of the blood, such as platelets and red blood cells. Leukemias can be classified as acute or chronic.

In this application, the term "CD47-positive solid tumor" generally refers to a solid tumor or a tangible tumor that expresses CD47, which can be detected as a tangible mass by clinical examination, such as X-ray, CT, ultrasound or palpation. The main categories may include carcinoma and sarcoma.

The present invention is further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments used is not specified, they are conventional products that can be purchased commercially.

The embodiments of the present invention are only exemplary and do not constitute any limitation on the scope of the present invention. A person with ordinary knowledge in the technical field to which the present invention belongs should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements fall within the scope of protection of the present invention.

Embodiment 1 SIRP Mutation of plastid architecture

SIRPα mutants with improved affinity were obtained using a variety of algorithms, and the corresponding sequences were designed and synthesized. The nucleic acid fragments encoding wild-type SIRPα IgV and the aforementioned mutants were cloned into pTT5 vectors with different Fc tags, and plasmids encoding SIRPα wild-type/variant and Fc fused proteins were prepared according to established standard molecular biology methods. The SIRPα wild type and variants used are as follows: SIRPα V1, SIRPα V2, Mut10, Mut31, Mut33, Mut35, Mut38, Mut66, Mut67, Mut68, Mut69, Mut70, Mut71, Mut72, Mut73, Mut75, Mut80, Mut81, Mut83, Mut84, Mut86, Mut87, Mut88, Mut89, Mut90, Mut150, Mut152, Mut153, Mut156, Mut157, Mut158, Mut160, Mut162, Mut164, Mut174, and Mut178.

The specific sequence information of the above fusion protein and each element is shown in Table 1, wherein "SIRPa V1" represents the IgV fragment of wild-type SIRPa V1, "SIRPa V2" represents the IgV fragment of wild-type SIRPa V2, "Fc" represents the Fc fragment of wild-type human IgG1, "inert Fc" represents the Fc mutant of human IgG1, "MutXX" represents a mutant SIRPa with mutations compared to the wild type, and "MutXX-Fc" represents a fusion protein of mutant SIRPa and Fc. "SIRPa V1/V2-Fc" represents a fusion protein of wild-type SIRPa V1/V2 and the wild-type Fc fragment shown in SEQ ID NO: 3. The sequence of TTI621 is referenced to WO2019084692A1, as shown in SEQ ID NO: 15.

Table 1 SIRPa mutant sequences Mutation Number Serial Number Sequence information SIRPa V1 SEQ ID NO: 1 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS SIRPa V2 SEQ ID NO: 2 EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS Fc SEQ ID NO: 3 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut10 (V2, N51R) SEQ ID NO: 4 EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYRQKEGHFPRVTTVSESTKRENMDFSISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS Mut31 (V1, N51G) SEQ ID NO: 5 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYGQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut33 (V1, N51R) SEQ ID NO: 6 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut35 (V1, I31M) SEQ ID NO: 7 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut38 (V1, I31Y) SEQ ID NO: 8 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut66 (V1, I31M/N51G) SEQ ID NO: 9 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYGQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut67 (V1, I31M/N51R) SEQ ID NO: 10 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut68 (V1, I31Y/N51G) SEQ ID NO: 11 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYGQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut69 (V1, I31Y/N51R) SEQ ID NO: 12 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut70 (V1, Q37T/N51G) SEQ ID NO: 13 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPITWFRGAGPGRELIYGQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut71 (V1, Q37T/N51R) SEQ ID NO: 14 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPITWFRGAGPGRELIYRQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS TTI621 SEQ ID NO: 15 EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Inert Fc SEQ ID NO: 16 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut72 (V1, I31Y/N51R/L66A) SEQ ID NO: 17 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDATKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut73 (V1, I31Y/N51R/L66V) SEQ ID NO: 18 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDVTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut75 (V1, I31Y/N51R/L66P) SEQ ID NO: 19 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDPTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut80 (V1, I31Y/N51R/L66M) SEQ ID NO: 20 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDMTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut81 (V1, I31Y/N51R/L66N) SEQ ID NO: 21 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut83 (V1, I31Y/N51R/L66E) SEQ ID NO: 22 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDETKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut84 (V1, I31Y/N51R/L66K) SEQ ID NO: 23 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDKTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut86 (V1, I31Y/N51R/L66H) SEQ ID NO: 24 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDHTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut87 (V1, I31M/N51G) SEQ ID NO: 25 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYGQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut88 (V1, I31M/N51R) SEQ ID NO: 26 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut89 (V1, I31Y/N51G) SEQ ID NO: 27 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYGQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut90 (V1, I31Y/N51R) SEQ ID NO: 28 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut10-Fc SEQ ID NO: 29 EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYRQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut31-Fc SEQ ID NO: 30 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYGQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut33-Fc SEQ ID NO: 31 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut35-Fc SEQ ID NO: 32 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut38-Fc SEQ ID NO: 33 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut66-Fc SEQ ID NO: 34 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYGQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut67-Fc SEQ ID NO: 35 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut68-Fc SEQ ID NO: 36 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYGQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut69-Fc SEQ ID NO: 37 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut70-Fc SEQ ID NO: 38 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPITWFRGAGPGRELIYGQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut71-Fc SEQ ID NO: 39 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPITWFRGAGPGRELIYRQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut72-Fc SEQ ID NO: 40 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDATKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut73-Fc SEQ ID NO: 41 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDVTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut75-Fc SEQ ID NO: 42 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDPTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut80-Fc SEQ ID NO: 43 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDMTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut81-Fc SEQ ID NO: 44 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut83-Fc SEQ ID NO: 45 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDETKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut84-Fc SEQ ID NO: 46 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDKTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut86-Fc SEQ ID NO: 47 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDHTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut87-Fc SEQ ID NO: 48 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYGQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut88-Fc SEQ ID NO: 49 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut89-Fc SEQ ID NO: 50 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYGQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut90-Fc SEQ ID NO: 51 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut150 (V1, N51A) SEQ ID NO: 55 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYAQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut152 (V1, N51E) SEQ ID NO: 56 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYEQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut153 (V1, N51F) SEQ ID NO: 57 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYFQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut156 (V1, N51K) SEQ ID NO: 58 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYKQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut157 (V1, N51L) SEQ ID NO: 59 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYLQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut158 (V1, N51M) SEQ ID NO: 60 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYMQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut160 (V1, N51Q) SEQ ID NO: 61 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYQQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut162 (V1, N51T) SEQ ID NO: 62 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYTQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut164 (V1, N51W) SEQ ID NO: 63 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYWQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut174 (V2, N51M) SEQ ID NO: 64 EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYMQKEGHFPRVTTVSESTKRENMDFSISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS Mut178 (V2, N51W) SEQ ID NO: 65 EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYWQKEGHFPRVTTVSESTKRENMDFSISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS Mut150-Fc SEQ ID NO: 66 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYAQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut152-Fc SEQ ID NO: 67 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYEQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut153-Fc SEQ ID NO: 68 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYFQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut156-Fc SEQ ID NO: 69 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYKQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut157-Fc SEQ ID NO: 70 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYLQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut158-Fc SEQ ID NO: 71 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYMQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut160-Fc SEQ ID NO: 72 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYQQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut162-Fc SEQ ID NO: 73 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYTQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut164-Fc SEQ ID NO: 74 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYWQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut174-Fc SEQ ID NO: 75 EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYMQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mut178-Fc SEQ ID NO: 76 EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYWQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Embodiment 2 SIRPα-Fc Production and purification of fusion proteins

HEK293 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were transiently transfected with the plasmid constructed in Example 1 (PEI, Polysciences) and cultured at 37°C using FreeStyle™ 293 Expression Medium (purchased from Gibco). After 7 days, the cell culture medium was collected and the cellular components were removed by centrifugation to obtain the culture supernatant containing SIRPa V1-Fc, SIRPa V2-Fc, Mut10-Fc, Mut31-Fc, Mut33-Fc, Mut35-Fc, Mut38-Fc, Mut66-Fc, Mut67-Fc, Mut68-Fc, Mut69-Fc, Mut70-Fc, Mut71-Fc, Mut72-Fc, Mut73-Fc, Mut75-Fc, Mut80-Fc, Mut81-Fc, Mut83-Fc, Mut84-Fc, Mut86-Fc, Mut87-Fc, Mut88-Fc, Mut89-Fc and Mut90-Fc.

Alternatively, add plasmids and transfection reagent (Thermofisher, Catalog No.: A29133) to OptiPRO SFM (Thermofisher, Catalog No.: 12309019), mix well, let stand for 5 minutes, add ExpiCHO-S™ cells (Manufacturer: Thermofisher, Catalog No.: A29127), and culture in a shaker at 5% CO ₂ , 120 rpm, and 37°C. On the second day of transfection, add feed and adjust the shaker temperature to 32°C for continued culture. On the 8th to 11th day after transfection, the cell supernatants containing Mut174-Fc, Mut178-Fc, Mut150-Fc, Mut152-Fc, Mut153-Fc, Mut156-Fc, Mut157-Fc, Mut158-Fc, Mut160-Fc, Mut162-Fc and Mut164-Fc were collected.

The fusion protein in the cell culture supernatant was purified using a 10 mL protein A column (purchased from Boglon). The protein A column was first equilibrated with 3 to 5 column volumes of equilibration buffer (PBS phosphate buffer, pH 7.4), and then the clarified culture supernatant sample was added to the protein A column, with the flow rate controlled at 10 mL/min. After the sample was added, the protein A column was washed with equilibration buffer, and the volume of equilibration buffer was 3 to 5 times the volume of the protein A column bed. The protein bound to the protein A column was eluted with elution buffer (0.02 M citric acid buffer, 0.1 M glycine, 0.1 M sodium chloride, pH 3.0), and the elution was monitored with a nucleic acid protein detector (A280 UV absorption peak). The eluted protein was collected, and the pH was neutralized by adding Tris buffer. After dialysis, the buffer system was PBS, and the target protein was collected. The purified SIRPa-Fc fusion protein was obtained by sterile filtration with a 0.22 μm filter and sterile storage.

The purified SIRPa-Fc fusion protein was tested for protein yield, protein concentration (A280/1.4), SEC purity, etc. The quality of the purified SIRPa-Fc fusion protein was qualified. The test information on protein yield, protein concentration and purity is shown in Table 2.

Table 2 Detection and analysis of SIRPa-Fc fusion protein Mutation Number Protein yield (mg/L) Protein SEC Purity Protein concentration (mg/mL) SIRPa V1-Fc 71.7 98.157% 3.01 SIRPa V2-Fc 125.2 98.910% 6.31 Mut10-Fc 162.0 98.748% 4.86 Mut174-Fc 233.3 98.997% 6.23 Mut178-Fc 226.7 99.299% 5.00 Mut31-Fc 69.9 99.022% 2.84 Mut33-Fc 35.9 98.995% 1.81 Mut35-Fc 39.0 99.046% 1.52 Mut38-Fc 25.0 99.071% 1.11 Mut150-Fc 193.3 99.195% 7.71 Mut152-Fc 186.7 99.179% 8.50 Mut153-Fc 181.7 99.005% 7.96 Mut156-Fc 238.3 99.409% 6.47 Mut157-Fc 265.0 98.977% 5.68 Mut158-Fc 228.3 99.295% 8.62 Mut160-Fc 168.3 99.359% 5.40 Mut162-Fc 160.0 99.460% 3.93 Mut164-Fc 135.0 99.230% 4.15 Mut66-Fc 54.0 98.915% 1.39 Mut67-Fc 85.4 98.878% 3.47 Mut68-Fc 76.7 98.854% 3.22 Mut69-Fc 61.9 99.034% 2.60 Mut70-Fc 79.0 98.956% 3.21 Mut71-Fc 60.7 99.021% 2.47 Mut72-Fc 20.3 97.693% 1.17 Mut73-Fc 64.8 97.932% 2.94 Mut75-Fc 62.4 97.774% 1.7 Mut80-Fc 55.0 97.812% 2.04 Mut81-Fc 66.5 97.745% 2.79 Mut83-Fc 69.7 98.001% 1.27 Mut84-Fc 64.3 99.747% 2.7 Mut86-Fc 66.7 97.565% 2.8 Mut87-Fc 68.8 97.597% 2.89 Mut88-Fc 65.3 97.689% 1.83 Mut89-Fc 48.8 97.831% 2.05 Mut90-Fc 40.3 97.862% 2.54

Embodiment 3 ELISA ( ELISA ) Detection SIRPα-Fc Fusion protein CD47 Protein binding degree

A. Production and purification of CD47 protein

(1) Production and purification of hCD47 ECD-His protein

The human CD47 extracellular domain (ECD) amino acid sequence (NCBI gene accession number Q08722, amino acids 19-139, the specific sequence is shown in SEQ ID NO: 52) was coupled with a 6×His sequence and cloned into the pTT5 vector.

SEQ ID NO: 52 QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSP

Plasmids and transfection reagent PEI (Polysciences, catalog number: 24765-1) were added to OPTI-MEM (Gibco, catalog number: 11058021) and mixed well. After standing for 15 minutes, they were added to Expi293 cells (manufacturer: Thermofisher, catalog number: A14527) and cultured in a shaker at 5% CO ₂ , 120 rpm, and 37°C. On the second day of transfection, OPM-293 ProFeed (Shanghai Aopumai, catalog number: F081918-001) and 6 g/L glucose (manufacturer: Sigma, catalog number: G7528) were added. On the sixth day of transfection, the cell supernatant was collected; the cell components were removed by centrifugation to obtain the culture supernatant containing CD47 ECD-His protein. The protein in the cell culture supernatant was purified using a Ni affinity chromatography column (GE, catalog number: 17371206). First, equilibrate with 3 to 5 times the column volume of equilibration buffer (PBS phosphate buffer, pH 7.4) (20× PBS buffer 500 ml, Bioengineering, catalog number B548117-0500), and then add the clarified culture supernatant sample to the Ni affinity chromatography column, controlling the flow rate at 5 mL/min. After the sample is added, wash the protein A column with equilibration buffer, and the volume of equilibration buffer is 3 to 5 times the volume of the Ni affinity chromatography column bed. Use 0-500 mM imidazole (Sinopharm, Catalog No.: 30104961) for gradient elution, and monitor the elution status with a nucleic acid protein detector (A280 UV absorption peak). Collect the eluted protein and dialyze the protein into PBS phosphate buffer at 4°C using a dialysis card (purchased from Thermo Scientific, Catalog No.: 88252). Use a 0.22 μm filter (Millipore, Catalog No. SLGVR13SL) for sterile filtration and sterile storage to obtain purified hCD47 ECD-His protein. The purified hCD47 ECD-His was analyzed for protein yield, protein concentration (A280/1.4), SEC purity, etc., and the purified protein quality was qualified. The test information about protein yield, protein concentration and purity is shown in Table 3.

(2) Production and purification of hCD47 ECD-mFc protein

The human CD47 extracellular domain (ECD) amino acid sequence (specific sequence as shown in SEQ ID NO: 52) was coupled to the mouse Fc sequence and cloned into the pTT5 vector.

Plasmids and transfection reagent PEI (Polysciences, catalog number: 24765-1) were added to OPTI-MEM (Gibco, catalog number: 11058021) and mixed well. After standing for 15 minutes, they were added to Expi293 cells (manufacturer: Thermofisher, catalog number: A14527) and cultured in a shaker at 5% CO ₂ , 120 rpm, and 37°C. On the second day of transfection, OPM-293 ProFeed (Shanghai Aopumai, catalog number: F081918-001) and 6 g/L glucose (manufacturer: Sigma, catalog number: G7528) were added. On the sixth day of transfection, the cell supernatant was collected; the cell components were removed by centrifugation to obtain the culture supernatant containing hCD47 ECD-mFc protein. Use 10 mL protein A column (GE, catalog number: 17549802) to purify the protein in the cell culture supernatant. The protein A column was first equilibrated with 3 to 5 times the column volume of equilibration buffer (PBS phosphate buffer, pH 7.4) (20× PBS buffer 500 ml, Bioengineering, catalog number B548117-0500), and then the clarified culture supernatant sample was added to the protein A column, and the flow rate was controlled at 10 mL/min. After the sample was added, the protein A column was washed with equilibration buffer, and the volume of equilibration buffer was 3 to 5 times the volume of the protein A column bed. The protein bound to the protein A column was eluted with elution buffer (0.02 M citric acid buffer, pH 3.5), and the elution was monitored with a nucleic acid protein detector (A280 UV absorption peak). The eluted protein was collected, and Tris buffer (Sinopharm, Catalog No.: 30188336) was added to neutralize the pH. After dialysis, the buffer system was PBS, and the target protein was collected. Aseptic filtration was performed with a 0.22 μm filter (Millipore, Catalog No. SLGVR13SL) and aseptic storage was performed to obtain the purified hCD47 ECD-mFc protein. The purified hCD47 ECD-mFc was analyzed for protein yield, protein concentration (A280/1.4), SEC purity, etc., and the purified protein quality was qualified. The test information about protein yield, protein concentration and purity is shown in Table 3.

Table 3 Detection and analysis of CD47 protein Protein name Protein yield (mg/L) Protein SEC Purity Protein concentration (mg/mL) hCD47 ECD-His 39.6 96.7% 1.13 hCD47 ECD-mFc 88.4 82.7% 2.21

B. Detection of binding between SIRPa-Fc fusion protein and hCD47 ECD-His protein

Biotin labeling of hCD47 ECD-His. According to the instructions of the Biotin Labeling kit (Cat. No. LK03, purchased from DO JINDO), add 100 µL WS buffer and 200 µg hCD47 ECD-His protein to the Filtration tube, mix well and centrifuge at 8000 g for 10 minutes; add 10 µL DMSO to the NH2-Reactive Biotin Tube and mix well for use; after centrifugation, add 100 µL reaction solution and 8 µL NH2-Reactive Biotin solution to the Filtration tube, mix well and place in a 37°C constant temperature incubator for 10 minutes, add 100 µL WS buffer and centrifuge at 8000 g for 10 minutes, discard the filtrate and add 200 µL WS buffer and centrifuge at 8000 g for 10 minutes. g for 10 minutes. Repeat the above step once and add 100 µL WS buffer to recover the labeled hCD47 ECD-His into a new centrifuge tube. Measure the concentration and set aside.

First, dilute streptavidin (SA, catalog number S4762, purchased from Sigma) with PBS to a final concentration of 3 µg/mL, then add 50 µL per well to a 96-well ELISA plate and incubate overnight at 4°C. The next day, discard the supernatant and add blocking solution, which is PBS buffer containing 5% (w/w) skimmed milk powder (purchased from Sangon, catalog number: A100777-0500), and block at 37°C for 2 hours. Pour off the blocking solution and wash the plate three times with PBS buffer (PBST) containing 0.1% (v/v) Tween20 (purchased from Sangon, catalog number A100777-0500). 2 µg/mL biotin-labeled hCD47 ECD-His was added to each well, 50 µL/well, incubated at 37°C for 1 hour, and washed 3 times with PBST. 50 µL of the purified SIRPa-Fc fusion protein obtained in Example 2 was added to each well, with a starting concentration of 50 nM, and a 5-fold gradient dilution. After incubation at 37°C for 1 hour, the plate was washed 3 times with PBST. 50 µL of a 1:5000 diluted secondary antibody labeled with horseradish peroxidase (HRP) (purchased from Bethyl, catalog number A80-304P) was added to each well, and the dilution solution was PBS containing 1% (w/w) BSA (purchased from Sangon, catalog number A500023-0100). After incubation at room temperature for 1 hour, the plate was washed 5 times with PBST. 50 µL TMB substrate was added to each well, incubated at room temperature for 5 minutes, and 50 µL stop solution (1.0N HCl) was added to each well. A450 nm values were read using a microplate reader (Ensight-HH3400, purchased from PerkinElmer). The results are shown in Figure 1 and Table 4. The results show that the binding activity of the purified SIRPa-Fc fusion protein to hCD47 ECD-His is better than that of wild-type V1 or V2, and better than that of TTI621. The negative control is an unrelated Fc fusion protein, the same below. The data in the table are OD450 nm values.

Table 4 ELISA detection of binding activity of SIRPa-Fc fusion protein and hCD47 ECD-His OD450 nm Protein concentration (nM) Protein name 50 10 2 0.4 0.08 0.016 0.0032 0.00064 Mut10-Fc 2.679 2.237 2.208 2.178 1.697 0.877 0.270 0.104 SIRPa V2-Fc 2.497 2.191 2.119 1.895 1.279 0.373 0.086 0.054 TTI621 2.329 2.352 2.170 1.945 1.255 0.359 0.088 0.055 Mut31-Fc 3.107 3.039 2.904 2.724 1.744 0.647 0.169 0.081 SIRPa V1-Fc 3.199 2.723 2.508 1.383 0.542 0.132 0.061 0.057 TTI621 3.176 3.082 2.933 2.302 1.107 0.258 0.076 0.067 Mut33-Fc 2.809 2.411 2.357 2.258 1.502 0.631 0.166 0.091 Mut35-Fc 2.701 2.295 2.292 2.139 1.322 0.449 0.127 0.070 Mut38-Fc 2.470 2.309 2.321 2.163 1.395 0.488 0.132 0.088 SIRPa V1-Fc 2.873 2.177 2.063 1.396 0.401 0.153 0.065 0.062 TTI621 2.438 2.240 2.150 1.905 0.960 0.233 0.076 0.062 Negative control 0.064 0.046 0.042 0.044 0.043 0.046 0.044 0.061 Mut33-Fc 1.730 1.714 1.693 1.540 1.032 0.491 0.147 0.102 Mut66-Fc 1.647 1.441 1.540 1.391 0.837 0.300 0.117 0.061 Mut67-Fc 1.698 1.563 1.476 1.351 0.888 0.336 0.116 0.067 Mut68-Fc 1.659 1.511 1.507 1.196 0.780 0.292 0.122 0.070 Mut69-Fc 1.649 1.615 1.638 1.340 0.809 0.291 0.113 0.078 TTI621 1.461 1.460 1.240 0.807 0.580 0.133 0.061 0.048 Negative control 0.050 0.044 0.039 0.045 0.039 0039 0.043 0.045 Mut72-Fc 2.081 1.983 1.971 1.746 1.039 0.411 0.132 0.073 Mut73-Fc 2.552 2.423 2.532 2.080 1.046 0.371 0.130 0.066 Mut75-Fc 2.268 2.206 1.985 1.913 1.119 0.379 0.126 0.070 Mut80-Fc 2.101 1.838 1.845 1.623 0.899 0.379 0.149 0.066 Mut81-Fc 1.931 1.768 1.796 1.564 1.009 0.369 0.117 0.069 TTI621 2.457 2.400 2.048 1.642 0.894 0.256 0.091 0.049 Mut83-Fc 1.372 1.518 1.491 1.441 0.843 0.349 0.119 0.075 Mut84-Fc 1.673 1.861 1.729 1.536 1.086 0.349 0.116 0.067 Mut86-Fc 1.630 1.280 1.600 1.445 0.789 0.307 0.107 0.067 Mut87-Fc 1.750 1.840 1.639 1.478 0.921 0.338 0.103 0.064 Mut88-Fc 1.391 1.709 1.729 1.337 0.949 0.360 0.116 0.058 Mut89-Fc 1.687 1.774 1.833 1.489 0.995 0.449 0.120 0.068 Mut90-Fc 1.508 1.542 1.541 1.307 1.016 0.425 0.144 0.072 TTI621 1.425 1.599 1.345 1.084 0.593 0.187 0.065 0.046 Negative control 0.047 0.046 0.038 0.038 0.039 0.041 0.046 0.047 Mut150-Fc 1.762 1.665 1.481 1.152 0.616 0.223 0.094 0.057 Mut152-Fc 1.683 1.596 1.571 1.360 0.817 0.274 0.091 0.052 Mut153-Fc 1.758 1.787 1.820 1.507 0.833 0.285 0.098 0.060 Mut156-Fc 1.719 1.827 1.791 1.554 0.914 0.254 0.093 0.058 Mut157-Fc 1.639 1.640 1.629 1.435 0.836 0.227 0.073 0.053 SIRPa V1-Fc 1.711 1.556 1.546 1.369 0.756 0.116 0.073 0.051 TTI621 1.762 1.714 1.537 1.249 0.641 0.152 0.073 0.054 Negative control 0.066 0.054 0.045 0.043 0.042 0.050 0.056 0.064 Mut158-Fc 1.794 1.800 1.785 1.621 1.031 0.420 0.145 0.072 Mut160-Fc 1.707 1.645 1.739 1.387 0.812 0.381 0.100 0.059 Mut162-Fc 1.667 1.892 1.681 1.448 0.822 0.486 0.164 0.056 Mut164-Fc 1.811 1.802 1.990 1.496 0.809 0.295 0.109 0.060 SIRPa V1-Fc 1.360 1.685 1.742 1.318 0.669 0.241 0.057 0.050 TTI621 1.607 1.609 1.690 1.363 0.781 0.253 0.075 0.051 Negative control 0.064 0.049 0.077 0.114 0.062 0.043 0.046 0.046 Mut174-Fc 1.833 1.807 1.733 1.396 0.816 0.293 0.090 0.059 Mut178-Fc 1.776 1.798 1.804 1.469 0.816 0.287 0.090 0.053 SIRPa V2-Fc 1.869 1.803 1.775 1.437 0.780 0.223 0.072 0.052 TTI621 1.772 1.728 1.776 1.393 0.782 0.244 0.085 0.051 Negative control 0.069 0.055 0.043 0.040 0.041 0.042 0.051 0.049

C. Detection of binding between SIRPa-Fc fusion protein and hCD47 ECD-mFc protein

Anti-mFc (purchased from Jackson, Cat. No. 115-006-071) was diluted with PBS to a final concentration of 2 µg/mL, then added to a 96-well ELISA plate at 50 µL per well and incubated at 4°C overnight. The next day, the supernatant was discarded and blocking solution, i.e. PBS buffer containing 5% (w/w) skimmed milk powder (purchased from Sangon, Cat. No. A600669-0250), was added and blocked at 37°C for 2 hours. The blocking solution was discarded and the plate was washed 3 times with PBST. 0.5 µg/mL hCD47 ECD-mFc was added to each well at 50 µL/well, incubated at 37°C for 1 hour, and washed 3 times with PBST. 50 μL of the purified SIRPa-Fc fusion protein obtained in Example 2 was added to each well, with a starting concentration of 50 nM, and then diluted 5-fold. After incubation at 37°C for 1 hour, the plate was washed 3 times with PBST. 50 μL of a 1:5000 diluted secondary antibody labeled with horseradish peroxidase (HRP) (purchased from Bethyl, catalog number A80-304P) was added to each well, and the dilution solution was PBS containing 1% (w/w) BSA (purchased from Sangon, catalog number A500023-0100). After incubation at room temperature for 1 hour, the plate was washed 5 times with PBST. 50 μL of TMB substrate was added to each well, incubated at room temperature for 5 minutes, and 50 μL of stop solution (1.0 N HCl) was added to each well. A450 nm values were read using a microplate detector (Ensight-HH3400, purchased from PerkinElmer). The results are shown in Figure 2 and Table 5. The results show that the binding activity of the purified SIRPa-Fc fusion protein to hCD47 ECD-mFc is better than that of wild-type V1 or V2 and TTI621. The data in the table are OD450 nm values.

Table 5 ELISA detection of the binding activity of SIRPa-Fc fusion protein and hCD47 ECD-mFc OD450 nm Protein concentration (nM) Protein name 50 10 2 0.4 0.08 0.016 0.0032 0.00064 Mut10-Fc 2.409 2.114 2.103 2.053 1.713 0.925 0.314 0.116 SIRPa V2-Fc 2.184 1.942 1.855 1.653 1.006 0.329 0.097 0.063 TTI621 1.995 1.987 1.935 1.724 1.013 0.336 0.090 0.062 Mut31-Fc 2.758 2.771 2.751 2.644 1.811 0.706 0.187 0.092 SIRPa V1-Fc 2.969 2.565 2.319 1.892 0.859 0.227 0.084 0.068 TTI621 2.608 2.668 2.550 2.215 1.233 0.383 0.136 0.068 Mut33-Fc 2.567 2.292 2.217 2.117 1.588 0.629 0.192 0.104 Mut35-Fc 2.658 2.100 2.005 1.939 1.244 0.510 0.145 0.100 Mut38-Fc 2.452 2.098 2.056 2.038 1.409 0.557 0.165 0.092 SIRPa V1-Fc 2.355 1.905 1.574 1.118 0.507 0.143 0.071 0.065 TTI621 2.188 1.966 1.848 1.588 0.799 0.228 0.087 0.078 Negative control 0.184 0.082 0.053 0.054 0.049 0.053 0.049 0.058 Mut33-Fc 1.669 1.468 1.518 1.301 0.987 0.417 0.155 0.092 Mut66-Fc 1.240 1.519 1.306 1.144 0.762 0.250 0.095 0.062 Mut67-Fc 1.329 1.430 1.593 1.415 0.920 0.309 0.108 0.061 Mut68-Fc 1.344 1.621 1.474 1.203 0.831 0.285 0.118 0.063 Mut69-Fc 1.328 1.612 1.418 1.347 0.907 0.333 0.116 0.078 TTI621 0.796 0.836 0.751 0.422 0.204 0.074 0.047 0.047 Negative control 0.048 0.037 0.036 0.037 0.0036 0.036 0.039 0.047 Mut72-Fc 1.267 1.463 1.142 1.227 0.964 0.415 0.145 0.076 Mut73-Fc 1.269 1.349 1.428 1.185 0.894 0.331 0.132 0.078 Mut75-Fc 1.432 1.506 1.397 1.277 1.078 0.373 0.147 0.069 Mut80-Fc 1.392 1.369 1.460 1.178 0.966 0.396 0.165 0.085 Mut81-Fc 1.267 1.291 1.281 1.295 0.993 0.373 0.150 0.071 TTI621 1.295 1.567 1.439 0.997 0.500 0.162 0.074 0.054 Mut83-Fc 1.139 1.430 1.288 1.384 0.731 0.288 0.127 0.062 Mut84-Fc 1.265 1.459 1.480 1.353 0.883 0.360 0.144 0.068 Mut86-Fc 1.309 1.571 1.479 1.454 0.863 0.296 0.125 0.062 Mut87-Fc 1.104 1.419 1.306 1.194 0.919 0.313 0.128 0.070 Mut88-Fc 1.251 1.258 1.287 1.263 0.838 0.308 0.121 0.066 Mut89-Fc 1.286 1.459 1.115 1.330 0.877 0.334 0.146 0.074 Mut90-Fc 1.334 1.374 1.159 1.252 0.816 0.382 0.158 0.087 TTI621 1.005 1.210 0.866 0.759 0.367 0.134 0.059 0.040 Negative control 0.046 0.038 0.036 0.035 0.037 0.043 0.043 0.039 Mut153-Fc 1.473 1.185 1.363 1.019 0.716 0.269 0.105 0.065 Mut156-Fc 1.405 0.978 1.153 0.7 0.404 0.146 0.075 0.061 Mut157-Fc 1.039 1.402 1.335 0.521 0.239 0.105 0.063 0.061 SIRPa V1-Fc 0.763 0.638 0.614 0.316 0.113 0.061 0.053 0.057 TTI621 1.219 0.949 0.957 0.56 0.308 0.116 0.064 0.07 Negative control 0.084 0.068 0.055 0.054 0.05 0.056 0.055 0.059 Mut158-Fc 1.844 1.74 1.603 1.44 1.079 0.47 0.174 0.097 Mut160-Fc 1.421 1.403 1.407 1.294 0.835 0.295 0.119 0.069 Mut162-Fc 1.222 1.26 1.469 1.105 0.697 0.229 0.115 0.067 Mut164-Fc 2.194 1.959 1.878 1.935 1.018 0.362 0.133 0.075 SIRPa V1-Fc 0.870 1.288 1.017 0.318 0.107 0.060 0.055 0.057 TTI621 1.133 1.008 1.128 0.778 0.33 0.115 0.069 0.059 Negative control 0.063 0.053 0.046 0.044 0.043 0.047 0.049 0.056 Mut178-Fc 1.625 1.73 1.872 1.557 0.898 0.304 0.116 0.061 SIRPa V2-Fc 1.638 1.568 1.386 0.764 0.384 0.139 0.067 0.056 TTI621 1.292 1.33 1.632 0.876 0.309 0.112 0.07 0.056 Negative control 0.073 0.063 0.059 0.052 0.049 0.052 0.058 0.053

Embodiment 4 Surface Plasmon Resonance (SPR) SPR ) Detection SIRPα-Fc Fusion protein CD47 Protein affinity

The affinity to the antigen was determined by multi-cycle kinetics using a BIAcore 8K instrument. First, according to the Human Antibody Capture Kit (purchased from cytiva, 29234600), the Anti-human IgG Fc antibody was immobilized on a CM5 chip (purchased from cytiva, BR100530), and according to the Amine Coupling Kit (purchased from cytiva, BR100633), HBS-EP+ pH7.4 was used as the mobile phase, and NHS and EDC were mixed to activate the chip for about 600 s, and the Anti-human IgG Fc was diluted to 15 µg/mL with 10 mM sodium acetate pH5.0 and injected for 420 s. Finally, the remaining activation sites were blocked with ethanolamine. Then, multi-cycle kinetics was used to determine the affinity with the antigen. In each cycle, the SIRPa-Fc fusion protein was first captured by an anti-human IgG Fc chip, and then a single concentration of antigen was injected to record the binding and dissociation process of the SIRPa-Fc fusion protein and the antigen protein. Finally, the chip was regenerated with 3M _MgCl2 , where the mobile phase was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20), the flow rate was 30 µL/min, the regeneration time was 30 s, and the detection temperature was 25°C. Finally, according to the 1:1 Binding model was used to analyze the data and fit the antibody-antigen binding kinetic parameters, including the association rate constant ka, dissociation rate constant kd, equilibrium dissociation constant KD, and maximum binding signal Rmax. The results are shown in Table 6, which show that the affinity of purified SIRPa-Fc fusion protein to hCD47 ECD-mFc is better than that of wild type or TTI621, among which Mut10-Fc, Mut33-Fc, Mut67-Fc, Mut69-Fc, Mut80-Fc, Mut81-Fc, Mut86-Fc, Mut153-Fc, Mut158-Fc, Mut160-Fc, and Mut164-Fc fusion proteins have a more obvious improvement in affinity.

Table 6 Affinity of SIRPa-Fc fusion protein and hCD47 ECD-mFc detected by SPR SIRPα-Fc fusion protein ka (1/Ms) kd (1/s) KD (M) Mut10-Fc 1.16E+06 8.66E-04 7.48E-10 TTI621 6.75E+05 1.57E-03 2.32E-09 Mut31-Fc 3.04E+05 1.19E-03 3.92E-09 Mut33-Fc 4.52E+05 8.74E-04 1.94E-09 Mut35-Fc 2.91E+05 1.19E-03 4.09E-09 Mut38-Fc 3.25E+05 1.17E-03 3.60E-09 SIRPa V1-Fc 1.36E+05 1.94E-03 1.42E-08 TTI621 1.80E+05 1.64E-03 9.13E-09 Mut33-Fc 1.68E+06 3.58E-03 2.13E-09 Mut66-Fc 1.28E+06 7.48E-03 5.82E-09 Mut67-Fc 1.62E+06 4.33E-03 2.66E-09 Mut68-Fc 1.42E+06 6.64E-03 4.68E-09 Mut69-Fc 1.57E+06 4.52E-03 2.88E-09 Mut71-Fc 1.39E+06 5.98E-03 4.29E-09 TTI621 7.18E+05 6.93E-03 9.65E-09 Mut72-Fc 8.45E+05 1.15E-03 1.36E-09 Mut73-Fc 8.28E+05 7.45E-03 9.00E-09 Mut75-Fc 8.78E+05 6.53E-03 7.45E-09 Mut80-Fc 8.47E+05 6.49E-04 7.67E-10 Mut81-Fc 8.29E+05 2.83E-04 3.42E-10 Mut83-Fc 8.28E+05 1.14E-03 1.37E-09 Mut84-Fc 8.81E+05 6.87E-03 7.80E-09 Mut86-Fc 8.18E+05 7.27E-04 8.89E-10 Mut87-Fc 7.63E+05 9.42E-03 1.24E-08 Mut88-Fc 8.46E+05 5.89E-03 6.97E-09 Mut89-Fc 9.09E+05 7.74E-03 8.51E-09 Mut90-Fc 1.17E+06 5.82E-03 4.99E-09 Mut150-Fc 4.36E+05 1.78E-03 4.09E-09 Mut152-Fc 2.90E+05 1.74E-03 5.98E-09 Mut153-Fc 7.10E+05 1.52E-03 2.15E-09 Mut156-Fc 4.96E+05 1.68E-03 3.39E-09 Mut157-Fc 4.06E+05 1.77E-03 4.35E-09 Mut158-Fc 8.66E+05 1.45E-03 1.67E-09 Mut160-Fc 6.60E+05 1.62E-03 2.45E-09 Mut162-Fc 5.15E+05 1.78E-03 3.46E-09 Mut164-Fc 9.34E+05 1.38E-03 1.48E-09 SIRPa V1-Fc 2.22E+05 1.63E-03 7.34E-09 TTI621 4.30E+05 1.80E-03 4.19E-09 Mut174-Fc 4.65E+05 1.79E-03 3.86E-09 Mut178-Fc 5.72E+05 1.68E-03 2.94E-09 SIRPa V2-Fc 3.88E+05 2.00E-03 5.15E-09

Embodiment 5 Flow cytometry experiment ( FACS ) Detection SIRPα-Fc Binding activity of fusion protein to cells

A. Detection of binding of SIRPa-Fc fusion protein to Jurkat cells

Jurkat cells were expanded and cultured in T-75 cell culture flasks, centrifuged at 1000 rpm for 5 minutes at room temperature, the culture medium was discarded, and the cells were washed twice with PBS buffer (purchased from Hyclone, catalog number SH30256.01), and the cells were resuspended in PBS and counted. 1×10 ⁵ cells per well, 50 µL per well were added to a U-bottom 96-well FACS reaction plate (purchased from Corning, catalog number 3795), and placed at 4°C for use. Dilute the SIRPa-Fc fusion protein to be tested in PBS containing 1% (w/w) BSA (purchased from Sangon, Cat. No. A500023-0100), add 50 µL per well to the cells, mix well, and incubate at 4°C for 1 hour. Add 200 µL of FACS buffer (PBS containing 1% (w/w) BSA) to each well, wash twice, and centrifuge at 1500 rpm for 5 minutes. After removing the supernatant, add 100 µL of diluted fluorescently labeled secondary antibody (purchased from Jackson ImmunoResearch, Cat. No. 109-605-098) to each well and incubate at 4°C for 1 hour. Wash twice by centrifugation with FACS buffer and suspend the cells in 100 µL of PBS. The results were detected and analyzed using a FACS instrument (FACS Canto II, purchased from BD). The results are shown in Figure 3 and Table 7. The results showed that the SIRPa-Fc fusion protein can bind to CD47 on the surface of Jurkat cells, and the binding activity of the SIRPa-Fc fusion protein to Jurkat is better than that of wild-type V1 or V2, and better than that of TTI621. The MFI data in the table is the average fluorescence intensity value of the measured cell population.

Table 7 FACS detection of binding activity of SIRPa-Fc fusion protein to Jurkat MFI Protein concentration (nM) Protein name 500 100 20 4 0.8 0.16 0.032 0.0064 Mut10-Fc 29421 29435 27944 26359 20067 5994 1359 342 SIRPa V2-Fc 23854 18974 15977 13447 9025 3268 811 248 TTI621 23468 19197 15960 13191 8722 3036 715 218 Negative control 247 94 45 33 36 31 54 38 Mut31-Fc 19581 20410 18106 16359 12517 4406 1092 241 Mut33-Fc 21417 24570 23371 22807 20429 6754 1472 334 Mut35-Fc 20879 21465 19055 17479 12776 4040 922 203 Mut38-Fc 20841 20669 17422 16337 13240 4716 1092 249 SIRPa V1-Fc 15935 11319 6613 3123 1222 311 98 31 TTI621 17556 14974 10285 8896 6198 2492 704 144 Negative control 34 29 twenty two twenty two 20 twenty three twenty four 209 MFI Protein concentration (nM) Protein name 1000 200 40 8 1.6 0.32 0.064 0.0128 Mut66-Fc 16478 27464 26727 25467 25216 23873 8938 2500 Mut67-Fc 13809 29021 27758 27161 26909 26165 8281 1792 Mut68-Fc 14670 27125 26078 25219 24530 23355 7932 2118 Mut69-Fc 14615 28442 28054 27286 26992 24980 7805 2020 Mut70-Fc 13997 19865 15690 12195 9923 6502 3526 2289 Mut71-Fc 13607 25644 25819 24005 23564 21379 9698 3135 Mut33-Fc 17151 26777 26336 24440 24129 21791 9270 3284 TTI621 15633 17641 14387 10577 8202 5294 1952 533 Negative control 163 91 38 30 twenty four twenty four twenty three twenty four

B. Detection of binding of SIRPa-Fc fusion protein to Raji cells

Raji cells were expanded and cultured in T-75 cell culture flasks, counted, centrifuged at 1000 rpm for 5 minutes at room temperature, the culture medium was discarded, washed twice with PBS buffer (purchased from Hyclone, catalog number SH30256.01), and the cells were resuspended with 25 μg/ml Fc blocking agent (purchased from BD, catalog number 564220) and blocked at room temperature for 20 minutes. 1×10 ⁵ cells per well, 50 μL per well were added to a U-bottom 96-well FACS reaction plate (purchased from Corning, catalog number 3795), and placed at 4°C for use. Dilute the SIRPa-Fc fusion protein to be tested in PBS containing 1% (w/w) BSA (purchased from Sangon, Cat. No. A500023-0100), add 50 µL per well to the cells, mix well, and incubate at 4°C for 1 hour. Add 200 µL of FACS buffer (PBS containing 1% (w/w) BSA) to each well, wash twice, and centrifuge at 1500 rpm for 5 minutes. After removing the supernatant, add 100 µL of diluted fluorescently labeled secondary antibody (purchased from Jackson ImmunoResearch, Cat. No. 109-605-098) to each well and incubate at 4°C for 1 hour. Wash twice by centrifugation with FACS buffer and suspend the cells in 100 µL of PBS. The results were detected and analyzed using a FACS instrument (FACS Canto II, purchased from BD). The results are shown in Figure 4 and Table 8. The results showed that the SIRPa-Fc fusion protein could bind to CD47 on the surface of Raji cells, and the binding activity of the SIRPa-Fc fusion protein to Raji was better than that of wild-type V1 or V2, and better than that of TTI621. The MFI data in the table is the average fluorescence intensity value of the measured cell population.

Table 8 FACS detection of binding activity of SIRPα-Fc fusion protein to Raji MFI Protein concentration (nM) Protein name 500 100 20 4 0.8 0.16 0.032 0.0064 Mut10-Fc 920 969 822 769 696 548 246 106 SIRPa V2-Fc 680 777 516 348 254 156 87 67 TTI621 724 792 557 392 268 175 97 64 Negative control 123 139 83 66 64 64 59 59 Mut31-Fc 204 207 158 137 120 79 37 twenty four Mut33-Fc 220 238 222 214 204 152 54 29 Mut35-Fc 208 215 169 154 132 79 39 26 Mut38-Fc 205 211 170 133 129 90 44 26 SIRPa V1-Fc 166 146 76 44 30 20 18 20 TTI621 178 169 114 82 63 44 31 twenty two Negative control 57 50 27 twenty three twenty two twenty two twenty two twenty four MFI Protein concentration (nM) Protein name 200 40 8 1.6 0.32 0.064 0.0128 0.00256 Mut33-Fc 205 208 204 194 173 103 60 50 Mut66-Fc 211 211 205 196 148 69 46 39 Mut67-Fc 218 223 220 215 178 84 52 45 Mut68-Fc 212 215 207 203 156 75 48 41 Mut69-Fc 222 230 224 217 185 88 52 48 Negative control 58 53 49 46 46 41 41 42 MFI Protein concentration (nM) Protein name 100 20 4 0.8 0.16 0.032 0.0064 0.00128 Mut72-Fc 194 197 201 194 152 71 52 37 Mut73-Fc 177 178 178 170 141 72 50 41 Mut75-Fc 193 200 199 199 162 73 54 42 Mut80-Fc 192 205 203 203 156 79 52 48 Mut81-Fc 192 197 205 201 162 83 60 46 TTI621 136 113 84 69 54 50 53 48 Negative control 48 46 46 46 48 45 49 - Mut83-Fc 192 190 193 189 137 68 46 37 Mut84-Fc 177 178 180 173 143 73 50 45 Mut86-Fc 194 200 196 199 159 76 50 46 Mut87-Fc 180 181 175 170 131 69 50 44 Mut88-Fc 181 193 189 188 158 78 57 50 Mut89-Fc 169 178 175 165 140 80 53 53 Mut90-Fc 178 188 190 190 158 80 56 50 TTI621 141 114 88 73 57 49 45 46 Negative control 42 48 50 48 49 42 46 46

Embodiment 6 Testing SIRPα-Fc Fusion protein blocking Raji and receptor SIRP Combination

A. Production and purification of SIRPa protein

The amino acid sequence of the human SIRPa V1 extracellular domain (ECD) (NCBI gene accession number NP_542970.1, amino acids 31-370, the specific sequence is shown in SEQ ID NO: 53) and the amino acid sequence of the human SIRPa V2 extracellular domain (ECD) (NCBI gene accession number AAH38510.1, amino acids 31-369, the specific sequence is shown in SEQ ID NO: 54) were coupled with 6×His sequences and cloned into the pTT5 vector.

SEQ ID NO: 53 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTL QGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNER

SEQ ID NO: 54 EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDP LRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNER

The obtained plasmid and transfection reagent PEI (Polysciences, Catalog No.: 24765-1) were added to OPTI-MEM (Gibco, Catalog No.: 11058021) and mixed well. After standing for 15 minutes, they were added to Expi293 cells (Manufacturer: Thermofisher, Catalog No.: A14527) and cultured in a shaker at 5% CO ₂ , 120 rpm, and 37°C. On the second day of transfection, OPM-293 ProFeed (Shanghai Aopumai, Catalog No.: F081918-001) and 6 g/L glucose (Manufacturer: Sigma, Catalog No.: G7528) were added. On the sixth day after transfection, the cell supernatant was collected; the cell components were removed by centrifugation to obtain the culture supernatant containing hSIRPa V1 ECD-His and hSIRPa V2 ECD-His proteins, respectively. The protein in the cell culture supernatant was purified using a Ni affinity chromatography column (GE, catalog number: 17371206). First, equilibrate with 3 to 5 times the volume of the column with equilibration buffer (PBS phosphate buffer, pH 7.4) (20× PBS buffer 500 ml, Bioengineering, catalog number B548117-0500), and then add the clarified culture supernatant sample to the Ni affinity chromatography column, controlling the flow rate at 5 mL/min. After the sample was added, the protein A column was washed with equilibration buffer, and the volume of the equilibration buffer was 3 to 5 times the volume of the Ni affinity chromatography column bed. Gradient elution was performed with 0-500 mM imidazole (Sinopharm, Catalog No.: 30104961), and the elution was monitored with a nucleic acid protein detector (A280 UV absorption peak). The eluted protein was collected and dialyzed into PBS phosphate buffer at 4°C using a dialysis card (purchased from Thermo Scientific Catalog No.: 88252). Aseptic filtration was performed using a 0.22 μm filter (Millipore, Catalog No. SLGVR13SL) and aseptic storage was performed to obtain purified hSIRPa V1 ECD-His and hSIRPa V2 ECD-His proteins. The purified hSIRPa V1 ECD-His and hSIRPa V2 ECD-His were tested for protein yield, protein concentration (A280/1.4), SEC purity, etc., and the purified proteins were of qualified quality. The test information on protein yield, protein concentration, and purity is shown in Table 9.

Table 9 Detection and analysis of SIRPa protein Protein name Protein yield (mg/L) Protein SEC Purity Protein concentration (mg/mL) hSIRPa V1 ECD-His 241.56 99.31 3.355 hSIRPa V2 ECD-His 234.24 99.39 2.928

B. Detection of the ability of SIRPα-Fc fusion protein to block the activity of Raji and receptor SIRPα V1

hSIRPa V1 ECD-His was labeled with biotin, and the method was the same as that in Example 3. Raji cells were expanded and cultured in T-75 cell culture flasks, the culture medium was collected, counted, centrifuged at 1000 rpm for 5 minutes at room temperature, the culture medium was discarded, and the cells were washed twice with PBS buffer (purchased from Hyclone, catalog number SH30256.01), and the cells were resuspended with 25 μg/ml Fc blocking agent (purchased from BD, catalog number 564220) and blocked at room temperature for 20 minutes. 1×10 ⁵ cells per well were added to a U-bottom 96-well FACS reaction plate (purchased from Corning, Cat. No. 3795) at 50 µL per well and placed at 4°C for use. 1% (w/w) BSA was added to PBS phosphate buffer as FACS buffer, and purified SIRPa-Fc fusion protein of different concentrations and biotinylated hSIRRPa V1 ECD-His were mixed to a final concentration of 4 µg/mL of hSIRRPa V1 ECD-His and incubated at room temperature for 15 minutes. Then 50 µL of the mixture was added to each well and incubated on ice for 1 hour. Wash twice with FACS buffer by centrifugation, add 100 μL fluorescently labeled streptavidin (purchased from Biolegend, catalog number 405243) to each well, and incubate on ice for 1 hour. Wash twice with FACS buffer by centrifugation. Suspend cells with 100 μL PBS, and detect and analyze the results by FACS (FACS Canto II, purchased from BD). The results are shown in Figure 5 and Table 10. SIRPa-Fc fusion protein can effectively block the binding activity of Raji to SIRPa V1, and the blocking activity is better than TTI621 or wild type. The MFI data in the table is the average fluorescence intensity value of the measured cell population.

Table 10 FACS detection of SIRPα-Fc fusion protein blocking the binding activity of Raji and SIRPα V1 MFI Protein concentration (nM) Protein name 200 40 8 1.6 0.32 0.064 0.0128 0.00256 Mut10-Fc 47 49 52 58 112 279 356 394 Mut31-Fc 48 48 57 95 194 333 371 389 Mut33-Fc 47 51 48 49 65 242 351 388 Mut35-Fc 47 48 56 89 197 346 380 401 Mut38-Fc 49 50 56 86 181 317 382 385 TTI621 48 52 86 183 304 378 389 398 Negative control 349 368 383 363 383 386 - - Mut33-Fc twenty three twenty four twenty three twenty three twenty four 76 99 102 Mut66-Fc 16 16 16 16 16 41 88 98 Mut67-Fc 18 18 18 18 16 54 95 102 Mut68-Fc 19 19 18 15 18 60 88 99 Mut69-Fc 20 19 19 16 15 49 90 103 TTI621 twenty three twenty two 31 53 84 95 99 102 Negative control 92 101 106 101 102 99 99 94 MFI Protein concentration (nM) Protein name 100 20 4 0.8 0.16 0.032 0.0064 0.00128 Mut150-Fc 25 45 104 186 237 256 270 235 Mut153-Fc twenty three twenty three 35 89 180 245 262 252 Mut156-Fc 25 42 102 184 222 248 271 247 Mut157-Fc 25 51 117 207 240 248 281 226 Mut158-Fc twenty one twenty three twenty three 39 150 240 266 242 Mut160-Fc twenty two 25 50 126 205 255 293 255 SIRPa V1-Fc 38 85 167 234 251 263 297 241 TTI621 twenty three 30 70 159 216 259 286 250 Negative control 278 282 270 261 264 273 279 261 Mut162-Fc twenty four 29 63 125 192 207 249 227 Mut164-Fc twenty one twenty two twenty three 40 153 193 229 229 SIRPa V1-Fc 37 80 151 193 219 204 231 222 Mut174-Fc twenty three 26 51 110 181 202 212 206 Mut178-Fc twenty two twenty three 38 89 170 206 217 218 SIRPa V2-Fc twenty four 30 67 134 190 194 221 210 TTI621 twenty four 31 68 132 193 203 220 221 Negative control 193 216 206 213 215 197 210 208

C. Detection of the ability of SIRPα-Fc fusion protein to block the activity of Raji and receptor SIRPα V2

hSIRPa V2 ECD-His was labeled with biotin, and the method was the same as that in Example 3. Raji cells were expanded and cultured in T-75 cell culture flasks, the culture medium was collected, counted, centrifuged at 1000 rpm for 5 minutes at room temperature, the culture medium was discarded, and the cells were washed once with PBS buffer (purchased from Hyclone, catalog number SH30256.01), and the cells were resuspended with 25 μg/ml Fc blocking agent (purchased from BD, catalog number 564220) and blocked at room temperature for 20 minutes. 1×10 ⁵ cells per well were added to a U-bottom 96-well FACS reaction plate (purchased from Corning, Cat. No. 3795) at 50 µL per well and placed at 4°C for use. 1% (w/w) BSA was added to PBS phosphate buffer as FACS buffer, and purified SIRPa-Fc fusion protein of different concentrations and biotinylated hSIRRPa V2 ECD-His were mixed to a final concentration of 4 µg/mL of hSIRRPa V2 ECD-His and incubated at room temperature for 15 minutes. Then 50 µL of the mixture was added to each well and incubated on ice for 1 hour. Wash twice with FACS buffer by centrifugation, add 100 μL fluorescently labeled streptavidin (purchased from Biolegend, catalog number 405243) to each well, and incubate on ice for 1 hour. Wash twice with FACS buffer by centrifugation. Suspend cells with 100 μL PBS, and detect and analyze the results by FACS (FACS Canto II, purchased from BD). The results are shown in Figures 6A-6B and Table 11. SIRPa-Fc fusion protein can effectively block the binding activity of Raji and SIRPa V2, and the blocking activity is better than TTI621. The MFI data in the table is the average fluorescence intensity value of the measured cell population.

Table 11 FACS detection of SIRPα-Fc fusion protein blocking the binding activity of Raji and SIRPα V2 MFI Protein concentration (nM) Protein name 200 40 8 1.6 0.32 0.064 0.0128 0.00256 Mut10-Fc 49 49 55 71 143 290 331 335 Mut31-Fc 51 50 68 120 204 318 346 362 Mut33-Fc 49 48 49 56 87 258 339 366 Mut35-Fc 51 50 64 114 220 347 362 372 Mut38-Fc 49 52 62 111 213 324 362 362 TTI621 50 65 116 215 327 368 377 381 Negative control 322 349 342 367 401 386 - - Mut33-Fc twenty two 26 twenty two twenty three 31 78 98 106 Mut66-Fc 26 twenty four twenty two twenty four 20 46 91 101 Mut67-Fc twenty three 26 27 twenty two twenty three 67 99 109 Mut68-Fc twenty two 27 twenty four 26 twenty three 64 101 109 Mut69-Fc twenty two 20 twenty two twenty four twenty four 61 99 99 TTI621 26 27 38 67 95 109 105 105 Negative control 98 95 103 105 114 95 102 97

Embodiment 7 SIRPα-Fc Fusion protein affinity maturation

Four plasmids, Mut87-Fc, Mut88-Fc, Mut89-Fc, and Mut90-Fc, were used as templates to construct four mutation libraries. Each library contained a combination of single-point amino acid mutations and multiple-point mutations targeting key mutation sites. Theoretical total number of mutations: 4.7×10 ⁸ , total library capacity > 3.8×10 ⁹ . After 2-3 rounds of panning, the phages from the second or third round were subjected to affinity identification.

Similar to Example 1, the nucleic acid fragment encoding the mutant was cloned into a pTT5 vector with an Fc tag, and plasmids were prepared according to established standard molecular biology methods. The SIRPα variants used are as follows: Mut113, Mut114, and Mut117. The specific sequence information of the aforementioned fusion protein and each element is shown in Table 12, wherein "MutXX-Fc" represents a fusion protein of mutant SIRPα and Fc.

Table 12 SIRPa mutant sequences Mutation Number Serial Number Sequence information Mut113 (Mut88+L66N+K68Q) SEQ ID NO: 77 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTQRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut114 (Mut88+L66N+K68L) SEQ ID NO: 78 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTLRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut117 (Mut88+L66N+K68I) SEQ ID NO: 79 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTIRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut113-Fc SEQ ID NO: 80 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTQRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut114-Fc SEQ ID NO: 81 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTLRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut117-Fc SEQ ID NO: 82 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTIRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Similar to Example 2, the mutant protein was expressed using the Expi293F system, and the purified SIRPa-Fc fusion protein was subjected to protein yield, protein concentration (A280/1.4), SEC purity and other tests and analyses, and the quality of the purified SIRPa-Fc fusion protein was qualified. The test information on protein yield, protein concentration and purity is shown in Table 13.

Table 13 Detection and analysis of SIRPa-Fc fusion protein Mutation Number Protein yield (mg/L) Protein SEC Purity Protein concentration (mg/mL) Mut113-Fc 76.3 98.191% 4.81 Mut114-Fc 66.3 97.992% 3.98 Mut117-Fc 91.3 97.990% 2.81

Similar to Example 4, the affinity of SIRPa-Fc fusion protein and CD47 protein was detected by surface plasmon resonance (SPR). The results are shown in Table 14. The results show that the affinity of SIRPa-Fc fusion protein and hCD47 ECD-mFc after affinity maturation is better than that of wild type or TTI621.

Table 14 Affinity of SIRPa-Fc fusion protein and hCD47 ECD-mFc detected by SPR SIRPα-Fc fusion protein ka (1/Ms) kd (1/s) KD (M) Mut88-Fc 1.11E+06 9.92E-04 8.97E-10 Mut113-Fc 9.47E+05 2.16E-04 2.28E-10 Mut114-Fc 1.25E+06 2.53E-04 2.03E-10 Mut117-Fc 1.35E+06 4.39E-04 3.25E-10 SIRPa V1-Fc 1.76E+05 7.64E-04 4.34E-09 TTI621 2.50E+05 1.19E-03 4.76E-09

Similar to Example 3C, the binding degree of SIRPα-Fc fusion protein to hCD47 ECD-mFc protein was detected. The results are shown in Figure 7 and Table 15, which show that the binding activity of SIRPα-Fc fusion protein to hCD47 ECD-mFc after affinity maturation is better than that of wild type or TTI621.

Table 15 ELISA detection of binding activity of SIRPa-Fc fusion protein and hCD47 ECD-mFc OD450 nm Protein concentration (nM) Protein name 50 10 2 0.4 0.08 0.016 0.0032 0.00064 Mut88-Fc 2.007 2.083 2.250 1.939 1.466 0.643 0.247 0.091 Mut113-Fc 1.892 1.949 2.164 2.098 1.315 0.625 0.355 0.091 Mut114-Fc 1.874 1.958 2.136 2.012 1.267 0.526 0.200 0.075 Mut117-Fc 2.021 2.013 2.185 1.976 1.294 0.528 0.216 0.071 SIRPa V1-Fc 1.556 1.348 1.300 0.864 0.400 0.143 0.061 0.057 TTI621 1.652 1.470 1.586 1.300 0.699 0.296 0.132 0.082 Negative control 0.056 0.053 0.046 0.044 0.042 0.048 0.051 0.067

Similar to Example 5A, flow cytometry (FACS) was used to detect the binding activity of SIRPa-Fc fusion protein to cells. The results are shown in Figure 8 and Table 16, which show that the binding degree of SIRPa-Fc fusion protein to Jurkat cells after affinity maturation is better than that of wild type or TTI621. The MFI data in the table is the average fluorescence intensity value of the measured cell population.

Table 16 FACS detection of binding activity of SIRPa-Fc fusion protein to Jurkat MFI Protein concentration (nM) Protein name 500 100 20 4 0.8 0.16 0.032 0.0064 Mut88-Fc 5300 5083 4986 4932 4586 1268 463 37 Mut113-Fc 5460 5261 5265 5235 3635 1192 367 34 Mut114-Fc 5563 5353 5292 5258 3921 1327 384 36 Mut117-Fc 5574 5357 5227 5260 3775 1242 390 36 SIRPa V1-Fc 2478 1322 576 211 90 47 36 30 TTI621 2928 2184 1360 792 429 173 73 31 Negative control 178 66 39 35 33 33 31 29

Embodiment 8 Screening for deglycosylation mutations

To ensure the controllability of the subsequent production process, we further mutated the amino acid at position 82 based on Mut81, Mut113, Mut114 and Mut117 to ensure that this site does not cause glycosylation modification. Similar to Example 1, the nucleic acid fragment encoding the mutant was copied to the pTT5 vector with an Fc tag, and the plasmid was prepared according to the established standard molecular biology method. The specific sequence information of the fusion protein and each element is shown in Table 17, where "MutXX-Fc" represents the fusion protein of the mutant SIRPa and Fc.

Table 17 SIRPa mutant sequence Mutation Number Serial Number Sequence information Mut92 (Mut81+T82F) SEQ ID NO: 83 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNIFPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut93 (Mut81+T82D) SEQ ID NO: 84 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNIDPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut94 (Mut81+T82N) SEQ ID NO: 85 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNINPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut95 (Mut81+T82E) SEQ ID NO: 86 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNIEPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut97 (Mut81+T82G) SEQ ID NO: 87 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNIGPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut98 (Mut81+T82H) SEQ ID NO: 88 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNIHPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut99 (Mut81+T82L) SEQ ID NO: 89 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNILPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut100 (Mut81+T82I) SEQ ID NO: 90 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNIIPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut105 (Mut81+T82V) SEQ ID NO: 91 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNIVPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut107 (Mut81+T82Y) SEQ ID NO: 92 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNIYPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut139 (Mut113+T82H) SEQ ID NO: 93 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTQRNNMDFSIRIGNIHPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut141 (Mut113+T82V) SEQ ID NO: 94 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTQRNNMDFSIRIGNIVPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut142 (Mut114+T82G) SEQ ID NO: 95 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTLRNNMDFSIRIGNIGPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut146 (Mut117+T82G) SEQ ID NO: 96 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTIRNNMDFSIRIGNIGPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS Mut92-Fc SEQ ID NO: 97 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNIFPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut93-Fc SEQ ID NO: 98 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNIDPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut94-Fc SEQ ID NO: 99 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNINPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut95-Fc SEQ ID NO: 100 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNIEPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut97-Fc SEQ ID NO: 101 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNIGPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut98-Fc SEQ ID NO: 102 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNIHPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut99-Fc SEQ ID NO: 103 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNILPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut100-Fc SEQ ID NO: 104 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNIIPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut105-Fc SEQ ID NO: 105 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNIVPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut107-Fc SEQ ID NO: 106 EEELQVIQPDKSVLVAAGETATLRCTATSLYPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTKRNNMDFSIRIGNIYPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut139-Fc SEQ ID NO: 107 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTQRNNMDFSIRIGNIHPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut141-Fc SEQ ID NO: 108 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTQRNNMDFSIRIGNIVPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut142-Fc SEQ ID NO: 109 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTLRNNMDFSIRIGNIGPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Mut146-Fc SEQ ID NO: 110 EEELQVIQPDKSVLVAAGETATLRCTATSLMPVGPIQWFRGAGPGRELIYRQKEGHFPRVTTVSDNTIRNNMDFSIRIGNIGPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS DKTHTCPPCPAPEAAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Similar to Example 2, the purified mutant proteins were expressed using the Expi293F or ExpiCHO system, wherein Mut92-Fc, Mut93-Fc, Mut94-Fc, Mut95-Fc, Mut97-Fc, Mut98-Fc, Mut99-Fc, Mut100-Fc, Mut105-Fc and Mut107-Fc were expressed using the Expi293F system, and Mut139-Fc, Mut141-Fc, Mut142-Fc and Mut146-Fc were expressed using the ExpiCHO system. The purified SIRPa-Fc fusion protein was analyzed for protein yield, protein concentration (A280/1.4), SEC purity, etc., and the quality of the purified SIRPa-Fc fusion protein was qualified. For information on protein yield, protein concentration, and purity, see Table 18.

Table 18 Detection and analysis of SIRPa-Fc fusion protein Mutation Number Protein yield (mg/L) Protein SEC Purity Protein concentration (mg/mL) Mut92-Fc 16.3 97.866% 1.03 Mut93-Fc 48.7 98.266% 1.98 Mut94-Fc 64.0 97.704% 1.72 Mut95-Fc 53.5 97.920% 3.38 Mut97-Fc 64.3 97.770% 4.06 Mut98-Fc 53.5 97.812% 2.18 Mut99-Fc 38.3 98.104% 2.42 Mut100-Fc 40.7 97.766% 1.32 Mut105-Fc 44.8 97.210% 2.46 Mut107-Fc 20.3 98.106% 0.92 Mut139-Fc 188.3 91.768% 5.58 Mut141-Fc 141.7 94.182% 3.70 Mut142-Fc 193.3 93.930% 5.88 Mut146-Fc 158.3 93.527% 4.35

Similar to Example 4, the affinity of SIRPa-Fc fusion protein and CD47 protein was detected by surface plasmon resonance (SPR). The results are shown in Table 19. The results show that the affinity of deglycosylated SIRPa-Fc fusion protein and hCD47 ECD-mFc is better than that of wild type or TTI621.

Table 19 Affinity of SIRPa-Fc fusion protein and hCD47 ECD-mFc detected by SPR SIRPα-Fc fusion protein ka (1/Ms) kd (1/s) KD (M) Mut81-Fc 1.06E+06 2.33E-04 2.20E-10 Mut92-Fc 9.23E+05 2.63E-04 2.85E-10 Mut93-Fc 9.70E+05 2.35E-04 2.42E-10 Mut94-Fc 9.06E+05 2.30E-04 2.54E-10 Mut95-Fc 9.61E+05 2.41E-04 2.50E-10 Mut97-Fc 9.19E+05 2.27E-04 2.47E-10 Mut98-Fc 9.41E+05 2.45E-04 2.60E-10 Mut99-Fc 9.98E+05 2.29E-04 2.29E-10 Mut100-Fc 9.32E+05 2.51E-04 2.70E-10 Mut105-Fc 9.60E+05 2.19E-04 2.28E-10 Mut107-Fc 8.59E+05 2.85E-04 3.31E-10 Mut88-Fc 1.11E+06 9.92E-04 8.97E-10 Mut139-Fc 1.14E+06 2.45E-04 2.14E-10 Mut141-Fc 1.16E+06 2.35E-04 2.02E-10 Mut142-Fc 8.99E+05 2.66E-04 2.96E-10 Mut146-Fc 9.82E+05 3.84E-04 3.90E-10 SIRPa V1-Fc 1.76E+05 7.64E-04 4.34E-09 TTI621 2.50E+05 1.19E-03 4.76E-09

Similar to Example 3C, the binding degree of SIRPα-Fc fusion protein to hCD47 ECD-mFc protein was detected. The results are shown in Figures 9A-9C and Table 20, which show that the binding activity of deglycosylated SIRPα-Fc fusion protein to hCD47 ECD-mFc is better than that of wild type or TTI621.

Table 20 ELISA detection of binding activity of SIRPα-Fc fusion protein and hCD47 ECD-mFc OD450 nm Protein concentration (nM) Protein name 50 10 2 0.4 0.08 0.016 0.0032 0.00064 Mut81-Fc 2.157 2.050 2.096 2.110 1.901 0.477 0.186 0.100 Mut92-Fc 2.158 2.039 2.175 2.038 1.115 0.401 0.155 0.112 Mut93-Fc 2.154 2.059 2.146 2.020 1.091 0.399 0.144 0.109 Mut94-Fc 2.128 2.099 2.111 2.073 1.164 0.462 0.193 0.113 Mut95-Fc 2.190 2.175 2.228 2.090 1.233 0.522 0.217 0.119 Mut97-Fc 2.109 2.252 2.185 1.874 0.946 0.345 0.143 0.120 Mut98-Fc 2.055 2.103 2.239 2.266 1.890 0.907 0.361 0.123 Mut99-Fc 2.164 2.138 2.060 2.130 1.854 0.852 0.363 0.124 SIRPa V1-Fc 1.689 1.550 1.423 0.882 0.406 0.121 0.063 0.117 TTI621 1.852 1.726 1.545 1.272 0.748 0.228 0.098 0.099 Negative control 0.061 0.056 0.048 0.045 0.049 0.048 0.055 0.098 Mut81-Fc 1.931 1.806 1.867 1.835 1.125 0.527 0.258 0.135 Mut100-Fc 1.952 1.784 1.989 1.662 0.877 0.321 0.135 0.122 Mut105-Fc 1.785 1.765 1.833 1.859 1.086 0.395 0.166 0.123 Mut107-Fc 1.839 1.876 1.919 1.913 1.112 0.446 0.185 0.124 SIRPa V1-Fc 1.431 1.239 0.973 0.775 0.367 0.136 0.077 0.117 TTI621 1.585 1.404 1.275 1.094 0.590 0.232 0.108 0.121 Negative control 0.046 0.045 0.039 0.040 0.037 0.069 0.048 0.125 Mut88-Fc 2.007 2.083 2.250 1.939 1.466 0.643 0.247 0.091 Mut139-Fc 2.062 2.004 2.120 2.033 1.358 0.583 0.228 0.067 Mut141-Fc 1.963 1.961 2.088 1.957 1.383 0.603 0.380 0.081 Mut142-Fc 1.959 2.010 2.105 2.056 1.347 0.684 0.335 0.060 Mut146-Fc 1.929 2.137 2.246 2.084 1.187 0.531 0.277 0.061 SIRPa V1-Fc 1.556 1.348 1.300 0.864 0.400 0.143 0.061 0.057 TTI621 1.652 1.470 1.586 1.300 0.699 0.296 0.132 0.082 Negative control 0.056 0.053 0.046 0.044 0.042 0.048 0.051 0.067

Similar to Example 5A, flow cytometry (FACS) was used to detect the binding activity of SIRPa-Fc fusion protein to cells. The results are shown in Figures 10A-10C and Table 21, which show that the binding degree of deglycosylated SIRPa-Fc fusion protein to Jurkat cells is better than that of wild type or TTI621.

Table 21 FACS detection of binding activity of SIRPa-Fc fusion protein to Jurkat MFI Protein concentration (nM) Protein name 500 100 20 4 0.8 0.16 0.032 0.0064 Mut81-Fc 5405 5316 5236 5179 4794 1342 421 36 Mut92-Fc 5248 5368 5278 5217 4284 1045 306 66 Mut93-Fc 5225 5030 5030 5105 4464 1076 393 36 Mut94-Fc 5294 5158 5152 5142 4120 957 372 36 Mut95-Fc 5421 5265 5233 5209 4177 1138 334 34 Mut97-Fc 5456 5117 5323 5218 4517 1128 459 34 Mut98-Fc 5486 5260 5229 5224 4523 1203 394 35 Mut99-Fc 5281 5394 5330 5272 4208 1109 458 34 SIRPa V1-Fc 2012 1231 543 214 88 47 36 33 TTI621 2759 1981 1255 758 392 165 72 35 Negative control 121 52 36 35 34 34 31 33 Mut81-Fc 5957 5738 5583 5551 5319 2468 755 48 Mut100-Fc 5547 5514 5506 5398 4605 1633 484 42 Mut105-Fc 5582 5519 5430 5314 5307 2763 850 39 Mut107-Fc 5469 5421 5424 5324 4980 1904 584 39 SIRPa V1-Fc 2020 1240 606 227 94 53 39 36 TTI621 2596 1943 1190 694 372 157 75 38 Negative control 134 53 42 36 38 36 35 33 Mut88-Fc 5300 5083 4986 4932 4586 1268 463 37 Mut139-Fc 5630 5405 5318 5337 4590 1521 514 36 Mut141-Fc 5780 5494 5365 5380 4479 1616 493 34 Mut142-Fc 5733 5441 5424 5337 4222 1394 533 34 Mut146-Fc 5607 5469 5366 5349 4399 1635 527 33 SIRPa V1-Fc 2478 1322 576 211 90 47 36 30 TTI621 2928 2184 1360 792 429 173 73 31 Negative control 178 66 39 35 33 33 31 29

Embodiment 9 Testing SIRPα-Fc Fusion protein-mediated phagocytosis of tumor cells

After PBMCs were taken out of liquid nitrogen, they were thawed in a 37°C water bath. PBMCs were resuspended in EasySep™ Buffer (purchased from Stemcell, catalog number 20144), centrifuged at 300xg for 5 minutes, and the supernatant was discarded. Monocytes were enriched with a monocyte sorting kit (purchased from Miltenyi Biotec, catalog number 130-096-537), and rhM-CSF (purchased from R&D systems, catalog number 216-MC-100) was used to induce differentiation. The medium was changed every two days until macrophages were collected on the seventh day for use.

Raji cells were expanded and cultured in T-75 flasks to 1×10 ⁶ cells/mL. The medium was removed by centrifugation, and the cells were washed twice with PBS (purchased from Hyclone, Cat. No. SH30256.01) to adjust the cell density to 1×10 ⁶ cells/mL. Raji cells were stained with CellTrace™ Violet (purchased from Thermo Fisher, Cat. No. C34557). Macrophages were stained with CellTrace™ Far Red (purchased from Thermo Fisher, Cat. No. C34572) in the same way. The stained Raji and macrophages were added to a 96-well plate (purchased from Corning, catalog number 3474) at an E:T ratio of 1:2 and incubated with SIRPa-Fc fusion protein for 3 hours.

After the experiment, PI (purchased from Thermo Fisher, catalog number P3566) was added to the cells, and after incubation for 5 minutes, flow cytometry was performed (FACS Canto Plus, purchased from BD). The results are shown in Figures 11A-11B and Table 22. The formula for calculating the phagocytic ratio of the data in the table is: CellTrace™ Violet+ CellTrace™ Far Red+ / CellTrace™ Far Red+. According to the results, SIRPa-Fc fusion protein can effectively mediate macrophage phagocytosis of Raji cells, and the phagocytic degree is better than that mediated by TTI621.

Table 22 FACS detection of phagocytosis of Raji cells mediated by SIRPa-Fc fusion protein Devouring ratio% Protein concentration (nM) Protein name 6 1.2 0.24 0.048 0.0096 0.0019 Mut10-Fc 43.9 37.6 30.9 16.4 3.4 1.3 Mut31-Fc 43.8 38.2 28.4 15.5 4.0 1.4 Mut33-Fc 43.5 42.9 36.3 22.1 10.4 2.2 Mut35-Fc 41.5 43.7 31.0 15.0 2.6 1.0 Mut38-Fc 42.9 38.8 31.3 15.3 3.4 1.3 TTI621 36.2 31.6 22.0 12.1 2.6 0.8 Negative control 1.7 - - - - - Mut33-Fc 14.7 14.2 14.3 12.4 7.0 3.4 Mut66-Fc 14.4 15.1 15.1 11.1 5.8 3.2 Mut67-Fc 15.6 15.2 14.9 13.2 8.2 4.3 Mut68-Fc 15.4 14 14.8 12.2 5.8 3.7 Mut69-Fc 16.8 16.6 14.9 12.1 7.2 4.6 TTI621 15.7 14.8 11.5 6.2 3.9 3.3 Negative control 3.5 - - - - -

DLD1 cells were expanded and cultured in T-75 flasks to 1×10 ⁶ cells/mL. The medium was removed by centrifugation, and the cells were washed twice with PBS (purchased from Hyclone, Cat. No. SH30256.01) to adjust the cell density to 1×10 ⁶ cells/mL. DLD1 cells were stained with CellTrace™ Violet (purchased from Thermo Fisher, Cat. No. C34557). Macrophages were stained with CellTrace™ Far Red (purchased from Thermo Fisher, Cat. No. C34572) in the same way. The stained DLD1 and macrophages were added to a 96-well plate (purchased from Corning, Cat. No. 3474) at an E:T ratio of 1:2 and incubated with SIRPa-Fc fusion protein and 0.04 nM Cetuximab for 3 hours.

After the experiment, PI (purchased from Thermo Fisher, catalog number P3566) was added to the cells, and after incubation for 5 minutes, flow cytometry was performed (FACS Canto Plus, purchased from BD). The results are shown in Figures 12A-12B and Table 23, and SIRPa-Fc fusion protein can effectively promote Cetuximab-mediated macrophage phagocytosis of DLD1 cells.

Table 23 Phagocytosis of DLD1 cells promoted by SIRPa-Fc fusion protein detected by FACS Devouring ratio% Protein concentration (nM) Protein name 20 4 0.8 0.16 0.032 0.0064 0.00128 0.000256 0.0000512 Mut81-Fc 53.8 55.1 56.6 53.3 49.6 48.7 50.3 51.7 48.9 Mut97-Fc 54.6 56.8 55.5 53.4 50.4 50.0 50.8 50.7 51.2 Mut98-Fc 55.9 55.5 56.6 54.1 50.4 52.5 51.4 51.1 51.1 Mut99-Fc 55.6 55.0 55.4 53.5 49.7 50.1 50.8 50.4 50.6 Mut105-Fc 54.5 56.3 55.4 52.8 50.6 49.1 50.8 50.8 49.2 Cetuximab (0.04 nM) 50.5 - - - - - - - - Mut98-Fc 41.8 42.9 40.9 40.1 36.4 33.4 33.4 31.5 33.5 Mut99-Fc 42.4 42.8 40.6 41.3 37.1 34.1 33.7 32.8 34.1 Mut139-Fc 43.1 42.0 41.5 39.3 34.6 33.9 33.8 33.2 32.6 Mut142-Fc 43.7 43.0 43.0 41.1 33.7 35.1 34.0 35.2 33.8 Mut146-Fc 42.3 40.0 41.8 40.9 35.9 34.1 33.0 34.8 35.1 Cetuximab (0.04 nM) 33.9 - - - - - - - -

Embodiment 10 Testing SIRPα-Fc Fusion protein-mediated inhibition of tumor growth

Female NOD-SCID mice (about 6-8 weeks old, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were acclimated for at least 1 week before the experiment. To ensure that the survival rate of OE19 cells was greater than 90%, 8×10 ⁶ /100 μL (1640:matrigel=1:1, corning, 356237) was inoculated subcutaneously on the right side of the trunk of NOD-SCID mice. After inoculation, the mice were divided into groups according to the tumor volume. SIRPa-Fc fusion protein alone group: mice were intraperitoneally injected with 30 mg/kg on days 0, 7, and 14 after grouping; Trastuzumab antibody alone group: mice were intraperitoneally injected with 10 mg/kg on days 0, 3, 7, and 14 after grouping (Group 1), or intraperitoneally injected with 5 mg/kg on days 0, 7, and 14 after grouping (Group 2); SIRPa-Fc fusion protein and Trastuzumab antibody combination group: according to the respective dosing methods of the above two drugs, combined administration. Mice were observed and their body weights were recorded every day, and the volume of inoculated tumors was measured 2-3 times a week. When the tumor volume of mice exceeded the endpoint, that is, the tumor volume was greater than 2000 mm ³ , the mice were anesthetized and sacrificed. The tumor volume was calculated by the following formula: tumor volume = (length × width ² )/2, where length is the long diameter of the tumor and width is the short diameter of the tumor. The tumor growth inhibition rate (TGI%) was calculated by the following formula: TGI% = (1-TVi/TVvi) × 100%, where TVi is the average tumor volume of mice in the drug administration group on a specific day, and TVvi is the average tumor volume of mice in the control group on a specific day. According to the results shown in Figures 13A-13B and Table 24, SIRPa-Fc fusion protein combined with Trastuzumab antibody can effectively inhibit tumor growth.

Table 24 Effects of SIRPa-Fc fusion protein on tumor growth Tumor inhibition rate, TGI% Group 1: D7 D10 D14 D17 D21 Trastuzumab antibody alone group 96.75% 98.02% 94.53% 89.50% 87.15% Mut81-Fc alone group -2.88% 3.65% 13.83% 8.28% 12.09% Mut98-Fc alone group -30.43% -2.49% 2.15% 0.74% -8.93% Mut99-Fc alone group -38.73% -16.28% -8.31% -12.63% -12.89% Mut81-Fc and Trastuzumab Antibody Combination 119.24% 117.02% 111.13% 108.63% 106.79% Mut98-Fc and Trastuzumab Antibody Combination 121.99% 119.17% 111.48% 108.96% 106.54% Mut99-Fc and Trastuzumab Antibody Combination 123.63% 118.43% 111.52% 108.79% 106.95% Group 2: D6 D10 D13 D17 D20 Trastuzumab antibody alone group 78.08% 89.09% 80.88% 73.17% 66.14% Mut139-Fc alone group -28.94% -9.40% -3.08% -6.28% -5.29% Mut142-Fc alone group 2.92% 16.25% 21.00% 22.14% 15.66% Mut146-Fc alone group 3.05% 9.71% 13.86% 17.18% 13.68% Mut139-Fc and Trastuzumab Antibody Combination 117.44% 116.29% 109.83% 102.56% 97.81% Mut142-Fc and Trastuzumab Antibody Combination 115.93% 105.47% 100.80% 95.05% 92.51% Mut146-Fc and Trastuzumab Antibody Combination 112.75% 112.32% 105.45% 98.34% 92.86%

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Figure 1. Enzyme-binding immunosorbent assay (ELISA) method was used to detect the binding activity of SIRPa-Fc fusion protein with hCD47 ECD-His; Figure 2. Enzyme-binding immunosorbent assay (ELISA) method was used to detect the binding activity of SIRPa-Fc fusion protein with hCD47 ECD-mFc; Figure 3. Flow cytometry (FACS) was used to detect the binding activity of SIRPa-Fc fusion protein with Jurkat cells; Figure 4. Flow cytometry (FACS) was used to detect the binding activity of SIRPa-Fc fusion protein with Raji cells; Figure 5. Detection of the binding activity of SIRPa-Fc fusion protein in blocking Raji and receptor SIRPa V1; Figure 6. Detection of the binding activity of SIRPa-Fc fusion protein in blocking Raji and receptor SIRPa V2; Figure 7, Enzyme-binding immunosorbent assay (ELISA) method to detect the binding activity of SIRPa-Fc fusion protein and hCD47 ECD-mFc; Figure 8, Flow cytometry (FACS) to detect the binding activity of SIRPa-Fc fusion protein and Jurkat cells; Figure 9, Enzyme-binding immunosorbent assay (ELISA) method to detect the binding activity of SIRPa-Fc fusion protein and hCD47 ECD-mFc; Figure 10, Flow cytometry (FACS) to detect the binding activity of SIRPa-Fc fusion protein and Jurkat; Figure 11, Detection of phagocytosis of Raji cells mediated by SIRPa-Fc fusion protein; Figure 12, Detection of phagocytosis of DLD1 cells mediated by SIRPa-Fc fusion protein; Figure 13. Detection of the inhibitory effect of SIRPa-Fc fusion protein on tumor growth.

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TWI859619B_111141360_SEQL.xml

Claims (14)

A polypeptide, which is a signal regulatory protein alpha (SIRPα) mutant, wherein the mutant further has an amino acid mutation selected from the following group relative to the wild-type SIRPα having SEQ ID NO: 1: (1) I31Y; N51R; L66N; and T82H; or (2) I31Y; N51R; L66N; and T82L. A polypeptide as described in claim 1, wherein the polypeptide is fused to the immunoglobulin Fc region at its N-terminus or C-terminus. In the polypeptide as described in claim 2, the immunoglobulin Fc region is selected from the Fc region of human IgG1, IgG2 or IgG4 or its mutant form. A polypeptide as described in claim 3, wherein the Fc mutant of human IgG1 comprises at least one of the following amino acid modifications relative to wild-type human IgG1: L234A, L235A, G237A and/or N297A. A polypeptide as described in claim 4, wherein the amino acid modification is specifically: (1) N297A mutation; or (2) L234A, L235A and G237A mutations; or (3) L234A, L235A, G237A and N297A mutations. A polypeptide as described in claim 3, wherein the Fc mutant exhibits eliminated or reduced effector function, eliminated or reduced C1q binding and Fcγ receptor binding compared to the wild-type human IgG Fc region. A polypeptide as described in any one of claims 1-6, wherein the polypeptide has an amino acid sequence of any one of SEQ ID NO: 88, 89, 102, 103. A nucleic acid molecule encoding a polypeptide as described in any one of claims 1-7. An expression vector comprising a nucleic acid molecule as described in claim 8. A host cell comprising the vector as described in claim 9. The host cell as described in claim 10, wherein the cell is a prokaryotic cell or a eukaryotic cell; the prokaryotic cell is Escherichia coli or yeast; the eukaryotic cell is a CHO cell or a 293T cell. A method for preparing a polypeptide as described in any one of claims 1 to 7, comprising culturing a host cell as described in claim 10 or 11, and isolating the polypeptide expressed by the host cell. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an amount of a polypeptide as described in any one of claims 1 to 7, or a nucleic acid molecule as described in claim 8, or a vector as described in claim 9 that can effectively inhibit the growth or proliferation of CD47+ disease cells. A method for detecting CD47 expression in a biological sample, characterized in that the method comprises contacting the biological sample with the polypeptide as described in any one of claims 1 to 7 under conditions where a complex can be formed between the polypeptide as described in any one of claims 1 to 7 and CD47.