WO2023072279A1 - Sirpa mutant and application thereof - Google Patents

Abstract

Provided is a high affinity SIRPa polypeptide, comprising at least one amino acid change relative to a wild-type protein; and having an increased affinity for CD47 relative to the wild-type protein. Also provided are a nucleic acid molecule encoding the SIRPa polypeptide and a vector expressing the polypeptide. Further provided are a pharmaceutical composition and a method for treating tumors expressing CD47.

Description

SIRPa Mutants and Their Applications

Cross References to Related Applications

This application claims the benefit and priority of the following 2 Chinese invention patent applications, the entire contents of which are hereby incorporated by reference in their entirety: Patent No. 202111283381.5 filed with the State Intellectual Property Office of China on November 1, 2021 application; and Patent Application No. 202210800901.3 filed with the State Intellectual Property Office of China on July 8, 2022.

technical field

The present invention relates to the field of biomedicine, in particular to SIRPa mutants and applications thereof.

Background technique

CD47 is a transmembrane protein with glycosylation. After activation, it can mediate a series of processes such as cell proliferation, migration, phagocytosis, apoptosis, and immune homeostasis. The ligands of CD47 include signal regulatory protein α (SIRPa), thrombospondin-1 (TSP-1) and integrins. CD47 expression is generally upregulated in a variety of malignant tumors, and high expression levels of CD47 are associated with treatment response and prognosis of cancer progression. 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 the subsequent phagocytosis of tumor cells. At present, some immunotherapies targeting CD47, including monoclonal antibodies, fusion proteins, CAR-T cells, and ADCs, have entered the clinical research stage, and the mechanisms of action, safety, and efficacy of various products are different.

Drugs that are currently in the clinical research stage include a variety of CD47 monoclonal antibodies, double antibodies and SIRPa proteins. Among them, magrolimab, the CD47 antibody with the earliest development and the fastest clinical progress, was developed by Forty Seven. Phase II/III clinical trials for hematological tumors and solid tumors are now being advanced. However, it causes certain safety problems. Side effects of grade 3 or above include about 14% of anemia events and about 16% of neutropenia events. The fastest clinical progress of SIRPa protein drugs includes 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 in phase I clinical trials for hematological tumors. The monotherapy of NHL has a certain objective response rate. Grade 3 or higher side effects include about 9% of anemia events, 20% of thrombocytopenia and about 9% of neutropenia events. TTI622 is a natural SIRPa IgV domain coupled to the Fc segment of human IgG4, and the existing efficacy and safety data are superior to TTI621. In addition, the combination or double antibody of various immune checkpoints and CD47 antibody, the combination or double antibody of tumor-associated antigen antibody and CD47 antibody, etc. have also entered clinical practice.

Contents of the invention

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

The invention provides a SIRPa variant with higher target binding activity and its fusion protein, which are used to detect the existence or level of CD47, block the combination of SIRPa and CD47, and realize tumor immunotherapy.

A first aspect of the present invention provides a polypeptide for inhibiting the growth and/or proliferation of CD47+ disease cells, which comprises a signal regulatory protein alpha (SIRPa) mutant or a fragment thereof, the mutant relative to wild-type SIRPa at selected from residue 31, residue 37, residue 51, residue 66, residue 68 or residue 82 with at least one amino acid modification; preferably, the amino acid modification is a substitution; preferably, the wild-type SIRPα has A sequence according to any one of SEQ ID NO: 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; N51T; N51W; (4) L66A; L66V; L66P; L66M; L66N; L66E; L66K; L66H; (5) K68D; K68M; K68Q; K68L; K68F; K68I; T82Q; T82G; T82H; T82L; T82I; T82K; T82M; T82P; T82R; T82V; T82W; T82Y.

In some embodiments, the polypeptide, relative to wild-type SIRPα having SEQ ID NO: 1, comprises an amino acid mutation selected from the group consisting of:

(1) N51G; (2) N51R; (3) I31M; (4) I31Y; (5) I31M; N51G; (6) I31M; N51R; (7) I31Y; N51G; (8) I31Y; )Q37T; N51G; (10) Q37T; N51R; (11) I31Y; N51R; L66A; (12) I31Y; )I31Y; N51R; L66N; (16) I31Y; N51R; L66E; (17) I31Y; N51R; L66K; (18) I31Y; 22) N51K; (23) N51L; (24) N51M; (25) N51Q; (26) N51T; (27) N51W; (28) I31M; N51R; L66N; K68D; (29) I31M; (30) I31Y; N51R; L66N; K68D; (31) I31M; N51R; L66N; K68Q; (32) I31M; N51R; L66N; K68I; (35) I31Y; N51R; L66N; T82F; (36) I31Y; (39) I31Y; N51R; L66N; T82Q; (40) I31Y; N51R; L66N; T82G; (41) I31Y; T82M; (46) I31Y; N51R; L66N; T82P; (47) I31Y; N51R; L66N; T82R; ( 48) I31Y; N51R; L66N; T82V; (49) I31Y; N51R; L66N; T82W; (50) I31Y; N51R; L66N; K68Q; T82H; (53) I31M; N51R; L66N; K68Q; T82L; (54) I31M; I31M; N51R; L66N; K68L; T82H; (57) I31M; N51R; L66N; K68L; T82L; (58) I31M; 60) I31M; N51R; L66N; K68I; T82H; (61) I31M; N51R; L66N; K68I; T82L; (62) I31M;

Alternatively, a N51R; N51M or N51W mutation is included relative to wild-type SIRPα having SEQ ID NO: 2.

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

In some embodiments, the Fc mutant type 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 modification comprises (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 wild type of the human IgG Fc region.

In some embodiments, the polypeptide has the 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 also linked with other functional molecules, and the other functional molecules 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 multimeric.

A second aspect of the present invention provides a chimeric antigen receptor (CAR), which at least comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, the extracellular antigen binding domain comprising Any one of the above polypeptides.

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

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

The 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 (Escherichia coli), fungus (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).

The seventh aspect of the present invention provides a method for preparing any one of the above-mentioned polypeptides, which comprises culturing the host cell, and isolating the polypeptide expressed by the host cell.

The eighth aspect of the present invention provides a method for preparing the above-mentioned immune effector cells, which includes introducing the nucleic acid fragment encoding the above-mentioned CAR into the immune effector cells, and optionally, further including enabling the immune effector cells 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 effector cells, nucleic acid molecules, and expression vectors in an amount that can effectively inhibit the growth or proliferation of CD47+ disease cells , or the product prepared by any one of the above methods.

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

The eleventh aspect of the present invention provides any of the above-mentioned polypeptides, immune effector cells, nucleic acid molecules, expression vectors, host cells, products prepared by the method, or pharmaceutical compositions for the preparation of drugs for treating tumors expressing CD47 Preferably, the tumor is a CD47-positive blood tumor or a CD47-positive solid tumor, and more preferably, the CD47-positive blood 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 tumors expressing CD47, the method comprising administering to a subject an effective amount of the polypeptide, immune effector cells, nucleic acid molecules, expression vectors, host cells, the The method prepares the obtained product and pharmaceutical composition; preferably, the tumor is a CD47-positive blood tumor or a CD47-positive solid tumor, and more preferably, the CD47-positive blood tumor includes leukemia, lymphoma and myeloma, and the CD47-positive Solid tumors include sarcomas.

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

The 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 making the biological The sample is contacted with the polypeptide; preferably, the method further comprises detecting the formation of the complex indicative of the presence or expression level of CD47 in the sample.

Definitions and Explanations of Terms

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

Also, unless otherwise specified herein, terms in the singular shall include pluralities, and terms in the plural shall include the singular, unless otherwise specified herein. 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 "comprising", "including" and "having" are used interchangeably herein and are intended to indicate the inclusiveness of the scheme, meaning that the scheme may have other elements besides the listed elements. At the same time, it should be understood that the terms "comprising", "comprising" and "having" are used herein to also provide "consisting of". Exemplarily, "a composition including A and B" should be understood as the following technical scheme: a composition composed of A and B, and a composition containing other components in addition to A and B, all fall into Into the scope of the aforementioned "a composition".

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

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

The terms "CD47+" and "CD47 positive" generally refer to the characteristic of expressing CD47 protein, its fragments, or its mutants with one or more amino acid substitutions on the surface of organisms or cells. CD47 positive cells may be cells overexpressing CD47. Said CD47-positive cells can usually be indicative of a disease. For example, in disease conditions, the CD47 protein density on the surface of the CD47-positive cells will exceed the CD47 protein density of the cells under normal conditions. In certain embodiments, the tumor or tumor cells may be positive for CD47. For example, said tumor may be selected from the group consisting of 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 (membrane integrins) and bind to its ligands thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRPα). CD47 protein is widely expressed on the cell membrane surface. In the present application, the CD47 protein may include any variant, isoform and species homologue of human CD47.

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

In the present invention, a certain amino acid residue in an amino acid sequence "relative to" a certain amino acid residue in another amino acid sequence usually refers to the corresponding relationship of amino acid residues obtained when amino acid sequence alignment is carried out under optimized conditions . The sequence alignment can be performed by means known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment across the full-length sequences being compared.

Amino acid substitutions described herein may be non-conservative substitutions. The non-conservative substitution may include changing amino acid residues in the target protein or polypeptide in a non-conservative manner, such as changing amino acid residues with a certain side chain size or a certain characteristic (for example, hydrophilicity) to have different Amino acid residues with side chain size or different properties (eg, hydrophobicity).

The amino acid substitutions may also be conservative substitutions. The conservative substitution may include changing amino acid residues in the target protein or polypeptide in a conservative manner, such as changing amino acid residues with a certain side chain size or a certain characteristic (for example, hydrophilicity) to have the same or similar Amino acid residues with side chain size or the same or similar properties (eg, still hydrophilic). Such conservative substitutions generally do not substantially affect the structure or function of the resulting protein. In the present application, amino acid sequence variants as said fusion protein, fragments thereof, or mutants thereof with one or more amino acid substitutions may include those that do not significantly change the structure of the protein or its function (for example, blocking CD47 and its Ligand binding ability) conservative amino acid substitutions.

By way of example, mutual substitutions between individual amino acids within each of the following groups may be considered conservative substitutions in this application:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), glutamic acid (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 fusion of two or more proteins or polypeptides. Fusion proteins can be artificially produced by recombinant DNA techniques. For example, the 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 may result in a single polypeptide, which may have the properties of at least one, or even each, of the two or more proteins or polypeptides prior to 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 may be directly or indirectly connected to the immunoglobulin Fc region . For example, the human SIRPα domain may be located at the N- or C-terminus of the immunoglobulin Fc region. For example, the C-terminal of the human SIRPα domain can be directly or indirectly connected to the N-terminal of the immunoglobulin Fc, or the C-terminal of the immunoglobulin Fc can be directly or indirectly connected to the N-terminal of the human SIRPα domain. For example, the human SIRPα domain can be linked to the immunoglobulin Fc via 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 can consist of two identical protein fragments derived from the second and third constant domains of the two heavy chains of the antibody; the Fc region of IgM and IgE can be found in each The polypeptide chain contains three heavy chain constant domains. The Fc region of IgG has highly conserved N-glycosylation sites. In certain embodiments, the immunoglobulin Fc region may comprise an IgG Fc region. In certain embodiments, the immunoglobulin Fc region may comprise the CH2 and CH3 regions of the heavy chain constant region. In certain embodiments, the immunoglobulin Fc region may comprise a hinge region. For example, the immunoglobulin Fc region may comprise an amino acid sequence selected from any of the following: SEQ ID NO: 3.

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

The term "IgG" herein generally refers to Immunoglobulin G (Immunoglobulin G). IgG is one of human immunoglobulins. According to the antigenic difference 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 a subtype with the highest proportion of IgG, which has a higher affinity with Fc receptors. For example, the IgG can be human IgG. For another example, the IgG can be selected from the group consisting of IgG1 and/or IgG4.

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

The term "antigen-binding molecule" is used herein in the broadest sense to refer to a molecule that specifically binds an antigen. Exemplary, antigen binding molecules include, but are not limited to, antibodies or antibody mimetics. "Antibody mimic" refers to an organic compound or binding domain that can specifically bind to an antigen, but has nothing to do with the structure of an antibody. Exemplary, antibody mimics include but are not limited to affibody, affitin, affilin, designed ankyrin repeat proteins (DARPins), aptamers or Kunitz-type domain peptides.

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

Herein the term "nucleic acid molecule" includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and phosphate groups. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is usually expressed 5' to 3'. In this context, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and synthetic forms of DNA or RNA comprising both Mixed polymers of one or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms. Furthermore, 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 suitable as vectors for direct expression of antibodies of the invention in vitro and/or in vivo, for example in a host or patient. Such DNA (eg cDNA) or RNA (eg 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 generate antibodies in vivo (see e.g. Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi: 10.1038/nm.4356 or EP 2 101 823B1). An "isolated" nucleic acid herein refers to a nucleic acid molecule that has been separated from components 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 which is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.

The term "expression vector" herein refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it has been 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 exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical to the parental cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

As used herein, the term "pharmaceutical composition" refers to a preparation that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain substances that are unacceptably toxic to the subject to which the pharmaceutical composition is administered. additional ingredients.

The term "treatment" herein refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) an undesired physiological change or pathology, such as the progression of cancer, in the subject being treated. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminished extent of disease, stable disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of disease state, and remission (whether partial response or complete response), whether detectable or undetectable. Those in need of treatment include those already with the condition or disease as well as those prone to have the condition or disease or those in which the condition or disease is to be prevented. When referring to the terms slow down, lessen, weaken, moderate, alleviate, etc., the meaning of eliminate, disappear, not occur, etc. is also included.

The term "subject" herein refers to an organism receiving treatment for a particular disease or condition as described herein. Examples of subjects and patients include mammals, such as humans, primate (eg, monkeys) or non-primate mammals, receiving treatment for a disease or disorder.

The term "effective amount" herein refers to an amount of a therapeutic agent effective to prevent or alleviate a disease condition or the progression of the 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, eg, treat, cure, prevent or alleviate the associated medical condition, or to increase the rate of treatment, cure, prevent or alleviate such condition. When the active ingredient is administered to a subject 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" herein refers to or describes the physiological condition in mammals typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. The term "tumor" or "neoplastic" herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when referred to herein.

In this application, the term "CD47-positive hematological tumor" generally refers to a hematological tumor overexpressing CD47, which may include various types of leukemia, lymphoma and myeloma. The term "leukemia" generally refers to a cancer of the blood in which too many white blood cells, which are ineffective at fighting infection, are produced, thereby crowding out other parts of the blood, such as platelets and red blood cells. Leukemia can be classified as acute or chronic.

In this application, the term "CD47-positive solid tumor" generally refers to a solid tumor or a solid tumor overexpressing CD47, which can be detected by clinical examination, such as X-ray film, CT scan, B-ultrasound or palpation. . Major categories may include carcinoma and sarcoma.

Description of drawings

Fig. 1, enzyme-linked immunoassay (ELISA) method detects the binding activity of SIRPa-Fc fusion protein and hCD47 ECD-His;

Fig. 2, the binding activity of SIRPa-Fc fusion protein and hCD47 ECD-mFc detected by enzyme-linked immunosorbent reaction (ELISA);

Fig. 3, flow cytometry (FACS) detects the binding activity of SIRPa-Fc fusion protein and Jurkat cell;

Fig. 4, flow cytometry experiment (FACS) detects the binding activity of SIRPa-Fc fusion protein and Raji cell;

Figure 5. Detecting the binding activity of SIRPa-Fc fusion protein blocking Raji and receptor SIRPa V1;

Figure 6. Detecting the binding activity of SIRPa-Fc fusion protein blocking Raji and receptor SIRPa V2;

Fig. 7, enzyme-linked immunosorbent reaction (ELISA) method detects the binding activity of SIRPa-Fc fusion protein and hCD47 ECD-mFc;

Fig. 8, flow cytometry experiment (FACS) detects the binding activity of SIRPa-Fc fusion protein and Jurkat cell;

Fig. 9, enzyme-linked immunoassay (ELISA) method detects the binding activity of SIRPa-Fc fusion protein and hCD47 ECD-mFc;

Fig. 10, flow cytometry (FACS) detects 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;

FIG. 13 . Detection of the inhibitory effect on tumor growth mediated by SIRPa-Fc fusion protein.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

The embodiments of the present invention are merely exemplary, and do not constitute any limitation to the scope of the present invention. Those skilled in the art 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 all fall within the protection scope of the present invention.

The plasmid construction of embodiment 1SIRPa mutation

Using a variety of algorithms to obtain SIRPa mutants with improved affinity, design and synthesize the corresponding sequences, clone the nucleic acid fragments encoding wild-type SIRPa IgV and the aforementioned mutants into pTT5 vectors with different Fc tags, and follow the established standard molecular biology Plasmids were prepared by scientific methods, and the plasmids encoded SIRPα wild-type/variant and Fc-fused proteins. The SIRPa wild type and variants used are as follows: SIRPa V1, SIRPa 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 aforementioned 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, and "Fc" represents the wild-type The Fc fragment of human IgG1, "inert Fc" indicates the Fc mutant of human IgG1, "MutXX" indicates the mutant SIRPa that is mutated compared with the wild type, and "MutXX-Fc" indicates the fusion protein of mutant SIRPa and Fc. "SIRPa V1/V2-Fc" represents the fusion protein of wild-type SIRPa V1/V2 and the wild-type Fc fragment shown in SEQ ID NO:3. The sequence of TTI621 refers to WO2019084692A1, as shown in SEQ ID NO:15.

Table 1 SIRPa mutant sequence

Example 2 Production and purification of SIRPa-Fc fusion protein

HEK293 cells (purchased from the Cell Bank of Chinese Academy of Sciences) were transiently transfected with the plasmid (PEI, Polysciences) constructed in Example 1 and expanded at 37°C using FreeStyle TM 293 Expression Medium (purchased from Gibco). After 7 days, the cell culture medium was collected, centrifuged to remove cell components, and cells 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, Culture supernatants of Mut87-Fc, Mut88-Fc, Mut89-Fc and Mut90-Fc.

Or add the plasmid and transfection reagent (Thermofisher, product number: A29133) to OptiPRO SFM (Thermofisher, product number: 12309019) and mix well, then let it stand for 5 minutes, then add it to ExpiCHO-S ^TM cells (manufacturer: Thermofisher, product number: A29127), put Into 5% CO ₂ , 120 rpm, 37°C shaker culture. On the second day after transfection, feed was added, and the shaker temperature was adjusted to 32°C to continue culturing. On the 8th to 11th day of transfection, collect cells containing Mut174-Fc, Mut178-Fc, Mut150-Fc, Mut152-Fc, Mut153-Fc, Mut156-Fc, Mut157-Fc, Mut158-Fc, Mut160-Fc, Mut162-Fc and Cell supernatant of Mut164-Fc.

Purify the fusion protein in the cell culture supernatant with a 10 mL protein A column (purchased from Bogeron). 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 was loaded onto the protein A column at a flow rate of 10 mL/min. After loading the sample, wash the protein A column with equilibration buffer, and the volume of the equilibration buffer is 3 to 5 times the volume of the protein A column bed. Use the eluent (0.02M citric acid buffer, 0.1M glycine, 0.1M sodium chloride, pH3.0) to elute the protein bound to the protein A column, and monitor the elution with a nucleic acid protein detector (A280 ultraviolet absorption peak). Collect the eluted protein, add Tris buffer to neutralize the pH, and the buffer system after dialysis is PBS to collect the target protein. Filter it with a 0.22 μm filter and store it aseptically to obtain the purified SIRPa-Fc fusion protein.

The purified SIRPa-Fc fusion protein was tested and analyzed for protein yield, protein concentration (A280/1.4), SEC purity, etc., and the quality of the purified SIRPa-Fc fusion protein was qualified. See Table 2 for the detection information about protein yield, protein concentration and purity.

Table 2 Detection and analysis of SIRPa-Fc fusion protein

Example 3 Enzyme-linked immunoassay (ELISA) detection of SIRPa-Fc fusion protein and CD47 protein binding level

A. Production and purification of CD47 protein

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

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

SEQ ID NO: 52

Add the plasmid and transfection reagent PEI (Polysciences, Cat. No.: 24765-1) to OPTI-MEM (Gibco, Cat. No.: 11058021) and mix well, then let it stand for 15 min, add it to Expi293 cells (manufacturer: Thermofisher, Cat. No.: A14527), and put Into 5% CO ₂ , 120 rpm, 37°C shaker culture. On the second day of transfection, add OPM-293ProFeed (Shanghai Opma, Cat. No.: F081918-001) and 6 g/L glucose (manufacturer: Sigma, Cat. No.: G7528). On the sixth day of transfection, the cell supernatant was collected; the cell components were removed by centrifugation, and the culture supernatant containing CD47 ECD-His protein was obtained. The protein in the cell culture supernatant was purified with a Ni affinity chromatography column (GE, catalog number: 17371206). Equilibrate with 3-5 column volumes of equilibration buffer (PBS phosphate buffer, pH7.4) (20×PBS buffer 500ml, Sanko, Cat. No. B548117-0500), then load the clarified culture supernatant To the Ni affinity chromatography column, control the flow rate at 5mL/min. After loading the sample, the protein A column is washed with an equilibration buffer, and the volume of the equilibration buffer is 3 to 5 times the volume of the column bed of the Ni affinity chromatography column. Gradient elution was carried out with 0-500mM imidazole (Sinopharm, product number: 30104961), and the elution was monitored with a nucleic acid protein detector (A280 ultraviolet absorption peak). The eluted protein was collected and dialyzed into PBS phosphate buffer at 4° C. with a dialysis card (purchased from Thermo Scientific, catalog number: 88252). Sterile filtration was performed with a 0.22 μm filter (Millipore, product number SLGVR13SL), and aseptically stored to obtain purified hCD47 ECD-His protein. The purified hCD47 ECD-His was tested and analyzed for protein yield, protein concentration (A280/1.4), SEC purity, etc., and the quality of the purified protein was qualified. The detection information about protein yield, protein concentration and purity can be found in Table 3.

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

The human CD47 protein extracellular domain (Extracellular Domain, ECD) amino acid sequence (the specific sequence is shown in SEQ ID NO: 52) coupled with the mouse Fc sequence was cloned into the pTT5 vector.

Add the plasmid and transfection reagent PEI (Polysciences, Cat. No.: 24765-1) to OPTI-MEM (Gibco, Cat. No.: 11058021) and mix well, then let it stand for 15 min, add it to Expi293 cells (manufacturer: Thermofisher, Cat. No.: A14527), and put Into 5% CO ₂ , 120 rpm, 37°C shaker culture. On the second day of transfection, add OPM-293ProFeed (Shanghai Opma, Cat. No.: F081918-001) and 6 g/L glucose (manufacturer: Sigma, Cat. No.: G7528). On the sixth day of transfection, the cell supernatant was collected; the cell components were removed by centrifugation, and the culture supernatant containing hCD47 ECD-mFc protein was obtained. Purify the protein in the cell culture supernatant with a 10 mL protein A column (GE, catalog number: 17549802). Protein A column was first equilibrated with 3-5 column volumes of equilibration buffer (PBS phosphate buffer, pH7.4) (20×PBS buffer 500ml, Sanko, Cat. No. B548117-0500), and then the clarified culture supernatant The solution was loaded onto the protein A column, and the flow rate was controlled at 10 mL/min. After loading the sample, wash the protein A column with equilibration buffer, and the volume of the equilibration buffer is 3 to 5 times the volume of the protein A column bed. The protein bound to the protein A column was eluted with an eluent (0.02M citric acid buffer, pH 3.5), and the elution was monitored with a nucleic acid protein detector (A280 ultraviolet absorption peak). The eluted protein was collected, and the pH was neutralized by adding Tris buffer (Sinopharm, product number: 30188336). After dialysis, the buffer system was PBS, and the target protein was collected. Sterile filtration was performed with a 0.22 μm filter (Millipore, product number SLGVR13SL), and aseptically stored to obtain purified hCD47 ECD-mFc protein. The purified hCD47 ECD-mFc was tested and analyzed for protein yield, protein concentration (A280/1.4), SEC purity, etc., and the quality of the purified protein was qualified. The detection information about protein yield, protein concentration and purity can be found in Table 3.

Table 3 Detection and analysis of CD47 protein

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

Biotinylated hCD47 ECD-His. Add 100 μL WS buffer and 200 μg hCD47 ECD-His protein to the Filtration tube according to the instructions of the Biotin Labeling kit (product number LK03, purchased from DO JINDO), mix well and centrifuge at 8000 g for 10 minutes; add 10 μL DMSO to the NH ₂ -Reactive Biotin Tube and mix well Standby; after centrifugation, add 100 μL Reaction Buffer and 8 μL NH ₂ -Reactive Biotin solution to the Filtration tube, mix well, place in a constant temperature incubator at 37 degrees for 10 minutes, then add 100 μL WS Buffer and centrifuge at 8000g for 10 minutes, discard the filtrate and then add 200 μL WS Buffer Centrifuge at 8000g for 10 minutes, repeat the above step once, add 100 μL WS Buffer to recover the marked hCD47 ECD-His into a new centrifuge tube, measure the concentration and set aside.

Firstly, streptavidin (streptavidin, hereinafter referred to as SA, product number S4762, purchased from Sigma) was diluted with PBS to a final concentration of 3 μg/mL, then added to 96-well ELISA plate at 50 μL per well, and incubated overnight at 4°C. The next day, the supernatant was discarded, and a blocking solution, namely PBS buffer solution containing 5% (w/w) skim milk powder (purchased from Sangon, product number: A100777-0500), was added to block at 37° C. for 2 hours. The blocking solution was discarded, and the plate was washed 3 times with PBS buffer solution PBST containing 0.1% (v/v) Tween20 (purchased from Sangon, Cat. No. A100777-0500). Add 2 μg/mL biotin-labeled hCD47 ECD-His to each well, 50 μL/well, incubate at 37°C for 1 hour, and wash 3 times with PBST. Add 50 μL of the purified SIRPa-Fc fusion protein obtained in Example 2 to each well, the initial concentration is 50 nM, 5-fold serial dilution, and after incubation at 37° C. for 1 hour, the plate is washed 3 times with PBST. Add 50 μL of horseradish peroxidase (HRP)-labeled secondary antibody (purchased from Bethyl, Cat. Product No. A500023-0100) in PBS, incubated at room temperature for 1 hour, and washed the plate 5 times with PBST. Add 50 μL of TMB substrate to each well, incubate at room temperature for 5 minutes, and add 50 μL of stop solution (1.0N HCl) to each well. Read the A450nm value with a microplate reader (Ensight-HH3400, purchased from PerkinElmer), and the results are shown in Figure 1 and Table 4. The results show that the purified SIRPa-Fc fusion protein has excellent binding activity to hCD47 ECD-His Compared with wild-type V1 or V2, it is better than TTI621. The negative control is non-related Fc fusion protein, the same below. The data in the table are OD450nm values.

Table 4 ELISA detection of binding activity of SIRPa-Fc fusion protein to hCD47 ECD-His

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

Anti-mFc (purchased from Jackson, Cat. No. 115-006-071) was first 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 overnight at 4°C. The supernatant was discarded the next day, and a blocking solution, namely PBS buffer solution containing 5% (w/w) skimmed milk powder (purchased from Sangon, product number A600669-0250), was added to block at 37° C. for 2 hours. Pour off the blocking solution and wash the plate 3 times with PBST. Add 0.5 μg/mL hCD47 ECD-mFc to each well, 50 μL/well, incubate at 37°C for 1 hour, and wash 3 times with PBST. Add 50 μL of the purified SIRPa-Fc fusion protein obtained in Example 2 to each well, the initial concentration is 50 nM, 5-fold serial dilution, and after incubation at 37° C. for 1 hour, the plate is washed 3 times with PBST. Add 50 μL of horseradish peroxidase (HRP)-labeled secondary antibody (purchased from Bethyl, Cat. Product No. A500023-0100) in PBS, incubated at room temperature for 1 hour, and washed the plate 5 times with PBST. Add 50 μL of TMB substrate to each well, incubate at room temperature for 5 minutes, and add 50 μL of stop solution (1.0N HCl) to each well. Read the A450nm value with a microplate reader (Ensight-HH3400, purchased from PerkinElmer), and 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 uniform. Better than wild-type V1 or V2, better than TTI621. The data in the table are OD450nm values.

Table 5 ELISA detection of binding activity of SIRPa-Fc fusion protein to hCD47 ECD-mFc

Example 4 Surface plasmon resonance (SPR) detection of SIRPa-Fc fusion protein and CD47 protein affinity

Using BIAcore 8K instrument, the affinity with antigen was determined by multi-cycle kinetic method. First, according to the method guidance of the Human Antibody Capture Kit (purchased from cytiva, 29234600), the Anti-human IgG Fc antibody was immobilized on the CM5 chip (purchased from cytiva, BR100530), and according to the Amine Coupling Kit kit (purchased from cytiva, 29234600), BR100633), use HBS-EP+pH7.4 as the mobile phase, mix NHS and EDC, activate the chip for about 600s, dilute Anti-human IgG Fc to 15μg/mL with 10mM sodium acetate pH5.0, and inject for 420s , and finally the remaining active sites were blocked with ethanolamine. Then, the affinity to the antigen was determined by a multi-cycle kinetic method, in each cycle, the SIRPa-Fc fusion protein was first captured with an Anti-human IgG Fc chip, and then a single concentration of antigen was injected to record the SIRPa-Fc fusion protein and antigen During the protein binding and dissociation process, the chip was regenerated with 3M MgCl ₂ at the end. The mobile phase was HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20), the flow rate was 30μL/min, and the regeneration time was 30s. The temperature was 25°C; finally, according to the 1:1binding model, the data was analyzed to fit the antibody-antigen binding kinetic parameters, including the binding rate constant ka, the dissociation rate constant kd, the equilibrium dissociation constant KD, and the maximum binding signal Rmax. The results are shown in Table 6. The results show that the affinity between the purified SIRPa-Fc fusion protein and hCD47ECD-mFc is better than that of wild type or TTI621, among which Mut10-Fc, Mut33-Fc, Mut67-Fc, Mut69-Fc, Mut80- The affinity of Fc, Mut81-Fc, Mut86-Fc, Mut153-Fc, Mut158-Fc, Mut160-Fc, Mut164-Fc fusion proteins increased significantly.

Table 6 SPR detection of affinity between SIRPa-Fc fusion protein and hCD47 ECD-mFc

Embodiment 5 Flow cytometry experiment (FACS) detects the binding activity of SIRPa-Fc fusion protein and cell

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

Expand the culture of Jurkat cells in a T-75 cell culture flask, centrifuge at 1000 rpm for 5 minutes at room temperature, discard the medium, wash twice with PBS buffer (purchased from Hyclone, product number SH30256.01), and resuspend the cells in PBS And count. Add 1×10 ⁵ cells per well, 50 μL per well into a U-bottom 96-well FACS reaction plate (purchased from Corning, Cat. No. 3795), and store at 4°C for later use. The SIRPa-Fc fusion protein to be tested was diluted with PBS containing 1% (w/w) BSA (purchased from Sangon, Cat. No. A500023-0100), added to the cells at 50 μL per well, mixed, and incubated at 4° C. for 1 hour. Add 200 μL of FACS buffer [PBS buffer containing 1% (w/w) BSA] to each well, wash twice, and centrifuge at 1500 rpm for 5 minutes. After the supernatant was removed, 100 μL of diluted fluorescently-labeled secondary antibody (purchased from Jackson ImmunoResearch, Cat. No. 109-605-098) was added to each well, and incubated at 4°C for 1 hour. Centrifuge and wash twice with FACS buffer, and suspend the cells with 100 μL of PBS. The results were detected and analyzed with a FACS instrument (FACS Canto II, purchased from BD Company). The results are shown in Figure 3 and Table 7. The results showed that SIRPa-Fc fusion protein can bind to CD47 on the surface of Jurkat cells, and the binding activity of SIRPa-Fc fusion protein to Jurkat was better than wild-type V1 or V2, and better than TTI621. The data MFI in the table is the mean fluorescence intensity value of the measured cell population.

Table 7 FACS detection of binding activity of SIRPa-Fc fusion protein to Jurkat

B. Detection of the binding level 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, discarded the medium, washed twice with PBS buffer (purchased from Hyclone, product number SH30256.01), and the cells were washed with 25 μg /ml of Fc blocker (purchased from BD, Cat. No. 564220) was resuspended, and blocked at room temperature for 20 minutes. Add 1×10 ⁵ cells per well, 50 μL per well into a U-bottom 96-well FACS reaction plate (purchased from Corning, Cat. No. 3795), and store at 4°C for later use. The SIRPa-Fc fusion protein to be tested was diluted with PBS containing 1% (w/w) BSA (purchased from Sangon, Cat. No. A500023-0100), added to the cells at 50 μL per well, mixed, and incubated at 4° C. for 1 hour. Add 200 μL of FACS buffer [PBS buffer containing 1% (w/w) BSA] to each well, wash twice, and centrifuge at 1500 rpm for 5 minutes. After the supernatant was removed, 100 μL of diluted fluorescently-labeled secondary antibody (purchased from Jackson ImmunoResearch, Cat. No. 109-605-098) was added to each well, and incubated at 4°C for 1 hour. Centrifuge and wash twice with FACS buffer, and suspend the cells with 100 μL of PBS. The results were detected and analyzed with a FACS instrument (FACS Canto II, purchased from BD Company). The results are shown in Figure 4 and Table 8. The results showed that SIRPa-Fc fusion protein can bind to CD47 on the surface of Raji cells, and the binding activity of SIRPa-Fc fusion protein to Raji was better than that of wild-type V1 or V2, and better than that of TTI621. The data MFI in the table is the mean fluorescence intensity value of the measured cell population.

Table 8 FACS detection of binding activity of SIRPa-Fc fusion protein to Raji

Example 6 Detecting that SIRPa-Fc fusion protein blocks the binding of Raji to the receptor SIRPa

A. Production and purification of SIRPa protein

The human SIRPa V1 protein extracellular domain (Extracellular Domain, ECD) amino acid sequence (the gene accession number in NCBI is NP_542970.1, amino acids 31-370, the specific sequence is shown in SEQ ID NO: 53) and SIRPa V2 The amino acid sequence of the extracellular domain (ECD) of the protein (the gene accession number in NCBI is AAH38510.1, amino acids 31-369, the specific sequence is shown in SEQ ID NO: 54) was cloned by coupling 6×His sequence respectively to the pTT5 vector.

SEQ ID NO: 53

SEQ ID NO: 54

Add the obtained plasmid and transfection reagent PEI (Polysciences, Cat. No.: 24765-1) into OPTI-MEM (Gibco, Cat. No.: 11058021) and mix well, then let it stand for 15 min, then add it into Expi293 cells (manufacturer: Thermofisher, Cat. No.: A14527), Put in 5% CO ₂ , 120 rpm, and culture on a shaker at 37°C. On the second day of transfection, add OPM-293ProFeed (Shanghai Opma, Cat. No.: F081918-001) and 6 g/L glucose (manufacturer: Sigma, Cat. No.: G7528). On the sixth day of transfection, the cell supernatant was collected; the cell components were removed by centrifugation, and culture supernatants containing hSIRPa V1 ECD-His and hSIRPa V2 ECD-His proteins were obtained respectively. The protein in the cell culture supernatant was purified with a Ni affinity chromatography column (GE, catalog number: 17371206). Equilibrate with 3-5 column volumes of equilibration buffer (PBS phosphate buffer, pH7.4) (20×PBS buffer 500ml, Sanko, Cat. No. B548117-0500), then load the clarified culture supernatant To the Ni affinity chromatography column, control the flow rate at 5mL/min. After loading the sample, the protein A column is washed with an equilibration buffer, and the volume of the equilibration buffer is 3 to 5 times the volume of the column bed of the Ni affinity chromatography column. Gradient elution was carried out with 0-500mM imidazole (Sinopharm, product number: 30104961), and the elution was monitored with a nucleic acid protein detector (A280 ultraviolet absorption peak). The eluted protein was collected, and the protein was dialyzed into PBS phosphate buffer at 4° C. with a dialysis card (purchased from Thermo Scientific, catalog number: 88252). The purified hSIRPa V1 ECD-His and hSIRPa V2 ECD-His proteins were obtained by sterile filtration with a 0.22 μm filter (Millipore, product number SLGVR13SL) and aseptically stored. The purified hSIRPa V1 ECD-His and hSIRPa V2 ECD-His were tested and analyzed for protein yield, protein concentration (A280/1.4), SEC purity, etc., and the quality of the purified protein was qualified. The detection information about protein yield, protein concentration and purity is shown in Table 9.

Table 9 Detection and analysis of SIRPa protein

B. Detection of SIRPa-Fc fusion protein blocking activity of Raji and receptor SIRPa V1

The method of biotin-labeling hSIRPa V1 ECD-His was the same as in Example 3. Raji cells were expanded and cultured in T-75 cell culture flasks, the medium was collected, counted, centrifuged at 1000 rpm for 5 minutes at room temperature, the medium was discarded, and washed twice with PBS buffer (purchased from Hyclone, product number SH30256.01). The cells were resuspended with 25 μg/ml Fc blocker (purchased from BD, Cat. No. 564220), and blocked at room temperature for 20 minutes. Add 1×10 ⁵ cells per well, 50 μL per well into a U-bottom 96-well FACS reaction plate (purchased from Corning, Cat. No. 3795), and store at 4°C for later use. Add 1% (w/w) BSA in PBS phosphate buffer as FACS buffer, mix the purified SIRPa-Fc fusion protein with different concentrations and biotinylated hSIRRPa V1 ECD-His, the final concentration of hSIRRPa V1 ECD-His 4 μg/mL, incubate at room temperature for 15 minutes. Afterwards, 50 μL of the mixture was added to each well and incubated on ice for 1 hour. Centrifuge and wash twice with FACS buffer, add 100 μL of fluorescently labeled streptavidin (purchased from Biolegend, catalog number 405243) to each well, and incubate on ice for 1 hour. Centrifuge and wash 2 times with FACS buffer. The cells were suspended with 100 μL of PBS, and the results were detected and analyzed by FACS (FACS Canto II, purchased from BD Company). The results are shown in Figure 5 and Table 10, SIRPa-Fc fusion protein can effectively block the binding activity of Raji and SIRPa V1, and the blocking activity is better than that of TTI621 or wild type. The data MFI in the table is the mean fluorescence intensity value of the measured cell population.

Table 10 FACS detection of SIRPa-Fc fusion protein blocking the binding activity of Raji and SIRPa V1

C. Detection of SIRPa-Fc fusion protein blocking activity of Raji and receptor SIRPa V2

The method of biotin-labeling hSIRPa V2 ECD-His was the same as in Example 3. Raji cells were expanded and cultured in T-75 cell culture flasks, the medium was collected, counted, centrifuged at 1000rpm for 5 minutes at room temperature, the medium was discarded, and washed once with PBS buffer (purchased from Hyclone, product number SH30256.01). The cells were resuspended with 25 μg/ml Fc blocker (purchased from BD, Cat. No. 564220), and blocked at room temperature for 20 minutes. Add 1×10 ⁵ cells per well, 50 μL per well into a U-bottom 96-well FACS reaction plate (purchased from Corning, Cat. No. 3795), and store at 4°C for later use. Add 1% (w/w) BSA in PBS phosphate buffer as FACS buffer, mix the purified SIRPa-Fc fusion protein with different concentrations and biotinylated hSIRRPa V2 ECD-His, the final concentration of hSIRRPa V2 ECD-His 4 μg/mL, incubate at room temperature for 15 minutes. Afterwards, 50 μL of the mixture was added to each well and incubated on ice for 1 hour. Centrifuge and wash twice with FACS buffer, add 100 μL of fluorescently labeled streptavidin (purchased from Biolegend, catalog number 405243) to each well, and incubate on ice for 1 hour. Centrifuge and wash 2 times with FACS buffer. The cells were suspended with 100 μL of PBS, and the results were detected and analyzed by FACS (FACS Canto II, purchased from BD Company). 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 that of TTI621. The data MFI in the table is the mean fluorescence intensity value of the measured cell population.

Table 11 FACS detection of SIRPa-Fc fusion protein blocking the binding activity of Raji and SIRPa V2

Example 7 SIRPa-Fc fusion protein affinity maturation

Using the four plasmids Mut87-Fc, Mut88-Fc, Mut89-Fc, and Mut90-Fc as templates, four mutation libraries were constructed respectively; each library contained a combination of single-point amino acid mutations and multiple-point mutations targeting key mutation sites. The total number of theoretical mutations: 4.7×10 ⁸ , and the total library capacity of the construction is >3.8×10 ⁹ . After 2-3 rounds of panning, the 2nd or 3rd round phages were subjected to affinity identification.

Similar to Example 1, the nucleic acid fragment encoding the mutant was cloned into the pTT5 vector with Fc tag, and the plasmid was prepared according to the established standard molecular biology method. The SIRPα variants used were 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" indicates a fusion protein of mutant SIRPa and Fc.

Table 12 SIRPa mutant sequence

Similar to Example 2, the Expi293F system was used to express the mutant protein, and the purified SIRPa-Fc fusion protein was tested for protein yield, protein concentration (A280/1.4), SEC purity, etc., and the quality of the purified SIRPa-Fc fusion protein was qualified. See Table 13 for the detection information about protein yield, protein concentration and purity.

Table 13 Detection and analysis of SIRPa-Fc fusion protein

Similar to Example 4, the results of surface plasmon resonance (SPR) detection of the affinity between SIRPa-Fc fusion protein and CD47 protein 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 SPR detects the affinity of SIRPa-Fc fusion protein and hCD47 ECD-mFc

SIRPa-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 level of SIRPα-Fc fusion protein to hCD47 ECD-mFc protein was detected. The results are shown in Figure 7 and Table 15. The results show that the binding activity of SIRPa-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 to hCD47 ECD-mFc

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

Table 16 FACS detection of binding activity of SIRPa-Fc fusion protein to Jurkat

Example 8 Screening for deglycosylation mutations

In order to ensure the controllability of the subsequent production process, we further mutated the 82nd amino acid on the basis of 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 cloned into the pTT5 vector with 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, wherein "MutXX-Fc" indicates the fusion protein of mutant SIRPa and Fc.

Table 17 SIRPa mutant sequence

Similar to Example 2, use the Expi293F or ExpiCHO system to express and purify mutant proteins, wherein Mut92-Fc, Mut93-Fc, Mut94-Fc, Mut95-Fc, Mut97-Fc, Mut98-Fc, Mut99-Fc, Mut100-Fc, Mut105- Fc and Mut107-Fc were expressed by Expi293F system, and Mut139-Fc, Mut141-Fc, Mut142-Fc and Mut146-Fc were expressed by ExpiCHO system. The purified SIRPa-Fc fusion protein was tested and analyzed for protein yield, protein concentration (A280/1.4), SEC purity, etc., and the quality of the purified SIRPa-Fc fusion protein was qualified. The detection information about protein yield, protein concentration and purity can be found in Table 18.

Table 18 Detection and analysis of SIRPa-Fc fusion protein

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

Table 19 SPR detects the affinity of SIRPa-Fc fusion protein and hCD47 ECD-mFc

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

Table 20 ELISA detects the binding activity of SIRPa-Fc fusion protein and hCD47 ECD-mFc

Similar to Example 5A, the binding activity of SIRPα-Fc fusion protein to cells was detected by flow cytometry (FACS). The results are shown in Figures 10A-10C and Table 21. The results show that the binding level of the 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

Example 9 Detection of phagocytosis of tumor cells mediated by SIRPa-Fc fusion protein

After PBMCs were taken out from liquid nitrogen, they were thawed in a water bath at 37°C. PBMCs were resuspended in EasySep ^TM Buffer (purchased from Stemcell, Cat. No. 20144), centrifuged at 300×g for 5 minutes, and the supernatant was discarded. Monocytes were enriched with a monocyte sorting kit (purchased from Miltenyi Biotec, product number 130-096-537), and induced to differentiate with rhM-CSF (purchased from R&D systems, product number 216-MC-100). The medium was changed every two days, and the macrophages were collected on the seventh day for use.

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

After the experiment, PI (purchased from Thermo Fisher, Cat. No. P3566) was added to the cells, and after incubation for 5 minutes, flow detection (FACS Canto Plus, purchased from BD Company) was performed. The results are shown in Figures 11A-11B and Table 22. The formula for calculating the phagocytosis ratio of the data in the table is: CellTrace ^TM Violet+CellTrace ^TM Far Red+/CellTrace ^TM Far Red+. According to the results, SIRPa-Fc fusion protein can effectively mediate macrophage phagocytosis of Raji cells, and the phagocytosis level is better than that mediated by TTI621.

Table 22 FACS detection of phagocytosis of Raji cells mediated by SIRPa-Fc fusion protein

Expand the DLD1 cells to 1×10 ⁶ cells/mL in a T-75 culture flask. The medium was removed by centrifugation, washed twice with PBS (purchased from Hyclone, product number SH30256.01), and the cell density was adjusted to 1×10 ⁶ cells/mL. DLD1 cells were stained with CellTrace ^™ Violet (purchased from Thermo Fisher, Cat. No. C34557). In the same way, macrophages were stained with CellTrace ^TM Far Red (purchased from Thermo Fisher, Cat. No. C34572). The stained DLD1 and macrophages were added to a 96-well plate (purchased from Corning, Cat. No. 3474) according to E:T=1:2, and incubated with SIRPa-Fc fusion protein and 0.04nM Cetuximab for 3 hours.

After the experiment, PI (purchased from Thermo Fisher, Cat. No. P3566) was added to the cells, and after incubation for 5 minutes, flow cytometric detection (FACS Canto Plus, purchased from BD Company) was performed. Results As shown in Figures 12A-12B and Table 23, SIRPa-Fc fusion protein can effectively promote Cetuximab-mediated macrophage phagocytosis of DLD1 cells.

Table 23 FACS detection of phagocytosis of DLD1 cells promoted by SIRPa-Fc fusion protein

Example 10 Detection of tumor growth inhibition mediated by SIRPa-Fc fusion protein

Female NOD-SCID mice (approximately 6-8 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), received and adapted for at least 1 week before starting the experiment. To ensure that the viability of OE19 cells was greater than 90%, the volume of 8×10 ⁶ /100 μL (1640:matrigel=1:1, corning, 356237) per mouse was inoculated subcutaneously on the right side of the trunk of NOD-SCID mice. Mice were grouped according to tumor volume after inoculation. SIRPa-Fc fusion protein single-use group: mice were intraperitoneally injected with 30 mg/kg on days 0, 7, and 14 after grouping; Trastuzumab antibody single-use group: mice were intraperitoneally injected with 10 mg on days 0, 3, 7, and 14 after grouping /kg (group 1), or intraperitoneal injection of 5 mg/kg (group 2) on the 0th, 7th, and 14th day after grouping; SIRPa-Fc fusion protein and Trastuzumab antibody combination group: according to the respective doses of the above two drugs The way of medicine is combined with administration. The mice were observed daily and their body weight was recorded, and the inoculated tumor volume was measured 2-3 times a week. When the tumor volume of the mice exceeds the end point, ie, the tumor volume is >2000mm ³ , the mice are 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 inhibition rate (Tumor growth inhibition rate, TGI%) is calculated by the following formula: TGI%=(1-TVi/TVvi)×100%, TVi is the average tumor volume of mice in the administration group on specific days, and TVvi is the specific days The average tumor volume of mice in the control group. 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 Effect of SIRPa-Fc fusion protein on tumor growth

Claims (22)

1. A polypeptide comprising a signal regulatory protein alpha (SIRPa) mutant or fragment thereof, said mutant having at least one amino acid modification selected from N51, I31, Q37, L66, K68 or T82 relative to wild-type SIRPa; preferably Preferably, the amino acid modification is a substitution; preferably, the wild-type SIRPα has a sequence selected from any one of SEQ ID NO: 1-2.
2. The polypeptide of claim 1, wherein the amino acid modification is at least one amino acid modification selected from the group consisting of: (1) N51R; N51G; N51A; N51E; N51F; N51K; N51L; N51M; N51Q; I31Y; (3) Q37T; (4) L66N; L66A; L66V; L66P; L66M; L66E; L66K; L66H; (5) K68Q; T82F; T82D; T82N; T82E; T82Q; T82I; T82K; T82M; T82P; T82R; T82V; T82W; T82Y.
3. The polypeptide of claim 2, relative to wild-type SIRPa having SEQ ID NO: 1, comprising an amino acid mutation selected from the group consisting of:

   (1) I31Y; N51R; L66N; (2) I31Y; N51R; L66N; T82H; (3) I31Y; ; L66N; K68L; T82G; (6) I31M; N51R; L66N; K68I; T82G; (7) N51G; (8) N51R; (9) I31M; N51G; (10) I31M; (12) I31Y; N51R; (13) Q37T; N51G; (14) Q37T; N51R; (15) I31Y; N51R; L66A; (16) I31Y; 18) I31Y; N51R; L66M; (19) I31Y; N51R; L66E; (20) I31Y; N51R; L66K; (21) I31Y; (25) N51K; (26) N51L; (27) N51M; (28) N51Q; (29) N51T; (30) N51W; (31) I31M; K68M; (33)I31Y; N51R; L66N; K68D; (34) I31M; N51R; L66N; K68Q; (35) I31M; N51R; N51R; L66N; K68I; (38) I31Y; N51R; L66N; T82F; (39) I31Y; N51R; L66N; T82D; (40) I31Y; N51R; L66N; (42) I31Y; N51R; L66N; T82Q; (43) I31Y; N51R; L66N; T82G; (44) I31Y; N51R; L66N; T82M; (47) I31Y; N51R; L66N; T82P; (48) I31Y; (51) I31Y; N51R; L66N; T82Y; (52) I31M; N51R; L66N; K68Q; T82G; (53) I31M; (55) I31M; N51R; L66N; K68L; T82H; (56) I31M; N51R; L66N; K68L; T82L; (57) I31M; T82H; (59)I31M;N51R;L66N;K68I;T82L;(60)I31M;N51R;L66N;K68I;T82V.
4. The polypeptide according to any one of claims 1-3, wherein the polypeptide is fused to an immunoglobulin Fc region at its N-terminus or C-terminus; preferably, the immunoglobulin Fc region is selected from human IgG1, IgG2 or IgG4 The Fc region of the immunoglobulin or its mutant type; more preferably, the immunoglobulin Fc region is selected from the Fc region of human IgG1 or its mutant type.
5. The polypeptide according to claim 4, wherein the Fc mutant type 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 modification comprises (1) N297A Mutations; (2) L234A, L235A, and G237A mutations; or (3) L234A, L235A, G237A, and N297A mutations.
6. The polypeptide of claim 5, wherein the Fc mutant exhibits abolished or reduced effector function, abolished or reduced C1q binding and Fcγ receptor binding compared to the wild type of the human IgG Fc region.
7. The polypeptide according to any one of claims 1-6, wherein said polypeptide has the amino acid sequence of any one of SEQ ID NO: 4-14, 17-51, 55-110.
8. The polypeptide of any one of claims 1-7, wherein said polypeptide has a KD for CD47 of at least 6 x 10 ^-9 M.
9. The polypeptide according to any one of claims 1-8, which is also connected with other functional molecules, and the other functional molecules can be selected from one or more of the following: signal peptides, protein tags, other antigen-binding molecules, leukocytes interkines, cytokines, steroids, anti-inflammatory agents, immunomodulators or cytotoxins.
10. The polypeptide of any one of claims 1-9, wherein the polypeptide is monomeric, or polymerized into multimers.
11. A chimeric antigen receptor (CAR), which at least comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, the extracellular antigen binding domain comprising any one of claims 1-10 of polypeptides.
12. An immune effector cell expressing the chimeric antigen receptor according to claim 11, or comprising a nucleic acid fragment encoding the chimeric antigen receptor according to claim 11; preferably, the immune effector cell is selected from T cells, NK cells (natural killer cell), NKT cells (natural killer T cell), DNT cells (double negative T cell), monocytes, macrophages, dendritic cells or mast cells, said T cells are preferably selected from cytotoxic T cells, regulatory T cells or helper T cells; preferably, the immune effector cells are autoimmune effector cells or allogeneic immune effector cells.
13. A nucleic acid molecule encoding the polypeptide of any one of claims 1-10, or the chimeric antigen receptor of claim 11.
14. An expression vector comprising the nucleic acid molecule of claim 13.
15. A kind of host cell, it comprises the carrier described in claim 14; Preferably, described cell is prokaryotic cell or eukaryotic cell, such as bacterium (Escherichia coli), fungus (yeast), insect cell or mammalian cell (CHO cell line or 293T cell line).
16. A method for preparing the polypeptide according to any one of claims 1-10, comprising culturing the host cell according to claim 15, and isolating the polypeptide expressed by the host cell.
17. A method for preparing immune effector cells according to claim 12, comprising introducing the nucleic acid fragment encoding the CAR according to claim 11 into the immune effector cells, optionally, further comprising enabling the immune effector cells to express claim 11 The CAR.
18. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the polypeptide according to any one of claims 1-10, or the immune effector cell of claim 12, in an amount capable of effectively inhibiting the growth or proliferation of CD47+ disease cells , or the nucleic acid molecule of claim 13, or the carrier of claim 14; or the product obtained by any method of claims 16-17.
19. The polypeptide according to any one of claims 1-10, the immune effector cell according to claim 12, the nucleic acid molecule according to claim 13, the expression vector according to claim 14, the host cell according to claim 15, the The product prepared by the method described in any one of 16-17, or the use of the pharmaceutical composition described in claim 18 for the preparation of a medicament for treating a tumor expressing CD47; preferably, the tumor is a CD47-positive blood tumor or a CD47 Positive solid tumors, more preferably, the CD47-positive blood tumors include leukemia, lymphoma and myeloma, and the CD47-positive solid tumors include sarcoma.
20. A method for treating a tumor expressing CD47, the method comprising administering to a subject an effective amount of the polypeptide of claims 1-10, the immune effector cell of claim 12, the nucleic acid molecule of claim 13, the The expression vector according to claim 14, the host cell according to claim 15, the product prepared by the method according to any one of claims 16-17, or the pharmaceutical composition according to claim 18; preferably, the tumor It is a CD47-positive blood tumor or a CD47-positive solid tumor. More preferably, the CD47-positive blood tumor includes leukemia, lymphoma and myeloma, and the CD47-positive solid tumor includes sarcoma.
21. The polypeptide according to any one of claims 1-10, the immune effector cell according to claim 12, the nucleic acid molecule according to claim 13, the expression vector according to claim 14, the host cell according to claim 15, the The product prepared by the method described in any one of 16-17, or the pharmaceutical composition described in claim 18 is used to treat tumors expressing CD47; preferably, the tumors are CD47-positive blood tumors or CD47-positive solid tumors, and then Preferably, the CD47-positive blood tumors include leukemia, lymphoma and myeloma, and the CD47-positive solid tumors include sarcoma.
22. A method for detecting the expression of CD47 in a biological sample, characterized in that the method comprises making the biological The sample is contacted with the polypeptide according to any one of claims 1-10; preferably, the method further comprises detecting the formation of the complex, indicating the presence or expression level of CD47 in the sample.

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