CN111253482A

CN111253482A - SIRPa variants, fusion proteins, and uses thereof

Info

Publication number: CN111253482A
Application number: CN202010099916.2A
Authority: CN
Inventors: 罗龙龙; 王志宏; 乔春霞; 王晶; 李新颖; 陈国江; 胡乃静; 周刘忠; 肖鹤; 冯健男; 沈倍奋
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS; Academy of Military Medical Sciences AMMS of PLA
Priority date: 2020-02-18
Filing date: 2020-02-18
Publication date: 2020-06-09
Anticipated expiration: 2040-02-18
Also published as: CN111253482B

Abstract

The invention discloses a SIRPa variant, a fusion protein and application thereof. The fusion protein is prepared by connecting the SIRPa variant and the Fc fragment of the human antibody. Experiments prove that compared with SIRPa and TTI-621, the fusion protein disclosed by the invention has stronger binding capacity with CD47, so that the interaction between wild type SIRPa and CD47 is more remarkably inhibited, the phagocytic capacity of macrophages is further enhanced, and the treatment of cancers and autoimmune diseases is facilitated. The fusion protein provided by the invention has great development potential as a candidate drug for treating cancer.

Description

SIRPa variants, fusion proteins, and uses thereof

Technical Field

The invention belongs to the technical field of genetic engineering and protein engineering, and relates to a SIRPa variant, a fusion protein and application thereof.

Background

CD47 is a widely distributed cell membrane protein, which is a key "eat me" signal of the innate immune system, and inhibits phagocytosis by combining with inhibitory immunoreceptor signal regulatory protein α (signal regulatory protein α α). CD47 has been widely concerned about various physiological and pathological functions of human body, especially about the occurrence and development of tumors.

CD47 belongs to the immunoglobulin superfamily, has a molecular weight of about 52kDa, is a widely expressed, highly glycosylated transmembrane protein with 1 IgV-like domain at its N-terminus and 5 transmembrane segments and an optionally spliced highly hydrophobic cytoplasmic C-terminus the CD47 molecule has 4 subtypes, CD47 subtype 2 is most widely expressed, distributed mainly in hematopoietic, vascular endothelial and epithelial cells, while subtype 1 is distributed in keratinocytes, with both subtypes 3 and 4 distributed in neuronal, intestinal mucosal and testicular cells the ligand for the CD47 molecule is thrombospondin 1(thrombospondin 1, TSP1) and SIRP α 47 binding to SIRP α on macrophages leading to activation of the intracellular Src2 tyrosine phosphatase domain and inhibition of myosin accumulation in the synapses of phagocytes, ultimately signaling "eat me" to macrophages.

CD47 has been found to be selectively expressed during hematopoietic stem cell migration to avoid phagocytosis, while low expression of CD47 results in macrophage clearance of senescent or damaged cells.it has been reported that, to maintain normal function of human erythrocytes, large expression of CD47 molecules on the surface of newborn erythrocytes to avoid phagocytosis of macrophages, while down-regulation of CD47 expression on the surface of senescent erythrocytes promotes macrophage clearance of senescent erythrocytes.furthermore, it has been found that the accumulation of Necrotic Cells (NCs) is found in human plaques, and NC surface over-expression of CD47 results in increased inflammation and necrosis formation in atherosclerotic lesions, presumably due to defective phagocytosis of macrophages by the immune system to avoid phagocytosis of macrophages.pathological function, down-regulation of CD47 on the surface of erythrocytes may lead to the development of anemia.during the development of tumorigenesis, CD47 shows a role which is widely expressed on the surface of tumor cells, such as ovarian cancer, gastric cancer and small cell tumor cells [ 5 inhibits tumor cell escape from the tumor cell system, inhibits tumor cells in vivo, inhibits tumor cell transplantation of natural immune cells, thereby effects of the immune T-phagocytosis in vivo, and thus, the immune cell transplantation of tumor cells inhibits the immune tumor cells, and the immune response of the immune system against the immune receptor for inhibiting the tumor phagocytosis of the tumor cell receptor.

Based on the important function of CD47 in natural immunity and acquired immunity, researchers are continuously dedicated to research on a CD47-SIRP α signal channel-related action mechanism and downstream related signals, and gradually define the key action of the CD 47-related signals in various diseases, particularly tumor-related diseases, so that a targeted CD47 molecular drug becomes one of the most popular tumor drug development targets.

More and more data show that the antibody or Fc fusion protein can block a CD47-SIRP α signal channel and activate phagocytosis of macrophages on tumor cells, and the phagocytosis mechanism provides a powerful theoretical basis for development of a tumor immunotherapy medicament targeting CD47 molecules.

TTI-621 is a SIRP α analog, belonging to recombinant Fc fusion protein medicine, obtained by fusing the N-terminal immunoglobulin-like V domain of human SIRP α to human IgG1 Fc region, the affinity of the molecule with CD47 molecule is at least 5 times higher than that of the whole extracellular region of human SIRP α, and the molecular weight is smaller (about 80kDa), so it has better tissue permeability and distribution ability, besides, compared with CD47 antibody medicine, TTI-621 has the greatest advantage of not combining with human red blood cells, but still combining with platelets and leukocytes, which improves the safety of the medicine in clinical application to some extent.

From the Trillium Therapeutic official network (https:// Trillium Therapeutic. com/pipeline/# TTI621), TTI-621 is conducting 2 phase I clinical trials, one is TTI-621 single drug (0.2mg/kg) or TTI-621(0.1mg/kg) in combination with rituximab administered intravenously to treat recurrent or refractory hematologic malignancies, and the results show that it has good objective reactivity and tolerance, with less than grade 3 thrombocytopenic adverse reactions in only 18% of patients. Another objective is to evaluate the therapeutic effect of TTI-621 on recurrent or refractory granulomatous mycosis and Szary syndrome, considering the safety of the drug, researchers use a local administration mode of percutaneous injection, and the results show that TTI-621 is well tolerated, and 91% (20/22) granulomatous mycosis patients have obvious improvement of local lesion and show a far-end and systemic therapeutic effect. The clinical development advantage of TTI-621 is obvious, and although slight platelet toxicity exists, the fact that the drug targeting CD47 molecule is expected is suggested, and reference is provided for further clinical development.

The invention content is as follows:

one of the purposes of the invention is to provide a SIRPa variant and a fusion protein thereof.

The second object of the present invention is to provide a DNA molecule, recombinant vector or host cell encoding the above variant or fusion protein.

It is a further object of the present invention to provide a pharmaceutical composition or kit comprising the above SIRPa variants and fusion proteins thereof.

The fourth object of the present invention is to provide the use of SIRPa variants and fusion proteins thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a SIRPa variant, the amino acid sequence of which is any one of the following groups:

(1) an amino acid sequence shown as SEQ ID NO. 1;

(2) the amino acid sequence shown in SEQ ID NO.1 is obtained by substituting and/or deleting and/or adding one or more amino acids to obtain the amino acid sequence with the same or similar functions.

(3) And (3) an amino acid sequence which has at least 99% homology with the amino acid sequence defined in (1) or (2) and has the same function.

Preferably, the amino acid sequence of the SIRPa variant is shown as SEQ ID NO. 1.

The number of one or more amino acids substituted and/or deleted and/or added in the amino acid sequence shown in SEQ ID NO.1 is not more than 10.

The "human antibody Fc fragment" in the present invention refers to a "human immunoglobulin heavy chain constant region" and can be derived from an antibody belonging to each immunoglobulin class called IgA, IgD, IgE, IgG and IgM. Furthermore, it is contemplated that the immunoglobulin heavy chain constant region may be derived from any of the IgG subclasses of antibodies known in the art as IgG1, IgG2, IgG3, and IgG 4.

Preferably, the human antibody Fc fragment of the invention is derived from the human antibody IgG 1.

Preferably, the amino acid sequence of the Fc fragment of the human antibody of the present invention is any one of the following groups:

(1) an amino acid sequence shown as SEQ ID NO. 2;

(2) an amino acid sequence with the same or similar functions obtained by substituting and/or deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 2;

(3) and (3) an amino acid sequence which has at least 98% homology with the amino acid sequence defined in (1) or (2) and has the same function.

More preferably, the amino acid sequence of the Fc fragment of the human antibody is shown in SEQ ID NO. 2.

The amino acid sequence of the SIRPa variant-Fc of the fusion protein is any one of the following groups:

(1) an amino acid sequence shown as SEQ ID NO. 3;

(2) an amino acid sequence with the same or similar functions obtained by substituting and/or deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 3;

Preferably, the amino acid sequence of the fusion protein SIRPa variant-Fc is shown in SEQ ID NO. 3.

A SIRPa variant-Fc sequence that is SIRPa due to one or more conservative amino acid substitutions or due to one or more non-conservative amino acid substitutions, deletions, or insertions, wherein the substitutions, deletions, or insertions do not abrogate the biological activity of the wild-type sequence. Conservative substitutions typically include the substitution of one amino acid with another having similar characteristics, such as within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative amino acid substitutions are known in the art and are included herein. Non-conservative substitutions, such as the replacement of a basic amino acid with a hydrophobic amino acid, are also well known in the art.

Modifications to the fusion protein SIRPa variant-Fc of the invention to increase protein or peptide stability are also included within the scope of the invention, such modifications including the inclusion of, for example, one or more non-peptide bonds in the protein or peptide sequence (to replace peptide bonds), and such modifications also including the inclusion of D-amino acids or non-naturally occurring or synthetic amino acids, such as β or gamma amino acids.

The SIRPa variants or fusion proteins thereof described herein can be used in vitro in binding assays, such as immunoassays. For example, in some embodiments, a SIRPa variant or fusion protein thereof described herein is utilized in a liquid phase to bind to a solid support. In some embodiments, the SIRPa variant or fusion protein thereof for use in an immunoassay is detectably labeled in various ways.

In some embodiments, SIRPa variants or fusion proteins thereof described herein are bound to various vectors and used to detect the presence of particular antigen expressing cells. Examples of carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon (nylon), amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The carrier may be soluble or insoluble in nature.

Various labels and labeling methods are known. Examples of labels include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds. Various techniques are available for binding labels to the polypeptides disclosed herein.

In some embodiments, the SIRPa variant or fusion protein thereof is coupled to a low molecular weight hapten. These haptens are then specifically detected by a second reaction. For example, in some embodiments, the hapten biotin is used with avidin, or the hapten dinitrophenol, pyridoxal, or fluorescein is detected with specific anti-hapten antibodies (e.g., anti-dinitrophenol antibodies, anti-pyridoxal antibodies, and anti-fluorescein antibodies, respectively).

The present invention provides a DNA molecule encoding the SIRPa variant or fusion protein described above.

Further, the nucleotide sequence of the DNA molecule is any one of the following group:

(1) the nucleotide sequence shown as SEQ ID NO.4 or a degenerate sequence thereof;

(2) a nucleotide sequence obtained by substituting and/or deleting and/or adding one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 4; the nucleotide sequence shown in SEQ ID NO.4 or the degenerate sequence thereof encodes proteins with the same or similar functions;

(3) a nucleotide sequence which is hybridized with the nucleotide sequence defined in (1) under strict conditions and has the same function;

(4) a nucleotide sequence which has at least 98 percent of homology with the nucleotide sequence defined in the step (1) and has the same function.

The "stringent conditions" described herein may be conditions in which specific hybridization with the nucleotide sequence shown in SEQ ID NO.4 occurs at 65 ℃ in a 6 XSSC solution of 0.5% SDS, and then membranes are washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS. A nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence defined in SEQ ID No.4 and has the same function is at least about 40% -50% homologous, about 60%, 65% or 70% homologous, or even at least 98% or more homologous to the sequence indicated in SEQ ID No. 4. I.e., the range of sequence identity is distributed over at least about 40% -50%, about 60%, 65%, or 70% homology, and even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence homology.

The DNA molecules described above can be prepared by a variety of methods. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. In some embodiments, the DNA molecule described above is obtained using standard techniques (e.g., gene synthesis). In some cases, DNA molecules are synthesized using nucleotide synthesizers or PCR techniques.

The present invention provides a recombinant vector comprising a DNA molecule as described above.

As used herein, the term "vector" is understood to mean any nucleic acid comprising a nucleotide sequence capable of being incorporated into and recombined with and integrated into a host cell genome, or autonomously replicating as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids (cosmids), RNA vectors, viral vectors, and the like.

Vectors suitable for use in the present invention include, but are not limited to: expression vectors, cloning vectors, for example, vectors suitable for use in prokaryotic (e.g., bacteria such as E.coli), lower eukaryotic (e.g., yeast), insect, plant, or mammalian cells. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host.

Viral vectors that can be used in the present invention include, but are not limited to, adenoviral vectors (adeno-associated viral vectors), retroviral vectors (retroviral vectors), herpes simplex viral-based vectors (herpes simplex vectors), and lentiviral vectors (lentiviral vectors).

The carrier may comprise various components or elements. In some embodiments, vector components include, but are not limited to, transcriptional and translational regulatory sequences, such as promoter sequences, ribosome binding sites, signal sequences, transcriptional initiation and termination sequences, translational initiation and termination sequences, 3 'and 5' untranslated regions (UTRs), and enhancer or activator sequences; an origin of replication; a selectable marker gene; and a nucleic acid sequence encoding a polypeptide of interest and a transcription termination sequence. In some embodiments, the expression vector comprises a protein operably linked to a control or regulatory sequence, a selectable marker, any fusion partner, an additional element, or any combination thereof. The term "operably linked" means that a nucleic acid is placed in a functional relationship with another nucleic acid sequence. Generally, these expression vectors include transcription and translation regulatory nucleic acids operably linked to a nucleic acid encoding an Fc variant, and are typically suitable for use in host cells for expression of the protein. A selection gene or marker, such as but not limited to an antibiotic resistance gene or a fluorescent protein gene, can be used to select for host cells containing an expression vector, for example, by antibiotic or fluorescent expression. Various selection genes are available.

In some embodiments, the components or elements of the vector are optimized to make the expression vector compatible with the host cell type. Expression vectors for use in the present disclosure include, but are not limited to, those that enable expression of proteins in mammalian cells, bacteria, insect cells, yeast, and in vitro systems.

The invention also provides a host cell comprising a DNA molecule or recombinant vector as described above.

Further, the host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.

Examples of mammalian cell types include, but are not limited to, Human Embryonic Kidney (HEK) (e.g., HEK293F) cells, Chinese Hamster Ovary (CHO) cells, HeLa, COS, PC3, Vero, MC3T3, NS0, Sp2/0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0 (murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030, and HsS78Bst cells. Examples of E.coli strains include, but are not limited to, E.coli 294(ATCC-31,446), E.coli lambda 1776(ATCC-31,537, E.coli BL21(DE3) (ATCC-BAA-1025), and E.coli RV308(ATCC-31,608).

Different host cells have characteristics and specific mechanisms for post-translational processing and modification (e.g., glycosylation) of protein products. In some embodiments, an appropriate cell line or host system is selected to ensure proper modification and processing of the expressed polypeptide. After the vector is introduced into a host cell to produce a protein, the host cell is cultured in an appropriately modified conventional nutrient medium to induce a promoter, select a transformant or amplify a gene encoding a desired sequence.

The invention also provides a preparation method of the fusion protein, which comprises the following steps:

(1) constructing the recombinant vector as described above;

(2) introducing the recombinant vector into a host cell for expression;

(3) and separating and purifying the fusion protein.

Further, there are various expression systems that can be used to express these fusion proteins, including eukaryotic cells and prokaryotic cells, including but not limited to mammalian cells, bacteria, yeast, insect cells, and the like. Since the amino acid sequence of the optimized fusion protein of the present invention comprises amino acids that can be glycosylated, mammalian cells are the preferred system for expressing the protein. There are various mammalian cells that can be used for large-scale expression of proteins, such as CHO cells, 293 cells, NS0 cells, COS cells, BHK cells, etc., and many other cells can be used for expression of proteins, and are included in the list of cells that can be used in the present invention. Expression systems other than mammalian cells, such as bacteria, yeast, insect cells, and the like, may also be used to express the optimized fusion proteins of the present invention, and are also encompassed by the list of host cells that can be used with the present invention. These expression systems have higher protein yields than mammalian cells, but the expressed proteins lack glycosylation or form sugar chain structures different from those of mammalian cells.

The recombinant plasmid containing the gene encoding the above fusion protein can be transfected into host cells by various methods including, but not limited to, electroporation, lipofection, calcium phosphate transfection, etc.

In some cases, the fusion protein is expressed in vitro using a cell-free translation system. In vitro translation systems derived from prokaryotic (e.g., e.coli) and eukaryotic (e.g., wheat germ, rabbit reticulocyte) cells are available and, in some embodiments, are selected based on the expression level and functional properties of the protein of interest. For example, in vitro translation is required for some display techniques, such as ribosome display, as will be appreciated by those skilled in the art. Additionally, in some embodiments, the SIRPa variant or fusion protein is produced by a chemical synthesis method, such as, but not limited to, solution phase peptide synthesis.

Since the fusion protein contains immunoglobulin Fc, protein a affinity chromatography can be used to purify the expressed fusion protein. In addition, the fusion protein of the present invention can be further purified by using it in combination with other protein purification methods such as ion exchange chromatography and the like.

The invention also provides a pharmaceutical composition comprising the SIRPa variant, the fusion protein, the DNA molecule, the recombinant vector, or the host cell.

Further, the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier, excipient or stabilizer, which includes buffers such as phosphate, citrate, HEPES, TAE and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (for example hexamethonium chloride; octadecyldimethylbenzylammonium chloride; benzalkonium chloride, benzethonium chloride; phenol alcohol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (e.g., less than about 10 residues) polypeptides; proteins such as human serum albumin, gelatin, dextran, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, histidine, and lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, sucrose, and sorbitol; chelating agents, such as EDTA sugars, such as sucrose, mannitol, trehalose, or sorbitol; sweeteners and other flavoring agents; fillers such as microcrystalline cellulose, lactose, corn starch, and other starches; a binding agent; an additive; a colorant; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); nonionic surfactants, such as TWEEN^TM、PLURONICS^TMAnd polyethylene glycol (PEG); or any combination thereof.

The pharmaceutical compositions of the present invention may be administered by a variety of means, such as orally, sublingually, buccally, parenterally, nasally, topically, rectally, transdermally, transmucosally, and the like.

Depending on the form of administration of the pharmaceutical composition, the pharmaceutical composition may be prepared into various corresponding dosage forms including, but not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols, suppositories.

In some embodiments, the pharmaceutical compositions described herein are comprised in a water-soluble form, such as in the form of a pharmaceutically acceptable salt, which is intended to include both acid addition salts and base addition salts. The term "pharmaceutically acceptable acid addition salts" refers to those salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, as well as organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, that retain the biological effectiveness of the free base and are not otherwise undesirable. The term "pharmaceutically acceptable base addition salts" includes those base addition salts derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are ammonium, potassium, sodium, calcium and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include primary, secondary, and tertiary amines; substituted amines, including naturally occurring substituted amines; a cyclic amine; and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and salts of ethanolamine. The formulation to be used for in vivo administration is preferably sterile. This may be accomplished by filtration through sterile filtration membranes or other methods.

In some embodiments, the pharmaceutical compositions of the present disclosure are administered parenterally in the form of injectable formulations, in some embodiments, sterile solutions or any pharmaceutically acceptable liquids are used as vehicles to formulate pharmaceutical compositions for injection, pharmaceutically acceptable vehicles include, but are not limited to, sterile water, saline, and cell culture media (e.g., Dulbecco's Modified Eagle's Medium (DMEM), α -modified eagle's medium (α -MEM), and F-12 medium), various formulation methods are available.

In some embodiments, the fusion proteins described herein are formulated as immunoliposomes. Liposomes are small vesicles containing various types of lipids, phospholipids, or surfactants, suitable for delivering therapeutic agents to mammals. Liposomes of fusion proteins can be prepared by various methods known in the art. In some embodiments, the components of the liposomes are arranged in a bilayer format, similar to the lipid arrangement of biological membranes. In some embodiments, the liposomes are produced by a reverse phase evaporation method in the presence of a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). In some embodiments, the liposomes are extruded through a filter of defined pore size to produce liposomes having a desired diameter. In some embodiments, the liposomes optionally contain a chemotherapeutic or other therapeutically active agent therein.

In some embodiments, the fusion proteins and other therapeutically active agents described herein are embedded in microcapsules prepared by methods including, but not limited to, coacervation techniques, interfacial polymerization (e.g., using hydroxymethylcellulose or gelatin microcapsules or poly (methylmethacylate) microcapsules), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), and macroemulsions.

In some embodiments, sustained release formulations are prepared. Examples of suitable sustained-release formulations include semipermeable matrices of solid hydrophobic polymers, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactide, copolymers of L-glutamic acid and γ -ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as LUPRON DEPOT)^TMInjectable microspheres consisting of lactic-glycolic acid copolymer and leuprolide acetate), poly D- (-) -3-hydroxybutyric acid and prolease (commercially available from Alkermes, a microsphere based delivery system consisting of the desired bioactive molecule incorporated into a matrix of poly DL-lactide-co-glycolide (PLG). Some sustained release formulations enable release of the molecule over several months, e.g., one to six months, while other formulations release the pharmaceutical compositions of the present disclosure over a shorter period of time (e.g., days to weeks).

Subjects for treatment include humans, non-human mammals, e.g., companion animals such as dogs, cats, horses, etc.; laboratory mammals such as rabbits, mice, rats, and the like.

It will be understood that the specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors including the activity of the pharmaceutical composition employed; the metabolic stability and length of action of the pharmaceutical composition; species, age, weight, general health, sex, and diet of the subject; the mode and time of administration; excretion rate and clearance rate; a pharmaceutical composition; and the severity of the particular condition.

The pharmaceutical composition of the present invention may be used alone or in combination with other therapeutic agents. For example, other therapeutic agents include immunooncology antibodies including, but not limited to: cetuximab, nixituzumab, pembrolizumab, nivolumab, pidiumtuzumab, MEDI0680, MED16469, atelizumab, avilluzumab, duvaluzumab, MEDI6383, RG7888, ipilimumab, tremelimumab, ureuzumab, PF-05082566, enoblituzumab, valtuzumab, myristylumab, moguzumab, SAR650984, daruzumab, trastuzumab-elmataxin, pertuzumab, rituximab, ofatumumab, oubizumab, RG7155, FPA008, panitumumab, bevacizumab-dimenstine, MSB001 0010718C, belimumab, bevacizumab, denozumab, panitumumab, ranizumab, nituzumab, niveumab, nivezumab, nivepavea, nivea 060606alemtuzumab, mevritumumab, mevriumtuzumab, meduus, mevriumtuzumab, meduum 6580, mezuzumab, or MED 16480, An anti-HER 2 antibody, an anti-CD 20 antibody, an anti-CD 19 antibody, an anti-CS 1 antibody, an anti-CD 38 antibody, an anti-EGFR antibody, an anti-PD 1 antibody, an anti-RANKL antibody, an anti-OX 40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CD 274 antibody, an anti-CTLA-4 antibody, an anti-CD 137 antibody, an anti-4-1 BB antibody, an anti-B7-H3 antibody, an anti-FZD 7 antibody, an anti-CD 27 antibody, an anti-CCR 4 antibody, an anti-CD 38 antibody, an anti-CSF 1R antibody, an anti-CSF antibody, an anti-CD 30 antibody, an anti-BAFF antibody, an anti-VEGF antibody, or an anti-VEGFR.

The invention also provides a kit comprising a SIRPa variant as defined above, a fusion protein as defined above, a DNA molecule as defined above, a recombinant vector as defined above, or a host cell as defined above.

Further, the kit comprises instructions for use of the SIRPa variant, the fusion protein, the DNA molecule, the recombinant vector, or the host cell described above.

Optionally, the kit further comprises at least one additional reagent. As a non-limiting example, a chemotherapeutic agent or an anti-tumor antibody may be used as at least one additional agent. In some embodiments, the kit includes a label indicating the intended use of the kit contents. The term label includes any written or recorded material provided on or with the kit or otherwise accompanying the kit.

The invention also provides the use of a SIRPa variant as defined above, a fusion protein as defined above, a DNA molecule as defined above, a recombinant vector as defined above, or a host cell as defined above, a pharmaceutical composition as defined above, comprising the use of any one of:

(1) use in the preparation of a kit as hereinbefore described;

(2) the application of the compound in preparing the medicine for inhibiting the interaction between SIRPa and CD 47;

(3) the application in preparing the medicament for enhancing the phagocytic function of macrophages;

(4) the application in preparing the medicine for eliminating regulatory T cells;

(5) the application in the preparation of the medicine for treating the diseases related to the SIRPa or CD47 activity.

Further, diseases associated with the SIRPa or CD47 activity include cancer, immune diseases;

still further, the cancer is selected from the group consisting of solid tumor cancer, hematologic cancer, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-hodgkin's lymphoma, multiple myeloma, bladder cancer, pancreatic cancer, cervical cancer, endometrial cancer, lung cancer, bronchial cancer, liver cancer, ovarian cancer, colon and rectal cancer, gastric cancer, gallbladder cancer, gastrointestinal stromal tumor cancer, thyroid cancer, head and neck cancer, oropharyngeal cancer, esophageal cancer, melanoma, non-melanoma skin cancer, mercker cell cancer, virus-induced cancer, neuroblastoma, breast cancer, prostate cancer, renal cell cancer, renal pelvis cancer, leukemia, lymphoma, sarcoma, glioma, brain tumor, carcinoma.

Still further, the immune disease includes autoimmune disease, inflammatory disease.

Preferably, the autoimmune disease or the inflammatory disease is selected from multiple sclerosis, rheumatoid arthritis, spondyloarthropathies, systemic lupus erythematosus, antibody-mediated inflammatory or autoimmune diseases, graft-versus-host disease, sepsis, diabetes, psoriasis, atherosclerosis, sjogren's syndrome, progressive systemic sclerosis, scleroderma, acute coronary syndrome, ischemia reperfusion, crohn's disease, endometriosis, glomerulonephritis, myasthenia gravis, idiopathic pulmonary fibrosis, asthma, Acute Respiratory Distress Syndrome (ARDS), vasculitis, inflammatory autoimmune myositis.

As used herein, the term "treatment" refers to the administration of an agent or the performance of a procedure for the purpose of obtaining an effect. In some embodiments, the effect is prophylactic in terms of preventing the disease or symptoms thereof, in whole or in part. In some embodiments, the effect is therapeutic in terms of effecting a partial or complete cure of the disease or disease symptoms.

The SIRPa protein used as a control in the present invention is a part of an extracellular domain of SIRPa (NCBI Reference Sequence: NP-001035111.1), and the amino acid Sequence thereof is represented by SEQ ID NO. 4.

The invention has the following advantages and beneficial effects:

compared with TTI-621, the fusion protein disclosed by the invention has a more remarkable effect of enhancing the phagocytic capacity of macrophages and has better blood safety.

Drawings

FIG. 1 shows an electrophoretogram of a fusion protein of the present invention detected by SDS-PAGE;

FIG. 2 shows a graph of the binding activity of the fusion protein of the present invention detected by ELISA;

FIG. 3 shows the results of detecting the binding kinetics and affinity of the fusion protein of the present invention and CD47-HIS antigen by Fortebio biofilm interference technique;

FIG. 4 is a graph showing the results of ELISA detection of the inhibition of wild type SIRPa interaction with CD47 by the fusion protein of the present invention;

FIG. 5 is a graph showing the effect of the fusion protein of the present invention on macrophage phagocytosis activity measured using a macrophage phagocytosis-promoting activity assay;

FIG. 6 is a graph showing the effect of the fusion protein of the present invention on blood coagulation reaction measured by hemagglutination assay.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

EXAMPLE 1 expression, purification and characterization of fusion proteins

1. Construction of SIRPa variant-Fc recombinant plasmid

Preparation of SIRP α variant DNA fragment, synthesizing gene sequence by the firm of Genisthio engineering bioengineering (Shanghai) and using PCR method to introduce enzyme cutting sites (Nhe I-HF and Xho I) and protective base (CCGT), and recovering target PCR fragment by Qiagen gel recovery kit, namely the DNA fragment of SIRP α variant.

Enzyme cutting conditions are as follows: the digestion system is as follows at 37 ℃ for 16 h:

connection conditions are as follows: at 16 ℃ for 4h, the ligation system was as follows:

and (3) transformation: the ligation product is transformed into TOP10 competent cells by heat shock for 90s at 42 ℃, plated and inverted in an incubator at 37 ℃ for 16 h; and picking the correctly identified monoclonal colony for culture and amplification.

Extraction of SIRPa variant-Fc recombinant plasmid: and selecting the identified correct monoclonal colony for culture and amplification, and obtaining the target recombinant vector by using the plasmid extraction kit.

2. Expression of SIRP α variant-Fc fusion protein (code: 106)

Cell recovery and culture: taking out the frozen 293T cells, quickly placing the cells into a 37 ℃ constant-temperature water bath for thawing, re-suspending the thawed cells into 10mL DMEM complete culture medium (10% of imported fetal calf serum is mixed into 90% of DMEM culture medium), centrifuging at 1200rpm for 4min, uniformly bouncing the cells at the bottom of the tube, re-suspending the cells with the DMEM complete culture medium, and culturing the cells in a 37 ℃ constant-temperature cell culture box.

Transfection and expression, 293T cells subcultured to the third generation were inoculated into 15cm cell culture dishes at an appropriate density, and when the cell density reached 60%, the fresh medium was replaced, and the SIRP α variant-Fc plasmid (No. 106) was transfected into 293T cells, when the cell density reached 100%, the serum-free protein expression medium (90% 293TGE (Cat. CM-1156-11) was replaced and 10% CD Feed X (Cat. CF1116-12) was mixed, and the above products were purchased from Beijing Behcet bioscience Co., Ltd.), cultured at 37 ℃ for 72h, and the supernatant was the expression product of the SIRP α variant-Fc recombinant plasmid.

3. SIRP α variant-Fc fusion protein (code: 106) purification

Collecting: centrifuging the expression product at 3500rpm for 30min, and filtering with 0.45 μm filter membrane to remove cells and cell debris;

and (3) purification and preservation: purifying by protein A affinity chromatography with AKTA prime plus purifier, adjusting pH to 6.5-7.0 with Tris, ultrafiltering the purified product with 50kD ultrafiltering tube, replacing with PBS for 2 times, measuring concentration with BCA protein quantification kit, packaging and storing at-80 deg.C.

SDS-PAGE 4. mu.g SIRP α variant-Fc fusion protein (code: 106), 4-20% gradient protein pre-gel, 200V, 0.5h, staining using an eStain L1 protein staining instrument (Nanjing Kingshi Biotech, Ltd.), scanning and storing the image of the protein gel, the result is shown in FIG. 1, wherein lane 1 represents protein Marker and lane 2 represents SIRP α variant-Fc fusion protein.

Example 2 binding and affinity assays for fusion proteins

1. Detection of binding Activity of SIRPa variant-Fc fusion protein (accession number: 106) to CD47 molecule by ELISA method

1.1 Experimental procedures

Coating: CD47 (1. mu.g/mL), 100. mu.L/well, 4 ℃, overnight, PBST washing 3 times;

and (3) sealing: 1.5% casein blocking solution, at 37 ℃, 1h, PBST washing 3 times;

sample adding: 15 mu g/mL, 3-fold dilution, 12 gradients, 100 mu L/well, 37 ℃, 1h, PBST washing 3 times;

adding a detection antibody: GAH-IgG-HRP, room temperature, 45min, PBST washing 3 times;

color development: TMB, 100. mu.L/well, 3min, stop: 2N H₂SO₄100 μ L/well;

reading: reading the value by an enzyme linked detector OD450nm, and storing the data;

data processing: GraphPad Prism 8

1.2 results of the experiment

The results are shown in FIG. 2, and the binding activity of SIRPa variant-Fc fusion protein (No. 106) to CD47 is better than that of SIRP α (the sequence is shown in SEQ ID NO: 4) and TTI-621.

2. Measurement of binding kinetics and affinity of SIRPa variant-Fc fusion protein and CD47-HIS antigen by Fortebio biofilm interference technology

2.1 Experimental procedures

Capturing high-affinity SIRPa-Fc by an Anti-Human IgG Fc Capture (AHC) chip, diluting a molecule to be detected to 10 mu g/mL by using a running buffer solution (PBS + 0.1% Tween 20+ 0.02% BSA), and loading for 90 s; the analyte CD47-HIS was also diluted with a running buffer gradient to the corresponding concentrations (50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.56nM, 0.78nM and 0nM), with a binding time of 100s for the test molecule to the analyte and a dissociation time of 600 s; the chip was regenerated by repeating 3 pulses of 10mM glycine HCl at pH 1.7 for 5 seconds. The data were fit to a 1:1 binding model to determine the equilibrium dissociation constant, KD.

2.2 results of the experiment

As a result, as shown in FIG. 3, the equilibrium dissociation constant KD was 1.341E-10.

3. Epitope competition (ELISA)

3.1 Experimental procedures

and (3) sealing: 1.5% casein blocking solution, 1h, PBST washing 3 times;

adding SIRP α -Biotion (0.25. mu.g/mL, wild type SIRP α marked by self-made biotin), 100. mu.L/hole, 1h, and washing with PBST for 3 times;

the concentration of the SIRPa variant-Fc fusion protein is added in turn as follows: 40. 7.7, 1.48, 0.28(μ g/mL), 100 μ L/well, 37 ℃, 1h, PBST washing 3 times;

adding detection antibody streptadinvin-HRP, washing for 3 times by PBST at room temperature for 45 min;

color development: TMB, 100. mu.L/well, 3min, stop: 2N H₂SO₄100 μ L/well;

reading: reading an OD450 value by an enzyme-linked detector, and storing data;

data processing: GraphPad Prism 8

3.2 results of the experiment

The result is shown in figure 4, the inhibition rate of the SIRPa variant-Fc fusion protein on the interaction of wild type SIRPa and CD47 is obviously higher than that of SIRP α and TTI-621, and the SIRP α variant has stronger activity of blocking the interaction of natural SIRPa and CD 47.

4. Macrophage phagocytosis-promoting activity assay

4.1 experimental principle: the CD47 antibody blocks a CD47-SIRPa signal axis, so that macrophages are inhibited from receiving 'do-me-eat' signals, the efficiency of the macrophages in phagocytosing tumor cells is increased, and fusion proteins with excellent phagocytosis promoting activity can be screened by using the flow cytometry result.

4.2 Experimental materials and reagents peripheral blood mononuclear cells, M-CSF, tryptic digest, 1640 medium, SIRPa variant-Fc fusion protein, SIRP α, TTI-621, Raji cell line, Anti-human CD14(APC), CFSE, serum of healthy volunteers.

4.3 Experimental procedure:

acquisition of macrophages: 1640 complete Medium + 10% FBS, 37 ℃, 5% CO containing M-CSF (50ng/mL)₂Inducing the monocyte to differentiate to the macrophage by the incubator, changing the liquid (half-changing) every 3 days, and obtaining the macrophage in 7-10 days;

cell preparation:

digesting the induced and differentiated macrophage by using digestive juice, washing twice by using a serum-free 1640 culture medium, and counting; cell density 7.7 x 10⁵one/mL (total 1 mL);

centrifuging Raji cells at 1200rpm for 4min, adding 2 μ l CFSE, labeling at 37 deg.C for 15min, adding 40% serum, and stopping at room temperature for 5 min;

diluting the SIRPa variant-Fc fusion protein, the SIRP α and the TTI-621 to 30 mu g/mL by serum-free 1640 medium;

cell samples were prepared and labeled: macrophages and Raji Cells (CFSE) were mixed together at 1:10 and dispensed into 1.5mL EP tubes at 100. mu.l each, ensuring a macrophage count of 2.5 x 10 per sample⁴A plurality of; raji (CFSE) cell number 2.5X 10⁵A plurality of;

adding 50 μ l of diluted SIRPa variant-Fc fusion protein, SIRP α and TTI-621 to the cell sample to a final concentration of 10 μ g/mL;

reacting at 37 ℃ for 4h, and adding FACS buffer solution to wash once;

adding Anti-human CD14(APC) antibody at 4 deg.C for 30 min;

FACS buffer washing twice;

fixing with 1% paraformaldehyde;

flow cytometry was used for detection analysis.

4.4 results of the experiment

The results are shown in table 1 and fig. 5.

Group of	Positive rate (Gate%)	Mean fluorescence intensity (Mean)
			Macrophage controls		20
CSFE control	55.5	406
			SIRPα	80.2	1089
TTI-621	67.5	581
			SIRPa variant-Fc fusion protein	87.5	1329

The results of the above experiments indicate that the SIRPa variant-Fc fusion protein (code: 106) has the promotion effect on the phagocytosis of Raji cells by macrophages, and the promotion effect is better than that of SIRP α and TTI-621.

5. Hemagglutination assay

Purpose of the experiment: detection of the Effect of fusion proteins on blood coagulation by hemagglutination assay

Experimental materials: whole blood of healthy volunteers, Ashi solution, PBS, round-bottom 96-well plate and fusion protein

The experimental steps are as follows:

preparation of erythrocyte suspension: collecting 1:1 whole blood of a healthy volunteer, putting the whole blood into the Ashi solution, washing the whole blood with PBS for 3-5 times, and preparing 2% erythrocyte suspension with the PBS for later use;

sample dilution: the SIRPa variant-Fc fusion protein, TTI-621, Hu5F9 were diluted with PBS to a final concentration starting from 100 μ g/mL, with 12 gradients of fold-by-fold dilution;

adding 100 mu L of prepared 2% erythrocyte suspension into a round-bottom 96-well plate, and adding 100 mu L of diluted sample into the erythrocyte suspension;

standing at 37 deg.C for 4 hr, and observing whether hemolysis occurs.

The experimental results are as follows:

as shown in FIG. 6, hemolysis was observed in the control group Hu5F9 (100. mu.g/mL, 50. mu.g/mL, 25. mu.g/mL), hemolysis was observed in the control group TTI-621 (100. mu.g/mL), hemolysis was not observed in the control group PBS, and hemolysis was not observed in all concentration gradients of the SIRPa variant-Fc fusion protein (code: 106).

As can be seen from hemagglutination experiments, the SIRPa variant-Fc fusion protein has no influence on blood agglutination reaction.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Sequence listing

<110> military medical research institute of military science institute of people's liberation force of China

<120> SIRPa variants, fusion proteins, and uses thereof

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>117

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly

1 5 10 15

Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Leu Pro Ile Gly

20 25 30

Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Val Leu Ile Tyr

35 40 45

Asn Gln Arg Asn Gly His Phe Pro Arg Val Thr Thr Ile Ser Glu Ser

50 55 60

Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Lys Ile Gln Asn Ile Thr

65 70 75 80

Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser

85 90 95

Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val

100 105 110

Arg Ala Lys Pro Ser

115

<210>2

<211>233

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Leu Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly PheTyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210>3

<211>350

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>3

Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly

1 5 10 15

Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Leu Pro Ile Gly

20 25 30

Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Val Leu Ile Tyr

35 40 45

Asn Gln Arg Asn Gly His Phe Pro Arg Val Thr Thr Ile Ser Glu Ser

50 55 60

Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Lys Ile Gln Asn Ile Thr

65 70 75 80

Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser

85 90 95

Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val

100 105 110

Arg Ala Lys Pro Ser Leu Glu Pro Lys Ser Cys Asp Lys Thr His Thr

115 120 125

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

130 135 140

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

145 150 155 160

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

165 170 175

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

180 185 190

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

195 200 205

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

210 215 220

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

225 230 235 240

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

245 250 255

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

260 265 270

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

275 280 285

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

290 295 300

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

305 310 315 320

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

325 330 335

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345 350

<210>4

<211>117

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>4

Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly

1 5 10 15

Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro Val Gly

20 25 30

Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu Ile Tyr

35 40 45

Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu

50 55 60

Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn Ile Thr

65 70 75 80

Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser

85 90 95

Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val

100 105 110

Arg Ala Lys Pro Ser

115

Claims

1. A SIRPa variant, wherein the amino acid sequence of the SIRPa variant is any one of the group consisting of:

(1) an amino acid sequence shown as SEQ ID NO. 1;

(2) an amino acid sequence with the same or similar functions obtained by substituting and/or deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 1;

2. A fusion protein comprising the SIRPa variant of claim 1 and a human antibody Fc fragment.

Preferably, the amino acid sequence of the Fc fragment of the human antibody is any one of the following group:

(1) an amino acid sequence shown as SEQ ID NO. 2;

(3) and (3) an amino acid sequence which has at least 98 percent of homology with the amino acid sequence defined in the step (1) or (2) and has the same function.

3. The fusion protein of claim 2, wherein the amino acid sequence of the fusion protein is any one of the group consisting of:

(1) an amino acid sequence shown as SEQ ID NO. 3;

(3) and (3) an amino acid sequence which has at least 98 percent of homology or homology with the amino acid sequence defined in (1) or (2) and has the same function.

4. A DNA molecule encoding the SIRPa variant of claim 1, or the fusion protein of claim 2 or 3.

5. A recombinant vector comprising the DNA molecule of claim 4.

6. A host cell comprising the DNA molecule of claim 4 or introduced with the recombinant vector of claim 5.

7. A pharmaceutical composition, comprising the SIRPa variant of claim 1, the fusion protein of claim 2 or 3, the DNA molecule of claim 4, the recombinant vector of claim 5, or the host cell of claim 6; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient or stabilizer.

8. A kit comprising the SIRPa variant of claim 1, the fusion protein of claim 2 or 3, the DNA molecule of claim 4, the recombinant vector of claim 5, or the host cell of claim 6; preferably, the kit further comprises instructions for use of the SIRPa variant of claim 1, the fusion protein of claim 2 or 3, the DNA molecule of claim 4, the recombinant vector of claim 5, or the host cell of claim 6.

9. The use of the SIRPa variant of claim 1, the fusion protein of claim 2 or 3, the DNA molecule of claim 4, the recombinant vector of claim 5, or the host cell of claim 6, the pharmaceutical composition of claim 7, wherein the use comprises the use of any one of:

(1) use in the preparation of a kit according to claim 8;

Preferably, the diseases associated with SIRPa or CD47 activity include cancer, immune diseases;

more preferably, the cancer is selected from solid tumor cancer, hematological cancer, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-hodgkin's lymphoma, multiple myeloma, bladder cancer, pancreatic cancer, cervical cancer, endometrial cancer, lung cancer, bronchial cancer, liver cancer, ovarian cancer, colon and rectal cancer, gastric cancer, gallbladder cancer, gastrointestinal stromal tumor cancer, thyroid cancer, head and neck cancer, oropharyngeal cancer, esophageal cancer, melanoma, non-melanoma skin cancer, merkel cell cancer, virus-induced cancer, neuroblastoma, breast cancer, prostate cancer, renal cell cancer, renal pelvis cancer, leukemia, lymphoma, sarcoma, glioma, brain tumor, carcinoma.

10. The use of claim 9, wherein the immune disease comprises an autoimmune disease, an inflammatory disease; preferably, the autoimmune disease or the inflammatory disease is selected from multiple sclerosis, rheumatoid arthritis, spondyloarthropathies, systemic lupus erythematosus, antibody-mediated inflammatory or autoimmune diseases, graft-versus-host disease, sepsis, diabetes, psoriasis, atherosclerosis, sjogren's syndrome, progressive systemic sclerosis, scleroderma, acute coronary syndrome, ischemia reperfusion, crohn's disease, endometriosis, glomerulonephritis, myasthenia gravis, idiopathic pulmonary fibrosis, asthma, Acute Respiratory Distress Syndrome (ARDS), vasculitis, inflammatory autoimmune myositis.