CN111808183B - High-affinity SIRP alpha mutant targeting CD47 and fusion protein thereof - Google Patents

Abstract

Experiments prove that compared with wild type SIRP alpha and TTI-621, the fusion protein has stronger binding capacity with CD47, thereby effectively inhibiting the interaction between the wild type SIRP alpha and CD47, further enhancing the phagocytic capacity of macrophages, and not causing coagulation reaction. The invention also discloses application of the SIRP alpha mutant and the fusion protein in a kit for detecting CD47 and a tumor treatment drug. The fusion protein has excellent tumor inhibiting effect and has wide development prospect in the application of tumor treating medicines.

Description

High-affinity SIRP alpha mutant targeting CD47 and fusion protein thereof

Technical Field

The invention belongs to the fields of tumor immunotherapy and molecular immunology, and relates to a high-affinity SIRP alpha mutant and a fusion protein formed by the same and human IgG 1-Fc. The fusion protein can specifically recognize CD47 molecules, block the interaction between CD47 and SIRPa, does not cause a coagulation reaction, and can inhibit the growth and/or proliferation of tumor cells.

Background

CD47 is a widely expressed highly glycosylated transmembrane Protein, also known as Integrin-Associated Protein (IAP), expressed on the cell surface, belonging to a member of the Immunoglobulin Superfamily (IgSF), first identified as a tumor antigen of human ovarian cancer in the 80 th 19 th century, and then CD47 was found to be expressed in a variety of human tumors, including Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), Acute Lymphocytic Leukemia (ALL), non-hodgkin's lymphoma (NHL), Multiple Myeloma (MM), bladder cancer and other solid tumors, and to date, CD47 has been shown to be highly expressed on almost ALL tumor cells and to play an important role in tumor growth, metastasis and recurrence.

The CD47 molecule interacts with its ligand molecule Signal regulatory protein alpha (SIRP alpha), thrombospondin (TSP 1) and integrins (integrins) to mediate a series of reactions such as apoptosis, proliferation and immunity. SIRP α is a cell surface glycoprotein belonging to the immunoglobulin superfamily, the extracellular domain of which comprises 3 immunoglobulin-like domains, the most distal of which is 1 IgV domain, and the membrane proximal of which is 2 IgC domains; the intracellular contains 2 immunoreceptor tyrosine-based inhibitory motifs (ITIMs). SIRP alpha is specifically expressed on the surfaces of nerve cells and hematopoietic cells of myeloid lineage, such as macrophages and Dendritic Cells (DCs), and exists in a monomeric form. The SIRP alpha on the surface of macrophage is combined with CD47, so that an ITIMs (tyrosine inhibitory motif of a cytoplasmic medial immunoreceptor of the SIRP alpha is phosphorylated, and can be combined with SH2 domain-containing tyrosine phosphatases SHP-1 and SHP-2, both of which can inhibit the accumulation of myosin IIA in synapses of phagocytes, so that an inhibition signal of 'eating alone' is transmitted, the phagocytosis of the macrophage is inhibited, the secretion of Tumor Necrosis Factor (TNF) is reduced, and the like. Studies have shown that most tumor cells also evade innate immune system surveillance and killing by highly expressing CD47 molecules. Therefore, blocking the interaction of CD47/SIRP alpha between tumor cells and macrophages can enhance the tumor phagocytosis ability of macrophages, and further inhibit and kill tumors.

More and more data show that the antibody or Fc fusion protein can block a CD47-SIRP alpha signal channel and activate phagocytosis of tumor cells by macrophages. Therefore, antibody drugs targeting this signaling pathway are expected to be potential drugs for tumor therapy. Currently, the clinical research antibody drugs targeting CD47 are mainly classified into 3 types according to structure, namely monoclonal antibodies (e.g. clinical research drug Hu5F9), sirpa-Fc fusion proteins (e.g. clinical research drug TTI-621, ALX148) and bispecific antibodies. However, on one hand, the natural SIRP alpha-Fc fusion protein has low affinity with CD47 protein and cannot effectively exert antitumor activity; on the other hand, the existing antibody drugs targeting CD47 have hematology-related side effects such as hemolytic anemia and thrombocytopenia, and development of a novel therapy with strong affinity and high safety is urgently needed.

Based on the technical problems of the prior art, the invention aims to provide a protein with high recognition and specific binding capacity to CD47 and a fusion protein capable of fully exerting the immunoregulatory activity.

Disclosure of Invention

Based on the above objects, the present invention provides a human sirpa mutant targeting CD47, based on the crystal structure of a complex acting between CD47 and sirpa, wherein the mutant site is derived from residues acting on CD47 (key acting site and participating acting site), and the amino acid residues are determined as follows: ile²⁹、Glu⁴⁵、Gln⁵⁰、Lys⁵¹、Glu⁵²、Val⁶¹、Asp⁶³、Leu⁶⁴、Asn⁶⁸、Arg⁷⁵、Gly⁷⁷And Val¹⁰⁰. The amino acid sequence of the SIRPa mutant is identical with the truncated sequence SEQ ID NO.1 of the extracellular segment of the natural SIRPa variant 1 (the sequence is derived from the mutation at one or more sites of 29 th, 45 th, 50 th, 51 th, 52 th, 61 th, 63 th, 64 th, 68 th, 75 th, 77 th or 100 th from Swiss Prot database P78324(SHPS1_ human).

The method comprises the steps of considering the physicochemical properties of amino acids of an extracellular segment of a natural SIRP alpha variant 1 and the mutual recognition mode of CD47, following the principle of similar volume and property and considering intermolecular force, carrying out mutant design under the action of hydrophilicity and hydrophobicity and polarity, and selecting a mutant with the minimum structural change and the maximum energy reduction of the interaction energy compared with the energy of a parent compound by analyzing the interaction energy and the structural characteristics of the mutant to obtain 3 mutant sequences, wherein the amino acid sequence of the human SIRP alpha mutant targeting CD47 is shown as any one of SEQ ID NO.2, 3 and 4.

Secondly, the invention provides a fusion protein containing the SIRP alpha mutant.

In a preferred embodiment, the fusion protein further comprises an immunoglobulin or a fragment thereof.

In a more preferred embodiment, the immunoglobulin fragment is an Fc fragment.

The immunoglobulin may be an IgG, preferably of the IgG1 or IgG4 subtype.

The amino acid sequence of the Fc fragment of IgG1 is shown in SEQ ID No. 5, and more preferably, the amino acid sequence of the Fc fragment of the immunoglobulin is shown in any one of SEQ ID No.6, 7 and 8. Among them, N of Fc fragment of IgG1²⁹⁷The amino acid sequence of the A mutant is shown as SEQ ID.6, and the L of the Fc fragment of IgG1²³⁴A；L²³⁵A；G²³⁷A；N²⁹⁷The amino acid sequence of the A mutant is shown as SEQ ID.7, and the amino acid sequence of the Fc fragment of IgG4 is shown as SEQ ID.8.

Particularly preferably, the human sirpa mutant is located at the N-terminus of the fusion protein. Preferably, the two proteins are directly linked.

In a preferred embodiment, the sequence combination of the human sirpa mutant of the fusion protein and the immunoglobulin Fc fragment is selected from any one of the following:

SEQ ID NO.2 and SEQ ID NO. 5 (defined as JY002-M1G1) or

SEQ ID NO.3 and SEQ ID NO. 5 (defined as JY002-M2G1) or

SEQ ID NO.4 and SEQ ID NO. 5 (defined as JY002-M3G1) or

SEQ ID NO.2 and SEQ ID NO.6 (defined as JY002-M1G1(N297A)) or

SEQ ID NO.3 and SEQ ID NO.6 (defined as JY002-M2G1(N297A)) or

SEQ ID NO.4 and SEQ ID NO.6 (defined as JY002-M3G1(N297A)) or

SEQ ID NO.2 and SEQ ID NO.7 (defined as JY002-M1G1(AAAA)) or

SEQ ID NO.3 and SEQ ID NO.7 (defined as JY002-M2G1(AAAA)) or

SEQ ID NO.4 and SEQ ID NO.7 (defined as JY002-M3G1(AAAA)) or

SEQ ID NO.2 and SEQ ID NO.8 (defined as JY002-M1G4) or

SEQ ID NO.3 and SEQ ID NO.8 (defined as JY002-M2G4) or

SEQ ID NO.4 and SEQ ID NO.8 (defined as JY002-M3G 4).

Thirdly, the present invention provides a polynucleotide encoding the above fusion protein, and in a preferred embodiment, the polynucleotide has a sequence shown in SEQ ID NO.10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21.

Fourthly, the invention also provides an expression vector containing the polynucleotide, wherein the expression vector is a secretory expression vector, the 5' end of the polynucleotide for coding the human SIRP alpha mutant is provided with the polynucleotide for coding the signal peptide, and the amino acid sequence of the signal peptide coded by the polynucleotide is shown in SEQ ID NO.9 (Immunoglobulin H chain V-region (V) precusor, partial [ Mus mulus ] GenBank: AAA 51634.1).

In a preferred embodiment, the polynucleotide encoding a signal peptide has the sequence shown in SEQ ID NO. 22. In a specific embodiment of the invention, the expression vector is PCDNA 3.4.

Fourth, the present invention also provides a host cell containing the above expression vector. In a specific embodiment of the invention, the host cell is an antigen transient transfection expression 293T cell, and the host cell can also be selected from mammalian host cells, such as CHO-S cells, which are mass-fermentatively produced with antigen.

Fifth, the present invention also provides a method for producing the above fusion protein, which comprises culturing the above host cell under conditions allowing the expression of the fusion protein.

Sixth, the invention provides an application of the human SIRP alpha mutant in preparing a CD47 detection kit.

Seventh, the invention provides an application of the human SIRP alpha mutant in preparing a medicine for treating tumor diseases.

Finally, the invention provides the application of the fusion protein in preparing medicaments for treating tumor diseases.

Such neoplastic diseases may include, but are not limited to: acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, non-hodgkin's lymphoma, multiple myeloma, melanoma, lung cancer, colorectal cancer, renal tumor, bladder cancer, gastrointestinal cancer, prostate cancer, liver cancer, ovarian cancer, pancreatic cancer, endometrial cancer, gastric cancer, prostate cancer, renal cancer, cervical cancer, thyroid cancer, uterine cancer, neuroendocrine cancer, head and neck cancer, nasopharyngeal cancer, testicular cancer, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protrusions, merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome.

Compared with the similar product TTI-621 (refer to CN105073780A), the SIRPa mutant and the fusion protein thereof can be more effectively combined with CD47, inhibit a CD 47-SIRPa signal channel and have stronger in-vivo tumor proliferation inhibition activity, and the fusion protein of the SIRPa mutant and the IgG1-Fc fusion protein can not cause hematology related side effects such as hemolysis and the like on the premise of retaining the combination activity with CD47 and inhibiting the growth of tumor cells and in-vivo oncolytic activity, and the fusion protein of the SIRPa mutant and the IgG1-Fc fusion protein variant can also greatly reduce in-vitro ADCC activity and has extremely high biological safety.

Drawings

FIG. 1 is a schematic diagram of the structure of SIRP alpha and its mutant fusion protein, in which A is fused with IgG1 Fc; b is fusion to IgG1Fc mutant (mutated residue N297A); c is fused with another mutant of IgG1Fc (mutant residues L234A; L235A; G237A; N297A); d is fusion to IgG4 Fc;

FIG. 2 is a graph of an assay for in vitro binding activity of a SIRP alpha truncated domain mutant IgG1Fc fusion protein;

FIG. 3 is a graph of a SIRP α truncated domain variant IgG1Fc fusion protein cross-binding assay with human, mouse, cynomolgus monkey CD47 antigen;

FIG. 4 is a graph of an assay for the specific recognition of target antigen binding activity by a SIRP α truncated domain variant IgG1Fc fusion protein;

FIG. 5 is a graph of an assay for specific blockade of the CD 47/SIRPa interaction by a SIRPa truncation domain variant IgG1Fc fusion protein;

FIG. 6 is a graph of the determination of CD47 molecules on the surface of SIRP alpha truncated domain variant IgG1Fc fusion protein specifically recognizing Raji cells, Jurkat cells and A549 cells of tumor cells;

FIG. 7 is an observation of coagulation response of a SIRP alpha truncated domain variant IgG1Fc fusion protein;

FIG. 8 is a graph of tumor growth over time in mice receiving a SIRP α truncated domain variant IgG1Fc fusion protein;

FIG. 9 is a graph comparing the binding activity of different Fc subtype fusion proteins of SIRP alpha truncated domain variants;

FIG. 10 is a graph comparing the blocking activity of different Fc subtype fusion proteins of SIRP alpha truncated domain variants;

FIG. 11 is a graph comparing the ADCC activity in vitro of different Fc subtype fusion proteins of a variant SIRP alpha truncated domain;

FIG. 12 is a graph of the in vivo tumor suppressor activity of JY002-M2G1(N297A) fusion protein of the present application.

Detailed Description

"SIRPa" refers to the amino acid sequence of a wild-type signal-regulating protein alpha, or of a recombinant or non-recombinant polypeptide having the amino acid sequence of a wild-type signal-regulating protein alpha, or of a naturally occurring mutant of a signal-regulating protein alpha. A "mutant" of SIRPa is defined as a SIRPa amino acid sequence having one or more amino acid modifications compared to a wild type SIRPa. Mutants may have "conservative" modifications, where the substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, mutants may have "non-conservative" modifications, e.g., replacement of glycine with tryptophan. Similar minor changes may also include amino acid deletions or insertions, or both.

In humans, sirpa is found in 10 mutant forms. The amino acid sequence of one mutant form (mutant 1 or V1 type) is listed as NCBI RefSeqNP _001035111.1 (residues 31-504 constitute the mature form). The other form (mutant 2 or V2 type) differs by 13 amino acids from the mutant 1 or V1 type, the amino acid sequence of which is listed as CAA71403.1 in GenBank. These two forms of sirpa constitute approximately 80% of the various types of sirpa present in humans.

The invention connects the first immunoglobulin V structural domain (the specific amino acid sequence is shown in SEQ ID NO.1) at the outer end of the SIRP alpha mutant 1 membrane with the Fc fragment of human immunoglobulin to form the fusion protein, and compared with the fusion protein formed by connecting the outer end of the SIRP alpha whole membrane with the Fc fragment of human immunoglobulin, the fusion protein has higher target binding activity. Further, in order to improve the binding ability of sirpa mutant 1 to human CD47 and thus more effectively block the interaction between native CD47 and sirpa, the present study designed 3 high affinity sirpa mutants by using site-directed mutagenesis and considering the interaction energy and structural variation characteristics through the crystal structure information of the interaction between CD47 and sirpa. The sequence protected by the application has stronger activity of blocking CD47/SIRP alpha and has certain advantages in the aspect of treating tumors.

In a first aspect, the invention provides a high affinity sirpa mutant comprising a modification at one or more positions 29, 45, 50, 51, 52, 61, 63, 64, 68, 75, 77 or 100 in the amino acid sequence shown as SEQ ID No.1 (the sequence is derived from NCBI RefSeqNP _ 001035111.1) of the extracellular stretch truncated sequence of native sirpa mutant 1. The modification may be deletion, substitution or insertion.

The SIRP alpha mutant and the fusion protein thereof have higher affinity to a tumor specific antigen CD47 on tumor cells; efficient antibody-dependent cellular phagocytosis (ADCP) is caused by a combination of blocking CD47 "eat me" signaling and Fc-dependent activation of immune effector cells.

As a preferred embodiment, the sirpa mutants of the present invention have one or more of the following substitutions: ile²⁹、Glu⁴⁵、Gln⁵⁰、Lys⁵¹、Glu⁵²、Val⁶¹、Asp⁶³、Leu⁶⁴、Asn⁶⁸、Arg⁷⁵、Gly⁷⁷And Val¹⁰⁰。

As a preferred embodiment, the mutant is selected from the group consisting of substitutions of: i is²⁹L；E⁴⁵L；K⁵¹R；E⁵²D；V⁶¹I；D⁶³S；L⁶⁴T；R⁷⁵S；G⁷⁷Q, the sequence is shown as SEQ ID NO. 2.

As another embodiment, the mutant is selected from the group consisting of: I.C. A²⁹L；E⁴⁵L；K⁵¹R；E⁵²Q；V⁶¹I；D⁶³Q；L⁶⁴T；N⁶⁸S；R⁷⁵Q, and the sequence is shown in SEQ ID NO. 3.

As another embodiment, the mutant is selected from the group consisting of: i is²⁹L；E⁴⁵L；V⁶¹I；D⁶³Q；L⁶⁴T；N⁶⁸D；R⁷⁵S；G⁷⁷S；V¹⁰⁰I, the sequence is shown in SEQ ID NO. 4.

In the application, on the basis of the extracellular immunoglobulin-like V truncated domain (the amino acid sequence is shown as SEQ ID NO: 1) of the human SIRP alpha mutant 1, the SIRP alpha mutants respectively comprising the amino acid substitutions of the above SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 can be named as M1, M2 and M3 in sequence.

In the present application, a natural fusion protein comprising a truncated domain of human SIRP alpha mutant 1 (amino acid sequence shown as SEQ ID NO: 1) and human IgG1-Fc (amino acid sequence shown as SEQ ID NO: 5) may be named JY002-WG 1.

In the application, on the basis of JY002-WG1, the natural fusion proteins respectively containing the amino acid substitutions of the above SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 and human IgG1-Fc (the amino acid sequence shown as SEQ ID NO: 5) can be named as JY002-M1G1, JY002-M2G1 and JY002-M3G1 in sequence, and the corresponding nucleotide sequences are JY ID NO.10(JY002-M1G1), JY ID NO.11(JY002-M2G1) and JY ID NO.12(JY002-M3G 1).

In the application, on the basis of the JY002-WG1, the amino acid substitutions of the SEQ ID NO.2, the SEQ ID NO.3 and the SEQ ID NO.4 and the human IgG1-Fc mutant (the mutation residue is N)²⁹⁷A, an amino acid sequence shown as SEQ ID NO: 6) can be sequentially named as JY002-M1G1(N297A), JY002-M2G1(N297A) and JY002-M3G1(N297A), and corresponding nucleotide sequences are shown as SEQ ID NO.13(JY002-M1G1(N297A)), SEQ ID NO.14(JY002-M2G1(N297A)) and SEQ ID NO.15(JY002-M3G1 (N297A)).

In the application, the JY002-WG1 respectively comprises the amino acid substitutions of the SEQ ID NO.2, the SEQ ID NO.3 and the SEQ ID NO.4 and a human IgG1Fc mutant (the mutation residue is L)²³⁴A；L²³⁵A；G²³⁷A；N²⁹⁷A mutant, such as an amino acid sequence shown in SEQ ID NO. 7) can be named as JY002-M1G1(AAAA), JY002-M2G1(AAAA), JY002-M3G1(AAAA) in sequence, and the corresponding nucleotide sequences are shown as JY ID NO.16(JY002-M1G1(AAAA)), JY002-M2G1(AAAA)), and SEQ ID NO.18(JY002-M3G1 (AAAA)).

In the application, on the basis of the JY002-WG1, the natural fusion proteins respectively containing the amino acid substitutions of the SEQ ID NO.2, the SEQ ID NO.3 and the SEQ ID NO.4 and the human IgG4 Fc (the amino acid sequence shown as SEQ ID NO: 8) can be named as JY002-M1G4, JY002-M2G4 and JY002-M3G4 in sequence, and the corresponding nucleotide sequences are shown as JY ID NO.19(JY002-M1G4), SEQ ID NO.20(JY002-M2G4) and SEQ ID NO.21(JY002-M3G 4).

Immunoglobulins

An "immunoglobulin" or "immunoglobulin molecule" refers to an antibody or antigen-binding fragment of an antibody as defined herein, but also includes portions of an antibody, such as heavy or light chain variable or constant regions. Immunoglobulin moieties such as C_H1，C _H2，C _H3 hinge (hinge), V_H，C_LAnd V_LDomains and Fc fragments. In addition, "immunoglobulin" includes Fc fragments or regions that have been engineered to include an antigen binding site ("Fcab"). In some embodiments, an "immunoglobulin" may be IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgD, or IgA, with preferred immunoglobulins being IgG1, IgG 4.

Expression vector

The expression vector of the present invention is not particularly limited, but may be a vector capable of replicating and/or expressing a polynucleotide in eukaryotic or prokaryotic cells including mammalian cells (e.g., human, monkey, rabbit, rat, hamster, or mouse cells), plant cells, yeast cells, insect cells, and bacterial cells (e.g., escherichia coli). Preferably, it may be a vector comprising at least one selectable marker operably linked to a suitable promoter such that transcription and expression of the protein may be achieved in the host cell. For example, the vector may comprise a polynucleotide introduced into a phage, plasmid, cosmid, minichromosome, viral or retroviral vector.

Host cell

Host cells for introducing the vector in the present invention include prokaryotic cells and eukaryotic cells, including, but not limited to, bacterial cells such as E.coli, Streptomyces and Salmonella typhimurium; a yeast cell; fungal cells such as pichia pastoris; insect cells such as Drosophila or Spodoptera Sf9 cells; animal cells such as CHO-K1 (Chinese hamster ovary cells), SP2/0 (mouse myeloma cells), human lymphoid mother cells, COS, NSO, 293T, Bowes melanoma cells, HT-1080, BHK (baby hamster kidney cells), HEK (human embryonic kidney cells), PERC.6 (human retinal cells), etc.; and plant cells. Any cell known to those skilled in the art to be useful as a mammalian host cell may be used in the art.

The term "introduction" refers to the delivery of a vector comprising a polynucleotide encoding a monoclonal antibody into a host cell. This introduction can be performed by various methods known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polybrene mediated transfection, electroporation, microinjection, liposome-mediated transfection, liposome fusion, lipofection, and protoplast fusion. In addition, transfection refers to the use of viral particles through infection to deliver the desired material into cells. In addition, the vector may be introduced into a host cell by gene bombardment. In the present invention, introduction and transfection may be used interchangeably.

The recombinant cells of the invention can then be used for expression as well as culture purposes for antibody expression for large scale drug production. Can also be used as active ingredient of pharmaceutical composition. Any suitable culture technique may be used, including but not limited to static culture, spinner flask culture, ascites fluid, hollow fiber-type bioreactor cartridges, modular mini-fermenters, stirred tanks, microcarrier culture, ceramic core perfusion, and the like.

Application of kit

As an alternative embodiment, the kit of the invention includes a SIRPa mutant or fusion protein prepared by the invention. As another alternative embodiment, the kits of the present invention comprise a diagnostic composition comprising at least one detectable label, such as a detectable moiety/agent. The tag may be non-covalently conjugated to a sirpa mutant of the invention or a fusion protein thereof. The tag may also be directly conjugated to the sirpa mutant or fusion protein thereof by a covalent bond. Alternatively, the tag may be conjugated to the sirpa mutant or fusion protein thereof described above using one or more linking compounds. The technique of conjugating tags is well known to those skilled in the art. The detectable moiety/agent as a label is preferably one selected from the group consisting of, but not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting materials and non-radioactive paramagnetic metal ions.

Pharmaceutical use

The SIRP alpha mutant and the fusion protein thereof can be applied to the preparation of medicaments for treating tumor diseases, the mutant and the fusion protein antigen thereof are prepared into a pharmaceutical composition, and the composition can also comprise a pharmaceutically acceptable carrier and/or an excipient.

The pharmaceutically acceptable carrier should not itself induce the production of antibodies harmful to the individual receiving the composition and should not be toxic. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive viral particles. Or a pharmaceutically acceptable salt, for example, an inorganic acid salt such as hydrochloride, hydrobromide, phosphate and sulfate, or an organic acid salt such as acetate, propionate, malonate and benzoate.

The pharmaceutically acceptable carrier may further comprise a liquid such as water, physiological saline, glycerol and ethanol. In addition, auxiliary substances (e.g., wetting or emulsifying agents or pH buffer substances) may be present in the composition. These carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by a patient.

The pharmaceutical compositions of the present invention can be prepared in different types. For example, the compositions may be prepared as injectable solutions or suspensions, or may be prepared in solid form (e.g., lyophilized compositions, sterile water for reconstitution with a preservative) prior to injection as solutions or suspensions in liquid carriers. The compositions may be prepared for topical administration, for example as an ointment, cream or powder. The compositions may be prepared for oral administration, for example as tablets or capsules, as a spray, or as a syrup (optionally flavored). The compositions may be prepared for pulmonary administration, for example as an inhalant, using a fine powder or a spray. The composition can be prepared as a suppository or pessary. The compositions may be prepared for nasal, otic or ocular administration, for example as drops. The composition may be in the form of a kit designed to reconstitute the mixed composition prior to administration to a patient. For example, lyophilized antibodies in kit form can be provided, as well as sterile water or sterile buffer.

The pharmaceutical compositions of the present invention may be administered by any of a variety of routes, including, but not limited to: oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intrathecal, intracerebral, transdermal, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal, or rectal routes. Needleless syringes (Hypospray) may also be used to administer the pharmaceutical compositions of the invention. Typically, the therapeutic composition may be prepared as an injectable liquid solution or suspension. Or in a pre-injection solid form suitable for solution or suspension in a liquid carrier.

Direct delivery of the composition is typically accomplished by injection, subcutaneous injection, intraperitoneal injection, intravenous injection, or intramuscular injection. The therapeutic dose may be a single dose schedule or a multiple dose schedule. Known fusion protein-based drugs provide instructions regarding the frequency of administration, e.g., whether the drug needs to be administered daily, weekly, monthly, etc. The frequency and dosage will also depend on the severity of the symptoms.

The pharmaceutical compositions of the present invention may be administered in a pharmaceutically effective amount. By "pharmaceutically effective amount" is meant an amount sufficient to treat the disease at a reasonable benefit/risk ratio applicable to any medical treatment. The effective dosage level of the composition may be determined according to the type of the subject, the severity of the disease, the age and sex of the subject, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration, the excretion rate, the treatment time, the drug to be used in combination with the composition, and other known factors in the medical field. The pharmaceutical compositions of the present invention may be used alone or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. The compositions may be administered in one or more dosage forms. In view of all the above factors, it is important to administer the composition at the minimum amount capable of exhibiting the maximum effect without causing side effects, which can be readily determined by one skilled in the art.

"treatment" in the context of the present invention refers to the treatment of a disease in a mammal, such as a human. This includes: (a) inhibiting the disease, i.e. arresting its development; (b) remission of the disease, i.e. causing regression of the disease state.

The pharmaceutical compositions of the invention may be used to treat various forms of cancer, including but not limited to acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, non-hodgkin's lymphoma, multiple myeloma, melanoma, lung cancer, colorectal cancer, renal tumor, bladder cancer, gastrointestinal cancer, prostate cancer, liver cancer, ovarian cancer, pancreatic cancer, endometrial cancer, stomach cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, uterine cancer, neuroendocrine cancer, head and neck cancer, nasopharyngeal cancer, testicular cancer, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protrusions, merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome.

Production of fusion proteins

In general, the fusion protein of the invention uses an expression vector containing nucleic acid modified to express the fusion protein, the vector comprises the fusion protein nucleic acid sequence in the embodiment case, and expression regulation elements of a prokaryotic and/or eukaryotic system, and the fusion protein is prepared by introducing the fusion protein into the prokaryotic and/or eukaryotic system in the way and carrying out the expression of the SIRPa mutant and the fusion protein thereof under specific production conditions.

The present invention will be described in further detail with reference to the drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention as defined by the claims.

Example 1 high affinity SIRP alpha mutant design

Experimental materials: conventional molecular reagents such as Swiss-Model, UCSF Chimera, site-directed mutagenesis kit, PCR, etc.

The experimental steps are as follows: by means of the crystal structure of the compound for the action of human CD47 and SIRPa, key amino acids mutually recognized by CD47 and SIRPa are predicted by using online analysis software Swiss-Model and UCSF Chimera, and finally the key amino acid residue position I of the SIRPa molecule involved in recognizing CD47 is determined⁶¹、V⁶³、E⁷⁷、Q⁸²、K⁸³、E⁸⁴、V⁹³、D⁹⁵、L⁹⁶、K⁹⁸、N¹⁰⁰、R¹⁰⁷、G¹⁰⁹And V¹³². The physical and chemical properties and the structural characteristics of amino acids are considered, the principles of similar volume and similar properties are followed, the crystal structure of the action compound of CD47 and SIRPa is utilized to analyze the secondary structure characteristics of SIRPa, and potential high-affinity SIRPa mutants are analyzed through action energy calculation on the basis of basically not influencing the secondary structure of SIRPa.

The experimental results are as follows: combining with the crystal structure analysis of the interaction complex of the CD47 and the SIRP alpha, performing rigid site-specific mutagenesis on the SIRP alpha structure through function energy calculation analysis, selecting proper force field parameters, and determining 3 potential mutants, wherein the function energy of the potential mutants is respectively reduced by 46.72kCal/mol, 52.18kCal/mol and 48.23kCal/mol compared with the functions of parent SIRP alpha and CD47, which indicates that the function capability of the potential mutants is enhanced compared with the functions of parent SIRP alpha and CD 47. Accordingly, the designed SIRP alpha mutants are named as M1, M2 and M3 respectively, and the amino acid sequences are shown as SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.

Example 2 determination of the binding Activity of high affinity SIRP α mutant-Fc fusion proteins to CD47

The truncated domain of the human SIRPa mutant 1 in example 1 (or named as wild type SIRPa truncated domain) and the SIRPa mutant determined in example 1 are respectively fused with human IgG1-Fc (the amino acid sequence is shown as SEQ ID NO: 5) to be expressed (the specific structure is shown as figure 1), the above sequences are cloned into a PCDNA3.4 (purchased from Thermo Fisher, Cat.: A14697) eukaryotic expression vector which is a secretory expression vector, a polynucleotide encoding a signal peptide is arranged at the 5' end of the polynucleotide encoding the human SIRP alpha mutant, the amino acid sequence of the signal peptide coded by the polynucleotide is shown in SEQ ID NO.9 (immunoglulin H chain V-region (V) precorsor, partial [ Mus musculus ] GenBank: AAA51634.1), the Protein was purified by transient transfection into 293T cells (purchased from ATCC) using affinity column Protein A. Wherein, the wild type SIRP alpha truncated Fc fusion protein is named as JY002-M1G1 (fused with SEQ ID NO.2 and SEQ ID NO: 5), the SIRP alpha mutant Fc fusion protein is named as JY002-M2G1 (fused with SEQ ID NO.3 and SEQ ID NO: 5), and JY002-M3G1 (fused with SEQ ID NO.4 and SEQ ID NO: 5).

The binding activity of JY002-WG1, JY002-M1G1, JY002-M2G1 and JY002-M3G1 fusion proteins and CD47 molecules is measured by an ELISA method.

Experimental materials: CD47-His was purchased from Beijing Yinzhou Biotechnology Ltd, goat anti-human Fc-HRP secondary antibody was purchased from Thermo, and TMB color developing solution was purchased from Ebioscience.

The experimental steps are as follows: the coating diluted CD47-His antigen to 1. mu.g/mL, 100. mu.L per well was added to the enzyme-linked plate, at 4 ℃ overnight. Washed 3 times with PBST (0.1% Tween 20 in PBS), and blocked by adding 200. mu.L of PBSM (4% milk in PBS) per well at 37 ℃ for 1 h. With PBSDiluting the fusion protein of SIRP alpha and mutant thereof to 10 mu g/mL, diluting with 2-fold gradient, adding 100 mu L of fusion protein into an enzyme-linked plate per well, reacting at 37 ℃ in a wet box for 1h, cleaning the enzyme-linked plate for 3 times, adding sheep anti-human Fc-HRP secondary antibody for reacting at room temperature for 45min, washing PBST for 5 times, adding 100 mu L of TMB substrate for color development, reacting for 3min, and then using 100 mu L of 2N H₂SO₄The reaction was stopped and the ELISA read at 450 nm. And (4) drawing an antibody-antigen binding curve by taking the concentration of the antibody as an abscissa and the OD value as an ordinate. Fitting a four parameter equation curve using Graphpad analysis software, the equation being y ═ A-D)/(1+ (X/C). sub.B) + D, where B represents slope and C represents EC₅₀。

The experimental results are as follows: the ELISA binding activity of the SIRPa wild type and its variant fusion protein to CD47 is shown in FIG. 2 and Table 1, and the results show that the binding activity of the SIRPa mutant fusion protein to CD47 is higher than that of the wild type SIRPa fusion protein.

TABLE 1 binding Activity of fusion protein of SIRP alpha and its mutant with IgG1-Fc with CD47

Fusion proteins	EC50(μg/mL)
		JY002-WG1	0.045
JY002-M1G1	0.029
		JY002-M2G1	0.009
JY002-M3G1	0.013

Compared with wild type SIRP alpha truncated Fc fusion protein JY002-WG1, the mutant Fc fusion protein has higher binding activity, particularly JY002-M2G1 is most prominent, and the binding activity is 5 times of JY002-WG 1.

Example 3 detection of the affinity of SIRP α mutant-Fc fusion protein to CD47

The affinity of JY002-WG1, JY002-M1G1, JY002-M2G1 and JY002-M3G1 fusion proteins and CD47 molecules is measured by using surface plasmon resonance SPR.

Experimental materials: the CM5 chip was purchased from GE, and the CD47-His was purchased from Beijing Yi Qiao Shen Biotechnology, Inc.

The experimental steps are as follows: JY002-WG1, JY002-M1G1, JY002-M2G1 and JY002-M3G1 fusion proteins are diluted to 1 mu G/ml by HBS-EP Buffer to be used as ligands. CD47/HIS was diluted with HBS-EP Buffer to 10nM, 5nM, 2.5nM, 1.25nM, 0.625nM, 0.3125nM, 0.15625nM and 0nM, respectively, as an analyte. The ligand was immobilized by indirect capture by covalently binding to the CM5 chip surface via amino coupling using Anti-IgG-FC at 50 μ g/ml, followed by binding of ligand and analyte. Affinity analysis experiments were performed in a Biacore Wizard model in a multicycle fashion with the fusion protein as ligand and the CD47/HIS sample as analyte. The test for each sample included 3 Start up, 1 zero concentration control, 5 gradient concentration samples, and 1 replicate, with the chips being regenerated with 10mM Glycine-HCl, pH 1.5 regenerant after the end of each cycle. Each concentration cycle of analyte set capture time 60s, ligand solution flow rate 10 μ l/min; the binding time of the ligand and the analyte is 180s, and the flow rate of the analyte solution is 30 mu L/min; the dissociation time is 180 s. Raw data were imported into biacore X100 analysis software, zero concentration controls were subtracted, and reference channels were subtracted to eliminate volume effects, graphs were fitted in steady state mode with affinity analysis methods, and data were collated.

The experimental results are as follows: the affinity of the SIRPa wild type and its mutant fusion protein to CD47 is shown in Table 2, and the results show that the binding activity of the SIRPa mutant fusion protein to CD47 is higher than that of the wild type SIRPa fusion protein.

TABLE 2 affinity of fusion proteins of SIRP alpha and its mutants with IgG1-Fc to CD47

Fusion proteins	kon(1/Ms)	kdis(1/s)	KD(M)
				JY002-WG1	1.18E+06	4.97E-04	4.22×10-10
JY002-M1G1	1.40E+06	1.88E-04	1.34×10-10
				JY002-M2G1	4.43E+06	3.05E-04	6.88×10-11
JY002-M3G1	1.49E+06	1.12E-04	7.55×10-11

Compared with wild type SIRP alpha truncated Fc fusion protein JY002-WG1, the mutant Fc fusion protein has higher binding activity, particularly JY002-M2G1 is most prominent, and the binding activity is more than 6 times of JY002-WG 1.

Example 4 Cross-binding of SIRP alpha mutant-Fc fusion proteins to different species of CD47

As an example, the binding of JY002-M2G1 fusion protein to CD47 molecule of different species was determined by ELISA method.

Experimental materials: HIS-tagged human CD47, mouse CD47, and cynomolgus monkey CD47 antigens were purchased from Beijing Yi-Qiao Shenzhou Biotechnology, Inc. Goat anti-human Fc-HRP was purchased from Thermo.

The experimental steps are as follows: the antigen was diluted to 1. mu.g/mL using coating buffer and incubated overnight at 4 ℃. Washed 3 times with PBST, 200. mu.L of PBSM (4% milk in PBS) per well was blocked for 1 hour at 37 ℃. Diluting the fusion protein of SIRP alpha and mutant thereof to 15 mu g/mL by PBS, diluting with 3-fold gradient, adding 100 mu L of fusion protein into an enzyme-linked plate per well, reacting for 1h at 37 ℃ in a wet box, cleaning the enzyme-linked plate for 3 times, adding a goat anti-human Fc-HRP secondary antibody for room temperature reaction for 45min, washing for 5 times by PBST, adding 100 mu L of TMB substrate for color development, reacting for 3min, and then using 100 mu L of 2N H₂SO₄The reaction is stopped, and the enzyme linked immunosorbent assay instrument reads 450 nm. And (4) drawing an antibody-antigen binding curve by taking the concentration of the antibody as an abscissa and the OD value as an ordinate. Fitting a four parameter equation curve using Graphpad analysis software, the equation being y ═ A-D)/(1+ (X/C). sub.B) + D, where B represents slope and C represents EC₅₀。

The experimental results are as follows: JY002-M2G1 fusion protein and different species CD47 molecule combined activity as shown in figure 3 and table 3, from the results, it is known that JY002-M2G1 fusion protein has similar combined activity with human and Cynomolgus monkey (Cynomolgus) CD47, and has weaker combined activity with murine CD 47.

TABLE 3 binding Activity of the fusion protein JY002-M2G1 with different species CD47 antigen

Example 5 specific recognition of target antigens by SIRP α mutant-Fc fusion proteins

As an example, the condition that JY002WG1 and JY002-M2G1 fusion proteins specifically recognize target antigens is determined by an ELISA method.

Experimental materials: human Epcam, CD47 and IL-17A, CD73 with HIS tags are purchased from Beijing Yiqiao Hoizhou Biotechnology Co., Ltd, and human TGF beta 1, Siglec15, ACE2, Trop2, Her2 and IL-6 with HIS tags are purchased from Shanghai Neishi science Co., Ltd. Sheep anti-human Fc-HRP was purchased from thermo.

The experimental steps are as follows: the antigen was diluted to 1. mu.g/mL with coating buffer and incubated overnight at 4 ℃. Washed 3 times with PBST, and blocked by adding 200. mu.L of BSM (4% milk in PBS) per well at 37 ℃ for 1 hour. Diluting the fusion protein of SIRP alpha and mutant thereof to 2 mu g/mL by PBS, adding 100 mu L of fusion protein into an enzyme linked plate per well, reacting for 1h at 37 ℃ in a wet box, cleaning the enzyme linked plate for 3 times, adding goat anti-human Fc-HRP secondary antibody for reacting for 45min at room temperature, washing for 5 times by PBST, adding 100 mu L of TMB substrate for color development, reacting for 3min, and then using 100 mu L of 2N H₂SO₄The reaction was stopped and the ELISA read at 450 nm. And drawing an antibody-antigen binding bar chart by taking the antigen name as an abscissa and the OD value as an ordinate.

The experimental results are as follows: as shown in FIG. 4, JY002-WG1 and JY002-M2G1 fusion proteins only recognize human CD47 antigen, and have no cross recognition with other human proteins.

Example 6 blocking of the CD47/SIRP α interaction by SIRP α mutant-Fc fusion proteins

As an example, JY002-WG1, JY002-M2G1 fusion proteins blocked CD47/SIRP alpha interaction by ELISA method.

Experimental materials: CD47-His, SIRP alpha-Fc-Biotin antigen was purchased from Beijing Quizhou Biotechnology Limited, Peroxidase-Labeled Streptavidin was purchased from American KPL company, TMB color developing solution was purchased from American bioscience company.

Experiment ofThe method comprises the following steps: the CD47/HIS antigen was diluted to 1. mu.g/mL in the coating solution and 100. mu.L was added to the plate and placed in a wet box overnight at 4 ℃. The plate washer washes the enzyme-linked plate 3 times, and 200 μm of lPBSM (4% milk in PBS) was added to each well and blocked at 37 ℃ for 1 hour. The fusion protein was diluted to 40. mu.g/mL with SIRP α -Fc-Biotin diluted with PBS to 3. mu.g/mL, and diluted 5-fold with the above solution as a diluent to obtain 4 dilution concentrations. Adding 100 μ L per well into wet box in enzyme-linked plate, reacting at 37 deg.C for 1h, washing enzyme-linked plate for 3 times, adding Peroxidase-Labeled Streptavidin, reacting at room temperature for 45min, washing enzyme-linked plate for 5 times, adding 100 μ L TMB substrate, developing, reacting for 5min, and reacting with 100 μ L2N H₂SO₄Terminating the reaction, reading 450nm by an enzyme linked immunosorbent assay (ELISA) detector and calculating IC₅₀The value is obtained.

The experimental results are as follows: JY002-WG1, JY002-M2G1 fusion protein blocked CD47/SIRP alpha interaction curve graph as shown in FIG. 5, IC calculated according to four parameter fitting curve₅₀The results of the value determination are shown in Table 4, the competitive activity of the SIRP alpha mutant JY002-M2G1 fusion protein is more than 17 times of that of the wild type SIRP alpha.

TABLE 4 SIRP α mutant-Fc fusion protein competitive activity

Fusion proteins	IC50(μg/mL)
		JY002WG1	＞20
JY002-M2G1	1.16

Example 7 specific recognition of the tumor cell surface CD47 molecule by SIRP alpha mutant-Fc fusion protein

As an example, the binding activity of JY002-WG1 and JY002-M2G1 fusion proteins and the CD47 molecule on the surface of tumor cells is measured by an ELISA method.

Experimental materials: raji cells, Jurkat cells and A549 tumor cell lines were purchased from ATCC, Inc. in the United states, PBS, DMEM, RPMI 1640 medium and imported fetal bovine serum from Gibco. Sheep anti-human IgG-Fc-PE was purchased from Biolegend.

The experimental steps are as follows: in vitro, cells are cultured for 3-5 passages in complete medium (10% fetal bovine serum in DMEM/RPMI 1640 medium) for routine cell culture, suspension cells are centrifuged such as Raji and Jurkat and counted in suspension with cell wash (2% fetal bovine serum in PBS), and pancreatin-treated adherent cells A549 are centrifuged and counted in suspension with cell wash until the cells are in logarithmic growth phase. 3X 10 per sample⁵Subpackaging into sample tubes, simultaneously diluting the fusion protein to be detected to 100 mu g/mL by using washing liquid, and carrying out 4-fold dilution to obtain 7 concentration gradient sites. Adding the fusion protein with corresponding concentration into a cell tube, reacting at 4 ℃ for 30 minutes, washing the cells for 1 time by using a cell washing solution, adding a goat anti-human IgG-Fc-PE secondary antibody with appropriate concentration, reacting at 4 ℃ for 30 minutes, washing the cells for 2 times by using the cell washing solution, adding an appropriate amount of the cell washing solution, and detecting by using a flow cytometer. The binding curve of the fusion protein and the CD47 on the surface of the tumor cell is plotted with the sample concentration as the abscissa and the Mean Fluorescence Intensity (MFI) as the ordinate.

The experimental results are as follows: JY002-WG1, JY002-M2G1 fusion protein and tumor cell (Raji, Jurkat, A549) surface CD47 combination curve is shown in figure 6-A (Raji), figure 6-B (Jurkat) and figure 6-C (A549), and combination EC₅₀The value is shown in Table 5, and the result shows that the binding activity of the SIRP alpha mutant JY002-M2G1 fusion protein and the tumor cell surface CD47 is higher than that of the wild type SIRP alpha, and the fold increase is between 2.5 and 5.1.

TABLE 5 binding Activity of SIRP alpha mutant-Fc fusion protein with tumor cell surface CD47

Example 8 detection of the SiRP alpha mutant-Fc fusion protein clotting response

As an example, the coagulation activity of JY002-WG1, JY002-M2G1 fusion protein was determined by using a coagulation experiment, and the American clinical in-research varieties TTI-621 (see CN105073780A), Hu5F9 and PBS are used as controls.

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

The experimental steps are as follows: collecting the whole blood of a healthy volunteer at a ratio of 1:1, putting the whole blood into the Alzheimer's disease solution, washing the whole blood with PBS for 3-5 times, and preparing the whole blood into 6% erythrocyte suspension with the PBS for later use. JY002WG1, JY002-M2G1 fusion protein, TTI-621 and Hu5F9 were diluted to 200. mu.g/mL with PBS, and 12 concentration gradients were obtained by 2-fold dilution. The prepared 6% red blood cell suspension was added to a round bottom 96 well plate at 100. mu.L/well, while the diluted sample was added to the red blood cell suspension at 100. mu.L/well such that the highest initial concentration of the sample was 100. mu.g/mL. Standing at 37 deg.C for 4 hr, and observing whether hemolysis occurs.

The experimental results are as follows: as shown in FIG. 7, hemolysis was observed in the control groups Hu5F9 (indicated by arrows in the figure) of 100. mu.g/mL, 50. mu.g/mL and 25. mu.g/mL, and no hemolysis was observed in the concentration gradients of JY002-WG1, JY002-M2G1 and TTI-621 as in the PBS control group. The SIRPa mutant-Fc fusion protein has no influence on blood agglutination reaction and has extremely high safety in use.

Example 9 in vivo tumor suppressor Activity of SIRP α mutant-Fc fusion proteins

As an example, JY002-WG1 and JY002-M2G1 fusion proteins were evaluated for in vivo tumor suppression activity by NOD-SCID mouse xenograft tumor model, and clinically developed variety Hu5F9 and PBS of Forty Seven company in the United states were used as controls.

Experimental materials: RAJI cells and NOD-SCID mice were obtained from Beijing Wintolite laboratory animals technologies, Inc., and fusion proteins, TTI-621 and HU5F9 were prepared by expression of their reference patent sequences.

The experimental steps are as follows: feeding NOD-SCID mice in SPF-level animal room for 7 days, and after the mice are adapted to the feeding environment, culturing Raji cells in logarithmic growth phase at 5 × 10⁷1/mL, 1mu.L/seed was inoculated to subcutaneous part of hind limb inguinal region of mouse, and after 7-10 days of observation, tumor size [ tumor volume (mm) ] was measured using vernier caliper³) 0.5 long diameter (mm) x short diameter²(mm)]. The tumor volume is about 250 +/-100 mm³In the meantime, based on the tumor volume and the animal body weight, random number notation was used to randomly group 5 mice into a PBS group, a JY002-WG1(3mg/kg) group, a JY002-WG1(10mg/kg) group, a JY002-M2G1(3mg/kg) group, a JY002-M2G1(10mg/kg) group and a Hu5F9(10mg/kg) group. The drug was diluted to the corresponding concentration with PBS, i.p., administered 2 times per week and tumor volume and mouse body weight were measured for a total of 5 administrations and measurements. At the end of dosing, mice were euthanized. The tumor size is used as the ordinate, and the days after administration is used as the abscissa to draw the tumor growth curve along with the time.

The experimental results are as follows: as shown in FIG. 8, compared with the PBS group, each administration group has certain tumor inhibition activity, and the JY002-M2G1 fusion protein with improved affinity has obviously enhanced tumor inhibition activity compared with the wild-type fusion protein JY002-WG 1. The statistical analysis result shows that JY002-M2G1 fusion protein (B) is obviously superior to JY002-WG1(D) which is dosed at the same dose under the condition of high dose (10mg/kg), the tumor inhibition activity is improved by more than 3.5 times, and is slightly superior to Hu5F9(E) which is a positive control medicament, and the tumor inhibition activity is improved by more than 1.5 times.

Example 10 comparison of binding Activity of different isoforms of SIRP alpha mutant-Fc fusion proteins

The antibody or fusion protein targeting CD47 is combined with human erythrocytes, platelets and the like to generate hemolytic anemia and thrombocytopenia, and has potential toxic and side effects. Research shows that the potential toxic and side effects of the medicine can be effectively reduced by changing the immunoglobulin Fc of the antibody into IgG4 subtype or mutating the site of IgG1Fc which is responsible for combining with Fc gamma R, the dosage of the medicine is greatly improved, and the specific mutation can be seen in three mutation strategies of SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 in a sequence table. Similarly, the American ALX Oncology company developed strain ALX148 in clinical practice to effectively balance the safety and efficacy of the high affinity fusion protein SIRP alpha, and mutated in the IgG1Fc region (4 alanine substitution mutations such as L are introduced into the Fc fragment of IgG 1)²³⁴A；L²³⁵A；G²³⁷A；N²⁹⁷A) Thereby inactivating Fc mediated killing effect to achieve the aim of clinical medication safety, and the ALX148 has outstanding treatment effect when being combined with other drugs (such as herceptin and Palbolizumab) clinically. Similarly, the present application also pertains to high affinity sirpa fusion proteins, and in order to expand the scope of the present application, the present example examines the binding activity of sirpa mutants to Fc-inactive fusion proteins.

According to the construction method of the fusion protein in the example 2, the SIRPa domain mutant (SEQ ID NO.2, 3 or 4) obtained in the example 1 is respectively combined with the human IgG1-Fc mutant (N)²⁹⁷A; the amino acid sequence is shown as SEQ ID NO: 6), and the fusion proteins are named JY002-M1G1(N297A), JY002-M2G1(N297A) and JY002-M3G1(N297A) respectively. Or a mutant of the SIRPa domain (SEQ ID NO.2, 3 or 4) is respectively combined with a human IgG1-Fc mutant (L)²³⁴A；L²³⁵A；G²³⁷A；N²⁹⁷A; the amino acid sequence is shown as SEQ ID NO: 7), and the fusion proteins are named JY002-M1G1(AAAA), JY002-M2G1(AAAA) and JY002-M3G1(AAAA) respectively.

As an example, the binding activity of JY002-M2G1, JY002-M2G1(N297A) and JY002-M2G1(AAAA) fusion proteins and CD47 molecules is measured by an ELISA method.

The coating diluted CD47-His antigen to 1. mu.g/mL, 100. mu.L per well was added to the enzyme-linked plate, at 4 ℃ overnight. Washed 3 times with PBST, 200. mu.L of PBSM (4% milk in PBS) per well was blocked for 1h at 37 ℃. JY002-M2G1, JY002-M2G1(N297A) and JY002-M2G1(AAAA) fusion proteins are diluted to 15 mu G/mL by PBS, 12 different dilution concentrations are obtained by 3 times of dilution, 100 mu L of fusion protein is added into an enzyme-linked plate per hole, the reaction is carried out for 1h at 37 ℃ in a wet box, the enzyme-linked plate is washed for 3 times, sheep anti-human Fc-HRP secondary antibody is added for reaction for 45min at room temperature, PBST is washed for 5 times, 100 mu L of TMB substrate is added for color development, and 100 mu L of 2N H is used for reaction after 3min₂SO₄The reaction was stopped and the ELISA read at 450 nm. And (4) drawing an antibody-antigen binding curve by taking the concentration of the antibody as an abscissa and the OD value as an ordinate. The four parameter equation curve was fitted using Graphpad analysis software,the equation is y ═ A-D)/(1+ (X/C) ^ B) + D, where B represents the slope and C represents EC₅₀。

As shown in FIG. 9, JY002-M2G1(N297A), JY002-M2G1(AAAA) fusion protein and CD47 antigen have good binding activity, and EC listed in Table 6₅₀The values are respectively 0.0088 mu G/mL, 0.0050 mu G/mL and the EC of JY002-M2G1₅₀(0.0064. mu.g/mL) remained essentially the same.

TABLE 6 binding Activity of different isoforms of SIRP alpha mutant-Fc fusion protein with CD47

Fusion proteins	EC50(μg/mL)
		JY002-M2G1	0.0064
JY002-M2G1(N297A)	0.0088
		JY002-M2G1(AAAA)	0.0050

Example 11 comparison of different subtypes of SIRP alpha mutant-Fc fusion protein blocking Activity

The activity of JY002-M2G1, JY002-M2G1(N297A) and JY002-M2G1(AAAA) fusion proteins for blocking CD47/SIRP alpha interaction was analyzed according to the analysis method for blocking CD47/SIRP alpha interaction in example 6, and the results are shown in FIG. 10. JY002-M2G1(N297A) and JY002-M2G1(AAAA) fusion proteins can effectively block CD47/SIRP alpha interaction, and IC of the fusion proteins₅₀The values are respectively 1.12. mu.g/mL, 1.16. mu.g/mL, and IC of JY002-M2G1₅₀The value (1.14. mu.g/mL) remained essentially the same.

Example 12 comparison of different isoforms of SIRP alpha mutant-Fc fusion proteins for in vitro ADCC Activity

JY002-M2G1, JY002-M2G1(N297A) and JY002-M2G1(AAAA) fusion proteins were analyzed for in vitro ADCC activity by using lactate dehydrogenase assay.

Experimental materials: RAJI cells, NK92/CD16a stably transfected with human Fc γ RIII (CD16a), cytoxicity Detection KitPLUS (LDH) (Roche Cat # 04744926001).

The experimental steps are as follows: configuring ADCC buffer, namely adding 2% FBS into MEM Alpha, collecting target cells in logarithmic phase by using a 50mL sterile tube, centrifuging for 4min at room temperature at 1200g, and then removing supernatant; after 10mL of ADCC buffer was taken to resuspend the cells, it was centrifuged again at 1200g for 4min at room temperature. The supernatant was discarded completely, the cells were resuspended in 10mL ADCC buffer and viable cells were counted using Trypan blue staining, and the viability rate of viable cells reached 95% or more and was used for the experiment. ADCC buffer was used to adjust RAJI target cell density to 2X 10⁵one/mL, 50uL per well was added to a 96 well cell culture plate. Preparing a sample to be detected, wherein the final concentration of the sample is as follows: 20. mu.g/mL, 2. mu.g/mL, 0.2. mu.g/mL, 0.02. mu.g/mL, 0.002. mu.g/mL, 0.0002. mu.g/mL, 0.00002. mu.g/mL, 0.000002. mu.g/mL, 8 dilutions of the antibody gradient, 50 uL/well into a 96-well plate that had been seeded with target cells, and incubated in a 37 ℃ cell incubator with 5% CO2 for 30 min. Resuspending NK92/CD16a cells by 10mL ADCC buffer and calculating the viable cells by trypan blue staining method, wherein the viable cell survival rate reaches more than 95 percent and can be used for experiments. Adjusting effector cell density to 1.2X 10 with ADCC buffer⁶one/mL (1:6), 50uL per well was added to a 96 well cell culture plate, gently mixed, and incubated for 6h in a 37 ℃ 5% CO2 incubator.

Each set of experiments was set up as follows:

control 1: ADCC buffer solution

Control 2: target cells + ADCC buffer

Control 3: target cells +3# lysate (provided by 10uL kit) + ADCC buffer

Control 4: target cell + effector cell + ADCC buffer

Experimental groups: target cells + effector cells + ADCC buffer solution + sample to be tested

15min before the cell incubation is finished, Detection is carried out according to the instruction recommended by the cytotoxin Detection KitPLUS kit. All OD492nm data were subtracted with the corresponding OD650nm background for data analysis, and% specific kill ═ ((experimental-control 4)/(control 3-control 2)) × 100%, dose-response curves were data analyzed using GraphPad Prism Version 5.

The experimental results are as follows: as shown in FIG. 11, JY002-M2G1, a fusion protein of a normal IgG1 subtype, has stronger ADCC activity, and the half-killing rate ED50 value is about 0.0102 mu G/mL, compared with JY002-M2G1(N297A) and JY002-M2G1(AAAA) fusion proteins, the ADCC activity disappears, and the directional mutation of an IgG1Fc domain can change the effector function of Fc. From the above results, it was found that the IgG1 constant region mutation did not significantly affect the binding activity and competitive activity of the sirpa domain mutant, but significantly affected the Fc-mediated ADCC effect.

Example 13 in vivo tumor suppressor Activity of different isoforms of SIRP α mutant-Fc fusion proteins

The high-affinity SIRP alpha fusion protein has stronger activity of blocking CD47/SIRP alpha, because IgG1 wild-type Fc can generate stronger hematological toxicity through ADCC and CDC, for the clinical use safety, IgG1-Fc is subjected to mutation inactivation, the Fc-mediated cytotoxic activities such as ADCC and CDC are reduced, and simultaneously the Fc inactivation can more effectively obtain the clinical use safety and greatly improve the drug safety dosage window. Therefore, in order to examine the influence of Fc inactivation on anti-tumor, JY-002M2G1(N297A) potential anti-tumor activity was evaluated by the animal model in reference example 9 in the present example, and as shown in FIG. 12, JY-002(N297A) had good anti-tumor activity at a dose of 5mg/kg compared with control group PBS.

The above description of the embodiments is only intended to understand the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

<110> Beijing Gill Maydi biomedical science and technology Co., Ltd

<120> high affinity SIRP alpha mutant targeting CD47 and fusion protein thereof

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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> 3

<211> 117

<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 Val Gly

20 25 30

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

35 40 45

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

50 55 60

Thr Lys Arg Ser Asn Met Asp Phe Ser Ile Gln 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

<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 Phe Pro Val Gly

20 25 30

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

35 40 45

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

50 55 60

Thr Lys Arg Asp Asn Met Asp Phe Ser Ile Ser Ile Ser 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 Ile Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val

100 105 110

Arg Ala Lys Pro Ser

115

<210> 5

<211> 233

<212> PRT

<213> Homo sapiens

<400> 5

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 Phe Tyr 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> 6

<211> 233

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

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 Ala 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 Phe Tyr 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> 7

<211> 233

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

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

1 5 10 15

Ala Pro Glu Ala Ala Gly Ala 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 Ala 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 Phe Tyr 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> 8

<211> 230

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Leu Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu

1 5 10 15

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

20 25 30

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

35 40 45

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

50 55 60

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

65 70 75 80

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

85 90 95

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

100 105 110

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

115 120 125

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

130 135 140

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

145 150 155 160

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

165 170 175

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

180 185 190

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

195 200 205

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

210 215 220

Ser Leu Ser Leu Gly Lys

225 230

<210> 9

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser

<210> 10

<211> 1050

<212> DNA

<213> Homo sapiens

<400> 10

gagctgcagg tgatccagcc cgacaagtcc gtgctggtgg ctgctggcga aaccgccacc 60

ctgaggtgca ccgctacaag cctgctgccc gtgggcccca tccagtggtt cagaggagcc 120

ggccccggca gactgctgat ctacaaccag agagacggcc acttccccag ggtgaccacc 180

atctccagca ccaccaagag gaacaacatg gacttcagca tcagcatcca gaacatcacc 240

cctgccgatg ccggcaccta ctactgcgtg aagttcagga agggcagccc cgacgacgtg 300

gagttcaaga gcggcgccgg cacagagctg agcgtgagag ccaagcccag cctcgagccc 360

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 420

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 480

gaggtcacgt gcgtggtggt ggacgtgagc cacgaagacc ccgaggtcaa gttcaactgg 540

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 600

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 660

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 720

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 780

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 840

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 900

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 960

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1020

cagaagagcc tctccctgtc tccgggtaaa 1050

<210> 11

<211> 1050

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gagctgcagg tgatccagcc cgacaagtcc gtgctggtgg ctgctggcga aaccgccacc 60

ctgaggtgca ccgctacaag cctgctgccc gtgggcccca tccagtggtt cagaggagcc 120

ggccccggca gactgctgat ctacaaccag agacagggcc acttccccag ggtgaccacc 180

atctcccaga ccaccaagag gagcaacatg gacttcagca tccagatcgg caacatcacc 240

cctgccgatg ccggcaccta ctactgcgtg aagttcagga agggcagccc cgacgacgtg 300

gagttcaaga gcggcgccgg cacagagctg agcgtgagag ccaagcccag cctcgagccc 360

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 420

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 480

gaggtcacgt gcgtggtggt ggacgtgagc cacgaagacc ccgaggtcaa gttcaactgg 540

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 600

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 660

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 720

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 780

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 840

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 900

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 960

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1020

cagaagagcc tctccctgtc tccgggtaaa 1050

<210> 12

<211> 1050

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gagctgcagg tgatccagcc cgacaagtcc gtgctggtgg ctgctggcga aaccgccacc 60

ctgaggtgca ccgctacaag cctgttcccc gtgggcccca tccagtggtt cagaggagcc 120

ggccccggca gactgctgat ctacaaccag aaggagggcc acttccccag ggtgaccacc 180

atctcccaga ccaccaagag ggacaacatg gacttcagca tcagcatcag caacatcacc 240

cctgccgatg ccggcaccta ctactgcgtg aagttcagga agggcagccc cgacgacatc 300

gagttcaaga gcggcgccgg cacagagctg agcgtgagag ccaagcccag cctcgagccc 360

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 420

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 480

gaggtcacgt gcgtggtggt ggacgtgagc cacgaagacc ccgaggtcaa gttcaactgg 540

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 600

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 660

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 720

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 780

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 840

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 900

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 960

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1020

cagaagagcc tctccctgtc tccgggtaaa 1050

<210> 13

<211> 1050

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gagctgcagg tgatccagcc cgacaagtcc gtgctggtgg ctgctggcga aaccgccacc 60

ctgaggtgca ccgctacaag cctgctgccc gtgggcccca tccagtggtt cagaggagcc 120

ggccccggca gactgctgat ctacaaccag agagacggcc acttccccag ggtgaccacc 180

atctccagca ccaccaagag gaacaacatg gacttcagca tcagcatcca gaacatcacc 240

cctgccgatg ccggcaccta ctactgcgtg aagttcagga agggcagccc cgacgacgtg 300

gagttcaaga gcggcgccgg cacagagctg agcgtgagag ccaagcccag cctcgagccc 360

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 420

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 480

gaggtcacgt gcgtggtggt ggacgtgagc cacgaagacc ccgaggtcaa gttcaactgg 540

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacgcc 600

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 660

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 720

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 780

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 840

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 900

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 960

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1020

cagaagagcc tctccctgtc tccgggtaaa 1050

<210> 14

<211> 1050

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gagctgcagg tgatccagcc cgacaagtcc gtgctggtgg ctgctggcga aaccgccacc 60

ctgaggtgca ccgctacaag cctgctgccc gtgggcccca tccagtggtt cagaggagcc 120

ggccccggca gactgctgat ctacaaccag agacagggcc acttccccag ggtgaccacc 180

atctcccaga ccaccaagag gagcaacatg gacttcagca tccagatcgg caacatcacc 240

cctgccgatg ccggcaccta ctactgcgtg aagttcagga agggcagccc cgacgacgtg 300

gagttcaaga gcggcgccgg cacagagctg agcgtgagag ccaagcccag cctcgagccc 360

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 420

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 480

gaggtcacgt gcgtggtggt ggacgtgagc cacgaagacc ccgaggtcaa gttcaactgg 540

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacgcc 600

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 660

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 720

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 780

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 840

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 900

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 960

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1020

cagaagagcc tctccctgtc tccgggtaaa 1050

<210> 15

<211> 1050

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gagctgcagg tgatccagcc cgacaagtcc gtgctggtgg ctgctggcga aaccgccacc 60

ctgaggtgca ccgctacaag cctgttcccc gtgggcccca tccagtggtt cagaggagcc 120

ggccccggca gactgctgat ctacaaccag aaggagggcc acttccccag ggtgaccacc 180

atctcccaga ccaccaagag ggacaacatg gacttcagca tcagcatcag caacatcacc 240

cctgccgatg ccggcaccta ctactgcgtg aagttcagga agggcagccc cgacgacatc 300

gagttcaaga gcggcgccgg cacagagctg agcgtgagag ccaagcccag cctcgagccc 360

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 420

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 480

gaggtcacgt gcgtggtggt ggacgtgagc cacgaagacc ccgaggtcaa gttcaactgg 540

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacgcc 600

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 660

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 720

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 780

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 840

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 900

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 960

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1020

cagaagagcc tctccctgtc tccgggtaaa 1050

<210> 16

<211> 1050

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gagctgcagg tgatccagcc cgacaagtcc gtgctggtgg ctgctggcga aaccgccacc 60

ctgaggtgca ccgctacaag cctgctgccc gtgggcccca tccagtggtt cagaggagcc 120

ggccccggca gactgctgat ctacaaccag agagacggcc acttccccag ggtgaccacc 180

atctccagca ccaccaagag gaacaacatg gacttcagca tcagcatcca gaacatcacc 240

cctgccgatg ccggcaccta ctactgcgtg aagttcagga agggcagccc cgacgacgtg 300

gagttcaaga gcggcgccgg cacagagctg agcgtgagag ccaagcccag cctcgagccc 360

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaagc tgcaggggct 420

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 480

gaggtcacgt gcgtggtggt ggacgtgagc cacgaagacc ccgaggtcaa gttcaactgg 540

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacgcc 600

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 660

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 720

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 780

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 840

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 900

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 960

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1020

cagaagagcc tctccctgtc tccgggtaaa 1050

<210> 17

<211> 1050

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gagctgcagg tgatccagcc cgacaagtcc gtgctggtgg ctgctggcga aaccgccacc 60

ctgaggtgca ccgctacaag cctgctgccc gtgggcccca tccagtggtt cagaggagcc 120

ggccccggca gactgctgat ctacaaccag agacagggcc acttccccag ggtgaccacc 180

atctcccaga ccaccaagag gagcaacatg gacttcagca tccagatcgg caacatcacc 240

cctgccgatg ccggcaccta ctactgcgtg aagttcagga agggcagccc cgacgacgtg 300

gagttcaaga gcggcgccgg cacagagctg agcgtgagag ccaagcccag cctcgagccc 360

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaagc tgcaggggct 420

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 480

gaggtcacgt gcgtggtggt ggacgtgagc cacgaagacc ccgaggtcaa gttcaactgg 540

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacgcc 600

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 660

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 720

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 780

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 840

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 900

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 960

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1020

cagaagagcc tctccctgtc tccgggtaaa 1050

<210> 18

<211> 1050

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gagctgcagg tgatccagcc cgacaagtcc gtgctggtgg ctgctggcga aaccgccacc 60

ctgaggtgca ccgctacaag cctgctgccc gtgggcccca tccagtggtt cagaggagcc 120

ggccccggca gactgctgat ctacaaccag agacagggcc acttccccag ggtgaccacc 180

atctcccaga ccaccaagag gagcaacatg gacttcagca tccagatcgg caacatcacc 240

cctgccgatg ccggcaccta ctactgcgtg aagttcagga agggcagccc cgacgacgtg 300

gagttcaaga gcggcgccgg cacagagctg agcgtgagag ccaagcccag cctcgagccc 360

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaagc tgcaggggct 420

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 480

gaggtcacgt gcgtggtggt ggacgtgagc cacgaagacc ccgaggtcaa gttcaactgg 540

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacgcc 600

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 660

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 720

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 780

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 840

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 900

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 960

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1020

cagaagagcc tctccctgtc tccgggtaaa 1050

<210> 19

<211> 1041

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gagctgcagg tgatccagcc cgacaagtcc gtgctggtgg ctgctggcga aaccgccacc 60

ctgaggtgca ccgctacaag cctgctgccc gtgggcccca tccagtggtt cagaggagcc 120

ggccccggca gactgctgat ctacaaccag agagacggcc acttccccag ggtgaccacc 180

atctccagca ccaccaagag gaacaacatg gacttcagca tcagcatcca gaacatcacc 240

cctgccgatg ccggcaccta ctactgcgtg aagttcagga agggcagccc cgacgacgtg 300

gagttcaaga gcggcgccgg cacagagctg agcgtgagag ccaagcccag cctcgagtcc 360

aagtacggcc ccccttgccc tccttgtccc gctcccgagt ttttaggagg acctagcgtg 420

ttcctcttcc cccccaagcc caaagacact ttaatgatta gccggacccc cgaagtgact 480

tgtgtggtgg tggacgtgag ccaagaagac cccgaagtgc agtttaactg gtacgtggac 540

ggcgtggagg tgcacaacgc taagaccaag ccccgggaag agcagttcaa ctccacctat 600

cgggtggtga gcgtgctcac cgtgctgcat caagattggc tgaacggcaa ggagtacaag 660

tgcaaggtga gcaacaaggg actgccctcc tccattgaga agaccattag caaggccaag 720

ggccagccta gggaacctca agtttacact ttaccccctt cccaagaaga gatgaccaag 780

aatcaagttt ctttaacttg tttagtcaaa ggcttctacc cctccgacat cgctgtggag 840

tgggagtcca acggccagcc cgagaacaac tacaagacca ccccccccgt tctggacagc 900

gatggcagct tttttttata cagccggctg acagtggaca agtctcgttg gcaagaaggc 960

aatgtcttca gctgctccgt gatgcacgag gctttacaca accactacac ccagaagtct 1020

ttatctttat ctttaggcaa g 1041

<210> 20

<211> 1041

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gagctgcagg tgatccagcc cgacaagtcc gtgctggtgg ctgctggcga aaccgccacc 60

ctgaggtgca ccgctacaag cctgctgccc gtgggcccca tccagtggtt cagaggagcc 120

ggccccggca gactgctgat ctacaaccag agacagggcc acttccccag ggtgaccacc 180

atctcccaga ccaccaagag gagcaacatg gacttcagca tccagatcgg caacatcacc 240

cctgccgatg ccggcaccta ctactgcgtg aagttcagga agggcagccc cgacgacgtg 300

gagttcaaga gcggcgccgg cacagagctg agcgtgagag ccaagcccag cctcgagtcc 360

aagtacggcc ccccttgccc tccttgtccc gctcccgagt ttttaggagg acctagcgtg 420

ttcctcttcc cccccaagcc caaagacact ttaatgatta gccggacccc cgaagtgact 480

tgtgtggtgg tggacgtgag ccaagaagac cccgaagtgc agtttaactg gtacgtggac 540

ggcgtggagg tgcacaacgc taagaccaag ccccgggaag agcagttcaa ctccacctat 600

cgggtggtga gcgtgctcac cgtgctgcat caagattggc tgaacggcaa ggagtacaag 660

tgcaaggtga gcaacaaggg actgccctcc tccattgaga agaccattag caaggccaag 720

ggccagccta gggaacctca agtttacact ttaccccctt cccaagaaga gatgaccaag 780

aatcaagttt ctttaacttg tttagtcaaa ggcttctacc cctccgacat cgctgtggag 840

tgggagtcca acggccagcc cgagaacaac tacaagacca ccccccccgt tctggacagc 900

gatggcagct tttttttata cagccggctg acagtggaca agtctcgttg gcaagaaggc 960

aatgtcttca gctgctccgt gatgcacgag gctttacaca accactacac ccagaagtct 1020

ttatctttat ctttaggcaa g 1041

<210> 21

<211> 1041

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gagctgcagg tgatccagcc cgacaagtcc gtgctggtgg ctgctggcga aaccgccacc 60

ctgaggtgca ccgctacaag cctgttcccc gtgggcccca tccagtggtt cagaggagcc 120

ggccccggca gactgctgat ctacaaccag aaggagggcc acttccccag ggtgaccacc 180

atctcccaga ccaccaagag ggacaacatg gacttcagca tcagcatcag caacatcacc 240

cctgccgatg ccggcaccta ctactgcgtg aagttcagga agggcagccc cgacgacatc 300

gagttcaaga gcggcgccgg cacagagctg agcgtgagag ccaagcccag cctcgagtcc 360

aagtacggcc ccccttgccc tccttgtccc gctcccgagt ttttaggagg acctagcgtg 420

ttcctcttcc cccccaagcc caaagacact ttaatgatta gccggacccc cgaagtgact 480

tgtgtggtgg tggacgtgag ccaagaagac cccgaagtgc agtttaactg gtacgtggac 540

ggcgtggagg tgcacaacgc taagaccaag ccccgggaag agcagttcaa ctccacctat 600

cgggtggtga gcgtgctcac cgtgctgcat caagattggc tgaacggcaa ggagtacaag 660

tgcaaggtga gcaacaaggg actgccctcc tccattgaga agaccattag caaggccaag 720

ggccagccta gggaacctca agtttacact ttaccccctt cccaagaaga gatgaccaag 780

aatcaagttt ctttaacttg tttagtcaaa ggcttctacc cctccgacat cgctgtggag 840

tgggagtcca acggccagcc cgagaacaac tacaagacca ccccccccgt tctggacagc 900

gatggcagct tttttttata cagccggctg acagtggaca agtctcgttg gcaagaaggc 960

aatgtcttca gctgctccgt gatgcacgag gctttacaca accactacac ccagaagtct 1020

ttatctttat ctttaggcaa g 1041

<210> 22

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

atgggctggt cctgtatcat cctgttcctg gtggctacag ccacaggagt gcattct 57

Claims (13)

1. A human SIRP alpha mutant targeting CD47,

the amino acid sequence of the mutant is shown in any sequence of SEQ ID NO.2, 3 and 4.

2. A fusion protein containing the human SIRPa mutant according to claim 1, wherein the fusion protein further contains an immunoglobulin fragment, and the immunoglobulin fragment is an Fc fragment.

3. The fusion protein of claim 2, wherein the amino acid sequence of the immunoglobulin Fc segment is as shown in any one of SEQ ID NO. 5, 6, 7, and 8.

4. The fusion protein of claim 3, wherein the human SIRPa mutant is located at the N-terminus of the fusion protein.

5. The fusion protein of claim 4, wherein the sequence combination of the human SIRPa mutant of the fusion protein and the immunoglobulin Fc segment is selected from any one of the following:

SEQ ID NO.2 and SEQ ID NO. 5 or

SEQ ID NO.3 and SEQ ID NO. 5 or

SEQ ID NO.4 and SEQ ID NO. 5 or

SEQ ID NO.2 and SEQ ID NO.6 or

SEQ ID NO.3 and SEQ ID NO.6 or

SEQ ID NO.4 and SEQ ID NO.6 or

SEQ ID NO.2 and SEQ ID NO.7 or

SEQ ID NO.3 and SEQ ID NO.7 or

SEQ ID NO.4 and SEQ ID NO.7 or

SEQ ID NO.2 and SEQ ID NO 8 or

SEQ ID NO.3 and SEQ ID NO 8 or

SEQ ID NO.4 and SEQ ID NO. 8.

6. A polynucleotide encoding the fusion protein of claim 5, the polynucleotide having a sequence selected from any one of the sequences shown in SEQ ID No.10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21.

7. An expression vector containing the polynucleotide of claim 6, wherein the expression vector is a secretory expression vector, and a polynucleotide encoding a signal peptide with the amino acid sequence shown in SEQ ID NO.9 is arranged at the 5' end of the polynucleotide encoding the human SIRPa mutant.

8. The expression vector of claim 7, wherein the polynucleotide encoding the signal peptide has the sequence shown in SEQ ID No. 22.

9. A host cell comprising the expression vector of claim 7 or 8.

10. A method of making the fusion protein of claim 5, the method comprising culturing the host cell of claim 9 under conditions such that the fusion protein is expressed.

11. Use of the human SIRPa mutant of claim 1 in the preparation of a CD47 test kit.

12. Use of the human SIRPa mutant according to claim 1 in the preparation of a medicament for the treatment of a neoplastic disease, which is a CD47+ associated neoplastic disease.

13. Use of the fusion protein according to any one of claims 2 to 5 for the preparation of a medicament for the treatment of a neoplastic disease, said neoplastic disease being a CD47+ associated neoplastic disease.

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