Serpina3 protein variant or coding sequence thereof, and use thereof

Abstract

A SERPINA3 protein variant or a coding sequence thereof, the use of a SERPINA3 protein, the SERPINA3 protein variant or the coding sequence thereof in the preparation of a drug for resisting inflammation, treating autoimmune diseases, and resisting transplantation rejection, and the use of the SERPINA3 protein variant in the preparation of an anti-tumor antibody.

Description

SERPINA3 protein variants or their coding sequences and their applications Technical Field

[0001] This invention relates to the field of biomedical technology, and more particularly to the application of SERPINA3 protein variants and the SERPINA3 protein, its variants, and genes in the preparation of anti-inflammatory, autoimmune, and transplant rejection drugs. This invention relates to the application of SERPINA3 protein variants, alone or in combination with anti-inflammatory drugs, in the preparation of anti-inflammatory drugs and anti-inflammatory drug compositions; the application of SERPINA3 protein variants, alone or in combination with immunosuppressive drugs, in the preparation of drugs for treating autoimmune diseases and compositions for treating autoimmune diseases; and the application of SERPINA3 protein variants, alone or in combination with immunosuppressive drugs, in the preparation of transplant rejection drugs and compositions for anti-transplant rejection. This invention also relates to the application of genes encoding SERPINA3 or its variants in the production of transgenic mesenchymal stem cells (MSCs) or stem cells with differentiation potential, to enhance the immunosuppressive function of regulatory cells derived from MSCs or stem cell differentiation. Furthermore, this invention relates to the application of SERPINA3 protein or its variants in the preparation and screening of anti-SERPINA3 antibodies with anti-tumor activity. Background Technology

[0002] SIRPα +T cells, including macrophages, monocytes, dendritic cells (DCs), granulocytes, and activated NK cells, play a crucial role in detecting infection and injury signals, regulating the recruitment, differentiation, and activation of adaptive immune cells, and maintaining tissue and immune homeostasis. Inappropriate activation of these cells can drive numerous pathological processes, such as chronic inflammation and autoimmune diseases (Greene JT, Brian BF 4th, Senevirinathne SE, et al. Curr Opin Immunol. 2021; 73:34-42.). In many cancers, macrophages dominate the tumor microenvironment (TME) and enhance neoantigen presentation by phagocytizing cancer cells, thereby promoting adaptive T cell immunity, but their antitumor potential is often suppressed. The activation of myeloid cells (including macrophages, monocytes, DCs, and granulocytes) is regulated by a balance of inhibitory and activating receptor signaling. CD47 protein is expressed in all healthy and cancer cells. It plays a crucial role in the balance of myeloid cells by binding to signal regulatory protein alpha (SIRPα) and activating phosphorylation of tyrosine residues in the cytoplasmic ITIM and ITSM sequences of SIRPα. This leads to the recruitment and activation of protein tyrosine phosphatases SHP-1 and SHP-2, which transmit a "don't eat me" signal to myeloid cells (Logtenberg MEW, Scheeren FA, Schumacher TN. Immunity. 2020; 52(5):742-752.). Natural killer (NK) cells also express SIRPα upon activation, and CD47 / SIRPα signaling can effectively inhibit NK cell function (Deuse T, Hu X, Agbor-Enoh S, et al. J Exp Med. 2021; 218(3):e20200839.).

[0003] SERPINA3 is a member of the serine protease inhibitor (SERPIN) superfamily. The mouse SERPINA3 family has four members: SERPINA3c, SERPINA3n, SERPINA3m, and SERPINA3k. SERPINA3n is considered a homolog of human SERPINA3 due to its highest homology (61%). J, S, et al. Biomedicines. 2023; 11(1):156.). Currently, SERPINA3 is classified as an acute-phase inflammatory response protein, mainly secreted into the blood by the liver. Under the stimulation of inflammatory cytokines such as TNFα, IL-1, and IL-6, the serum concentration of SERPINA3 can increase 2-5 times during the immune response. It is also known as α-1-antichymotrypsin (AACT / ACT), and its main function is to prevent tissue damage during the lysis and phagocytosis process caused by neutrophils in damaged tissues. Cathepsin G is its main target (de Mezer M, J, S, et al. Biomedicines. 2023; 11(1):156.). A urinary proteomics study of patients with acute allogeneic kidney transplant rejection indicated that SERPINA3 may be a potential early biomarker of transplant rejection (Ziegler ME, Chen T, LeBlanc JF, et al. Transplantation. 2011; 92(4):388-95.), but its true role in transplant rejection remains unclear. Although SERPINA3 has been used as an inflammatory biomarker, it cannot be ruled out that its release into the plasma during inflammation is intended to signal cells involved in defense mechanisms, and whether it possesses receptors on immune cells is unknown.

[0004] Whether SERPINA3n / SERPINA3 induces pathogenesis or provides protection in inflammation remains controversial. In the central nervous system (CNS), some studies have proposed that SERPINA3n acts as a protective molecule by inhibiting the activity of serine proteases. Neuronal-derived SERPINA3n can counteract neuroinflammation in ischemic brain injury (Zhu M, Lan Z, Park J, et al. Neuropathol Appl Neurobiol. 2024; 50(2):e12980.). However, intracranial delivery of recombinant SERPINA3n in healthy mice induces neuroinflammation and causes widespread damage to vascular integrity (Kim H, Leng K, Park J, et al. Nat Commun. 2022; 13(1):6581.), suggesting that SERPINA3n may have a highly pro-inflammatory effect on the central nervous system and cerebral vascular endothelium. Furthermore, knockdown of SERPINA3n reduced LPS-induced cardiomyocyte inflammation (Xie W, Zhang A, Huang X, et al. Shock. 2023 May 1; 59(5):791-802.), suggesting a possible pro-inflammatory effect in the heart. However, SERPINA3-deficient mice exhibited delayed inflammation resolution in a chemically induced colitis model (Ho YT, Shimbo T, Wijaya E, et al. Cell Mol Gastroenterol Hepatol. 2021; 12(2):547-566.), suggesting a possible anti-inflammatory effect in inflammatory bowel disease. Recombinant SERPINA3k reduced corneal neovascularization and inflammation in a mouse model of corneal alkali burn by negatively regulating corneal angiogenesis and inflammatory factor production (Liu X, Lin Z, Zhou T, et al. PLoS One. 2011; 6(1):e16712.). In summary, the role of SERPINA3 in central or peripheral inflammation is contradictory. Further research is needed to determine whether it possesses functionally distinct domains and how it can eliminate pro-inflammatory effects on cardiomyocytes and cerebral vascular endothelial cells while exerting its anti-inflammatory effects. Furthermore, there are no reports on the correspondence between mouse SERPINA3k and human SERPINA3.

[0005] Mesenchymal stem cells (MSCs) are known to possess unique immunomodulatory capabilities. In inflamed tissues, MSCs can directly influence innate and adaptive immune cells, blocking the immune response cascade and thus restoring immune homeostasis. They hold great potential in the treatment of tissue damage, organ transplant rejection, inflammatory diseases, and autoimmune diseases. However, the immunomodulatory properties of MSCs are not innate but are induced by a group of inflammatory cytokines. Different inflammatory microenvironments lead to different immunomodulatory functions of MSCs. The use of anti-inflammatory drugs and immunosuppressants may alter the established inflammatory tissue microenvironment, resulting in highly unstable efficacy of MSC therapy. How to enable MSCs to stably exert immunosuppressive effects and enhance their immune function is one of the key issues that need to be addressed in this research field in the future (Li P, Ou Q, Shi S, et al. Cell Mol Immunol. 2023; 20(6):558-569.; Wang Y, Fang J, Liu B, et al. Cell Stem Cell. 2022; 29(11):1515-1530.). Since the immunomodulatory function of SERPINA3 protein is contradictory, it is unknown whether its gene overexpression can be used to create more functional MSCs.

[0006] Although SERPINA3 is not present in the nuclei of normal tissue cells, it has been detected in the nuclei of pancreatic cancer, gastric cancer, breast cancer, liver cancer, and lymphoma cells. Serum SERPINA3 levels are elevated in cancer patients (Sánchez-Navarro A, González-Soria I, ...). R, et al. Am J Physiol Cell Physiol. 2021; 320(1):C106-C118.). To date, it has been used as a diagnostic factor for colon cancer, breast cancer, lung cancer, and gastric cancer (de Mezer M, J, S, et al. Biomedicines. 2023; 11(1):156.). Furthermore, increased SERPINA3 expression in endometrial cancer and melanoma is associated with high mortality (Zhou J, Cheng Y, Tang L, et al. Oncotarget. 2017; 8(12):18712-18725.; Zhou ML, Chen FS, Mao H. World J Clin Cases. 2019; 7(15):1996-2002.). In melanoma and colon cancer, SERPINA3 is closely associated with the migration and invasion of malignant cells (Zhou J, Cheng Y, Tang L, et al. Oncotarget. 2017; 8(12):18712-18725.; Cao LL, Pei XF, Qiao X, et al. Dig Dis Sci. 2018; 63(9):2309-2319.). Knockdown of SERPINA3 inhibits the invasion and migration of melanoma cells (Zhou J, Cheng Y, Tang L, et al. Oncotarget. 2017; 8(12):18712-18725.; Kulesza DW, Ramji K, Maleszewska M, et al. Lab Invest. 2019; 99(11):1607-1621.). Although SERPINA3 levels are abnormally elevated in malignant tumors, its specific role in these tumors remains unclear. If an antibody capable of effectively blocking SERPINA3 is needed, the targeting sequence of this antibody against the SERPINA3 protein is a key area of ​​research.

[0007] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0008] This application aims to discover substances other than CD47 that can bind to and regulate SIRPα. + Ligands in the activated state of cells to regulate SIRPα + Cellular function, for SIRPα + Treatment of diseases caused by abnormal cell function (including inflammation, autoimmune diseases, transplant rejection, and tumors).

[0009] This study found that mouse SERPIA3k is a ligand for mouse SIRPα (mSIRPα); SERPIA3k binds to mSIRPα, activates downstream inhibitory signaling, and inhibits SIRPα. + SERPINA3k protein has phagocytic, inflammatory factor secretion, and killing functions in cells. It can alleviate LPS-induced pneumonia and has therapeutic effects on mouse models of autoimmune diseases such as experimental allergy encephalomyelitis (EAE) and rheumatoid arthritis (RA). It also reduces allergic organ transplant rejection and graft-versus-host disease (GVHD) in bone marrow transplantation. Overexpression of the SERPINA3k gene in mouse MSCs enhances the immunosuppressive function of MSCs and improves the therapeutic effect on inflammatory diseases. The serine protease inhibitor superfamily 1 (SERPIN-SF1) of the SERPINA3k protein (amino acids 130–323 from the first amino acid of the SERPINA3k protein signal peptide) is responsible for binding to mSIRPα, thereby mediating the aforementioned functions of the SERPINA3k protein, but has no pro-inflammatory effect on mouse cardiomyocytes and cerebral vascular endothelial cells. Human SERPINA3 (hSERPINA3) is the human counterpart of mSERPINA3k and a ligand for human SIRPα (hSIRPα), possessing similar functions. Like SERPINA3k, hSERPINA3 also possesses a SERPIN SF1 domain (amino acids 202-366 from the first amino acid of the SERPINA3 signal peptide) and an RCL domain (amino acids 369-394 from the first amino acid of the SERPINA3 signal peptide). Its SERPIN SF1 domain is responsible for binding to hSIRPα, thereby mediating the activation of downstream inhibitory signals of hSIRPα by hSERPINA3, inhibiting SIRPα. +The functions of cellular phagocytosis, antigen presentation, and secretion of inflammatory factors; amino acid residues P223, D225, Q228, E310, and Y312 are crucial for maintaining the interaction between SERPINA3 and hSIRPα; the SERPINA3D2 variant containing only the SERPIN SF1 domain (amino acids 202-366 from the first amino acid of the SERPINA3 protein signal peptide) lacks the pro-inflammatory effects of full-length SERPINA3 on human cardiomyocytes and cerebral vascular endothelial cells, which is unexpected under the current technology. SERPINA3D2 variants have therapeutic potential for human autoimmune diseases. Human MSCs or their derivatives (cellular vesicles, exosomes) overexpressing human SERPINA3D2 variants have stronger immunosuppressive functions than natural human MSCs. Stem cells with differentiation potential overexpressing SERPINA3D2 (such as human stem cells with differentiation potential), including but not limited to human pluripotent stem cells or embryonic stem cells, and induced pluripotent stem cells (iPSCs) have stronger immunosuppressive functions than immunoregulatory cells (including but not limited to mesenchymal stem cells, regulatory T cells (Treg cells), regulatory B cells, etc.) differentiated from normal stem cells. Xenograft rejection of porcine cells or organs expressing human SERPINA3D2 is reduced. Antibodies made by immunizing rabbits with SERPINA3D2 variants as antigens showed significant anti-human tumor effects in humanized mouse tumor-bearing models.

[0010] Based on the applicant's research findings above, the applicant requests protection for the following technical solutions:

[0011] To address the shortcomings of existing technologies, the first aspect of this invention provides SERPINA3 protein variants (including their derived polypeptide fragments such as mutant polypeptide fragments) and nucleotide sequences encoding these SERPINA3 protein variants (sometimes simply referred to as "coding nucleotide sequences").

[0012] According to a preferred embodiment, the SERPINA3 protein variant comprises the functional domain portion of the natural human SERPINA3 protein that is essential for binding to human SIRPα, and relative to the natural human SERPINA3 protein, the SERPINA3 protein variant has at least one amino acid mutation at at least one of the following three positions in the natural human SERPINA3 protein: (1) other parts besides the essential functional domain portion; (2) other positions in the essential functional domain portion besides the amino acids at positions P223, D225, Q228, E310 and Y312; (3) amino acids at positions P223, D225, Q228, E310 and Y312; and wherein the mutation at position (3) is a substitution mutation selected from the group consisting of P223I, P223L, P223V, D225E, Q228C, Q228M, Q228S, Q228Y, E310D, Y312C, Y312T and Y312W.

[0013] According to some preferred embodiments, the mutation is a deletion mutation, a substitution mutation, or an insertion mutation, preferably a deletion mutation or a substitution mutation.

[0014] According to some preferred embodiments, mutations at positions other than P223, D225, Q228, E310, and Y312 are deletion mutations. In this application, deletion mutations include one or more of the following: (1) deletion of a single amino acid; (2) deletion of multiple non-adjacent (discrete) amino acids; (3) deletion of two or more consecutive amino acids. For example, when the SERPINA3 protein variant is SEQ ID NO. 1, 2, or 3 below, SEQ ID NO. 1, 2, or 3 can be considered as the deletion of the portion of the natural human SERPINA3 protein other than the region of SEQ ID NO. 1, 2, or 3.

[0015] According to some preferred embodiments, the functional domain portion necessary for binding to human SIRPα is the region corresponding to SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 on the natural human SERPIA3 protein.

[0016] According to some preferred embodiments, the mutation is caused by human intervention, such as through genetic engineering.

[0017] According to some particularly preferred embodiments, the SERPINA3 protein variant comprises or has the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3 below:

[0018] As shown in SEQ ID NO. 1-3, P223 can be mutated to I (P223I), L (P223L) or V (P223V), D225 can be mutated to E (D225E), Q228 can be mutated to C (Q228C), M (Q228M), S (Q228S) or Y (Q228Y), E310 can be mutated to D (E310D), and Y312 can be mutated to C (Y312C), T (Y312T) or W (Y312W). The resulting variants still have the ability to bind to hSIRPα. Except for the P / D / Q and E / Y amino acids in the PXDXXQ and / or EXY amino acid sequences, which can be mutated to the above amino acids, the other amino acids can be substituted, deleted, or inserted to produce different protein mutants. These mutants preferably have more than 80% homology with the corresponding regions of the SERPINA3 protein mutants disclosed in this application.

[0019] According to some preferred embodiments, the SERPINA3 protein variant comprises at least one amino acid sequence from the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.

[0020] According to some preferred embodiments, the SERPINA3 protein variant comprises the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 and has undergone at least one substitution mutation, wherein the at least one substitution mutation is selected from the group consisting of P223I, P223L, P223V, D225E, Q228C, Q228M, Q228S, Q228Y, E310D, Y312C, Y312T, and Y312W. That is, the SERPINA3 protein variant comprises at least SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3 with at least one substitution mutation.

[0021] According to some preferred embodiments, the SERPINA3 protein variant has an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0022] According to some preferred embodiments, the SERPINA3 protein variant has the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 and has undergone at least one substitution mutation, wherein the at least one substitution mutation is selected from the group consisting of P223I, P223L, P223V, D225E, Q228C, Q228M, Q228S, Q228Y, E310D, Y312C, Y312T, and Y312W. That is, the SERPINA3 protein variant is SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3 with at least one substitution mutation.

[0023] According to some other preferred embodiments, the SERPINA3 protein variant has at least one amino acid mutation in only the parts other than the essential functional domain.

[0024] According to some other preferred embodiments, the SERPINA3 protein variant has at least one amino acid mutation at a position other than the amino acids at positions P223, D225, Q228, E310, and Y312 in the essential functional domain.

[0025] According to some other preferred embodiments, the SERPINA3 protein variant has at least one substitution mutation at the amino acid positions P223, D225, Q228, E310, and Y312, selected from the group consisting of P223I, P223L, P223V, D225E, Q228C, Q228M, Q228S, Q228Y, E310D, Y312C, Y312T, and Y312W.

[0026] It should be understood that SERPINA3 protein variants can be truncated forms of the natural human SERPINA3 protein. In other words, truncated forms of the natural human SERPINA3 protein can be considered as SERPINA3 protein variants with deletion mutations of several amino acids. SERPINA3 protein variants can be natural human SERPINA3 protein with point mutations, i.e., point-mutated forms of the SERPINA3 protein variant.

[0027] The above SERPINA3 protein variants can be linked to 6-9 histidine residues, the Fc fragment of human IgG, or other tags at their amino or carboxyl ends, or they can be part of other fusion proteins.

[0028] The first aspect of the present invention also provides nucleotide sequences (sometimes referred to as “coding nucleotide sequences”) that encode variants of the SERPINA3 protein. These encoding nucleotide sequences may be the corresponding nucleotide sequences of the original genes in an existing gene library, or they may be nucleotide sequences that encode the same SERPINA3 protein variants after single or multiple base substitutions.

[0029] A second aspect of the present invention provides a vector, cell, virus, or bacterium containing a nucleotide sequence.

[0030] According to a preferred embodiment, SERPINA3 protein variants can be obtained through synthesis, transgenic or recombinant techniques.

[0031] According to a preferred embodiment, the coding nucleotide sequence of the SERPINA3 protein variant can be obtained through synthesis and gene recombination techniques.

[0032] According to a preferred embodiment, microorganisms such as vectors, cells, viruses, or bacteria containing these encoding nucleotide sequences can be obtained through genetic engineering techniques such as construction or transfection.

[0033] A third aspect of this invention provides the use of SERPINA3 or a SERPINA3 protein variant in the prevention or treatment of a disease, or the use of SERPINA3 or a SERPINA3 protein variant in the preparation of a reagent or drug for the prevention or treatment of a disease, wherein the disease is SIRPα. + Diseases caused by abnormal cell function or SIRPα + Diseases in which cells are involved in the pathogenesis.

[0034] According to some preferred embodiments, the diseases include, but are not limited to, inflammation, autoimmune diseases, and transplant rejection-related diseases (such as graft-versus-host disease).

[0035] According to a preferred embodiment, the inflammation includes, but is not limited to, inflammation caused by bacterial / viral infection or aseptic inflammation.

[0036] According to a preferred embodiment, autoimmune diseases include, but are not limited to, multiple sclerosis, arthritis, inflammatory bowel disease, systemic lupus erythematosus, and autoimmune liver disease.

[0037] According to a preferred embodiment, transplantation in transplant rejection-related diseases includes, but is not limited to, cell transplantation (such as solid cell transplantation), tissue transplantation (such as bone marrow transplantation), or organ transplantation (including but not limited to multi-organ transplantation and cell and organ transplantation).

[0038] A fourth aspect of the present invention provides a formulation comprising SERPIA3 protein or a variant of SERPIA3 protein and a pharmaceutically acceptable carrier thereof, for the treatment of SIRPα. + Diseases in which cells are involved in the pathogenesis.

[0039] According to a preferred embodiment, the formulation further comprises one or more selected from anti-inflammatory agents, immunosuppressants, immunomodulators, anti-transplant rejection agents, antimicrobial agents, and cytotoxic drugs.

[0040] A fifth aspect of the present invention provides a pharmaceutical composition comprising the formulation provided in the fourth aspect of the present invention. Preferably, the pharmaceutical composition further comprises one or more anti-inflammatory agents, immunosuppressants, immunomodulators, anti-transplant rejection agents, antimicrobial agents, cytotoxic drugs, or combinations thereof.

[0041] The sixth aspect of the present invention provides a pharmaceutical composition comprising SERPINA3 protein or a variant of SERPINA3 protein, and the pharmaceutical composition further comprising one or more anti-inflammatory agents, immunosuppressants, immunomodulators, anti-transplant rejection agents, antimicrobial agents, cytotoxic drugs or combinations thereof.

[0042] The seventh aspect of the present invention provides the use of the nucleotide sequence encoding the SERPINA3 protein or the nucleotide sequence encoding a variant of the SERPINA3 protein in the preparation of a medicament for conditions related to inflammation and / or unwanted immune responses.

[0043] The eighth aspect of the present invention provides a nucleotide sequence encoding the SERPIA3 protein or a variant of the SERPIA3 protein for the preparation of a treatment for the prevention or treatment of SIRPα. + Application in substances involved in the pathogenesis of diseases (such as autoimmune diseases and / or other related diseases requiring suppression of the immune system).

[0044] The ninth aspect of the present invention provides the use of a nucleotide sequence encoding a SERPINA3 protein or a nucleotide sequence encoding a variant of the SERPINA3 protein in cells prepared for genetic modification for use as a drug, wherein the cells are gene-edited cells that overexpress a nucleotide sequence encoding a SERPINA3 protein or a nucleotide sequence encoding a variant of the SERPINA3 protein.

[0045] According to a preferred embodiment, the cells are gene-edited cells that overexpress the nucleotide sequence encoding the human SERPINA3 protein or the nucleotide sequence encoding a variant of the SERPINA3 protein.

[0046] According to a preferred embodiment, the gene-edited cells are mesenchymal stem cells or stem cells with differentiation potential (e.g., human stem cells with differentiation potential).

[0047] According to a preferred embodiment, the cells are mesenchymal stem cells or stem cells with differentiation potential that overexpress a nucleotide sequence encoding a SERPINA3 protein or a nucleotide sequence encoding a variant of the SERPINA3 protein.

[0048] The tenth aspect of the present invention provides a vector comprising a nucleotide sequence encoding a SERPINA3 protein or a nucleotide sequence encoding a variant of the SERPINA3 protein.

[0049] According to some preferred embodiments, the vector is used to deliver SERPINA3 protein or SERPINA3 protein variants, which can enhance the stability and / or bioavailability of SERPINA3 protein or SERPINA3 protein variants.

[0050] The eleventh aspect of the present invention provides a mesenchymal stem cell or a stem cell with differentiation potential, wherein the mesenchymal stem cell or the stem cell with differentiation potential contains exogenous nucleic acid, the exogenous nucleic acid containing a nucleotide sequence encoding SERPINA3 protein or a nucleotide sequence encoding a SERPINA3 variant, wherein the mesenchymal stem cell or the stem cell with differentiation potential overexpresses the nucleotide sequence encoding SERPINA3 protein or the nucleotide sequence encoding a SERPINA3 variant to enhance the immunosuppressive function of immunomodulatory cells differentiated from the mesenchymal stem cell or the stem cell with differentiation potential.

[0051] According to a preferred embodiment, the nucleotide sequence encoding the SERPINA3 protein or the nucleotide sequence encoding a SERPINA3 variant is operatively linked to the nucleotide sequence encoding the signal peptide, the promoter, and / or the enhancer.

[0052] The twelfth aspect of the present invention provides the use of a nucleotide sequence encoding a SERPINA3 protein or a variant of the SERPINA3 protein in the preparation of transgenic cells, transgenic organs, or transgenic animals for providing cell or organ donors for transplantation, wherein the transgenic cells are transgenic cells expressing the exogenous nucleotide sequence, the transgenic organs are transgenic organs expressing the exogenous nucleotide sequence, and the animals are transgenic animals expressing the exogenous nucleotide sequence.

[0053] According to a preferred embodiment, the transgenic animal can be a transgenic pig.

[0054] According to a preferred embodiment, the nucleotide sequence encoding the SERPINA3 protein or the nucleotide sequence encoding a variant of the SERPINA3 protein is operatively linked to the nucleotide sequence encoding the signal peptide, the promoter, and / or the enhancer.

[0055] The thirteenth aspect of the present invention provides a treatment for SIRPα + Methods for treating diseases involving cell involvement include administering an effective amount of SERPINA3 protein or a variant of SERPINA3 protein to a subject.

[0056] The fourteenth aspect of the present invention provides a method for preventing or treating SIRPα. + A method for the pathogenesis of a disease involving cells, comprising administering to a subject an effective amount of a reagent or pharmaceutical composition containing SERPINA3 protein or a variant of SERPINA3 protein.

[0057] The fifteenth aspect of the present invention provides a method for preventing or treating SIRPα. + A method for cellular involvement in the pathogenesis of a disease, comprising introducing a therapeutically effective number of cells into the blood or tissue of a subject, wherein the cells are gene-edited cells that overexpress a nucleotide sequence encoding a SERPINA3 protein or a variant of the SERPINA3 protein.

[0058] According to a preferred embodiment, the introduction of the subject's blood or tissue includes, but is not limited to, the introduction of the mesenchymal stem cells provided in the eleventh aspect of the present invention into one of the subject's veins or arteries by injection.

[0059] The sixteenth aspect of this invention provides the use of SERPINA3 protein variants as antigens in the preparation or screening of anti-SERPINA3 antibodies. Preferably, the anti-SERPINA3 antibody can be used in pharmaceutical applications as an anti-tumor drug, and can be used alone or in combination with immunotherapy drugs.

[0060] The seventeenth aspect of this invention provides SERPIA3 as a target in the development, screening, or preparation of drugs for the prevention and / or treatment of SIRPα. + Application in drugs for diseases in which cells are involved in the pathogenesis.

[0061] The eighteenth aspect of this invention provides an inhibitor (including an inhibitor or antibody) targeting SERPIA3 in the preparation of a product for the prevention or treatment of SIRPα. + Application in drugs for diseases in which cells are involved in the pathogenesis.

[0062] According to some preferred embodiments, the inhibitor has at least one of the following effects:

[0063] Inhibit the expression or activity of the SERPINA3 gene;

[0064] Inhibit the transcription of the SERPINA3 gene into mRNA;

[0065] Inhibit the translation of the SERPINA3 gene into protein;

[0066] Inhibit the activity or function of SERPINA3 protein;

[0067] Inhibit the binding of SERPIA3 protein to SIRPα.

[0068] According to some preferred embodiments, the inhibitor is one or more selected from nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, peptides, proteins, gene editing vectors, lentiviruses, or adeno-associated viruses that inhibit SERPINA3 expression.

[0069] The nineteenth aspect of the present invention provides a treatment for SIRPα. + Preparations for diseases involving cell pathogenesis, comprising: small molecule inhibitors that block the binding function of SERPIA3 protein to SIRPα, neutralizing / blocking antibodies, peptide-based inhibitors, nucleic acid interference molecules, competitive inhibitors of proteins or peptides and / or aptamers; and their pharmaceutically acceptable carriers.

[0070] The twentieth aspect of the present invention provides a pharmaceutical composition comprising the formulation of the nineteenth aspect of the present invention and one or more selected from immunotherapeutic drugs, immune checkpoint inhibitors and immune agonists.

[0071] The twenty-first aspect of the present invention provides a targeted drug comprising an inhibitor targeting SERPINA3, wherein the drug is capable of selectively acting on tumor cells while having minimal impact on normal cells.

[0072] The twenty-second aspect of the present invention provides a carrier for delivering a SERPINA3 inhibitor; preferably, the carrier is a liposome, nanoparticle, or polymer matrix.

[0073] According to some preferred embodiments, the carrier is a nanoparticle carrier coated with a SERPINA3 inhibitor, which is used to improve its targeted delivery efficiency in tumor tissue.

[0074] According to some preferred embodiments, the carrier is a biodegradable polymer carrier capable of releasing SERPINA3 inhibitors in the tumor microenvironment.

[0075] According to some preferred embodiments, the carrier comprises a liposome carrier of a SERPINA3 inhibitor to enhance its permeability and bioavailability in cells at the disease-affected site.

[0076] The twenty-third aspect of this invention provides a drug development kit for screening SERPINA3 inhibitors, wherein the kit includes: SERPINA3 protein or SERPINA3 protein variants, a purified sample of a SERPINA3 inhibitor, pharmacological reagents, and instructions for experimental procedures.

[0077] The twenty-fourth aspect of the present invention provides a controlled release system, wherein the system is used to regulate the release rate and time of a SERPINA3 inhibitor in vivo.

[0078] The twenty-fifth aspect of the present invention provides a method for releasing an antitumor drug, wherein the method includes administering to a subject an effective amount of a formulation or pharmaceutical composition containing a SERPINA3 inhibitor.

[0079] According to some preferred embodiments, the method includes introducing an effective amount of a carrier delivering a SERPINA3 inhibitor into a subject.

[0080] The twenty-sixth aspect of the present invention provides a combination therapy, wherein the method comprises: simultaneously or alternately using a drug containing a SERPIA3 inhibitor with other drugs used to treat SIRPα. + Drugs that involve cells in the pathogenesis of diseases, in order to enhance the therapeutic effect.

[0081] The twenty-seventh aspect of the present invention provides a method for preparing a drug containing a SERPINA3 inhibitor, wherein the method includes: mixing the SERPINA3 inhibitor with at least one excipient to form a formulation or pharmaceutical composition; and subjecting it to appropriate physical or chemical treatment to enhance its pharmacological activity.

[0082] SIRPα as described in various aspects of this invention + Cellular involvement in pathogenesis includes, but is not limited to, inflammation (such as inflammatory responses that exceed physiological benefits), autoimmune diseases, transplant rejection-related diseases (such as graft-versus-host disease and transplant rejection), and tumors.

[0083] According to a preferred embodiment, the inflammatory response includes, but is not limited to, inflammation caused by bacterial or viral infection as well as aseptic inflammation.

[0084] According to a preferred embodiment, autoimmune diseases include, but are not limited to, multiple sclerosis, arthritis, inflammatory bowel disease, systemic lupus erythematosus, and autoimmune liver disease.

[0085] According to a preferred embodiment, anti-transplant rejection includes, but is not limited to, anti-transplant rejection during cell transplantation, tissue transplantation, and organ transplantation.

[0086] According to a preferred embodiment, the tumor includes, but is not limited to, melanoma, bladder cancer, prostate cancer, breast cancer, colon cancer, lung cancer, liver cancer, ovarian cancer, pancreatic cancer, esophageal cancer, lymphoma, brain tumor, sarcoma, cervical cancer, prostate cancer, osteosarcoma, head and neck cancer, renal cell carcinoma, or gastric cancer.

[0087] This application demonstrates that SERPIA3 / SERPINA3k can communicate with SIRPα. + SIRPα on cells binds and activates its inhibitory function, thus inhibiting SIRPα at its source. + Cells exert their anti-inflammatory effects by secreting inflammatory factors, presenting antigens to activate T cells, and killing target cells. This action is related to the SERPIN SF1 domain of the SERPINA3 protein, but not to its RCL domain (amino acids 369–394 from the first amino acid of the signal peptide). Furthermore, the SERPINA3 protein variant containing only the SERPIN SF1 domain (SERPINA3D2, amino acids 202–366 from the first amino acid of the signal peptide) exerts its anti-inflammatory effect but lacks the pro-inflammatory effects of the full-length SERPINA3 protein on cardiomyocytes and cerebral vascular endothelial cells. Transfection of mesenchymal stem cells with this SERPINA3 protein variant gene and its derived polypeptide genes can enhance the immunosuppressive function of mesenchymal stem cells; transgenic individuals created using this SERPINA3 protein variant gene and its derived polypeptide genes can reduce xenograft rejection in these individuals. Antibodies made using this SERPINA3 protein variant have significant anti-tumor effects against a variety of tumors. The SERPINA3 protein variant can be used as a target protein for the production and screening of anti-SERPINA3 antibodies with anti-tumor effects.

[0088] Technical Effects: The SERPINA3 protein variant containing only the region binding to human SIRPα exerts anti-inflammatory effects similar to natural SERPINA3 without the pro-inflammatory side effects of natural SERPINA3 on cardiomyocytes and cerebral vascular endothelial cells, thus improving the safety of systemic application. Furthermore, this variant can be used as an antigen to prepare or screen anti-SERPINA3 antibodies, which exhibit significant anti-tumor activity. Genes of the SERPINA3 protein variant or its derived peptides can be used to modify MSCs or stem cells with differentiation potential. Regulatory cells differentiated from human MSCs or stem cells overexpressing the human SERPINA3 protein variant or its derived peptides exhibit stronger immunosuppressive functions and more potent therapeutic effects against inflammatory diseases. Attached Figure Description

[0089] Figure 1 (Example 1): Mouse SERPIA3k is a ligand for mSIRPα;

[0090] Figure 2 (Example 2): SERPIA3k inhibits SIRPα by binding to mSIRPα + The function of immune cells;

[0091] Figure 3 (Example 3): SERPINA3k can reduce LPS-induced lung inflammation;

[0092] Figure 4 (Example 4): SERPINA3k has a therapeutic effect on autoimmune diseases such as multiple sclerosis and rheumatoid arthritis;

[0093] Figure 5 (Example 5): SERPINA3k can alleviate allogeneic organ transplant rejection and GVHD in bone marrow transplantation;

[0094] Figure 6 (Example 6): Overexpression of the SERPINA3k gene in mouse MSCs can enhance the immunosuppressive function of MSCs and enhance the therapeutic effect of MSCs on EAE mice;

[0095] Figure 7 (Example 7): mSIRPα binds to the SERPIN-SF1 domain (A3kD2) of SERPINA3k, and SERPINA3kD2 has no pro-inflammatory effect on mouse cardiomyocytes and cerebral vascular endothelial cells;

[0096] Figure 8 (Example 8): Anti-SERPINA3k antibody prepared using mouse SERPINA3kD2 protein as antigen has anti-tumor effects on various tumors;

[0097] Figure 9 (Example 9): Human SERPINA3 protein is the human counterpart of mouse SERPINA3k, and is a ligand of human SIRPα, with similar functions to mouse SERPINA3k protein;

[0098] Figure 10 (Example 10): The SERPIN SF1 domain (A3D2) of human SERPIA3 is recognized by hSIRPα;

[0099] Figure 11 (Example 10): SERPINA3D2 J1, SERPINA3D2 J2 and hSIRPα with some key amino acid mutations still have affinity;

[0100] Figure 12 (Example 11): SERPINA3D2 has no pro-inflammatory effect on AC16 and HCMEC / D3 cells;

[0101] Figure 13 (Example 12): SERPINA3D2 has therapeutic potential for human multiple sclerosis;

[0102] Figure 14 (Example 13): Human MSCs overexpressing human SERPINA3D2 have stronger immunosuppressive function than natural human MSCs;

[0103] Figure 15 (Example 14): Stem cells overexpressing SERPINA3D2 have stronger immunosuppressive function than regulatory T cells (Treg cells) differentiated from normal stem cells;

[0104] Figure 16 (Example 15): Reduced rejection response to xenograft of porcine cells or organs expressing human SERPINA3D2;

[0105] Figure 17 (Example 16): Anti-human SERPINA3 antibody prepared using human SERPINA3D2 protein as antigen has anti-tumor effects on various human tumors. Detailed Implementation

[0106] The following is a detailed explanation with reference to the accompanying drawings.

[0107] In the accompanying drawings of this invention, NS represents no statistical significance, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0108] In this invention, "inflammation" preferably refers to inflammation in the subject exceeding the level of a physiologically beneficial inflammatory response, resulting in damage to cells, tissues, and / or organs at the site of inflammation. "An immune response requiring suppression" preferably refers to an alteration in the subject's immune system reactivity that has detrimental effects on their health and may involve the stimulation and / or production of cytokines and the recruitment of immune cells. Immune responses requiring suppression occur, for example, in autoimmune diseases, transplant rejection, allergies, or inflammatory diseases.

[0109] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0110] In this invention, the SERPINA3 protein variant or its derived polypeptide can be used alone, in combination with other anti-inflammatory drugs, or in a mixture with other anti-inflammatory drugs for the anti-inflammatory treatment of inflammation caused by bacterial or viral infections.

[0111] In this invention, the SERPINA3 protein variant or its derived polypeptide can be used alone, in combination with other anti-inflammatory drugs, or in a mixture with other anti-inflammatory drugs for the anti-inflammatory treatment of aseptic inflammation.

[0112] In this invention, the SERPINA3 protein variant or its derived polypeptide can be used alone, in combination with other anti-inflammatory / cytotoxic drugs, or in a mixture with other anti-inflammatory / cytotoxic drugs for the treatment of autoimmune diseases. Preferred autoimmune diseases include, but are not limited to, multiple sclerosis, arthritis, inflammatory bowel disease, systemic lupus erythematosus, and autoimmune liver diseases.

[0113] In this invention, the SERPINA3 protein variant or its derived polypeptide can be used alone, in combination with other anti-inflammatory / cytotoxic drugs, or in a mixture with other anti-inflammatory / cytotoxic drugs for the treatment of transplant rejection or the induction of transplant tolerance during cell transplantation and organ transplantation.

[0114] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0115] Main reagent sources:

[0116] Anti-mSIRPα (clone number P84) was purchased from BioXcell (West Lebanon, NH), USA; RAW264.7 (mouse macrophage cell line) and THP-1 cells (human macrophage cell line) were purchased from the American Type Culture Collection (ATCC) cell bank, USA; Pierce TMHis protein interaction pull-down kit was purchased from Thermo Fisher (Massachusetts, USA); anti-mouse CD11b (clone M1 / 70) was purchased from BD Biosciences (State of New Jersey, USA); anti-mouse Ly6G (clone 1A8), anti-mouse CD45 (clone 30-F11), anti-mouse CD19 (clone 6D5), anti-mouse CD3 (clone 17A2), anti-mouse TNFα-PE Cy7 (clone MP6-XT22), complete Freund's adjuvant, and PTX (Pertussis toxin) were purchased from Biolegend (SAN FRANK). DIEGO, CA92121, USA); anti-mouse p-SHP-1 (clone number tyr536) was purchased from Sigma-Aldrich (Massachusetts, USA); anti-Flag (clone number L5), anti-His (clone number J095G46) and anti-mouse SHP-1 (clone number Y47) were purchased from Abcam (Cambridge, England); CFSE was purchased from Thermo Fisher Scientific (Massachusetts, United States); anti-human CD14-FITC (clone number M5E2) and anti-human TNFα-APC (clone number MAb11) were purchased from BD Biosciences (State of New Jersey, USA); anti-Phosphotyrosine (clone number... Platinum was purchased from Millipore, Massachusetts, USA; MOG35-55 peptide was purchased from GenScript, China; LPS was purchased from Aladdin, China; and human IFNγ and human M-CSF were purchased from R&D, Minnesota, USA.

[0117] Example 1

[0118] Mouse SERPINA3k is a ligand for mSIRPα.

[0119] CD47 is a known ligand of SIRPα. Using surface plasmon resonance (SPR) technology, with mouse recombinant CD47 extracellular domain (mCD47) as a control, it was found that mouse SERPIA3k (A3k) can bind to mSIRPα with an affinity of approximately 2.393 × 10⁻⁶. -7 M is approximately the affinity of the mCD47 molecule for mSIRPα, which is 2.763 × 10⁻⁶. -6The M-value is 9 times that of mSIRPα, and it is difficult to dissociate after binding to mSIRPα (part A in Figure 1). Co-incubation of RAW264.7 (mouse macrophage) cells with different concentrations of SERPIA3k followed by staining with anti-mSIRPα antibody revealed that SERPIA3k could block the binding of anti-mSIRPα antibody to mSIRPα on the surface of RAW264.7 cells in a dose-dependent manner (part B in Figure 1). These results demonstrate a specific interaction between SERPIA3k and mSIRPα on mouse macrophages.

[0120] Next, we experimentally evaluated the effect of SERPIA3k binding on downstream mSIRPα signaling. RAW264.7 cells were incubated with 6×His-tagged mCD47 and SERPIA3k at 37°C for 30 min, and then cytoplasmic and cell membrane proteins were extracted using EBC buffer (containing a phosphatase inhibitor cocktail). A portion of the total protein was added to protein loading buffer and heated at 100°C for 5 min. After SDS-PAGE electrophoresis, the proteins were analyzed by Western blotting using anti-SHP-1 and anti-phosphorylated (p)SHP-1 antibodies. For the detection of mSIRPα phosphorylation, cell lysate was incubated overnight at 4°C with Protein A magnetic beads conjugated with mSIRPα antibody for co-immunoprecipitation (Co-IP). The magnetic beads were washed with PBS under magnetic adsorption, and the proteins were eluted with 100 mM Glycine solution at pH 2.5. After SDS-PAGE electrophoresis, the proteins were analyzed by Western blotting using mSIRPα antibody and phosphorylation antibody (4G10). SERPINA3k was found to increase the phosphorylation of mSIRPα and its downstream molecule SHP-1, and this effect was stronger than that of mCD47 (part C in Figure 1). These results indicate that SERPINA3k binding can activate downstream inhibitory signaling of mSIRPα.

[0121] Example 2

[0122] SERPINA3k inhibits mSIRPα by binding to it. + Functions of immune cells.

[0123] Next, we use several methods to verify the binding effect of SERPIA3k and mSIRPα on SIRPα. +Effects on Cellular Function. First, erythrocytes (RBCs) from mCD47KO mice or wild-type (WT) mice were stained with CFSE. RAW264.7 cells were cultured in serum-free medium for 2 hours, then treated with either anti-mSIRPα antibody (final concentration 2 μg / mL), or SERPINA3k protein (final concentration 10 μg / mL), or first treated with anti-mSIRPα antibody (final concentration 2 μg / mL) and then with SERPINA3k protein (final concentration 10 μg / mL), for 1 hour each time. The CFSE-stained RBCs were then co-incubated with the treated RAW264.7 cells for 1 hour. After lysing the RBCs with erythrocyte lysis buffer, slides were fixed with paraformaldehyde and mounted with DAPI. Phagocytosis was observed using a confocal microscope, and the phagocytosis of mCD47KO or WT RBCs by RAW264.7 cells was analyzed using ImageJ software. SERPINA3k was found to significantly reduce the phagocytosis of RBCs by RAW264.7 cells. mCd47KO Phagocytosis was observed, and pre-treatment with anti-mSIRPα blocking of mSIRPα inhibited this effect of SERPINA3k (part A in Figure 2). Furthermore, we in vitro used Phrodo red fluorescently labeled mCD47KO B16 tumor cell line (B16...). mCd47KO After co-incubating with RAW264.7 cells treated as described above for 4 hours, flow cytometry analysis showed that tumor cells labeled with Phrodo red fluorescent dye changed from non-fluorescent to emitting red fluorescence after being phagocytosed by macrophages. This indicated that SERPINA3k significantly reduced the susceptibility of RAW264.7 cells to B16. mCd47KO Phagocytosis of tumor cells was inhibited by pre-blocking mSIRPα with anti-mSIRPα antibody (part B in Figure 2). In summary, these results confirm that SERPIA3k can inhibit macrophage phagocytosis of mCD47KO erythrocytes or mCD47KO tumor cells by binding to mSIRPα.

[0124] RAW264.7 cells were treated with lipopolysaccharide (LPS, final concentration 100 ng / mL) for 4 hours under standard culture conditions (37℃, 5% CO2). After treatment with SERPINA3k (final concentration 10 μg / mL), anti-mSIRPα (final concentration 2 μg / mL), or anti-mSIRPα (final concentration 2 μg / mL) + SERPINA3k (final concentration 10 μg / mL) for 4 hours, the expression levels of IL-1β and TNFα in RAW264.7 cells were detected by flow cytometry. The results showed that SERPINA3k significantly inhibited LPS-induced IL-1β and TNFα production, and anti-mSIRPα effectively blocked this inhibitory effect (part C and part D of Figure 2).

[0125] The femur and tibia of euthanized B6 mice were aseptically removed, and bone marrow cells were washed out with PBS. Red blood cells were then lysed with erythrocyte lysis buffer (3-5 minutes, room temperature). After washing, the cells were resuspended in complete RPMI 1640 medium containing 10% fetal bovine serum at a concentration of 0.5 × 10⁻⁶ cells / mL. 6 Dendritic cells (DCs) were seeded at a density of 10 cells / mL in culture flasks containing 20 ng / mL mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF, R&D, 415-ML-01M) and 10 ng / mL IL-4 (PeproTech, 214-14-500). On days 2 and 4, half of the culture medium was replaced with fresh medium containing mGM-CSF. Dendritic cells (DCs) were harvested on day 6, washed twice with PBS, and cultured at 5 × 10⁻⁶ cells / mL. 4 Cells were resuspended in culture medium at a density of 100 cells / mL for subsequent experiments. Flow cytometry analysis revealed that mature differentiated BMDCs were mSIRPα positive (part E in Figure 2).

[0126] In the phagocytosis assay, Phrodo red dye-labeled B16 was used. mCd47KO Tumor cells and BMDCs were co-cultured at a 1:1 ratio, with the BMDCs density being 5 × 10⁶ cells / year. 4 Cells / mL, incubated at 37°C and 5% CO2 for 2 hours, then phagocytic efficiency of dendritic cells (DCs) was determined by flow cytometry. To assess changes in antigen presentation pathways during BMDC phagocytosis, CD11c was sorted using magnetic beads. + DCs were collected, and RNA was subsequently extracted for RT-qPCR analysis. The primer sequences used are as follows:

[0127] The results showed that SERPINA3k inhibited the engulfment of B16 by BMDCs. mCd47KO It has an inhibitory effect on the activity of tumor cells and downregulates the expression of antigen-presenting related molecules (including Tap1, B2M, PSMB9 and H2Ab1) in BMDCs. Anti-mSIRPα can effectively block this inhibitory effect of SERPIA3k (part F in Figure 2, part G in Figure 2).

[0128] Mouse peripheral blood neutrophils were isolated using a mouse peripheral blood neutrophil isolation kit (P9201, Solarbio) according to the manufacturer's protocol: anticoagulated whole blood was placed on the separation medium and centrifuged at 500g for 30 minutes at room temperature with the brake off. The neutrophil layer was collected, washed twice with PBS, and resuspended in RPMI-1640 complete medium. After treatment with LPS (final concentration 100 ng / mL) for 4 hours, SERPINA3k (final concentration 10 μg / mL), anti-mSIRPα (final concentration 2 μg / mL), or anti-mSIRPα (final concentration 2 μg / mL) + SERPINA3k (final concentration 10 μg / mL) were added for 4 hours. Flow cytometry was then used to detect TNFα expression in neutrophils. The results showed that SERPINA3k significantly inhibited LPS-induced TNFα expression in neutrophils, and anti-mSIRPα effectively blocked this inhibitory effect (part H in Figure 2).

[0129] C57BL / 6 mice were euthanized, and spleens were aseptically harvested to prepare single-cell suspensions. NK cells were positively selected and purified using anti-mouse CD335(NKp46)-PE antibody (Miltenyi) and anti-PE magnetic beads (Miltenyi, 130-048-801). The purified NK cells were cultured in complete RPMI 1640 medium containing a final concentration of 50 ng / mL recombinant mouse IL-2. NK cell expression was detected at different time points. The results showed that although... mSIRPα expression was not detected on NK cells, but it was detected on IL-2 activated NK cells (part I in Figure 2). Activated NK cells (effective cells) at the time point of highest mSIRPα expression were compared with CFSE-labeled B16. mCd47KO Cells (target cells) were mixed at a 1:1 ratio (total cell count 8 × 10⁻⁶). 4 7-AAD was co-cultured in U-bottom 96-well plates with or without SERPINA 3k (final concentration 10 μg / mL). Flow cytometry was used to detect 7-AAD after 4 hours. + CFSE + The proportion of target cells (left of part J in Figure 2) and according to the formula ([%7-AAD) + CFSE + E+T ]-[%7-AAD + CFSE + The specific killing rate of NK cells against target cells was calculated using [targets alone] (right side of J in Figure 2). SERPINA3k was found to significantly inhibit IL-2-activated NK cells' effect on B16. mCd47KO Cytotoxic activity of tumor cells (part J in Figure 2).

[0130] In summary, these findings indicate that SERPIA3k is a functional ligand for mSIRPα and can inhibit mSIRPα. + The function of immune cells.

[0131] Example 3

[0132] SERPINA3k can reduce LPS-induced lung inflammation.

[0133] To simulate lung inflammation caused by bacterial or viral infections, we established an LPS-induced mouse pneumonia model: 6-8 week old B6 mice were anesthetized and fixed on an inclined board at a 60° angle to the operating table. The upper incisors of the mice were suspended from the top of the board. The mice's mouths were opened with forceps, and the tongues were pulled out from one side. A sterilized tubing was inserted into the pharynx. The required LPS dose was calculated at 5 mg (LPS) / kg (mouse body weight). The required LPS was dissolved in 25 μL PBS solution with or without 50 μg SERPINA3k protein and administered via tracheal nebulization. Control mice received an equal volume of PBS. Four days later, tests showed that SERPINA3k significantly reduced the number of neutrophils (Ly6G) in bronchoalveolar lavage fluid and lung tissue. + CD11b + ) in CD45 + CD19 - CD3 - The proportion and number of cells (part A in Figure 3); pathological sections showed that in the LPS-induced group, the lungs exhibited alveolar structural disorder, extensive thickening of alveolar walls, and partial alveolar destruction, leading to widening of alveolar septa and obvious inflammatory cell infiltration around the bronchi; after treatment with SERPINA3k protein, inflammatory cell infiltration was significantly reduced and alveolar septal thickening was alleviated (part B in Figure 3). Mouse lung tissue was digested into single-cell suspensions and cultured in RPMI 1640 complete medium containing LPS at a final concentration of 3 μg / mL and phorbol 12-myristate 13-acetate (PMA) at a final concentration of 100 ng / mL. SERPINA3k, mCD47 at a final concentration of 50 μg / mL, and SERPINA3k + mCD47 at a final concentration of 50 μg / mL were added or omitted to the medium, respectively. After 3 hours of culture, flow cytometry analysis showed that SERPINA3k could alleviate LPS-induced alveolar (CD11b) cytotoxicity. - SiglecF + ) and lung interstitium (CD11b + F4 / 80 +The expression of TNFα in macrophages was inhibited (part C in Figure 3), and the inhibitory effect of SERPINA3k was stronger than that of mCD47. In the presence of mCD47, the addition of SERPINA3k could further inhibit LPS-induced TNFα expression in macrophages (part C in Figure 3), which is consistent with the result in part A of Figure 1 that SERPINA3k has a higher affinity for mSIRPα than mCD47.

[0134] Example 4

[0135] SERPINA3k has therapeutic effects on autoimmune diseases such as multiple sclerosis and rheumatoid arthritis.

[0136] SERPINA3k can alleviate the severity of multiple sclerosis: Experimental allergic encephalomyelitis (EAE) has an immunopathogenesis and lesions similar to those of human multiple sclerosis (MS). To investigate the effect of SERPINA3k on MS, we established an EAE model: MOG35-55 peptide was fully dissolved and diluted to 2 mg / mL in sterile PBS, and then mixed with complete Freund's adjuvant containing 6 mg / mL Mycobacterium tuberculosis at a 1:1 volume ratio and fully emulsified to obtain a water-in-oil antigen emulsion. The final concentration of MOG35-55 peptide in the emulsion was 1 mg / mL. Approximately 8-week-old B6 mice were anesthetized and injected subcutaneously with 200 μL of the antigen emulsion at four points on both sides of the spine. Thirty minutes after the injection of the antigen emulsion, the mice were intraperitoneally injected with 200 μL of 2 μg / mL PTX (Pertussis toxin) solution (i.e., 400 ng / mouse). Forty-eight hours later, 200 μL of 2 μg / mL PTX solution was injected intraperitoneally again. The start of modeling was recorded as Day 0. From Day 0, the mice were weighed and their condition observed daily. Neurological function was assessed using a 5-point scoring system: 0 points: asymptomatic; 1 point: tail weakness or staggering gait; 2 points: unilateral hind limb weakness or paralysis; 3 points: bilateral hind limb paralysis; 4 points: complete bilateral hind limb paralysis accompanied by forelimb paralysis; 5 points: near-death state or death. Mice were divided into a SERPINA3k treatment group and a disease control group, with 5 mice in each group. At the onset of disease (clinical score approximately 0.5), the treatment group received an intraperitoneal injection of 0.2 mg SERPINA3k protein every 3 days, while the disease control group received an equal volume of PBS solution intraperitoneally until the mice were sacrificed. Mice were weighed daily and scored according to the EAE model clinical scoring criteria for 30 consecutive days. Data were analyzed using GraphPad 8.0 and corresponding charts were generated. The results showed that SERPINA3k significantly reduced the severity of EAE in diseased mice (part A in Figure 4). Flow cytometry showed IL-17 infiltrating the spinal cord. + and IFNγ + CD4 + The proportion and number of T cells decreased (part B in Figure 4). Furthermore, the SERPINA3k treatment group showed a decrease in CD4 count. + MOG-Class II tetramer in T cells + The proportion of cells was significantly lower than that of the PBS control group (part C in Figure 4). These findings are consistent with the results showing that the SERPINA3k protein inhibits the phagocytosis of BMDCs, thereby suppressing antigen presentation (part G in Figure 2).

[0137] SERPINA3k can alleviate the symptoms of rheumatoid arthritis (RA): Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovial inflammation of symmetrical small joints, with persistent and recurrent inflammatory attacks as its main feature. The tissue and immunological changes of the experimental arthritis mouse model (Collagen-induced arthritis, CIA) are most similar to those of human RA, making it one of the better animal models for mimicking human RA. An 8-week-old B6 (H-2b) mouse model of CIA was induced. 2 mg / mL of chicken type II collagen was mixed with an equal volume of complete Freund's adjuvant (CFA) containing 5 mg / mL Mycobacterium tuberculosis, and emulsified thoroughly on ice using a three-way valve. After anesthetizing the mice, the hair at the base of the tail was removed and disinfected with 75% alcohol. 100 μL of the emulsion (containing 100 μg of collagen) was injected into the base of the tail. A booster immunization was administered 21 days after the initial immunization, with a subcutaneous injection of 100 μL of collagen CFA emulsion. The booster injection was performed at a different site than the initial injection. Arthritis appeared approximately 23-35 days after the initial immunization. Between 42-56 days, the incidence of arthritis in these mice reached 50-70%. Mice that successfully developed the arthritis model were selected on day 23 after the initial immunization and randomly divided into two groups of five mice each: a PBS group and a treatment group (SERPINA3k protein 200 μg / mouse / dose). Five unimmunized mice served as a control group. Starting on day 23 after the initial immunization, mice in the treatment group received intraperitoneal injections of SERPINA3k protein every 3 days for 21 consecutive days, while mice in the PBS group received an equal volume of PBS. Every 3 days after treatment, the thickness of the toes and the width of the ankle joint were measured using calipers, and the Arthritis Index (AI) was recorded. The AI ​​scoring criteria are as follows: 0 points: no redness or swelling; 1 point: erythema or mild swelling of the little toe joint; 2 points: moderate swelling of the toe joint; 3 points: swelling of the paw below the ankle joint; 4 points: swelling of the entire paw, including the ankle joint. A cumulative score of 6-8 points or higher for all four limb joints indicates severe arthritis. The effect of SERPINA3k protein on paw inflammation in CIA mice was comprehensively evaluated by combining changes in hind paw thickness with AI scores. After modeling, the hind paws of the model group mice began to swell slowly, and the hind paw thickness gradually increased, exceeding that of the non-model group. Compared with the PBS group, the hind paw thickness of the SERPINA3k protein treatment group mice was significantly reduced from day 30 after initial immunization to the end of the treatment period. The AI ​​scores of the treatment group mice were reduced, and from day 3 after administration to the end of the treatment period, the AI ​​scores were significantly lower than those of the model group (part D in Figure 4).

[0138] In summary, in EAE and CIA mouse models, treatment with SERPIA3k inhibited mSIRPα. + Cellular antigen presentation and inflammatory factor secretion reduce autoimmune inflammatory damage and slow disease progression.

[0139] Example 5

[0140] SERPINA3k can alleviate rejection in allogeneic organ transplants and GVHD in bone marrow transplants.

[0141] SERPINA3k can alleviate host rejection of transplanted organs: Next, we examined whether SERPINA3k helps reduce transplant rejection. Heart grafts were obtained from 8-10 week old Balb / c mice. The aorta and pulmonary artery of the grafts were anastomosed end-to-side with the aorta and vena cava of 8-10 week old B6 recipient mice, respectively, and then transplanted into the peritoneal cavity of the recipient mice. The survival status of the transplanted hearts was monitored daily by abdominal palpation, and transplant rejection was defined as palpable cardiac arrest (confirmed by exploratory laparotomy). Mice were divided into a SERPINA3k treatment group and a disease control group, with 5-8 mice in each group. The treatment group received intraperitoneal injections of 200 μg SERPINA3k protein / mouse / 4 days starting from the day of transplantation. The disease control group received an equal volume of PBS solution intraperitoneally until the mice were sacrificed. The survival status of the transplanted hearts was observed daily until the heartbeat of the transplanted hearts stopped. The results showed that SERPINA3k significantly reduced host rejection of the transplanted hearts and prolonged the survival time of the transplanted hearts (part A in Figure 5).

[0142] SERPINA3k can alleviate GVHD in allogeneic bone marrow transplantation: 1×10⁻⁶ SERPINA3k from B6 mice. 7 Bone marrow cells and 3×10 6 An acute GVHD model of allogeneic bone marrow transplantation was established in 8-week-old Balb / c mice after T-cell transplantation followed by lethal dose irradiation. These mice were divided into two groups. Starting from day 7 post-transplantation, mice were administered 200 μg / mouse of SERPINA3k protein via intraperitoneal injection / no injection every 3 days, with one group receiving irradiation only and the other receiving only 1×10⁻⁶ transplanted mice. 7 Balb / c mice were used as a control group after irradiation of B6 mouse bone marrow cells. Recipient mice fed under SPF conditions were given sterile water containing gentamicin for 1 week before and 2 weeks after bone marrow transplantation. The results showed that injection of SERPINA3k protein could significantly reduce acute GVHD in allogeneic bone marrow transplantation and prolong the survival of mice (part B in Figure 5).

[0143] Example 6

[0144] Overexpression of the SERPINA3k gene in mouse MSCs can enhance the immunosuppressive function of MSCs and improve the therapeutic effect of MSCs on EAE mice.

[0145] To explore whether SERPINA3k gene overexpression could be used to create more functional MSCs for the treatment of inflammatory diseases, we inserted the SERPINA3k gene into a lentivirus plasmid vector (part A in Figure 6). The recombinant lentivirus vector and the empty vector were transfected into 293 cells to package lentivirus containing the SERPINA3k gene and a control virus containing only the empty vector, respectively. Mouse MSCs (mMSCs) were then infected with the lentivirus containing the SERPINA3k gene and the control lentivirus to obtain SERPINA3k-overexpressing mMSCs. A3k and control mMSC NC Cells, adjust cell concentration to 2×10 5 / mL for later use. An acute GVHD model of B6→Balb / c bone marrow transplantation was established according to Example 5 above. The mice were divided into 3 groups (n=5 per group). Starting on day 7 post-transplantation, mMSCs were injected via tail vein every 7 days with or without injection. NC or mMSC A3k 5×10 cells 4 One, irradiated only and transplanted only 1×10 7 Balb / c mice were used as a control group after irradiation of B6 mouse bone marrow cells. Recipient mice fed under SPF conditions were given sterile water containing gentamicin one week before and two weeks after bone marrow transplantation. The results showed that mMSCs A3k Cells exhibited better performance than mMSCs NC Stronger immunosuppressive function, injected with mMSCs A3k The survival time of mice injected with mMSCs was longer. NC The mice showed a significant increase in length (part B in Figure 6).

[0146] A mouse EAE model was established according to the method in Example 4. The start of model establishment was recorded as Day 0. Starting from Day 0, the mice were weighed daily and their condition was observed. Neurological function was assessed using the 5-point scoring method described in Example 4. The mice were divided into a disease control group and a mMSC group. NC Treatment group and mMSC A3k Treatment group (n=5 per group). Mice were intravenously injected with 5×10⁻⁶ mg / L solution every 7 days starting on day 8 post-immunization. 4 mMSC NC Or mMSC A3k The control group received an equal volume of PBS intravenously at the same time point until the mice were sacrificed. Mice were weighed daily and scored according to the EAE clinical scoring criteria. Results showed that mMSCs... A3k It showed better performance than mMSC NC Stronger immunosuppressive function, injected with mMSCs A3k Mice injected with mMSCs NCThe EAE symptoms in mice were significantly reduced (part C in Figure 6).

[0147] These results demonstrate that the SERPINA3k gene can be used to modify cells (such as MSCs) to overexpress SERPINA3k and have a stronger immunosuppressive function than natural cells, thereby being used to treat inflammatory diseases or other related diseases that require immunosuppression (such as autoimmune diseases and GVHD).

[0148] Example 7

[0149] mSIRPα binds to the SERPIN-SF1 domain (A3kD2) of SERPINA3k, and SERPINA3kD2 has no pro-inflammatory effect on mouse cardiomyocytes and cerebral vascular endothelial cells.

[0150] The 3D structure of the SERPIA3k protein predicted using AlphaFold contains two key regions: the serine protease inhibitor superfamily domain 1 (SERPIN-SF1) (amino acids 130-323 starting from the first amino acid of the SERPIA3k protein signal peptide) and the reaction center loop (RCL) (amino acids 369-394 starting from the first amino acid of the SERPIA3k protein signal peptide) (part A in Figure 7). The RCL exerts its inhibitory effect by binding to the active site of the serine protease and is the main domain responsible for enzyme inactivation. AlphaFold3 simulations show that SERPIA3k mainly binds to the S50 / R107, R91, S109, V111, H174, N110, and V154 residues of mSIRPα through amino acid residues D132, Q186, Q188, K215, D221, E371, and K384 (mainly located in SERPIN-SF1) (part A and part B in Figure 7).

[0151] To investigate the interaction between SERPIA3k and mSIRPα, we expressed different fragments of the SERPIA3k protein containing a 6×hi tag: SERPIA3kD1 (amino acids 22-125 from the first amino acid of the SERPIA3k signal peptide), SERPIN-SF1 (SERPINIA3kD2) (amino acids 130-395 from the first amino acid of the SERPIA3k signal peptide), and SERPIA3kD3 (amino acids 396 to the terminal amino acid from the first amino acid of the SERPIA3k signal peptide). mSIRPα-Fc was diluted to 2 μg / mL with coating buffer (50 mM carbonate-bicarbonate buffer, pH 9.6) and coated onto ELISA plates overnight at 4°C. The plate was washed five times with TBST, blocked with TBST solution containing 1% BSA at room temperature for 1 hour, and then 100 μL of 30 nM full-length SERPINA3k with 6 His tags (typically 6-9 histidines) and each peptide fragment were added and incubated at 37°C for 1 hour. Protein interactions were detected using rabbit anti-His (JPAR-2; Abcam, ab245114) and horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (Proteintech, SA00001-2), with color development using TMB substrate (Absin, abs9288) and termination of the reaction using stop solution (Absin, abs9472). Absorbance was measured at 450 nm, with a reference wavelength of 570 nm. The results showed that only SERPINA3kD2 of different SERPINA3k protein fragments bound to mSIRPα (part C in Figure 7), and its affinity was comparable to that of the full-length SERPINA3k / mSIRPα (part D in Figure 7 and part A in Figure 1). Furthermore, SERPINA3kD2 induced phosphorylation of mSIRPα and SHP-1, inhibited the phagocytosis of mCD47KO erythrocytes by RAW264.7 cells, and reduced IL-1β production in LPS-stimulated RAW264.7 cells. Anti-mSIRPα effectively blocked these inhibitory effects (part EG in Figure 7).

[0152] HL-1 (mouse cardiomyocyte cell line) and bEnd.3 (mouse brain microvascular endothelial cell line) cells were treated with LPS (final concentration 100 ng / mL), SERPINA3k, SERPINA3kD2 (10 nM), or control protein (293 cell culture supernatant transfected with an empty vector containing an equal amount of protein) for 48 hours under standard culture conditions (37℃, 5% CO2). Total RNA was then extracted and reverse transcribed into cDNA. The expression of mouse TNFα, IL-6, and VCAM-1 was detected by qRT-PCR. The primer sequences used are as follows:

[0153] Consistent with reports of pro-inflammatory effects of full-length SERPINA3 on cardiomyocytes and cerebral vascular endothelial cells (Xie W, Zhang A, Huang X, et al. Shock. 2023 May 1; 59(5):791-802.; Kim H, Leng K, Park J, et al. Nat Commun. 2022; 13(1):6581.), full-length SERPINA3k upregulated the mRNA levels of TNFα and IL-6 in HL-1 cells and the mRNA levels of TNFα, IL-6, and VCAM1 in bEnd.3 cells in in vitro experiments. However, SERPINA3kD2 failed to induce upregulation of TNFα, IL-6, and VCAM1 mRNA levels in these cell lines (parts H and I in Figure 7). These results indicate that SERPINA3kD2, unlike full-length SERPINA3k, has no pro-inflammatory effect on mouse cardiomyocytes and cerebral vascular endothelial cells.

[0154] Example 8

[0155] Anti-SERPINA3k antibodies prepared using mouse SERPINA3kD2 protein as an antigen have anti-tumor effects on various tumors.

[0156] Given the abundance of myeloid cells in the tumor microenvironment, and the often suppressed anti-tumor functions of myeloid cells and NK cells, combined with the aforementioned findings on the immunosuppressive effects of SERPINA3k on macrophages and activated NK cells, we were prompted to investigate the expression pattern of SERPINA3k in tumor tissue and whether SERPINA3k is involved in tumor immune escape. Tumor tissue or surrounding tissue (more than 1 cm from the tumor mass) was homogenized using high-speed electrophoresis and then added to reducing SDS-PAGE gel electrophoresis loading buffer. The samples were denatured by boiling at 100°C for 10 minutes. Approximately 30-40 μg of protein sample was loaded onto a 4-12% pre-prepared gel for electrophoresis, and the protein bands on the gel were then transferred to a polyvinyl fluoride (PVDF) membrane. The transferred PVDF membrane was blocked at room temperature for 1 hour in TBST (10 mM Tris pH 8.0, 150 mM NaCl, and 0.1% Tween-20) buffer containing 5% skim milk powder. The membrane was then incubated overnight in 5% BSA-TBST buffer containing either anti-SERPINA3k (1:3000 dilution) or anti-GAPDH (1:5000 dilution). The membrane was washed and incubated at room temperature for 1 hour with HRP-labeled anti-rabbit IgG (1:2000 dilution). After rinsing the membrane, it was then treated with Pierce... TMECL Western blotting. Western blot results confirmed that SERPINA3k was widely expressed in various mouse tumor tissues, with expression levels in tumor tissues being much higher than in adjacent tissues (part A in Figure 8). Combined with the high affinity and poor dissociation of SERPINA3k / mSIRPα, which differs from the binding of mCD47 / mSIRPα (part A in Figure 1), this suggests that SERPINA3k may have a stronger pro-tumor effect than CD47.

[0157] Next, we prepared anti-mouse SERPINA3k antibodies (A3k Ab) by immunizing New Zealand white rabbits with mouse SERPINA3kD2 protein. Three healthy 6-week-old female New Zealand white rabbits (weighing 2.5-3 kg) were allowed to acclimatize to their environment for several days before immunization. Each 2 mg of SERPINA3kD2 protein (500 μL) was emulsified with an equal volume (500 μL) of complete Freund's adjuvant via a three-way stopcock to obtain an emulsion. A total of 1.2 mL of the emulsion was injected subcutaneously at multiple sites on the back for primary immunization (day 0). Booster immunizations were administered on days 14, 28, and 42 with incomplete Freund's adjuvant (antigen concentration of 1 mg / mL, 1.2 mL per rabbit). On days 35 and 56, 2-10 mL of peripheral blood was collected from the marginal ear vein and centrifuged at 10,000 rpm for 15 minutes to separate the serum. When the antibody titer reaches 1:64000 (usually on day 35), a final booster immunization is performed, followed by terminal blood sampling via cardiac puncture on days 56-70. After overnight coagulation at 4°C, serum is collected by centrifugation at 3500 rpm for 10 minutes. Rabbit serum antibodies are purified using a HiTrap Protein GHP column (Sartorius, 17040401) via acidic elution (0.1 mol glycine hydrochloride, pH 2.7). All eluents are immediately neutralized and desalted to PBS or other physiological buffers using a HiTrap desalting column (Sartorius, 29048684). Protein concentration is determined using the BCA method (Thermo Fisher Scientific, 23227), and purity (>95%) is verified by SDS-PAGE and Coomassie Brilliant Blue staining. Purified antibodies are aliquoted and stored at -80°C for later use.

[0158] Next, we evaluated the anti-tumor effects of the prepared anti-SERPINA3k antibody in different types of mouse tumor models. First, 5 × 10⁵ antibodies were subcutaneously injected into the backs of B6 mice (8-10 weeks old, n = 5 / group). 5 Five B16 (melanoma), MB49 (bladder cancer), and RM1 (prostate cancer) tumor cells were injected subcutaneously into the backs of Balb / c mice (8-10 weeks old, n=5 / group) at a dose of 5 × 10⁸ cells. 5 One CT26 (colon cancer) or 5×10 CT26 (in situ injection into the breast) 54T1 (triple-negative breast cancer) tumor cells were inoculated. Mice were monitored daily after inoculation, and the tumor was measured with calipers every 2-4 days. Tumor volume = 0.5 × length × width. 2 Treatment was initiated approximately 7-10 days after inoculation when the tumor became palpable. The regimen was as follows: intraperitoneal injection of 250 μg of anti-SERPINA3k, anti-mSIRPα (P84), or 250 μg of anti-SERPINA3k + 250 μg of anti-PD1 every 4 days. Control group mice were injected with the same amount of isotype IgG. In B16, MB49, RM1, CT26, or 4T1 tumors, tumor growth was significantly reduced in the anti-SERPINA3k antibody group compared to the control group (part BD in Figure 8). These results indicate that anti-SERPINA3k antibodies prepared using SERPINA3kD2 protein as an antigen have broad-spectrum anti-tumor activity. In MB49 tumor-bearing mice, the tumor growth of mice injected with anti-SERPINA3k or anti-PD1 was significantly lower than that of the control group. Furthermore, the tumor growth of mice in the combined anti-SERPINA3k and anti-PD1 treatment group was significantly lower than that of the two antibody treatment groups alone. These results indicate that anti-SERPINA3k antibodies made with SERPINA3kD2 protein as an antigen can be used in combination with anti-PD1 antibodies for anti-tumor therapy (part C in Figure 8).

[0159] Example 9

[0160] Human SERPINA3 protein is the human counterpart of mouse SERPINA3k. It is a ligand of hSIRPα and has similar functions to mouse SERPINA3k protein.

[0161] In the human genome, only one gene, SERPINA3, corresponds to the mouse SERPINA3k family. To determine whether human SERPINA3 is also a ligand for hSIRPα, we expressed the full-length proteins of hSIRPα, hCD47, and human SERPINA3. SPR assays showed that SERPINA3 (A3) had a higher affinity for the major hSIRPα isoforms (including V1, V2, and V8) than hCD47 had for its corresponding hSIRPα isoform, and was also more difficult to dissociate after binding (part AD in Figure 9). These results indicate that SERPINA3 is a ligand for hSIRPα.

[0162] Fresh human peripheral blood was diluted 1:1 with PBS and added to a centrifuge tube containing 20 mL of Ficoll solution, with the blood dilution on top of the Ficoll solution. Centrifugation was performed at 1600 rpm for 20 minutes at room temperature, with the brake off during centrifugation. After centrifugation, the intermediate white membrane layer cells were aspirated, centrifuged again, and washed with PBS to obtain human peripheral blood mononuclear cells (PBMCs). The isolated human PBMCs were stained with anti-human CD14-FITC antibody, washed with PBS (containing 1% bovine serum albumin) to remove free antibodies, and then incubated with anti-FITC-labeled magnetic beads. CD14 cells were then separated using a magnetic sorting column. + Mononuclear cells, at 5 × 10 5 Cells were seeded in 24-well plates and cultured at 37°C with 5% CO2 for 6 days. Afterward, LPS (100 ng / mL), human IFNγ (20 ng / mL), and human M-CSF (100 ng / mL) were added to polarize the cells for 24 hours. In phagocytosis assays, SERPINA3 (10 μg / mL) was added or not added during human monocyte polarization. After 24 hours of culture, macrophages were labeled with CFSE and 1 × 10⁶ Phrodo red-stained human erythrocytes pre-blocked with anti-hCD47 were added. 7 Two hours later, flow cytometry was used to detect the phagocytosis of erythrocytes by monocytes. The results showed that hSERPINA3 significantly inhibited the phagocytosis of pre-blocked anti-hCD47 human erythrocytes by polarized human monocytes, and the use of anti-hSIRPα before adding hSERPINA3 blocked this function of SERPINA3 (part E in Figure 9). In the detection of inflammatory factors, SERPINA3 was added or not added at a final concentration of 10 μg / mL to polarized human monocytes, and cells were collected 24 hours later for flow cytometry detection of TNFα expression. The results showed that SERPINA3 reduced TNFα expression in monocytes. + The proportion of cells (part F in Figure 9) increased the phosphorylation levels of hSIRPα and SHP-1 (part G in Figure 9). These results indicate that human SERPINA3 is the human counterpart of mouse SERPINA3k, a ligand of hSIRPα, and has similar functions to mouse SERPINA3k.

[0163] Example 10

[0164] The SERPIN SF1 domain (SERPINA3D2) of human SERPIA3 is recognized by hSIRPα.

[0165] To determine which domain of SERPIA3 is recognized by hSIRPα, we first compared the structural similarity between mouse SERPIA3k and human SERPIA3. We found that human SERPIA3 possesses a SERPIN SF1 domain (amino acids 202-366 from the first amino acid of the SERPIA3 signal peptide) and an RCL domain (amino acids 369-394 from the first amino acid of the SERPIA3k signal peptide) similar to mouse SERPIA3k (part A in Figure 10). AlphaFold simulations showed that SERPIA3 primarily binds to hSIRPα residues R99, K126, N100, R125, V63, and E31 via amino acid residues P223, D225, Q228, E310, Y312, and N396, respectively (parts A and B in Figure 10). Therefore, we expressed different fragments of the SERPINA3 protein containing a 6×his tag: A3D1 (amino acids 24-200 from the first amino acid of the SERPINA3 signal peptide), A3D2 (amino acids 202-366 from the first amino acid of the SERPINA3k signal peptide), and A3D3 (amino acids 369-418 from the first amino acid of the SERPINA3k signal peptide). The hSIRPα-Fc fusion protein was diluted to 2 μg / mL with coating buffer (50 mM carbonate-bicarbonate, pH 9.6) and coated onto ELISA plates overnight at 4°C. The plates were washed five times with TBST, blocked with TBST solution containing 1% BSA at room temperature for 1 hour, and then 100 μL of 30 nM His-tagged full-length SERPINA3 and each peptide fragment were added and incubated at 37°C for 1 hour. Protein interactions were detected using rabbit anti-His (JPAR-2, Abcam, ab245114) and horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (Proteintech, SA00001-2). Colorimetric development was performed using TMB substrate (Absin, abs9288), and the reaction was terminated with stop solution (Absin, abs9472). Absorbance was measured at 450 nm, with a reference wavelength of 570 nm. ELISA affinity analysis showed that only SERPINA3D2 among different SERPINA3 protein fragments exhibited strong affinity for hSIRPα, comparable to the affinity of full-length SERPINA3 and hSIRPα (parts C and D in Figure 10).Furthermore, using the method in Example 9, it was found that SERPINA3D2 induced the same degree of phosphorylation of hSIRPα and SHP-1 in THP-1 cells as full-length SERPINA3, exhibited similar inhibitory effects on THP-1 cells phagocytosis of human erythrocytes coated with anti-hCD47, and had the same inhibitory effect on the expression of IL-1β and TNFα in LPS-activated THP-1 cells. The above-mentioned effects of SERPINA3D2 could be inhibited by anti-hSIRPα (EH part in Figure 10).

[0166] Based on the structure and residue distribution simulated by AlphaFold, SERPINA3D2 was further truncated into two fragments: SERPINA3D2 J1 (amino acids 218-270 from the first amino acid of the SERPINA3 protein signal peptide) and SERPINA3D2 J2 (amino acids 282-332 from the first amino acid of the SERPINA3 protein signal peptide). ELISA affinity analysis confirmed that SERPINA3D2 J1 and SERPINA3D2 J2 had similar affinity for hSIRPα to the full-length SERPINA3 (part I in Figure 10). Using the method in Example 9, it was found that the SERPINA3D2 J1 fragment inhibited the production of TNFα in LPS-activated THP-1 cells and inhibited the phagocytosis of hCD47-coated erythrocytes by THP-1 cells, with effects comparable to the full-length SERPINA3. SERPINA3D2 J2 inhibits the secretion of TNFα by LPS-activated THP-1 cells, but does not inhibit the phagocytosis of anti-hCD47-coated erythrocytes by THP-1 cells (parts J and K in Figure 10). To determine which amino acid residues mediate the binding, we sequentially mutated specific amino acid residues in SERPINA3D2 J1 and SERPINA3D2 J2. The results showed that mutating any of the amino acid residues P223, D225, and Q228 of SERPINA3D2 J1 or E310 and Y312 of SERPINA3D2 J2 to alanine (A) resulted in the loss of the binding ability of the corresponding truncated variant to hSIRPα (part L in Figure 10). These findings suggest that amino acid residues P223, D225, Q228, E310, and Y312 are crucial for maintaining the interaction between SERPINA3 and hSIRPα.

[0167] Next, we mutated any one of the amino acid residues P223, D225, and Q228 of SERPINA3D2 J1 or E310 and Y312 of SERPINA3D2 J2 to the same amino acid (nonpolar hydrophobic amino acid mutated to another nonpolar hydrophobic amino acid, polar neutral amino acid mutated to another polar neutral amino acid, acidic amino acid mutated to another acidic amino acid, and basic amino acid mutated to another basic amino acid). We then used the above ELISA method to detect the affinity of each mutant (containing a 6×his tag) to hSIRPα, using the affinity of the full-length SERPINA3 (containing a 6×his tag) and SERPINA3D2J1 (containing a 6×his tag) to hSIRPα as controls. The results showed that when the P223 amino acid residue of SERPINA3D2J1 was mutated to I, L, or V, the D225 amino acid residue was mutated to E, and the Q228 amino acid residue was mutated to C, M, S, or Y, SERPINA3D2J1 still had a significant affinity for hSIRPα; when the E310 amino acid residue of SERPINA3D2J2 was mutated to D, and the Y312 amino acid residue was mutated to C, T, or W, SERPINA3D2J2 still had a significant affinity for hSIRPα (part A and part B of Figure 11). Furthermore, we performed simultaneous two-site or three-site mutations on key amino acids such as P223, D225, and Q228 of SERPINA3D2J1 or E310 and Y312 of SERPINA3D2J2 according to the mutable amino acid sequences obtained in Figure 11B. We found that the obtained SERPINA3D2J1 or SERPINA3D2J2 mutants still have the ability to bind to hSIRPα (parts C and D in Figure 11).

[0168] Example 11

[0169] SERPINA3D2 has no pro-inflammatory effect on AC16 and HCMEC / D3 cells.

[0170] AC16 (human cardiomyocyte cell line) and HCMEC / D3 (human cerebral vascular endothelial cell line) cells were treated with LPS (final concentration 100 ng / mL), SERPINA3 (10 nM), SERPINA3D2 (10 nM), or control protein (293 cell culture supernatant transfected with an empty vector containing an equal amount of protein) for 48 hours under standard culture conditions (37℃, 5% CO2). Total RNA was then extracted and reverse transcribed into cDNA. qRT-PCR was performed to detect the expression of human TNFα, IL-6, and VCAM-1. The primer sequences used are as follows:

[0171] Consistent with reports of pro-inflammatory effects of full-length SERPIA3 on cardiomyocytes and cerebral vascular endothelial cells (Xie W, Zhang A, Huang X, et al. Shock. 2023 May 1; 59(5):791-802.; Kim H, Leng K, Park J, et al. Nat Commun. 2022; 13(1):6581.), full-length SERPIA3 upregulated the mRNA levels of TNFα and IL-6 in A16 cells and the mRNA levels of TNFα, IL-6, and VCAM1 in HCMEC / D3 cells in in vitro experiments. However, SERPIA3D2 failed to induce upregulation of TNFα, IL-6, and VCAM1 mRNA levels in these cell lines (part A in Figure 12, part B in Figure 12). These results indicate that SERPIA3D2 containing only the region interacting with hSIRPα lacks the pro-inflammatory effects of full-length SERPIA3 on human cardiomyocytes and cerebral vascular endothelial cells.

[0172] Example 12

[0173] SERPINA3D2 has therapeutic potential for treating multiple sclerosis in humans.

[0174] We systematically evaluated the potential of SERPINA3D2 for treating human autoimmune diseases. Notably, ELISA assays performed according to the method described in Example 7 revealed no cross-reactivity between mouse SERPINA3k and hSIRPα, and between human SERPINA3 and mSIRPα (part A of Figure 13). Initially, CD14 isolated from peripheral blood of newly diagnosed multiple sclerosis patients... + Monocytes were treated with SERPINA3D2 in vitro according to the methods in Examples 9 and 10. The results showed that SERPINA3D2 significantly inhibited the expression of TNFα and IFNγ in LPS-activated monocytes (part B and part C of Figure 13) and induced an increase in the phosphorylation levels of hSIRPα and SHP-1 in these cells (part D of Figure 13). The above-mentioned effects of SERPINA3D2 could be inhibited by the pre-added anti-hSIRPα.

[0175] Humanized mice with the immune system were purchased from Jilin Qianhe Model Biotechnology Co., Ltd. The specific steps for establishing this mouse system were as follows: 8-10 week old NCG mice were given a sublethal dose (2-3 Gy) of whole-body irradiation. Within 3 days after irradiation, approximately 1 mm of... 3 Human fetal thymus tissue was implanted subcapsularly into the recipient's kidney, and purified CD34 from the same donor was intravenously injected on the same day as the human thymus transplant. + Artificial hematopoietic stem cells (1-5×10) 5(Each mouse) was used to reconstruct the human immune system. Approximately 8 weeks later, flow cytometry analysis was performed on the levels of artificial blood cells in the transplanted mice. Mice with more than 75% hCD45 cells in their peripheral blood total lymphocytes at 8 weeks showed good reconstruction of the human immune system in mice, and these mice were suitable for further experiments (part E in Figure 13). After an 8-week reconstruction period, an EAE model was established using immune system-humanized mice according to the method in Example 4. During disease flare-ups, these mice received either SERPINA3-his or SERPINA3D2-his treatment every 3 days for 21 consecutive days, at a dose of 200 μg / mouse / treatment. Compared to the PBS control group, both SERPINA3-his and SERPINA3D2-his showed significant therapeutic effects, with significantly reduced clinical disease scores, and the therapeutic effects were similar (part F in Figure 13). These results indicate that SERPINA3D2 has a therapeutic effect on inflammation-based autoimmune diseases such as multiple sclerosis in humans.

[0176] Example 13

[0177] Human MSCs overexpressing human SERPINA3D2 have stronger immunosuppressive function than natural human MSCs.

[0178] The SERPINA3D2 gene was ligated to the IL-2 signal peptide gene and then inserted between AscⅠ and SalⅠ in the lentiviral plasmid vector (part A in Figure 6). The recombinant lentiviral vector and the empty vector were transfected into 293 cells to package lentivirus containing the SERPINA3D2 gene and control virus containing only the empty vector, respectively. The two lentiviruses were then transfected into human MSC cells to obtain MSCs overexpressing SERPINA3D2. A3D2 and MSC NC Control cells. The experiment was divided into the following 5 groups: THP-1 group, THP-1+MSC group, etc. NC Group, THP-1+MSC A3D2 Group, THP-1+MSC A3D2 +IgG group, THP-1+MSC A3D2 +A3 Ab group, THP-1 cells (1.5×10) 5 MSCs were seeded in the lower chamber of a Transwell 12-well plate (0.4 μm pore size) and induced for 24 hours with 1 mL of RPMI-1640 complete medium (containing 100 ng / mL PMA) until the cells adhered to the plate. The medium was then replaced with RPMI-1640 complete medium containing 100 ng / mL LPS and 20 ng / mL IFNγ. After 24 hours, MSCs were added to the upper chamber of the Transwell plate. A3D2 or MSC NC (1.5×10 5( / well), and replace the lower chamber medium with fresh complete medium, THP-1+MSC A3D2 +IgG group and THP-1+MSC A3D2 The culture medium of the +A3 Ab group was supplemented with the corresponding antibody (control IgG or anti-SERPINA3) to a final antibody concentration of 50 μg / mL. After co-culturing for 48 hours, the cell culture supernatant was collected, and the concentration of TNFα in the supernatant was detected by ELISA. The results are shown in part A of Figure 14. THP-1+ MSCs NC The concentration of TNFα in the supernatant of the group was significantly lower than that of the THP-1 group. The concentration of TNFα in the supernatant of the group was significantly lower than that of THP-1+MSC. NC Group, and THP-1+MSC A3D2 The +IgG group was comparable, both were lower than THP-1+MSC. A3D2 +A3 Ab group. THP-1+MSC A3D2 The concentration of TNFα in the supernatant of the +A3 Ab group was slightly higher than that of THP-1+MSC. NC The levels in the MSC group were lower than those in the THP-1 group. These results indicate that the functional role of MSCs in inhibiting activated monocytes and macrophages is partly borne by SERPINA3. A3D2 Compared to MSC NC The control cells had a stronger ability to inhibit the secretion of TNFα by activated human monocytes and macrophages.

[0179] MSC therapy for EAE in humanized mice: An immune system-humanized mouse EAE model was established according to the method in Example 12. Mice were divided into a disease control group and an MSC-treated group. NC Treatment group and MSC A3D2 Treatment group (n=5 per group). Mice were intravenously injected with 1×10⁻⁶ mg / L on day 8 post-immunization. 6 MSC NC Or MSC A3D2 Cells (cells diluted in Ca2+-free solution) + and Mg2 + In the control group, mice were intravenously injected with an equal volume of PBS at the same time point until they were sacrificed. Mice were scored daily according to the EAE model clinical scoring criteria. Results showed that mice injected with either type of MSC cell had milder EAE symptoms than the PBS group, but the MSC-injected group… A3D2 Mice injected with MSCs NC The EAE symptoms in the cell-mediated mice were significantly reduced (part B of Figure 14).

[0180] These results demonstrate that the SERPINA3D2 gene can be used to modify human MSC cells. These MSC cells overexpressing SERPINA3D2 have stronger immunosuppressive functions than natural MSC cells and have better therapeutic effects on autoimmune diseases such as EAE.

[0181] Example 14

[0182] Stem cells overexpressing SERPINA3D2 have stronger immunosuppressive function than regulatory T cells (Treg cells) derived from the differentiation of normal stem cells.

[0183] In recent years, stem cells have shown great potential in regenerative medicine and immunotherapy, capable of differentiating into various immune cells and immunomodulatory cells for the treatment of various immune-related diseases. To explore whether SERPINA3D2 overexpression can enhance the therapeutic effect of Treg cells obtained from stem cell differentiation on GVHD, we first obtained induced pluripotent stem cells (iPSCs) from the National Stem Cell Resource Center of China (NSCRC). Following the method in Example 13, we cloned the SERPINA3D2-puro gene into the pRRLSIN plasmid (EF1α promoter) to construct a lentiviral core vector. SERPINA3D2-puro and the empty puro vector (control group) were co-transfected into HEK293T cells with packaging plasmids (psPAX2, pMD2.G), respectively. The supernatant was collected to concentrate the virus, yielding SERPINA3D2 virus and the empty vector control virus. iPSCs at low passage numbers (within 15 passages) were infected with the virus and screened with 1 μg / mL Puromycin for 10 days to obtain iPSCs. A3D2 and iPSC NC Cells. Following the experimental method of Hisashi Yano's team (Yano H, Koga K, Sato T, et al. Cell Stem Cell. 2024; 31(6):795-802.; Bézie S, Meistermann D, Boucault L, et al. Front Immunol. 2018; 8:2014.), iPSCs were first differentiated into CD4 cells. +T cells were cultured in ultra-low adhesion dishes with StemFit AK03N medium containing Y-27632 (a ROCK inhibitor) and CHIR99021 (a Wnt pathway activator) for 24 hours to form embryonic bodies (EBs). The medium was then replaced with EB basal medium (containing BMP-4, VEGF, and bFGF cytokines) to induce EB differentiation into a mixed cell population containing hematopoietic progenitor cells (HPCs). After 5 days of culture, CD34 cells were collected. + CD43 + HPCs were used for subsequent T cell differentiation. MS5-hDLL4 mouse stromal cells expressing human DLL4 (Notch ligand) (purchased from ATCC) were mixed with HPCs at a 4:1 ratio and seeded in Transwell chambers containing ATO medium. The ATO medium formulation (50 mL) was: RPMI 1640 medium (47 mL), 4% B27 (Thermo Fisher Scientific) (2 mL), 50 mg / mL polyacrylic acid (Sigma-Aldrich) (50 mL of 50 mg / mL), 1% L-glutamine and penicillin-streptomycin solution (Sigma-Aldrich), 5 ng / mL recombinant human FLT3 ligand (rhFLT3L) (PeproTech) (1.25 mL of 200 mg / mL stock), and 5 ng / mL recombinant human IL-7 (rhIL-7) (PeproTech) (2.5 mL of 100 mg / mL stock). After culturing for 6-9 weeks, HPCs are induced to differentiate into CD4. + T cells. CD4 sorting + T cells were used to expand CD4 cells in vitro using anti-CD3 / CD28 magnetic beads (Dynabeads) and IL-2 (10 ng / mL). + T cells were expanded by adding the AMRT combination: AS2863619 (CDK8 / 9 inhibitor, 32.52 nM); MR2-1 (anti-TNFR2 agonist antibody, 2.5 μg / mL); rapamycin (mTOR inhibitor, 100 nM); and TGFβ1 (5 ng / mL). After 14 days of continuous culture, Treg cells were obtained. NC or Treg A3D2 Flow cytometry analysis showed FOXP3 + The cell ratio can reach over 95%.

[0184] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy individuals and CD8+ cells were sorted using immunomagnetic beads. +T is combined with IL-2 and anti-human CD3 / CD28 magnetic beads (Dynabeads). CD8 + T and Treg NC ,Treg A3D2 After being cultured in a 1:4 ratio in a U-shaped bottom 96-well plate for 4 days, CD8 was detected by CCK8 assay. + T proliferation status. The results showed that Treg... NC It can significantly inhibit CD8 + T proliferates, but overexpression of SERPINA3D2 does not enhance Treg's response to CD8. + The inhibitory effect on T cell proliferation (part A in Figure 15) is consistent with the results in Example 2 where SERPIA3k inhibited DC antigen presentation. This indicates that in the absence of SIRPα... + The presence of antigen-presenting cells (mainly monocytes / macrophages and dendritic cells), and CD8 + In the absence of SIRPα expression in T cells, SERPIA3D2 secretion cannot enhance the Treg response to activated CD8+. + Inhibition of T cell proliferation. Similar to Example 13, THP-1 cells were seeded in the lower chamber of a Transwell 12-well plate and cultured for 24 hours until adherence. After stimulation for 24 hours with RPMI-1640 medium containing 100 ng / mL LPS and 20 ng / mL IFNγ, the lower chamber medium was replaced with fresh complete medium. Treg cells were added to the upper chamber of the Transwell at a cell ratio of 1:4 (THP-1:Treg). NC ,Treg A3D2 After culturing for 2 days, the cell culture supernatant was collected, and the concentration of TNFα in the supernatant was detected by ELISA. The results showed that Treg... A3D2 Treg NC It more significantly inhibited the ability of activated THP-1 cells to secrete TNFα (part B of Figure 15). These results indicate that SIRPα... + In the presence of cells, Treg A3D2 Treg NC It has a stronger immunosuppressive function.

[0185] After irradiating 11-week-old male NSG mice with 2.2 Gy, they were then subjected to 5.0 × 10⁻⁶ irradiation. 6 One PBMC and 5.0 × 10 6 Treg NC or Treg A3D2 Mice were injected via tail vein, and their body weight and survival rate were monitored regularly. Results showed that, compared to Treg... NC Compared to the group of mice, Treg A3D2The Treg group mice experienced less weight loss and longer survival time. A3D2 It has a better therapeutic effect on GVHD (n=6, part C in Figure 15).

[0186] These results indicate that the SERPINA3 gene can be used to modify stem cells with differentiation potential, enabling the differentiated immunomodulatory cells to have stronger immunosuppressive functions.

[0187] Example 15

[0188] The rejection response to xenograft of porcine cells or organs expressing human SERPINA3D2 is reduced.

[0189] Pig xenotransplantation is a promising strategy to address clinical organ shortages, and xenogeneic immune rejection is one of the key issues to be addressed in xenotransplantation. Based on the above results, we anticipate that modifying pig cells or organs with the human SERPINA3D2 gene will help reduce rejection responses during pig cell or organ xenotransplantation. To verify this hypothesis, we first used the lentiviral system from Example 13 to infect AOC cells (pig endothelial cell line) with lentivirus containing the SERPINA3D2 gene and control virus containing only an empty vector to prepare SERPINA3D2 overexpressing cells (AOC-A3D2) or control cells (AOC-NC). These virus-transfected AOCs were pretreated with 25 μg / mL mitomycin C (Selleck, USA) at room temperature for 30 minutes, followed by washing three times with sterile PBS. These pretreated AOCs will be used as cell stimulators. Freshly collected human peripheral blood samples were gently diluted with PBS at a 1:1 ratio and slowly added along the tube wall to centrifuge tubes containing Lymphoprep separation medium, stacked on top of the separation medium. Place the centrifuge tubes in a horizontal centrifuge and centrifuge at 400×g for 40 minutes at room temperature, with the brake off during centrifugation. After centrifugation, four layers will be visible inside the centrifuge tube. Carefully aspirate the white membrane layer located at the interface between the plasma and Ficoll separation solution using a sterile pipette and transfer it to a new sterile centrifuge tube. Wash the cells 2-3 times with an appropriate amount of PBS buffer to obtain purified human peripheral blood mononuclear cells (PBMCs) for subsequent experiments. Adjust the concentration of human PBMCs to 1×10⁻⁶. 6 / mL as the reaction cells. 1×10 5 One group of reactive cells was seeded into the wells of a 96-well plate with a round bottom. The plate was set up with no stimulated cells and with 1×10⁶ cells added. 5Six replicates were set up for each stimulated cell group. Cells were cultured at 37°C and 5% CO2 for 3 days. Lymphocyte proliferation was assessed using a CCK-8 cell assay kit (Beyotime, China). CCK-8 solution was added to cell wells, incubated for 2 hours, and then the optical density (OD) was measured at 450 nm using a microplate reader. The proliferation index was calculated as: OD value of the responding cells in the well containing the stimulated cells / OD value of the well containing only the responding cells. The results in Part A of Figure 16 show that when stimulated with AOC endothelial cells, the proliferation index of human PBMCs was more than twice that of the unstimulated group, exhibiting a strong proliferative response. However, when AOC endothelial cells overexpressed SERPINA3D2, the proliferation index of human PBMCs decreased significantly, indicating that porcine cells overexpressing SERPINA3D2 showed a weakened response to human lymphocyte stimulation, and that overexpression of SERPINA3D2 has a certain protective effect on the graft.

[0190] Furthermore, using gene editing technology, the SERPINA3D2 gene with the SERPINA3 promoter and the SERPINA3D2 gene with the porcine IL-6 specific promoter were knocked into porcine fibroblasts. IL-6 Transgenic pigs were obtained through nuclear transfer using pPBMC-A3D2. Peripheral blood was collected from different types of transgenic pigs, and porcine PBMCs (control pPBMC-NC, pPBMC-A3D2, pPBMC-P) were isolated using the method described above. IL-6 A3D2), porcine PBMCs were treated with mitomycin C as described above. The treated porcine PBMCs were then co-cultured with human PBMCs for a mixed lymphocyte reaction (MLR). After 5 days of culture at 37°C and 5% CO2, CCK8 assays revealed that porcine PBMCs overexpressing SERPINA3D2 (pPBMC-A3D2) reduced the stimulatory effect on human PBMCs and decreased immune rejection. Meanwhile, P... IL-6 The stimulatory effect of A3D2 pig PBMCs on human PBMCs is comparable to that of pPBMC-NC under normal culture conditions, but can be reduced to the level of pPBMC-A3D2 under LPS stimulation (part B in Figure 16). This indicates that if a IL-6 promoter-specifically activated SERPINA3D2 gene-edited pig graft is rejected by the host after transplantation, the IL-6 produced by the rejection response will stimulate the local secretion of human SERPINA3D2 to inhibit the rejection response and protect the graft.

[0191] Example 16

[0192] Anti-human SERPINA3 antibodies (A3 Ab) prepared using human SERPINA3D2 protein as an antigen have anti-tumor effects against various human tumors.

[0193] Anti-human SERPINA3 antibodies were prepared and purified by immunizing New Zealand white rabbits with SERPINA3D2 protein according to the method in Example 8. Humanized mice with immune systems prepared according to the method in Example 12 were purchased. 5 × 10⁶ 6 TNBC MDA-MB-231 cells (human triple-negative breast cancer cell line) suspended in a 1:1 mixture of Matrigel and PBS were injected into the mammary fat pads of humanized mice, or 5 × 10⁻⁶ cells were injected into the mammary fat pads of humanized mice. 6 A375 cells (human melanoma cell line) were suspended in a 1:1 mixture of Matrigel and PBS and injected subcutaneously into the right back of mice. Tumor volume was measured and calculated according to the method in Example 8. When the tumor diameter reached 4-5 mm (approximately 6-8 days post-inoculation), the mice were randomly divided into groups of 5 and then received intraperitoneal injections of anti-human SERPINA3 (250 μg each time) or an equivalent amount of isotype IgG every 5 days. In both A375 and MDA-MB-231 tumor-bearing mice, tumor growth in mice injected with anti-SERPINA3 was significantly reduced compared to the IgG control group (Figure 17). These results indicate that the anti-SERPINA3 antibody prepared using SERPINA3D2 as the antigen has a significant anti-tumor effect.

[0194] It should be noted that the specific embodiments described above are exemplary. Those skilled in the art can devise various solutions inspired by the disclosure of this invention, and these solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents. This specification contains multiple inventive concepts; phrases such as "preferredly" or "according to a preferred embodiment" indicate that the corresponding paragraph discloses an independent concept. The applicant reserves the right to file divisional applications based on each inventive concept.

Claims

1. SERPINA3 protein variants, in which: The SERPINA3 protein variant contains the functional domain portion essential for the binding of the natural human SERPINA3 protein to human SIRPα. and Compared to the natural human SERPINA3 protein, the SERPINA3 protein variant has at least one amino acid mutation at at least one of the following three positions in the natural human SERPINA3 protein: (1) other parts besides the essential functional domain; (2) The other positions in the required functional domains besides the amino acids at positions P223, D225, Q228, E310 and Y312; (3) Amino acids at positions P223, D225, Q228, E310, and Y312; Furthermore, the mutation at position (3) is a substitution mutation selected from the group consisting of P223I, P223L, P223V, D225E, Q228C, Q228M, Q228S, Q228Y, E310D, Y312C, Y312T and Y312W.

2. The SERPINA3 protein variant according to claim 1, wherein: Except for the positions P223, D225, Q228, E310, and Y312, the mutations at other positions are insertion, substitution, or deletion mutations.

3. The SERPINA3 protein variant according to claim 1, wherein: The functional domain necessary for binding to human SIRPα is the region corresponding to SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3; or The mutation was caused by artificial intervention through genetic engineering.

4. The SERPINA3 protein variant according to claim 1, wherein: The SERPINA3 protein variant comprises at least one amino acid sequence from the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3; or The SERPINA3 protein variant comprises the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 and has undergone at least one substitution mutation, wherein the at least one substitution mutation is selected from the group consisting of P223I, P223L, P223V, D225E, Q228C, Q228M, Q228S, Q228Y, E310D, Y312C, Y312T, and Y312W.

5. The SERPINA3 protein variant according to claim 1, wherein: The SERPINA3 protein variant has an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3, or the SERPINA3 protein variant has an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 and has undergone at least one substitution mutation, wherein the at least one substitution mutation is selected from the group consisting of P223I, P223L, P223V, D225E, Q228C, Q228M, Q228S, Q228Y, E310D, Y312C, Y312T and Y312W.

6. The encoding nucleotide sequence of the SERPINA3 protein variant according to any one of claims 1 to 5.

7. A vector, cell, virus, or bacterium comprising the encoding nucleotide sequence according to claim 6.

8. The use of SERPIA3 or a variant of the SERPIA3 protein according to any one of claims 1 to 5 in the prevention or treatment of a disease or in the preparation of a reagent or medicament for the prevention or treatment of said disease, said disease being SIRPα. + Diseases in which cells are involved in the pathogenesis.

9. A formulation or pharmaceutical composition, wherein, The formulation or pharmaceutical composition comprises: An effective amount of SERPINA3, or a SERPINA3 protein variant as described in any one of claims 1 to 5, or the encoding nucleotide sequence as described in claim 6, and Its pharmaceutically acceptable carrier; The formulation or pharmaceutical composition is used to treat SIRPα. + Diseases in which cells are involved in the pathogenesis.

10. The formulation or pharmaceutical composition according to claim 9, wherein: The formulation or pharmaceutical composition further comprises one or more selected from anti-inflammatory agents, immunosuppressants, immunomodulators, anti-transplant rejection agents, antimicrobial agents, and cytotoxic drugs.

11. The nucleotide sequence encoding the SERPIA3 protein or the nucleotide sequence encoding the SIRPα protein as described in claim 6, in the preparation of a vaccine for the prevention or treatment of SIRPα. + Application of substances in diseases in which cells participate in the pathogenesis.

12. The application according to claim 11, wherein: The substance is a gene-edited cell used as a drug for genetic modification, wherein the gene-edited cell overexpresses the nucleotide sequence encoding the SERPINA3 protein or the nucleotide sequence encoding the protein as described in claim 6.

13. The application according to claim 12, wherein: The gene-edited cells are mesenchymal stem cells or stem cells with differentiation potential.

14. A cell, said cell being a mesenchymal stem cell or a stem cell with differentiation potential, wherein: The mesenchymal stem cells overexpress the nucleotide sequence encoding the SERPINA3 protein introduced as an exogenous nucleic acid or the nucleotide sequence encoding the protein as described in claim 6 to enhance immunosuppressive function; or The stem cells with differentiation potential overexpress the nucleotide sequence encoding SERPINA3 protein introduced as an exogenous nucleic acid or the nucleotide sequence encoding as described in claim 6 to enhance the immunosuppressive function of regulatory cells obtained from stem cell differentiation.

15. The mesenchymal stem cells or stem cells with differentiation potential according to claim 14, wherein: The encoding nucleotide sequence is operatively linked to the encoding nucleotide sequence of the signal peptide, the promoter, and / or the enhancer.

16. The application of the nucleotide sequence encoding the SERPINA3 protein or the nucleotide sequence encoding the protein according to claim 6 in the preparation of transgenic cells, transgenic organs, or transgenic animals providing cell or organ donors for transplantation, wherein, The transgenic cell is a transgenic cell expressing the exogenous encoding nucleotide sequence, the transgenic organ is a transgenic organ expressing the exogenous encoding nucleotide sequence, and the animal is a transgenic animal expressing the exogenous encoding nucleotide sequence.

17. The application according to claim 16, wherein the transgenic animal is a transgenic pig.

18. A treatment for SIRPα + Methods involving cell involvement in the pathogenesis of diseases, among which, The method includes administering an effective amount of SERPINA3 protein to a subject, a variant of the SERPINA3 protein according to any one of claims 1 to 5, a nucleotide sequence encoding the SERPINA3 protein or the nucleotide sequence encoding the SERPINA3 protein according to claim 6, and a vector expressing the nucleotide sequence encoding the protein.

19. The use of the SERPINA3 protein variant as an antigen in the preparation or screening of anti-SERPINA3 antibodies according to any one of claims 1 to 5.

20. SERPINA3 as a target in the development, screening, or preparation of drugs for the prevention and / or treatment of SIRPα + Application in drugs for diseases in which cells are involved in the pathogenesis.

21. Inhibitors targeting SERPIA3 are being developed for the prevention or treatment of SIRPα. + Application in drugs for diseases in which cells are involved in the pathogenesis.

22. The application according to claim 21, wherein: The inhibitor is selected from one or more of the following: nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, peptides, proteins, gene editing vectors, lentiviruses, or adeno-associated viruses that inhibit SERPINA3 expression.

23. The application according to the preceding claim, wherein: The SIRPα + Diseases in which cells are involved include inflammation, autoimmune diseases, transplant rejection-related diseases, and tumors.

24. The application according to claim 23, wherein: The inflammation includes inflammation caused by bacterial or viral infection and aseptic inflammation; The autoimmune diseases mentioned include multiple sclerosis, arthritis, inflammatory bowel disease, systemic lupus erythematosus, and autoimmune liver disease; The transplantation includes cell transplantation, tissue transplantation, and / or organ transplantation; The tumors include melanoma, bladder cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, ovarian cancer, pancreatic cancer, esophageal cancer, lymphoma, brain tumor, sarcoma, cervical cancer, prostate cancer, osteosarcoma, head and neck cancer, renal cell carcinoma, and stomach cancer.

Images