Pharmaceutical use of stem cell modified based on single-domain antibody genes for various diseases

Abstract

Provided is a pharmaceutical use of a stem cell modified based on single-domain antibody genes for various diseases, belonging to the technical field of stem cells. The stem cell contains, expresses o

Description

Pharmaceutical applications of stem cells modified with single-domain antibodies for various diseases

[0001] This application claims priority to a Chinese patent application filed with the Patent Office of China on March 12, 2024, with application number "202410276792.9" and invention name "Pharmaceutical use of stem cells modified with single-domain antibody genes for a variety of diseases", the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application belongs to the field of stem cell technology, and specifically relates to the pharmaceutical use of stem cells modified with single-domain antibody genes for a variety of diseases. Background Art

[0003] Stem cells are immature cells that are not fully differentiated and have the potential to regenerate various tissues, organs, and the human body. Stem cell therapy has been attempted in a variety of diseases, including diabetes, Parkinson's disease, lupus erythematosus, and leukemia. The preparation technology and application of induced pluripotent stem cells (iPSCs) have brought stem cell therapy into a new era. With the continuous development of molecular biology technology, especially gene editing technology, gene modification is playing an increasingly important role in stem cell therapy.

[0004] As a regenerative medicine technology, stem cell therapy aims to replace dysfunctional cells with cells with specific functions, restore the corresponding cell functions, and improve the health of the body. Currently, there are many types of stem cells used in clinical practice. Among them, mesenchymal stem cells (MSC) have certain immunomodulatory functions and are used to treat immune diseases such as graft-versus-host disease, achieving certain therapeutic effects. The prior art has disclosed the autologous HSC drug Strimvelis for the treatment of adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID) and the autologous MSC drug AstroStem for the treatment of Alzheimer's disease.

[0005] MSCs are multipotent stem cells, multipotent cells with the ability to self-replicate. Under certain conditions, they can differentiate into a variety of functional cells, such as APSCs. They originate from the mesoderm during embryonic development. During the body's normal tissue damage and repair process, MSCs are an important cell reservoir involved in tissue regeneration. In response to specific signals caused by tissue damage, MSCs migrate to the damaged area, aggregate and proliferate locally, and differentiate along different pathways based on the different damage signals. MSCs are easy to isolate and amplify, have a strong in vitro multiplication capacity, and can maintain their multidirectional differentiation ability even after amplification of 100 million times. Therefore, MSCs are a practical seed cell for tissue repair.

[0006] IL-17 is a key cytokine for host protection against mucosal infections and is also a major pathogenic cytokine and drug target for a variety of autoimmune and inflammatory diseases. The IL-17 family includes six members: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F, each of which mediates its biological function through the IL-17 receptor (IL-17RA to IL-17RE). Among them, the most studied IL-17 family member is IL-17A, which promotes its biological activity by binding to IL-17RA and IL-17RC. The prior art has disclosed that research on the pathological role of IL-17A in human diseases ultimately led to the development of monoclonal antibodies (mAbs) against IL-17A (IL-17A and IL-17F, IL-17RA, or IL-23).

[0007] Existing technologies studying the interaction between IL-17 and stem cells include Chinese patent application number CN105079792A, which discloses the use of IL-17 to enhance the immunosuppressive function of mesenchymal stem cells. Specifically, the invention provides the use of interleukin-17, interleukin-17 derivatives, or agonists thereof for preparing a preparation or kit for enhancing the immunosuppressive function of mesenchymal stem cells; upregulating the expression of immunosuppressive factors in mesenchymal stem cells; enhancing the stability of immunosuppressive factor mRNA; reducing the expression level of the RNA-binding protein AUF1; inhibiting T cell proliferation; and treating hepatitis or liver damage.

[0008] However, the prior art does not disclose the use of IL-17-related antibodies to modify stem cells, particularly the role of IL-17A-specific tandem antibodies in stem cell modification. Further research and development of the application of modified stem cells is needed in this field. Summary of the Invention

[0009] To address the above issues, the present application provides an application of stem cells modified with IL-17A-specific tandem antibodies. This application is a fourth-generation technology development based on the anti-IL-17A single-domain antibody development project.

[0010] To facilitate the review, a brief introduction to the technical background of the R&D project is provided: the applicant first developed nine single-domain antibodies (a separate application has been filed); based on the single-domain antibodies, the applicant developed an antibody combination consisting of two single-domain antibodies in series (a separate application has been filed); based on the single-domain antibodies, the applicant developed gene-modified stem cells composed of antibody combinations (a separate application has been filed); based on the gene-modified stem cells, the applicant developed an application technology for modifying stem cells, and this application is one of the application technologies. The above patent applications are filed separately based on the relevant provisions of the Patent Law on unity. To facilitate the understanding of this application, you may optionally refer to other patent application documents of this project.

[0011] This application is based on screening of immunized alpacas, and obtained a variety of nanoantibodies with IL-17A specific binding ability, and selected two of them for series connection to achieve stem cell gene modification applications.

[0012] The IL-17 family consists of seven members, designated IL-17A to IL-17F. The amino acid sequences of the IL-17 family are highly similar at the C-terminus, primarily consisting of five spatially conserved cysteine ​​groups. The cells that most abundantly express IL-17 are thymus-dependent lymphocytes, including αβ T cells in adaptive immunity, γδ T cells in innate immunity, invariant natural killer T cells, and lymphoid tissue inducer-like cells (LTi-like cells). The IL-17 family plays a crucial role in host antimicrobial responses and the development of inflammatory diseases. IL-17 promotes the synthesis and secretion of numerous cytokines that regulate its various functions. These cytokines include chemokines (such as CXCL1, CXCL8, and CCL2); proinflammatory cytokines (such as IL-6, TNF-α, and IL-1β); proinflammatory cytokine regulators (such as NOS and COX); GM-CSF and G-CSF growth factors; and tissue remodeling factors (such as MMP1, MMP3, and RANKL).

[0013] Whether secreted by innate or adaptive immune cells, IL-17A and IL-17F have the most significant function of inducing neutrophil migration to the site of infection. This function of IL-17A and IL-17F is crucial for the clearance of microorganisms. Some studies have shown that IL-17A and IL-17F induced by bacterial infection of epithelial and mucosal barriers play an important role in host defense. The first study to demonstrate the role of IL-17 in host defense was the Klebsiella pneumonia lung infection model in IL-17R-deficient mice (Ye P, et al. Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense. J Exp Med. 2001; 194(4): 519-527.). Studies using IL-17RA- and IL-17-deficient mouse models or using neutralizing antibodies to IL-17 receptors or ligands to block IL-17 signaling have shown that mice are more susceptible to infection with a variety of extracellular and intracellular bacteria, including Salmonella, Salmonella typhimurium, Streptococcus pneumoniae, Mycobacterium tuberculosis, Listeria monocytogenes, Staphylococcus aureus, and Helicobacter pylori (Pappu R, et al. The interleukin-17 cytokine family: critical players in host defense and inflammatory diseases. Immunology. 2011; 134(1): 8-16.; Iwakura Y, et al. Functional specialization of interleukin-17 family members. Immunity. 2011; 34(2): 149-162.; McGeachy MJ, et al. The IL-17 Family of Cytokines in Health and Disease. Immunity 2019;50(4):892-906.). IL-17 was identified as one of the molecules responsible for host defense against fungi, and these molecules were shown to be associated with human susceptibility to fungi through genetic mutations.Congenital defects in the IL-17 pathway, including defects in the genes encoding IL-17F or IL-17RA, have been found to be associated with increased susceptibility to fungal infections, especially chronic mucocutaneous candidiasis, which clearly indicates that the IL-17 pathway plays an important role in antifungal immunity (Cypowyj, et al. Immunity to infection in IL-17-deficient mice and humans. European Journal of Immunology 42.9(2012):2246-2254.). IL-17 has been shown to protect mice from various fungal infections, including those caused by Pneumocystis jiroveci, Histoplasma capsulatum, and Aspergillus fumigatus (Gladiator A, et al. Innate lymphoid cells: new players in IL-17-mediated antifungal immunity. PLoS Pathog. 2013;9(12):e1003763.).

[0014] IL-17 promotes tumor development through two pathways. One pathway is to reduce the local immune response to the tumor by maintaining an inflammatory environment (He D, et al. IL-17 promotes tumor development through the induction of tumor promoting microenvironments at tumor sites and myeloid-derived suppressor cells. J Immunol. 2010; 184(5): 2281-2288.); the other pathway is to promote the expression of anti-apoptotic genes by activating the STAT3 and NF-kB pathways (Grivennikov SI, et al. Dangerous liaisons: STAT3 and NF-kappaB collaboration and crosstalk in cancer. Cytokine Growth Factor Rev. 2010; 21(1): 11-19.) IL-17 plays a pro-cancer role in inflammation-related cancers mainly by relying on its pro-angiogenic properties. IL-17 acts on stromal cells and fibroblasts to induce them to secrete angiogenesis regulatory factors, including VEGF. These secreted angiogenesis regulatory factors can significantly enhance the inflammatory response and promote tumor angiogenesis.

[0015] Members of the IL-17 cytokine family, particularly IL-17A, are associated with a variety of autoimmune diseases, including multiple sclerosis (MS), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), type 1 diabetes mellitus (TIDM), inflammatory bowel disease (IBD), and psoriasis (Zhu S, et al. IL-17 / IL-17 receptor system in autoimmune disease: mechanisms and therapeutic potential. Clin Sci (Lond). 2012; 122(11): 487-511.). IL-17 cytokines and IL-17 receptor expression are elevated in many human autoimmune diseases. Transgenic and knockout mouse models targeting IL-17 and IL-17 receptor genes have linked IL-17 cytokines to the development of autoimmunity. Studies in experimental autoimmune encephalomyelitis (EAE) have shown that IL-17A is a key pathogenic cytokine in the pathology of T cell-mediated autoimmune diseases (Langrish, C. Let al. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J. Exp. Med. 201, 233-240 (2005).; Sutton, C., Brereton, C., Keogh, B., Mills, KH & Lavelle, E.C.A crucial role for interleukin (IL)-1 in the induction of IL-17-producing T cells that mediate autoimmune encephalomyelitis. J. Exp. Med. 203, 1685-1691 (2006).). Subsequent studies have shown that in EAE, IL-17A is secreted by TH17 cells and IL-17A-secreting γδT (γδT17) cells (Sutton, CE et al. Interleukin-1 and IL-23 induce innate IL-17 production from gammadelta T cells, amplifying Th17 responses and autoimmunity. Immunity 31, 331-341 (2009).).

[0016] These studies, along with others that detailed the pathological role of IL-17A in human disease, ultimately led to the development of monoclonal antibodies (mAbs) targeting IL-17A (IL-17A and IL-17F, IL-17RA, or IL-23). ​​Currently, several IL-17-blocking antibody drugs are marketed worldwide, including mAbs that neutralize IL-17A (Secukinumab and Ixekizumab) or IL-17RA (Brodalumab). In China, over 50 antibody drugs or small molecules targeting IL-17 are under development for the treatment of autoimmune diseases. Although antibody drugs generally have better efficacy and tolerability than chemotherapy drugs, they are associated with numerous side effects in clinical use, such as nasopharyngitis, headache, nausea, and diarrhea, or more serious side effects such as infection, cardiotoxicity, and severe immune reactions. Furthermore, the complex structure and relatively poor stability of mAbs necessitate repeated, high-dose injections for optimal efficacy.

[0017] In one aspect, the present application provides an application of stem cells in diseases.

[0018] The application is pharmaceutical use, use in the preparation of diagnostic products, adjunctive therapy, adjunctive diagnosis of therapy and / or diagnostic method;

[0019] The disease is an IL-17A-related disease; preferably, the disease is an inflammatory disease, an infectious disease, an autoimmune disease, a nervous system disease and / or a tumor.

[0020] The stem cells are stem cells modified based on single-domain antibody genes, and the stem cells contain: (1) an amino acid sequence shown in SEQ ID NO: 1-6 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1-6; and / or; (2) a nucleotide sequence encoding an amino acid sequence shown in SEQ ID NO: 1-6 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1-6; SEQ ID NO: 1: GEKLDYFA; SEQ ID NO: 2: VTSSGSST; SEQ ID NO: 3: ASTILLCSDYISAFGT; SEQ ID NO: 4: GFSIHIYA; SEQ ID NO: 5: ITRGGVT; SEQ ID NO: 6: NAGGTNGGY.

[0021] In some understandings, the aforementioned SEQ ID NOs: 1-6 represent CDR regions. Those skilled in the art are aware that the CDR region is the region of an antibody that specifically recognizes an antigen and has the primary binding activity. Therefore, based on the CDR regions described in this application, those skilled in the art can theoretically design a variety of antibodies for application, such as in the form of nanobodies.

[0022] Preferably, the present application provides a use of stem cells in preparing a drug, wherein the stem cells contain, express or secrete a first antibody and a second antibody, wherein the first antibody contains HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 1-3; and the second antibody contains HCDR4, HCDR5, and HCDR6 as shown in SEQ ID NOs: 4-6.

[0023] Preferably, the stem cells comprise: (1) an amino acid sequence shown in SEQ ID NO: 7-14 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 7-14; and / or; (2) a nucleotide sequence encoding an amino acid sequence shown in SEQ ID NO: 7-14 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 7-14.

[0024] Preferably, the first antibody described in the present application further comprises the amino acid sequences shown in SEQ ID NOs: 7-10 as FR regions; and the second antibody further comprises the amino acid sequences shown in SEQ ID NOs: 11-14 as FR regions.

[0025] Further preferably, the stem cells comprise: (1) an amino acid sequence shown in SEQ ID NO: 16-17 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 16-17; and / or; (2) a nucleotide sequence encoding an amino acid sequence shown in SEQ ID NO: 16-17 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 16-17.

[0026] The terms "hypervariable region", "hypervariable region", "complementarity determining region", "HVR" or "CDR" as used herein refer to regions in the variable domain region of an antibody that are highly variable in sequence and / or form structurally defined loops ("hypervariable loops"). Typically, a natural four-chain antibody comprises six HVRs or CDRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Based on the Chothia definition, exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3) are located at amino acid residues L26-L32 (L1), L50-L52 (L2), L91-L96 (L3), H26-H32 (H1), H52-H56 (H2) and H96-H101 (H3) (Chothia et al., J. Mol. Biol. 196:901-917 (1987)). Based on the Kabat definition rules, exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3) are located at amino acid residues L24-L34 (L1), L50-L56 (L2), L89-L97 (L3), H31-H35 (H1), H50-H65 (H2) and H95-H102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, the fifth edtion, Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Based on the IMGT definition rules, exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are located at amino acid residues L27-L32 (L1), L50-L51 (L2), L89-L97 (L3), H26-H33 (H1), H51-H56 (H2), and H93-H102 (H3) (Honjo, T. and Alt, FW (1995) Immunoglobulin genes. Academic Press pp. 3-443). It is well known in the art that the CDRs of antibodies can be defined in the art by a variety of methods, such as the Kabat definition rules based on sequence variability, the Chothia definition rules based on the position of the structural loop region, and the reference tool for antibody humanization design based on CDR grafting (see J Mol Biol. 273: 927-48, 1997).It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions defined by any of the known schemes described herein. Although the CDRs disclosed herein are sequences based on the IMGT definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this application.

[0027] The "sequence identity" described in this application can be understood to a certain extent as sequence similarity, sequence identity, etc. Within a category with a certain sequence identity, any variation thereof will be understood by those skilled in the art to not affect the functional properties (e.g., a functional variant of an antibody has the same or similar affinity as the source antibody, or can achieve the same or similar biological function, such as specifically binding to IL-17A and blocking the binding of IL-17A to its receptor). The scope covered by the "at least 80% sequence identity" mentioned above includes, but is not limited to, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity. When achieving sequence identity, the technical means that can be used include, but are not limited to, the addition, deletion, modification and / or substitution of at least one, preferably one, two, three, four or five, amino acids.

[0028] In some embodiments, the amino acid substitution is a conservative substitution, which is preferably a substitution in which one amino acid in the following groups (a) to (e) is replaced by another amino acid residue in the same group: (a) small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0029] In some preferred embodiments, conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu.

[0030] Preferably, the first antibody and the second antibody are directly linked or linked through a linker.

[0031] Optionally, the stem cells may further include a linker, which may be selected from (GS)n, (GGS)n, (GGGS)n, (GGGGS)n, or / and AS(GGGGS)n, preferably, n is 1, 2, 3, 4, 5, or 6.

[0032] As an example, the linker may be (GGGGS)n, where n is an integer ≥ 1, more preferably 3. As an example, SEQ ID NO: 16 and SEQ ID NO: 17 are linked via SEQ ID NO: 15.

[0033] Optionally, the stem cells may further include a hinge region and a CH region.

[0034] In some specific embodiments, the stem cells comprise: (1) an amino acid sequence as represented by SEQ ID NO:16-SEQ ID NO:15-SEQ ID NO:17-SEQ ID NO:26, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence; and / or; (2) a nucleotide sequence encoding an amino acid sequence as represented by SEQ ID NO:16-SEQ ID NO:15-SEQ ID NO:17-SEQ ID NO:26, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence.

[0035] According to the general understanding of those skilled in the art, the stem cells described herein include vectors expressing IL-17A-specific tandem antibodies. However, the meaning of stem cells herein is not limited to the aforementioned form. Any methods, both disclosed and undisclosed in the art, for modifying stem cells with antibodies should be understood by those skilled in the art, and these methods can be used to implement the present application.

[0036] As is well known to those skilled in the art, due to the problem of codon degeneracy, when an amino acid sequence is clear, its corresponding nucleic acid sequence has countless possibilities. The preferred nucleic acid sequence of this application is only used as a better example, not the only possibility.

[0037] Specifically, the stem cells further include a biologically active protein or a functional fragment thereof that assists in expression and / or secretion, or prolongs the in vivo half-life; the biologically active protein or functional fragment thereof is selected from at least one of an immunoglobulin Fc domain, serum albumin, an albumin-binding polypeptide, prealbumin, a carboxyl-terminal peptide, an elastin-like polypeptide, a His tag, a GST tag, an MBP tag, a FLAG tag, and a SUMO tag; the immunoglobulin Fc domain is derived from a human antibody, a mouse antibody, a primate antibody, or a camelid antibody, or a variant thereof; preferably, the immunoglobulin Fc domain is derived from a human IgG antibody, such as IgG1 Fc, IgG2 Fc, IgG3 Fc, or IgG4 Fc, preferably IgG1 Fc.

[0038] Preferably, the biologically active protein or a functional fragment thereof is connected to the tandem single domain antibody via a linker, preferably a peptide linker;

[0039] Preferably, the connecting peptide comprises G (glycine), S (serine) and A (alanine) or a flexible polypeptide composed of G and S, preferably a flexible polypeptide of 2-30 amino acid residues; more preferably, the connecting peptide includes (GS)n, (GGS)n, (GGGS)n, (GGGGS)n and AS(GGGGS)n, preferably n is 1, 2, 3, 4, 5 or 6.

[0040] Preferably, the type of vector includes, but is not limited to, a viral vector or a non-viral vector. Further preferably, the viral vector comprises at least one of a lentiviral vector, an adenoviral vector, a baculoviral vector, a retroviral vector, a poxvirus vector, a Sendai virus vector, and a herpes simplex virus vector. Further preferably, the non-viral vector includes, but is not limited to, a eukaryotic vector or a prokaryotic vector.

[0041] In the application of this application, the types of stem cells described can be distinguished according to their differentiation and developmental potential and include: totipotent stem cells: can differentiate into a complete individual, such as embryonic stem cells (ES cells); pluripotent stem cells: can differentiate into a variety of cell tissues and organs, such as hematopoietic stem cells, germline stem cells, mesenchymal stem cells, neural stem cells, liver stem cells, pancreatic stem cells, etc.; unipotent stem cells: refers to those with the potential to differentiate into one or two related cell types, such as myoblasts in muscles and stem cells in the basal layer of epithelial tissue.

[0042] In the application of the present application, the types of stem cells described may include, according to the developmental stage of the stem cells: embryonic stem cells: highly undifferentiated cells with developmental omnipotence, capable of differentiating into all tissues and organs of an adult; adult stem cells: under specific conditions, adult stem cells either generate new stem cells or differentiate according to a certain procedure to form new functional cells, thereby maintaining a dynamic balance of growth and decline of tissues and organs.

[0043] The adult stem cells include but are not limited to: hematopoietic stem cells: a type of adult stem cell with the longest and most in-depth research history, which has the potential to differentiate into various cells of the blood system and has great application potential in the fields of disease treatment, anti-aging and health care; reproductive stem cells: have the potential to differentiate into reproductive cells and various supporting cells of the gonads, enabling adults to exert their reproductive function and delaying the aging of the gonads; mesenchymal stem cells: have the potential to differentiate into the body's bone, cartilage and various organ cells, and also have unique immune regulation functions; neural stem cells: have the potential to differentiate into various cells of the nervous system; retinal stem cells: have the potential to differentiate into various cells of the retina; cardiac stem cells: have the potential to differentiate into various cells of the heart; liver stem cells: have the potential to differentiate into various cells of the liver; pancreatic stem cells: have the potential to differentiate into various cells of the pancreas, and under specific conditions, can differentiate into pancreatic islet cells; lung stem cells: have the potential to differentiate into various cells of the lungs; kidney stem cells: have the potential to differentiate into various cells of the kidneys.

[0044] Preferably, the stem cells are embryonic stem cells, adult stem cells, mesenchymal stem cells, umbilical cord blood stem cells, hematopoietic stem cells, neural stem cells, adipose stem cells, skin stem cells and / or muscle stem cells.

[0045] Preferably, the stem cells are mesenchymal stem cells; the stem cells are isolated from umbilical cord blood, umbilical cord, placenta, adipose tissue, skin, neural tissue, bone marrow or embryo.

[0046] In the application of the present application, the inflammatory diseases include, but are not limited to, polymyositis, dermatomyositis, periarteritis nodosa, aortitis syndrome, malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, ANCA-associated vasculitis, chronic atrophic gastritis, rapidly progressive glomerulonephritis, lupus nephritis, psoriatic arthritis, temporal arteritis, eosinophilic fasciitis, nonalcoholic steatohepatitis, eosinophilic chronic sinusitis, ankylosing spondylitis, inclusion body myositis, neuromyelitis optica, giant cell arteritis, chronic inflammatory demyelinating polyneuropathy, inflammatory bowel disease, vasculitis, allergic granulomatosis with vasculitis, hypersensitivity vasculitis, rheumatoid vasculitis and / or large vessel vasculitis. Based on the expression characteristics of the stem cells, those skilled in the art can selectively apply them to suitable indications.

[0047] In the application of this application, the infectious diseases include but are not limited to: sepsis and toxic shock, infectious diarrhea, abdominal infection, urinary tract infection, skin and soft tissue infection;

[0048] Preferably, the infectious diseases include but are not limited to: streptococcal infection, staphylococcal infection, streptococcal infection, enterococcal infection, meningococcal infection, Neisseria gonorrhoeae infection, cholera and Vibrio infection, Salmonella infection, Shigella infection, Pseudomonas and Gram-negative enterobacteriaceae infection, Legionella infection, pertussis, diphtheria, anaerobic bacteria infection, Helicobacter pylori infection, tuberculosis, Yersinia pestis infection, anthrax, syphilis, leptospirosis, Lyme disease, rickettsial disease, mycoplasma infection, chlamydia infection, influenza, avian influenza, SARS, viral hemorrhagic fever, epidemic encephalitis B, rabies, viral hepatitis, chronic hepatitis B and / or chronic hepatitis C.

[0049] In the application of the present application, the autoimmune diseases include but are not limited to Behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, systemic scleroderma, progressive systemic sclerosis, scleroderma, mixed connective tissue disease, Castleman's disease, Sjögren's syndrome, adult Still's disease, Cogan syndrome, RS3PE syndrome, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, IgG4-related disease, Guillain-Barré syndrome, myasthenia gravis, autoimmune hepatitis, primary biliary cirrhosis, Good-pasture syndrome, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Basedow's disease, Hashimoto's disease, autoimmune adrenal insufficiency, primary hypothyroidism, Addison's disease, idiopathic Addison's disease, type 1 diabetes mellitus, slowly progressive type 1 diabetes mellitus, localized scleroderma, psoriasis, bullous pemphigoid, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, alopecia areata, vitiligo, vitiligo vulgaris, multifocal motor neuropathy, sarcoidosis, amyotrophic lateral sclerosis, Harada disease, autoimmune optic neuropathy, idiopathic azoospermia, recurrent miscarriage, celiac disease, severe asthma, chronic urticaria, transplant immunization, familial Mediterranean fever, dilated cardiomyopathy, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, lupus nephritis, and / or systemic mastocytosis;

[0050] Preferably, the autoimmune disease is plaque psoriasis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis and / or lupus nephritis.

[0051] In the application of the present application, the tumor includes malignant tumors and / or non-malignant tumors.

[0052] According to the general understanding of those skilled in the art, the malignant tumors and / or non-malignant tumors may include at least the following according to the human body system: malignant tumors and / or non-malignant tumors of the musculoskeletal system, circulatory system, digestive system, respiratory system, nervous system, urinary system, endocrine system, reproductive system and / or immune system.

[0053] In some specific embodiments, the malignant tumor includes but is not limited to: basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, breast cancer, peritoneal cancer, cervical cancer, bile duct cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, glioblastoma, liver cancer, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rectal cancer, respiratory system cancer, salivary gland cancer, skin cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, urinary system cancer, B-cell lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell leukemia and / or chronic myeloblastic leukemia. The non-malignant tumors include, but are not limited to, lipoma, neurofibroma, sebaceous cyst, breast cyst, fibroma, leiomyoma, thyroid papilloma, hamartoma, hepatic hemangioma, renal cyst, gastrointestinal polyp and / or serous cystadenoma.

[0054] Preferably, the disease is arthritis or psoriasis; the arthritis includes but is not limited to rheumatoid arthritis, psoriatic arthritis or joint synovitis. In some specific embodiments, the drug has the effect of reducing inflammatory factors.

[0055] The application is preferably pharmaceutical use or use in preparing diagnostic products.

[0056] When the application is for preparing medicine, the medicine also includes pharmaceutically acceptable excipients.

[0057] Beneficial effects of this application:

[0058] This application improves the therapeutic efficacy of stem cell therapy by modifying stem cells with tandem nanoantibodies. The resulting modified stem cells exhibit the following characteristics: high expression of IgG4, with an expression level as high as 10.43±0.52μg / mL; high expression of IL-17Nb, with a concentration of 4106±185.84ng / mL; and stable and sustained expression and secretion of IL-17Nb. The secreted IL-17Nb can block the binding of IL-17A / IL17RA, with an inhibition rate of up to 60% after a 5-fold dilution. The modified stem cells of this application have been validated in experimental animal models, demonstrating their promising application prospects in IL-17A-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0060] FIG1 shows the results of the blocking experiment using the positive control antibody Ixekizumab.

[0061] FIG2 shows the SDS-PAGE results of the tandem antibody G4-H10.

[0062] FIG3 shows the antibody affinity test results of the positive control antibody ixekizumab and the tandem antibody G4-H10 in Basic Experimental Example 3. The left figure shows the positive control antibody ixekizumab, and the right figure shows the tandem antibody G4-H10.

[0063] FIG4 shows the antibody blocking experiment results of the tandem antibody G4-H10 in Basic Experimental Example 4.

[0064] FIG5 shows the titer detection results of the lentivirus prepared in Basic Experimental Example 6.

[0065] FIG6 shows the FITC channel signal detection results of mesenchymal stem cells in Basic Experimental Example 7. The reading value of hUC-MSC is 0.353%, and the reading value of G4-H10-MSC is 63.329%.

[0066] Figure 7 shows the results of ELISA detection of IgG4 expression and IL-17Nb expression in G4-H10-MSC cells.

[0067] FIG8 shows the results of ELISA assay showing that IL-17Nb blocks the binding of IL-17A to IL-17RA.

[0068] Figure 9 shows the expression of IL17Nb in mesenchymal stem cells at different culture times. *** indicates a significant difference between the hUC-MSC group supplemented with G4-H10 and the IL17Nb-MSC group (P < 0.001).

[0069] Figure 10 shows the statistical results of animal weights in Example 1. *** indicates a significant difference between the model control group and the normal control group (P < 0.001); ## indicates a significant difference between the G4-H10-MSC treatment group and the model control group (P < 0.01); && indicates a significant difference between the G4-H10-MSC treatment group and the positive antibody treatment group (P < 0.01); @ indicates a significant difference between the C3-H10-MSC treatment group and the hUC-MSC treatment group (P < 0.05).

[0070] Figure 11 shows the statistical results of paw thickness in animals from Example 1. *** indicates a significant difference between the model control group and the normal control group (P < 0.001); ## indicates a significant difference between the three treatment groups and the model control group (P < 0.01); & indicates a significant difference between the G4-H10-MSC treatment group and the positive antibody treatment group (P < 0.05).

[0071] Figure 12 shows the pathological tissue staining scoring results of Example 1. # indicates a significant difference between the hUC-MSC treatment group and the model control group (P < 0.05); ## indicates a significant difference between the G4-H10-MSC treatment group and the model control group (P < 0.01); & indicates a significant difference between the G4-H10-MSC treatment group and the positive antibody treatment group (P < 0.05); @ indicates a significant difference between the G4-H10-MSC treatment group and the hUC-MSC treatment group (P < 0.05). The hUC-MSC group refers to the hUC-MSC treatment group, the positive antibody group refers to the positive antibody treatment group, and the G4-H10-MSC group refers to the G4-H10-MSC treatment group.

[0072] FIG13 is the histopathological staining results of Example 1.

[0073] Figure 14 shows the statistical results of animal weights in Example 2. ** indicates a significant difference between the model control group and the normal control group (P < 0.01); ## indicates a significant difference between the G4-H10-MSC treatment group and the model control group (P < 0.01); && indicates a significant difference between the G4-H10-MSC treatment group and the positive antibody treatment group (P < 0.01).

[0074] Figure 15 is a photograph of the animal skin in Example 2. The hUC-MSC group is the hUC-MSC treatment group, the positive antibody group is the positive antibody treatment group, and the G4-H10-MSC group is the G4-H10-MSC treatment group.

[0075] Figure 16 shows the clinical scoring results of animal skin in Example 2. **** indicates a significant difference between the model control group and the normal control group (P < 0.0001); ### indicates a significant difference between each of the three treatment groups and the model control group (P < 0.001); && indicates a significant difference between the G4-H10-MSC treatment group and the positive antibody treatment group (P < 0.01); @@ indicates a significant difference between the G4-H10-MSC treatment group and the hUC-MSC treatment group (P < 0.01).

[0076] Figure 17 shows the statistical results of animal skin thickness in Example 2. *** indicates a significant difference between the model control group and the normal control group (P < 0.001); ## indicates a significant difference between each of the three treatment groups and the model control group (P < 0.01); && indicates a significant difference between the G4-H10-MSC treatment group and the positive antibody treatment group (P < 0.01). The hUC-MSC group refers to the hUC-MSC treatment group, the positive antibody group refers to the positive antibody treatment group, and the G4-H10-MSC group refers to the G4-H10-MSC treatment group.

[0077] Figure 18 shows the statistical results of animal weights in Example 3. ** indicates a significant difference between the model control group and the normal control group (P < 0.01); ## indicates a significant difference between the G4-H10-MSC treatment group and the model control group (P < 0.01); && indicates a significant difference between the G4-H10-MSC treatment group and the positive antibody treatment group (P < 0.01).

[0078] Figure 19 shows the paw joint scoring results of the animals in Example 3. **** indicates a significant difference between the model control group and the normal control group (P < 0.0001); ### indicates a significant difference between each of the three treatment groups and the model control group (P < 0.001); && indicates a significant difference between the G4-H10-MSC treatment group and the positive antibody treatment group (P < 0.01); @ indicates a significant difference between the G4-H10-MSC treatment group and the hUC-MSC treatment group (P < 0.05).

[0079] Figure 20 shows the clinical scoring results of animal skin in Example 3. **** indicates a significant difference between the model control group and the normal control group (P < 0.0001); ## indicates a significant difference between each of the three treatment groups and the model control group (P < 0.01); & indicates a significant difference between the G4-H10-MSC treatment group and the positive antibody treatment group (P < 0.05); @ indicates a significant difference between the C3-H10-MSC treatment group and the hUC-MSC treatment group (P < 0.05).

[0080] Figures 21 to 23 are the results of inflammatory factor detection in the serum of each group of animals in Example 3. The hUC-MSC group is the hUC-MSC treatment group, the positive antibody group is the positive antibody treatment group, and the G4-H10-MSC group is the G4-H10-MSC treatment group:

[0081] Figure 21 shows the results of TNF-α detection in the serum of each group of animals in Example 3, where: ** represents a significant difference between the model control group and the normal control group (P<0.01); # represents a significant difference between the hUC-MSC treatment group and the model control group (P<0.05); ## represents a significant difference between the G4-H10-MSC treatment group and the model control group (P<0.01); & represents a significant difference between the G4-H10-MSC treatment group and the positive antibody treatment group (P<0.05); @ represents a significant difference between the G4-H10-MSC treatment group and the hUC-MSC treatment group (P<0.05).

[0082] Figure 22 shows the IL-6 detection results in the serum of each group of animals in Example 3, where: ** indicates that there was a significant difference between the model control group and the normal control group (P<0.01); # indicates that there was a significant difference between the hUC-MSC treatment group and the model control group (P<0.05); ## indicates that there was a significant difference between the G4-H10-MSC treatment group and the model control group (P<0.01); & indicates that there was a significant difference between the G4-H10-MSC treatment group and the positive antibody treatment group (P<0.05).

[0083] Figure 23 shows the IL-23 detection results in the serum of each group of animals in Example 3, where: * indicates that there was a significant difference between the model control group and the normal control group (P<0.01); # indicates that there was a significant difference between the positive antibody and G4-H10-MSC treatment groups and the model control group (P<0.05); @ indicates that there was a significant difference between the G4-H10-MSC treatment group and the hUC-MSC treatment group (P<0.05). DETAILED DESCRIPTION

[0084] Below in conjunction with specific embodiment, this application is further elaborated in detail, and the following embodiment is not intended to limit this application, but is only intended to illustrate this application. Unless otherwise specified, the experimental methods used in the following examples, the experimental methods for which specific conditions are not specified in the examples, are usually in accordance with conventional conditions, and the materials and reagents used in the following examples, etc., can be obtained from commercial channels unless otherwise specified. Some of the reagents, materials and equipment that may be involved in this application are shown in Table 1.

[0085] Table 1 Main reagents, materials and equipment involved in this application

[0086] Basic Experimental Example 1 IL-17 Nanobody Screening

[0087] As embodied in the invention patent of the same filing date as this application, the present application screens antibodies by immunizing animals with antigens, including the following steps:

[0088] (1) Preparation of recombinant antigens

[0089] A 6×His tag was added to the C-terminus of the IL-17 antigen, and after prokaryotic codon optimization, gene synthesis and subcloning into the pET28a vector were performed. After verification by Sanger sequencing, plasmid extraction was performed. The recombinant plasmid was transformed into BL21 competent cells, induced overnight with 0.5 mM IPTG, and the bacterial culture was collected and lysed. The recombinant protein was purified using a nickel column to a purity greater than 90%.

[0090] (2) Preparation of positive control antibody Ixekizumab

[0091] Ixekizumab heavy chain variable region: SEQ ID NO. 20; light chain variable region: SEQ ID NO. 21.

[0092] The purity of the positive control antibody protein was detected by SDS-PAGE and the purity was >95%.

[0093] (3) Construction and verification of IL-17A reporter gene cell lines

[0094] Based on the amino acid sequence information of IL-17RA (UniProtKB: Q96F46) and IL-17RC (UniProtKB: Q8NAC3), a lentiviral expression vector was constructed and packaged into lentivirus. 293 cells were co-infected with these cells, and recombinant 293 cells overexpressing both receptors were screened. Furthermore, the NFκB-Luciferase and ACT1 genes were stably transfected to construct the IL-17A reporter cell line 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc. Recombinant antigen was added for activation, and the positive control antibody ixekizumab was added for blocking assays. This established cell line for in vitro pharmacodynamic evaluation of candidate antibodies targeting IL-17A. The constructed IL-17A receptor-overexpressing cell line was able to bind to IL-17A, and the recombinant IL17A protein effectively activated luciferase expression in the 293F-IL17Ra / IL17Rc-NFκB-Luc reporter cell line.

[0095] The positive control antibody ixekizumab was added to 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc cells together with the IL-17A recombinant protein. The positive control antibody ixekizumab inhibited the binding of the IL17A protein to its membrane receptor and inhibited the intracellular NFκB signal, showing a dose-effect relationship, as shown in Figure 1.

[0096] (4) Alpaca Immunity

[0097] Alpaca were immunized with the recombinant antigen protein prepared above, with an immunization interval of 21 days. Ten days after the last immunization, some peripheral blood was collected, and the serum was separated and the immune effect was detected by ELISA.

[0098] (5) Detection of immune titer

[0099] After 6 rounds of immunization, the immune titers of the alpacas all reached the requirements (see Table 2-3 below).

[0100] Table 2 Immune titer test results

[0101] Table 3 Immune titer test results

[0102] (6) PBMC isolation and VHH antibody fragment cloning

[0103] PBMCs were isolated, RNA was extracted and reverse transcribed, and two rounds of PCR were performed. The products were recovered from gel and the concentration was determined.

[0104] (7) Construction and selection of single domain antibody yeast display library

[0105] The linearized vector and PCR product are co-electroporated into a competent yeast strain. A single-domain antibody yeast display library is selected; after panning, individual yeast colonies are detected by flow cytometry. Overlap PCR products are amplified, transiently transfected and detected, and the product is recovered and transfected. Positive clones are selected by ELISA, and the VHH antibody sequence is obtained and gene synthesis is performed. After genetically engineered expression, protein A purification is performed.

[0106] Positive clones were enriched; selected monoclonal clones were identified by Phage ELISA and sequenced to obtain nucleic acid and amino acid sequence information for candidate single-domain antibodies. Twenty monoclonal clones were randomly selected and sequenced, revealing significant sequence diversity and good library diversity. In silico analysis of the amino acid sequences in the CDR regions of candidate single-domain antibodies was used to identify potential post-translational modification sites.

[0107] Based on the results of yeast monoclonal flow cytometry, positive clones binding to IL-17A-His were selected for genomic DNA extraction and antibody sequences were obtained through PCR. Differential clones were selected for overlapping PCR amplification. A signal peptide was added to the N-terminus of the VHH and an IgG1-Fc was added to the C-terminus. The PCR products were transiently transfected into HEK293 cells. The expressed antibody supernatant was assayed by ELISA: 100 μL of transfection supernatant was added to a 96-well plate pre-coated with IL-17A recombinant antibody and incubated. ELISA was performed using HRP-Protein A as a secondary antibody. Differential clones were then used to construct eukaryotic expression vectors. The constructed single-domain antibody expression vectors were transiently transfected into 293F cells, and the recombinant antibodies were affinity purified using Protein A.

[0108] (8) ELISA was used to detect the binding of the recombinant antibody to the target protein, and FACS was used to detect the binding of the single-domain antibody to the reporter gene cell line to complete the single-domain antibody blocking function experiment, and the ForteBio OCTET R2 system was used for kinetic characterization analysis. The ForteBio OCTET R2 system was used for kinetic characterization analysis. Screening was performed based on the results of all antibodies. This application further expands on the above-mentioned basic screening method. In the antibody blocking activity test, it was found that although some single-domain antibodies were able to block the activation of downstream target proteins by the Human IL-17A protein, the blocking effect was weaker than that of the positive antibody Ixekizumab. Therefore, two of the anti-IL-17A single-domain antibodies were connected in series to form a bivalent antibody to enhance its blocking effect. Specifically, this application protects the application of stem cells prepared by the tandem antibodies constructed based on 2-G4 (abbreviated as G4) and 3-H10 (abbreviated as H10), and the subsequent tandem antibodies are referred to as G4-H10.

[0109] In this application:

[0110] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly used in the field to which this application belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0111] Unless the context clearly dictates otherwise, as used herein, the expressions "a" and "an" include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0112] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0113] As used herein, the term "comprising" or "including" means "including but not limited to". The term is intended to be open-ended to specify the presence of any of the described features, elements, integers, steps or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components or groups thereof. Therefore, the term "comprising" includes the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" used throughout the invention, particularly in the claims, may be replaced by the term "consisting of". The three-letter and one-letter codes for amino acids used herein are known to those skilled in the art, or as described in J Biol. Chem, 243, p3558 (1968).

[0114] As used herein, the terms "optionally," "either," "any," or "any" mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprising an antibody heavy chain variable region" means that an antibody heavy chain variable region of a specific sequence may but need not be present.

[0115] As used herein, the term "stem cell" refers to undifferentiated cells capable of self-renewal and differentiation at the single-cell level to produce daughter cells, including self-renewing progenitor cells, non-renewing progenitor cells, and terminally differentiated cells. Stem cells are cells with unlimited self-renewal, proliferation, and differentiation capabilities under certain conditions. They can produce daughter cells with identical phenotypes and genotypes, as well as specialized cells that make up tissues and organs. They play a major role in the body's growth, maintenance, renewal, and injury repair.

[0116] As used herein, the term "mesenchymal stem cells," also referred to in the art as "mesenchymal stem cells (MSC)" or "mesenchymal stromal cells (MSC)," refers to a population of multipotent stromal cells derived from the mesoderm that possess a certain differentiation potential and can differentiate into a variety of cell types. They primarily originate from and reside in the bone marrow, but also include multipotent cells derived from a wide range of other "non-bone marrow" tissues, such as placenta, umbilical cord blood, adipose tissue, adult muscle, corneal stroma, and dental pulp of deciduous teeth.

[0117] As used herein, the term "IL-17A," "interleukin-17A," or "IL-17" refers to a cytokine that belongs to the interleukin 17 family, is produced by T cells and other types of immune cells, and plays an important role in the immune system. IL-17A is primarily produced by Th17 cells, and other cells, including CD8+ T cells, γδ T cells, NK cells, and neutrophils, mast cells, and macrophages, also express IL-17A. It primarily acts on immune cells, such as macrophages, neutrophils, and endothelial cells, to induce an inflammatory response. In some instances, the term includes variants, homologs, orthologs, and paralogs. For example, an antibody specific for human IL-17A may, under certain circumstances, cross-react with an IL-17A protein from another species, such as a monkey. In other embodiments, antibodies specific for human IL-17A protein may be completely specific for human IL-17A protein and not cross-react with proteins from other species or other types, or may cross-react with IL-17A proteins from some other species but not all other species.

[0118] As used herein, the term "anti-IL-17A single domain (nano) antibody", "IL-17A single domain (nano) antibody" or "tandem single domain (nano) antibody that specifically binds to IL-17A" refers to an antibody that specifically binds to IL-17A and partially or completely neutralizes, inhibits or weakens IL-17A activity, and / or inactivates IL-17A, prevents IL-17A response or downstream pathways mediated by IL-17A or other IL-17A-mediated functions.

[0119] As used herein, the term "antibody" refers to a glycoprotein comprising heavy chains (H) and light chains (L) interconnected by disulfide bonds (SS). Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as VH). The light chain constant region is composed of one domain, CL. Light chains are divided into two categories: kappa-type light chains and lambda-type light chains (for example, the light chain constant region Cκ / λ in this application indicates that the light chain constant region is a kappa-type light chain or a lambda-type light chain). The VH region and the VL region can be further divided into hypervariable regions (also called complementarity determining regions (CDRs)), with relatively conserved framework regions or framework regions (FRs) interposed therebetween. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Antibodies include monospecific antibodies, bispecific antibodies, and multispecific antibodies, as long as they exhibit the desired biological activity or function.

[0120] As used herein, the term "single domain antibody" (sdAb) or "nanoantibody" has its general meaning in the art and refers to an antibody fragment with a molecular weight of only 1215 kDa, which is composed of a single monomeric variable antibody domain derived from a heavy chain. Such single domain antibodies (named VHH) can be found in camelid mammals and naturally lack light chains. For a general description of (single) domain antibodies, reference is also made to the above-mentioned prior art and EP 0368684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al, Trends Biotechnol, 2003, 21 (11): 484-490; and WO 06 / 030220, WO 06 / 003388. The amino acid sequence and structure of a single-domain antibody can be considered to be composed of four framework regions or "FRs," which are referred to in the art as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively. The framework regions are separated by three complementarity-determining regions or "CDRs," which are referred to in the art as "complementarity-determining region 1" or "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3," respectively. Thus, a single-domain antibody can be defined as an amino acid sequence having the following general structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 and FR4 refer to framework regions 1-4, respectively, and CDR1-CDR3 refer to complementarity-determining regions 1-3. In the context of the present disclosure, the amino acid residues of single domain antibodies are numbered according to the common numbering scheme for VH domains given by the International ImMunoGeneTics information system amino acid numbering (http: / / imgt.cmes.fr / ).

[0121] As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are amino acids encoded by the genetic code, as well as modified amino acids such as hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., carbon is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids.

[0122] As used herein, the terms "activity," "functional activity," or "biological activity," or the terms "biological property" or "biological characteristic" are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize IL-17A activity in vivo or in vitro, IC50, in vivo stability of the antibody, and the immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the target peptide, or with other proteins or tissues), and the ability to maintain high protein expression levels in mammalian cells. The aforementioned properties or characteristics can be observed, measured, or assessed using techniques known in the art, including, but not limited to, ELISA, FACS, or BIACORE plasma resonance analysis, in vitro or in vivo neutralization assays, receptor binding, cytokine or growth factor production and / or secretion, signal transduction, and immunohistochemistry of tissue sections from various sources (including humans, primates, or any other source).

[0123] As used herein, the term "Fc," "Fc region," or "Fc fragment" refers to a polypeptide consisting of the CH2 and CH3 domains of IgA, IgD, and IgG, or the CH2, CH3, and CH4 domains of IgE and IgM, connected by a hinge region. Although the breakdown of the Fc fragment varies, the heavy chain Fc fragment of human IgG typically refers to the polypeptide extending from A231 to its carboxyl terminus.

[0124] As used herein, the term "epitope" refers to a protein determinant that is capable of specific binding to an antibody. An epitope is typically composed of surface clustered molecules, such as amino acids or sugar side chains, and typically has specific three-dimensional structural characteristics, as well as specific charge characteristics. The difference between conformational and non-conformational epitopes is that the binding to the former, but not the latter, is lost in the presence of a denaturing solvent. An epitope may include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked or covered by a specific antigen-binding peptide (in other words, the amino acid residue is within the footprint of the specific antigen-binding peptide).

[0125] As used herein, the term "affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is a product of the dissociation rate constant and the association rate constant (KD, respectively). off and K on Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0126] As used herein, the term "high affinity" or "high avidity" refers to an IgG antibody with a KD of 1.0 × 10 -6 M or less, preferably 5.0×10 -8 M or less, more preferably 1.0×10 -8 M or lower, 5.0×10 -9 M or less, more preferably 1.0×10 -9 M or lower. For other antibody subtypes, “high affinity” binding may vary. For example, “high affinity” binding for the IgM subtype is defined as a KD of 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or lower.

[0127] As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and a polymer thereof in single-stranded or double-stranded form. Unless explicitly limited, the term includes nucleic acids with similar binding properties to reference nucleic acids and analogs containing known natural nucleotides that are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer, Nucleic Acid Res. 19:5081 (1991); Ohtsuka, J. Biol. Chem. 260:2605-2608 (1985); Rossolini, Mol. Cell. Probes 8:91-98 (1994)).

[0128] As used herein, the term "construct" refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule), derived from any source, capable of integrating with a genome or autonomously replicating, constituting a polynucleotide molecule in which one or more polynucleotide molecules have been connected (i.e., operably linked) in a functionally operational manner. Recombinant constructs will typically comprise a polynucleotide of the present invention operably linked to a transcription initiation regulatory sequence that directs transcription of the polynucleotide in a host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acid of the present invention.

[0129] As used herein, the term "vector" refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (i.e., introducing heterologous DNA into a host cell). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial origins of replication). After being introduced into the host cell, other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell and are therefore replicated together with the host genome. In addition, certain vectors are capable of directing the expression of operatively connected genes. Such vectors are referred to herein as "expression vectors."

[0130] As used herein, the term "expression vector" refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected, or transduced into a host cell. Expression vectors contain one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, to provide for amplification within the host.

[0131] As used herein, the term "pharmaceutical composition" generally refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is to be administered. The composition is sterile.

[0132] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio, within the scope of reasonable medical judgment. As used herein, the term "pharmaceutically acceptable carrier, excipient and / or diluent" refers to a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Pharmaceutically acceptable materials, compositions, or vehicles, such as liquid or solid fillers, diluents, excipients, solvents, media, encapsulating materials, manufacturing aids, or solvent encapsulating materials, are involved in maintaining the stability, solubility, or activity of the antibodies or antigen-binding fragments thereof disclosed herein, and include, but are not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein), or degradation products thereof (such as lactalbumin hydrolysate), etc.

[0133] Basic Experimental Example 2 Preparation and Purification of IL-17A-Specific Tandem Antibodies

[0134] The sequence of 2-G4 is SEQ ID NO. 16, and the CDR regions are SEQ ID NO. 1-3;

[0135] The sequence of 3-H10 is SEQ ID NO. 17, and the CDR regions are SEQ ID NOs. 4-6, respectively.

[0136] The tandem antibody in this example also includes a linker (GGGGS) 3, a hinge region, and a CH region.

[0137] The sequence of the tandem antibody constructed in this example is SEQ ID NO. 18. The gene encoding the tandem antibody is SEQ ID NO. 19.

[0138] The above-mentioned tandem antibodies can be prepared by referring to the antibody preparation method in the prior art, as follows:

[0139] (1) The gene of SEQ ID NO. 19 was synthesized and concatenated with the human IgG1 Fc region gene sequence and subcloned into the expression vector pcDNA3.4-hIgG1-Fc. After the vector was verified by sequencing, the endotoxin-free plasmid was prepared using the Qiagen Plasmid Extraction Kit (CAT#: DP117).

[0140] (2) Remove the LVTransm transfection reagent and single-chain antibody expression vector from the refrigerator. After thawing at room temperature, pipette up and down to mix thoroughly. Remove the PBS buffer and warm it to room temperature. Take 2 mL of PBS to one well of a 6-well plate and add 130 μg of the antibody expression vector to each well. Pipet up and down to mix thoroughly. Then add 400 μL of LVTransm and immediately pipette up and down to mix thoroughly. Let it stand at room temperature for 10 minutes to obtain the DNA / LVTransm complex.

[0141] (3) Add the DNA / LVTransm complex to 30 mL of 293F cells and gently shake to mix thoroughly. Incubate the cells at 37°C, 5% CO2, and 130 rpm for 6-8 hours. Add 50 mL of fresh 293 cell culture medium and return the cells to the incubator for continued culture.

[0142] After 7 days of continuous culture, the culture supernatant was collected by centrifugation and filtered through a 0.45 μm filter membrane. The filtrate was transferred to a sterile centrifuge tube, and the antibody was purified using Protein A to obtain the purified target antibody.

[0143] The purity of the target antibody protein was detected by SDS-PAGE. The SDS-PAGE results of the tandem antibody G4-H10 are shown in Figure 2. The results showed that the protein purity was >95%.

[0144] Basic Experimental Example 3: Affinity Detection of IL-17A-Specific Tandem Antibodies

[0145] Using ixekizumab as a positive control antibody, the heavy and light chain variable regions of ixekizumab were synthesized. The heavy chain variable region was subcloned into the pcDNA3.4-hIgG4 vector (hIgG4 amino acid sequence is SEQ ID NO. 22), and the light chain variable region was subcloned into the pcDNA3.4-hIgKc vector (hIgKc amino acid sequence is SEQ ID NO. 23). After verification by Sanger sequencing, an endotoxin-free plasmid was prepared using a plasmid extraction kit for later use. Other steps were similar to those in Basic Experimental Example 2 to prepare the positive control antibody, with a purity of >95%.

[0146] ELISA to detect the binding of recombinant antibodies to target proteins:

[0147] The purified test antibody (2 μg / mL) was used to coat the ELISA plate, and Biotin-IL-17A-His was added at a starting concentration of 10 μg / mL. The plate was diluted 5-fold to 7 points. HRP-Streptavdin was used for ELISA detection. The OD450 values ​​measured at each concentration were plotted to read the EC50 value.

[0148] The results showed that the EC50 value of the control antibody ixekizumab was 10.06 μg / mL, while the EC50 value of the tandem antibody was 1.177 μg / mL (see Figure 3).

[0149] Basic Experiment Example 4 Antibody Blocking Function Experiment

[0150] Blocking activity was tested using the 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc reporter cell line, testing the tandem antibodies described in Basic Experimental Example 2 and the positive control antibody ixekizumab described in Basic Experimental Example 3.

[0151] Based on the amino acid sequence information of IL-17RA (UniProtKB: Q96F46) and IL-17RC (UniProtKB: Q8NAC3), a lentiviral expression vector was constructed and packaged with lentivirus. 293 cells were co-infected, and recombinant 293 cells that simultaneously overexpressed these two receptors were screened. The IL-17A reporter gene cell line 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc was further stably transfected with IL-17A (SEQ ID NO. 24) and ACT1 genes (SEQ ID NO. 25).

[0152] Ixekizumab and the tandem antibody can block the activation of 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc by human IL-17A protein, with IC50 values ​​of 2.235nM and 0.5888nM, respectively. The tandem antibody is superior to the control antibody. See Figures 1 and 4.

[0153] Basic Experiment Example 5 Stability Experiment Process and Result Analysis:

[0154] Experimental process: Fluorescence changes are detected by micro-differential scanning fluorescence technology (nanoDSF), which can detect protein thermal denaturation and chemical denaturation under natural conditions, and accurately determine the temperature (Tm) when the protein is 50% unfolded and the temperature (Tagg) when aggregation begins to occur; the higher the thermal denaturation Tm value and Tagg, the more stable the antibody protein.

[0155] Take 100 μL of the target antibody and centrifuge at 4°C, 12,000 × g for 10 minutes. Then, draw up the sample using a capillary tube. Prepare two capillaries for each sample as a parallel control. Place them into the corresponding slots in order, ensuring that the capillaries are fully aspirated and free of bubbles before analysis. The temperature range is 20°C-95°C, at a rate of 1°C / min, and the test time is 75 minutes.

[0156] Tm is defined as the melting temperature, Tonset is defined as the temperature at which unfolding begins, and Tagg is defined as the aggregation temperature.

[0157] The antibodies to be detected were the tandem antibodies involved in Basic Experimental Example 2 and the positive control antibody ixekizumab in Basic Experimental Example 3.

[0158] Ixekizumab results: T m1 The value is 56.10±0.02℃; T m2 The value is 79.84±0.13℃; T onset The value is 47.50±0.08℃; T agg The value was 61.86±0.23℃; the result of tandem antibody: T m1 The value is 58.13±0.06℃; T m2 The value is 82.43±0.48℃; T onset The value is 51.28±0.22℃; T agg The value is 58.48±0.00℃.

[0159] Basic Experimental Example 6 LV-G4-H10: Fc Lentivirus Preparation and Detection Method

[0160] 1. Construction of Lentiviral Shuttle Plasmid

[0161] A G4-H10:Fc fusion protein lentiviral shuttle plasmid was constructed. The VHH sequences of two candidate antibodies (IL-17Nb), G4 and H10 (the amino acid sequences of G4 and H10 are shown in SEQ ID NOs. 16 and 17), were linked via (GGGGS)3 and then tandemly linked with IgG4 Fc (the amino acid sequence of IgG4 Fc is shown in SEQ ID NO: 26, and the nucleotide sequence is shown in SEQ ID NO: 27) to form a VHH-(GGGGS)3-VHH-IgG4Fc sequence, which was located downstream of the EF-1alpha promoter, thereby obtaining the G4-H10:Fc lentiviral shuttle plasmid.

[0162] 2. Lentivirus Preparation (Viral Packaging)

[0163] 24 h before lentiviral packaging, prepare shake flasks and adjust the density of HEK293T cells (purchased from ATCC, catalog number CRL-3216) to 1.0 × 10 6 / mL, 60mL per bottle for standby use.

[0164] Prepare transfection reagent / DNA complex: Take 7.5 mL of 293T culture medium to a 15 mL centrifuge tube, add 40 μg of G4-H10:Fc lentiviral shuttle plasmid and 80 μg of helper plasmid (purchased from addGene, catalog number: 12253, 12259), mix thoroughly by pipetting up and down, then add 360 μL of transfection reagent and immediately mix by pipetting up and down with a 1 mL pipette. Let it stand at room temperature for 10 min (no more than 15 min).

[0165] Add the transfection reagent / DNA complex dropwise to the cells prepared the day before, shaking the flask while adding. After thorough mixing, place the flask on a shaker at 37°C, 5% CO2, and 120 rpm for incubation.

[0166] After culturing for 24 h, the supernatant was collected and filtered through a 0.45 μm syringe filter into a virus centrifuge tube, and centrifuged at 45,000 × g and 4°C for 90 min.

[0167] After centrifugation, pour off the supernatant and remove the remaining supernatant with a pipette. Resuspend the pellet with 1 mL of PBS and dispense it into virus aliquot tubes, 100 μL per tube, which is the LV-G4-H10:Fc lentivirus.

[0168] 3. Lentiviral Titer Detection

[0169] 293T cells were seeded into 24-well plates and cultured overnight. 20 μL of LV-G4-H10:Fc virus stock solution, 10-fold diluted virus solution, and 100-fold diluted virus solution were added, respectively. The culture was continued for 24 h. Fresh medium was replaced after 24 h. After 9 days of continuous culture, the cells were harvested and genomic DNA was extracted (the kit was purchased from Thermo, product number K0721). The plasmid template copy number was adjusted with ddH2O (double-distilled water) and the calibration range was 1×10 9 -1×10 3 Primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. 2× PCR Mix (purchased from Applied Biosystems, ABI, Catalog No. A25742), primers, DNA, and PCR water (purchased from Thermo Fisher Scientific, Catalog No. R0582) were mixed thoroughly and added to the corresponding PCR reaction wells for PCR reaction. Virus titer was calculated according to the following formula.

[0170] Lentivirus titer = number of cells × number of copies / virus volume (mL) × dilution factor.

[0171] The results of lentivirus titer detection are shown in Figure 5. Regardless of whether the virus is diluted 10-fold or 100-fold, the virus titer ranges from 2.3E+7 to 2.6E+7, and there is no significant difference among the three groups. Therefore, the lentivirus titer is 2.45E+7TU / mL.

[0172] Basic Experimental Example 7 Preparation of G4-H10 Gene-Modified Stem Cells (G4-H10-MSC) and Infection Efficiency Detection

[0173] The prepared LV-G4-H10:Fc lentivirus was added at an MOI of 10 to cultured mesenchymal stem cells (P2 mesenchymal stem cells were isolated from the umbilical cord of newborns using an enzymatic hydrolysis method and purified by passage and expansion) with a confluency of 70-80%. The cells were cultured at 37°C and CO2, and then passaged after the cell density reached 100%. Mesenchymal stem cells infected with LV-G4-H10:Fc (G4-H10-MSC) were successfully obtained.

[0174] The obtained G4-H10-MSC and hUC-MSC cells were divided into 1×10 4 pieces / cm 2The cells were cultured in T25 cell culture flasks using DMEM / F12 (containing 10% fetal bovine serum, both products from Thermo Fisher Scientific). When the cell density reached 80-90%, a transport inhibitor (purchased from BD Biosciences, USA, catalog number 555029) was added. After fixation, washing, and staining with FITC-Protein A (stained with FITC-Protein A, purchased from BOSTER Biosciences, catalog number BA1120), the cells were tested on an immunohistochemical analyzer for the FITC channel signal of mesenchymal stem cells (see Figure 6). The FITC channel signal positivity rate of G4-H10-MSC cells exceeded 60%, while normal mesenchymal stem cells (hUC-MSC) had no signal in the FITC channel, indicating that the LV-G4-H10:Fc lentivirus can successfully infect mesenchymal stem cells.

[0175] Basic Experimental Example 8 Detection of IgG4 and IL-17Nb Expression in G4-H10-MSC Cells by ELISA

[0176] The G4-H10-MSCs and hUC-MSCs obtained in Basic Experimental Example 7 were respectively divided into 1×10 4 pieces / cm 2 The cells were cultured in T25 cell culture flasks using DMEM / F12 (containing 10% fetal bovine serum). After 72 hours of culture, the cell supernatant was harvested, aliquoted and frozen for detection of IgG4 and IL-17Nb antibody content and blocking efficiency.

[0177] 1. ELISA detection of IgG4 expression in G4-H10-MSC cells prepared in basic experimental example 7

[0178] The Invitrogen Human IgG4 ELISA Kit (purchased from Thermo Fisher Scientific, Catalog No. BMS2095) was used to detect the amount of secreted IgG4 fusion protein in cell culture supernatants. This kit utilizes a human IgG4 solid-phase sandwich ELISA (enzyme-linked immunosorbent assay) to detect the amount of target bound between matched antibody pairs. IgG4-specific antibodies are pre-coated in ELISA plates. Cell supernatant samples, standards, or controls are then added to these wells and bind to the immobilized (capture) antibody. A secondary antibody is added to form a sandwich structure, and a substrate solution is added to react with the enzyme-antibody-target complex to produce a measurable signal. The intensity of this signal is proportional to the target concentration present in the original sample.

[0179] The cell culture supernatant obtained above (after different dilutions, it was found that 25-fold dilution was the best) was used to detect the IgG4 protein content in the fusion protein using the above-mentioned human IgG4 ELISA kit. The results showed that mesenchymal stem cells (G4-H10-MSC) infected with G4-H10 lentivirus highly expressed IgG4, with an expression level as high as 10.43±0.52μg / mL, while normal hUC-MSC did not express IgG4 (see Figure 7).

[0180] 2. IL-17Nb antibody content detection

[0181] The expression of IL-17A nanoantibodies was detected by IL-17A protein binding assay. IL-17A protein (prepared in Basic Experimental Example 1) with a final concentration of 2 μg / mL was coated on an ELISA plate at 4°C overnight. After blocking with BSA, the G4-H10-MSC supernatant obtained above was detected (after performing different dilutions, a 20-fold dilution was found to be optimal). The standard was G4-H10 fusion protein (prepared in Basic Experimental Example 2). The standard curve concentration range was 0-250 ng / mL. The standard was added to the corresponding wells, and the cell supernatant obtained above (20-fold dilution) was added to the sample wells and incubated for 1 hour. Then, HRP-labeled Protein A antibody (purchased from Boster, catalog number BA1080) was used as an enzyme-labeled antibody and incubated for 1 hour. Finally, TMB was added to develop color in the dark for 20 minutes. After termination, the OD450nm value of each well was detected by a microplate reader. The experimental results are shown in Figure 7. Through the IL-17A binding experiment, G4-H10-MSC can be identified, indicating that IL-17Nb expressed by G4-H10-MSC can bind to IL-17A, while normal mesenchymal stem cells do not express IL-17Nb.

[0182] Basic Experimental Example 9: ELISA to determine the ability of IL-17Nb to block the binding of IL-17A and IL-17RA

[0183] The ability of G4-H10-MSC cells to block IL-17A / IL-17RA binding was assessed using the ACRO Biosystems IL-17A [Biotinylated]:IL-17RA Inhibitor Screening ELISA Kit (Cat. No. EP-139). The kit is coated with IL-17RA and uses an anti-IL-17A neutralizing antibody as a standard to block the binding of IL-17RA to biotinylated IL-17A. The blocking ability is determined by measuring the OD450nm value. The stronger the blocking ability, the lower the OD450nm value, and the blocking ability is inversely proportional to the OD450nm value. The supernatant obtained in Basic Experiment 8 was diluted (a 5-fold dilution was found to be optimal) and the ability of IL-17Nb in the supernatant to block IL-17A / IL17RA binding was assessed using the IL-17A / IL17RA blocking kit.

[0184] The IL-17A / IL17RA binding inhibition rate was calculated using the following formula:

[0185] Binding inhibition rate (%) = [OD450 (positive well) - OD450 (sample well)] / OD450 (positive well) x 100%.

[0186] All samples were diluted 5-fold according to the instructions and tested. The results are shown in Figure 8. Normal hUC-MSC cells cannot block the binding of IL-17A / IL17RA, while IL-17Nb secreted by G4-H10-MSC cells can block the binding of IL-17A / IL17RA, with an inhibition rate of up to 60% after 5-fold dilution.

[0187] Basic Experimental Example 10: Stem Cell Stability Study

[0188] According to the results obtained in Basic Experimental Example 8 (ELISA method to detect the expression of IL-17Nb), the IL-17Nb-modified mesenchymal stem cells G4-H10-MSC and the control hUC-MSC obtained in Basic Experimental Example 7 were added at a concentration of 1×10 4 pieces / cm 2Inoculate 24-well plates, inoculate 8 wells and 16 wells respectively. After overnight culture, randomly select 8 wells of hUC-MSC and replace the complete culture medium containing G4-H10 fusion protein (target antibody protein prepared in Basic Experimental Example 2) with a final concentration of 2000 ng / mL. Continue culture, and harvest the supernatant at 24h, 48h, 72h and 96h of culture, respectively. Detect the IL17Nb content in the supernatant according to the method of Basic Experimental Example 8 (ELISA method for detecting IL17Nb expression). The results are shown in Figure 9. Over time, from 24 to 96 hours, the IL17Nb content detected in the hUC-MSC supplemented with G4-H10 group decreased from 1986±79.90 ng / mL to 1620±37.54 ng / mL, indicating a gradual decrease in concentration. However, the IL17Nb expression in the IL17Nb-MSC group increased from 1333±334.79 at 24 hours to 5554±508.12 ng / mL at 96 hours. At 72 and 96 hours, there was a highly significant difference between the hUC-MSC supplemented with G4-H10 and IL17Nb-MSC groups (P<0.001). These results indicate that IL-17Nb-MSC can stably and continuously express and secrete IL-17Nb, with both expression concentration and stability superior to recombinant G4-H10 protein.

[0189] Example 1 Representative Indication 1: Construction of Rheumatoid Arthritis Animal Model and Test Substance Evaluation Results

[0190] A rheumatoid arthritis (RA) model was established using B-hIL17A transgenic mice (Biocytogen Incorporated) by collagen induction.

[0191] On the first day (Day 1), the RA model group received an intradermal injection of 100 μg of bovine type II collagen (CII, Chondrex) and an emulsion of Freund's complete adjuvant (CFA, Chondrex) containing 200 μg of Mycobacterium tuberculosis H37Ra at the base of the tail for initial immunization. On Day 21 after the initial immunization, the animals received a booster immunization of CII and an emulsion of Freund's incomplete adjuvant (IFA). Twenty-two days after the initial immunization (Day 22), the RA model mice (modeling criteria: RA model clinical score ≥ 2) were randomly divided into four groups: MSC treatment group, positive antibody treatment group, G4-H10-MSC treatment group, and model control group. On the same day of grouping, hUC-MSC (2×10 6 / ), positive antibody Ixekizumab (1 mg / Kg) and G4-H10-MSC (2×10 6Body weight and paw thickness were assessed every other day from Day 20 to the end of the experiment (Day 42) for a total of 23 days. All animals were euthanized at the end of the experiment (Day 42), and joint tissue was obtained for pathological staining and scoring.

[0192] RA model clinical scoring criteria:

[0193] 0: Normal; 1: Mild redness and swelling of ankles and wrists; 2: Moderate redness and swelling of ankles or wrists; 3: Severe redness and swelling of paws, including fingertips; 4: Maximum inflammation of limbs, including multiple joints.

[0194] Scoring criteria for pathological tissue staining:

[0195] Representative images of the three groups of H&E-stained limbs were scored independently by two experimenters using a double-blind method, with individual scores ranging from 0 to 5, and a total score of 20 points. (1) Inflammatory cell infiltration: 0, no inflammatory cell infiltration; 1, a small amount of inflammatory cell infiltration; 2, mild inflammatory cell infiltration; 3, moderate inflammatory cell infiltration; 4, severe inflammatory cell infiltration; 5, very severe inflammatory cell infiltration. (2) Pannus formation: 0, no pannus; 1, a few pannus; 2, mild pannus (involving less than 1 / 4 of the metacarpophalangeal joints); 3, moderate pannus (involving 1 / 4 to 1 / 2 of the metacarpophalangeal joints); 4, severe pannus (involving 1 / 2 to 3 / 4 of the metacarpophalangeal joints); 5, very severe pannus (involving more than 3 / 4 of the metacarpophalangeal joints) (3) Cartilage erosion: 0, no cartilage erosion; 1, mild cartilage erosion; 2, mild cartilage erosion (superficial or focal chondrocyte reduction and collagen destruction); 3, moderate cartilage erosion (multifocal or deep to 1 / 2 of the cartilage layer chondrocyte reduction and collagen destruction); 4, severe cartilage erosion (involving more than 1 / 2 of the cartilage surface, one or multiple tarsal joint cartilage surfaces are completely destroyed); 5, extremely severe cartilage erosion (severe chondrocyte reduction and collagen destruction, deep to the tide line) (4) Bone destruction: 0, no bone destruction; 1, mild bone destruction, not obvious under low power microscope; 2, mild bone destruction (involving less than 1 / 4 of the metacarpophalangeal joints); 3, moderate bone destruction, obvious trabeculae and cortical bone absorption, but not involving the full thickness of the cortex (involving 1 / 4 to 1 / 2 of the metacarpophalangeal joints); 4, severe bone destruction, partial involvement of the full thickness of the cortex, cortical deformation, and trabecular bone absorption (involving 1 / 2 to 3 / 4 of the metacarpophalangeal joints); 5, extremely severe bone destruction, involving the full thickness of the cortex, cortical deformation, and trabecular bone absorption (involving more than 3 / 4 of the metacarpophalangeal joints).

[0196] As shown in Figure 10, the weight of the model group decreased significantly compared to the normal control group starting with the booster immunization (D21). Both the MSC- and G4-H10-MSC-treated groups recovered their weight, while the positive antibody-treated group showed no significant weight recovery. At the end of the experiment, the weight of the mice in the G4-H10-MSC-treated group was significantly higher than that in the positive antibody-treated group. As shown in Figure 11, the average paw thickness of the animals shows rapid swelling of the paws after the booster immunization (D21). However, paw thickness decreased significantly after intravenous injection of hUC-MSC, the positive antibody ixekizumab, and G4-H10-MSC. The G4-H10-MSC-treated group showed a significantly better effect than the positive antibody ixekizumab. The results of histopathological staining and scoring are shown in Figures 12-13. The model mice showed tissue inflammatory cell infiltration, joint synovitis and / or pannus formation, articular cartilage destruction, disappearance of the joint cavity, and bone tissue fusion. After treatment, it was found that the pathological tissue staining scores of the mice decreased significantly. Among them, G4-H10-MSC treatment (G4-H10-MSC group) was significantly better than the positive antibody Ixekizumab (positive antibody group) and hUC-MSC treatment (hUC-MSC group).

[0197] Example 2 Representative Indication 2: Psoriasis Animal Model Construction Method and Construction Results

[0198] The psoriasis (Ps) model was established using B-hIL17A transgenic mice by imiquimod application. After shaving the back of all mice, 50 mg of imiquimod ointment (IMQ, Mingxinlidi, Sichuan Mingxin Pharmaceutical Co., Ltd.) was applied to the back daily. The first application day was designated as D0, and the application was continued for 7 days (D6) to establish the model. The Ps model group was randomly divided into four groups: hUC-MSC treatment group (hUC-MSC group, 2×10 6 / ), positive antibody Ixekizumab treatment group (1 mg / Kg), G4-H10-MSC treatment group (2×10 6 The drug treatment group received subcutaneous injections of the drug on days 1 and 4. During the experiment, the animals were weighed daily, their survival and health were observed, their skin was photographed, and skin clinical scores were performed based on the degree of keratinization and inflammatory cell infiltration to assess skin inflammation and related indicators. On day 7, the animals were euthanized and tested: skin from the modeled mice was collected, and skin thickness was measured using a vernier caliper for each group.

[0199] Clinical skin scoring standard: The animal skin (ears and front and back paws) is scored, and a comprehensive score is given based on erythema, scaling, and thickness. Each indicator is scored on a 5-point scale of 0-5, where 0 indicates no related symptoms; 1 indicates mild symptoms; 2 indicates general symptoms; 3 indicates significant symptoms; and 4 indicates very significant or severe symptoms. The total score of the three indicators is calculated as the final score.

[0200] As shown in Figure 14, the weight of the model group after imiquimod induction was significantly reduced compared to the normal control group. Both the hUC-MSC- and G4-H10-MSC-treated groups recovered their weight, while the positive antibody-treated group showed no significant weight recovery. At the endpoint of the experiment, the weight of the mice in the G4-H10-MSC-treated group was significantly higher than that in the positive antibody-treated group.

[0201] Skin photographs and skin clinical scores are shown in Figures 15 and 16. The results indicate that IMQ can cause skin damage in mice, with increased rash and desquamation, thickening of the epidermis, and histopathological findings showing parakeratosis and inflammatory leukocyte infiltration in the dermis, indicating successful modeling. The skin clinical score in the model group was significantly elevated, while the skin clinical score was significantly reduced after subcutaneous injection of hUC-MSC, the positive antibody ixekizumab, and G4-H10-MSC. The therapeutic effect of G4-H10-MSC was significantly better than that of the positive antibody ixekizumab.

[0202] Skin thickness was measured at the end of the experiment. As shown in Figure 17, it was found that IMQ could significantly increase the skin thickness of the model mice. After subcutaneous injection of hUC-MSC, positive antibody Ixekizumab and G4-H10-MSC, the skin thickness of the mice was significantly reduced. Among them, the therapeutic effect of G4-H10-MSC was significantly better than that of the positive antibody Ixekizumab.

[0203] Example 3 Representative Indication 3: Psoriatic Arthritis Animal Model Construction Method and Construction Results

[0204] A psoriatic arthritis (PsA) model was established using B-hIL17A transgenic mice (Biocytogen Incorporated) via intraperitoneal injection of mannan. Mice were selected for modeling, with the first intraperitoneal injection date designated D0. Mannan was then injected three times intraperitoneally at D0, D4, and D8. The model was established with three intraperitoneal injections of mannan (SIGMA, M7504-5G), each administered at 100 mg / mL in a 200 μL injection volume, for a total of 20 mg of mannan per animal.

[0205] The PsA model group was randomly divided into four groups: hUC-MSC treatment group (hUC-MSC group, 2×10 6 / ), positive antibody Ixekizumab treatment group (positive antibody group, 1 mg / Kg), G4-H10-MSC treatment group (G4-H10-MSC group, 2×10 6 / rat) and model control group;

[0206] The drug-treated group received intravenous administration of the drug on days 3 and 7. Animal body weight was measured every two days throughout the experiment, and their survival and health were observed. The skin and fore and hind paws were observed and scored based on relevant indicators. On day 14, the animals were euthanized, and peripheral blood was collected. Serum was isolated and assayed for cytokines such as mIL-6, mIL-23, and mTNF-α (all kits were from Biolegend).

[0207] Clinical skin scoring standard: The animal skin (ears and front and back paws) is scored, and a comprehensive score is given based on erythema, scaling, and thickness. Each indicator is scored on a 5-point scale of 0-5, where 0 indicates no related symptoms; 1 indicates mild symptoms; 2 indicates general symptoms; 3 indicates significant symptoms; and 4 indicates very significant or severe symptoms. The total score of the three indicators is calculated as the final score.

[0208] The paw (joint) scoring criteria are as follows: 0 = normal; 1 = erythema and swelling of one finger in the paw; 2 = erythema and swelling of two fingers in the paw; 3 = erythema and swelling of more than two fingers in the paw and / or swelling of the ankle joint. The total score of the four paws is calculated as the final score.

[0209] As shown in Figure 18, the weight of the animals in the PsA model group decreased significantly compared with the normal control group after mannan modeling. Both the hUC-MSC treatment group and the G4-H10-MSC treatment group were able to recover the weight of the mice, while the positive antibody group did not recover the weight of the mice significantly. At the end of the experiment, the weight of the mice in the G4-H10-MSC treatment group was significantly higher than that in the positive antibody group.

[0210] The animal paw scores and skin clinical scores are shown in Figures 19 and 20. After mannan modeling, the skin and paw clinical scores of the PsA model group were significantly increased. The positive antibody, hUC-MSC treatment group and G4-H10-MSC treatment group could significantly reduce the paw and skin clinical scores of the model animals, and the body weight of the mice in the G4-H10-MSC treatment group was significantly better than that in the positive antibody group.

[0211] As shown in Figures 21-23, the detection of inflammatory factors showed that after mannan modeling, the serum levels of IL-6, IL-23, TNF-α and other cytokines in the PsA model group animals were significantly increased, while the hUC-MSC treatment group and the G4-H10-MSC treatment group could significantly reduce the serum IL-6 and TNF-α levels of the model animals, and the positive antibody group and the G4-H10-MSC treatment group could significantly reduce the serum IL-23 level of the model animals.

[0212] The above embodiments are only used to illustrate the present application and are not intended to limit the present application. In particular, with respect to the types of indications, those skilled in the art may replace and optimize conventional technical means based on the disclosure of the present application. For example, technical solutions applicable to other indications may be inferred based on a certain correlation between the disease and IL-17A, which are also within the scope of protection of the present application.

[0213] Although the above embodiment provides a detailed description of the present application, it is only a part of the embodiments of the present application, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present application.

Claims

1. Use of stem cells modified with single domain antibody genes in the preparation of medicines, characterized in that: The stem cells comprise: (1) an amino acid sequence shown in SEQ ID NO: 1-6 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1-6; and / or; (2) a nucleotide sequence encoding an amino acid sequence shown in SEQ ID NO: 1-6 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1-6.

2. Use of stem cells modified with single domain antibody genes in the preparation of medicines, characterized in that: The stem cells contain, express or secrete a first antibody and a second antibody; the first antibody contains HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 1-3; the second antibody contains HCDR4, HCDR5, and HCDR6 with amino acid sequences as shown in SEQ ID NOs: 4-6.

3. The use according to claim 1, characterized in that The stem cells further comprise: (1) an amino acid sequence as shown in SEQ ID NO:7-14 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:7-14; and / or; (2) a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO:7-14 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:7-14.

4. The use according to claim 2, characterized in that The first antibody further comprises the amino acid sequences shown in SEQ ID NOs: 7-10 as FR regions; the second antibody further comprises the amino acid sequences shown in SEQ ID NOs: 11-14 as FR regions.

5. The use according to claim 4, characterized in that The first antibody and the second antibody are linked directly or through a linker.

6. The use according to claim 5, characterized in that The linker is selected from (GS)n, (GGS)n, (GGGS)n, (GGGGS)n or AS(GGGGS)n, and n is selected from 1, 2, 3, 4, 5 or 6.

7. The use according to claim 2, characterized in that The stem cells also include a hinge region and a CH region.

8. [Corrected 22.05.2025 according to Rule 26] The use according to claim 5, characterized in that The stem cells comprise: (1) an amino acid sequence as shown in SEQ ID NO: 16-17 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 16-17; and / or; (2) a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 16-17 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 16-17.

9. The use according to claim 6, characterized in that The stem cells contain, express or secrete: (1) an amino acid sequence represented by SEQ ID NO:16-SEQ ID NO:15-SEQ ID NO:17-SEQ ID NO:26 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence; and / or; (2) a nucleotide sequence encoding an amino acid sequence represented by SEQ ID NO:16-SEQ ID NO:15-SEQ ID NO:17-SEQ ID NO:26 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence.

10. The use according to claim 2, characterized in that The stem cells also include biologically active proteins or functional fragments thereof that assist in expression and / or secretion, or prolong the half-life in vivo.

11. The use according to claim 2, characterized in that The medicine is used for preventing and / or treating IL-17A related diseases.

12. The use according to claim 2, characterized in that The medicine is used for preventing and / or treating inflammatory diseases, infectious diseases, autoimmune diseases, nervous system diseases or tumors.

13. The use according to claim 12, characterized in that The medicine is used for preventing and / or treating inflammatory diseases.

14. The use according to claim 13, characterized in that The inflammatory diseases include one or more of arthritis, psoriasis, ankylosing spondylitis, polymyositis, dermatomyositis, periarteritis nodosa, aortitis syndrome, chronic atrophic gastritis, rapidly progressive glomerulonephritis, lupus nephritis, temporal arteritis, eosinophilic fasciitis, nonalcoholic steatohepatitis, eosinophilic chronic sinusitis, ankylosing spondylitis, inclusion body myositis, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, inflammatory bowel disease and vasculitis.

15. The use according to claim 14, characterized in that The arthritis is malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, psoriatic arthritis or joint synovitis; the vasculitis includes one or more of ANCA-associated vasculitis, giant cell arteritis, allergic granulomatosis with vasculitis, hypersensitivity vasculitis, rheumatoid vasculitis and large vessel vasculitis.

16. The use according to claim 15, characterized in that The medicine is used for reducing inflammatory factors.

17. The use according to any one of claims 1 to 16, characterized in that: The stem cells are embryonic stem cells, adult stem cells, mesenchymal stem cells, umbilical cord blood stem cells, hematopoietic stem cells, neural stem cells, adipose stem cells, skin stem cells or muscle stem cells.

18. The use according to claim 17, characterized in that The stem cells are mesenchymal stem cells.

19. The use according to claim 18, characterized in that The mesenchymal stem cells are isolated from umbilical cord blood, umbilical cord, placenta, adipose tissue, skin, neural tissue, bone marrow or embryo.

20. The use according to claim 1 or 2, characterized in that The medicine also includes pharmaceutically acceptable excipients.

21. Use of stem cells modified with single domain antibody genes in the preparation of diagnostic products, characterized in that: The stem cells contain, express or secrete a first antibody and a second antibody; the first antibody contains HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 1-3; the second antibody contains HCDR4, HCDR5, and HCDR6 with amino acid sequences as shown in SEQ ID NOs: 4-6.

22. A stem cell modified based on a single domain antibody gene, characterized in that: The stem cells contain, express or secrete a first antibody and a second antibody; the first antibody contains HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 1-3; the second antibody contains HCDR4, HCDR5, and HCDR6 with amino acid sequences as shown in SEQ ID NOs: 4-6.

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