Application of gene-modified stem cells in IL-17 target-related diseases

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to the application of a genetically modified stem cell (IL17Nb-MSC) in IL-17 target-related diseases. The genetically modified stem cells described in the present invention can secrete a bivalent single-domain antibody that effectively binds to IL-17A, namely, an IL-17A nanobody (IL17Nb), which includes the amino acid sequence shown in SEQ ID NO.1-6, and can effectively block the binding of IL-17A to its receptor. The genetically modified stem cells described in the present invention can effectively treat diseases such as rheumatoid arthritis, psoriasis and / or psoriatic arthritis caused by IL-17A, and the therapeutic effect is significantly better than the positive antibody Ixekizumab. Provide technical support for the clinical treatment of diseases such as rheumatoid arthritis, psoriasis and / or psoriatic arthritis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application of gene-modified stem cells in IL-17 target-related diseases. Background Art

[0002] IL-17A is an inflammatory cytokine mainly produced by activated T cells. It acts on downstream effector cells to mediate a variety of physiological processes, such as inflammatory response, coagulation process and bone remodeling, and is closely related to infectious diseases, immune diseases, tumor diseases, etc. Excessive expression of IL-17A will lead to the occurrence and development of a variety of diseases such as spondyloarthritis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, inflammatory bowel disease, etc. There are few specific drugs available for this type of disease, and traditional treatments have the disadvantages of slow onset and poor compliance.

[0003] Stem cell therapy refers to the process of using stem cells from autologous or allogeneic sources, implanting them into the human body after in vitro manipulation, and treating diseases. That is, through stem cell separation, culture, directional induction, or even gene modification, new, normal, or younger cells, tissues, or organs are regenerated in vitro, and clinical diseases are treated through them. A large number of clinical studies have shown that some types of stem cells have significant therapeutic effects on many intractable diseases. For example, autoimmune diseases, degenerative diseases, metabolic diseases, tissue defect diseases, traumatic diseases, inflammatory diseases, radiation diseases, toxic diseases, ischemic diseases, tumors, insomnia, sub-health conditions, anti-aging, beauty, etc., involving the human nervous, circulatory, respiratory, digestive, endocrine, urinary, reproductive, motor and other major systems.

[0004] Most of the existing studies on antibody secretion in cells focus on antibodies of general molecular weight or large molecular weight, and there are few reports on the cell secretion of small molecular weight antibodies, especially nano-level antibodies. Patent CN112941028A discloses a mesenchymal stem cell modified with a nano-antibody gene and its preparation method and application. The mesenchymal stem cell contains, and / or expresses, and / or secretes nano-antibodies. The mesenchymal stem cell or the culture extract of the mesenchymal stem cell, while migrating to the target target, enables the nano-antibody to accurately reach the lesion area in a shorter time, thereby increasing the concentration and effective amount of the nano-antibody in the lesion area, and reducing the exhaustion phenomenon of the nano-antibody in the lesion area, and also promoting the secretion of other beneficial components by mesenchymal stem cells, improving the cellular immunotherapy mediated by mesenchymal stem cells, and improving the treatment effect of immune-related diseases.

[0005] The combination of antibodies and immune cells is a hot topic in current research, but immune cells do not have the function of improving and repairing, which has become a defect of current immunotherapy. Therefore, the present invention envisions combining nano antibodies with better performance with mesenchymal stem cells with targeting and repairing activity, so that antibodies are secreted at high density at the disease site, and the small molecular weight of nano antibodies increases the concentration and effective amount of antibodies at the disease site, while the repair activity of mesenchymal stem cells repairs the diseased lesions, improves the microenvironment, and enhances the killing effect of immune cells. The interaction of various factors improves the cure rate of the disease, providing a new approach for the treatment of the disease.

[0006] There is a need for further research and development in the field of modified stem cells.

[0007] The present invention was created under the above background. The present invention is the fourth-generation technology development carried out by the inventors based on the anti-IL-17A single domain antibody development project.

[0008] To facilitate the review, a brief introduction to the technical background of the R&D project is given below:

[0009] The inventors first developed 9 single-domain antibodies (which have been filed separately);

[0010] Based on the single-domain antibody, the inventors have developed an antibody combination consisting of two single-domain antibodies (which has been applied for separately);

[0011] Based on single-domain antibodies, the inventors have developed stem cells genetically modified by antibody combinations (another application has been filed);

[0012] The inventors have developed an application technology for modified stem cells based on genetically modified stem cells. This application is a patent application for one of the application technologies.

[0013] As mentioned above, based on the relevant provisions of the Patent Law on unity, patent applications are filed separately.

[0014] To facilitate the understanding of this application, it is optional to refer to other patent application documents of this project. Summary of the invention

[0015] In order to solve the above problems, the present invention provides the use of genetically modified stem cells in IL-17 target-related diseases, wherein the stem cells can secrete bivalent single-domain antibodies that effectively bind to IL-17A, and are used to treat IL-17A-mediated rheumatoid arthritis, psoriasis and / or psoriatic arthritis and other diseases.

[0016] In the present invention, single-domain antibodies, also known as nano antibodies, are antibodies whose complementary determining regions are part of single-domain polypeptides. Therefore, single-domain antibodies contain a single complementary determining region. Single-domain antibodies are antibodies with only heavy chains, which naturally do not contain light chains, and are derived from single-domain antibodies and engineered antibodies of conventional antibodies. Single-domain antibodies can be derived from any species, including mice, humans, camels, llamas, goats, rabbits and cattle. For example, naturally occurring VHH molecules can be derived from antibodies provided by camelid species (e.g., camels, dromedaries, llamas and guanacos). Like complete antibodies, single-domain antibodies can selectively bind to specific antigens. Single-domain antibodies can contain only the variable domains of immunoglobulin chains, which have CDR1, CDR2 and CDR3 and framework regions.

[0017] In the present invention, the bivalent single-domain antibody against IL-17A, i.e., the bivalent single-domain antibody against IL-17A, includes not only the complete bivalent single-domain antibody, but also the fragments, derivatives and analogs of the bivalent single-domain antibody against IL-17A. Among them, fragments, derivatives and analogs have the same meaning, and all refer to polypeptides that substantially maintain the same biological function or activity as the antibody of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or polypeptides having substitution groups in one or more amino acid residues, or polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with an Fc tag).

[0018] In the present invention, sequence homology means the extent to which two (nucleotide or amino acid) sequences have the same residue at the same position in the alignment, and is usually expressed as a percentage. Preferably, homology is determined over the entire length of the sequence being compared. Therefore, two copies of the same sequence have 100% homology. In some embodiments, a sequence that replaces only one or a few amino acids compared to the aforementioned sequence, for example, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions, can also achieve the purpose of the invention. These variant forms include but are not limited to: one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. In fact, when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2 and CDR3 in a single domain antibody, a technician may consider so-called "conservative" amino acid substitutions, in which case the substitution will preferably be a conservative amino acid substitution. The conservative amino acid, which can generally be described as an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and the substitution has little or substantially no effect on the function, activity or other biological properties of the polypeptide. The conservative amino acid substitution is common in the art, for example, a conservative amino acid substitution is a substitution in which one or a few amino acids in the following (a)-(d) groups are replaced by another or a few amino acids in the same group: (a) polar negatively charged residues and their uncharged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. Particularly preferred conservative amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg, Gln; Phe is replaced by Met, Leu, Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe, Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser.In addition, those skilled in the art know that the creativity of single-domain antibodies is embodied in the CDR1-3 regions, while the framework region sequences FR1-4 are not immutable, and the sequences of FR1-4 can be conservative sequence variants of the sequences disclosed in the present invention.

[0019] In the present invention, antibody fusion protein refers to the product obtained by fusing antibody fragments with other biologically active proteins using genetic engineering technology. Due to the differences in fusion proteins, such antibody fusion proteins have multiple biological functions, and the expressed recombinant protein neither affects the antigen binding ability of the single-chain antibody nor the biological properties of the protein fused therewith.

[0020] In the present invention, stem cells are a type of cell with unlimited or immortal self-renewal ability, capable of producing at least one type of highly differentiated daughter cells. Functionally, stem cells are cells with multidirectional differentiation potential and self-renewal ability, are the most primitive cells at the top of the cell lineage origin, and can differentiate into cells of a certain specific tissue type in the body. The stem cells of the present invention have the following biological characteristics: (a) they are non-terminally differentiated cells, which maintain undifferentiated or low-differentiated characteristics throughout life and lack differentiation markers; (b) their number and position in the body are relatively constant; (c) they have the ability to self-renew; (d) they can divide and proliferate indefinitely, can remain in a dormant state for a long time, and stem cells can divide continuously for several generations; (e) they have multidirectional differentiation potential and can differentiate into various different types of tissue cells; they also have the plasticity of differentiation and development, and under certain circumstances, they can be induced to differentiate into developmentally unrelated cell types, and their differentiation is affected by the surrounding microenvironment (stem cell niche); (f) they have slow periodicity of division; (g) stem cells grow in two ways, one is symmetrical division to form two identical stem cells, and the other is asymmetric division. In asymmetric division, one daughter cell maintains the characteristics of the parent and remains as a stem cell, while the other daughter cell irreversibly moves to the terminal of differentiation and becomes a differentiated cell with specific functions.

[0021] In the present invention, mesenchymal stem cells (MSC), also known as mesenchymal stromal cells, are subpopulations of non-hematopoietic adult stem cells derived from the mesoderm. They have self-renewal ability and multidirectional differentiation, and not only differentiate into mesodermal lineages, such as chondrocytes, osteocytes and adipocytes, but also differentiate into ectoderm cells and endoderm cells. Due to the absence of ethical issues and teratoma formation, MSC is the main stem cell type for cell therapy for the treatment of immune and non-immune diseases. They can be easily separated from bone marrow, adipose tissue, umbilical cord, fetal liver, muscle and lung, and can be successfully amplified in vitro. In addition, MSC has a tendency to home to damaged tissue sites. When MSC is exogenously delivered and systemically administered to humans and animals, they particularly migrate to damaged tissue sites with inflammation. The homing of MSC directed by inflammation involves several important cell transport-related molecules, including chemokines, adhesion molecules and matrix metalloproteinases (MMPs).

[0022] In the present invention, cell culture refers to a method of simulating the in vivo environment (sterility, suitable temperature, pH and certain nutritional conditions, etc.) in vitro to enable it to survive, grow, reproduce and maintain its main structure and function. The present invention improves the success rate of cell culture from the following aspects: (1) Aseptic operation: During the cell culture process, it is very important to maintain aseptic operation. Use aseptic techniques, including wearing appropriate laboratory clothing, gloves and masks, using sterile operating tables or incubators and other equipment, and using sterile culture vessels and culture reagents to avoid contamination by bacteria, fungi or other microorganisms. (2) Treatment of culture vessels: Before use, ensure that the culture vessels (such as culture dishes, centrifuge tubes, test tubes, etc.) are properly cleaned and disinfected. The culture dishes can be irradiated with ultraviolet light before use, and the test tubes and centrifuge tubes can be treated by high-temperature baking or automatic cleaning procedures. (3) Preparation of culture medium: Accurate preparation of culture medium is the key to ensuring cell growth and health. Accurately weigh and mix the culture medium components according to the manufacturer's instructions or the laboratory's standard operating procedures to ensure the correct concentration and pH value. (4) Cell density control: Before cell passage or experiment, determine the appropriate cell density according to the cell type and experimental requirements. Too low a density may lead to slow cell growth, while too high a density may lead to overcrowding and cell death. (5) Optimization of culture conditions: Different cell types have different requirements for culture conditions, including culture temperature, CO2 concentration, humidity, and culture medium formula. Understanding and optimizing the culture conditions suitable for specific cell types can increase the successful culture rate of cells. (6) Cell detection and identification: Regularly identify and detect cells to ensure their purity and authentication. Use but are not limited to cell-specific markers or PCR methods for identification to avoid cell contamination or cell line mixing. (7) Cryopreservation backup: Regularly freeze backup cell lines to prevent cell loss or contamination. The method and conditions for freezing cells need to be determined according to the cell type and laboratory requirements. (8) Observation and recording: Closely observe and record the growth and status of cells, including cell morphology, proliferation rate, and cell death. Promptly detect and deal with any abnormal phenomena, such as cell contamination or cell aging.

[0023] In the present invention, a nucleic acid construct contains the nucleic acid sequence of the antibody of the present invention, and one or more regulatory sequences operably linked to these sequences. The operably linked means that some parts of the linear DNA sequence can regulate or control the activity of other parts of the same linear DNA sequence. For example, if a promoter controls the transcription of a coding sequence, then it is operably linked to a coding sequence.

[0024] The regulatory sequence may be a suitable promoter sequence. The promoter sequence is usually operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, including mutant, truncated and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0025] The regulatory sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in the present invention.

[0026] In the present invention, recombinant vector. Specifically, the nucleic acid sequence of the antibody can be cloned into a vector, for example, a vector includes but is not limited to a plasmid, a phagemid, a phage derivative, an animal virus and a cosmid. The vector can be an expression vector (also referred to as a recombinant vector). The expression vector can be provided to the cell in a viral vector form or a non-viral form, preferably a non-viral vector. The expression vector may contain one or more repeated antibody nucleic acid sequences. Typically, a suitable vector comprises a replication origin that works in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers.

[0027] Suitable promoters include but are not limited to the immediate early cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto to express at a high level. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoter, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, it is also possible to consider the use of inducible promoters. The use of inducible promoters provides a molecular switch that can open the expression of a polynucleotide sequence operably connected to an inducible promoter when the deadline is expressed, and close the expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0028] Selectable markers include any one or both of selectable marker genes or reporter genes to facilitate identification and selection of expressing cells from a cell population infected by a viral vector. Useful selectable marker genes include, for example, antibiotic resistance genes, such as neo, etc. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein genes.

[0029] In the present invention, pharmaceutically acceptable excipients refer to excipients and additives used in the production of drugs and the preparation of prescriptions; they are substances other than active ingredients that have been reasonably evaluated in terms of safety and are included in pharmaceutical preparations. In addition to excipients, carriers, and stability enhancement, pharmaceutical excipients also have important functions such as solubilization, dissolution assistance, and sustained and controlled release, and are important components that may affect the quality, safety, and effectiveness of drugs.

[0030] In one aspect, the present invention provides a use of stem cells in preparing a drug for preventing and / or treating a disease.

[0031] Specifically, the stem cells include the following (1) and / or (2):

[0032] (1) the amino acid sequence shown in SEQ ID NO. 1-6;

[0033] (2) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1-6.

[0034] Specifically, the diseases include, but are not limited to, inflammatory diseases, infectious diseases, autoimmune diseases, nervous system diseases and / or tumors.

[0035] Further specifically, the inflammatory disease includes, but is not limited to: Hashimoto's thyroiditis, systemic lupus erythematosus, rheumatoid arthritis and / or primary biliary cirrhosis;

[0036] The infectious diseases include, but are not limited to: bacterial infection, viral infection, fungal infection, mycoplasma infection, chlamydia infection and / or rickettsial infection;

[0037] The autoimmune diseases include, but are not limited to, Behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, systemic sclerosis, progressive systemic sclerosis, scleroderma, polymyositis, dermatomyositis, periarteritis nodosa, aortitis syndrome, malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Castleman's disease, Sjögren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, rheumatoid vasculitis, large vessel blood vasculitis, ANCA-associated vasculitis, Cogan syndrome, RS3PE syndrome, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, IgG4-related disease, Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, nonalcoholic steatohepatitis, primary biliary cirrhosis, Good-pasture syndrome, rapidly progressive glomerulonephritis, lupus nephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia neutropenia, idiopathic thrombocytopenic purpura, Basedu disease, Hashimoto's disease, autoimmune adrenocortical insufficiency, primary hypothyroidism, Addison's disease, idiopathic Addison's disease, type I diabetes, slowly progressive type I diabetes, focal scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, acquired epidermolysis bullosa, alopecia areata, vitiligo, vitiligo vulgaris, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy disease, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harada disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, inflammatory bowel disease, celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria transplant immunity, familial Mediterranean fever, eosinophilic chronic sinusitis, dilated cardiomyopathy, systemic mastocytosis and / or inclusion body myositis; preferably, the autoimmune disease is plaque psoriasis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis and / or lupus nephritis;

[0038] The nervous system diseases include, but are not limited to, central nervous system infections, cerebrovascular diseases, movement disorders, peripheral neuropathy, and / or nerve and muscle junction and muscle diseases.

[0039] The tumors include, but are 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, digestive system 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, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, 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, chronic myeloblastic leukemia

[0040] More specifically, the disease includes rheumatoid arthritis, psoriasis and / or psoriatic arthritis.

[0041] Specifically, the stem cells may be 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.

[0042] Preferably, the stem cells are mesenchymal stem cells.

[0043] Further preferably, the mesenchymal stem cells can be isolated from umbilical cord blood, umbilical cord, placenta, adipose tissue, skin, neural tissue or bone marrow.

[0044] More preferably, the mesenchymal stem cells can be isolated from umbilical cord blood or umbilical cord.

[0045] Specifically, the stem cells secrete anti-interleukin antibodies.

[0046] More specifically, the stem cells secrete anti-interleukin 17 antibodies.

[0047] More specifically, the stem cells secrete anti-IL-17A antibodies.

[0048] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0049] More specifically, the pharmaceutically acceptable excipients include, but are not limited to, any one or more of excipients, stabilizers, diluents, adhesives, preservatives, lubricants, and antioxidants.

[0050] Preferably, the pharmaceutically acceptable excipient can be selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0051] In another aspect, the present invention provides a use of stem cells in the preparation of a drug for preventing and / or treating a disease. Specifically, the stem cells include the following (1) and / or (2):

[0052] (1) the amino acid sequence shown in SEQ ID NO. 1-14;

[0053] (2) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1-14;

[0054] Specifically, the diseases include, but are not limited to, inflammatory diseases, infectious diseases, autoimmune diseases, nervous system diseases and / or tumors.

[0055] Further specifically, the inflammatory disease includes, but is not limited to: Hashimoto's thyroiditis, systemic lupus erythematosus, rheumatoid arthritis and / or primary biliary cirrhosis;

[0056] The infectious diseases include, but are not limited to: bacterial infection, viral infection, fungal infection, mycoplasma infection, chlamydia infection and / or rickettsial infection;

[0057] The autoimmune diseases include, but are not limited to, Behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, systemic sclerosis, progressive systemic sclerosis, scleroderma, polymyositis, dermatomyositis, periarteritis nodosa, aortitis syndrome, malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Castleman's disease, Sjögren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, rheumatoid vasculitis, large vessel blood vasculitis, ANCA-associated vasculitis, Cogan syndrome, RS3PE syndrome, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, IgG4-related disease, Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, nonalcoholic steatohepatitis, primary biliary cirrhosis, Good-pasture syndrome, rapidly progressive glomerulonephritis, lupus nephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia neutropenia, idiopathic thrombocytopenic purpura, Basedu disease, Hashimoto's disease, autoimmune adrenocortical insufficiency, primary hypothyroidism, Addison's disease, idiopathic Addison's disease, type I diabetes, slowly progressive type I diabetes, focal scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, acquired epidermolysis bullosa, alopecia areata, vitiligo, vitiligo vulgaris, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy disease, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harada disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, inflammatory bowel disease, celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria transplant immunity, familial Mediterranean fever, eosinophilic chronic sinusitis, dilated cardiomyopathy, systemic mastocytosis and / or inclusion body myositis; preferably, the autoimmune disease is plaque psoriasis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis and / or lupus nephritis;

[0058] The nervous system diseases include, but are not limited to, central nervous system infections, cerebrovascular diseases, movement disorders, peripheral neuropathy, and / or nerve and muscle junction and muscle diseases.

[0059] The tumors include, but are 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, digestive system 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, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, 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, chronic myeloblastic leukemia

[0060] More specifically, the disease includes rheumatoid arthritis, psoriasis and / or psoriatic arthritis.

[0061] Specifically, the stem cells may be 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.

[0062] Preferably, the stem cells are mesenchymal stem cells.

[0063] Further preferably, the mesenchymal stem cells can be isolated from umbilical cord blood, umbilical cord, placenta, adipose tissue, skin, neural tissue or bone marrow.

[0064] More preferably, the mesenchymal stem cells can be isolated from umbilical cord blood or umbilical cord.

[0065] Specifically, the stem cells secrete anti-interleukin antibodies.

[0066] More specifically, the stem cells secrete anti-interleukin 17 antibodies.

[0067] More specifically, the stem cells secrete anti-IL-17A antibodies.

[0068] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0069] More specifically, the pharmaceutically acceptable excipients include, but are not limited to, any one or more of excipients, stabilizers, diluents, adhesives, preservatives, lubricants, and antioxidants.

[0070] Preferably, the pharmaceutically acceptable excipient can be selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0071] In yet another aspect, the present invention provides a use of stem cells in preparing a drug for preventing and / or treating a disease.

[0072] Specifically, the stem cells include the following (1) and / or (2):

[0073] (1) the amino acid sequence shown in SEQ ID NO.15-16;

[0074] (2) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.15-16;

[0075] Specifically, the diseases include, but are not limited to, inflammatory diseases, infectious diseases, autoimmune diseases, nervous system diseases and / or tumors.

[0076] Further specifically, the inflammatory disease includes, but is not limited to: Hashimoto's thyroiditis, systemic lupus erythematosus, rheumatoid arthritis and / or primary biliary cirrhosis;

[0077] The infectious diseases include, but are not limited to: bacterial infection, viral infection, fungal infection, mycoplasma infection, chlamydia infection and / or rickettsial infection;

[0078] The autoimmune diseases include, but are not limited to, Behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, systemic sclerosis, progressive systemic sclerosis, scleroderma, polymyositis, dermatomyositis, periarteritis nodosa, aortitis syndrome, malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Castleman's disease, Sjögren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, rheumatoid vasculitis, large vessel blood vasculitis, ANCA-associated vasculitis, Cogan syndrome, RS3PE syndrome, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, IgG4-related disease, Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, nonalcoholic steatohepatitis, primary biliary cirrhosis, Good-pasture syndrome, rapidly progressive glomerulonephritis, lupus nephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia neutropenia, idiopathic thrombocytopenic purpura, Basedu disease, Hashimoto's disease, autoimmune adrenocortical insufficiency, primary hypothyroidism, Addison's disease, idiopathic Addison's disease, type I diabetes, slowly progressive type I diabetes, focal scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, acquired epidermolysis bullosa, alopecia areata, vitiligo, vitiligo vulgaris, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy disease, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harada disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, inflammatory bowel disease, celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria transplant immunity, familial Mediterranean fever, eosinophilic chronic sinusitis, dilated cardiomyopathy, systemic mastocytosis and / or inclusion body myositis; preferably, the autoimmune disease is plaque psoriasis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis and / or lupus nephritis;

[0079] The nervous system diseases include, but are not limited to, central nervous system infections, cerebrovascular diseases, movement disorders, peripheral neuropathy, and / or nerve and muscle junction and muscle diseases.

[0080] The tumors include, but are 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, digestive system 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, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, 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, chronic myeloblastic leukemia

[0081] More specifically, the disease includes rheumatoid arthritis, psoriasis and / or psoriatic arthritis.

[0082] Specifically, the stem cells may be 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.

[0083] Preferably, the stem cells are mesenchymal stem cells.

[0084] Further preferably, the mesenchymal stem cells can be isolated from umbilical cord blood, umbilical cord, placenta, adipose tissue, skin, neural tissue or bone marrow.

[0085] More preferably, the mesenchymal stem cells can be isolated from umbilical cord blood or umbilical cord.

[0086] Specifically, the stem cells secrete anti-interleukin antibodies.

[0087] More specifically, the stem cells secrete anti-interleukin 17 antibodies.

[0088] More specifically, the stem cells secrete anti-IL-17A antibodies.

[0089] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0090] More specifically, the pharmaceutically acceptable excipients include, but are not limited to, any one or more of excipients, stabilizers, diluents, adhesives, preservatives, lubricants, and antioxidants.

[0091] Preferably, the pharmaceutically acceptable excipient can be selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0092] In yet another aspect, the present invention provides a use of stem cells in preparing a drug for preventing and / or treating a disease.

[0093] Specifically, the stem cells include the following (1) and / or (2):

[0094] (1) the amino acid sequence shown in SEQ ID NO.17;

[0095] (2) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.17;

[0096] Specifically, the diseases include, but are not limited to, inflammatory diseases, infectious diseases, autoimmune diseases, nervous system diseases and / or tumors.

[0097] Further specifically, the inflammatory disease includes, but is not limited to: Hashimoto's thyroiditis, systemic lupus erythematosus, rheumatoid arthritis and / or primary biliary cirrhosis;

[0098] The infectious diseases include, but are not limited to: bacterial infection, viral infection, fungal infection, mycoplasma infection, chlamydia infection and / or rickettsial infection;

[0099] The autoimmune diseases include, but are not limited to, Behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, systemic sclerosis, progressive systemic sclerosis, scleroderma, polymyositis, dermatomyositis, periarteritis nodosa, aortitis syndrome, malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Castleman's disease, Sjögren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, rheumatoid vasculitis, large vessel blood vasculitis, ANCA-associated vasculitis, Cogan syndrome, RS3PE syndrome, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, IgG4-related disease, Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, nonalcoholic steatohepatitis, primary biliary cirrhosis, Good-pasture syndrome, rapidly progressive glomerulonephritis, lupus nephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia neutropenia, idiopathic thrombocytopenic purpura, Basedu disease, Hashimoto's disease, autoimmune adrenocortical insufficiency, primary hypothyroidism, Addison's disease, idiopathic Addison's disease, type I diabetes, slowly progressive type I diabetes, focal scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, acquired epidermolysis bullosa, alopecia areata, vitiligo, vitiligo vulgaris, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy disease, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harada disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, inflammatory bowel disease, celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria transplant immunity, familial Mediterranean fever, eosinophilic chronic sinusitis, dilated cardiomyopathy, systemic mastocytosis and / or inclusion body myositis; preferably, the autoimmune disease is plaque psoriasis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis and / or lupus nephritis;

[0100] The nervous system diseases include, but are not limited to, central nervous system infections, cerebrovascular diseases, movement disorders, peripheral neuropathy, and / or nerve and muscle junction and muscle diseases.

[0101] The tumors include, but are 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, digestive system 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, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, 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, chronic myeloblastic leukemia

[0102] More specifically, the disease includes rheumatoid arthritis, psoriasis and / or psoriatic arthritis.

[0103] Specifically, the stem cells may be 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.

[0104] Preferably, the stem cells are mesenchymal stem cells.

[0105] Further preferably, the mesenchymal stem cells can be isolated from umbilical cord blood, umbilical cord, placenta, adipose tissue, skin, neural tissue or bone marrow.

[0106] More preferably, the mesenchymal stem cells can be isolated from umbilical cord blood or umbilical cord.

[0107] Specifically, the stem cells secrete anti-interleukin antibodies.

[0108] More specifically, the stem cells secrete anti-interleukin 17 antibodies.

[0109] More specifically, the stem cells secrete anti-IL-17A antibodies.

[0110] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0111] More specifically, the pharmaceutically acceptable excipients include, but are not limited to, any one or more of excipients, stabilizers, diluents, adhesives, preservatives, lubricants, and antioxidants.

[0112] Preferably, the pharmaceutically acceptable excipient can be selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0113] In yet another aspect, the present invention provides a use of stem cells in preparing a drug for preventing and / or treating a disease.

[0114] Specifically, the diseases include, but are not limited to, inflammatory diseases, infectious diseases, autoimmune diseases, nervous system diseases and / or tumors.

[0115] Further specifically, the inflammatory disease includes, but is not limited to: Hashimoto's thyroiditis, systemic lupus erythematosus, rheumatoid arthritis and / or primary biliary cirrhosis;

[0116] The infectious diseases include, but are not limited to: bacterial infection, viral infection, fungal infection, mycoplasma infection, chlamydia infection and / or rickettsial infection;

[0117] The autoimmune diseases include, but are not limited to, Behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, systemic sclerosis, progressive systemic sclerosis, scleroderma, polymyositis, dermatomyositis, periarteritis nodosa, aortitis syndrome, malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Castleman's disease, Sjögren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, rheumatoid vasculitis, large vessel blood vasculitis, ANCA-associated vasculitis, Cogan syndrome, RS3PE syndrome, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, IgG4-related disease, Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, nonalcoholic steatohepatitis, primary biliary cirrhosis, Good-pasture syndrome, rapidly progressive glomerulonephritis, lupus nephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia neutropenia, idiopathic thrombocytopenic purpura, Basedu disease, Hashimoto's disease, autoimmune adrenocortical insufficiency, primary hypothyroidism, Addison's disease, idiopathic Addison's disease, type I diabetes, slowly progressive type I diabetes, focal scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, acquired epidermolysis bullosa, alopecia areata, vitiligo, vitiligo vulgaris, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy disease, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harada disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, inflammatory bowel disease, celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria transplant immunity, familial Mediterranean fever, eosinophilic chronic sinusitis, dilated cardiomyopathy, systemic mastocytosis and / or inclusion body myositis; preferably, the autoimmune disease is plaque psoriasis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis and / or lupus nephritis;

[0118] The nervous system diseases include, but are not limited to, central nervous system infections, cerebrovascular diseases, movement disorders, peripheral neuropathy, and / or nerve and muscle junction and muscle diseases.

[0119] The tumors include, but are 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, digestive system 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, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, 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, chronic myeloblastic leukemia

[0120] More specifically, the disease includes rheumatoid arthritis, psoriasis and / or psoriatic arthritis.

[0121] Specifically, the stem cells contain, express and / or secrete an antibody having an amino acid sequence as shown in SEQ ID NO.21.

[0122] Specifically, the antibody also contains a biologically active protein or a functional fragment thereof that assists its expression and / or secretion, or prolongs its half-life in the body.

[0123] More specifically, the biologically active protein or its functional fragment is selected from the group consisting of an immunoglobulin Fc domain, an elastin-like polypeptide, serum albumin, an albumin-binding polypeptide, prealbumin, a carboxyl-terminal peptide, a His tag, a FLAG tag, a c-Myc tag, a HA tag, a GST tag, an MBP tag and / or a SUMO tag.

[0124] Preferably, the biologically active protein or a functional fragment thereof may be a human immunoglobulin Fc domain, preferably a human IgG Fc domain, such as a human IgG1, IgG2, IgG3, or IgG4 Fc domain.

[0125] Further preferably, the biologically active protein or its functional fragment is the Fc domain of IgG1.

[0126] Specifically, the stem cells may be 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.

[0127] Preferably, the stem cells are mesenchymal stem cells.

[0128] Further preferably, the mesenchymal stem cells can be isolated from umbilical cord blood, umbilical cord, placenta, adipose tissue, skin, neural tissue or bone marrow.

[0129] Specifically, the stem cells secrete anti-interleukin antibodies.

[0130] More specifically, the stem cells secrete anti-interleukin 17 antibodies.

[0131] More specifically, the stem cells secrete anti-IL-17A antibodies.

[0132] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0133] More specifically, the pharmaceutically acceptable excipients include, but are not limited to, any one or more of excipients, stabilizers, diluents, adhesives, preservatives, lubricants, and antioxidants.

[0134] Preferably, the pharmaceutically acceptable excipient can be selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0135] In yet another aspect, the present invention provides a method for treating a disease using stem cells, the method comprising administering a therapeutically effective amount of the stem cells to a subject in need thereof.

[0136] Specifically, the stem cells are mesenchymal stem cells;

[0137] Specifically, the diseases include, but are not limited to, inflammatory diseases, infectious diseases, autoimmune diseases, nervous system diseases and / or tumors.

[0138] Further specifically, the inflammatory disease includes, but is not limited to: Hashimoto's thyroiditis, systemic lupus erythematosus, rheumatoid arthritis and / or primary biliary cirrhosis;

[0139] The infectious diseases include, but are not limited to: bacterial infection, viral infection, fungal infection, mycoplasma infection, chlamydia infection and / or rickettsial infection;

[0140] The autoimmune diseases include, but are not limited to, Behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, systemic sclerosis, progressive systemic sclerosis, scleroderma, polymyositis, dermatomyositis, periarteritis nodosa, aortitis syndrome, malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Castleman's disease, Sjögren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, rheumatoid vasculitis, large vessel blood vasculitis, ANCA-associated vasculitis, Cogan syndrome, RS3PE syndrome, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, IgG4-related disease, Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, nonalcoholic steatohepatitis, primary biliary cirrhosis, Good-pasture syndrome, rapidly progressive glomerulonephritis, lupus nephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia neutropenia, idiopathic thrombocytopenic purpura, Basedu disease, Hashimoto's disease, autoimmune adrenocortical insufficiency, primary hypothyroidism, Addison's disease, idiopathic Addison's disease, type I diabetes, slowly progressive type I diabetes, focal scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, acquired epidermolysis bullosa, alopecia areata, vitiligo, vitiligo vulgaris, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy disease, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harada disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, inflammatory bowel disease, celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria transplant immunity, familial Mediterranean fever, eosinophilic chronic sinusitis, dilated cardiomyopathy, systemic mastocytosis and / or inclusion body myositis; preferably, the autoimmune disease is plaque psoriasis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis and / or lupus nephritis;

[0141] The nervous system diseases include, but are not limited to, central nervous system infections, cerebrovascular diseases, movement disorders, peripheral neuropathy, and / or nerve and muscle junction and muscle diseases.

[0142] The tumors include, but are 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, digestive system 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, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, 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, chronic myeloblastic leukemia

[0143] More specifically, the disease includes rheumatoid arthritis, psoriasis and / or psoriatic arthritis.

[0144] Further specifically, the stem cells secrete or express fusion antibodies;

[0145] More specifically, the fusion antibody comprises a first antibody and a second antibody;

[0146] The amino acid sequence of the first antibody is SEQ ID NO.15;

[0147] The amino acid sequence of the second antibody is SEQ ID NO.16.

[0148] The technical effects achieved by the present invention are:

[0149] (1) The C3-G4-MSC of the present invention can restore the body weight of mice with rheumatoid arthritis, effectively reduce the paw thickness of mice with rheumatoid arthritis, and significantly reduce the pathological tissue score, and the therapeutic effect is significantly better than the positive antibody Ixekizumab.

[0150] (2) The C3-G4-MSC of the present invention can restore the body weight of mice with psoriasis and significantly reduce the skin clinical score and skin thickness of mice, and the effects are significantly better than the positive antibody Ixekizumab.

[0151] (3) The C3-G4-MSCs of the present invention can restore the body weight of mice with psoriatic arthritis, significantly reduce the clinical scores of the model paws and skin, and significantly reduce the levels of mIL-6, mTNF-α and mIL-23 in the serum of mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0152] Figure 1 This is a graph showing the affinity ELISA test results for the bivalent single-domain antibody (C3-G4) and the positive control Ixekizumab.

[0153] Figure 2 This is a graph showing the affinity test results of the bivalent single-domain antibody (C3-G4) and the positive control Ixekizumab, where the values ​​from top to bottom in the graph are 25, 12.5, 6.25, and 3.13, respectively.

[0154] Figure 3 This is a graph showing the results of the blocking function experiment of the bivalent single-domain antibody (C3-G4) and the positive control Ixekizumab.

[0155] Figure 4 The stability results of the bivalent single-domain antibody (C3-G4).

[0156] Figure 5 The stability results of the positive control Ixekizumab.

[0157] Figure 6 The figure shows the transduction multiplicity results of lentiviral particles.

[0158] Figure 7 This is the result of lentivirus titer detection.

[0159] Figure 8 This is the result of FITC channel signal of mesenchymal stem cells.

[0160] Fig. 9 This is the expression result of IgG4 in mesenchymal stem cells.

[0161] Fig.10 This is the expression result of IL-17Nb in mesenchymal stem cells.

[0162] Fig.11 is the blocking rate of mesenchymal stem cells blocking the binding of IL-17A and IL-17RA.

[0163] Fig.12 These are the experimental results of stem cell stability in Example 9.

[0164] Fig.13 This is a trend chart of the weight change of mice in Example 10, where ***P<0.001 indicates that there is a significant difference between the model control group and the normal control group; ## P<0.01 indicates a significant difference between the C3-G4-MSC treatment group and the model control group; && P<0.01 indicates that there is a significant difference between the C3-G4-MSC treatment group and the positive antibody treatment group.

[0165] Fig.14 This is a graph showing the change trend of the mouse paw thickness in Example 10, where ***P<0.001 indicates that there is a significant difference between the model control group and the normal control group; ##P<0.01 indicates that there are significant differences between the three treatment groups and the model control group; && P<0.01 indicates that there is a significant difference between the C3-G4-MSC treatment group and the positive antibody treatment group.

[0166] Fig.15 This is a diagram of the mouse joint tissue pathology results in Example 10.

[0167] Fig.16 is the clinical score of mouse joint pathology in Example 10, wherein, # P < 0.05 indicates a significant difference between the hUC-MSC treatment group and the model control group; ## P<0.01 indicates a significant difference between the C3-G4-MSC treatment group and the model control group; & P<0.05 indicated a significant difference between the C3-G4-MSC treatment group and the positive antibody treatment group; @ P<0.05 indicated that there was a significant difference between the C3-G4-MSC treatment group and the hUC-MSC treatment group.

[0168] Fig.17 This is a trend chart of the weight change of mice in Example 11, where **P<0.01 indicates that there is a significant difference between the model control group and the normal control group; ## P<0.01 indicates a significant difference between the C3-G4-MSC treatment group and the model control group; && P<0.01 indicates that there is a significant difference between the C3-G4-MSC treatment group and the positive antibody treatment group.

[0169] Fig.18 This is a photo of mouse skin in Example 11.

[0170] Fig.19 is the clinical score of mouse skin in Example 11, wherein ****P<0.0001 indicates that there is a significant difference between the model control group and the normal control group; ### P<0.001 indicates that there are significant differences between the three treatment groups and the model control group; & P<0.05 indicated a significant difference between the C3-G4-MSC treatment group and the positive antibody treatment group; @ P<0.05 indicated that there was a significant difference between the C3-G4-MSC treatment group and the hUC-MSC treatment group.

[0171] Fig. 20 The results of the mouse skin thickness test in Example 11, where ***P<0.001 indicates that there is a significant difference between the model control group and the normal control group; ##P<0.01 indicates that there are significant differences between the three treatment groups and the model control group; & P<0.05 indicates that there is a significant difference between the C3-G4-MSC treatment group and the positive antibody treatment group.

[0172] Fig.21 This is a trend chart of the weight change of mice in Example 12, where **P<0.01 indicates that there is a significant difference between the model control group and the normal control group; # P<0.05 indicated a significant difference between the C3-G4-MSC treatment group and the model control group; & P<0.05 indicates that there is a significant difference between the C3-G4-MSC treatment group and the positive antibody treatment group.

[0173] Fig. 22 is the clinical score of the mouse paw in Example 12, wherein ****P<0.0001 indicates that there is a significant difference between the model control group and the normal control group; ### P<0.001 indicates that there are significant differences between the three treatment groups and the model control group; & P<0.05 indicated a significant difference between the C3-G4-MSC treatment group and the positive antibody treatment group; @ P<0.05 indicated that there was a significant difference between the C3-G4-MSC treatment group and the hUC-MSC treatment group.

[0174] Fig.23 is the clinical score of mouse skin in Example 12, wherein ****P<0.0001 indicates that there is a significant difference between the model control group and the normal control group; ## P<0.01 indicates that there are significant differences between the three treatment groups and the model control group; & P<0.05 indicates that there is a significant difference between the C3-G4-MSC treatment group and the positive antibody treatment group.

[0175] Fig.24 is the TNF-α level in the mouse serum in Example 12, wherein **P<0.01 indicates that there is a significant difference between the model control group and the normal control group; # P<0.05 indicates that there are significant differences between the hUC-MSC and C3-G4-MSC treatment groups and the model control group.

[0176] Fig.25 is the IL-6 level in the mouse serum in Example 12, wherein **P<0.01 indicates that there is a significant difference between the model control group and the normal control group; #P<0.05 indicates that there are significant differences between the hUC-MSC and C3-G4-MSC treatment groups and the model control group.

[0177] Fig.26 is the IL-23 level in the mouse serum in Example 12, wherein *P<0.05 indicates that there is a significant difference between the model control group and the normal control group; # P<0.05 indicates that there are significant differences between the positive antibody treatment group and the C3-G4-MSC treatment group and the model control group. DETAILED DESCRIPTION

[0178] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0179] The main instruments of the present invention are:

[0180] Electroporator (Eppendorf Multiporator);

[0181] Centrifuge (Thermo FRESCO-17);

[0182] Constant temperature incubator (Shanghai Jinghong DNP-9052);

[0183] Constant temperature shaking incubator (Jingqi CO-O6U);

[0184] Clean bench (Sujing Antai SW-CJ-1FD);

[0185] PCR instrument (Applied Biosystems ABI2720);

[0186] Biological safety cabinet (Haier, HR40-IIA2);

[0187] Flow cytometer (Thermo Attune Nxt flow cytometer);

[0188] Thermo 3111CO2 incubator;

[0189] ForteBio OCTET R2.

[0190] The main reagents of the present invention are:

[0191] SfiI (NEB, CAT#: R0123L);

[0192] T4 DNA ligase(TaKaRa,CAT#:2011A);

[0193] PrimeScript TM II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT#:6210B);

[0194] NuHi power mix (Xinhai Biotechnology, CAT#: NH9303);

[0195] 3M sodium acetate (pH 5.2-6) (Sigma, CAT#: 126-96-5);

[0196] DNA fragment recovery kit (TakaRa, CAT#: 9761);

[0197] Gel recovery kit (Qiagen, CAT#: 28706);

[0198] Tiangen Plasmid Extraction Kit (Tiangen, CAT#: DP117);

[0199] HRP-Anti-M13 (iCarTab);

[0200] PE-anti-Human IgG (eBioscience, Cat#:12-4998-82);

[0201] Rabbit anti-Llama IgG(H+L)Secondary Antibody[HRP](Novus, CAT#NBP1-75095);

[0202] SS320 receptive state (iCarTab);

[0203] pComF phage display vector (iCarTab);

[0204] NHS-biotin(APExBIO,CAT#:A8002);

[0205] HRP-Streptavidin(Boster,CAT#:BA1088);

[0206] HRP-ProteinA (Boster, BA1080);

[0207] ProA Biosensors(Sartorius,CAT#:18-5010);

[0208] PBS (Gbico, CAT#14190-250);

[0209] DMEM (Gbico, CAT#41965-062);

[0210] RPMI1640(Gbico,CAT#61870044);

[0211] FBS(VivaCell,CAT#C04001-500);

[0212] Genomic DNA Purification Kit (Lifetech, CAT#K0512);

[0213] Mouse-IL-17A-His (ACRO, CT8-M5240).

[0214] The bivalent single domain antibody in the present invention is a tandem single domain antibody.

[0215] Example 1 Preparation of Antibodies

[0216] 1.1 Screening of anti-IL-17A single domain antibodies

[0217] Preparation of IL-17A protein: Add 6xHis tag to the C-terminus of IL-17 protein (SEQ ID NO.30), perform gene synthesis and subclone into pET28a vector after prokaryotic codon optimization; perform plasmid extraction after Sanger sequencing verification; transform recombinant plasmid into BL21 competent cells, induce overnight with 0.5mM IPTG, collect bacterial solution for lysis; purify recombinant protein with nickel column; detect the purity of target protein by SDS-PAGE. After purification, the purity of IL-17A antigen protein is greater than 90%.

[0218] SEQ ID NO:30:

[0219] MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA.

[0220] The inventors used the prepared IL-17A protein to immunize alpacas and screen yeast libraries, and obtained 1-C3 and 2-G4 single-domain antibodies, respectively. In the antibody blocking activity test, it was found that although the single-domain antibody can block the activation of downstream target proteins by Human IL-17A protein, the blocking effect is weaker than that of the positive antibody Ixekizumab. Therefore, the inventors connected two anti-IL-17A single-domain antibodies in series to form a bivalent antibody to enhance its blocking effect. The two anti-IL-17A single-domain antibodies are 1-C3 and 2-G4.

[0221] The amino acid sequence of the CDR region of the first single-domain antibody (1-C3) is SEQ ID NO. 1-3; the amino acid sequence of the FR region is SEQ ID NO. 7-10; the amino acid sequence of the single-domain antibody (1-C3) is SEQ ID NO. 15;

[0222] The amino acid sequence of the CDR region of the second single-domain antibody (2-G4) is SEQ ID NO.4-6; the amino acid sequence of the FR region is SEQ ID NO.11-14; and the amino acid sequence of the single-domain antibody (2-G4) is SEQ ID NO.16.

[0223] SEQ ID NO.1: GEDLGYYA;

[0224] SEQ ID NO.2: VTSSGSST;

[0225] SEQ ID NO.3:ASTILLCSDYISAFGT;

[0226] SEQ ID NO.4: GEKLDYFA;

[0227] SEQ ID NO.5: VTSSGSST;

[0228] SEQ ID NO.6:ASTILLCSDYISAFGT;

[0229] SEQ ID NO.7: DVQLVESGGGLVEPGESLRLSCAAP;

[0230] SEQ ID NO.8: IAWFRQAPGKEREVVSC;

[0231] SEQ ID NO.9:NYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAVYYC;

[0232] SEQ ID NO.10: WGQGTQVTVAS;

[0233] SEQ ID NO.11: QVQLVESGGGLVQPGGSLRLSCAAS;

[0234] SEQ ID NO.12: IGWFRQAPGKEREVVSC;

[0235] SEQ ID NO.13: NYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAIYYC;

[0236] SEQ ID NO.14: WGQGTQVTVAS;

[0237] SEQ ID NO.15:

[0238] DVQLVESGGGLVEPGESLRLSCAAPGEDLGYYAIAWFRQAPGKEREVVSCVTSGSSTNYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAVYYCASTILLCSDYISAFGTWGQGTQVTVAS;

[0239] SEQ ID NO.16:

[0240] QVQLVESGGGLVQPGGSLRLSCAASGEKLDYFAIGWFRQAPGKEREVVSCVTSSGSTNYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAIYYCASTILLCSDYISAFGTWGQGTQVTVAS.

[0241] 1.2 Preparation of bivalent single-domain antibodies (C3-G4)

[0242] The bivalent single-domain antibody (1-C3-2-G4) of the present invention is referred to as bivalent single-domain antibody (C3-G4) or C3-G4 for short.

[0243] The bivalent single-domain antibody of this embodiment is a first single-domain antibody (1-C3) connected to a second single-domain antibody (2-G4) through a linker (GGGGSGGGGSGGGGS) (the amino acid sequence after connection is SEQ ID NO.17; the corresponding nucleic acid sequence is SEQ ID NO.18), and then the hinge region (SEQ ID NO.19) and the CH region (SEQ ID NO.20) are connected. The amino acid sequence of the obtained bivalent single-domain antibody (C3-G4) is SEQ ID NO.21, and the corresponding nucleic acid sequence is SEQ ID NO.22.

[0244] 1) The above-mentioned bivalent single-domain antibody (C3-G4) sequence (SEQ ID NO.22) was synthesized and subcloned in tandem with humanIgG1Fc into the expression vector pcDNA3.4-hIgG1-Fc (IgG1 constant region amino acid sequence SEQ ID NO.26). After the vector was sequenced and verified to be correct, the Qiagen plasmid extraction kit was used to prepare the endotoxin-free plasmid for use;

[0245] 2) Take out the LVTransm transfection reagent and single-chain antibody expression vector from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting up and down. Take out the PBS buffer and warm it to room temperature. Take 2mL PBS to one well of a 6-well plate, add 130μg of antibody expression vector respectively, pipette up and down to mix thoroughly, add 400μL LVTransm, and immediately pipette up and down to mix thoroughly, and let stand at room temperature for 10 minutes.

[0246] 3) Add the above DNA / LVTransm complex to 30 mL of 293F cells and gently shake to mix thoroughly. Place the cells in a 37°C, 5% CO2 incubator at 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.

[0247] 4) After 7 days of continuous culture, the culture supernatant was collected by centrifugation, filtered with a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube. The antibody was purified using a Protein A column to finally obtain a bivalent single-domain antibody (C3-G4).

[0248] SEQ ID NO.17:

[0249] DVQLVESGGGLVEPGESLRLSCAAPGEDLGYYAIAWFRQAPGKEREVVSCVTSGSSTNYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAVYYCASTILLCSDYISAFGTWGQGTQVTVASGGGGSGGG GSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGEKLDYFAIGWFRQAPGKEREVVSCVTSSGSSTNYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAIYYCASTILLCSDYISAFGTWGQGTQVTVAS;

[0250] SEQ ID NO.18:

[0251] gatgtgcagctggtggagtctgggggaggcttggtcgagcctggggaatctctgaggctctcctgtgcagcccctggagaggatttgggttattacgccatagcctggttccgccaggccccagggaaggagcgtgaggtagtctcatgtgtcacaagtagtggtagtagcacaaactatttaagttccgtgaaggaccgattcaccatctccatagacaacgccaagaacacggtatatctgcaaatgaacagcctgaaacctgaggacacagccgtttattactgtgcgtccactattctcctctgttcagattatatctctgcctttggcacctggggccaggggacccaggtcaccgtcgcctcgggaggcggaggatctggcggaggtggaagtggcggaggcggttctcaggtgcagctcgtggagtcggggggaggcttggtgcagcccgggggatctctgaggctctcgtgtgcagcctctggagagaaattggattattttgccataggctggttccgccaggccccagggaaggagcgtgaggtagtctcatgtgtcacaagtagtggtagtagcacaaactatttaagttccgtgaaggaccgattcaccatctccatagacaacgccaagaacacggtatatctgcaaatgaacagcctgaaacctgaggacacagccatttattactgtgcgtccactattctcctctgttcagattatatctctgcctttggcacctggggccaggggacccaggtcaccgtcgcctcg;

[0252] SEQ ID NO.19:DKTHTCP;

[0253] SEQ ID NO.20:

[0254] PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0255] SEQ ID NO.21:

[0256] DVQLVESGGGLVEPGESLRLSCAAPGEDLGYYIAWFRQAPGKEREVVSCVTSSGSSTNYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAVYYCASTILLCSDYISAFGTWGQGTQVTVASGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGEKLDYFAIGWFRQAPGKEREVVSCVTSSGSSTNYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAIYYCASTILLCSDYISAFGTWGQGTQVTVASDKTHCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0257] SEQ ID NO.22:

[0258]

[0259] SEQ ID NO.26:

[0260] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0261] Example 2 Affinity detection experiment

[0262] 2.1 Preparation of positive control antibody ixekizumab

[0263] (a) Gene synthesis of ixekizumab heavy chain and light chain variable regions (heavy chain variable region sequence SEQ ID NO.23, light chain variable region sequence SEQ ID NO.24), subclone the heavy chain variable region into pcDNA3.4-hIgG4 (IgG4 constant region amino acid sequence SEQ ID NO.25) vector, and subclone the light chain variable region into pcDNA3.4-hIgKc (hIgKc constant region amino acid sequence SEQ ID NO.27) vector; after verification by Sanger sequencing, endotoxin-free plasmid was prepared using a plasmid extraction kit for use.

[0264] (b) Take out LVTransm transfection reagent and heavy chain and light chain expression vectors from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting up and down. Take out PBS buffer and warm it to room temperature. Take 2 mL PBS to one well of a 6-well plate, add 50 μg of heavy chain and light chain expression vectors respectively, mix thoroughly by pipetting up and down, then add 300 μL LVTransm, immediately mix thoroughly by pipetting up and down, and let stand at room temperature for 10 minutes.

[0265] (c) Add the above DNA / LVTransm complex to 100 mL of 293F cells, gently shake to mix thoroughly, and place the cells in a 37°C, 5% CO2 incubator at 130 RPM for further culture.

[0266] (d) After continuous culture for 5-7 days, the culture supernatant was collected by centrifugation, filtered through a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube, and the antibody was purified using a Protein A column.

[0267] (e) The purity of the target antibody protein was detected by SDS-PAGE, and the purity was >95%.

[0268] The binding ability of the recombinant antigen was tested using a positive control antibody, and the results showed that the positive antibody bound well to the IL-17A antigen protein and could be used for subsequent immunization.

[0269] The steps for protein A purification of antibodies are as follows:

[0270] 1) Add the sample containing the target antibody into the EP tube and gently invert the tube to mix.

[0271] 2) Incubate the EP tubes at room temperature (1-4 hours or overnight) or mix on a rotator. 100 mM PMSF may be added to prevent protein degradation.

[0272] 3) Collect the beads using a magnetic separation rack and discard the supernatant. If necessary, retain the supernatant for analysis.

[0273] 4) Add 1 mL of binding / washing buffer to the EP tube and mix thoroughly. Collect the beads using a magnetic stand and discard the supernatant. Repeat the washing step three times.

[0274] 5) Add 500 μL of elution buffer to the EP tube, quickly resuspend by pipetting or vortexing, and then incubate for 5 minutes at room temperature (about 25° C.) in an inverting mixer or by gently inverting the EP tube manually.

[0275] 6) Collect the magnetic beads using a magnetic separation stand and transfer the supernatant containing the eluted antibody to a clean EP tube.

[0276] 7) Repeat steps 1) and 2) twice.

[0277] 8) Add 1 / 10 of the neutralization buffer to each 500 μL of the eluate to neutralize the pH to maintain the biological activity of the antibody and avoid antibody inactivation. If necessary, buffer exchange can be performed by dialysis or desalting.

[0278] 9) Binding / washing buffer: 1× PBS, pH 7.0.

[0279] Elution buffer: (1) 0.1 M glycine, pH 2-3; (2) 0.1 M NaAc-HAc, pH 3.6.

[0280] Neutralization buffer: 1 M Tris, pH 8.5.

[0281] Magnetic bead regeneration buffer: 0.1M NaOH.

[0282] SEQ ID NO.23:

[0283] QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSS;

[0284] SEQ ID NO.24:

[0285] DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIK;

[0286] SEQ ID NO.25:

[0287] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0288] SEQ ID NO.27:

[0289] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0290] 2.2 Detection of the binding activity of human IL-17A recombinant protein and control antibody by ELISA

[0291] (1) Use sterile CBS to dilute the IL-17A recombinant protein to a final concentration of 2 μg / mL. Take a new 96-well plate and add 100 μL / well to coat overnight at 4°C.

[0292] (2) Remove the antigen coating solution and wash three times with PBST (containing 0.5% Tween).

[0293] (3) Add 200 μL / well of 3% MPBS and block at 37°C for 2 hours;

[0294] (4) After removing the blocking buffer, wash the plate three times with PBST;

[0295] (5) The positive control antibody Ixekizumab was diluted to 10 μg / ml with PBS, 5-fold diluted to 7 points, added to the ELISA plate at 100 μL / well, and incubated at room temperature for 1 hour. The control wells were PBS;

[0296] (6) Remove the liquid in the wells and wash three times with PBST;

[0297] (7) Add the secondary antibody HRP-Protein A (Boster, BA1080) at a dilution of 1:10000, add 100 μL / well to the ELISA plate, and incubate at room temperature for 1 hour;

[0298] (8) After removing the liquid in the wells, wash the plate three times with PBST;

[0299] (9) Add 100 μL / well TMB colorimetric solution;

[0300] (10) Incubate at room temperature in the dark for 15 minutes;

[0301] (11) Add 50 μL / well stop solution (2 M HCL);

[0302] (12) Use a microplate reader to read the OD450 value in the well.

[0303] The results are shown in Table 1: The positive antibodies bind well to the IL-17A antigen protein and can be used for immunization.

[0304] Table 1. Human IL17A binding activity test with positive antibodies

[0305]

[0306] 2.3 Affinity detection method and results

[0307] 2.3.1 ELISA detection of HRP-Streptavdin

[0308] The purified single domain antibody 2ug / mL was coated on a 96-well ELISA plate, and Biotin-IL-17A-His was added with a starting concentration of 10ug / mL, 5-fold gradient dilution at 7 points, and ELISA detection was performed using HRP-Streptavdin. The test results showed that the EC50 of the bivalent single domain antibody (C3-G4) was 1.231ug / mL, and the EC50 of the positive antibody (Ixekizumab) was 10.06ug / mL, indicating that the bivalent single domain antibody (C3-G4) can bind to the target protein, and the binding ability is higher than that of the positive antibody (Ixekizumab). Figure 1 .

[0309] 2.3.2 Antibody affinity determination using the ForteBio OCTET R2 instrument

[0310] (1) Antibody affinity was measured using the ForteBio OCTET R2 instrument, HIS1K sensor ( ProABiosensors) were immobilized with IL7A-His at a concentration of 5ug / mL.

[0311] (2) The buffer was PBST (PBS+0.02% tween 20), and the candidate antibody samples were diluted to 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, and 0 nM.

[0312] (3) Affinity detection: equilibrium 60 s, binding 180 s, dissociation 180 s, detection temperature 25°C.

[0313] (4) Kinetic characterization analysis was performed using the ForteBio OCTET R2 system.

[0314] The results show that the KD of the bivalent single-domain antibody (C3-G4) is 1.391×10 -9 M, KD of positive antibody (Ixekizumab) = 3.910 × 10 -10 M( Figure 2 ).

[0315] Example 3 Blocking function experiment

[0316] Add gradient dilutions of detection antibodies (positive antibody: Ixekizumab; antibody to be detected: bivalent single domain antibody (C3-G4)) to a 96-well plate, dilute the antibody in a 10-fold gradient, and dilute 10 gradients in succession. The final concentrations are 100 μg / mL, 10 μg / mL, 1 μg / mL, 0.1 μg / mL, 0.01 μg / mL, 0.001 μg / mL, 0.0001 μg / mL, 0.00001 μg / mL, 0.000001 μg / mL, 0.0000001 μg / mL, 0 μg / mL, and 50 μL of the diluted gradient concentration antibodies are added to a 96-well plate, with 2 replicates for each gradient. Then, add 50 μL to each well of the corresponding wells. 0.4 μg / mL IL-17A protein (final concentration 0.1 μg / mL). After mixing, place in a 37°C incubator and incubate for 1 hour. Pipette 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc(A3) cells cultured to the logarithmic growth phase into a 96-well plate and inoculate 2×10 cells per well. 4 After 18 h of co-culture, 20 μL of Bright-Glo™ detection reagent was added to each well, and the luciferase activity in the well was detected using a Tecan M1000 pro microplate reader.

[0317] The results showed that the IC50 of the positive control Ixekizumab was 2.235nM, and the IC50 of the bivalent single-domain antibody (C3-G4) was 1.192nM. Both of them could block the activation of 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc ( Figure 3 ).

[0318] Example 4 Stability Test

[0319] NanoDSF technology can be used to detect fluorescence changes, detect thermal and chemical denaturation of proteins under native conditions, and accurately determine the temperature (T) at which 50% of the protein is in an unfolded state. m ) and the temperature at which aggregation begins (T agg ); thermal denaturation T m , T onset Value and T agg The higher the value, the more stable the antibody protein.

[0320] 4.1 Experimental procedures

[0321] Take 100 μL of candidate antibodies and Ixekizumab (sample concentration greater than 200 μg / mL) prepared in the previous project, centrifuge at 4°C, 12000×g for 10 min, and use a capillary to absorb the sample. Prepare two capillaries for each sample as parallel controls and place them in the corresponding card slots in order to ensure that the capillaries are full of samples without bubbles for detection and analysis.

[0322] 4.2 Results

[0323] The stability results of the bivalent single-domain antibody (C3-G4) and the positive control Ixekizumab are shown in Figure 4-Figure 5 The results showed that the T m1 is 62.20℃, T m3 is 81.58℃, T onset is 47.54℃, T agg The positive control Ixekizumab had a T m1 is 56.10℃, T m2 is 79.84℃, T onset is 47.50℃, T agg The results showed that the stability of the bivalent single-domain antibody (C3-G4) was significantly stronger than that of the positive control Ixekizumab.

[0324] Example 5 Preparation method of genetically modified mesenchymal stem cells

[0325] 5.1 Construction of lentiviral shuttle plasmid

[0326] A lentiviral shuttle plasmid of C3-G4:Fc fusion protein was constructed. The two candidate antibody VHH sequences 1-C3 and 2-G4 (the amino acid sequences of 1-C3 and 2-G4 are shown in SEQ ID NOs: 15 and 16) were linked by (GGGGS)3 and then connected in series with IgG4 Fc (the amino acid sequence of IgG4 Fc is shown in SEQ ID NO: 28, and the nucleotide sequence is shown in SEQ ID NO: 29) to form a VHH-(GGGGS)3-VHH-IgG4 Fc sequence, which is located downstream of the EF-1alpha promoter, thereby obtaining a C3-G4:Fc lentiviral shuttle plasmid.

[0327] SEQ ID NO.28:

[0328] APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0329] SEQ ID NO.29:

[0330] GCCCCCGAGTTTCTGGGAGGACCTAGCGTCTTTCTGTTCCCCCCCAAACCCAAGGACACACTGATGATCTCTAGGACCCCCGAGGTGACATGCGTCGTGGTGGACGTGAGCCAAGAGGACCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAAGTGCACAATGCCAAGACCAAACCTAGAGAAGAGCAGTTCAACAGCACCTATAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGACTGGCTGAACGGCAAGGAGTACAAGTGCAAAGTGAGCAACAAGGGCCTCCCCTCCTCCATCGAGAAAACCATCTCCAAGGCCAAGGGACAGCCTAGAGAGCCCCAAGTGTATACACTGCCCCCCAGCCAAGAGGAGATGACCAAGAACCAAGTGTCTCTGACATGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCTGTGGAGTGGGAGAGCAACGGCCAGCCCGAAAACAACTATAAGACCACCCCCCCCGTGCTGGACTCCGATGGCAGCTTCTTTCTGTACTCCAGACTGACCGTGGACAAAAGCAGATGGCAAGAGGGCAACGTGTTTAGCTGCTCCGTGATGCATGAGGCTCTGCACAACCACTATACCCAGAAGTCCCTCTCTCTGAGCCTCGGCAAGTGA。

[0331] 5.2 Lentivirus preparation

[0332] (1) 24 h before virus packaging, prepare the shake flask and adjust the 293F cell density to 1.0×10 6 / mL, 60mL per bottle, place the shake flask in a shaker at 37°C, 5% CO2, and 120rpm for culture and set aside.

[0333] (2) Preparation of transfection reagent / DNA complex: Take 7.5 mL of 293F medium to a 15 mL centrifuge tube, add 40 μg of C3-G4:Fc lentiviral shuttle plasmid, 80 μg of auxiliary plasmid (purchased from addGene, catalog number: 12253, 12259), pipette up and down to mix thoroughly, add 360 μL of transfection reagent, immediately pipette up and down to mix, and let stand at room temperature for 10 min (no more than 15 min).

[0334] (3) Add the transfection reagent / DNA complex dropwise to the cells prepared the day before, shake the shaker while adding, and after fully mixing, place the shaker on a shaker at 37°C, 5% CO2, and 120rpm for culture. Harvest the supernatant after 72 hours, centrifuge the cell culture supernatant at 8000g low temperature (2-8°C), filter through 2.5μm and 0.45μm pore size filters respectively, treat the concentrated virus suspension with nuclease to remove nucleic acid impurities, and then obtain the purified virus suspension by ion exchange chromatography and gel filtration chromatography respectively, and dispense into virus dispensing tubes, 100μL per tube. Attach a label, and write the virus name, volume, lot and other information on the label.

[0335] 5.3 Lentiviral titer detection

[0336] 293T cells were inoculated into 24-well plates. After overnight culture, 20uL of lentiviral stock solution, 10-fold diluted lentiviral solution, and 100-fold diluted lentiviral solution were added, respectively. The culture was continued for 24h. Fresh medium was replaced after 24h. After continuous culture for 9d, the cells were harvested and genomic DNA was extracted using a kit (purchased from Thermo, catalog number K0721). The plasmid template copy number was adjusted with ddH2O, and the calibration range was 1×10 9 -1×10 3 , the primers are LTR and WPRE (synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.), and 2×PCRMix (purchased from Applied Biosystems, catalog number A25742), primers, DNA, and PCR water (purchased from Thermo, catalog number R0582) are mixed according to the PCR premix and added to the corresponding PCR reaction wells for PCR reaction. The virus titer is calculated according to the following formula.

[0337] Lentivirus titer = number of cells × number of copies / virus volume (mL) × dilution factor.

[0338] The results of lentiviral titer detection are shown in Figure 6 Regardless of whether the lentivirus was diluted 10 times or 100 times, the lentivirus titer was between 2.2E+7 and 2.4E+7, and there was no significant difference among the three groups. Therefore, the lentivirus titer was 2.3E+7TU / mL.

[0339] 5.4 Preparation of Gene-modified Mesenchymal Stem Cells and Detection of Infection Efficiency

[0340] The prepared C3-G4:Fc lentivirus was added to cultured mesenchymal stem cells (isolated from the umbilical cord of a newborn using an enzymatic hydrolysis method, and P2 mesenchymal stem cells were obtained by passage amplification and purification) with a fusion degree of 70-80% at an MOI of 10, and cultured at 37° C., CO2. After the cell density reached 100%, the cells were passaged to successfully obtain mesenchymal stem cells infected with the C3-G4:Fc lentivirus, i.e., mesenchymal stem cells infected with IL-17Nb (abbreviated as: IL17Nb-MSC or C3-G4-MSC, referred to as C3-G4-MSC in the following embodiments).

[0341] The MOI kit (manufacturer: System Biosciences, catalog number: LV961A-1) was used for detection. The kit can determine the transduction multiplicity (MOI) of the lentiviral particles by comparing the ratio of the endogenous conserved structure (UCR1) in the transduced umbilical cord mesenchymal stem cells and the specific WPRE element in the lentiviral vector with the standard curve provided in the kit.

[0342] The obtained C3-G4-MSC and hUC-MSC were inoculated into 24-well plates at 50,000 cells / well, and the culture medium was DMEM / F12 (containing 10% fetal bovine serum). After 24 hours of adherence culture, the sample DNA was extracted according to the instructions, and the CT value of each sample was obtained by PCR. The MOI value of each sample was calculated according to the linear equation of the standard curve. The results are shown in the figure. Figure 7 As shown, the value of hUC-MSC is 0, while the MOI value of C3-G4-MSC is 9.90±0.68, which is close to the theoretical MOI value, indicating that C3-G4:Fc lentivirus successfully infected mesenchymal stem cells.

[0343] Example 6 Detection of Gene-Modified Stem Cells by Flow Cytometry

[0344] The cells obtained in Example 5 were divided into 1×10 4 / cm 2 Culture in T25 cell culture flasks until the cell density reaches 80-90%, add transport inhibitor (purchased from BD, catalog number 555029), fix, wash, and stain with FITC-Protein A (purchased from BOSTER, catalog number BA1120), and then test the FITC channel signal of mesenchymal stem cells on the microscope (see the results). Figure 8 ), it was found that the signal positivity rate of C3-G4-MSC in the FITC channel exceeded 50%, while normal mesenchymal stem cells (hUC-MSC) that were not infected with the virus had no signal in the FITC channel, indicating that the infection rate of C3-G4-MSC reached more than 50%.

[0345] Example 7 ELISA method to detect the expression of IgG4 and IL-17Nb in gene-modified mesenchymal stem cells

[0346] The cells obtained in Example 5 were cultured at a rate of 1×10 4 Pieces / cm 2 The cells were cultured in T25 cell culture flasks in DMEM / F12 (containing 10% fetal bovine serum) as culture medium. After 72 hours of adherent culture, the cell supernatant was harvested and aliquoted and frozen for later use.

[0347] 7.1 ELISA to detect the expression of IgG4 in gene-modified mesenchymal stem cells

[0348] The human IgG4 ELISA Kit (Manufacturer: Thermo, Catalog No.: BMS2095) was used to detect the fusion protein IgG4 content in the cell culture supernatant. The kit uses a human IgG4 solid phase sandwich ELISA (enzyme-linked immunosorbent assay) to detect the amount of target bound between matching antibody pairs. IgG4-specific antibodies have been pre-coated in the ELISA plate, and then cell supernatant samples, standards or controls are added to these wells and bound to the fixed (capture) antibody, a sandwich structure is formed by adding a secondary antibody, 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.

[0349] The above cell supernatant (diluted 25 times) was added to the IgG4 ELISA kit to detect the IgG4 protein content in the fusion protein. The results showed that C3-G4-MSC highly expressed IgG4, with an expression level of up to 6843.33±845.33ng / mL, while normal hUC-MSC did not express IgG4 (see the results). Fig. 9 ).

[0350] 7.2 IL-17Nb antibody content detection

[0351] The IL-17A protein binding experiment was used to detect the expression of IL-17A nanoantibodies. The IL-17A protein (prepared by Example 1) with a final concentration of 2ug / mL was coated on an ELISA plate at 4°C overnight. After BSA blocking, the above cell supernatant was detected. The standard was C3-G4 fusion protein (synthesized by Aconde), and the standard curve concentration range was 0-250ng / mL. The standard was added to the corresponding wells, and the above C3-G4-MSC and hUC-MSC cell supernatants (diluted 20 times) were added to the sample wells and incubated for 1h. Then, HRP-labeled Protein A antibody (purchased from Boster, product number BA1080) was used as an enzyme-labeled antibody for incubation for 1h. Finally, TMB was added to develop color in the dark for 20min. After termination, the OD450nm value of each well was detected by an ELISA instrument. The experimental results are as follows Fig.10 The results showed that, through the IL-17A binding experiment, it was determined that C3-G4-MSC highly expressed IL-17Nb with a concentration of 2212.33±214.65 ng / mL, indicating that IL-17Nb expressed by C3-G4-MSC can bind to IL-17A, while normal mesenchymal stem cells (hUC-MSC) do not express IL-17Nb.

[0352] Example 8 ELISA method to detect the ability of gene-modified mesenchymal stem cells to block the binding of IL-17A and IL-17RA

[0353] The IL-17A [Biotinylated]: IL-17RA Inhibitor Screening ELISA Kit (Cat. No. EP-139) from ACRO Biosystems was used to detect the ability of genetically modified mesenchymal stem cells to block IL-17A / IL-17RA binding. The kit was coated with IL-17RA, and the neutralizing antibody against IL-17A was used as the standard to block the binding of IL-17RA with biotinylated IL-17A. The strength of the blocking ability was determined by detecting the OD450nm value. The stronger the ability to block IL-17A / IL17RA binding, the lower the OD450nm value, and the blocking ability was inversely proportional to the OD450nm value. The cell supernatant described in Example 7 was used to detect the ability of IL-17Nb in the supernatant to block IL-17A / IL17RA binding using the IL-17A / IL17RA blocking kit.

[0354] The IL-17A / IL17RA binding inhibition rate was calculated using the following formula:

[0355] Binding inhibition rate (%) = [OD450 (Positive well) - OD450 (sample well)] / OD450 (Positive well) × 100%

[0356] The cell supernatant described in Example 7 was diluted 4 times according to the instructions and tested using the ACRO kit. The results were as follows: Fig.11 It showed that normal mesenchymal stem cells (hUC-MSC) could not block the binding of IL-17A and IL17RA, while IL-17Nb secreted by C3-G4-MSC cells could block the binding between IL-17A and IL17RA, with an inhibition rate of up to 51% after dilution 4 times.

[0357] Example 9 Stem Cell Stability Study

[0358] According to the results obtained in 7.2 of Example 7 (ELISA method for detecting the expression of IL17Nb), the C3-G4-MSC and hUC-MSC cells obtained in Example 5 (C3-G4 gene modified stem cell preparation) were cultured at 1×10 4 / cm 2 A 24-well plate was inoculated, 8 wells and 16 wells were inoculated, and after overnight culture, 8 wells of hUC-MSC were randomly selected to replace the complete medium containing C3-G4 nanobody (C3-G4 nanobody, i.e., bivalent single domain antibody (C3-G4) prepared in Example 1) with a final concentration of 1000 ng / mL, and culture was continued. The supernatant was harvested at 24h, 48h, 72h and 96h of culture, and the IL17Nb content in the supernatant was detected according to the method of 7.2 (ELISA method for detecting the expression of IL17Nb) in Example 7. The results are shown in FIG. Fig.12 As shown, with the extension of time, from 24h to 96h, the IL17Nb content detected in the hUC-MSC+C3-G4 group decreased from 993±43ng / mL to 709±39ng / mL, and the concentration gradually decreased, while C3-G4-MSC could stably express IL17Nb. With the extension of culture time, the concentration of IL17Nb gradually increased, from 715±37ng / mL at 24h to 3193±117ng / mL at 96h. Among them, at 24h and 48h, there were significant differences between the hUC-MSC+C3-G4 group and the C3-G4-MSC group (P<0.01), and at 72h and 96h, there were extremely significant differences between the hUC-MSC+C3-G4 and C3-G4-MSC groups (P<0.001). It can be concluded from the results that C3-G4-MSC can stably and continuously express and secrete IL-17Nb. Therefore, the stability of C3-G4-MSC in expressing IL17Nb is better than that of recombinant C3-G4 protein.

[0359] Example 10 Construction of animal model of rheumatoid arthritis and evaluation results of test animals

[0360] B-hIL17A transgenic mice (Biocytogen Biopharmaceuticals Co., Ltd.) were used to establish a rheumatoid arthritis (RA) model by collagen induction. The RA model group animals were immunized with 100 μg bovine type II collagen (CII, Chondrex) and an emulsion of Freund's complete adjuvant (CFA, Chondrex) containing 200 μg Mycobacterium tuberculosis H37Ra by intradermal injection at the base of the tail for initial immunization on the starting day (Day 1). CII and Freund's incomplete adjuvant (IFA) emulsion were used for booster immunization on the 21st day (Day 21) after the initial immunization. 22 days after the initial immunization (Day 22), the modeled RA mice (modeling standard: Clinicalscore ≥ 2) were randomly divided into 4 groups: hUC-MSC treatment group, positive antibody treatment group, C3-G4-MSC treatment group and model control group; and hUC-MSC (2x10 6 / ), positive antibody Ixekizumab (1 mg / Kg) and C3-G4-MSC (2x10 6 / ), the model group and normal control were injected with normal saline. From Day 20 to the end of the experiment (Day 42), body weight and paw thickness were evaluated every other day for 23 days. All animals were euthanized at the end of the experiment (Day 42), and joint tissues were taken for histopathological examination and clinical scoring.

[0361] The clinical score standard is shown in Table 2:

[0362] Table 2

[0363] Score Disease 0 normal 1 Mild redness and swelling of the ankle and wrist 2 Moderate redness or swelling in the ankle or wrist 3 Severe redness and swelling of the paws, including the tips of the fingers 4 Maximum inflammation in the extremities, including multiple joints

[0364] The criteria for arthritis histopathological scoring are as follows:

[0365] The representative images of the three groups of H&E-stained limbs were scored independently by two experimenters using a double-blind method, with a single-item score ranging from 0 to 5 and a total score of 20 points.

[0366] (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.

[0367] (2) Pannus formation: 0, no pannus; 1, a few pannus formations; 2, mild pannus formation (involving less than 1 / 4 of the metacarpophalangeal joints); 3, moderate pannus formation (involving 1 / 4 to 1 / 2 of the metacarpophalangeal joints); 4, severe pannus formation (involving 1 / 2 to 3 / 4 of the metacarpophalangeal joints); 5, very severe pannus formation (involving more than 3 / 4 of the metacarpophalangeal joints).

[0368] (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 chondrocyte reduction and collagen destruction as deep as 1 / 2 of the cartilage layer); 4, severe cartilage erosion (involving more than 1 / 2 of the cartilage surface, complete destruction of one or more tarsal joint cartilage surfaces); 5, very severe cartilage erosion (severe chondrocyte reduction and collagen destruction, deep to the tide line).

[0369] (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 bone 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 bone deformation, and trabecular bone absorption (involving more than 3 / 4 of the metacarpophalangeal joints).

[0370] Animal weight Fig.13 As shown in the figure, the weight of the model group decreased significantly compared with the normal control group at the beginning of the booster immunization. The hUC-MSC treatment group and the C3-G4-MSC treatment group both restored the weight of the mice, while the positive antibody group did not restore the weight of the mice significantly. At the end of the experiment, the weight of the mice in the C3-G4-MSC treatment group was significantly higher than that in the positive antibody group. Fig.14 As shown in the figure, the paws of mice swelled rapidly after booster immunization (D21), while the paw thickness of mice significantly decreased after intravenous injection of hUC-MSC, positive antibody Ixekizumab and C3-G4-MSC, among which the therapeutic effect of C3-G4-MSC was significantly better than that of positive antibody Ixekizumab. Fig.15 As shown in the results, the model mice had tissue inflammatory cell infiltration, joint synovitis and / or pannus formation, articular cartilage destruction, disappearance of joint cavity, and bone tissue fusion; after treatment, the pathological tissue scores of the mice ( Fig.16 The therapeutic effect of C3-G4-MSC was significantly better than that of the positive antibody Ixekizumab.

[0371] Example 11 Method for constructing an animal model of psoriasis and its results

[0372] B-hIL17A transgenic mice were used to establish a psoriasis (Ps) model by applying imiquimod (IMQ). After shaving the back of all mice, 50 mg of imiquimod ointment (Mingxinlidi, Sichuan Mingxin Pharmaceutical Co., Ltd.) was applied to the back every day. The first application day was recorded as D0, and the model was established for 7 days (D6); the Ps model group was randomly divided into four groups: hUC-MSC treatment group (2x10 6 / ), positive antibody Ixekizumab treatment group (1 mg / Kg), C3-G4-MSC treatment group (2x10 6 / ) and model control group. The drug treatment group was treated with subcutaneous medication on D1 and D4. During the experiment, the animal weight was measured every day, the survival and health of the animals were observed, and the skin inflammation and related indicators were clinically scored according to the degree of keratinization and inflammatory cell infiltration of the skin. The animals were euthanized on D7 and related tests were performed: the skin of the mouse model was collected, and the skin thickness of each group of animals was measured using a vernier caliper.

[0373] 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 means no related symptoms; 1 means mild symptoms; 2 means general symptoms; 3 means significant symptoms; 4 means very significant or severe symptoms. The total score of the three indicators is calculated as the final score.

[0374] Animal weight Fig.17 As shown in the figure, the body weight of the model group after imiquimod modeling decreased significantly compared with the normal control group. The hUC-MSC treatment and C3-G4-MSC treatment groups could restore the body weight of mice, while the positive antibody group did not restore the body weight of mice significantly. At the end of the experiment, the body weight of mice in the C3-G4-MSC treatment group was significantly higher than that in the positive antibody group. The skin photos are shown in the figure. Fig.18 As shown in the figure, the clinical skin scores were based on the degree of skin keratinization, dandruff and skin lesions. Fig.19 As shown in the results, IMQ can cause skin damage in mice, with increased rash and desquamation, thickening of the epidermis, and histopathological findings of the dermis with parakeratosis and inflammatory leukocyte infiltration. The clinical score of the skin in the model group increased significantly, while the clinical score of the skin in mice decreased significantly after subcutaneous injection of hUC-MSC, positive antibody Ixekizumab and C3-G4-MSC, among which the therapeutic effect of C3-G4-MSC was significantly better than that of positive antibody Ixekizumab. Skin thickness was tested at the end of the experiment, such as Fig. 20As shown, it was found that IMQ could significantly increase the skin thickness of model mice, while after subcutaneous injection of hUC-MSC, positive antibody Ixekizumab and C3-G4-MSC, the skin thickness of mice was significantly reduced, among which the therapeutic effect of C3-G4-MSC was significantly better than that of positive antibody Ixekizumab.

[0375] Example 12 Method for constructing an animal model of psoriatic arthritis and its results

[0376] B-hIL17A transgenic mice (Biocytogen Incorporated) were used to construct a psoriatic arthritis (PsA) model by intraperitoneal injection of mannan (SIGMA, M7504-5G). Modeling mice were taken, and the day of the first intraperitoneal injection was marked as D0. Mannan was injected intraperitoneally three times at D0, D4 and D8. Each animal was intraperitoneally injected with 100 mg / mL mannan each time, and the injection volume was 200 μL, that is, each animal was intraperitoneally injected with 20 mg mannan each time. The PsA model group was randomly divided into four groups: hUC-MSC treatment group (2x10 6 / ), positive antibody Ixekizumab treatment group (1 mg / Kg), C3-G4-MSC treatment group (2x10 6 / ) and model control group; the drug treatment group was treated by tail vein administration on D3 and D7. During the experiment, the weight of the animals was measured every 2 days, the survival and health of the animals were observed, and the skin and front and back paws of the animals were photographed, observed and scored according to relevant indicators. On D14, the animals were euthanized, and the skin of the mouse model was collected for relevant tests. The thickness of the skin of each group of animals was measured with a vernier caliper; the scores were based on the degree of keratinization and inflammatory cell infiltration of the skin; the peripheral blood of the mice was collected, and the serum was separated and sent to the immunoassay department to detect cytokines such as mIL-1β, mIL-6, hIL-17A and mTNF-α (the kits were all purchased from Biolegend).

[0377] Animal Paw Scoring Criteria:

[0378] The arthritis of all front and rear paws of the animals was scored according to the following criteria: 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 was calculated as the final score.

[0379] 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 means no related symptoms; 1 means mild symptoms; 2 means general symptoms; 3 means significant symptoms; 4 means very significant or severe symptoms. The total score of the three indicators is calculated as the final score.

[0380] Animal weight Fig.21 As shown, the body weight of the PsA model group after mannan modeling was significantly decreased compared with the normal control group. The hUC-MSC treatment and C3-G4-MSC treatment groups could restore the weight of mice, while the positive antibody group did not restore the weight of mice significantly. At the end of the experiment, the weight of mice in the C3-G4-MSC treatment group was significantly higher than that in the positive antibody group.

[0381] Clinical scoring of animal paws and skin Figure 22-Figure 23 As shown, after mannan modeling, the clinical scores of the skin and paws of the PsA model group were significantly increased, and the positive antibody, hUC-MSC treatment and C3-G4-MSC treatment groups could significantly reduce the clinical scores of the model paws and skin, while the body weight of mice in the C3-G4-MSC treatment group was significantly better than that in the positive antibody group.

[0382] Inflammatory factor detection Figure 24-26 As shown in the results, after mannan modeling, the levels of IL-6, IL-23, TNF-α and other cytokines in the serum of PsA model group animals were significantly increased, while the levels of IL-6 and TNF-α in the serum of model animals could be significantly reduced in both hUC-MSC treatment and C3-G4-MSC treatment groups, and the levels of IL-23 in the serum of model animals could be significantly reduced in both the positive antibody group and C3-G4-MSC treatment groups.

Claims

1. Use of umbilical cord mesenchymal stem cells in the preparation of drugs for treating diseases, characterized in that: The umbilical cord mesenchymal stem cells express or secrete the following (1) and (2): (1) a first antibody, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the first antibody are SEQ ID NOs. 1-3; (2) a second antibody, wherein the amino acid sequences of HCDR4, HCDR5 and HCDR6 of the second antibody are SEQ ID NOs. 4-6; The first antibody and the second antibody are connected via a linker having an amino acid sequence of (GGGGS)3; The first antibody and the second antibody both comprise single domain antibodies that specifically recognize IL-17A; The diseases include rheumatoid arthritis, psoriasis and / or psoriatic arthritis.

2. The use of umbilical cord mesenchymal stem cells in the preparation of drugs for treating diseases, characterized in that: The umbilical cord mesenchymal stem cells express or secrete the following (1) and (2): (1) a first antibody, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the first antibody are SEQ ID NOs. 1-3, and the amino acid sequences of FR1, FR2, FR3 and FR4 of the first antibody are SEQ ID NOs. 7-10; (2) a second antibody, wherein the amino acid sequences of HCDR4, HCDR5 and HCDR6 of the second antibody are SEQ ID NOs. 4-6, and the amino acid sequences of FR5, FR6, FR7 and FR8 of the second antibody are SEQ ID NOs. 11-14; The first antibody and the second antibody are connected via a linker having an amino acid sequence of (GGGGS)3; The first antibody and the second antibody both comprise single domain antibodies that specifically recognize IL-17A; The diseases include rheumatoid arthritis, psoriasis and / or psoriatic arthritis.

3. Use of umbilical cord mesenchymal stem cells in the preparation of drugs for treating diseases, characterized in that: The umbilical cord mesenchymal stem cells express or secrete the following (1) and (2): (1) a first antibody, wherein the amino acid sequence of the first antibody is SEQ ID NO.15; (2) a second antibody, wherein the amino acid sequence of the second antibody is SEQ ID NO.16; The SEQ ID NO.15 and SEQ ID NO.16 are connected by a linker having an amino acid sequence of (GGGGS)3; The first antibody and the second antibody both comprise single domain antibodies that specifically recognize IL-17A; The diseases include rheumatoid arthritis, psoriasis and / or psoriatic arthritis.

4. Use of umbilical cord mesenchymal stem cells in the preparation of drugs for treating diseases, characterized in that: The umbilical cord mesenchymal stem cells express or secrete an antibody whose amino acid sequence is shown in SEQ ID NO.17; The antibody comprises a single domain antibody that specifically recognizes IL-17A; The diseases include rheumatoid arthritis, psoriasis and / or psoriatic arthritis.

5. The use according to claim 4, characterized in that: The umbilical cord mesenchymal stem cells are isolated from umbilical cord blood, umbilical cord or placenta.

6. Use of umbilical cord mesenchymal stem cells in the preparation of drugs for treating diseases, characterized in that: The umbilical cord mesenchymal stem cells express or secrete an antibody whose amino acid sequence is shown in SEQ ID NO.21; The antibody comprises a single domain antibody that specifically recognizes IL-17A; The diseases include rheumatoid arthritis, psoriasis and / or psoriatic arthritis.

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