CN117866902B

CN117866902B - Genetically modified stem cells with anti-IL-17A activity, preparation method thereof and pharmaceutical composition

Info

Publication number: CN117866902B
Application number: CN202410276784.4A
Authority: CN
Inventors: 刘广洋; 刘拥军; 李欣; 王荷蕊; 张晨亮
Original assignee: Beijing Beilai Biotechnology Co ltd
Current assignee: Beijing Beilai Biotechnology Co ltd
Priority date: 2024-03-12
Filing date: 2024-03-12
Publication date: 2024-06-04
Anticipated expiration: 2044-03-12
Also published as: CN117866902A

Abstract

The invention belongs to the field of biological medicine, and in particular relates to a genetically modified stem cell with anti-IL-17A activity, a preparation method thereof and a pharmaceutical composition. The invention provides an anti-IL-17A nanobody (IL 17 Nb) gene modified mesenchymal stem cell (IL 17 Nb-MSC), wherein the IL17Nb-MSC comprises an HCDR region with an amino acid sequence shown in SEQ ID NO. 1-6. The genetically modified stem cell can highly express IgG4 and IL-17Nb, wherein the IL-17Nb can effectively block the combination of IL-17A/IL17RA, provides a new choice for the treatment of various diseases such as IL-17A mediated rheumatoid arthritis, psoriasis, psoriatic arthritis and the like, and has wide clinical application prospect.

Description

Genetically modified stem cells with anti-IL-17A activity, preparation method thereof and pharmaceutical composition

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a genetically modified stem cell with anti-IL-17A activity, a preparation method thereof and a pharmaceutical composition.

Background

Mesenchymal Stem Cells (MSCs) are derived from mesoderm at early embryonic development and are a heterogeneous population of cells, and MSCs in the organism comprise stem cells and differentiated progeny from different stages of embryonic development both early and later. Human tissues contain MSCs, and various tissues and organs contain rich MSCs, such as bone marrow, fat, dental pulp, umbilical cord, placenta, etc. MSCs are of low immunogenicity and are not recognized by the immune system of the recipient after repeated injections, which is one of the most significant advantages of MSCs for clinical treatment. MSCs can also secrete a plurality of bioactive molecules and a plurality of immune regulation factors, and can play a role in repairing injury, inflammation and the like of organisms. Currently, MSCs are used in clinical studies to treat a variety of diseases, including multiple sclerosis, myocardial infarction, organ transplantation, cirrhosis and liver failure, heart failure, GVHD, left ventricular dysfunction, leukemia, and crohn's disease.

MSCs are ideal gene therapy cell vectors, are easy to be transfected by all virus vector systems commonly applied in clinic, and after being infused, the MSCs subjected to gene modification can spread over the whole body, so that the effects of stem cell homing and repair can be exerted, target genes such as cytokines can be expressed in a large amount, and the MSCs and the target genes have synergistic effects, so that the disease range of MSCs treatment can be enlarged, and the local repair probability of disease focus parts is greatly improved. These advantages make MSCs one of the stem cells that has the greatest potential for gene therapy research and clinical therapeutic applications.

Patent CN 112941028A discloses a nanometer antibody gene modified mesenchymal stem cell, a preparation method and application thereof. The mesenchymal stem cells contain, and/or express, and/or secrete nanobodies. The mesenchymal stem cells or the culture extract of the mesenchymal stem cells migrate to a target position, and simultaneously, the nano antibodies can accurately reach a focus area in a short time, so that the concentration and the effective amount of the nano antibodies in the focus area are improved, the failure phenomenon of the nano antibodies in the focus area is weakened, the secretion of other beneficial components by the mesenchymal stem cells is promoted, the cell immunotherapy mediated by the mesenchymal stem cells is improved, and the treatment effect of immune-related diseases is improved.

IL-17A is an inflammatory cytokine produced mainly by activated T cells, and acts on downstream effector cells to mediate various physiological processes, such as inflammatory reaction, coagulation process, bone remodeling and the like, and is closely related to various diseases of the body. The excessive expression of IL-17A can cause the occurrence and development of various diseases such as rachitis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, inflammatory bowel disease and the like. The specific medicines clinically available for the diseases are few, and the traditional treatment has the defects of slower effect, poor compliance and the like. In view of the above therapeutic advantages of genetically modified stem cells, there is a need to develop a genetically modified stem cell for use in the treatment of the above-mentioned diseases.

The present inventors have focused on anti-IL-17A antibody development and, for ease of examination, now briefly introduced the technical background of the development project:

The inventors have further developed techniques based on the 9 single domain antibodies screened (filed in the alternative), resulting in a combination of 12 single domain antibodies with relatively improved affinity, blocking effect and stability (filed in the alternative).

Based on the technical scheme, the inventor continues to develop the technology of the single domain antibody combined gene modified stem cell, and based on the relevant regulations of the uniqueness of the patent laws, the invention patent application of one of the technology of the single domain antibody combined gene modified stem cell requests protection to 3 different gene modified stem cells.

For ease of understanding the application, reference is optionally made to other patent application documents of this project.

Disclosure of Invention

In order to solve the problems, the invention provides a stem cell containing, expressing and/or secreting an anti-IL-17A antibody, which is characterized in that the anti-IL-17A antibody is a bivalent single domain antibody, and the amino acid sequence of the bivalent single domain antibody is SEQ ID NO.1.

In the present invention, a single domain antibody is also called a nanobody as an antibody in which the complementarity determining region is a part of a single domain polypeptide. Thus, a single domain antibody comprises a single complementarity determining region. Single domain antibodies are heavy chain-only antibodies that naturally do not contain a light chain, single domain antibodies derived from conventional antibodies, and engineered antibodies. The single domain antibodies may be derived from any species including mice, humans, camels, llamas, goats, rabbits, and cattle. For example, naturally occurring VHH molecules may be derived from antibodies provided by camelidae species (e.g. camels, dromedaries, llamas and dromedaries). Like whole antibodies, single domain antibodies are capable of selectively binding to a particular antigen. A single domain antibody may contain only the variable domains of an immunoglobulin chain, which domains have CDR1, CDR2 and CDR3, as well as framework regions.

In the present invention, the anti-IL-17A bivalent single domain antibody, i.e., anti-IL-17A bivalent single domain antibody, includes not only the intact bivalent single domain antibody but also fragments, derivatives and analogues of the anti-IL-17A bivalent single domain antibody. Wherein fragments, derivatives and analogs are synonymous, all refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the present invention may be a polypeptide having one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, and such substituted amino acid residues may or may not be a polypeptide encoded by the genetic code or having a substituent in one or more amino acid residues, or a polypeptide formed by fusion of a mature polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol), or a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a pro-protein sequence for purification of the polypeptide, or a fusion protein with an Fc tag).

In the present invention, sequence homology means the degree to which two (nucleotide or amino acid) sequences have identical residues at identical positions in an alignment, and is generally expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Thus, two copies with identical sequences have 100% homology. In some embodiments, sequences that replace only one or a few amino acids, e.g., comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, as compared to the preceding sequences, may also achieve the object. These variants include, but are not limited to: deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. In fact, the skilled person may consider so-called "conservative" amino acid substitutions, which in the case of substitution would preferably be conservative amino acid substitutions, in determining the degree of sequence homology between two amino acid sequences or in determining the CDR1, CDR2 and CDR3 combinations in a single domain antibody. The conserved amino acid, which may be generally described as an amino acid substitution of an amino acid residue with another amino acid residue having a similar chemical structure, has little or no effect on the function, activity, or other biological property of the polypeptide. Such conservative amino acid substitutions are common in the art, e.g., conservative amino acid substitutions are those in which one or a few amino acids in the following groups (a) - (d) are substituted for another or a few amino acids in the same group: (a) a polar negatively charged residue and an uncharged amide thereof: asp, asn, glu, gln; (b) Polar positively charged residues: his, arg, lys; (c) aromatic residues: phe, trp, tyr; (d) aliphatic nonpolar or low polar residues: ala, ser, thr, gly, pro, met, leu, ile, val, cys. Particularly preferred conservative amino acid substitutions are as follows: asp is substituted with Glu; asn is substituted with Gln or His; glu is substituted with Asp; gln is substituted with Asn; his is substituted with Asn or Gln; arg is replaced by Lys; lys is substituted by Arg, gln; phe is substituted with Met, leu, tyr; trp is substituted with Tyr; tyr is substituted with Phe, trp; substitution of Ala with Gly or Ser; ser is substituted by Thr; thr is replaced by Ser; substitution of Gly with Ala or Pro; met is substituted with Leu, tyr or Ile; leu is substituted with Ile or Val; lie is substituted with Leu or Val; val is substituted with Ile or Leu; cys is replaced by Ser. In addition, those skilled in the art will recognize that the creativity of single domain antibodies is represented in the CDR1-3 regions, while the framework region sequences FR1-4 are not immutable, and that the sequences of FR1-4 may take the form of conservative sequence variants of the sequences disclosed herein.

In the present invention, the antibody fusion protein refers to a product obtained by fusing an antibody fragment with other bioactive proteins using genetic engineering techniques. Due to the differences in fusion proteins, such antibody fusion proteins have a variety of biological functions, and the expressed recombinant protein does not affect the antigen binding capacity of the single chain antibody nor the biological properties of the protein to which it is fused.

In the present invention, stem cells are a class of cells having unlimited or immortalized self-renewing capacity, capable of producing at least one type of highly differentiated daughter cells. Functionally, stem cells are cells with multipotent differentiation potential and self-renewal capacity, the most primitive cells at the top of the cell line origin, and are capable of differentiating in vivo to produce a specific tissue type. The stem cells of the present invention have the following biological characteristics: (a) The non-terminally differentiated cells remain undifferentiated or poorly differentiated throughout life, lacking differentiation markers; (b) relatively constant in the number and position of the bodies; (c) having self-renewing capability; (d) Can divide and proliferate infinitely, can be in a static state for a long time, and stem cells can divide for several generations continuously; (e) Has multidirectional differentiation potential and can differentiate into various tissue cells; also has the plasticity of differentiation and development, and can be induced to differentiate into cell types irrelevant to development under a specific environment, and the differentiation is influenced by the surrounding microenvironment (stem cell niche); (f) slow periodicity of cleavage; (g) Stem cells grow in two ways, one is symmetrically split to form two identical stem cells, the other is asymmetrically split, one of which maintains the characteristics of the parent and remains as a stem cell, and the other daughter cell irreversibly goes to the terminal end of differentiation to become a functionally specific differentiated cell.

In the present invention, mesenchymal Stem Cells (MSCs), also referred to as mesenchymal stromal cells, are a subset of non-hematopoietic adult stem cells derived from mesoderm. They have self-renewing ability and multipotent differentiation into not only mesodermal lineages such as chondrocytes, bone cells and adipocytes, but also ectodermal cells and endodermal cells. MSCs are the major stem cell type for cell therapies for the treatment of immune and non-immune diseases due to lack of ethical issues and teratoma formation. They can be easily isolated from bone marrow, adipose tissue, umbilical cord, fetal liver, muscle and lung and can be successfully amplified in vitro. In addition, MSCs have a tendency to home to damaged tissue sites. When MSCs are exogenously delivered and systematically administered to humans and animals, they migrate specifically to the site of damaged tissue with inflammation. Inflammation-directed MSC homing involves several important cell transport-related molecules, including chemokines, adhesion molecules, and Matrix Metalloproteinases (MMPs).

In the present invention, cell culture refers to a method of simulating in vitro an in vivo environment (sterility, proper temperature, pH value, certain nutritional conditions, etc.), so as to survive, grow, reproduce and maintain the main structure and function.

The invention improves the success rate of cell culture (1) aseptic technique from the following aspects: during cell culture, it is important to maintain aseptic manipulation. Aseptic techniques are used, including wearing appropriate laboratory clothing, donning gloves and masks, using aseptic stations or incubators, and the like, and using aseptic culture devices and culture reagents to avoid contamination by bacteria, fungi, or other microorganisms. (2) treatment with a culture tool: prior to use, the culture vessel (e.g., petri dish, centrifuge tube, test tube, etc.) is ensured to undergo proper cleaning and sterilization treatments. The culture dish can be irradiated by ultraviolet rays before use, and the test tube and the centrifuge tube can be treated by high-temperature baking or automatic cleaning procedures. (3) preparation of a culture medium: accurate preparation of the culture medium is critical to ensuring cell growth and health. The media components are accurately weighed and mixed according to manufacturer's instructions or standard laboratory procedures to ensure proper concentration and pH. (4) cell density control: the appropriate cell density is determined based on the cell type and experimental requirements prior to cell passage or experiment. Too low a density may result in slow cell growth, while too high a density may result in overcrowding and cell death. (5) optimization of culture conditions: the requirements of different cell types on culture conditions are different, including culture temperature, CO2 concentration, humidity, culture medium formula and the like. Knowing and optimizing the culture conditions appropriate for a particular cell type can increase the successful culture rate of the cells. (6) cell detection and identification: cells are periodically identified and tested to ensure purity and authentication. Identification is performed using, but not limited to, cell specific markers or PCR methods to avoid cell contamination or mixing of cell lines. (7) freezing and storing back-up: the backup cell lines are frozen periodically to prevent loss or contamination of cells. The method and conditions for cryopreserving cells need to be determined according to the cell type and laboratory requirements. (8) observation and recording: the growth and status of the cells, including cell morphology, proliferation rate, and cell death, are closely observed and recorded. Any abnormal phenomenon such as cell pollution or cell aging can be found and treated in time.

In the present invention, nucleic acid constructs comprising the nucleic acid sequences of the antibodies of the invention, and one or more regulatory sequences operably linked to these sequences. By operably linked is meant that some portion of a linear DNA sequence is capable of modulating or controlling the activity of other portions of the same linear DNA sequence. For example, if a promoter controls transcription of a coding sequence, it is operably linked to the coding sequence.

The regulatory sequence may be a suitable promoter sequence. The promoter sequence is typically 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 host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

The regulatory sequences may also be suitable transcription terminator sequences, sequences recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

In the present invention, the recombinant vector. In particular, the nucleic acid sequences of the antibodies may be cloned into vectors, such as vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses and cosmids. The vector may be an expression vector (also referred to as a recombinant vector). The expression vector may be provided to the cell in the form of a viral vector or a non-viral vector, preferably a non-viral vector. The expression vector may contain 1 or more repeated antibody nucleic acid sequences thereon. In general, suitable vectors comprise an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers.

Suitable promoters include, but are not limited to, the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV 40) early promoter, the mouse mammary carcinoma virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the epstein barr virus immediate early promoter, the ruses sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, the myosin promoter, the heme promoter, and the creatine kinase promoter. Further, the use of inducible promoters is also contemplated. The use of an inducible promoter provides a molecular switch that is capable of switching on expression of a polynucleotide sequence operably linked to the inducible promoter when expressed for a period of time and switching off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

Selectable markers include either or both selectable marker genes or reporter genes to facilitate identification and selection of expressing cells from a population of cells infected with the viral vector. Useful selectable marker genes include, for example, antibiotic resistance genes, such as neo and the like. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein genes.

In the invention, pharmaceutically acceptable auxiliary materials refer to excipients and additives used in the production of medicines and the preparation of prescriptions; are substances which, apart from the active ingredient, have been reasonably evaluated in terms of safety and are contained in pharmaceutical preparations. The pharmaceutical excipients not only form, serve as carriers and improve stability, but also have important functions of solubilization, dissolution assistance, sustained and controlled release and the like, and are important components which can influence the quality, safety and effectiveness of the medicine.

In one aspect, the present invention provides a modified stem cell comprising, expressing and/or secreting the following (1) and (2):

(1) A first antibody comprising a single domain antibody that specifically recognizes IL-17A;

(2) The second antibody comprises a single domain antibody that specifically recognizes IL-17A.

Specifically, the first single domain antibody comprises HCDR1, HCDR2 and HCDR3; the amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are SEQ ID NO.1-3;

the second single domain antibody comprises HCDR4, HCDR5 and HCDR6; the amino acid sequences of the HCDR4, the HCDR5 and the HCDR6 are SEQ ID NO.4-6.

In yet another aspect, the present invention provides a stem cell comprising, expressing and/or secreting the following (1) and (2):

(1) A first protein, the structure of said first protein comprising:

FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4；

(2) A second protein, the structure of said second protein comprising:

FR5-HCDR4-FR6-HCDR5-FR7-HCDR6-FR8。

Specifically, the HCDR1-6 is selected from amino acid sequences shown in SEQ ID NO. 1-6;

the FR1-8 is selected from amino acid sequences shown in SEQ ID NO. 7-14.

In yet another aspect, the present invention provides a stem cell comprising, expressing and/or secreting a fusion protein;

the fusion protein comprises the following structure:

Amino acid sequence of FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4-linker-FR5-HCDR4-FR6-HCDR5-FR7-HCDR6-FR 8.

the FR1-8 is selected from amino acid sequences shown in SEQ ID NO. 7-14;

The amino acid sequence of the linker is (GGGGS) n, wherein n is 1,2, 3, 4, 5 or 6.

In yet another aspect, the present invention provides a modified stem cell comprising, expressing and/or secreting the following (1) and (2):

(1) A first protein, wherein the sequence of the first protein comprises an amino acid sequence shown as SEQ ID NO. 15;

(2) And the second protein comprises an amino acid sequence shown as SEQ ID NO. 16.

In yet another aspect, the invention provides a stem cell comprising, expressing and/or secreting a fusion protein.

Specifically, the sequence of the fusion protein comprises a sequence shown as SEQ ID NO.17.

In yet another aspect, the present invention provides a stem cell comprising the following (1) and (2):

(1) A first nucleic acid molecule;

(2) A second nucleic acid molecule.

Specifically, the first nucleic acid molecule encodes a nucleotide sequence comprising the amino acid sequence shown in SEQ ID NO. 1-3;

The second nucleic acid molecule encodes a nucleotide sequence comprising the amino acid sequence shown in SEQ ID NO. 4-6.

Further specifically, the first nucleic acid molecule encodes a nucleotide sequence comprising the amino acid sequence shown in SEQ ID NO. 7-10;

The second nucleic acid molecule encodes a nucleotide sequence comprising the amino acid sequence shown in SEQ ID NO. 11-14.

In yet another aspect, the invention provides a stem cell, wherein the first nucleic acid molecule and the second nucleic acid molecule are linked by a nucleotide sequence encoding an amino acid sequence comprising (GGGGS) n, wherein n is 1,2, 3,4, 5 or 6.

Preferably, the n is 3.

(1) A first nucleic acid molecule;

(2) A second nucleic acid molecule;

The first nucleic acid molecule codes for a nucleotide sequence comprising the amino acid sequence shown in SEQ ID NO. 15;

the first nucleic acid molecule encodes a nucleotide sequence comprising the amino acid sequence shown in SEQ ID NO. 16.

In yet another aspect, the present invention provides a stem cell comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 17.

In particular, the stem cells further comprise, express and/or secrete biologically active proteins or functional fragments thereof capable of extending the half-life of the antibody in vivo.

Further, the biologically active protein or functional fragment thereof is selected from at least one of an immunoglobulin Fc domain, serum albumin, albumin binding polypeptide, prealbumin, carboxy terminal peptide, elastin-like polypeptide, his tag, GST tag, MBP tag, FLAG tag, and SUMO tag.

Preferably, the biologically active protein or functional fragment thereof is a human immunoglobulin Fc domain.

Further preferably, the biologically active protein or functional fragment thereof is an Fc domain of human IgG, e.g., an Fc domain of human IgG1, igG2, igG3, igG 4.

Further preferably, the biologically active protein or functional fragment thereof is the Fc domain of human IgG 1.

Specifically, the stem cells are selected from adult stem cells, mesenchymal stem cells, umbilical cord blood stem cells, hematopoietic stem cells, neural stem cells, adipose stem cells, skin stem cells or muscle stem cells.

Preferably, the stem cells are mesenchymal stem cells.

In particular, the stem cells are isolated from cord blood, umbilical cord, placenta, adipose tissue, skin, neural tissue or bone marrow.

In particular, the stem cells secrete anti-interleukin antibodies.

Further specifically, the stem cells secrete anti-interleukin 17 antibodies.

Still more particularly, the stem cells secrete anti-IL-17A antibodies.

In yet another aspect, the invention provides a cell culture comprising the aforementioned stem cells.

In yet another aspect, the invention provides an extract obtained from a cell culture as described above.

Specifically, the extract is cell culture supernatant, cell lysate and/or exosome.

Further specifically, the cell supernatant refers to an original untreated or simply treated culture solution rich in various cytokines produced by stem cells during the culture process;

The cell lysate refers to stem cells which are crushed by ultrasonic methods and the like to release soluble proteins and other soluble contents in the cells;

the extracellular exosomes are extracellular vesicles with complete membrane structures and are mainly responsible for substance transportation and information transmission among cells, and the diameters of the extracellular vesicles are 30-150nm.

In yet another aspect, the invention provides a pharmaceutical composition comprising the aforementioned stem cells or cell cultures or extracts.

Specifically, the pharmaceutical composition also comprises pharmaceutically acceptable auxiliary materials.

Further specifically, the pharmaceutically acceptable excipients include, but are not limited to: any one or more of excipients, stabilizers, diluents, binders, preservatives, lubricants, antioxidants.

Preferably, the pharmaceutically acceptable auxiliary material may be at least one selected from lactose, mannose, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

In yet another aspect, the invention provides a kit comprising the aforementioned stem cells or cell cultures or extracts.

Specifically, the kit also comprises a solid phase carrier, a detection label, a detection substrate and/or a buffer solution.

Further specifically, the solid support may be a material having affinity that immobilizes the specific antibody on the surface.

The detection label may be an enzyme label, which is an enzyme capable of binding to an antibody, for detecting binding of the antibody to an antigen.

The detection substrate may be a reaction product of an enzyme label capable of producing a measurable signal under enzymatic catalysis.

In yet another aspect, the present invention provides a method for preparing the stem cells described above, the method comprising: the nucleic acid molecules are introduced into stem cells by viral transfection, liposome transfection, electrotransfer, gene editing or mRNA transfection.

In yet another aspect, the invention provides the use of the aforementioned stem cells or cell cultures or extracts or pharmaceutical compositions for the preparation of a medicament for the prevention and/or treatment of a disease.

In particular, the diseases include, but are not limited to: inflammatory diseases, infectious diseases, autoimmune diseases, neurological diseases and/or tumors.

Further specifically, the inflammatory diseases include, but are not limited to: hashimoto thyroiditis, systemic lupus erythematosus, rheumatoid arthritis and/or primary biliary cirrhosis;

such infectious diseases include, but are not limited to: bacterial infection, viral infection, fungal infection, mycoplasma infection, chlamydia infection and/or rickettsia infection;

Such autoimmune diseases include, but are not limited to: behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, systemic scleroderma, progressive systemic sclerosis, scleroderma, polymyositis, dermatomyositis, perinodular arteritis, aortitis syndrome, malignant rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, kalman's disease, sjogren's syndrome, adult Steve's disease, vasculitis, allergic granulomatous vasculitis, allergic vasculitis, rheumatoid vasculitis, macrovasculitis, ANCA-related vasculitis, cogan syndrome, RS3PE syndrome, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, igG 4-related diseases, guillain-Barre syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, inflammatory bowel disease non-alcoholic steatohepatitis, primary biliary cirrhosis, good-pasture syndrome, acute glomerulonephritis, lupus nephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, barcedo's 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, pregnancy herpes, linear IgA bullous dermatoses, acquired bullous epidermolysis, alopecia areata, white spot, neuromyelitis, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, former 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;

such neurological disorders include, but are not limited to: central nervous system infections, cerebrovascular diseases, dyskinesia diseases, peripheral neuropathy, and/or nerve and muscle junctions and muscle diseases.

Such tumors include, but are not limited to: basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, bone carcinoma, breast carcinoma, peritoneal carcinoma, cervical cancer, cholangiocarcinoma, choriocarcinoma, colorectal cancer, connective tissue carcinoma, digestive system cancer, endometrial carcinoma, esophageal carcinoma, eye carcinoma, head and neck carcinoma, gastric cancer, glioblastoma, liver cancer, renal carcinoma, laryngeal carcinoma, 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 carcinoma, sarcoma, skin carcinoma, squamous cell carcinoma, testicular carcinoma, thyroid carcinoma, uterine cancer, urinary system cancer, B-cell lymphoma, chronic lymphoblastic leukemia, acute lymphoblastic leukemia, hairy cell leukemia, chronic myeloblastic leukemia

Still more particularly, the disease comprises rheumatoid arthritis, psoriasis and/or psoriatic arthritis.

Specifically, the medicine also comprises pharmaceutically acceptable auxiliary materials.

In yet another aspect, the invention provides a method for in vitro detection of IL-17A in a sample for non-diagnostic purposes, the method comprising the steps of:

s1: contacting the stem cells or cell cultures or extracts with a sample to be tested;

s2: detecting the antigen-antibody complex;

S3: and judging the result.

The invention has the technical effects that:

(1) The genetically modified mesenchymal stem cells (IL 17 Nb-MSC) prepared by the invention express high IgG4, and the expression quantity is up to 6843.33 +/-845.33 ng/mL; normal hUC-MSC does not express IgG4.

(2) The genetically modified mesenchymal stem cells (IL 17 Nb-MSC) prepared by the invention highly express IL-17Nb, and the concentration is 2212.33 +/-214.65 ng/mL; normal mesenchymal stem cells (hUC-MSC) do not express IL-17Nb.

(3) The IL-17Nb secreted by the genetically modified mesenchymal stem cells (IL 17 Nb-MSC) prepared by the invention can block the combination of IL-17A/IL17RA, and the inhibition rate is up to 51% after 4 times dilution; normal mesenchymal stem cells (hUC-MSC) cannot block IL-17A/IL17RA binding.

Drawings

FIG. 1 is a graph showing the results of ELISA detection of bivalent single domain antibody (C3-G4) and positive control Ixekizumab.

FIG. 2 is a graph showing the results of affinity detection of bivalent single domain antibody (C3-G4) and positive control Ixekizumab, wherein the graphs are 25 and 12.5,6.25,3.13 from top to bottom respectively.

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

FIG. 4 shows the stability results of bivalent single domain antibodies (C3-G4).

FIG. 5 shows the stability of the positive control Ixekizumab.

FIG. 6 is a graph of the results of transduction complex of lentiviral particles.

FIG. 7 shows the results of lentiviral titer assays.

Fig. 8 is a graph showing the results of FITC channel signaling of mesenchymal stem cells.

FIG. 9 is a graph showing the results of expression of mesenchymal stem cell IgG 4.

FIG. 10 is a graph showing the results of expression of mesenchymal stem cells IL-17 Nb.

FIG. 11 shows the blocking rate of mesenchymal stem cells blocking IL-17A binding to IL-17 RA.

FIG. 12 is the results of the stem cell stability test of example 9.

Fig. 13 is a graph of the trend of the body weight change of the mice in example 10, wherein P <0.01 represents that the model control group has significant differences from the normal control group; ^## P <0.01 represents a significant difference between the C3-G4-MSC treated group and the model control group; ^&& P <0.01 represents a significant difference between the C3-G4-MSC treated group and the positive antibody treated group.

FIG. 14 is a photograph of the skin of a mouse in example 10.

Fig. 15 is a plot of the clinical scores of the mice skin in example 10, wherein P <0.0001 represents a significant difference between the model control group and the normal control group; ^### P <0.001 represents a significant difference between the 3 treatment groups and the model control group; ^& P <0.05 represents a significant difference between the C3-G4-MSC treated group and the positive antibody treated group; ^@ P <0.05 represents a significant difference between the C3-G4-MSC treated group and the hoc-MSC treated group.

Fig. 16 is a graph showing the results of the skin thickness test of the mice in example 10, wherein P <0.001 represents a significant difference between the model control group and the normal control group; ^## P <0.01 represents a significant difference between the 3 treatment groups and the model control group; ^& P <0.05 represents a significant difference between the C3-G4-MSC treated group and the positive antibody treated group.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of antibodies

1.1 Screening of anti-IL-17A Single-Domain antibodies

Preparation of IL-17A protein: adding a 6xHis tag to the C end of the IL-17 protein (SEQ ID NO. 30), performing gene synthesis according to prokaryotic codon optimization, and subcloning the gene into a pET28a vector; after being verified by Sanger sequencing, the plasmid is extracted; transforming the recombinant plasmid into BL21 competent, inducing overnight with 0.5mM IPTG, and collecting bacterial liquid for cleavage; purifying the recombinant protein using a nickel column; purity of the target protein was checked by SDS-PAGE. The IL-17A antigen protein is refined and purified to have the purity of more than 90 percent.

SEQ ID NO:30：

MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA.

The inventor adopts the prepared IL-17A protein to immunize alpaca and yeast library to screen, and respectively obtains 1-C3 and 2-G4 single domain antibodies. In the detection of the blocking activity of the antibody, the single domain antibody can block the Human IL-17A protein from activating the downstream target protein, but the blocking effect is weaker than that of the positive antibody Ixekizumab. Thus, the inventors tandem two IL-17A single domain antibodies against different epitopes to form a bivalent antibody to enhance its blocking effect. Two anti-IL-17A single domain antibodies were 1-C3 and 2-G4.

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;

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; the amino acid sequence of the single domain antibody (2-G4) is SEQ ID NO.16.

SEQ ID NO. 1：GEDLGYYA；

SEQ ID NO. 2：VTSSGSST；

SEQ ID NO. 3：ASTILLCSDYISAFGT；

SEQ ID NO. 4：GEKLDYFA；

SEQ ID NO. 5：VTSSGSST；

SEQ ID NO. 6：ASTILLCSDYISAFGT；

SEQ ID NO. 7：DVQLVESGGGLVEPGESLRLSCAAP；

SEQ ID NO. 8：IAWFRQAPGKEREVVSC；

SEQ ID NO. 9：NYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAVYYC；

SEQ ID NO. 10：WGQGTQVTVAS；

SEQ ID NO. 11：QVQLVESGGGLVQPGGSLRLSCAAS；

SEQ ID NO. 12：IGWFRQAPGKEREVVSC；

SEQ ID NO. 13：NYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAIYYC；

SEQ ID NO. 14：WGQGTQVTVAS；

SEQ ID NO. 15：

DVQLVESGGGLVEPGESLRLSCAAPGEDLGYYAIAWFRQAPGKEREVVSCVTSSGSSTNYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAVYYCASTILLCSDYISAFGTWGQGTQVTVAS;

SEQ ID NO. 16：

QVQLVESGGGLVQPGGSLRLSCAASGEKLDYFAIGWFRQAPGKEREVVSCVTSSGSSTNYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAIYYCASTILLCSDYISAFGTWGQGTQVTVAS.

1.2 Preparation of bivalent Single-domain antibody (C3-G4)

The bivalent single domain antibody (1-C3-2-G4) of the present invention is simply referred to as a bivalent single domain antibody (C3-G4) or C3-G4.

The bivalent single domain antibody of this example was a first single domain antibody (1-C3) linked to a second single domain antibody (2-G4) via linker (GGGGSGGGGSGGGGS) (the amino acid sequence after linkage was SEQ ID NO.17; the corresponding nucleic acid sequence was SEQ ID NO. 18), followed by the hinge region (SEQ ID NO. 19) and CH region (SEQ ID NO. 20). 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.

1) The bivalent single domain antibody (C3-G4) sequence (SEQ ID NO. 22) was subjected to gene synthesis and subcloned in tandem with human IgG1Fc into the expression vector pcDNA3.4-hIgG1-Fc (IgG 1 constant region amino acid sequence SEQ ID NO. 26). After the vector is verified to be correct by sequencing, preparing endotoxin-removing plasmids for later use by using a Qiagen plasmid large-pump kit;

2) Taking out LVTransm transfection reagent and single-chain antibody expression vector from refrigerator, thawing at room temperature, and blowing with pipetting gun. The PBS buffer was removed and warmed to room temperature. Taking one hole of a2 mL PBS-6-hole plate, respectively adding 130 mug antibody expression vector, blowing up and down by a pipette, fully and uniformly mixing, adding 400 mu L LVTRANSM, immediately blowing up and down by the pipette, uniformly mixing, and standing for 10 minutes at room temperature.

3) The DNA/LVTransm complex was added to 30mL of 293F cells and mixed thoroughly with gentle shaking. After culturing the cells in a 5% CO ₂ incubator at 37℃for 6-8 hours in 130 rpm, 50mL fresh 293 cell medium was added and the cells were returned to the incubator for continued culture.

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, and the antibody was purified using a Protein A column, to finally obtain a bivalent single domain antibody (C3-G4).

SEQ ID NO. 17：

DVQLVESGGGLVEPGESLRLSCAAPGEDLGYYAIAWFRQAPGKEREVVSCVTSSGSSTNYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAVYYCASTILLCSDYISAFGTWGQGTQVTVASGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGEKLDYFAIGWFRQAPGKEREVVSCVTSSGSSTNYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAIYYCASTILLCSDYISAFGTWGQGTQVTVAS;

SEQ ID NO. 18：

gatgtgcagctggtggagtctgggggaggcttggtcgagcctggggaatctctgaggctctcctgtgcagcccctggagaggatttgggttattacgccatagcctggttccgccaggccccagggaaggagcgtgaggtagtctcatgtgtcacaagtagtggtagtagcacaaactatttaagttccgtgaaggaccgattcaccatctccatagacaacgccaagaacacggtatatctgcaaatgaacagcctgaaacctgaggacacagccgtttattactgtgcgtccactattctcctctgttcagattatatctctgcctttggcacctggggccaggggacccaggtcaccgtcgcctcgggaggcggaggatctggcggaggtggaagtggcggaggcggttctcaggtgcagctcgtggagtcggggggaggcttggtgcagcccgggggatctctgaggctctcgtgtgcagcctctggagagaaattggattattttgccataggctggttccgccaggccccagggaaggagcgtgaggtagtctcatgtgtcacaagtagtggtagtagcacaaactatttaagttccgtgaaggaccgattcaccatctccatagacaacgccaagaacacggtatatctgcaaatgaacagcctgaaacctgaggacacagccatttattactgtgcgtccactattctcctctgttcagattatatctctgcctttggcacctggggccaggggacccaggtcaccgtcgcctcg;

SEQ ID NO. 19：DKTHTCP；

SEQ ID NO. 20：

PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

SEQ ID NO. 21：

DVQLVESGGGLVEPGESLRLSCAAPGEDLGYYAIAWFRQAPGKEREVVSCVTSSGSSTNYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAVYYCASTILLCSDYISAFGTWGQGTQVTVASGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGEKLDYFAIGWFRQAPGKEREVVSCVTSSGSSTNYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAIYYCASTILLCSDYISAFGTWGQGTQVTVAS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

SEQ ID NO. 22：

gatgtgcagctggtggagtctgggggaggcttggtcgagcctggggaatctctgaggctctcctgtgcagcccctggagaggatttgggttattacgccatagcctggttccgccaggccccagggaaggagcgtgaggtagtctcatgtgtcacaagtagtggtagtagcacaaactatttaagttccgtgaaggaccgattcaccatctccatagacaacgccaagaacacggtatatctgcaaatgaacagcctgaaacctgaggacacagccgtttattactgtgcgtccactattctcctctgttcagattatatctctgcctttggcacctggggccaggggacccaggtcaccgtcgcctcgggaggcggaggatctggcggaggtggaagtggcggaggcggttctcaggtgcagctcgtggagtcggggggaggcttggtgcagcccgggggatctctgaggctctcgtgtgcagcctctggagagaaattggattattttgccataggctggttccgccaggccccagggaaggagcgtgaggtagtctcatgtgtcacaagtagtggtagtagcacaaactatttaagttccgtgaaggaccgattcaccatctccatagacaacgccaagaacacggtatatctgcaaatgaacagcctgaaacctgaggacacagccatttattactgtgcgtccactattctcctctgttcagattatatctctgcctttggcacctggggccaggggacccaggtcaccgtcgcctcggacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcacgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaataa;

SEQ ID NO.26：

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Example 2 affinity assay

2.1 Preparation of positive control antibody Ixekizumab

(A) Gene synthesis of Ixekizumab heavy and light chain variable regions (heavy chain variable region sequence SEQ ID No.23, light chain variable region sequence SEQ ID No. 24), subcloning the heavy chain variable region into pcdna3.4-hig 4 (IgG 4 constant region amino acid sequence SEQ ID No. 25) vector, and subcloning 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, the plasmid megapump kit is used for preparing the endotoxin-removing plasmid for standby.

(B) Taking the LVTransm transfection reagent and the heavy chain and light chain expression vector out of the refrigerator, thawing at room temperature, and blowing up and down by a pipetting gun to mix completely. The PBS buffer was removed and warmed to room temperature. Taking 2mL of PBS to one hole of a 6-hole plate, respectively adding 50 mug heavy chain and light chain expression vectors, fully and uniformly mixing the mixture up and down by a pipetting gun, adding 300 mug L LVTRANSM, immediately and uniformly mixing the mixture up and down by a pipetting device, and standing for 10 minutes at room temperature.

(C) The DNA/LVTransm complex was added to 100mL of 293F cells, gently swirled and thoroughly mixed, and the cells were placed in a 5% CO ₂ incubator at 37℃and incubated at 130 RPM.

(D) After continuous cultivation for 5-7 days, 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 and the antibody was purified using a Protein A column.

(E) SDS-PAGE detects purity of target antibody protein, purity >95%.

The positive control antibody is used for detecting the binding capacity of the recombinant antigen, and the result shows that the positive antibody and the IL-17A antigen protein are well combined and can be used for subsequent immunization.

The procedure for purifying antibodies by Protein A is as follows:

1) Samples containing the target antibodies were added to the EP tube and mixed by gently inverting the tube.

2) EP tubes were mixed at room temperature or incubated on a rotator, (1-4 hours or overnight) 100mM PMSF could be added to prevent protein degradation.

3) The magnetic beads were collected using a magnetic separation rack and the supernatant was discarded. The supernatant was retained for analysis, if necessary.

4) Add 1 mL binding/washing buffer to the EP tube and mix well, collect the beads using a magnetic rack and discard the supernatant, repeat the washing step three times.

5) To the EP tube, 500. Mu.L of elution buffer was added, and resuspended rapidly with pipetting or vortexing, and then incubated at room temperature (about 25 ℃) for 5 minutes either in a tumble mixer or by manually gently tumbling the EP tube.

6) Magnetic beads were collected using a magnetic separation rack and the supernatant containing the eluted antibodies was transferred to a clean EP tube.

7) Steps 1) and 2) were repeated twice.

8) To each 500. Mu.L of eluate, 1/10 of a neutralization buffer was added to neutralize the pH in order to maintain the biological activity of the antibody and avoid inactivation of the antibody. Buffer exchange can be performed by dialysis or desalting, if desired.

9) Binding/washing buffer: 1 XPBS, pH 7.0.

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

Neutralization buffer: 1M Tris, pH 8.5.

Magnetic bead regeneration buffer: 0.1 M NaOH.

SEQ ID NO.23：

QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSS;

SEQ ID NO.24：

DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIK;

SEQ ID NO.25：

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

SEQ ID NO. 27：

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

2.2 Detection of ELISA binding Activity of recombinant Human IL-17A protein and control antibody

(1) The IL-17A recombinant protein was diluted with sterile CBS to a final concentration of 2. Mu.g/mL. A new 96-well plate was taken and 100. Mu.L/well coated overnight at 4 ℃.

(2) The antigen coating was removed and washed 3 times with PBST (0.5% tween).

(3) Blocking was performed at 37℃for 2 hours with the addition of 200. Mu.L/well of 3% MPBS;

(4) After removal of the blocking buffer, the well plate was washed 3 times with PBST;

(5) The positive control antibody Ixekizumab is diluted to 10 mug/ml by PBS, 7 points are diluted 5 times, 100 mug/well is added into an ELISA plate, and the incubation is carried out for 1 hour at room temperature, and the control well is PBS;

(6) Remove the liquid in the wells and wash 3 times with PBST;

(7) Adding secondary antibody HRP-ProteinA (Boster, BA 1080) for 1:10000 dilution, adding into enzyme label plate according to 100 mu L/hole, and incubating for 1 hour at room temperature;

(8) After removing the liquid from the wells, the well plate was washed 3 times with PBST;

(9) Adding 100 mu L/hole TMB color development liquid;

(10) Incubating for 15 minutes at room temperature in a dark place;

(11) Add 50. Mu.L/Kong Zhongzhi of liquid (2M HCl);

(12) OD450 values within wells were read using a microplate reader.

The results are shown in Table 1: the positive antibody is well combined with IL-17A antigen protein, and can be used for immunization.

TABLE 1 detection of binding Activity of Human IL17A to Positive antibodies

2.3 Affinity detection methods and results

2.3.1 ELISA detection by HRP-STREPTAVDIN

Coating the purified single domain antibody 2ug/mL on a 96-well ELISA plate, adding Biotin-IL-17A-His, diluting 7 points with a 5-fold gradient with the initial concentration of 10ug/mL, and performing ELISA detection by using HRP-STREPTAVDIN. The detection result is EC 50=1.231 ug/mL of bivalent single-domain antibody (C3-G4), EC 50=10.06 ug/mL of positive antibody (Ixekizumab), which indicates that the bivalent single-domain antibody (C3-G4) can bind to the target protein, and the binding capacity is higher than that of the positive antibody (Ixekizumab) as shown in fig. 1.

2.3.2 Determination of antibody affinity by ForteBio OCTET R2 instrument

(1) Antibody affinity was determined using a ForteBio OCTET R2 instrument, and the HIS1K sensor (Octet cube ProA Biosensors) was immobilized at a concentration of 5ug/mL for IL 7A-His.

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

(3) Affinity detection: equilibrium 60 s, binding 180s, dissociation 180s, detection temperature 25 ℃.

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

From the results, kd=1.391× ^-9 M of bivalent single domain antibody (C3-G4), kd= 3.910 × ^-10 M of positive antibody (Ixekizumab) (fig. 2).

Example 3 blocking function experiment

A gradient diluted detection antibody (positive antibody: ixekizumab; to-be-detected antibody: bivalent single domain antibody (C3-G4) (prepared in example 1) was added to a 96-well plate, the antibody was diluted 10-fold in a 10-fold gradient, 50. Mu.L of the diluted gradient concentration antibody was added to the 96-well plate in the final concentration of 100μg/mL,10μg/mL,1μg/mL,0.1μg/mL,0.01μg/mL,0.001 μg/mL,0.0001 μg/mL,0.00001μg/mL,0.000001 μg/mL,0.0000001 μg/mL,0μg/mL, in this order, 2 multiplex wells per gradient, then 50. Mu.L of 0.4. Mu.g/mL of IL-17A protein (final concentration of 0.1. Mu.g/mL) was added to the corresponding well, and after mixing, the mixture was placed in a 37℃incubator and incubated for 1 hour, 293F-IL-17 RA-17 Rc-ACT 1-NF. Kappa.B-Luc (A3) cells cultured to the logarithmic phase were aspirated in the 96-well plate, and after 2X 10X ⁴ cells were inoculated for 18 hours, 20. Mu.L of a test reagent was added to each well, and the value of the luciferase activity in the wells was measured using a Tecan 1000 protease marker.

As a result, the positive control Ixekizumab had an IC50 of 2.235nM and the bivalent single domain antibody (C3-G4) had an IC50 of 1.192nM, both of which blocked the Human IL-17A protein from activating 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc (FIG. 3).

Example 4 stability experiment

By detecting fluorescence change through a micro-differential scanning fluorescence technique (nanoDSF), thermal denaturation and chemical denaturation of the protein can be detected under natural conditions, and the temperature (T _m) when 50% of the protein is in an unfolded state and the temperature (T _agg) when aggregation begins to occur can be accurately determined; the higher the heat denaturation T _m、T_onset value and T _agg indicates the more stable the antibody protein.

4.1 Experimental procedure

Taking 100 mu L of candidate antibody prepared in the earlier stage and Ixekizumab (the concentration of a sample is greater than 200 mu g/mL), centrifuging at 4 ℃ and 12000 Xg for 10min, sucking the sample by using a capillary tube, preparing two capillaries for each sample, taking the capillaries as parallel control, putting the capillaries into corresponding clamping grooves in sequence, ensuring that the capillaries are full of the sample, and carrying out detection analysis.

4.2 Results

The stability results of the bivalent single domain antibody (C3-G4) and the positive control Ixekizumab are shown in FIGS. 4-5, and the results show that the T _m1 of the bivalent single domain antibody (C3-G4) is 62.20 ℃, the T _m3 is 81.58 ℃, the T _onset is 47.54 ℃, and the T _agg is 81.73 ℃; the positive control Ixekizumab had T _m1 of 56.10 ℃, T _m2 of 79.84 ℃, T _onset of 47.50 ℃, and T _agg of 61.86 ℃. The results show that the stability of the bivalent single domain antibody (C3-G4) is obviously higher than that of the positive control Ixekizumab.

Example 5 preparation method of genetically modified mesenchymal Stem cells

5.1 Construction of lentiviral shuttle plasmid

Construction of a lentiviral shuttle plasmid of a C3-G4 Fc fusion protein two candidate antibody VHH sequences (1-C3 and 2-G4 amino acid sequences shown in SEQ ID NO: 15 and 16) were linked by (GGGGS) ₃ and then serially connected to an IgG4 Fc (IgG 4 Fc amino acid sequence shown in SEQ ID NO: 28 and nucleotide sequence shown in SEQ ID NO: 29) to construct a VHH- (GGGGS) ₃ -VHH-IgG4 Fc sequence downstream of the EF-1alpha promoter, thereby obtaining a C3-G4 Fc lentiviral shuttle plasmid.

SEQ ID NO.28：

APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

SEQ ID NO.29：

GCCCCCGAGTTTCTGGGAGGACCTAGCGTCTTTCTGTTCCCCCCCAAACCCAAGGACACACTGATGATCTCTAGGACCCCCGAGGTGACATGCGTCGTGGTGGACGTGAGCCAAGAGGACCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAAGTGCACAATGCCAAGACCAAACCTAGAGAAGAGCAGTTCAACAGCACCTATAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGACTGGCTGAACGGCAAGGAGTACAAGTGCAAAGTGAGCAACAAGGGCCTCCCCTCCTCCATCGAGAAAACCATCTCCAAGGCCAAGGGACAGCCTAGAGAGCCCCAAGTGTATACACTGCCCCCCAGCCAAGAGGAGATGACCAAGAACCAAGTGTCTCTGACATGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCTGTGGAGTGGGAGAGCAACGGCCAGCCCGAAAACAACTATAAGACCACCCCCCCCGTGCTGGACTCCGATGGCAGCTTCTTTCTGTACTCCAGACTGACCGTGGACAAAAGCAGATGGCAAGAGGGCAACGTGTTTAGCTGCTCCGTGATGCATGAGGCTCTGCACAACCACTATACCCAGAAGTCCCTCTCTCTGAGCCTCGGCAAGTGA.

5.2 Lentivirus preparation

(1) Before virus packaging, 24 h, shake flasks were prepared, 293F cell density was adjusted to 1.0X10: 10 ⁶/mL, 60 per flask mL, shake flasks were placed on a 5% CO ₂, 120 rpm shaker at 37℃for further use.

(2) Preparation of transfection reagent/DNA complexes: taking 7.5 mL of 293F culture medium to a 15 mL centrifuge tube, adding 40 mug of C3-G4 Fc lentiviral shuttle plasmid and 80 mug of auxiliary plasmid (purchased from addGene, product numbers: 12253 and 12259), adding 360 mug of transfection reagent after fully and uniformly mixing by up-and-down blowing of a pipette, immediately blowing and uniformly mixing up-and-down by using a 1mL pipette, and standing for 10min (not more than 15 min) at room temperature

(3) The transfection reagent/DNA complex is added dropwise to the cells prepared the day before, shake the shake flask while adding, and after thoroughly mixing, the shake flask is placed on a shaking table of 5% CO ₂, 120 rpm at 37 ℃ for culture. And (3) harvesting the supernatant after 72 hours, centrifuging the cell culture supernatant at 8000g at low temperature (2-8 ℃), filtering the supernatant by a 2.5 mu m pore size filter and a 0.45 mu m pore size filter respectively, treating the obtained concentrated virus suspension by nuclease to remove nucleic acid impurities, and then respectively carrying out ion exchange chromatography and gel filtration chromatography to obtain purified virus suspension, and packaging the purified virus suspension into virus separation tubes, wherein each tube is 100 mu L. And (5) labeling, namely writing information such as virus names, volumes, lots and the like on the label.

5.3 Lentiviral titer assay

The 293T cells were inoculated into 24-well plates, cultured overnight, then 20uL of lentiviral stock solution, 10-fold slow virus solution and 100-fold slow virus solution were added, the culture was continued for 24 hours, fresh medium was changed after 24 hours, the cells were harvested after 9 days of continuous culture, genomic DNA was extracted from the kit (purchased from Thermo, cat No. K0721), the copy number of the plasmid template was adjusted with dd H ₂ O, the standard curve range was 1X 10 ⁹-1×10³, primers were LTR and WPRE (synthesized by Souzhou Jin Weizhi Biotech Co., ltd.), and 2X PCR Mix (purchased from Applied Biosystems, cat No. A25742), primers, DNA and PCR water (purchased from Thermo, cat No. R0582) were added to the corresponding PCR reaction wells after mixing uniformly according to the PCR premix solution, and PCR reaction was performed. And the titer of the virus was calculated according to the following formula.

Lentiviral titer = cell number x copy number/viral volume (mL) x dilution.

The results of lentiviral titer measurements are shown in FIG. 6, and no matter whether lentivirus is primary or diluted 10-fold or 100-fold, lentiviral titers are between 2.2E+7-2.4E+7, and there is no significant difference between the three groups, so lentiviral titers are 2.3E+7TU/mL.

5.4 Preparation of genetically modified mesenchymal Stem cells and detection of infection efficiency

The prepared mesenchymal stem cells which are cultured and have the fusion degree of 70-80% (which are separated from neonatal umbilical cords by using an enzymolysis method) and contain the C3-G4 Fc lentivirus are added according to MOI=10, the P2 generation mesenchymal stem cells are obtained by means of generation amplification and purification, the mesenchymal stem cells are cultured at 37 ℃ and CO ₂, and after the cell density reaches 100%, the mesenchymal stem cells infected by the Fc lentivirus are passaged, so that the mesenchymal stem cells infected by the Fc lentivirus (IL-17 Nb-MSC or C3-G4-MSC) are successfully obtained, wherein the following examples are unified as C3-G4-MSC.

Detection was performed using a MOI kit (manufacturer: system Biosciences, cat# LV 961A-1) which can determine the transduction complex (MOI) of lentiviral particles by detecting the ratio of endogenous conserved structure (UCR 1) in transduced umbilical cord mesenchymal stem cells to specific WPRE elements in lentiviral vectors and comparing the ratios to a standard curve self contained in the kit.

Inoculating the obtained C3-G4-MSC and hUC-MSC into 24 pore plates according to 50000 cell/pore, culturing the culture solution of DMEM/F12 (containing 10% of fetal calf serum) for 24 hours, extracting cell sample DNA according to the operation of the specification, obtaining CT values of all samples through PCR, calculating according to a standard curve linear equation, obtaining MOI values of all samples, and as shown in figure 7, the hUC-MSC has a value of 0, and the MOI value of C3-G4-MSC is 9.90+/-0.68, which is close to the theoretical MOI value, and indicating that the C3-G4 is that the Fc slow virus successfully infects mesenchymal stem cells.

EXAMPLE 6 flow cytometry detection of genetically modified Stem cells

The cells obtained in example 5 were cultured in a T25 cell culture flask at 1X 10 ⁴/cm², after the cell density reached 80-90%, a transport inhibitor (purchased from BD, cat. No. 555029) was added, and after fixation, washing and staining of FITC-Protein A (purchased from BOSTER, cat. No. BA 1120), the mesenchymal stem cells were subjected to FITC channel signaling (see FIG. 8 for results), and found that the positive rate of C3-G4-MSC in FITC channel signaling exceeded 50%, while normal mesenchymal stem cells (hUC-MSC) that did not infect viruses had no signaling in FITC channel, indicating that the infection rate of C3-G4-MSC reached over 50%.

EXAMPLE 7 ELISA detection of expression of genetically modified mesenchymal Stem cells IgG4, IL-17Nb

The cells obtained in example 5 were cultured in accordance with C3-G4-MSC and hUC-MSC at 1X 10 ⁴ cells/cm ² in T25 cell culture flasks, the culture medium was DMEM/F12 (containing 10% fetal bovine serum), and after 72 hours of adherent culture, the cell supernatants were harvested and sub-packaged for frozen storage.

7.1ELISA method for detecting expression of genetically modified mesenchymal stem cell IgG4

Human IgG4 ELISA Kit (manufacturer: thermo, cat# BMS 2095) was used to detect the amount of secreted fusion protein IgG4 in the cell culture supernatant. The kit adopts a human IgG4 solid-phase sandwich ELISA (enzyme-linked immunosorbent assay) to detect the amount of the target bound between the matched antibody pair. IgG 4-specific antibodies have been pre-coated in an elisa plate, and then cell supernatant samples, standards or controls are added to these wells and bound to immobilized (capture) antibodies, forming a sandwich structure by adding secondary antibodies, and the added substrate solution is reacted with the enzyme-antibody-target complex to produce a measurable signal. The intensity of the signal is proportional to the target concentration present in the original sample.

The above cell supernatants (diluted 25-fold) were added to an IgG4 ELISA kit to detect the content of IgG4 protein in the fusion protein, and as a result, it was found that C3-G4-MSC highly expressed IgG4 up to 6843.33.+ -. 845.33ng/mL, whereas normal hUC-MSC did not express IgG4 (see FIG. 9).

7.2IL-17Nb antibody content detection

IL-17A nanobody expression was detected by IL-17A protein binding assay, and the cell supernatant was detected after the ELISA plate was coated with IL-17A protein (prepared in example 1) at a final concentration of 2. Mu.g/mL overnight at 4℃and BSA blocked. Standard was C3-G4 fusion Protein (prepared from example 1) at standard curve concentration ranging from 0-250ng/mL, standard was added to the corresponding wells, the sample wells were incubated for 1h with the above cell supernatants (diluted 20-fold), then incubated with HRP-labeled Protein a antibody (purchased from bordetella, cat No. BA 1080) as enzyme-labeled antibody for 1h, finally TMB was added and developed for 20min in the dark, and after termination the OD450nm value of each well was detected by an enzyme-labeled instrument. As shown in FIG. 10, the high expression of IL-17Nb by C3-G4-MSC can be determined by the IL-17A binding experiment, the concentration is 2212.33 +/-214.65 ng/mL, which shows that the IL-17Nb expressed by C3-G4-MSC can bind to IL-17A, while the IL-17Nb is not expressed by normal mesenchymal stem cells (hUC-MSC).

Example 8 ELISA method for detecting the ability of genetically modified mesenchymal Stem cells to block IL-17A binding to IL-17RA

IL-17A [ biotinylated ]: IL-17RA Inhibitor Screening ELISA Kit (cat. EP-139) kit from ACRO Biosystems was used to test the ability of C3-G4-MSC to block IL-17A binding to IL-17 RA. The kit coats IL-17RA, takes a neutralizing antibody of anti-IL-17A as a standard substance, blocks the combination of the IL-17RA and biotinylated IL-17A, judges the blocking capacity by detecting the OD450nm value, and has the inverse relation between the blocking capacity and the OD450nm value, wherein the stronger the blocking capacity of the IL-17A/IL17RA is, the lower the OD450nm value is. The cell supernatants of example 7 were tested for the ability of IL-17Nb to block IL-17A/IL17RA binding by an IL-17A/IL17RA blocking kit.

IL-17A/IL17RA binding inhibition was calculated using the following formula:

binding inhibition (%) = [ OD450 (Positive well) -OD450 (sample well) ]/OD450 (Positive well) ×100% was measured

After 4-fold dilution of the cell supernatant described in example 7 was performed, the results were examined using the ACRO kit described above, as shown in FIG. 11, in which normal mesenchymal stem cells (hUC-MSC) were unable to block the binding of IL-17A to IL17RA, whereas IL-17Nb secreted by C3-G4-MSC cells was able to block the binding of IL-17A to IL17RA, and the inhibition rate was as high as 51% after 4-fold dilution.

EXAMPLE 9 Stem cell stability study

According to the results obtained in 7.2 of example 7 (ELISA method for detecting IL17Nb expression), C3-G4-MSC and hUC-MSC obtained in example 5 (C3-G4 gene modified stem cells were inoculated in 24-well plates at 1X 10 ⁴/cm², 8-well and 16-well plates were inoculated, respectively, after overnight culture, 8-well hUC-MSC was randomly selected to replace the complete medium containing C3-G4 nanobody (C3-G4 nanobody, bivalent single domain antibody (C3-G4) prepared in example 1) at a final concentration of 1000ng/mL, culture was continued, supernatants were harvested at 24h, 48h, 72h and 96h, respectively, and IL17Nb content in the supernatants was detected according to the method of 7.2 (ELISA method for detecting IL17Nb expression) in example 7. As a result, as shown in FIG. 12, the IL17Nb content measured from 24h to 96h, the hUC-MSC+C3-G4 group was reduced from 993+ -43 ng/mL to 709+ -39 ng/mL, the concentration was gradually decreased, and the C3-G4-MSC was able to stably express IL17Nb, and the concentration of IL17Nb was gradually increased from 715+ -37 ng/mL of 24h to 3193+ -117 ng/mL of 96h over the course of the culture period, wherein there was a significant difference (P < 0.01) between the 24h and 48h, the hUC-MSC+C3-G4 group and the C3-G4-MSC group, and there was a very significant difference (P < 0.001) between the 72h and 96h, the hUC-MSC+C3-G4 and the C3-G4-MSC group. From the results, it can be seen that the C3-G4-MSC can stably and continuously express and secrete IL-17Nb, and therefore, the stability of the expression of IL17Nb by the C3-G4-MSC is superior to that of recombinant C3-G4 protein.

Example 10 construction method and construction results of animal model of psoriasis

A B-hIL17A transgenic mouse is adopted to construct a psoriasis (Ps) model by an Imiquimod (IMQ) smearing method. After shaving the backs of all mice, 50mg imiquimod ointment (Ming Xin Li Di, sichuan Ming Xin pharmaceutical Co., ltd.) is applied to the backs of all mice every day, and the first time when the diary is D0, the molding is carried out for 7 days (D6); the Ps model group was randomly divided into four groups: hUC-MSC treated group (2X 10 ⁶ /), positive antibody Ixekizumab treated group (1 mg/Kg), C3-G4-MSC treated group (2X 10 ⁶/only), and model control group. Drug treatment groups were treated with D1 and D4 subcutaneous drug administration. In the experimental process, animal weight is measured every day, animal survival condition and health condition are observed, and clinical scores are carried out on skin inflammation and related indexes according to skin keratinization degree and inflammatory cell infiltration degree. D7 euthanized animals, and related assays were performed: the skin of the molded part of the mice was collected and the skin thickness of each group of animals was measured using a vernier caliper.

Skin scoring criteria: scoring animal skin (ear, front and rear paws), comprehensively scoring according to erythema, scales and thickness, wherein each index score is divided into 5 grades and 0-5 grades, wherein 0 represents no related symptoms; score 1 represents mild symptoms; score 2 indicates symptoms are general; score 3 indicates significant symptoms; score 4 indicates very significant or severe and a total score of 3 indicators was calculated as the final score.

As shown in fig. 13, the body weight of the model group after imiquimod modeling is significantly reduced compared with that of the normal control group, the mice body weight can be recovered by both the hUC-MSC treatment and the C3-G4-MSC treatment groups, and the recovery of the body weight of the mice by the positive antibody group is not obvious; the body weight of the mice in the C3-G4-MSC treatment group at the test end point is significantly higher than that of the mice in the positive antibody group; skin photographs are shown in fig. 14, and clinical scores of skin according to skin keratinization degree and skin scaling lesion are shown in fig. 15, IMQ can cause skin injury, rash and scaling degree increase of mice, skin epidermis is thickened, and dermis mainly consisting of keratinization and inflammatory leukocyte infiltration is histopathologically seen; the clinical scores of the skin of the model group are obviously increased, and the clinical scores of the skin of the mice are obviously reduced after the hUC-MSC, the positive antibody Ixekizumab and the C3-G4-MSC are injected subcutaneously, wherein the treatment effect of the C3-G4-MSC is obviously better than that of the positive antibody Ixekizumab. The experimental end point was tested for skin thickness, as shown in fig. 16, and IMQ was found to significantly increase the skin thickness of model mice, while after subcutaneous injection of the hic-MSC, positive antibody Ixekizumab and C3-G4-MSC, the skin thickness of mice was significantly reduced, wherein the C3-G4-MSC treatment effect was significantly better than positive antibody Ixekizumab.

The C3-G4-MSC can be effectively used for treating rheumatoid arthritis, and can restore the weight of a mouse after being treated by the C3-G4-MSC, reduce the thickness of the paw of the mouse with rheumatoid arthritis, and obviously reduce the symptoms of inflammatory cell infiltration, joint synovitis and/or pannus formation, joint cartilage destruction, joint cavity disappearance, bone tissue fusion and the like of the mouse with rheumatoid arthritis. The above therapeutic effect is significantly better than that of the positive antibody Ixekizumab.

The C3-G4-MSC can also be used for treating psoriasis and arthritis, and the therapeutic department of the C3-G4-MSC can recover the weight of a rheumatoid arthritis mouse, remarkably reduce the skin thickness, the keratinization degree and the inflammatory cell infiltration degree, and remarkably reduce cytokines such as IL-6, IL-23, TNF-alpha and the like in serum. The above therapeutic effect is significantly better than that of the positive antibody Ixekizumab.

Claims

1. A modified umbilical cord mesenchymal stem cell, characterized in that the umbilical cord mesenchymal stem cell expresses and/or secretes the following (1) and (2):

(1) A first antibody comprising HCDR1, HCDR2 and HCDR3; the amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are SEQ ID NO.1-3;

(2) A second antibody comprising HCDR4, HCDR5 and HCDR6; the amino acid sequences of the HCDR4, the HCDR5 and the HCDR6 are SEQ ID NO.4-6;

The first antibody and the second antibody are connected through a linker with an amino acid sequence of (GGGGS) ₃;

both the first antibody and the second antibody comprise a single domain antibody that specifically recognizes IL-17A.

2. The umbilical cord mesenchymal stem cell of claim 1, wherein the structure of the first antibody comprises: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4;

The structure of the second antibody comprises: FR5-HCDR4-FR6-HCDR5-FR7-HCDR6-FR8;

The FR1-8 is the amino acid sequence shown in SEQ ID NO. 7-14.

3. The umbilical cord mesenchymal stem cells of claim 1, wherein the umbilical cord mesenchymal stem cells express and/or secrete a fusion protein;

the fusion protein is an amino acid sequence shown as SEQ ID NO.15- (GGGGS) ₃ -SEQ ID NO.16-SEQ ID NO. 28.

4. The umbilical cord mesenchymal stem cells of any one of claims 1-3, wherein the umbilical cord mesenchymal stem cells secrete anti-interleukin antibodies.

5. The umbilical cord mesenchymal stem cells of claim 4, wherein the umbilical cord mesenchymal stem cells secrete anti-interleukin 17 antibodies.

6. The umbilical cord mesenchymal stem cells of claim 5, wherein the umbilical cord mesenchymal stem cells secrete anti-IL-17A antibodies.

7. An umbilical cord mesenchymal stem cell, characterized in that the umbilical cord mesenchymal stem cell comprises the following (1) and (2):

(1) A first nucleotide sequence, wherein the first antibody coded by the first nucleotide sequence comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are shown as SEQ ID NO. 1-3;

(2) A second nucleotide sequence encoding a second antibody comprising HCDR4, HCDR5 and HCDR6; the amino acid sequences of the HCDR4, the HCDR5 and the HCDR6 are shown in SEQ ID NO. 4-6;

The first nucleotide sequence and the second nucleotide sequence are connected by a nucleotide sequence of an amino acid sequence shown as a code (GGGGS) ₃;

8. The umbilical cord mesenchymal stem cell of claim 7, wherein the first nucleotide sequence encodes an amino acid sequence as set forth in SEQ ID No. 15; the second nucleotide sequence codes for an amino acid sequence shown as SEQ ID NO. 16.

9. The umbilical cord mesenchymal stem cell of claim 8, comprising a third nucleotide sequence encoding the amino acid sequence set forth in SEQ ID No.15- (GGGGS) ₃ -SEQ ID No.16-SEQ ID No. 28.

10. A pharmaceutical composition comprising the umbilical mesenchymal stem cell of claim 9.

11. A method for preparing umbilical cord mesenchymal stem cells according to any one of claims 7 to 9, comprising: the nucleotide sequence is introduced into umbilical cord mesenchymal stem cells through virus transfection, liposome transfection, electrotransfer, gene editing or mRNA transfection.

12. A method for in vitro detection of IL-17A in a sample for non-diagnostic purposes, characterized in that the method comprises the steps of:

S1: contacting the umbilical cord mesenchymal stem cells of any one of claims 1-9 with a sample to be tested;

s2: detecting the antigen-antibody complex;

S3: and judging the result.