CN118001428A - Application of genetically modified stem cells in IL-17 target related diseases - Google Patents
Application of genetically modified stem cells in IL-17 target related diseases Download PDFInfo
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention belongs to the technical field of biological medicine, and particularly relates to application of a genetically modified stem cell (IL 17 Nb-MSC) in IL-17 target related diseases. The genetically modified stem cell can secrete a bivalent single-domain antibody which can effectively combine with IL-17A, namely an IL-17A nanobody (IL 17 Nb), and the antibody comprises an amino acid sequence shown in SEQ ID NO.1-6, so that the combination of IL-17A and a receptor thereof can be effectively blocked. The genetically modified stem cell can effectively treat diseases such as rheumatoid arthritis, psoriasis and/or psoriatic arthritis caused by IL-17A, and the treatment effect is obviously better than that of a positive antibody Ixekizumab. Provides technical support for clinical treatment of diseases such as rheumatoid arthritis, psoriasis and/or psoriatic arthritis.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to application of genetically modified stem cells in IL-17 target related diseases.
Background
IL-17A is an inflammatory cytokine mainly produced 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 infectious diseases, immune diseases, neoplastic diseases and the like of organisms. 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.
Stem cell therapy refers to a process of using autologous or allogeneic stem cells, implanting them into a human body after in vitro manipulation, and treating diseases, i.e., regenerating new, normal or younger cells, tissues or organs in vitro through stem cell isolation, culture, directed induction and even genetic modification, and treating clinical diseases through the same. A large number of clinical studies show that some types of stem cells have obvious curative effects on a plurality of refractory diseases. Such as autoimmune diseases, degenerative diseases, metabolic diseases, tissue defect diseases, traumatic diseases, inflammatory diseases, radioactive diseases, toxic diseases, ischemic diseases, tumors, insomnia, sub-health states, aging resistance, beauty treatment, etc., and relates to various large systems of human body such as nerves, circulation, respiration, digestion, endocrine, urinary, reproduction, exercise, etc.
Most of the research on antibody secretion in the existing cells is focused on antibodies with general molecular weight or large molecular weight, and few researches on cell secretion of small molecular weight antibodies, particularly nano-scale antibodies, are reported. Patent CN 112941028A discloses a nano-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.
The combination of antibodies and immune cells is a hotspot of the existing research, but immune cells do not have the effects of improving and repairing, and become a defect of the existing immunotherapy. Therefore, the invention envisages that the nanometer antibody with better performance is combined with the mesenchymal stem cells with targeting and repairing activities, so that the antibody is secreted in a concentrated way at a disease part at high density, the small molecular weight of the nanometer antibody improves the concentration and the effective amount of the antibody at the disease part, the repairing activity of the mesenchymal stem cells is carried out at the same time, the disease part is repaired, the microenvironment is improved, the killing effect of immune cells is improved, the disease cure rate is improved by the interaction of all factors, and a new way is provided for the treatment of the disease.
There is a need in the art for intensive research and development of the use of modified stem cells.
The present invention was made in the above background, and the present invention was developed based on the fourth generation technology of the anti-IL-17A single domain antibody development project.
For the convenience of examination, the technical background of the research project will be briefly described:
The inventors first developed 9 single domain antibodies (filed another application);
the present inventors developed an antibody combination composed of 2 single domain antibodies on the basis of single domain antibodies (filed another application);
The inventors developed stem cells modified by antibody combinatorial genes on the basis of single domain antibodies (filed another application);
The inventor develops an application technology of the modified stem cells on the basis of the genetically modified stem cells, and the application is one of the patent applications of the application technology.
The patent applications are each filed based on the relevant regulations of the uniqueness of the patent laws.
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 application of genetically modified stem cells in IL-17 target related diseases, wherein the stem cells can secrete bivalent single domain antibodies which can effectively bind IL-17A, and the bivalent single domain antibodies can be used for treating IL-17A mediated diseases such as rheumatoid arthritis, psoriasis and/or psoriatic arthritis.
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) a polar positively charged residue: 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 invention provides an application of stem cells in preparing a medicament for preventing and/or treating diseases.
Specifically, the stem cells comprise the following (1) and/or (2):
(1) Amino acid sequences shown in SEQ ID NO. 1-6;
(2) Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1-6.
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.
In particular, the stem cells may be embryonic stem cells, adult stem cells, mesenchymal stem cells, umbilical cord blood stem cells, hematopoietic stem cells, neural stem cells, adipose stem cells, skin stem cells and/or muscle stem cells.
Preferably, the stem cells are mesenchymal stem cells.
Further preferably, the mesenchymal stem cells may be isolated from umbilical cord blood, umbilical cord, placenta, adipose tissue, skin, neural tissue, bone marrow or embryo.
Still further preferably, the mesenchymal stem cells may be isolated from umbilical cord blood or umbilical cord.
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.
Specifically, the medicine 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 the use of a stem cell in the manufacture of a medicament for the prevention and/or treatment of a disease. Specifically, the stem cells comprise the following (1) and/or (2):
(1) Amino acid sequences shown in SEQ ID NO. 1-14;
(2) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1-14;
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.
In particular, the stem cells may be embryonic stem cells, adult stem cells, mesenchymal stem cells, umbilical cord blood stem cells, hematopoietic stem cells, neural stem cells, adipose stem cells, skin stem cells and/or muscle stem cells.
Preferably, the stem cells are mesenchymal stem cells.
Further preferably, the mesenchymal stem cells may be isolated from umbilical cord blood, umbilical cord, placenta, adipose tissue, skin, neural tissue, bone marrow or embryo.
Still further preferably, the mesenchymal stem cells may be isolated from umbilical cord blood or umbilical cord.
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.
Specifically, the medicine 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 the use of a stem cell in the manufacture of a medicament for the prevention and/or treatment of a disease.
Specifically, the stem cells comprise the following (1) and/or (2):
(1) Amino acid sequences shown in SEQ ID NO. 15-16;
(2) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 15-16;
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.
In particular, the stem cells may be embryonic stem cells, adult stem cells, mesenchymal stem cells, umbilical cord blood stem cells, hematopoietic stem cells, neural stem cells, adipose stem cells, skin stem cells and/or muscle stem cells.
Preferably, the stem cells are mesenchymal stem cells.
Further preferably, the mesenchymal stem cells may be isolated from umbilical cord blood, umbilical cord, placenta, adipose tissue, skin, neural tissue, bone marrow or embryo.
Still further preferably, the mesenchymal stem cells may be isolated from umbilical cord blood or umbilical cord.
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.
Specifically, the medicine 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 the use of a stem cell in the manufacture of a medicament for the prevention and/or treatment of a disease.
Specifically, the stem cells comprise the following (1) and/or (2):
(1) An amino acid sequence shown in SEQ ID NO. 17;
(2) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 17;
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.
In particular, the stem cells may be embryonic stem cells, adult stem cells, mesenchymal stem cells, umbilical cord blood stem cells, hematopoietic stem cells, neural stem cells, adipose stem cells, skin stem cells and/or muscle stem cells.
Preferably, the stem cells are mesenchymal stem cells.
Further preferably, the mesenchymal stem cells may be isolated from umbilical cord blood, umbilical cord, placenta, adipose tissue, skin, neural tissue, bone marrow or embryo.
Still further preferably, the mesenchymal stem cells may be isolated from umbilical cord blood or umbilical cord.
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.
Specifically, the medicine 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 the use of a stem cell in the manufacture 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 stem cells comprise, express and/or secrete antibodies with the amino acid sequences shown in SEQ ID NO. 21.
In particular, the antibodies also comprise a biologically active protein or functional fragment thereof that aids in its expression and/or secretion, or that extends its half-life in vivo.
Further specifically, the biologically active protein or functional fragment thereof is selected from the group consisting of an immunoglobulin Fc domain, an elastin-like polypeptide, serum albumin, an albumin binding polypeptide, prealbumin, a carboxy terminal peptide, a His tag, a FLAG tag, a c-Myc tag, an HA tag, a GST tag, an MBP tag, and/or a SUMO tag.
Preferably, the biologically active protein or functional fragment thereof may be a human immunoglobulin Fc domain, preferably an Fc domain of human IgG, such as an Fc domain of human IgG1, igG2, igG3, igG 4.
Further preferably, the biologically active protein or functional fragment thereof is an Fc domain of IgG 1.
In particular, the stem cells may be embryonic stem cells, adult stem cells, mesenchymal stem cells, umbilical cord blood stem cells, hematopoietic stem cells, neural stem cells, adipose stem cells, skin stem cells and/or muscle stem cells.
Preferably, the stem cells are mesenchymal stem cells.
Further preferably, the mesenchymal stem cells may be isolated from umbilical cord blood, umbilical cord, placenta, adipose tissue, skin, neural tissue, bone marrow or embryo.
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.
Specifically, the medicine 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 method of treating a disease using stem cells, the method comprising administering to a subject in need thereof a therapeutically effective amount of the stem cells.
Specifically, the stem cells are mesenchymal stem cells;
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.
Further specifically, the stem cells secrete or express a fusion antibody;
Still more particularly, the fusion antibody comprises a first antibody and a second antibody;
The amino acid sequence of the first antibody is SEQ ID NO.15;
The amino acid sequence of the second antibody is SEQ ID NO.16.
The invention has the technical effects that:
(1) The C3-G4-MSC can restore the weight of a mouse suffering from rheumatoid arthritis, effectively reduce the paw thickness of the mouse suffering from the rheumatoid arthritis, obviously reduce the pathological tissue score, and has the treatment effect obviously superior to that of a positive antibody Ixekizumab.
(2) The C3-G4-MSC can restore the weight of a mouse suffering from psoriasis, obviously reduce the clinical score and the thickness of the skin of the mouse, and has the effect obviously superior to that of a positive antibody Ixekizumab.
(3) The C3-G4-MSC of the invention can restore the weight of mice suffering from psoriatic arthritis, remarkably reduce clinical scores of the paw and skin of a model, and remarkably reduce serum mIL-6, mTNF-alpha and mIL-23 levels of the mice.
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 <0.001 represents that the model control group has a significant difference 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 graph showing the trend of the thickness change of the paw 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 <0.01 represents a significant difference between the C3-G4-MSC treated group and the positive antibody treated group.
FIG. 15 is a graph showing pathological results of joint tissue of mice in example 10.
FIG. 16 is a clinical score of joint histopathology in mice in example 10, wherein # P <0.05 represents a significant difference between the hUC-MSC treated group and the model control group; ## P <0.01 represents a significant difference between the C3-G4-MSC treated group 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. 17 is a graph of the trend of the body weight change of the mice in example 11, 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. 18 is a photograph of the skin of the mouse in example 11.
Fig. 19 is a plot of the clinical scores of the mice skin in example 11, 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. 20 is a graph showing the results of the skin thickness test of the mice in example 11, 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.
Fig. 21 is a graph of the trend of the body weight change of the mice in example 12, wherein P <0.01 represents a significant difference between the model control group and the normal control group; # P <0.05 represents a significant difference between the C3-G4-MSC treated group 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.
Fig. 22 is a plot of the clinical scores of the mice paw in example 12, 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. 23 is a plot of the clinical scores of the mice skin in example 12, wherein P <0.0001 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.
Fig. 24 is a graph of TNF- α levels in the serum of mice in example 12, wherein P <0.01 represents a significant difference between the model control group and the normal control group; # P <0.05 represents a significant difference between the hUC-MSC and C3-G4-MSC treated groups, respectively, and the model control group.
Fig. 25 is a graph of IL-6 levels in mouse serum in example 12, wherein P <0.01 represents a significant difference between the model control group and the normal control group; # P <0.05 represents a significant difference between the hUC-MSC and C3-G4-MSC treated groups, respectively, and the model control group.
Fig. 26 is the IL-23 level in the serum of mice in example 12, wherein P <0.05 represents a significant difference between the model control group and the normal control group; # P <0.05 represents a significant difference between the positive antibody treated group and the C3-G4-MSC treated group, respectively, and the model control 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.
The main instrument of the invention:
an electrotransport device (Eppendorf Multiporator);
A centrifuge (Thermo FRESCO-17);
constant temperature incubator (Shanghai extract macro DNP-9052);
Constant temperature shake incubator (refined Qi CO-O6U);
Ultra clean bench (Sujing Antai SW-CJ-1 FD);
PCR instrument (Applied Biosystems ABI 2720);
biosafety cabinet (halr, HR40-IIA 2);
a flow cytometer (Thermo Attune Nxt flow cytometer);
thermo 3111CO2 incubator;
ForteBio OCTET R2。
The main reagent of the invention:
SfiI(NEB,CAT#:R0123L);
T4 DNA ligase(TaKaRa,CAT#:2011A);
PrimeScriptTMII 1st Strand cDNA Synthesis Kit(TaKaRa,CAT#:6210B);
NuHi power mix (Xinhai organism, cat#: NH 9303);
3M sodium acetate (pH 5.2-6) (Sigma, CAT#: 126-96-5);
DNA fragment recovery kit (TakaRa, CAT#: 9761);
glue recovery kit (Qiagen, CAT#: 28706);
the Tiangen plasmid large drawing kit (Tiangen, CAT#: DP 117);
HRP-Anti-M13(iCarTab);
PE-anti-Human IgG(eBioscience,Cat#:12-4998-82);
Rabbit anti-Llama IgG(H+L)Secondary Antibody[HRP](Novus,CAT#NBP1-75095);
SS320 competence (iCarTab);
pComF phage display vector (iCarTab);
NHS-biotin(APExBIO,CAT#:A8002);
HRP-Streptavidin(Boster,CAT#:BA1088);
HRP-ProteinA(Boster,BA1080);
ProA Biosensors(Sartorius,CAT#:18-5010);
PBS(Gbico,CAT#14190-250);
DMEM(Gbico,CAT#41965-062);
RPMI1640(Gbico,CAT#61870044);
FBS(VivaCell,CAT#C04001-500);
Genomic DNA Purification Kit(Lifetech,CAT#K0512);
Mouse-IL-17A-His(ACRO,CT8-M5240)。
the bivalent single domain antibody in the invention is the serial single domain antibody.
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:
MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRTVMVNLNI HNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINAD GNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA.
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 have tandem two anti-IL-17A single domain antibodies to make up 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:
DVQLVESGGGLVEPGESLRLSCAAPGEDLGYYAIAWFRQAPGKEREVVS CVTSSGSSTNYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAVYYCASTILLC SDYISAFGTWGQGTQVTVAS;
SEQ ID NO.16:
QVQLVESGGGLVQPGGSLRLSCAASGEKLDYFAIGWFRQAPGKEREVVS CVTSSGSSTNYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAIYYCASTILLC SDYISAFGTWGQGTQVTVAS.
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 2mL of PBS to one hole of a 6-hole plate, respectively adding 130 mug antibody expression vector, blowing up and down by a pipette, fully and uniformly mixing, adding 400 mug 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 the cells were placed in a 37℃5% CO 2 incubator at 130rpm for 6-8 hours, 50mL of 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:
DVQLVESGGGLVEPGESLRLSCAAPGEDLGYYAIAWFRQAPGKEREVVSCVTSSGSSTNYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAVYYCASTILLCSDYISAFGTWGQGTQVTVASGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGEKLDYFAIGWFRQAPGKEREVVSCVTSSGSSTNYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAIYYCASTILLCSDYISAFGTWGQGTQVTVASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;
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) and 100mM PMSF was 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) To the EP tube, 1mL of binding/washing buffer was added and thoroughly mixed, the beads were collected using a magnetic rack and the supernatant was discarded, and the washing step was repeated 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: 0.1M NaOH.
SEQ ID NO.23:
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEW MGVINPMYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARY DYFTGTGVYWGQGTLVTVSS;
SEQ ID NO.24:
DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQ LLIYKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFG QGTKLEIK;
SEQ ID NO.25:
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;
SEQ ID NO.27:
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC.
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
2.3.1HRP-STREPTAVDIN ELISA detection
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.
2.3.2ForteBio OCTET R2 Instrument for determining antibody affinity
(1) Antibody affinity determination using ForteBio OCTET R2 instrument, HIS1K sensor ProA Biosensors) solidified IL7A-His at a concentration of 5ug/mL.
(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 is carried out for 60s, binding for 180s, dissociation for 180s, and detection temperature is 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
Adding gradient diluted detection antibody (positive antibody: ixekizumab; to-be-detected antibody: bivalent single domain antibody (C3-G4), diluting the antibody according to 10 times of gradient, continuously diluting 10 gradients, adding 50 mu L of diluted gradient concentration antibody into 96-well plates with final concentration of 100μg/mL,10μg/mL,1μg/mL,0.1μg/mL,0.01μg/mL,0.001μg/mL,0.0001μg/mL,0.00001μg/mL,0.000001μg/mL,0.0000001μg/mL,0μg/mL, in sequence, adding 2 multiple wells of each gradient, adding 50 mu L of IL-17A protein (final concentration of 0.1 mu G/mL) into corresponding wells, uniformly mixing, placing in a 37 ℃ incubator, incubating for 1 hour, absorbing 293F-IL-17RA-IL-17Rc-ACT1-NF kappa B-Luc (A3) cells cultured to logarithmic phase into the 96-well plates, inoculating 2X 10 4 cells into each well, co-culturing for 18 hours, adding 20 mu L of Bright-GloTM detection reagent into each well, and detecting luciferase activity value in the wells by using a Tecan M1000pro enzyme 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、Tonset 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) 24H before virus packaging, shake flasks were prepared, 293F cell density was adjusted to 1.0X10 6/mL, 60mL per flask, and shake flasks were placed on a shaker at 37℃with 5% CO 2, 120rpm for further use.
(2) Preparation of transfection reagent/DNA complexes: taking 7.5mL of 293F culture medium to a 15mL 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), blowing up and down by a pipette to mix well, adding 360 mug of transfection reagent, immediately blowing up and down by a 1mL pipette to mix well, and standing for 10min (not more than 15 min) at room temperature
(3) The transfection reagent/DNA complex was added dropwise to the cells prepared the day before, shake the shake flask while adding, and after thoroughly mixing, the shake flask was placed on a shaker at 37℃with 5% CO 2, 120rpm for cultivation. The supernatant was harvested 72h later and 8000g of low temperature (2-8deg.C) centrifuged cell culture supernatant was filtered through 2.5 μm and 0.45 μm pore filters, respectively, the resulting concentrated viral suspension was nuclease treated to remove nucleic acid impurities, and then purified viral suspensions were obtained by ion exchange chromatography and gel filtration chromatography, respectively, and packaged into viral packaging tubes, each 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 (separated from neonatal umbilical cords by using an enzymolysis method and subjected to generation amplification and purification to obtain P2 generation mesenchymal stem cells) containing C3-G4 Fc lentivirus are added according to MOI=10, the P2 generation mesenchymal stem cells are cultured at 37 ℃ and CO 2, and after the cell density reaches 100%, the mesenchymal stem cells are passaged, and the mesenchymal stem cells infected by the Fc lentivirus, namely the mesenchymal stem cells infected by IL-17Nb (IL 17Nb-MSC or C3-G4-MSC for short) 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 a 24-well plate according to 50000 cells/well, culturing the culture solution of DMEM/F12 (containing 10% of fetal calf serum) for 24 hours, extracting sample DNA according to the operation of the specification, obtaining CT values of all samples through PCR, and calculating according to a standard curve linear equation to obtain MOI values of all samples, wherein the result is shown in figure 7, the value of hUC-MSC is 0, the MOI value of C3-G4-MSC is 9.90+/-0.68, and the value is close to the theoretical MOI value, so 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 7ELISA method for detecting 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
The cell culture supernatant was assayed for the content of the fusion protein IgG4 using the human IgG4 ELISA Kit (manufacturer: thermo, cat# BMS 2095). 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 2ug/mL overnight at 4℃and BSA blocked. Standard is C3-G4 fusion Protein (synthesized by Aikangde), standard curve concentration range is 0-250ng/mL, standard is added into corresponding hole, sample hole is added with the above C3-G4-MSC and hUC-MSC cell supernatant (diluted 20 times) for incubation for 1h, HRP marked Protein A antibody (purchased from doctor, product number BA 1080) is used as enzyme-labeled antibody for incubation for 1h, TMB is added for 20min in dark color, and after termination, the enzyme-labeled instrument detects OD450nm value of each hole. 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 8ELISA method for detecting the ability of genetically modified mesenchymal Stem cells to block IL-17A binding to IL-17RA
IL-17A from ACRO Biosystems [ biotinylated ] was used: the IL-17RA Inhibitor Screening ELISA Kit (cat. EP-139) kit detects the ability of genetically modified mesenchymal stem cells to block IL-17A/IL-17RA binding. 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 between IL-17A and 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 animal model construction of rheumatoid arthritis and evaluation results of tested animals
A B-hIL17A transgenic mouse (Baioesai medicine technologies Co., ltd.) was used for constructing a Rheumatoid Arthritis (RA) model by a collagen induction method. The animals of the RA model group were initially immunized on the initial Day (Day 1) by intradermal injection of tail root with 100. Mu.g of bovine type II collagen (CII, chondrex company) and an emulsifier containing 200. Mu.g of Freund's complete adjuvant (CFA, chondrex company) with Mycobacterium tuberculosis H37Ra, and boosted with an emulsifier of CII and Freund's incomplete adjuvant (IFA) on Day21 after the initial immunization. Molded RA mice (molding criteria: clinical score. Gtoreq.2) were randomly divided into 4 groups 22 days after primary immunization (Day 22): a hUC-MSC treated group, a positive antibody treated group, a C3-G4-MSC treated group and a model control group; and on the day of grouping, hUC-MSC (2 x10 6/dose), positive antibody Ixekizumab (1 mg/Kg) and C3-G4-MSC (2 x10 6/dose) were injected tail vein, model group and normal control were injected with physiological saline, respectively. Body weight, paw thickness were assessed every other Day for 23 days from Day 20 (Day 20) to the end of the experiment (Day 42). All animals were euthanized at the end of the experiment (Day 42) and joint tissue was taken for histopathological examination and clinical scoring.
Clinical score (Clinical score) criteria are shown in table 2:
TABLE 2
Score of | Disease conditions |
0 | Normal state |
1 | Mild redness, swelling of ankle and wrist |
2 | Moderate redness and swelling of ankle or wrist |
3 | The paws are severely reddish and swollen, including the finger tips |
4 | Maximum inflammation of limbs, including multiple joints |
The criteria for the pathology of arthritis tissue were as follows:
representative images of three groups of H & E stained limbs were each independently scored by 2 panelists using a double-blind method, with a single scoring range of 0-5, and a total score of 20.
(1) Inflammatory cell infiltration: 0, no inflammatory cell infiltration; 1, infiltration of a small number of inflammatory cells; 2, mild inflammatory cell infiltration; 3, moderately inflammatory cell infiltration; 4, severe inflammatory cell infiltration; 5, infiltration of extremely severe inflammatory cells.
(2) Pannus formation: 0, no pannus; 1, a minority pannus formation; 2, mild pannus formation (less than 1/4 of the metacarpophalangeal joint involved); 3, moderate pannus formation (involving 1/4-1/2 metacarpophalangeal joints); 4, severe pannus formation (involving 1/2-3/4 metacarpophalangeal joints); 5, extremely severe pannus formation (involvement of the metacarpophalangeal joint greater than 3/4).
(3) Cartilage erosion: 0, no cartilage erosion; 1, slight cartilage erosion; 2, mild cartilage erosion (superficial or focal chondrocyte depletion and collagen destruction); 3, moderate cartilage erosion (multifocal or chondral cytopenia and collagen destruction deep to cartilage layer 1/2); 4, severe cartilage erosion (involving greater than 1/2 depth of cartilage surface, complete destruction of one or more tarsal articular cartilage surfaces); 5, extremely severe cartilage erosion (severe chondrocyte depletion and collagen destruction, down to the tidal line).
(4) Bone destruction: 0, no bone destruction; 1, slight bone destruction, insignificant under low power lens; 2, mild bone destruction (less than 1/4 of the metacarpophalangeal joint involved); 3, moderate bone destruction, obvious bone trabecula and cortical bone absorption, but not reaching the full cortex (involving 1/4-1/2 of the metacarpophalangeal joint); 4, severe bone destruction, local involvement of the full cortex, cortical deformation, trabecular absorption (involvement of 1/2-3/4 of the metacarpophalangeal joint); 5, extremely severe bone destruction, involvement of the full layer of cortical bone, cortical bone deformation, trabecular bone resorption (involvement of the metacarpophalangeal joint greater than 3/4).
As shown in fig. 13, the body weight of the animals was significantly reduced in the boost starting model group compared with the normal control group, and the mice body weight was recovered in both the hoc-MSC treatment and the C3-G4-MSC treatment groups, whereas the positive antibody group did not significantly recover the mice body weight; the mice in the end-point C3-G4-MSC treated group had significantly higher body weight than the positive antibody group. The thickness of the animal paw is shown in fig. 14, the mouse paw rapidly swells after the boost (D21), and the thickness of the mouse paw is significantly reduced after intravenous injection of the hoc-MSC, positive antibody Ixekizumab and C3-G4-MSC, wherein the C3-G4-MSC treatment effect is significantly better than that of the positive antibody Ixekizumab. The histopathological results are shown in figure 15, and the model mice have histoinflammatory cell infiltration, joint synovitis and/or pannus formation, joint cartilage destruction, joint cavity disappearance and bone tissue fusion; and after treatment, the mice pathological tissue score (shown in figure 16) is obviously reduced, wherein the C3-G4-MSC treatment effect is obviously better than that of the positive antibody Ixekizumab.
Example 11 animal model construction method of psoriasis and construction results
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. 17, the body weight of the model group after imiquimod modeling is significantly reduced compared with the normal control group, the hUC-MSC treated group and the C3-G4-MSC treated group can recover the body weight of the mice, and the positive antibody group does not significantly recover the body weight of the mice; 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. 18, and clinical scores of skin according to skin keratinization degree and skin scaling lesion are shown in fig. 19, 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. 20, and it was found that IMQ significantly increased 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 that of positive antibody Ixekizumab.
Example 12 animal model construction method of psoriatic arthritis and construction results
A model of psoriatic arthritis (PsA) was constructed by intraperitoneal injection of mannan (SIGMA, M7504-5G) using B-hIL17A transgenic mice (Bai Chart pharmaceutical technologies Co., ltd.). Taking a model mouse, performing three times of intraperitoneal injections of mannans at D0, D4 and D8 respectively when the first intraperitoneal injection is marked as D0. Each animal was intraperitoneally injected with 100mg/mL of mannan in a volume of 200. Mu.L, i.e., 20mg of mannan per animal. The PsA model group was randomly divided into four groups: hUC-MSC treated group (2 x10 6 /), positive antibody Ixekizumab treated group (1 mg/Kg), C3-G4-MSC treated group (2 x10 6/only) and model control group; drug treatment groups D3 and D7 were treated by tail vein administration. In the experimental process, animal weight is measured every 2 days, animal survival and health conditions are observed, animal skin, front and rear paws are photographed, and scoring is performed according to related indexes. D14 euthanizing animals, performing relevant detection, collecting the skin of the modeling part of the mice, and measuring the skin thickness of each group of animals by using a vernier caliper; scoring according to the degree of keratinization of the skin and the degree of inflammatory cell infiltration; mouse peripheral blood was collected, serum was isolated and sent to the immunoassay department for detection of cytokines such as mIL-1β, mIL-6, hIL-17A and mTNF- α (kits were all purchased from Biolegend).
Animal paw scoring criteria:
All cases of front and rear paw arthritis were scored and evaluated as follows: 0 = normal; 1 = single finger erythema and swelling in the paw; 2 = erythema and swelling of two fingers in the paw; 3 = more than two digits erythema and swelling in the paw and/or ankle swelling. The total score of 4 paws was calculated as the final score.
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.
Animal body weight as shown in figure 21, the PsA model group body weight after mannan modeling was significantly reduced compared to the normal control group, both the hoc-MSC treated and the C3-G4-MSC treated groups restored mouse body weight, while the positive antibody group restored mouse body weight less significantly, and the test endpoint C3-G4-MSC treated group mouse body weight was significantly higher than the positive antibody group.
Animal paw and skin clinical scores as shown in fig. 22-23, both skin and paw clinical scores of the PsA model group were significantly increased after mannan modeling, positive antibody, hic-MSC treatment and C3-G4-MSC treatment groups were able to significantly reduce model paw and skin clinical scores, while the C3-G4-MSC treated group mice body weight was significantly better than the positive antibody group.
As shown in FIGS. 24-26, the serum of PsA model animals after mannan modeling is significantly increased in cytokines such as IL-6, IL-23 and TNF-alpha, while both hUC-MSC treatment and C3-G4-MSC treatment can significantly reduce the serum IL-6 and TNF-alpha levels of model animals, and both positive antibody and C3-G4-MSC treatment can significantly reduce the serum IL-23 levels of model animals.
Claims (11)
1. Use of stem cells for the preparation of a medicament for the prevention and/or treatment of a disease, characterized in that said stem cells comprise (1) and/or (2) as follows:
(1) Amino acid sequences shown in SEQ ID NO. 1-6;
(2) Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1-6;
the disease includes inflammatory disease, infectious disease, autoimmune disease, nervous system disease and/or tumor.
2. Use of stem cells for the preparation of a medicament for the prevention and/or treatment of a disease, characterized in that said stem cells comprise (1) and/or (2) as follows:
(1) Amino acid sequences shown in SEQ ID NO. 1-14;
(2) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1-14;
the disease includes inflammatory disease, infectious disease, autoimmune disease, nervous system disease and/or tumor.
3. Use of stem cells for the preparation of a medicament for the prevention and/or treatment of a disease, characterized in that said stem cells comprise (1) and/or (2) as follows:
(1) Amino acid sequences shown in SEQ ID NO. 15-16;
(2) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 15-16;
the disease includes inflammatory disease, infectious disease, autoimmune disease, nervous system disease and/or tumor.
4. Use of stem cells for the preparation of a medicament for the prevention and/or treatment of a disease, characterized in that said stem cells comprise (1) and/or (2) as follows:
(1) An amino acid sequence shown in SEQ ID NO. 17;
(2) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 17;
the disease includes inflammatory disease, infectious disease, autoimmune disease, nervous system disease and/or tumor.
5. The use according to claim 4, wherein the stem cells are embryonic stem cells, adult stem cells, mesenchymal stem cells, umbilical cord blood stem cells, hematopoietic stem cells, neural stem cells, adipose stem cells, skin stem cells and/or muscle stem cells.
6. The use according to claim 5, wherein the stem cells are mesenchymal stem cells.
7. The use according to claim 6, wherein the mesenchymal stem cells are isolated from umbilical cord blood, umbilical cord, placenta, adipose tissue, skin, neural tissue, bone marrow or embryo.
8. The application of stem cells in preparing a medicament for preventing and/or treating diseases is characterized in that the stem cells comprise, express and/or secrete antibodies with amino acid sequences shown as SEQ ID NO. 21; the disease includes inflammatory disease, infectious disease, autoimmune disease, nervous system disease and/or tumor.
9. The use according to claim 8, wherein the antibody further comprises a biologically active protein or functional fragment thereof that aids in its expression and/or secretion, or that extends its half-life in vivo.
10. The use according to claim 9, wherein the biologically active protein or functional fragment thereof is selected from the group consisting of an immunoglobulin Fc domain, an elastin-like polypeptide, serum albumin, an albumin binding polypeptide, prealbumin, a carboxy terminal peptide, a His tag, a FLAG tag, a c-Myc tag, an HA tag, a GST tag, an MBP tag and/or a SUMO tag.
11. The use according to any one of claims 8 to 10, wherein the disease comprises rheumatoid arthritis, psoriasis and/or psoriatic arthritis.
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