CN117866905A - Stem cell based on nanobody gene modification and preparation method and product thereof - Google Patents

Abstract

The invention provides a stem cell based on nanobody gene modification, a preparation method and a product thereof, belonging to the technical field of stem cells. The stem cell based on nano antibody gene modification comprises, expresses or secretes a first antibody and a second antibody, wherein the first antibody comprises HCDR1, HCDR2 and HCDR3 with sequences shown as SEQ ID NO. 1-3; the second antibody comprises HCDR4, HCDR5 and HCDR6 with sequences shown in SEQ ID NO. 4-6. Compared with a single-domain antibody before tandem, the tandem antibody based on the stem cell expression modified by the nano antibody gene has better IL-17A antigen binding capacity and blocking activity, better stability compared with the single-domain antibody before tandem, and better disease treatment effect compared with a positive antibody. The serial nano antibody provided by the invention can be used for modifying stem cells, especially MSC, and has good clinical application prospect.

Description

Stem cell based on nanobody gene modification and preparation method and product thereof

Technical Field

The invention belongs to the technical field of stem cells, and particularly relates to a stem cell based on nano antibody gene modification, a preparation method and a product thereof.

Background

Stem cells are immature cells that are not sufficiently differentiated to have the potential to regenerate various tissues and organs and the human body. Stem cell therapy has been attempted in various diseases such as diabetes, parkinsonism, lupus erythematosus, leukemia, and the like. The preparation technology and application of induced pluripotent stem cells (induced pluripotent stem cell, iPSC) bring stem cell treatment into a brand new era. With the continued development of molecular biology techniques, particularly gene editing techniques, genetic modification has played an increasingly important role in stem cell therapy.

Stem cell therapy is a regenerative medicine technology, which aims to replace abnormal cells with cells having specific functions, restore the functions of the corresponding cells and improve the health state of the body. There are a variety of stem cells currently in clinical use, where HSC transplantation has improved prognosis for a variety of immune and blood system diseases, such as congenital immunodeficiency, multiple myeloma, thalassemia, and the like. Research data show that the implantation of allogeneic HSC can improve the 4-year survival rate of r/rAML (IDH 1/IDH2 mutated acute myelogenous leukemia) patients to 20% -35%. In addition, MSC has a certain immunoregulatory function, and thus is used for treating immune diseases, such as graft versus host disease, and the like, and achieves a certain curative effect. Autologous HSC drug Strimvelis for the treatment of adenosine deaminase deficient severe combined immunodeficiency disease (ADA-SCID) and autologous MSC drug Astro stem for the treatment of alzheimer's disease have been disclosed in the prior art.

Mesenchymal stem cells (mesenchymal stem cell, MSC) are multipotent stem cells, which are multipotent cells with self-replicating capacity. Under certain conditions, it can differentiate into a variety of functional cells like APSC pluripotent cells. Derived from mesoderm during embryonic development. MSCs are an important cell bank involved in tissue regeneration during normal tissue injury repair in the body. Under the action of special signals caused by tissue injury, MSC migrate to the injured part, locally accumulate and proliferate, and differentiate along different paths according to different injury signals. MSC is easy to separate and amplify, has vigorous in vitro multiplication capacity, and can maintain the multi-directional differentiation capacity even if amplified by 1 hundred million times. Therefore, MSC is a practical seed cell for tissue repair.

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

The prior art for studying the mode of action of IL-17 with stem cells is, for example, the China patent application with publication No. CN105079792A, which discloses the application of IL-17 in improving the immunosuppressive function of mesenchymal stem cells. In particular to the application of interleukin-17, interleukin-17 derivative or an agonist thereof in preparation of a preparation or a kit for improving the immunosuppressive function of mesenchymal stem cells; upregulating expression of immunosuppressive factors in mesenchymal stem cells; enhancing the stability of the mRNA of the immunosuppressive factor; reducing the expression level of the RNA binding protein AUF 1; inhibit proliferation of T cells; treating hepatitis or liver injury.

However, the prior art does not disclose the modification of stem cells by IL-17 related antibodies, in particular the role of IL-17A specific tandem antibodies in stem cell modification.

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

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

Based on the technical scheme, the invention continues to develop the technology of combining single-domain antibodies and genetically modified stem cells, and based on the relevant regulations of the singleness of the patent law, a plurality of different genetically modified stem cells are respectively claimed for protection.

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

Disclosure of Invention

In order to solve the problems, the invention provides a modified stem cell, which is screened based on immune alpaca to obtain a plurality of nano antibodies with IL-17A specific binding capacity, and two of the nano antibodies are selected to be connected in series so as to realize stem cell modification to prepare the genetically modified stem cell.

Stem cells of the invention, expression and/or secretion:

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

(2) A second antibody comprising a single domain antibody that specifically recognizes IL-17A;

the amino acid sequences of the first antibody and the second antibody are different.

The first antibody comprises HCDR1, HCDR2 and HCDR3 with sequences shown as SEQ ID NO. 1-3; and/or; an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 1-3;

SEQ ID NO: 1：GEKLDYFA；

SEQ ID NO: 2：VTSSGSST；

SEQ ID NO: 3：ASTILLCSDYISAFGT。

The second antibody comprises HCDR4, HCDR5 and HCDR6 with the sequences shown in SEQ ID NO. 4-6; and/or; an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 4-6;

SEQ ID NO: 4：GFSIHIYA；

SEQ ID NO: 5：ITRGGVT；

SEQ ID NO: 6：NAGGTNGGY。

those skilled in the art know that CDR regions are regions of antibodies specifically recognizing antigens and have major binding activity, and thus, those skilled in the art can theoretically design various antibodies or fusion proteins for use according to the CDR regions described in the present invention. Such as in the form of nanobodies.

Alternatively, the first and second antibodies are linked directly or through a linker, preferably through a linker.

Preferably, the first antibody and the second antibody are linked by a linker, the linker being (GGGGS) _n And n is an integer not less than 1, more preferably 3.

Preferably, the stem cells comprise, express and/or secrete:

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

FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4；

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

FR5-HCDR4-FR6-HCDR5-FR7-HCDR6-FR8；

HCDR1-6 is selected from the amino acid sequences shown in SEQ ID NO 1-6; or; amino acid sequences having a difference of 1, 2, 3, 4 or 5 amino acids compared to SEQ ID NO. 1-6;

FR1-8 is selected from the amino acid sequences shown in SEQ ID NO. 7-14; or; amino acid sequences having a difference of 1, 2, 3, 4 or 5 amino acids compared to SEQ ID NO. 7-14.

SEQ ID NO:7：QVQLVESGGGLVQPGGSLRLSCAAS；

SEQ ID NO:8：IGWFRQAPGKEREVVSC；

SEQ ID NO:9：NYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAIYYC；

SEQ ID NO:10：WGQGTQVTVAS；

SEQ ID NO:11：EVQLVESGGGLVQPGGSLRLSCAAS；

SEQ ID NO:12：MGWYRQAPGKQRELVAT；

SEQ ID NO:13：NNADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYC；

SEQ ID NO:14：WGQGTQVTVSS。

In the present invention, the amino acid difference may be achieved by at least one of addition, deletion, modification and/or substitution of an amino acid.

In some embodiments, the amino acid substitution is a conservative substitution, which is preferably a substitution of one amino acid in the following groups (a) to (e) with another amino acid residue in the same group: (a) small aliphatic, non-polar or weakly polar residues: ala, ser, thr, pro and Gly; (b) Polar, negatively charged residues and (uncharged) amides: asp, asn, glu and Gln; (c) polar, positively charged residues: his, arg and Lys; (d) large aliphatic, nonpolar residues: met, leu, he, val and Cys; and (e) an aromatic residue: phe, tyr and Trp.

In some preferred embodiments, conservative substitutions are as follows: ala to Gly or to Ser; arg to Lys; asn to Gln or to His; asp to Glu; cys to Ser; gln to Asn; glu to Asp; gly to Ala or to Pro; his to Asn or to Gln; lie to Leu or to Val; leu to Ile or to Val; lys to Arg, to gin, or to Glu; met to Leu, to Tyr or to Ile; phe to Met, to Leu, or to Tyr; ser to Thr; thr to Ser; trp to Tyr; tyr to Trp; and/or Phe to Val, to Ile or to Leu.

Further preferred, said stem cells comprise, express and/or secrete fusion proteins;

the fusion protein comprises an amino acid sequence with the structure of FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4-linker-FR5-HCDR4-FR6-HCDR5-FR7-HCDR6-FR 8;

HCDR1-6 is selected from: amino acid sequences shown in SEQ ID NO. 1-6; or; amino acid sequences having a difference of 1, 2, 3, 4 or 5 amino acids compared to SEQ ID NO. 1-6;

FR1-8 is selected from the group consisting of: amino acid sequence shown in SEQ ID NO. 7-14; or; amino acid sequence having a difference of 1, 2, 3, 4 or 5 amino acids compared to SEQ ID NO. 7-14;

the linker is selected from: the amino acid sequence shown in SEQ ID NO. 15.

SEQ ID NO: 15：GGGGSGGGGSGGGGS。

Still further preferred, said stem cells comprise, express and/or secrete:

(1) A first protein, wherein the sequence of the first protein comprises an amino acid sequence shown as SEQ ID NO. 16;

(2) And the second protein comprises an amino acid sequence shown as SEQ ID NO. 17.

SEQ ID NO:16：

QVQLVESGGGLVQPGGSLRLSCAASGEKLDYFAIGWFRQAPGKEREVVSCVTSSGSSTNYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAIYYCASTILLCSDYISAFGTWGQGTQVTVAS。

SEQ ID NO:17：

EVQLVESGGGLVQPGGSLRLSCAASGFSIHIYAMGWYRQAPGKQRELVATITRGGVTNNADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCNAGGTNGGYWGQGTQVTVSS。

In some embodiments, presented as examples, the stem cells comprise, express and/or secrete fusion proteins:

the sequence of the fusion protein comprises: SEQ ID NO. 16-SEQ ID NO. 15-SEQ ID NO. 17-SEQ ID NO. 26.

SEQ ID NO: 26：

APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

Based on the above, it will be appreciated by those skilled in the art that the present invention provides a stem cell comprising:

(1) A first nucleic acid molecule; and; (2) a second nucleic acid molecule;

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

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

The first nucleic acid molecule codes for a nucleotide sequence comprising the amino acid sequence shown in SEQ ID NO. 7-10;

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

Preferably, the first nucleic acid molecule and the second nucleic acid molecule are linked by a nucleotide sequence encoding an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO. 15.

Further preferably, the stem cell comprises:

(1) A first nucleic acid molecule; and; (2) a second nucleic acid molecule;

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

the first nucleic acid molecule encodes a nucleotide sequence comprising the amino acid sequence shown in SEQ ID NO. 17.

In some specific embodiments, the stem cells described by way of example comprise: nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO. 16-SEQ ID NO. 15-SEQ ID NO. 17-SEQ ID NO. 26.

In fact, as is well known to the person skilled in the art, due to the problem of codon degeneracy, in the case of defined amino acid sequences, there are numerous possibilities for their corresponding nucleic acid sequences, the preferred nucleic acid sequences mentioned above being only a preferred example and not the only possibility.

The stem cells may also comprise, express and/or secrete biologically active proteins or functional fragments thereof capable of extending the in vivo half-life of the antibody, according to techniques already disclosed in the art.

Alternatively, the stem cells comprise a nucleotide sequence encoding a biologically active protein or functional fragment thereof that is capable of extending the half-life of the antibody in vivo.

In some preferred embodiments, the biologically active protein or functional fragment thereof is selected from at least one of an immunoglobulin Fc domain, a serum albumin (e.g., human Serum Albumin (HSA)), an albumin binding polypeptide (e.g., HAS binding polypeptide), a prealbumin (also known as transthyretin), a carboxy terminal peptide (e.g., human chorionic gonadotrophin β subunit (CTP)), an elastin-like polypeptide (ELP), a His tag (preferably 6 xhis), a GST (glutathione-mercaptotransferase) tag, an MBP (maltose binding protein) tag, a FLAG tag, a SUMO (ubiquitin-like modifying protein) tag, and the like.

Preferably, the biologically active protein or functional fragment thereof is a human immunoglobulin Fc domain, preferably an Fc domain of human IgG, such as an Fc domain of human IgG1, igG2, igG3, igG4, more preferably an Fc domain of human IgG 1.

One or more amino acid modifications may be introduced in the Fc region or Fc domain of an antibody provided herein, thereby producing an Fc region variant. An Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, igG2, igG3, or IgG4 Fc region) comprising amino acid modifications (e.g., substitutions, deletions, and insertions) at one or more amino acid positions.

In some embodiments, any of the foregoing nucleic acid molecules are independent of, or integrated into, the genome of the modified stem cell.

In some embodiments, any of the foregoing nucleic acid molecules are operably linked to a promoter, such as a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

The stem cells are selected from 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, muscle stem cells and the like;

in the present invention, the differentiation of the stem cell type according to differentiation development potential may include:

Totipotent stem cells: can differentiate into intact individuals, such as embryonic stem cells (embryonic stem cell, ES cells); pluripotent stem cells: can differentiate into various cell tissue organs such as hematopoietic stem cells, germ stem cells, mesenchymal stem cells, neural stem cells, liver stem cells, pancreatic stem cells, etc.; monoenergetic stem cells: refers to stem cells that have the potential to differentiate into one or two related cell types, such as myoblasts in muscle, basal layer of epithelial tissue.

In the present invention, the differentiation of the types of stem cells according to the developmental stages of the stem cells may include:

embryonic stem cells: highly undifferentiated cells, having a developmental totipotent, capable of differentiating into all tissue organs of an adult; adult stem cells: under specific conditions, adult stem cells either produce new stem cells or differentiate to form new functional cells according to a program, thereby maintaining the dynamic balance of growth and deterioration of tissues and organs.

The adult stem cells include, but are not limited to:

hematopoietic stem cells: the adult stem cells with the longest and deepest research history have the potential of differentiating into various cells of a blood system, and have great application potential in the fields of disease treatment, anti-aging health care and the like; germ stem cells: the preparation has the potential of differentiating into germ cells and various support cells of gonads, so that adults can play a fertility function and the aging of the gonads can be delayed; mesenchymal stem cells: has the potential of differentiating into organism bones, cartilages and cells of various organs, and has the characteristic immunoregulation function; neural stem cells: has the potential of differentiating into various cells of the nervous system; retinal stem cells: has the potential of differentiating into various cells of retina; cardiac stem cells: has the potential of differentiating into various cells of the heart; liver stem cells: has the potential of differentiating into various cells of the liver; pancreatic stem cells: has the potential of differentiating into various cells of pancreas, and can differentiate into islet cells under specific conditions; lung stem cells: has the potential of differentiating into various cells of the lung; renal stem cells: has the potential of differentiating into various cells of the kidney.

Preferably, the stem cells are mesenchymal stem cells.

The stem cells are isolated from cord blood, umbilical cord, placenta, adipose tissue, skin, neural tissue, bone marrow, or embryo.

According to the above, the stem cells secrete anti-interleukin antibodies.

Preferably, the stem cells secrete anti-interleukin 17 antibodies.

Further preferably, the stem cells secrete anti-IL-17A antibodies.

Based on the above stem cells, the present invention also provides:

a cell culture obtained by culturing stem cells according to the foregoing.

An extract obtained by extraction of the aforementioned cell culture.

A pharmaceutical composition comprising the aforementioned stem cells, cell cultures or extracts; optionally, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

In some embodiments, the adjuvant is selected from at least one of solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickening agents, emulsifiers, preservatives, stabilizers, hydration agents, emulsifying accelerators, buffers, absorbents, colorants, flavorants, sweeteners, ion exchangers, mold release agents, coating agents, flavoring agents, or antioxidants.

A kit comprising the aforementioned stem cell, cell culture pharmaceutical composition.

The invention also provides a preparation method of the stem cells, which comprises the following steps:

the nucleic acid molecules are introduced into stem cells by viral transfection, liposome transfection, electrotransfer, gene editing or mRNA transfection.

In some embodiments, the stem cells may be genetically modified by methods and techniques known in the art, such as physical, chemical, or biological methods, or a combination thereof, by one of skill in the art. For example, the biological methods include the use of viral vectors such as lentiviruses, retroviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. For example, the chemical means include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and the like; lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles or liposomes, and the like. For example, the physical methods include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.

The invention also relates to the protection of uses including, but not limited to:

the use of said stem cells, said cell culture, said extract or said pharmaceutical composition in the manufacture of a medicament for inflammatory diseases, infectious diseases, autoimmune diseases, neurological diseases and/or tumors.

The use of said stem cells, said cell culture, said extract or said pharmaceutical composition for the preparation of a diagnostic product for inflammatory diseases, infectious diseases, autoimmune diseases, neurological diseases and/or tumors.

Based on the foregoing, the present invention also provides a method for in vitro detection of IL-17A in a sample for non-diagnostic purposes.

The method comprises the following steps:

d1: the stem cells, the cell culture or the extract are contacted with a sample to be tested;

d2: detecting the antigen-antibody complex;

d3: and judging the result.

Based on the foregoing, the present invention also provides a method for preventing and/or treating a disease, the method comprising:

administering to a subject in need thereof a therapeutically effective amount of said stem cells, said cell culture, said extract or said pharmaceutical composition;

the disease is inflammatory disease, infectious disease, autoimmune disease, nervous system disease and/or tumor.

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, barcup'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, leukoplakia, neuromyelitis optica, 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 transplantation immunity, familial mediterranean fever, eosinophilic chronic sinusitis, dilated cardiomyopathy, systemic mastocytosis or inclusion body myositis.

Preferably, the autoimmune disease is plaque psoriasis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis or lupus nephritis.

Such tumors include, but are not limited to: basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, bone cancer, breast cancer, peritoneal carcinoma, cervical cancer, cholangiocarcinoma, choriocarcinoma, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, ocular cancer, head and neck cancer, gastric cancer, glioblastoma, liver cancer, renal cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, cancer of the respiratory system, salivary gland carcinoma, sarcoma, skin cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, cancer of the urinary system, B-cell lymphoma, chronic lymphoblastic leukemia, acute lymphoblastic leukemia, hairy cell leukemia or chronic myelogenous leukemia.

The invention has the beneficial effects that:

according to the invention, the recombinant antigen immune alpaca is prepared, the nano antibody with IL-17A specificity is screened, and two nano antibodies are pertinently selected for series connection, so that the obtained IL-17A specificity serial antibody has better IL-17A antigen binding capacity and blocking activity compared with a single domain antibody before series connection, can be used for modifying stem cells, particularly MSC, and has good clinical application prospect.

The modified stem cells of the present invention exhibit superior effects in a variety of animal models, including but not limited to:

(1) Collagen-induced Rheumatoid Arthritis (RA) mouse model, at equivalent injection levels (2×10) relative to treatment regimen of positive antibodies Ixekizumab (1 mg/Kg) and hUC-MSC (pre-modification) ⁶ Under the condition of individual/individual), the weight of the mice can be recovered by the MSC treatment and the G4-H10-MSC treatment, and the weight recovery of the mice by the positive antibody group is not obvious; test endpoint G4-H10-MSC treated mice had significantly higher body weight than the positive antibody group. In terms of the average paw thickness of animals, the G4-H10-MSC treatment effect of the invention is obviously better than that of positive antibodies Ixekizumab and hUC-MSC; pathological tissue staining scores also show that the treatment effect of G4-H10-MSC is significantly better than that of positive antibodies Ixekizumab and hUC-MSC;

(2) In a psoriasis (Ps) model constructed by an imiquimod smearing method, the treatment effect of G4-H10-MSC is obviously better than that of positive antibodies Ixekizumab and hUC-MSC in animal weight, skin clinical score and skin thickness;

(3) In the model of psoriatic arthritis (PsA) constructed by injecting mannans into the abdominal cavity, the treatment effect of G4-H10-MSC is obviously better than that of positive antibodies Ixekizumab and hUC-MSC in terms of animal weight, paw joint score and skin clinical score; and also shows superior effect (with significant difference) with respect to positive antibodies Ixekizumab and hUC-MSC in terms of animal serum inflammatory factors (TNF-alpha, IL-6, IL-23).

Drawings

FIG. 1 shows the result of SDS-PAGE of IL-17A recombinant protein (antigen).

FIG. 2 shows the results of a positive control antibody Ixekizumab blocking assay.

FIG. 3 shows SDS-PAGE results of the tandem antibodies G4-H10.

FIG. 4 shows the results of the detection of the antibody affinity of the positive control antibody Ixekizumab and the tandem antibodies G4-H10 in basic experimental example 3, wherein the left panel shows the positive control antibody Ixekizumab and the right panel shows the tandem antibodies G4-H10.

FIG. 5 shows the results of the antibody blocking test of the tandem antibodies G4 to H10 in basic test example 4.

FIG. 6 shows the results of lentiviral titer assays in example 1.

Fig. 7 shows FITC channel signal detection results of the mesenchymal stem cells of example 2: the hUC-MSC reading value is 0.353%, and the G4-H10-MSC reading value is 63.329%.

FIG. 8 shows the results of measurement of the amount of IgG4 expressed in example 3.

FIG. 9 shows the results of detection of IL-17Nb antibody content in example 3.

FIG. 10 is a graph showing the results of the ability of G4-H10-MSC cells to block IL-17A/IL-17RA binding assay in example 4.

FIG. 11 shows statistics of IL17Nb expression levels of mesenchymal stem cells of example 5 at different culture times. The hUC-MSC has extremely obvious difference between the two groups of G4-H10 and G4-H10-MSCP＜0.001）。

FIG. 12 shows the weight statistics of each group of animals in example 6. * Representative model control group has significant difference compared with normal control group P<0.01 A) is provided; # represents a significant difference between the G4-H10-MSC treated group and the model control groupP<0.01）；&&Representing a significant difference between the G4-H10-MSC treated group and the positive antibody treated groupP<0.01）。

Fig. 13 is a graph showing the results of clinical scores of paw joints of animals in each group of example 6. * Significant difference between representative model control group and normal control groupP<0.0001 A) is provided; # # represents a significant difference between the 3 treatment groups and the model control group, respectivelyP<0.001）；&&Representing a significant difference between the G4-H10-MSC treated group and the positive antibody treated groupP<0.01 A) is provided; @represents the G4-H10-MSC treatment groupSignificant differences exist between hUC-MSC treatment groupsP<0.05）。

FIG. 14 shows the results of the skin clinical scores of the animals of example 6. * Significant difference between representative model control group and normal control groupP<0.0001 A) is provided; # represents a significant difference between the 3 treatment groups and the model control group, respectivelyP<0.01；&Representing a significant difference between the G4-H10-MSC treated group and the positive antibody treated groupP<0.05 A) is provided; @ represents a significant difference between the C3-H10-MSC treated group and the hUC-MSC treated group @P<0.05）。

FIGS. 15-17 show the results of detection of inflammatory factors in serum of animals of example 6, wherein the hUC-MSC group is a hUC-MSC treated group, the positive antibody group is a positive antibody treated group, and the G4-H10-MSC group is a G4-H10-MSC treated group.

FIG. 15 shows the results of TNF- α detection in the serum of each group of animals of example 6. Wherein: * Representative model control group has significant difference compared with normal control groupP<0.01 A) is provided; # represents significant difference between hUC-MSC treated group and model control groupP<0.05 A) is provided; # represents a significant difference between the G4-H10-MSC treated group and the model control groupP<0.01）；&Representing a significant difference between the G4-H10-MSC treated group and the positive antibody treated groupP<0.05 A) is provided; @ represents a significant difference between the G4-H10-MSC treated group and the hUC-MSC treated groupP<0.05）。

FIG. 16 shows the results of IL-6 detection in serum of animals of each group of example 6. Wherein: * Representative model control group has significant difference compared with normal control groupP<0.01 A) is provided; # represents significant difference between hUC-MSC treated group and model control groupP<0.05 A) is provided; # represents a significant difference between the G4-H10-MSC treated group and the model control groupP<0.01）；&Representing a significant difference between the G4-H10-MSC treated group and the positive antibody treated groupP<0.05）。

FIG. 17 shows the results of IL-23 detection in serum of animals of each group of example 6. Wherein: * Compared with the normal control group, the representative model control group has significant differenceP<0.01）The method comprises the steps of carrying out a first treatment on the surface of the # represents a significant difference between the positive antibody and the G4-H10-MSC treated group and the model control group P<0.05 A) is provided; @ represents a significant difference between the G4-H10-MSC treated group and the hUC-MSC treated groupP<0.05）。

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 reagents, materials and equipment that may be involved in the present invention are shown in Table 1.

TABLE 1 Main reagents, materials and apparatus according to the invention

The primers used for screening and cloning VHH fragments and constructing nanobodies are designed by referring to the following documents:

Maass DR, Sepulveda J, Pernthaner A, Shoemaker CB. Alpaca (Lama pacos) as a convenient source of recombinant camelid heavy chain antibodies (VHHs). J Immunol Methods. 2007;324(1-2):13-25.

Lin, J, Gu, Y, Xu, Y et al.Characterization and applications of nanobodies against Pseudomonas aeruginosa exotoxin a selected from single alpaca B cells. Biotechnol Biotechnol Equip 2020; 34: 1028–37.

Studies on design of single domain antibodies by AlpacaVHH phage library and high throughput sequencing toconstruct Fab antibody purification system（http: //hdl.handle.net/10232/00030916）.

unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.

The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.

As used herein, the term "include" or "comprising" means "including but not limited to. The term is intended to be open ended to specify the presence of any stated features, elements, integers, steps, or components, but does not preclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" includes the more limiting terms "consisting of … …" and "consisting essentially of … …". In one embodiment, the term "comprising" as used throughout the invention, and in particular in the claims, may be replaced by the term "consisting of … …". The amino acid three-letter codes and one-letter codes used herein are as known to those skilled in the art, or as described in J biol. Chem, 243, p3558 (1968).

As used herein, the terms "optional," "any," or "any" mean that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1 antibody heavy chain variable region" means that an antibody heavy chain variable region of a particular sequence may be, but is not required to be, present.

As used herein, the term "about" means a range of ±20% of the numerical value thereafter. In some embodiments, the term "about" means a range of ±10% of the numerical value following that. In some embodiments, the term "about" means a range of ±5% of the numerical value following that.

As used herein, the term "stem cell" refers to an undifferentiated cell that self-renews and differentiates at the single cell level to produce daughter cells, including self-renewing progenitor cells, non-renewing progenitor cells, and terminally differentiated cells. The stem cells have unlimited self-renewal, proliferation and differentiation capacity under certain conditions, can generate daughter cells with the same phenotype, genotype and self, can also generate specialized cells composing tissues and organs of the organism, and plays a main role in body growth, maintenance, renewal and injury repair.

As used herein, the term "mesenchymal stem cells", also known in the art as "mesenchymal stem cells (mesenchymal stem cell, MSC)", refers to a group of multipotent stromal cell populations derived from mesoderm that have a certain differentiation potential and can differentiate into a variety of cell types. It is derived mainly from and is present in bone marrow, and in addition includes multipotent cells that are widely derived from other "non-bone marrow" tissues, such as: placenta, umbilical cord blood, adipose tissue, adult muscle, cornea stroma, tooth pulp of deciduous teeth, etc.

As used herein, the term "IL-17A", "interleukin-17A" or "IL-17" refers to a cytokine, belonging to the interleukin 17 family, produced by T cells and other types of immune cells, and playing an important role in the immune system. IL-17A is produced primarily by Th17 cells, and other cells including CD8+ T cells, γδ T cells, NK cells and neutrophils, mast cells and macrophages also express IL-17A. It acts primarily on immune cells, such as macrophages, neutrophils and endothelial cells, inducing an inflammatory response. In some examples, the term includes variants, homologs, orthologs, and paralogs. For example, antibodies specific for human IL-17A may in some cases cross-react with IL-17A protein of another species, such as a monkey. In other embodiments, antibodies specific for human IL-17A protein may be completely specific for human IL-17A protein without cross-reacting with other species or other types of proteins, or may cross-react with IL-17A proteins of some other species but not all other species.

As used herein, the terms "anti-IL-17A single domain (nanobody," IL-17A single domain (nanobody) "or" tandem single domain (nanobody) that specifically binds IL-17A refer to an antibody that specifically binds IL-17A and partially or fully neutralizes, inhibits or attenuates IL-17A activity, and/or inactivates IL-17A, prevents IL-17A responses, or downstream pathways mediated by IL-17A or other IL-17A mediated functions.

As used herein, the term "antibody" refers to a glycoprotein comprising a heavy chain (H) and a light chain (L) interconnected by disulfide bonds (S-S). Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of 3 domains, CH1, CH2 and CH 3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as VH). The light chain constant region consists of one domain CL. Light chains fall into two categories, kappa-type light chains and lambda-type light chains, respectively (e.g., light chain constant regions Ckappa/lambda in the present invention mean that the light chain constant regions are kappa-type light chains or lambda-type light chains). The VH and VL regions may be further subdivided into hypervariable regions (also known as Complementarity Determining Regions (CDRs)) with more conserved framework or Framework Regions (FR) interposed therebetween. Each VH and VL consists of three CDRs and 4 FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Antibodies include monospecific antibodies, bispecific antibodies, and multispecific antibodies so long as they exhibit the desired biological activity or function.

As used herein, the term "single domain antibody" (sdAb) or "nanobody" has its ordinary meaning in the art and refers to an antibody fragment having a molecular weight of only 12-15 kDa, which consists of a single monomeric variable antibody domain derived from a heavy chain. Such single domain antibodies (designated VHH) can be found in camelidae mammals and naturally lack the light chain. For a general description of (single) domain antibodies, reference is also made to the above-mentioned prior art and EP 0368684, ward et al (Nature 1989 Oct 12;341 (6242): 544-6), holt et al Trends Biotechnol, 2003, 21 (11): 484-490; and WO 06/030220, WO 06/003388. The amino acid sequence and structure of a single domain antibody can be considered to consist of four framework regions or "FR", which are referred to in the art as "framework region 1" or "FR1", respectively; "frame region 2" or "FR2"; is "frame region 3" or "FR3"; "frame region 4" or "FR4"; the framework regions are separated by three complementarity determining regions or "CDRs", referred to in the art as "complementarity determining region 1" or "CDR1", respectively; "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3". Thus, a single domain antibody can be defined as an amino acid sequence having the general structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1-FR4 refers to framework regions 1-4, respectively, and wherein CDR1-CDR3 refers to complementarity determining regions 1-3. In the context of the present disclosure, the amino acid residues of a single domain antibody are numbered according to the general numbering of the VH domains given by amino acid numbering International ImMunoGeneTics information system (http:// imgt. Cmes. Fr /).

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

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

As used herein, the term "Fc" or "Fc region" or "Fc fragment" refers to a polypeptide consisting of the CH2 and CH3 domains of IgA, igD, and IgG, or the CH2, CH3, and CH4 domains of IgE and IgM through a hinge region. Although the breakdown of the Fc fragment is variable, the heavy chain Fc fragment of human IgG generally refers to the polypeptide from A231 to its carboxy terminus.

As used herein, the term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes are typically composed of surface-clustered molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural features, as well as specific charge features. Conformational and non-conformational epitopes differ in that binding to the former but not the latter is lost in the presence of denaturing solvents. Epitopes can include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked or covered by a specific antigen binding peptide (in other words, amino acid residues are within the footprint of a specific antigen binding peptide).

As used herein, the term "affinity" or "binding affinity" refers to an inherent binding affinity that reflects interactions between members of a binding pair. The affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is the dissociation rate constant and the binding rate constant (K, respectively _off And K _on ) Is a ratio of (2). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

As used herein, the term"high affinity" or "high affinity" for an IgG antibody means a KD of 1.0X10 for an antigen ^-6 M or less, preferably 5.0X10 ^-8 M or less, more preferably 1.0X10 ^-8 M or less, 5.0X10 s ^-9 M or less, more preferably 1.0X10 ^-9 M or lower. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding of IgM subtype refers to KD of 10 ^-6 M or less, preferably 10 ^-7 M or less, more preferably 10 ^-8 M or lower.

As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single or double stranded form. Unless specifically limited, the term includes nucleic acids containing known analogues of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Pat. No. 8278036 to Kariko et al, which discloses mRNA molecules with uridine replaced by pseudouridine, methods of synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues (Batzer, Nucleic Acid Res. 19: 5081 (1991); Ohtsuka,J.Biol.Chem. 260: 2605-2608 (1985); Rossolini,Mol.Cell.Probes8: 91-98 (1994))。

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

As used herein, the term "vector" refers to any recombinant polynucleotide construct that can be used for transformation purposes (i.e., introducing heterologous DNA into a host cell). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Upon introduction into a host cell, other vectors (e.g., non-episomal mammalian vectors) integrate into the genome of the host cell and thereby replicate together with the host genome. In addition, certain vectors are capable of directing the expression of genes that are operably linked. Such vectors are referred to herein as "expression vectors".

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

As used herein, the term "pharmaceutical composition" generally refers to a formulation that exists in a form that allows for the biological activity of the active ingredient to be effective and that does not contain additional ingredients that have unacceptable toxicity to the subject to which the composition is to be administered. The composition is sterile.

As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. As used herein, the term "pharmaceutically acceptable carrier, excipient and/or diluent" refers to a carrier that is pharmacologically and/or physiologically compatible with the subject and active ingredient, and is well known in the art (see, e.g., remington's Pharmaceutical sciences Edited by Gennaro AR, 19th ed. Pennsylvania: mack Publishing Company, 1995). A pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, medium, encapsulating material, manufacturing aid or solvent encapsulating material, which is involved in maintaining the stability, solubility or activity of an antibody or antigen binding fragment thereof of the present disclosure, and includes, but is not limited to: pH modifiers, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugar, naCl, and the like. Agents that delay absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning commonly understood by those skilled in the art and are capable of stabilizing the desired activity of the active ingredient in a medicament, including but not limited to sodium glutamate, gelatin, SPGA, saccharides (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin or casein) or degradation products thereof (e.g., lactalbumin hydrolysate), and the like.

Basic Experimental example 1IL-17A nanobody screening

As shown in the invention patent on the same date as the application, the invention screens antibodies by immunizing animals with antigen, and comprises the following steps:

(1) Preparation of recombinant antigens

Adding a 6 XHis tag at the C end of the IL-17A antigen, performing gene synthesis after 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; the recombinant protein was purified to a purity of greater than 90% using a nickel column. (FIG. 1)

(2) Positive control antibody: preparation of Enoki monoclonal antibody (Ixekizumab)

Ixekizumab heavy chain variable region: SEQ ID NO. 20; the light chain variable region SEQ ID NO. 21.

SDS-PAGE detects the purity of the positive control antibody protein >95%.

SEQ ID NO:20：

QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG；

SEQ ID NO:21：

DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

(3) Construction and verification of IL-17A reporter gene cell strain

Based on the amino acid sequence information of IL-17RA (UniProtKB: Q96F 46) and IL-17RC (UniProtKB: Q8NAC 3), a lentiviral expression vector is constructed, lentivirus is packaged, 293 cells are co-infected, recombinant 293 cells which simultaneously overexpress the two receptors are screened, NFKB-Luciferase and ACT1 genes are further stably transformed, and an IL-17A reporter cell strain 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc is constructed. And adding a recombinant antigen for activation, and simultaneously adding a positive control antibody Ixekizumab for detection of a blocking experiment, so as to establish a candidate antibody in vitro pharmacodynamics evaluation cell strain targeting IL-17A. The constructed IL-17A receptor over-expression cell strain can be combined with IL-17A, and the IL17A recombinant protein can effectively activate luciferase expression in 293F-IL17Ra/IL17 Rc-NFkB-Luc reporter gene cell strain.

The positive control antibody Ixekizumab and the IL-17A recombinant protein are added into 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc cells together, and the positive control antibody Ixekizumab can inhibit the combination of IL17A protein and a membrane receptor thereof and inhibit the signal of intracellular NFkB and present a dose effect. The results are shown in FIG. 2.

(4) Alpaca immunity

Immunization is carried out on Alpaca (Alpaca) by using the prepared recombinant antigen protein, the immunization interval is 21 days, partial peripheral blood is collected after 10 days of the last immunization, and ELISA detection is carried out on the immune effect of the separated serum.

(5) Detection of immune titers

The immunological titers of alpaca reached the requirements after 6 rounds of immunization (see tables 2-3 below).

TABLE 2 results of immunotiter assays

TABLE 3 results of immunotiter assays

(6) PBMC isolation and VHH antibody fragment cloning

PBMC were isolated, RNA was extracted and subjected to two rounds of PCR after reverse transcription, the products were recovered, and the concentration was determined.

(7) Construction and panning of Single-Domain antibody Yeast display libraries

The linear vector and the PCR product together electrically transformed the yeast competent strain. Single domain antibody yeast display library panning; after panning, yeast monoclonal was subjected to flow detection. Amplification of overlapping PCR (Overlap PCR) products, transient transfection and detection, recovery of the products, transfection. ELISA was used to select positive clones, VHH antibody sequences were obtained and gene synthesis was performed, and Protein A (Protein A) was purified after genetic engineering expression.

Enriching positive clones; and selecting the enriched monoclonal, performing Phage ELISA identification, and performing sequencing analysis on the clone to obtain the nucleic acid and amino acid sequence information of the candidate single domain antibody. 20 monoclonals are randomly selected for sequencing analysis, the sequence difference is large, and the library diversity is good. The potential post-translational modification sites were analyzed by the In silico method against the amino acid sequence information of the CDR regions of the candidate single domain antibodies.

According to the result of the monoclonal flow detection of the yeast, positive clones combined with IL-17A-His are selected to extract genome DNA, and the antibody sequence is obtained by PCR. Selecting differential clones for overlapping PCR amplification, adding a signal peptide to the N end of VHH, adding IgG1-Fc to the C end, and transiently transfecting HEK293 cells with PCR products; ELISA detection was performed on the expressed antibody supernatants: 100 mu L of transfection supernatant is added into a 96-well plate pre-coated by IL-17A recombinant antibody for incubation, ELISA detection is carried out by adopting HRP-Protein A as a secondary antibody, and differential cloning is arranged for constructing eukaryotic expression vectors. The constructed single domain antibody expression vector is transiently transfected into 293F cells, and recombinant antibodies are affinity purified by using Protein A.

(8) ELISA detects the combination of the recombinant antibody and the target protein, FACS detects the combination of the single domain antibody and the reporter gene cell strain to complete the single domain antibody blocking function experiment, and the ForteBio OCTET R2 system carries out dynamic characterization analysis. Kinetic characterization analysis was performed using the ForteBio OCTET R2 system. Screening was performed based on the results of all antibodies. The invention further extends to the basis of the basic screening method described above. In the detection of the blocking activity of the antibody, the blocking effect of a part of single domain antibodies is weaker than that of a positive antibody Ixekizumab although the single domain antibodies can block the Human IL-17A protein from activating a downstream target protein. Therefore, two anti-IL-17A single domain antibodies are serially connected to form a bivalent antibody so as to enhance the blocking effect, and relevant stem cells are prepared on the basis and the application of the stem cells in preparing medicines is verified. Specifically, the invention protects stem cells prepared by serial antibodies constructed by serial numbers of 2-G4 (G4 for short) and 3-H10 (H10 for short), and the subsequent serial antibodies are called G4-H10 for short.

Basic Experimental example 2 preparation and purification of IL-17A-specific tandem antibodies

2-G4 has the sequence of SEQ ID NO 16, and CDR regions have the sequence of SEQ ID NO 1-3;

3-H10 has the sequence of SEQ ID NO. 17, and the CDR regions have the sequence of SEQ ID NO. 4-6.

The tandem antibody also includes a linker (GGGGS) ₃ A hinge region and a CH region.

The sequence of the constructed tandem antibody is SEQ ID NO. 18. The coding gene of the tandem antibody is SEQ ID NO. 19.

SEQ ID NO: 18：

QVQLVESGGGLVQPGGSLRLSCAASGEKLDYFAIGWFRQAPGKEREVVSCVTSSGSSTNYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAIYYCASTILLCSDYISAFGTWGQGTQVTVASGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSIHIYAMGWYRQAPGKQRELVATITRGGVTNNADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCNAGGTNGGYWGQGTQVTVSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

SEQ ID NO:19：

CAGGTGCAGCTCGTGGAGTCGGGGGGAGGCTTGGTGCAGCCCGGGGGATCTCTGAGGCTCTCGTGTGCAGCCTCTGGAGAGAAATTGGATTATTTTGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGTAGTCTCATGTGTCACAAGTAGTGGTAGTAGCACAAACTATTTAAGTTCCGTGAAGGACCGATTCACCATCTCCATAGACAACGCCAAGAACACGGTATATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCATTTATTACTGTGCGTCCACTATTCTCCTCTGTTCAGATTATATCTCTGCCTTTGGCACCTGGGGCCAGGGGACCCAGGTCACCGTCGCCTCGGGAGGCGGAGGATCTGGCGGAGGTGGAAGTGGCGGAGGCGGTTCTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCGGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTTAGTATCCACATCTATGCCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGCTGGTCGCAACTATTACTAGAGGTGGTGTAACAAATAATGCAGACTCCGTGAAGGGGCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGCGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGCAGGTGGGACGAACGGGGGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA。

The tandem antibodies can be prepared by reference to the prior art antibody preparation methods, specifically as follows:

(1) The SEQ ID NO. 19 gene is synthesized and subcloned into an expression vector pcDNA3.4-hIgG1-Fc in series with the human IgG1 Fc segment gene sequence. After the vector was verified by sequencing, the endotoxin-free plasmid was prepared using the Qiagen plasmid large-sample kit (CAT#: DP 117) for use.

(2) And taking out the LVTransm transfection reagent and the single-chain antibody expression vector from the refrigerator, thawing at room temperature, and blowing up and down by a pipetting gun to completely mix uniformly. 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 pipetting gun, fully and uniformly mixing, adding 400 mug LVTransm, immediately blowing up and down by a pipetting device, uniformly mixing, and standing for 10 minutes at room temperature to obtain the DNA/LVTransm compound.

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

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 Protein A to obtain a purified single domain antibody.

SDS-PAGE results of the tandem antibodies G4-H10 are shown in FIG. 3.

SDS-PAGE detects the purity of the target antibody protein, and the result shows that the protein purity is >95%.

Basic Experimental example 3IL-17A specific tandem antibody affinity assay

Using Ixekizumab as a positive control antibody, synthesizing Ixekizumab heavy chain and light chain variable regions by genes, subcloning the heavy chain variable regions into pcDNA3.4-hIgG4 (hIgG 4 amino acid sequence is SEQ ID NO: 22) vectors, and subcloning the light chain variable regions into pcDNA3.4-hIgKc (hIgKc amino acid sequence is SEQ ID NO: 23) vectors; after verification by Sanger sequencing, the plasmid megapump kit is used for preparing the endotoxin-removing plasmid for standby. Other steps reference basic Experimental example 2, control antibodies were prepared with purity >95%.

SEQ ID NO:22：

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK；

SEQ ID NO:23：

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

ELISA detects binding of recombinant antibodies to target proteins:

coating an ELISA plate with the purified antibody to be detected (2 ug/mL), adding Biotin-IL-17A-His, diluting 7 points with a 5-fold gradient, performing ELISA detection with HRP-strepitavdin, and drawing a curve of OD450 values measured by each concentration to read EC50 values.

The results showed that the positive control antibody Ixekizumab had an EC50 value of 10.06 μg/mL; the EC50 of the tandem antibody was 1.177. Mu.g/mL. See fig. 4.

Basic Experimental example 4 antibody blocking function experiment

Blocking activity assays were performed using the 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc reporter cell line. The tandem antibody of basic experimental example 2 and the positive control antibody Ixekizumab of basic experimental example 3 were detected.

Based on the amino acid sequence information of IL-17RA (UniProtKB: Q96F 46) and IL-17RC (UniProtKB: Q8NAC 3), a lentiviral expression vector is constructed, lentivirus is packaged, 293 cells are co-infected, recombinant 293 cells which simultaneously overexpress the two receptors are screened, NFKB-Luciferase (SEQ ID NO: 24) and ACT1 gene (SEQ ID NO: 25) are further stably transformed, and an IL-17A reporter cell strain 293F-IL-17RA-IL-17Rc-ACT1-nfκB-Luc is constructed.

SEQ ID NO:24：

atggaagatgccaaaaacattaagaagggcccagcgccattctacccactcgaagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatcgcctttaccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaagcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtgttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaacgtgcaaaagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtacaccttcgtgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtaccggattgcccaagggcgtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctcagcgtggtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgctggtgcccacactatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggcggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctacggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtggtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctgtgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggacggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacaccccaacatcttcgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggtaaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattctcattaaggccaagaagggcggcaagatcgccgtg；

SEQ ID NO:25：

ATGCCACCTCAGTTGCAGGAAACTCGGATGAATAGAAGCATCCCCGTGGAAGTGGACGAGAGCGAGCCGTACCCTAGTCAGCTGCTGAAGCCGATCCCTGAGTACTCCCCGGAAGAGGAATCCGAACCACCAGCCCCCAACATTCGCAATATGGCCCCCAATAGCTTGTCCGCACCAACAATGCTGCACAACTCTTCTGGCGACTTCTCTCAGGCCCACTCCACCCTGAAACTGGCGAATCACCAGCGGCCTGTATCCCGGCAGGTGACCTGTCTGAGAACTCAGGTGCTTGAAGACTCCGAGGACTCTTTCTGTAGGCGGCATCCAGGTTTGGGCAAGGCGTTTCCGTCCGGCTGTTCCGCGGTTTCAGAGCCCGCTTCCGAAAGTGTCGTGGGCGCCCTGCCAGCCGAGCACCAGTTCTCCTTCATGGAAAAGCGGAACCAGTGGCTGGTCAGTCAGCTGAGCGCCGCGTCACCTGATACAGGTCACGATTCCGACAAGTCTGACCAGTCTCTGCCCAATGCGTCAGCCGATAGTCTCGGGGGCTCCCAGGAGATGGTGCAGAGACCACAGCCGCACAGAAACCGGGCCGGGCTTGATCTGCCCACCATTGATACAGGCTACGATTCCCAGCCCCAGGACGTCCTTGGCATTCGCCAGCTGGAAAGGCCTCTGCCCTTGACCTCCGTGTGTTACCCCCAGGACCTGCCCCGCCCTTTGAGAAGCCGGGAGTTTCCCCAGTTTGAGCCCCAACGATACCCTGCCTGTGCTCAGATGCTGCCTCCGAACCTGAGCCCACACGCTCCCTGGAACTACCACTATCACTGTCCCGGCAGCCCCGATCACCAGGTGCCTTATGGACACGACTACCCGCGGGCTGCATACCAGCAGGTCATACAGCCTGCCTTGCCGGGTCAGCCGCTGCCCGGAGCTTCTGTGCGCGGCCTGCACCCCGTTCAGAAAGTGATCCTGAACTATCCAAGCCCATGGGACCATGAAGAGAGACCAGCCCAAAGAGATTGCTCTTTTCCTGGGTTGCCTAGACACCAAGACCAGCCTCACCACCAGCCTCCCAATCGGGCAGGCGCCCCAGGCGAAAGTCTCGAGTGCCCCGCCGAACTCAGACCACAGGTCCCTCAGCCCCCTTCCCCCGCGGCAGTACCCAGACCCCCCTCTAACCCACCCGCCCGGGGAACGCTCAAGACTTCAAATCTCCCAGAAGAGCTGCGCAAAGTGTTCATAACCTACAGCATGGACACCGCTATGGAGGTGGTTAAGTTCGTCAACTTCCTGCTGGTCAATGGGTTCCAGACTGCAATCGACATTTTTGAGGATAGAATTCGGGGAATCGACATCATCAAGTGGATGGAGAGATACCTGCGGGATAAGACAGTGATGATTATCGTGGCCATTAGTCCCAAGTACAAGCAAGATGTGGAGGGCGCAGAATCACAGTTGGACGAAGACGAGCACGGACTCCATACAAAATATATCCACAGGATGATGCAGATCGAGTTCATTAAACAAGGCTCCATGAATTTCCGCTTCATACCGGTCCTGTTTCCAAACGCAAAAAAAGAGCATGTACCCACTTGGCTCCAGAATACCCATGTCTACTCCTGGCCCAAGAACAAGAAGAATATCCTGCTGCGCTTGCTCAGAGAAGAAGAGTATGTCGCCCCTCCAAGGGGGCCCCTCCCCACACTCCAAGTAGTGCCACTT。

The results show that Ixekizumab and the tandem antibody can block Human IL-17A protein from activating 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc, the IC50 values are 2.235nM and 0.5888nM respectively, and the tandem antibody is superior to the control antibody. Ixekizumab blocking assay results are shown in FIG. 2 and tandem antibody blocking assay results are shown in FIG. 5.

Basic experimental example 5 stability experimental procedure and result analysis:

the experimental process comprises the following steps: by detecting fluorescence change through a micro-differential scanning fluorescence (nanoDSF) technique, thermal denaturation and chemical denaturation of proteins can be detected under natural conditions, and the temperature (Tm) at which 50% of the proteins are in an unfolded state and the temperature (Tagg) at which aggregation begins to occur can be accurately determined; the higher the thermal denaturation Tm value and Tagg, the more stable the antibody protein.

Taking 100 mu L of antibody to be detected, centrifuging at 4 ℃ for 10min at 12000 Xg, sucking samples by using capillaries, preparing two capillaries for each sample, taking the capillaries as parallel control, sequentially placing the capillaries into corresponding clamping grooves, ensuring that the capillaries are full of the samples, and carrying out detection analysis. The temperature rising range is 20-95 ℃, the temperature rising rate is 1 ℃/min, and the detection time is 75min.

Tm is defined as the melting temperature, tonset is defined as the temperature at which unfolding begins, tagg is defined as the aggregation temperature.

The antibody to be detected was the tandem antibody according to basic experimental example 2, and the positive control antibody Ixekizumab according to basic experimental example 3.

Ixekizumab results: t (T) _m1 The value is 56.10 plus or minus 0.02 ℃; t (T) _m2 The value is 79.84+/-0.13 ℃; t (T) _onset The value is 47.50 plus or minus 0.08 ℃; t (T) _agg The value is 61.86 plus or minus 0.23 ℃; results of tandem antibodies: t (T) _m1 The value is 58.13 plus or minus 0.06 ℃; t (T) _m2 The value is 82.43 +/-0.48 ℃; t (T) _onset The value is 51.28 +/-0.22 ℃; t (T) _agg The value was 58.48.+ -. 0.00 ℃.

EXAMPLE 1LV-G4-H10 Fc lentivirus preparation and detection methods

1. Lentiviral shuttle plasmid construction

Construction of a lentiviral shuttle plasmid of the G4-H10: fc fusion protein two candidate antibodies (IL-17 Nb) VHH sequences (SEQ ID NO: 16-17) were passed (GGGGS) ₃ The linked form is connected with IgG4 Fc (the amino acid sequence of the IgG4 Fc is shown as SEQ ID NO: 26, the nucleotide sequence is shown as SEQ ID NO: 27) in series and constructed on a lentiviral shuttle plasmid to form VHH- (GGGGS) ₃ The VHH-IgG4 Fc sequence was downstream of the EF-1alpha promoter, resulting in a G4-H10 Fc lentiviral shuttle plasmid.

SEQ ID NO: 27：

GCCCCCGAGTTTCTGGGAGGACCTAGCGTCTTTCTGTTCCCCCCCAAACCCAAGGACACACTGATGATCTCTAGGACCCCCGAGGTGACATGCGTCGTGGTGGACGTGAGCCAAGAGGACCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAAGTGCACAATGCCAAGACCAAACCTAGAGAAGAGCAGTTCAACAGCACCTATAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGACTGGCTGAACGGCAAGGAGTACAAGTGCAAAGTGAGCAACAAGGGCCTCCCCTCCTCCATCGAGAAAACCATCTCCAAGGCCAAGGGACAGCCTAGAGAGCCCCAAGTGTATACACTGCCCCCCAGCCAAGAGGAGATGACCAAGAACCAAGTGTCTCTGACATGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCTGTGGAGTGGGAGAGCAACGGCCAGCCCGAAAACAACTATAAGACCACCCCCCCCGTGCTGGACTCCGATGGCAGCTTCTTTCTGTACTCCAGACTGACCGTGGACAAAAGCAGATGGCAAGAGGGCAACGTGTTTAGCTGCTCCGTGATGCATGAGGCTCTGCACAACCACTATACCCAGAAGTCCCTCTCTCTGAGCCTCGGCAAGTGA。

2. Lentivirus preparation (Virus Package)

Lentivirus 24. 24 h before packaging, shake flasks were prepared and HEK293T (available from ATCC under the trade designation CRL-3216) cell density was adjusted to 1.0X10 ⁶ Per mL, 60 per bottle mL.

Preparation of transfection reagent/DNA complexes: taking 7.5 mL of 293T culture medium to a 15 mL centrifuge tube, adding 40 [ mu ] g g G-H10 of Fc lentiviral shuttle plasmid and 80 [ mu ] g of auxiliary plasmid (purchased from addGene, product numbers: 12253 and 12259), adding 360 [ mu ] L of transfection reagent after fully and uniformly mixing by up-and-down blowing of a pipette, immediately and uniformly mixing by up-and-down blowing of a 1 mL pipette, and standing for 10 min (not more than 15 min) at room temperature.

Dropwise adding transfection reagent/DNA complex into cells prepared the day before, shaking shake flask while adding, mixing thoroughly, placing shake flask at 37deg.C and 5% CO ₂ Culturing on a shaking table at 120 rpm.

After 24h incubation, the supernatant was collected and filtered into a virus centrifuge tube using a 0.45 μm needle filter, centrifuged at 45000 Xg, 4℃for 90 min.

After centrifugation, the supernatant was decanted and the residual supernatant was removed using a pipettor, the pellet was resuspended in 1 mL PBS and split-packed into virus split-tubes, 100 μl each, the LV-G4-H10: fc lentivirus.

3. Lentivirus titer detection

Inoculating 293T cells into a 24-well plate, culturing overnight, adding 20 mu L LV-G4-H10 (Fc virus stock solution, 10 times diluted virus solution and 100 times diluted virus solution respectively, continuously culturing for 24H, replacing fresh culture medium after 24H, continuously culturing for 9d, harvesting cells, and extracting genome DNA (the kit is purchased from Thermo, cat No.)K0721) with ddH ₂ O (double distilled water) is used for regulating the copy number of plasmid template, and the standard curve range is 1 multiplied by 10 ⁹ -1×10 ³ The primers were synthesized by the Souzhou Jin Weizhi biotechnology Co., ltd, and 2 XPCR Mix (purchased from Applied Biosystems, ABI, applied biosystems, USA, cat# A25742), primers, DNA, and PCR water (purchased from Thermo, siemens, cat# R0582) were mixed together according to the PCR premix and added to the corresponding PCR wells to perform the PCR reaction. 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 assays are shown in FIG. 6, and no matter whether the virus was primary or diluted 10-fold or 100-fold, the virus titer was between 2.3E+7 and 2.6E+7, with no significant differences between the three groups, so the lentiviral titer was 2.45E+7 TU/mL.

Example 2 preparation of G4-H10 Gene-modified Stem cells (G4-H10-MSC) and detection of infection efficiency

The prepared LV-G4-H10 is prepared by adding Fc slow virus into cultured mesenchymal stem cells with fusion degree of 70-80% according to MOI=10 (separating from neonatal umbilical cord by enzymolysis method, performing generation amplification and purification to obtain P2 generation mesenchymal stem cells), subjecting to 37deg.C, and CO ₂ Culturing, and after the cell density reaches 100%, passaging to successfully obtain mesenchymal stem cells (G4-H10-MSC) infected with LV-G4-H10: fc.

The obtained G4-H10-MSC and hUC-MSC cells were cultured at a ratio of 1X 10 ⁴ Individual/cm ² Culturing to T25 cell culture flask, adding transport inhibitor (purchased from BD Co., USA, cat# 555029), fixing, washing, staining FITC-Protein A (stained FITC-Protein A, purchased from BOSTER doctor, cat# BA 1120), loading, detecting FITC channel signal of mesenchymal stem cells (result, see figure 7), finding that G4-H10-MSC cells have positive rate of more than 60% in FITC channel signal, and normal mesenchymal stem cells (hUC-MSC) have no signal in FITC channel, which indicates that LV-G4-H10:Fc lentivirus can successfully infect mesenchymal stem cells, and the infection rate is close to 70%.

EXAMPLE 3ELISA method for detecting the expression of IgG4 and IL-17Nb in G4-H10-MSC cells

G4-H10-MSC and hUC-MSC cells obtained in example 2 were isolated according to 1X 10 ⁴ Individual/cm ² Culturing to a T25 cell culture flask, culturing for 72 hours, harvesting cell supernatant, sub-packaging, freezing, and detecting the content of IgG4 and IL-17Nb antibodies and blocking efficiency.

1. ELISA method for detecting the expression of IgG4 in G4-H10-MSC cell prepared in example 2

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

The cell culture supernatant prepared in this example was subjected to the above-mentioned human IgG4 ELISA kit to detect the content of IgG4 protein in the fusion protein, and as a result, it was found that G4-H10-MSC (detected after 25-fold dilution) highly expressed IgG4 up to 10.43.+ -. 0.52. Mu.g/mL, whereas normal hUC-MSC did not express IgG4 (see FIG. 8 for the results).

2. IL-17Nb antibody content detection

IL-17A nanobody expression was detected by IL-17A protein binding assay, and the cell supernatants obtained in this example were examined (20-fold best dilution was found after various-fold dilutions were made) after blocking with BSA by coating the ELISA plate with IL-17A protein (prepared in basic Experimental example 1) at a final concentration of 2. Mu.g/mL at 4℃overnight. Standard is G4-H10 fusion Protein (target antibody Protein prepared in basic experimental example 2), standard is added to corresponding wells at concentration ranging from 0-250ng/mL, sample wells are added to hUC-MSC and G4-H10-MSC cell supernatant (20-fold dilution) and incubated for 1H, HRP-labeled Protein a antibody (purchased from doctor, cat No. BA 1080) is then used as an enzyme-labeled antibody for 1H, TMB is finally added to develop color for 20min in the dark, and the enzyme-labeled instrument detects OD450nm values of each well after termination. As shown in FIG. 9, the high expression of IL-17Nb by G4-H10-MSC can be determined by the IL-17A binding experiment, the concentration is 4106+/-185.84 ng/mL, which shows that the IL-17Nb expressed by G4-H10-MSC can bind with IL-17A, while the IL-17Nb is not expressed by normal mesenchymal stem cells hUC-MSC.

EXAMPLE 4ELISA determination of IL-17Nb ability to block IL-17A binding to IL-17RA

IL-17A [ biotinylated ] of ACRO Biosystems (Beepxoles) IL-17RA Inhibitor Screening ELISA Kit (IL-17A/IL-17 RA blocking kit, cat. EP-139) was used to examine the ability of G4-H10-MSC 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. After the supernatant obtained in example 3 was subjected to a certain dilution (dilution of 5-fold was found to be optimal), the ability of IL-17Nb to block IL-17A/IL17RA binding in the supernatant was examined 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%.

All samples were tested after 5-fold dilution according to the instructions, as shown in FIG. 10, normal hUC-MSC cells could not block IL-17A/IL17RA binding, whereas G4-H10-MSC cells secreted IL-17Nb could block IL-17A/IL17RA binding, with a 5-fold inhibition as high as 60%.

EXAMPLE 5 Stem cell stability Studies

IL-17Nb expression was detected by ELISA, and the obtained IL-17 Nb-modified mesenchymal stem cells G4-H10-MSC and control hUC-MSC were expressed at 1X 10 ⁴ Individual/cm ² Inoculating 24-well plate, inoculating 8-well and 16-well respectively, culturing overnight, randomly selecting 8-well hUC-MSC, and replacing the hUC-MSC with 2000 final concentrationComplete medium of ng/mL of the G4-H10 fusion protein (antibody protein of interest prepared in basic Experimental example 2) was continued to culture, supernatants were harvested at 24H, 48H, 72H and 96H, respectively, and the content of IL17Nb in the supernatant was examined by the method of example 3 (ELISA method for detecting the expression of IL17 Nb). As shown in FIG. 11, the IL17Nb content detected in the hUC-MSC-added G4-H10 group was reduced from 1986.+ -. 79.90ng/mL to 1620.+ -. 37.54ng/mL over time, the concentration was gradually decreased, and the IL17Nb expressed by the IL17Nb-MSC (G4-H10-MSC) increased from 1333.+ -. 334.79 for 24H to 5554.+ -. 508.12ng/mL for 96H over time, with a very significant difference between the two groups of hUC-MSC-added G4-H10 and G4-H10-MSC for 72H and 96H, with a prolonged culture timeP< 0.001). The result shows that the G4-H10-MSC can stably and continuously express and secrete IL-17Nb, and the expression concentration and stability are better than those of the recombinant G4-H10 protein.

Example 6 validation of treatment efficacy in mice inflammatory disease models

This example uses the psoriatic arthritis (PsA) model for efficacy verification.

A model of psoriatic arthritis (PsA) was constructed by intraperitoneal injection of mannan in B-hIL17A transgenic mice (Bai Chart pharmaceutical technologies Co., ltd., cat No. 110053). 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. The molding was performed by three intraperitoneal injections of mannan (SIGMA, SIGMA, M7504-5G) at a volume of 200. Mu.L per animal per 100mg/mL of mannan per abdominal cavity, i.e., 20mg of mannan per animal per abdominal cavity.

The PsA model group was randomly divided into four groups: hUC-MSC treatment group (2X 10) ⁶ Individual/individual), positive antibody Ixekizumab (1 mg/kg body weight), G4-H10-MSC (2X 10) ⁶ Individual/individual) and model control groups;

drug treatment groups were D3 and D7 intravenous administration treatments. During the experiment, animal body weight was measured every 2 days, animal survival and health were observed, and animal skin and fore-and-aft paw were scored according to relevant indicators. D14 euthanized animals, peripheral blood from mice was collected, serum was isolated, and cytokines such as mIL-6, mIL-23, mTNF- α, etc., were detected (kit brands were all Biolegend).

Skin clinical 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.

Paw (joint) scoring criteria were 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.

As shown in fig. 12, the body weight of the PsA model group after the mannan modeling was significantly reduced compared with the normal control group, the hUC-MSC treated group and the G4-H10-MSC treated group both restored the body weight of the mice, while the positive antibody group restored the body weight of the mice less significantly, and the test endpoint G4-H10-MSC treated group had significantly higher body weight than the positive antibody group.

As shown in fig. 13-14, the clinical scores of animal paw and skin were significantly increased in both the PsA model group and paw after mannan modeling, and positive antibody, hic-MSC treatment and G4-H10-MSC treatment groups significantly reduced the clinical scores of the paw and skin in the model animals, while the weight of the G4-H10-MSC treated mice was significantly better than that of the positive antibody group.

The inflammatory factor detection results are shown in fig. 15-17, and the serum of the PsA model animal is obviously increased in cytokines such as IL-6, IL-23 and TNF-alpha after the mannan modeling, while the serum IL-6 and TNF-alpha levels of the model animal can be obviously reduced in both the hUC-MSC treatment and the G4-H10-MSC treatment, and the serum IL-23 level of the model animal can be obviously reduced in both the positive antibody group and the G4-H10-MSC treatment.

Finally, it should be noted that the above description is only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and that the simple modification and equivalent substitution of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present invention.

Claims (18)

1. The stem cell modified based on the nano antibody gene is characterized by comprising, expressing or secreting a first antibody and a second antibody, wherein the first antibody comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO. 1-3; the second antibody comprises HCDR4, HCDR5 and HCDR6 with sequences shown in SEQ ID NO. 4-6.

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

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

wherein the sequence of FR1-FR8 comprises the amino acid sequence shown in SEQ ID NO. 7-14.

3. The stem cell of claim 2, wherein the sequence of the first antibody comprises the amino acid sequence shown in SEQ ID No. 16; the sequence of the second antibody comprises an amino acid sequence shown as SEQ ID NO. 17.

4. The stem cell of claim 1, wherein the first antibody and the second antibody are linked directly or via a linker; the linker comprises an amino acid sequence shown in SEQ ID NO. 15.

5. The stem cell of claim 4, wherein the sequence of the fusion protein comprises the amino acid sequence as set forth in SEQ ID NO. 16-SEQ ID NO. 15-SEQ ID NO. 17-SEQ ID NO. 26.

6. The stem cell of claim 1, comprising: a nucleic acid molecule 1 encoding a first antibody and a nucleic acid molecule 2 encoding a second antibody.

7. The stem cell of claim 6, wherein the nucleic acid molecule 1 and the nucleic acid molecule 2 are linked by a nucleotide sequence encoding an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO. 15.

8. The stem cell of claim 7, wherein the nucleic acid molecule 1 encodes a nucleotide sequence comprising the amino acid sequence set forth in SEQ ID No. 16; the nucleic acid molecule 2 codes for a nucleotide sequence comprising the amino acid sequence shown in SEQ ID NO. 17.

9. The stem cell of claim 8, comprising a nucleic acid molecule 3, wherein said nucleic acid molecule 3 comprises a nucleotide sequence encoding an amino acid sequence as set forth in SEQ ID NO. 16-SEQ ID NO. 15-SEQ ID NO. 17-SEQ ID NO. 26.

10. The stem cell of claim 1, further comprising, expressing or secreting a biologically active protein or functional fragment thereof capable of extending the half-life of an antibody in vivo.

11. The stem cell of claim 10, comprising a nucleic acid molecule encoding a biologically active protein or functional fragment thereof capable of extending the half-life of the antibody in vivo.

12. The stem cell of claim 11, wherein the biologically active protein or functional fragment thereof is selected from at least one of an immunoglobulin Fc domain, serum albumin, albumin binding polypeptide, prealbumin, carboxy terminal peptide, elastin-like polypeptide, his tag, GST tag, MBP tag, FLAG tag, and SUMO tag.

13. The stem cell of any one of claims 1-12, wherein the stem cell is selected from embryonic stem cells, adult stem cells, mesenchymal stem cells, umbilical cord blood stem cells, hematopoietic stem cells, neural stem cells, adipose stem cells, skin stem cells, or muscle stem cells.

14. Cell culture, characterized in that it is obtained by culturing stem cells according to any one of claims 1 to 13.

15. A pharmaceutical composition, characterized in that it comprises a stem cell according to any one of claims 1-13 or a cell culture according to claim 14.

16. Kit, characterized in that it comprises a stem cell according to any one of claims 1 to 13 or a cell culture according to claim 14.

17. A method of preparing a stem cell according to any one of claims 1 to 13, comprising: introducing the nucleic acid molecule into the stem cell by viral transfection, liposome transfection, electrotransfer, gene editing or mRNA transfection; the nucleic acid molecules comprise a nucleic acid molecule 1 encoding a first antibody and a nucleic acid molecule 2 encoding a second antibody.

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

D1: contacting the stem cell of any one of claims 1-13 or the cell culture of claim 14 with a test sample;

d2: detecting the antigen-antibody complex;

d3: and judging the result.