CN114349857B

CN114349857B - Treg cell preparation method and application in aspect of autoimmune diseases

Info

Publication number: CN114349857B
Application number: CN202210248939.4A
Authority: CN
Inventors: 李少波; 黄昱; 亓爱杰; 马润林
Original assignee: Nuosa Union Beijing Biomedical Technology Co ltd
Current assignee: Nuosa Union Beijing Biomedical Technology Co ltd
Priority date: 2022-03-15
Filing date: 2022-03-15
Publication date: 2022-05-24
Anticipated expiration: 2042-03-15
Also published as: CN114349857A

Abstract

The invention relates to a preparation method of Treg cells and application in autoimmune diseases, the invention also provides a PDE9A monoclonal antibody, the antibody can promote the proliferation of Treg cells and improve the activity of the Treg cells, and the proliferated Treg cells have better effect of treating immune-related diseases.

Description

Treg cell preparation method and application in aspect of autoimmune diseases

Technical Field

The invention relates to the field of biology, in particular to a preparation method of Treg cells and application of the Treg cells in autoimmune diseases.

Background

Regulatory T cells (Tregs) are a subset of T cells that control autoimmune reactivity in vivo, and are also referred to earlier as supressor T cells. The regulatory T cells can be divided into naturally-occurring natural regulatory T cells (n T-regs) and induced adaptive regulatory T cells (a T-regs or i T-regs), such as Th3 and Tr1, and further, CD8 Treg, NKT cells and the like are closely related to the occurrence of autoimmune diseases, and abnormal expression of the regulatory T cells can cause the autoimmune diseases.

Regulatory T cells (Regulatory T cells) are one of the important factors for maintaining immune tolerance of the body, are exported to the periphery after being produced by the thymus, and inhibit the activation and proliferation of potential autoreactive T cells existing in the normal body by means of active regulation, thereby regulating the immunity of the body, such as preventing the occurrence of autoimmune diseases. The reduction in the number or dysfunction of CD4 CD 25'/FOXP 3 cells may lead to the development of autoimmune diseases, and the number and function of regulatory T cells such as multiple sclerosis, active rheumatoid arthritis, type I diabetes, etc. may vary. A significant increase in regulatory T cells has been found in a number of malignant diseases such as lung, pancreas and breast cancer. Regulatory T cells also block the anti-tumor immune response leading to increased mortality and can also be used for post-operative follow-up before and after bone marrow transplantation.

Autoimmune diseases (AIDs) are diseases caused by the damage of body tissues, organs and systems due to the immune reaction of immune dysfunction to autoantigens, and include various diseases such as autoimmune liver disease (AILD), autoimmune thyroid disease, autoimmune hemolytic anemia, systemic sclerosis, systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, multiple sclerosis, myasthenia gravis, scleroderma, mixed connective tissue disease and the like. The pathogenesis of AID is unclear, the clinical manifestations and the diagnostic methods are different, a plurality of cytokines participate in the pathogenesis, but the immune response with the participation of effector T cells is more obvious, and the treatment of AID by adrenocortical hormone and/or immunosuppressant has certain curative effect. T cells comprise two new subgroups of Th17 and Treg besides subgroups Th1 and Th2, IL-17 which is a main effector of Th17 cells has a proinflammatory effect, IL-4, IL-10, transforming growth factor beta (TGF-beta) and the like which are secreted by Treg cells have an immunosuppressive effect, and the effects of the two in AID become hot spots in recent years.

Treg cells are T cell subsets except Th1, Th2 and Th17, have an immunoregulation function, can secrete cytokines such as IL-4, IL-10, TGF-beta and the like, and play an important role in immune homeostasis and immune tolerance induction. Treg cells can be divided into natural regulatory T cells (nTregs) and inducible regulatory T cells (iTregs), wherein the nTregs are formed by differentiating thymocytes, and the iTregs are formed by differentiating peripheral mature T cells under the stimulation of specific antigens and under the induction of cytokines. Tregs exert immunosuppressive effects through a variety of mechanisms, such as the production of suppressive cytokines, disruption of the metabolism of targeted T cells, cytolysis, and regulation of dendritic cell differentiation and function. The transcription factor forkhead box P3(Foxp3) is involved in the differentiation and specific high expression of Treg cells, and is the most specific Treg cell marker at present. The transcriptional activator STAT5 is another important factor involved in Treg cell differentiation and survival.

Treg cells play a role in immune balance. Among the most severe Treg deficiencies, IPEX (immunoregulation, polyendocrinopathy, enteropathy, X-linked) syndrome is the most prominent, with mutations in the key Treg lineage specific transcription factor FOXP3 (forkhead box P3) leading to Treg deficiency, leading to fatal multi-organ inflammation and autoimmunity. Importantly, a reduction in the number and/or function of Tregs is associated with the pathology of many common autoimmune diseases. Indeed, in some disease environments, Treg function is deficient or the production of Treg growth and survival factors such as interleukin-2 (IL-2), plus the production of high concentrations of inflammatory cytokines such as IL-6, IL-12, IL-1 in the tissue environment destabilizes the Treg, leading to uncontrolled inflammation and excessive tissue damage.

Therefore, much ongoing research is beginning to re-use previously approved drugs, such as Treg growth factor (e.g., IL-2) and Treg stabilizing factor (e.g., rapamycin), to enhance Treg function as a drug that can be used as a means to control various autoimmune diseases, GvHD and organ transplant rejection. These results highlight the opportunity to directly alter the pathological immune response by enhancing Treg activity, which is also based on the ACT target of tregs.

The first preclinical proof-of-concept study for the treatment of autoimmune diseases was performed using polyclonal regulatory T cells (Tregs), referred to as suppressor cells, isolated based on the expression of a variety of cell surface markers including CD4, CD25, and CD 62L. These efforts, and subsequent studies using purified FOXP3 + Tregs, were successful, facilitating several clinical trials in a variety of disease settings, including organ transplant rejection, GvHD, T1D, and autoimmune syndrome.

Research shows that the PDE9A inhibitor can be used for preparing and increasing the content of Tregs (namely Regulatory T cells) and can be applied to the treatment of autoimmune diseases. The PDE9A inhibitor can be used for preparing a reagent for increasing the content of Tregs. However, at present, the number of isolated Treg cells is small, so that how to research how to obtain Treg cells with high yield is the current research direction.

Disclosure of Invention

The inventor finds that the PDE9A inhibitor can improve the content of Treg cells, and on the basis, develops the monoclonal antibody inhibitor aiming at the PDE9A and a corresponding method for improving the yield of the Treg cells.

Further, the present invention provides a monoclonal antibody 1A3 of PDE9A, wherein the light chain variable regions of said antibody are respectively as follows:

light chain variable region (SEQ ID NO: 1)

DIVITQRPALMAASPGEKVTITCALALVYWPPKYFWYQQKSGISPKPWIYDYTDIKWGVPARFSGSGSGTSYSLTITSMEAEDAATYYCQKDILDGNLFGAGTKLELK

Heavy chain variable region (SEQ ID NO: 2)

EVQLEESATELARPGASVKLSCKASGYIFSKDSNVWIKQRPGQGLEWIGLEKMEANIREPYCDMEGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGSHSTYLSWGLGTTLAVSS

Further, the present invention provides a gene encoding the monoclonal antibody against PDE9A as described above.

Furthermore, an expression vector of a gene encoding a monoclonal antibody against PDE 9A.

In addition, another mode of the antibody of the present invention is as a "derivative" of any of the following antibodies and antigen binding fragments thereof. The term "derivative" refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to an antigen, but which includes one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to the "parent" (or wild-type) molecule. Such amino acid substitutions or additions may introduce naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. The term "derivative" includes, for example, variants having altered CH1, hinge, CH2, CH3, or CH4 regions, such as to form antibodies and the like having variant Fc regions that exhibit enhanced or impaired effector or binding properties. The term "derivative" also includes non-amino acid modifications, e.g., amino acids that can be glycosylated (e.g., altered levels of mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-ethanoneuraminic acid, etc.), acetylated, pegylated, phosphorylated, amidated, derivatized with known protecting/blocking groups, proteolytically cleaved, linked to cellular ligands or other proteins, and the like. In some embodiments, the altered carbohydrate modification modulates one or more of the following: solubilization of the antibody, promotion of subcellular transport and secretion of the antibody, promotion of antibody assembly, conformational integrity and antibody-mediated effector functions. In particular embodiments, the altered carbohydrate modification enhances antibody-mediated effector function relative to an antibody lacking the carbohydrate modification. Carbohydrate modifications that result in antibody-mediated changes in effector function are well known in the art.

Alternatively, the coding sequence of the antibody may define the sequence homologously, optionally using the methods discussed in more detail below. For example, the nucleic acid sequence may differ from the sequences described above in that (a) the variable region may be separated from the constant domains of the light and heavy chains, (b) the nucleic acid may differ from the nucleic acids described above without affecting the residues it encodes, (c) the nucleic acid may differ from the nucleic acids described above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, (d) the nucleic acid may differ from the nucleic acids described above due to its ability to hybridize under high stringency conditions, exemplified by low salt and/or high temperature conditions, e.g., provided by about 0.02M to about 0.15M NaCl at a temperature of about 50 ℃ to about 70 ℃), (e) the amino acids may vary from those described above by a given percentage, for example 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, or (f) amino acids may be altered from those described above by allowing conservative substitutions

Furthermore, the invention provides the application of the monoclonal antibody of PDE9A in preparing medicines for treating immune diseases.

Still further, the present invention provides a pharmaceutical composition comprising a monoclonal antibody drug of PDE9A or Treg cells cultured with a monoclonal antibody of PDE9A and a suitable carrier.

The present disclosure provides compositions comprising a prophylactically or therapeutically effective amount of Treg cells cultured with an antibody or fragment thereof or a monoclonal antibody to PDE9A and a suitable carrier. In a particular embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, excipient or vehicle to which therapy is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.

The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may include standard carriers such as Pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, examples of suitable Pharmaceutical preparations being described in "Remington's Pharmaceutical Sciences". Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with an appropriate amount of carrier to provide the patient with a suitable form of administration. The formulation should be adapted for administration, which may be oral, intravenous, intraarterial, buccal, intranasal, nebulized, bronchial inhaled, rectal, intravaginal, delivered topically or by mechanical ventilation.

The enriched TREG cells expanded by the methods of the present invention are preferably administered to the subject for a period of at least one day, preferably 7-180 days, more preferably 30-90 days.

The preferred dosing regimen is a constant infusion of at least one month equivalent to 10⁶-10⁸One/kg/day, but can reach 10⁷-10⁸One/kg/day. Alternatively, daily injections (1 or 2 per day) may be given, up to 10 per day⁶-10⁸One/kg/day, but can reach 10⁸One/kg/day.

In the case of local delivery, higher concentrations may be used due to lack of toxicity, but lower doses may also be used. For local delivery, the range may vary depending on the extent of absorption, ability to reach the target organ (from pulmonary, oral or topical application), and therapeutic efficacy. Thus, in topical application, the dosage may be in the range of 0.001g to 50 mg/kg/day.

In a preferred embodiment, the Treg cells are expanded by extracting a mixed population of T cells from the patient, isolating a subpopulation of T cells enriched for Treg cells from the population by negative and positive immunoselection and/or cell sorting, and expanding the subpopulation of Treg cells by contacting the subpopulation with an effective amount of a PDE9A monoclonal antibody and IL-2. Preferably, the enriched T cell subpopulation comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or greater than 98% CD4+ CD25+ T cells (Treg cells). More preferably, the enriched subpopulation comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or greater than 98% CD4+ CD25+ T cells (Treg cells). The most preferred subpopulation comprises greater than 98% CD4+ CD25+ T cells (Treg cells).

After expansion of the Treg cells, the cells are preferably transplanted or reintroduced into the patient. This is typically performed as known in the art, and typically involves injecting or introducing the treated cells into a patient by intravenous administration, as will be understood by those skilled in the art. For example, the cells may be placed in an infusion bag by injection using a sterile syringe or other sterile transfer mechanism. The cells can then be infused immediately by intravenous administration over a period of time, for example 15 minutes. Additional reagents such as buffers or salts may also be added to the cells. Alternatively, local transplantation of the treated cells may be performed when necessary.

Another example of a disease in which the invention may be used is a disease with an autoimmune component, including those genetic diseases such as Wiskott-Aldrich syndrome in which Treg cells exhibit a significant deficiency in their activation, survival, migratory proliferation and/or suppressive function.

Previous studies have demonstrated a clear role for tregs in controlling intestinal inflammation and suggest that loss of Treg cell function is responsible for the lack of immune modulation observed in patients with inflammatory bowel disease. Inflammatory bowel disease (crohn's disease, ulcerative colitis) is an idiopathic chronic inflammatory disease that results in a variety of clinical symptoms such as abdominal pain, severe diarrhea, rectal bleeding and wasting. Several models of IBD have been developed to increase our understanding of the mechanisms of these inflammatory diseases. Some of these models paralleled the amplified pro-inflammatory T cell responses observed in some human forms of IBD. In fact, chronic intestinal inflammation is mediated primarily by T lymphocytes. Adoptive transfer models have demonstrated that T cells initiate and maintain intestinal and/or colonic inflammation. They also demonstrate a clear role for TREG in controlling intestinal inflammation. Tregs appear to mediate some of their effects through the cytokines IL-10 and TGF-13. Therefore, enhancing the number and activity of CD4+ CD25+ Treg cells is a clear target for treating IBD and inhibiting inflammation.

Advantageous effects

The invention relates to the proliferation of Treg cells and the corresponding treatment for immune-related diseases, and more particularly provides a PDE9A monoclonal antibody, which can promote the proliferation and improve the activity of the Treg cells, and the proliferated Treg cells have a better effect of treating the immune-related diseases.

Drawings

FIG. 1 treatment outcome scoring plot

Detailed Description

The following detailed description refers, by way of illustration, to specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and structural and logical changes may be made without departing from the scope of the present invention. The various embodiments are not necessarily mutually exclusive, as some embodiments may be combined with one or more other embodiments to form new embodiments.

EXAMPLE 1 preparation of PDE9A monoclonal antibody

Specific antigenic sequences were designed and obtained for the co-conserved regions and the highly immunogenic regions based on the protein sequence of human and murine PDE 9A: the antigen sequence is expressed and synthesized by Emberson (Beijing) biotechnology limited. The purity of the peptide is more than 98.5 percent through HPLC analysis, and the synthetic peptide is used as immunogen for standby after being coupled with hemocyanin KLH.

3 mice are immunized once, 50ug of PDE9A polypeptide KLH conjugate is taken each time, a mouse subcutaneous multipoint injection method is adopted, 3 weeks are separated, blood is taken after the third immunization for 1 week to measure the titer, a person with high titer is selected for boosting immunization, cell fusion is prepared after 3 days of boosting immunization, and hybridoma cell strains are obtained by cloning culture according to the traditional monoclonal antibody preparation technology by a limiting dilution method. And after repeated cryopreservation and resuscitation, identifying the chromosome and monoclonal antibody secretion stabilizer to establish 3 hybridoma cell strains in total, namely three hybridoma cell strains 1A3, 4D3 and 6E 2.

Preparing ascites from BALB/c mice aged 8 weeks by a conventional method. The monoclonal antibody ascites was treated with 1: dilution is carried out at a ratio of 100, the reaction titer is detected by indirect ELISA, and the titer of 3 hybridoma cell strains is more than 1: 12800. And simultaneously, carrying out subclass identification on the monoclonal antibodies in the purified ascites by using an HRP-labeled goat anti-mouse antibody subclass identification kit, wherein 3 monoclonal antibodies are IgG1, and light chains are kappa chains.

PDE9A protein detection was performed by indirect ELISA. Three monoclonal antibodies were able to specifically bind to PDE9A protein (ab 125666) by indirect ELISA. The protein is not combined with BSA protein and KLH, and shows better specificity.

Example 2 characterization of the PDE9A monoclonal antibody 1A3

Antibody binding and dissociation constants were determined using Fortibio. The affinity of the anti-1A 3 antibody was determined using an octetRED (Fortebio, USA) instrument. The concentration of PBS diluted antibody is 10 mug/mL, and an AHC sensor is coated; PBS was a control, diluted 1A3 was at a concentration of 0.1. mu.g/mL, 1. mu.g/mL, 3. mu.g/mL, 10. mu.g/mL, and the binding and dissociation curves for the interaction of anti-1A 3 antibody with PDE9A protein were determined; and (5) calculating the dissociation constant of the interaction of the two by using a GlobeFitting fitting curve. As a result, the dissociation constant of the 1A3 monoclonal antibody obtained by the invention reaches 1.21nM, and the monoclonal antibody has a better binding effect.

Example 3 isolation and validation of proliferative Activity of human peripheral blood CD4+ CD25+ regulatory T cells (Tregs)

Collecting peripheral blood of healthy people, separating PBMC by density gradient centrifugation, positively sorting CD8+ T cells in the PBMC by Dynabeads, and then removing beads on CD8+ T cells by DERAChaBEAD; when sorting CD4+ T cells, Miltenyi magnetic beads were used for negative sorting. The remaining depleted T cells (including CD8+ T cells and CD4+ T cells) are used⁶⁰Co irradiation (30 Gy) as Antigen Presenting Cells (APC). Total CD4+ cells were then sorted positively by anti-CD 25 magnetic beads to separate CD4+ CD25+ T cells from CD4+ CD25-T cells.

The separated CD4+ CD25+ Treg is inoculated in a 96-well U-shaped bottom culture plate, and the number of cells per well is 1 multiplied by 10⁴The final volume was 200. mu.1. The culture medium is RPMI1640 complete culture medium (containing 10% inactivated fetal calf serum, 50U/ml each of cyan and streptomycin), the ratio of Dynal Beads coated with CD 3/CD 28 monoclonal antibody to cells in the culture system is 4:1, and the concentration of Dynal Beads is addedIL-2 at 200U/ml, 5% CO at 37 ℃₂The cultivation was carried out under saturated humidity conditions. The experiments were divided into 2 groups: group 1, in a CD4+ CD25+ Treg culture system, PDE9A monoclonal antibody 1A3 is not added, two rounds of cocultivation are carried out, DynalBeads coated by CD 3/CD 28 monoclonal antibodies are removed on day 6, only 20U/ml IL-2 is added, and the DynalBeads coated by CD 3/CD 28 monoclonal antibodies are used for stimulating again on day 8 to continue the second round of cultivation; the culture system of group 2 was cultured under the same conditions as those of group 1 except that 100 mg/L of the PDE9A monoclonal antibody 1A3 was added. Each experimental group was supplemented with exogenous human IL-2 and RPMI1640 complete medium every 2 to 3 days. The results show that the expression of the second group of CD4+ CD25+ reaches 99.7%, which is obviously improved compared with the 94.3% of the first group; meanwhile, the number of CD4+ CD25+ cells in the second group is 34.2% higher than that in the first group, which shows that the addition of the PDE9A monoclonal antibody 1A3 can promote the proliferation of CD4+ CD25+ Treg cells.

Example 4 cellular vs. CIA mouse experiments

The sorted purified CD4+ CD25+ Treg cells were resuspended in complete medium containing 1000 u/ml IL-2 and 100 mg/L PDE9A monoclonal antibody 1A3, seeded into 96-well plates and incubated at saturated humidity, 5% CO₂Culturing in an incubator at 37 ℃, replacing the culture medium every 3d, continuously culturing for 9d, and collecting cells, namely the proliferated and activated CD4+ CD25+ Treg cells, which are named as cells 1.

The sorted purified CD4+ CD25+ Treg cells were resuspended in complete medium containing 1000 u/ml IL-2, seeded into 96-well plates and incubated at saturated humidity, 5% CO₂And culturing in an incubator at 37 ℃, replacing the culture medium every 3d, continuously culturing for 9d, and collecting cells, namely the control CD4+ CD25+ Treg cells after proliferation and activation, which are named as cells 2.

Establishing and evaluating a CIA mouse model: type II collagen solution (2 mg/ml in 0.05 mol/L acetic acid) in volume 1:1, completely mixing and emulsifying, and injecting 50ul of mixed solution (containing 50ug of II type collagen) into the root of the tail of the mouse subcutaneously, and marking as the day 0; on day 21, 50ul of the mixed solution was used for the second immunization. Treatment was performed on day 22. CD4+ CD25+ Tregs-treatment CIA mice after secondary immunization were randomly divided into control group, cell 1 treatment group, cell 2 treatment group, cell 1 combination 1A3 treatment group, and 1A3 treatment group. The cell 1 and cell 2 treatment groups were separately treated by intravenous infusion of 100ul Tregs cell suspension (1X 10) into the tail root of the mouse⁷Rat); 1A3 treatment group was injected intraperitoneally with 100ul of 1.0 mg/kg mab; cell 1 in combination with 1a3 treatment group the combination treatment group was given two treatments simultaneously: the control group was injected intravenously with 100ul of PBS solution simultaneously with the intraperitoneal injection. Treatment was once per week.

Scoring was started on day 21, once a week, and arthritis severity was assessed using the following scoring criteria: 0 point, normal joints and no obvious symptoms; 1 point, inflammation and swelling of single toe joint; 2 minutes, more than two (including two) toe joints are red and swollen but not reach the whole sole, or the whole sole is slightly red and swollen; inflammation and swelling of the whole sole in 3 minutes; 4 points, severe redness and swelling of the entire sole, or deformation and stiffness of the ankle joint. The severity of arthritis in each mouse was expressed as a 4-foot cumulative total.

The scoring results for each group of CIA mice are shown in fig. 1.

As can be seen from the results in FIG. 1, each treatment group delayed the onset of arthritic symptoms in CIA mice. Compared with a control group, the cell 1 combined 1A3 treatment group can significantly improve the clinical score of arthritis, the score is only (3.12 +/-0.19) at 49 days, and the score is significantly reduced (P is less than 0.05) compared with that of the control group (10.2 +/-0.20); from the experimental results, the treatment effect of the treatment group with the cell 1 obtained after the treatment with the monoclonal antibody is better than that of the treatment group with the Treg cells not treated with the monoclonal antibody, which shows that the activity of the Treg cells obtained after the treatment with the monoclonal antibody is stronger. And after the 1A3 monoclonal antibody is singly used for treating the mice, the proliferation of Treg cells in the mice can be stimulated to further inhibit arthritis symptoms, but the effect is slightly worse than that of the treatment of directly using the Treg cells, which also shows that the monoclonal antibody has certain hysteresis for stimulating the proliferation of the Treg cells.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> Nosai Union (Beijing) biomedical science and technology Co., Ltd

<120> preparation method of Treg cells and application of Treg cells in aspect of autoimmune diseases

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Asp Ile Val Ile Thr Gln Arg Pro Ala Leu Met Ala Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ala Leu Ala Leu Val Tyr Trp Pro Pro

20 25 30

Lys Tyr Phe Trp Tyr Gln Gln Lys Ser Gly Ile Ser Pro Lys Pro Trp

35 40 45

Ile Tyr Asp Tyr Thr Asp Ile Lys Trp Gly Val Pro Ala Arg Phe Ser

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Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Thr Ser Met Glu

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Glu Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

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115

Claims

1. Monoclonal antibody 1A3 of PDE9A, wherein the variable region of the light chain of the monoclonal antibody 1A3 is as shown in SEQ ID NO: 1 is shown in the specification; the heavy chain variable region sequence is shown as SEQ ID NO: 2, respectively.

2. An expression vector encoding monoclonal antibody 1A3 of PDE9A of claim 1.

3. IncreaseUse of post-colonized CD4+ CD25+ Treg cells in the manufacture of a medicament for the treatment of arthritis, wherein said post-colonized CD4+ CD25+ Treg cells are isolated CD4+ CD25+ Treg cells in 5% CO in complete medium containing 1000 u/ml IL-2 and monoclonal antibody 1A3 of 100 mg/L PDE9A₂And culturing in an incubator at 37 ℃, replacing the culture medium every 3d, and continuously culturing the proliferated and activated CD4+ CD25+ Treg cells collected after 9d, wherein the light chain variable region of the monoclonal antibody 1A3 is shown as SEQ ID NO: 1 is shown in the specification; the heavy chain variable region sequence is shown as SEQ ID NO: 2, respectively.

4. The use of claim 3, wherein the medicament further comprises a pharmaceutically acceptable carrier.

5. The use of claim 4, wherein the pharmaceutically acceptable carrier comprises an excipient.

6. The use as claimed in claim 5, wherein the excipient comprises starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk, glycerol or propylene glycol.