CN109864992B

CN109864992B - Vitamin D3 or its active form 1 alpha, 25(OH)2D3Application in inhibiting PD-1 expression

Info

Publication number: CN109864992B
Application number: CN201711248940.2A
Authority: CN
Inventors: 高云飞; 尹芝南; 黎鹏; 郑良焰
Original assignee: Jinan University
Current assignee: Beijing Deyan Technology Co.,Ltd.
Priority date: 2017-12-01
Filing date: 2017-12-01
Publication date: 2021-07-27
Anticipated expiration: 2037-12-01
Also published as: CN109864992A

Abstract

The present invention discloses vitamin D3 or its active form 1 alpha, 25(OH)₂D₃The application of the compound in inhibiting the expression of PD-1. 1 α,25(OH)₂D₃The expression of PD-1 on the surfaces of activated CD8T cells and gamma delta T cells can be inhibited through VDR, meanwhile, VDR can promote the expression of Blimp1, and Blimp1 can effectively inhibit the expression of PD-1, so that the tumor killing function of T cells is enhanced. 1 α,25(OH)₂D₃The vitamin D3 can be combined with cytokines such as IL-2 and IL-15, can effectively reduce the expression of immune cells PD-1, promote the expression of CD28, further enhance the killing function and the metabolic function of T cells and maintain the steady state of the T cells, and can promote the T cells to secrete I-type interferon due to the fact that the vitamin D3 has little toxic and side effect and a large tolerance range. Thus, it can be used for the treatment of various diseases, including tumors and infectious diseases.

Description

Vitamin D3 or its active form 1 alpha, 25(OH)2D3Application in inhibiting PD-1 expression

Technical Field

The invention belongs to the technical field of biomedicine of tumor immunocyte treatment and antivirus, and particularly relates to vitamin D3 or an active form 1 alpha, 25(OH) thereof₂D₃The application of the compound in inhibiting the expression of PD-1.

Background

Tumors are one of the major diseases that are at present threatening human health and life, and the tumor-based treatments are mainly surgical therapy, chemotherapy, radiation therapy and immunotherapy. Immunotherapy is currently well-established and occupies a large market share. In the immunotherapy of tumors, PD-1 monoclonal antibodies are mainly used by the most famous pharmaceutical companies worldwide, among which valuable Opdivo (El-Khouuery AB, Sangro B, Yau T, Crocenzi TS, Kudo M, Hsu C, Kim TY, Choo SP, Trojan J, Welling TH Rd, Meyer T, Kang YK, Yeo W, Chorra A, Anderson J, Dela Cruz C, Lang L, Neely J, Tang H, Datani HB, Melero I. Nivolumab in tissues with advanced pharmaceutical cells, Carcinoma (CheckMate): 040 an open-labell, Schnon-complex, phase 1/2 patent application 20178, Lancek J20192, J, Young J, Hakkenk J, K S, K, lorigan P, Neyns B, Blank C, Petrella TM, Hamid O, Zhou H, Ebbinghaus S, Ibrahim N, Robert C.Pembrolizumab versuls ipilimumab for advanced melanoma final overview of multi overview survivals of a multicentre, randomised, open-label phase 3study (KEYNOTE-006), Lancet.2017Aug 16. pi: S0140-6736) 31601-X; schachter J, Ribas A, Long GV, Arance A, Grob JJ, Mortier L, Daud A, Carlino MS, McNeil C, Lotem M, Larkin J, Lorigan P, Neyns B, Blank C, Petrella TM, Hamid O, Zhou H, Ebbinghaus S, Ibrahim N, Robert C. Pembrolizumab verized ab verisimi for advanced melanoma. final overall surval results of a multicentre, randomid, open-label Auphase 3study (KEYNE-006). Lancet.2017 g 16.pii: S0140-6736. (17) PD-601. target point is PD-1; the term "environmental impact" refers to environmental impact of magnesium alloy, magnesium alloy, magnesium alloy, magnesium alloy, magnesium alloy, magnesium 1/2Open-label study, JAMA Oncol.2017Sep 14; 3, (9) e172411.) all obtain U.S. FDA approval targeting PD-L1. These monoclonal antibodies all achieved good clinical results. However, the research and development of the PD-1 small molecule inhibitor are few, and the main reason is that no effective screening method is found at present, and in addition, the potential toxic and side effects of the artificially synthesized compound on the body are also large.

Programmed cell death receptor (PD-1) proteins are inhibitory members of the CD28 receptor family, whereas CD28 family members include CD28, CTLA-4, ICOS and the like, and are expressed predominantly on activated T cells, B cells and bone marrow cells (Agata Y, Kawasaki A, Nishimura H, Ishida Y, Tsubata T, Yagita H, Honjo T. expression of the PD-1 anti-orientation on the surface of stimulated mouse T and B lymphocytes. int Immunol. 1996; 8(5) 765-72.; Okazaki T, Iwai Y, Honjo T. New expression co-antigens: inductive PD-stimulation 1. and J. Opunti J. E.J., J.E.J.E.J.P.P.P.E.P.K.R.P.S. H, C.S. J.P.S. K.S. J.S. K.S. J.R.S. J.P.S. E.S. K.S. J.S. 7, J.S. K.S. 7, J.S. K.S. K. 7, J.S. K.S. K. 7, collins M, Carreno BM. program depth-1 engage expression up TCR activation has effects on simulation and cytokine-drive promotion, initiation of ICOS, IL-4, and IL-21, but not CD28, IL-7, and IL-15responses. J Immunol.2003Jan 15; 170(2):711-8.).

PD-1 is a 55kDa transmembrane protein, a member of the immunoglobulin superfamily of the same genus as CD28, ICOS, CTLA-4, but lacks the MYPPY motif essential for binding B7-1/B7-2, but PD-1 contains the membrane proximal Immunoreceptor Tyrosine Inhibition Motif (ITIM) and the membrane distal tyrosine turnover motif (ITSM) (Thomas ML1.of ITAMs and ITIMs: turning on and the off B cell antigen receptor. J Exp Med.1995 Jun 1; 181(6): 1953-6.). Two ligands for PD-1 have been identified, PD-L1(B7-H1) and PD-L2 (B7-DC). Current studies have demonstrated that PD-1 binding to PD-L1 leads to down-regulation of T cell activation and to a reduction in T cell proliferation and cytotoxicity, leading to tumor immune escape (Latchman Y1, Wood CR, Chernova T, Chaudhary D, Borde M, Chernova I, Iwai Y, Long AJ, Brown JA, Nunes R, Greenfield EA, Bourque K, Boussiositis VA, Carter, Carreno BM, Malenkovich N, Nishimura H, Okazaki T, Honjo T, Shae AH, Freeman GJ. PD-L2 a second ligand for PD-1and inhibits T cell activity. Nat Immunol. 2001; 2(3) 261-8; Cheng H, Dong H7-L7H-281J. 11. mu. 11. beta. 7. and 7. the. 9. moderate experiment J. (beer J.) (beer J. 2003, 14. 7. 11. born) of molecular animal experiment).

It has been demonstrated that the immune system can be reactivated using monoclonal antibodies against PD-1 or PD-L1 (Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, Drake CG, Camacho LH, Kauh J, Odunsi K, Pitot HC, Hamid O, Bhatia S, Martins R, Eaton K, Chen S, Salay TM, Alaparthy S, Grosso JF, Korman AJ, Parsm, Agrawal S, Goldberg SM, Pardoll DM, Gupta A, Wiggon JM.safety and activity of anti-PD-L1 antibodies in tissues with attached cancer. N Engl J.201Jun 28; 2Jun 366; 2-26-65). Furthermore, interference with the PD-1/PD-L1 interaction in a mouse model of long-term infection can improve viral clearance and restore immune function in mice with chronic lymphocytic choriomeningitis virus infection (Barber DL, where EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, Freeman GJ, Ahmed R. restoring function in infected CD8T cell reduced viral infection. Nature.2006Feb 9; 439(7077):682-7. Epub. Dec 28.). Recent studies have found that Metabolic activity has a significant impact on tumor therapy, and that Warburg metabolism provides tumor cells with AN extracellular advantage in terms of accelerated depletion of extracellular glucose and tumor infiltration of T Cell dysfunction (Ho PC, Bihuniak JD, Macintyre AN, Staron M, Liu X, Amezquita R, Tsui YC, Cui G, Micevic G, metals JC, Kleinstein SH, Abel ED, Insoga KL, Feske S, Locasale JW, Bosenberg MW, RathJC, Kaech SM. phospho virocyte Is a Metabolic Checkpoint of Anti-tumor T responses. Cell.2015 10; 162 (28) (1217-28.); in addition, PD-1 blocking therapy activated T cells also require CD 28-dependent costimulation (Hui E, Cheung J, Zhu J, Su X, Taylor MJ, Wallweber HA, Sasmal DK, Huang J, Kim JM, Mellman I, Vale RD.T cell diagnostic receptor CD28is a primary target for PD-1-mediated inhibition. science.2017Mar 31; 355(6332): 1428-.

However, the PD-1 antibody used at home and abroad has high cost, potential toxic and side effects on human bodies and off-target effect. Multiple use of PD-1 antibody will produce anti-antibody, severely affecting the effect of PD-1 antibody. The main reasons for the research and development of the PD-1 small-molecule inhibitor are that no effective screening method is found at present, and in addition, the potential toxic and side effects of the artificially synthesized compound on organisms are large.

Disclosure of Invention

The object of the present invention is to overcome the drawbacks and disadvantages of the prior art by providing vitamin D3 or its active form 1 alpha, 25(OH)₂D₃The application of the compound in inhibiting the expression of PD-1.

The purpose of the invention is realized by the following technical scheme: vitamin D3 or its active form 1 alpha, 25(OH)₂D₃The application of the compound in inhibiting the expression of PD-1.

The 1 alpha, 25(OH)₂D₃Is an active form of vitamin D3(VD3), and can be purchased directly or converted by VD 3.

Vitamin D3 or its active form 1 alpha, 25(OH)₂D₃Application in preparing PD-1 inhibitor.

A PD-1 inhibitor comprises vitamin D3 or 1 α,25(OH)₂D₃(ii) a The PD-1 inhibitor also comprises IL-2 (interleukin 2), the IL-2 is used for maintaining the activation and proliferation of T cells, and the core component 1 alpha, 25(OH)₂D₃The peptide is used for reducing the expression of a T cell immunosuppressive molecule PD-1, promoting the expression of CD28 and promoting the expression of Blimp1 so as to enhance immune response.

The use concentration of IL-2 in the PD-1 inhibitor is 2-100 ng/mL, 1 alpha, 25(OH)₂D₃The use concentration of (A) is 10-1000 nmol/L; the concentration of IL-2 used is preferably 10 to 40ng/mL, 1 alpha, 25(OH)₂D₃The concentration of (B) is preferably 50 nmol/L.

The IL-2 is preferably recombinant human interleukin 2.

The PD-1 inhibitor also comprises IL-15 (interleukin 15), and the combined cytokine can effectively enhance the killing function and the metabolic function of T cells.

The IL-15 is preferably recombinant human interleukin 15.

The using concentration of the IL-15 is 2-40 ng/mL; preferably 10 ng/mL.

The dosage forms of the PD-1 inhibitor comprise injection preparations, oral preparations and the like.

The PD-1 inhibitor is applied to the preparation of antitumor drugs.

The anti-tumor drug is a tumor immunotherapy drug, a tumor radiotherapy drug or a tumor chemotherapy drug.

The immunotherapy comprises CAR-T, TCR-T, gamma delta T cell and CIK cell therapy, and antibody drug therapy.

The tumor comprises all tumor types such as lung cancer, gastric cancer, laryngeal cancer, esophageal cancer, liver cancer, kidney cancer, colorectal cancer, lymphoma, bladder cancer, head and neck cancer, pancreatic cancer, breast cancer, neuroblastoma, leukemia or melanoma and the like, and is specifically shown in the aspects of down-regulating the expression of T cell PD-1 of the immune system of a cancer patient and up-regulating the expression of CD 28.

The PD-1 inhibitor is applied to the preparation of medicines for promoting the expression of CD28 and/or promoting the expression of Blimp 1.

The PD-1 inhibitor is applied to the preparation of medicines for preventing and treating infectious diseases, inhibiting the expression of CCR5 and/or promoting the secretion of type I interferon.

Vitamin D3 or its active form 1 alpha, 25(OH)₂D₃The application of the polypeptide in preparing the medicines for promoting the expression of CD28, promoting the expression of Blimp1, preventing and treating infectious diseases, inhibiting the expression of CCR5 and/or promoting the secretion of type I interferon.

The infectious diseases comprise viral infectious diseases, 1 alpha, 25(OH)₂D₃Can down-regulate the expression of CCR5 on CD4T cells, can activate a natural antiviral signaling pathway (I-type interferon signaling pathway), and can be applied to the anti-infection field.

The research of the invention discovers a formula, the core component of which is the activity of vitamin D3Form 1 α,25(OH)₂D₃The expression of PD-1 on the surfaces of activated CD8T cells and gamma delta T cells is inhibited through VDR (vitamin D receptor), meanwhile, the VDR can promote the expression of Blimp1, and the Blimp1 can effectively inhibit the expression of PD-1, so that the tumor killing function of the T cells is enhanced; vitamin D in the formulation₃The compound can also promote the high expression of CD28 by T cells so as to strengthen a second signal required by T cell activation, and the proliferation and metabolic functions of the T cells are enhanced. The active form of vitamin D3 also initiates the type I IFN signaling pathway, with potential antiviral effects. The vitamin D3 in the formula is a necessary nutrient element for human bodies, has extremely small toxic and side effects and wide tolerance range, can be orally taken and injected in safe dose, and further effectively avoids the toxic effect of a conventional small molecule inhibitor, so that the immunotherapy can be performed by taking vitamin D3 and a combined PD-L1antibody or using T cells treated by vitamin D3 in the future antitumor therapy (including antibody therapy and cell therapy), and in addition, the immunity function of the organism can be enhanced by independently using the vitamin D3 in tumor patients.

Compared with the prior art, the invention has the following advantages and effects:

1. the invention provides a small molecule inhibitor formula, which can effectively reduce the expression of immune cell PD-1and enhance the function of T cells by singly or jointly using cell factors (such as interleukin 2 and interleukin 15), thereby being used for treating various diseases, including tumors and infectious diseases.

2. The small molecule inhibitor formula of the invention is shown to reduce the expression of PD-1 of T cells and promote the expression of CD28 in biochemical and cell-based experimental systems, thereby enhancing the killing function and metabolic function of the T cells and maintaining the steady state of the T cells. These results are consistent with the regulatory points of current immunotherapies for cancer and long-term infectivity.

3. The small molecule inhibitor in the formula can inhibit the expression of PD-1 at the transcription and translation levels through receptor mediation, and enhances the function of T cells. The formula has no toxic and side effects in normal organism range, and the core components are essential nutrient elements for human bodyThe body has better tolerance. The core component of the formula is vitamin D3 and its active form 1 alpha, 25(OH)₂D₃Can activate T cells in a short time, and start tumor killing and antiviral effects.

4. Vitamin D3 or its active form 1 alpha, 25(OH) in the present invention₂D₃Can also regulate the expression of CCR5 on CD4T cells, and can activate a natural antiviral signaling pathway: type I interferon signaling pathway. In combination with the existing clinical antiviral treatment method, the vitamin D3 also has great application value in the anti-infection field.

5. Existing immunotherapy, including CAR-T, TCR-T, CIK, etc., has significant drawbacks. Such as: they have difficulty entering solid tumors; even if they enter the interior of the tumor, they hardly exert tumor killing action due to the inhibitory action of the tumor microenvironment. Immune cell therapy also presents the problem of inflammatory storms. The formulation can effectively reduce the expression of inflammatory storm and immunosuppressive molecules (such as PD-1), so that the immunotherapy can more effectively eliminate tumors.

6. The PD-1 small molecular compound inhibitor has the following advantages: (1) the PD-1 antibody used at home and abroad at present has high cost and potential toxic and side effects on human bodies. The vitamin D3 is a necessary nutrient element for human bodies, has good tolerance and low price for the human bodies, and can be widely popularized and used; (2) the T cells activated by the PD-1 antibody blocking therapy also need to be co-stimulated by CD28, while the expression of the CD28 of the T cells can be greatly increased while the PD-1 is down-regulated by vitamin D3, so that the requirements of the T cells for down-regulating PD-1and up-regulating CD28 can be met simultaneously by using vitamin D3 in the tumor immunotherapy process; (3) at present, no effective specific medicine for resisting virus exists at home and abroad, vitamin D3 can also down-regulate the expression of CCR5 on CD4T cells and greatly up-regulate the secretion of T cell type I interferon, so that the antiviral function of T cells is enhanced; (4) the above functions of vitamin D3 are discovered firstly, and are the independent intellectual property rights of China, which will make us occupy a place internationally in the hot field of PD-1 resistance; (5) can effectively enhance the function of killing tumors by T cells and enhance the metabolic function of the T cells; (6) the vitamin D3 in the active form of the core component can effectively reduce the expression of the immune cells PD-1; (7) the formula is different from the known monoclonal antibody for blocking the combination of PD-1and PD-L1 and the known small molecule inhibitor for inhibiting the combination activity of PD-1and PD-L1, but plays a role in the transcription and translation stages and has no toxic or side effect; (8) the product has good tolerance in human body, is an essential nutrient element in human body, can be effectively applied in a safe dosage range, and has high safety factor, wide application range, huge market and wide prospect.

Drawings

FIG. 1 shows Azole Re-phosphoric acid and 1 α,25(OH) in example 2₂D₃And (3) a graph of the amplification results of V gamma 9V delta 2T cells.

FIG. 2 shows 1. alpha. 25(OH) in example 3₂D₃The effect of inhibiting the expression of PD-1 in V gamma 9V delta 2T cells is shown.

FIG. 3 shows 1. alpha. 25(OH) in example 4₂D₃Flow-through histogram of PD-1 inhibition on V γ 9V δ 2T cells.

FIG. 4 shows 1. alpha. 25(OH) in example 5₂D₃Results of CD28 expression from V gamma 9V delta 2T cells.

FIG. 5 shows 1. alpha. 25(OH) in example 6₂D₃Results are shown for apoptosis of V γ 9V δ 2T cells.

FIG. 6 shows 1. alpha. 25(OH) in example 7₂D₃Influence profile on purified V γ 9V δ 2T cells; wherein, graph A shows the number change of V gamma 9V delta 2T cells; panel B shows the results of V γ 9V δ 2T cell proliferation.

FIG. 7 shows 1. alpha. 25(OH) in example 8₂D₃Flow chart of PD-1 inhibition on CD8T cells.

FIG. 8 shows 1. alpha. 25(OH) in example 9₂D₃Graph of results of killing jurkat by treated V γ 9V δ 2T cells.

FIG. 9 shows 1. alpha. 25(OH) in example 10₂D₃Graph of results of detecting the transcriptional levels of PD-1and PRDM1 β (Blimp1) after treatment of V.gamma.9 V.delta.2T cells 12.

FIG. 10 shows 1. alpha. 25(OH) in example 10₂D₃Results of PRDM1 beta (Blimp1)

protein changes

48h, 72h, 96h after treatment of V gamma 9V delta 2T cells.

FIG. 11 shows 1. alpha. 25(OH) in example 11₂D₃Flow chart of PD-1 expression inhibition on CD8T cells from lung cancer patient samples.

FIG. 12 shows 1. alpha. 25(OH) in example 12₂D₃Results of the ability to enhance glycolysis in V gamma 9V delta 2T cells and CD8T cells; wherein, the A is 1 alpha, 25(OH)₂D₃Enhancement of glycolytic capacity of V gamma 9V delta 2T cells (changes in mRNA levels of glycolytic key enzyme genes), FIG. B shows 1 alpha, 25(OH)₂D₃Enhancing the results of changes in energy metabolism (glycolysis) of CD8T cells; FIG. C shows 1. alpha.25 (OH)₂D₃Enhancing the change of mRNA level of the glycolytic key enzyme gene of CD8T cells.

FIG. 13 shows 1. alpha. 25(OH)₂D₃Results of interferon signaling pathway capable of up-regulating V gamma 9V delta 2T cells.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

In the present example, all cells were cultured in DMEM or 1640 medium supplemented with 10% (v/v) fetal bovine serum, 100. mu.g/ml ampicillin, 100 units/ml streptomycin. The cell culture environment was 37 degrees Celsius, 5% (v/v) carbon dioxide.

Example 1:

1α,25(OH)₂D₃the structural formula of (Calcitriol) is shown below:

the compound is used as a PD-1 inhibitor. This compound was purchased from Sigma.

Example 2: vgamma 9 Vdelta 2T cell expansion

Separating normal human blood leucocyte layer at Guangzhou blood station center, purchasing separating liquid (ficoll) from GE Healthcare, gradient centrifuging (600g, 30min, centrifuging condition: rise 6 and fall 2), and performing selective amplification culture on the obtained peripheral blood mononuclear cells (culture condition: day 0, oxazole Re-phosphate: (2))Zol, from Sigma) 30-50 (μ M), 40(μ M) recombinant human interleukin 2(IL-2), 40ng/mL, 1 α,25(OH)₂D₃The addition amount is 50 (nmol/L); on the third day, the culture medium is changed without adding Zol, the recombinant human interleukin 2 is changed into 10ng/mL, 1 alpha, 25(OH)₂D₃The addition amount is 50 (nmol/L); the ratio of V gamma 9V delta 2/CD3 was flow-detected on day 12. The results are shown in FIG. 1, and show that V.gamma.9Vdelta.2T cells are highly pure.

Example 3: the V gamma 9V delta 2T cells of example 2 were subjected to protein lysis (RIPA) and 20ug were subjected to protein gel electrophoresis to detect the protein level of PD-1, actin was used as an internal reference. The results are shown in FIG. 2 and indicate 1. alpha., 25(OH)₂D₃Has good inhibition effect on V gamma 9V delta 2T cell PD-1.

Example 4: flow detection and statistics of PD-1 were performed on V γ 9V δ 2T cells in example 2. Flow-through antibodies were purchased from Biolegend. The results are shown in FIG. 3, and indicate 1. alpha., 25(OH)₂D₃Has good inhibition effect on V gamma 9V delta 2T cell PD-1.

Example 5: the V gamma 9V delta 2T cells in the example 2 are detected by CD28, and the expression of CD28is counted by flow. The results are shown in FIG. 4, and indicate 1. alpha., 25(OH)₂D₃Has great promotion effect on the expression of V gamma 9V delta 2T cell CD 28.

Example 6: the V.gamma.9 V.delta.2T cells of example 2 were cultured for 12 days, and 1. alpha.25 (OH) was detected in 20 days₂D₃Apoptosis on V γ 9V δ 2T cells. The apoptosis kit was purchased from biotool and the main index was anxin V-FITC. The results are shown in FIG. 5, and indicate 1. alpha., 25(OH)₂D₃No apoptosis was caused on V γ 9V δ 2T cells.

Example 7: cell count statistics were performed on V.gamma.9 V.delta.2T cells of example 2 on

days

16, 18, 20 and 22, respectively, and the results are shown in FIG. 6A, which indicates 1. alpha.25 (OH)₂D₃There was no effect on the expansion of V γ 9V δ 2T cells. Twelve-day-old V.gamma.9 V.delta.2T cells were labeled with CFSE (purchased from Thermofisher) and observed for proliferation. The results are shown in FIG. 6B, which shows 1. alpha., 25(OH)₂D₃Has one to the proliferation of gamma 9V delta 2T cellsThe function of the medicine is determined.

Example 8: magnetic beads enriched normal human PBMC (peripheral blood mononuclear cells from Guangzhou blood station center; magnetic beads available from BD Co.) with purity CD8/CD3 by flow assay after enrichment>98% of flow antibody was purchased from Biolegend, BD. Activated culture on day 0, culture conditions: wrapping alpha CD 35 ug/mL at 4 ℃ overnight, stimulating alpha CD 281 ug/mL for 3 days, and detecting the expression of PD-1 by WB on the fifth day; and (3) flow-detecting the expression of PD-1 of the CD8T cells. The results are shown in FIG. 7, and indicate 1. alpha., 25(OH)₂D₃Can down-regulate PD-1 of CD8T cells.

Example 9: reference example 2, the dose of the combination IL-2 was 10ng/mL (2ng/mL-50ng/mL), 1. alpha., 25(OH)₂D₃The dosage is 50nmol/L (range of 50 nmol/L-500 nmol/L), and the IL-15 dosage is 10 ng/mL. The cultured V.gamma.9 V.delta.2T cells were co-cultured with jurkat cells. The method comprises the following specific steps:

1. tumor cell (Jurkat) serum (Jurkat cells from ATCC in usa) was collected and resuspended in culture medium, 100ul was taken and left as blank control (blank), and 100ul was left as PI stain;

2. 1ml of the resuspended tumor cells were removed into a 1.5ml EP tube, 0.4ul CFSE (5 mM concentration) was added to give a final concentration of 2. mu.M, and incubated at 37 ℃ for 10 min. The remaining CFSE-free cells were plated back into the culture dish;

3. the EP tubes were removed, 3 volumes of ice medium containing serum was added, placed on ice for 5min, staining terminated, then washed twice with PBS buffer, and media added for resuspension counting. Leaving a little of the tumor cells stained with CFSE as a CFSE single staining tube;

4. collecting gamma delta T cells (taking 10 ten thousand cells to add to the blank in step 1);

5. tumor cell sampling 1X 10⁵2 control groups without gamma delta T cells and CFSE stained tumor cells are set, and then co-culture is carried out by setting different effective target ratios of 1:1, 5:1, 10: 1and 20: 1;

6. the final volume of the medium was 200ul during co-cultivation;

7. culturing for 6 hours, taking out and washing once, and dyeing PI up-flow type; cells which are double positive for CFSE and PI are killed target cells;

8. calculating the formula: specific cytotoxic activity (%) - (experimental group target cell death rate-natural target cell death rate)/(100-natural target cell death rate) × 100%.

As a result, as shown in FIG. 8, the results were 1. alpha., 25(OH)₂D₃The cultured V gamma 9V delta 2T cells can enhance the tumor killing function.

Example 10

1. Referring to example 2, the PD-1 transcript levels were measured 12h after stimulation of selectively expanded V γ 9V δ 2T cells. Extracting V gamma 9V delta 2T cells, using a Tiangen biochemical technology RNA extraction kit, determining total RNA, using a Takara reverse transcription kit for inversion, and using a Biotool fluorescent dye for real-time PCR analysis. The results are shown in FIG. 9 and show 1. alpha., 25(OH)₂D₃Has inhibitory effect on PD-1 of V gamma 9V delta 2T cells at the transcription level and promotes the expression of PRDM1 beta (Blimp 1).

2. For V.gamma.9 V.delta.2T cells in example 2, 1. alpha.25 (OH)₂D₃After stimulation, protein gel electrophoresis is carried out, and detection is carried out at 48h, 72h and 96h respectively. The results are shown in FIG. 10, and show 1. alpha., 25(OH)₂D₃Can stimulate the V gamma 9V delta 2T cells to express PRDM1 beta (Blimp 1).

Example 11: blood of lung cancer patient (derived from Oncology of Chinese hospital of river university) was isolated by the method of example 2, and PBMC obtained by the method contains 1 α,25(OH) in an amount of 50-500 nmol/L₂D₃The culture was performed under stimulation, and the expression of PD-1 on CD8, CD4T cells was flow-assayed on the third and fifth days using 100 nmol/L. The results are shown in FIG. 11, and indicate 1. alpha., 25(OH)₂D₃Inhibit the expression of PD-1 of peripheral blood CD8T cells of lung cancer patients.

Example 12: energy metabolism assays and glycolytic metabolic key enzyme assays were performed on the V γ 9V δ 2T cells of examples 2 and 8. See Hippocampus, USA for energy metabolism procedure, and for glycolytic key enzyme expression reference example 10. As a result, as shown in FIG. 12, 1. alpha., 25(OH) was found₂D₃Can enhance glycolytic ability of V gamma 9V delta 2T cells and CD8T cells.

Example 13: referring to the procedure of example 2, the level of change in interferon-related genes was measured after extracting RNA based on the experiment of example 2, and the results are shown in FIG. 13, indicating 1. alpha., 25(OH)₂D₃Can up-regulate interferon signal pathway of V gamma 9V delta 2T cell.

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.

Claims

1. Vitamin D3 or its active form 1 alpha, 25(OH)₂D₃The application in preparing PD-1 in vitro inhibitors is characterized in that: the PD-1 in vitro inhibitor also contains IL-2.

2. Use according to claim 1, characterized in that: the use concentration of IL-2 in the PD-1 in-vitro inhibitor is 2-100 ng/mL, 1 alpha, 25(OH)₂D₃The use concentration of (A) is 10 to 1000 nmol/L.

3. Use according to any one of claims 1 to 2, characterized in that: the PD-1 in vitro inhibitor also contains IL-15.

4. Use according to claim 3, characterized in that: the using concentration of the IL-15 is 2-40 ng/mL.

5. Use according to claim 1, characterized in that: vitamin D3 or its active form 1 alpha, 25(OH)₂D₃The purpose of inhibiting the expression of PD-1 is realized by promoting the expression of CD28 and/or promoting the expression of Blimp 1.