Search

CN-121975733-A - Method for preparing induced pluripotent stem cell-derived gamma delta T cells and application thereof

CN121975733ACN 121975733 ACN121975733 ACN 121975733ACN-121975733-A

Abstract

The present invention provides a method for preparing induced pluripotent stem cell-derived gamma delta T cells and its application, belonging to the field of cell immunotherapy technology. In the method, induced pluripotent stem cells can be efficiently differentiated and expanded through 3D suspension culture, and a universal "spot type" gamma delta T cell production scheme can be prepared on a large scale. The method provided by the present invention has a short differentiation cycle, high purity, and does not require nourishing cells for differentiation. The differentiation process is stable and can be used as a process platform for large-scale production of uniform quality gamma delta T cells, with broad prospects for industrialization and clinical application.

Inventors

  • TIAN LINJIE
  • XU JING
  • GUO TINGTING
  • REN KEYONG

Assignees

  • 杭州济元基因科技有限公司

Dates

Publication Date
20260505
Application Date
20260408

Claims (13)

  1. 1. A method of making an induced pluripotent stem cell-derived γδ T cell, the method comprising: (S1) differentiating for 0 day, culturing induced pluripotent stem cells by adopting a 3D suspension culture mode, wherein a culture medium is taken StemFit TM AK03N as a basic culture medium, and Y-27632 and CHIR99021 are added; (S2) differentiating for 1 day, replacing the culture medium for 3D suspension culture, wherein the replaced culture medium takes any one of ADVANCE DMEM/F12, stempro, SFM or Essential 6 as a basal culture medium, and adding BMP-4, VEGF and bFGF; (S3) differentiating for 2 days, replacing the culture medium for 3D suspension culture, wherein the replaced culture medium takes ADVANCE DMEM/F12, stempro, SFM or Essential 6 as a basic culture medium, and adding BMP-4, VEGF, bFGF and SB431542; (S4) differentiating for 4-7 days, replacing a culture medium for 3D suspension culture, culturing to obtain hematopoietic progenitor cells, wherein the replaced culture medium takes any one of ADVANCE DMEM/F12, stempro 34 SFM or Essential 6 as a basic culture medium, and adding SCF, VEGF and bFGF; (S5) differentiating for 7-30 days, inducing the hematopoietic progenitor cells to differentiate into γδ T cells; (S6) amplifying the gamma delta T cells, and performing large-scale amplification of the gamma delta T cells after 30 days of differentiation induction.
  2. 2. The method of preparing induced pluripotent stem cell-derived γδ T cells according to claim 1, wherein in (S1), the final concentration of Y-27632 in the basal medium is 1-50 μΜ, and the final concentration of CHIR99021 is 1-20 μΜ; In the step (S2), the final concentration of BMP-4 in the basal medium is 1-200 ng/mL, the final concentration of VEGF is 1-200 ng/mL, and the final concentration of bFGF is 1-200 ng/mL; In the step (S3), the final concentration of BMP-4 in the basal medium is 1-200 ng/mL, the final concentration of VEGF is 1-200 ng/mL, the final concentration of bFGF is 1-200 ng/mL, and the final concentration of SB431542 is 1-20 mu M; In the step (S4), the final concentration of SCF in the basal medium is 1-200 ng/mL, the final concentration of VEGF is 1-200 ng/mL, and the final concentration of bFGF is 1-200 ng/mL.
  3. 3. The method of claim 1, wherein in (S2) - (S4), any one or a combination of at least two of GlutaMAX, vitamin C injection, monothioglycerol, or insulin-transferrin-selenium is further added to the basal medium; The basic culture medium comprises 1-20 mM percent of Glutamax, 10-100 mug/mL of vitamin C injection, 0.1-10 mM percent of monothioglycerol, and 100 times of dilution of insulin-transferrin-selenium; in (S1) to (S5), the 3D suspension culture method is carried out under the condition that the suspension culture is carried out within 500 rpm.
  4. 4. The method for producing induced pluripotent stem cell-derived γδ T cells according to claim 1, wherein in (S5), the culture medium for the induced culture of the hematopoietic progenitor cells is DMEM/F12 as a basal medium, and at least one of human serum or serum replacement, SCF, IL-7, FLT3L, TPO, IL-2 or L-ascorbic acid is added; In the basal medium, the final concentration of human serum or serum replacement is 5% -30%, the final concentration of SCF is 0.1-200 ng/mL, the final concentration of IL-7 is 0.1-200 ng/mL, the final concentration of FLT3L is 0.1-200 ng/mL, the final concentration of TPO is 0-200 ng/mL, the final concentration of IL-2 is 0.1-200 ng/mL, and the final concentration of L-ascorbic acid is 1-1000 mug/mL.
  5. 5. The method of claim 1, wherein the culturing in (S1) - (S4) is performed under anaerobic conditions in which the concentration of O 2 is less than 20% (v/v).
  6. 6. The method of claim 1, wherein the step of inducing culture of said hematopoietic progenitor cells in (S5) comprises differentiating said hematopoietic progenitor cells into γδ T cells by co-culture with feeder cells.
  7. 7. The method for producing induced pluripotent stem cell-derived γδ T cells according to claim 1, wherein (S5) the induction culture is performed by an adherent culture or a 3D suspension culture; inducing the hematopoietic progenitor cells by an adherent culture method, wherein the hematopoietic progenitor cells are differentiated into gamma delta T cells by using a culture plate coated with vascular cell adhesion molecule-1 or recombinant human fibrin fragment and delta-like protein 4 or delta-like protein 1; The step of inducing the culture of the hematopoietic progenitor cells by 3D suspension culture comprises culturing using a medium mixed with beads to which delta-like protein 4 or delta-like protein 1 is coupled.
  8. 8. The method for producing induced pluripotent stem cell-derived γδ T cells according to claim 1, wherein the condition for the mass expansion is (S6) that the induced γδ T cells are mass-expanded in a medium containing γδ T stimulators and one or more selected from IL-2 and/or IL-15, wherein the γδ T stimulators are selected from the group consisting of trophoblasts, phosphate compounds or derivatives thereof which are metabolites of the mevalonate pathway or the non-mevalonate pathway, and specific inhibitors of biosynthesis pathway rate-limiting enzyme farnesyl pyrophosphate synthases; The trophoblasts comprise K562 trophoblasts or PBMC trophoblasts with functional factors; the phosphate compounds of the metabolic products of the mevalonate pathway or the non-mevalonate pathway or derivatives thereof include (E) -4-hydroxy-3-methyl-but-2-enyl pyrophosphate, isopentenyl diphosphate or bromohydrin pyrophosphate; specific inhibitors of the biosynthetic pathway rate-limiting enzyme farnesyl pyrophosphate synthase include zoledronic acid or pamidronate; The addition amount of the specific inhibitor of the biosynthesis pathway speed-limiting enzyme farnesyl pyrophosphate synthase is 0.01-100 mu M; The culture mode of the large-scale amplification comprises static culture or 3D suspension culture.
  9. 9. The method for preparing induced pluripotent stem cell-derived γδ T cells according to claim 1, wherein the source of the induced pluripotent stem cells comprises any one of iPS cells derived from CD34 + cells, iPS cells derived from αβ T cells or γδ T cells isolated from mononuclear PBMCs after reprogramming, and iPS cells engineered by knockout or knock-in.
  10. 10. An induced pluripotent stem cell-derived γδ T cell or cell population, wherein the γδ T cell is prepared by the method for preparing an induced pluripotent stem cell-derived γδ T cell of any one of claims 1-9.
  11. 11. A non-antigen specific cellular immunotherapeutic agent, wherein the active ingredient of the agent comprises γδ T cells or cell populations derived from the induced pluripotent stem cells of claim 10.
  12. 12. A pharmaceutical composition comprising the induced pluripotent stem cell-derived γδ T cell or cell population of claim 10.
  13. 13. Use of any one or a combination of at least two of the induced pluripotent stem cell-derived γδ T cells or cell populations of claim 10, the non-antigen specific cellular immunotherapeutic agent of claim 11 or the pharmaceutical composition of claim 12 in the manufacture of a medicament for the treatment of cancer, infectious disease or autoimmune disease.

Description

Method for preparing induced pluripotent stem cell-derived gamma delta T cells and application thereof Technical Field The invention belongs to the technical field of cellular immunotherapy, and particularly relates to a method for preparing induced pluripotent stem cell-derived gamma delta T cells and application thereof. Background The gamma delta T cells have congenital and adaptive immune functions, are independent of MHC, have no GVHD side reaction, and are widely applied to the fields of hematoma, solid tumor, autoimmune diseases and the like, and are used as allogeneic 'spot' cell therapy products. γδ T cells are usually present in the peripheral blood only in a proportion of 1% to 5%, and therefore there is a problem that the purity and the number of cells sufficient for treatment cannot be ensured. Furthermore, there is a problem in that when the amount of blood to be collected is increased in order to secure the purity and the number of cells sufficient for the treatment, a great burden is imposed on the person collecting the blood. Methods involving in vitro expansion of γδ T cells isolated from peripheral blood have failed to achieve adequate expansion and activation due to difficulty in ensuring the number of cells and due to depletion of cells. At present, most gamma delta T cells are prepared by PBMC (positive-negative) amplification, and because of heterogeneity, high difficulty in gene modification and high cost for producing CAR-gamma delta T, the iPS cells have certain advantages, and the iPS cells can be used for producing and preparing large-batch uniform general 'spot' gamma delta T cells at one time after gene modification. In the prior art, a method for preparing gamma delta T by PBMC or induced pluripotent stem cells exists, but the gamma delta T prepared by the induced pluripotent stem cells in the prior art is in an adherent culture state, and has the disadvantages of long differentiation period, low cell yield and no benefit to industrialization. The prior art still has the following defects that (1) gamma delta T cells prepared by PBMC have heterogeneity, and have large gene modification difficulty and high cost, and cannot ensure batch detection stability. (2) The induced pluripotent stem cells are used for preparing gamma delta T in an adherence mode, so that industrialization is not facilitated. (3) The efficiency of inducing the differentiation and the expansion of the pluripotent stem cells is low, and the industrialization is not facilitated. (4) induced pluripotent stem cell differentiation γδT has low purity. (5) The induced pluripotent stem cells differentiate γδt with a long cycle, resulting in weak cell function. (6) Inducing pluripotent stem cells to differentiate γδ T cells has poor batch-to-batch stability. (7) Inducing pluripotent stem cells to differentiate γδ T cells requires feeder cells to differentiate. (8) induced pluripotent stem cell differentiation γδ T cell production is low. The scheme of inducing the differentiation gamma delta T cells in the differentiation gamma delta T cells is in an adherence mode for differentiation, and the differentiation gamma delta T has low efficiency, low purity, low yield, weak cell functions caused by long period, poor batch-to-batch stability and no contribution to the later industrialization. Therefore, the prior art is lack of a scheme for efficiently differentiating and amplifying induced pluripotent stem cells in a 3D suspension culture mode, the differentiated γδ T cells have short cycle, high purity, high yield, freeze-resistant performance, strong cell functions, stable batch, and capability of preparing universal 'spot' γδ T cells in a large scale, and the method is applied to the technical field of cell therapy. Disclosure of Invention Aiming at the defects of the prior art, the invention aims to provide a method for preparing induced pluripotent stem cell-derived gamma delta T cells and application thereof. The method of the invention obviously improves the production efficiency of the gamma delta T cells and shortens the production period, thereby providing higher quality and more stability for cell therapy products and realizing the general 'spot' gamma delta T cells which can be produced in large scale. In order to achieve the aim of the invention, the invention adopts the following technical scheme: In a first aspect, the invention provides a method of preparing induced pluripotent stem cell-derived γδ T cells, the method comprising: (S1) differentiating for 0 day, culturing induced pluripotent stem cells by adopting a 3D suspension culture mode, wherein a culture medium is taken StemFit TM AK03N as a basic culture medium, and Y-27632 and CHIR99021 are added; (S2) differentiating for 1 day, replacing the culture medium for 3D suspension culture, wherein the replaced culture medium takes any one of ADVANCE DMEM/F12, stempro, SFM or Essential 6 as a basal culture medium, and adding BMP-4, VEGF and bFGF; (S3) d