Synergistic and depletion-resistant chimeric antigen receptor T cell and application thereof in preparation of medicine for treating tumors

A technology of chimeric antigen receptors and antigens, applied in the direction of antibody medical components, genetically modified cells, antibody mimics/scaffolds, etc., can solve the problems of B cell function loss, tumor recurrence, poor persistence, etc., and achieve a strong immune response , good anti-tumor, strong anti-tumor effect

Active Publication Date: 2021-10-12
NANJING CART MEDICAL TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are still some problems in the current traditional CAR-T for hematological tumors, which limit its clinical application
The currently FDA-approved CD19 CAR-T cell drugs use CD28 or 4-1BB as costimulatory domains respectively (Forsberg MH, et al. The potential of CAR T therapy for relapsed or refractory pediatric and young adult B-cell ALL.Ther Clin Risk Manag ,2018,14:1573-1584.Hombach AA,et al.Theweal and woe of costimulation in the adoptive therapy of cancer with chimericantigen receptor (CAR)-redirected T cells.Curr Mol Med,2013,13(7):1079- 1088.) The two CAR-T cells have their own advantages and disadvantages. The CAR-T cells with the costimulatory domain of CD28 proliferate faster in the early stage of the response, and can mediate stronger tumor killing capabilities, but at the same time have stronger side effects , and the persistence in the late stage of the immune response is poor; while the proliferation ability of 4-1BB-based CAR-T cells is poorer than that of CD28 CAR in the early stage of the response, but the persistence is better, and it can also maintain a high level in the late stage of the immune response Cell number (Salter AI, et al. Phosphoproteomic analysis of chimeric antigen receptor signaling reveals kinetic and quantitative differences that affect cell function. Sci Signal, 2018, 11(544): eaat6753. Maloney DG. Anti-CD19 CAR T cell therapy for lymphoma-off to theraces!.Nat Rev Clin Oncol,2019,16(5):279-280.Kawalekar OU,et al. Distinct signaling by chimeric antigen receptors(CARs) containing CD28 signaling domainversus 4-1BB in primary human T cells.Blood,2013 ,122(21):2902.)
In addition, it is worth noting that CAR-T cells also have some side effects during treatment, such as cytokine release syndrome (Cytokine release syndrome, CRS), neurotoxicity, loss of B cell function, and tumor recurrence (Bonifant CL, et al.Toxicity and management in CAR T-cell therapy.Mol Ther-Oncolytics,2016, 3:16011.Porter DL,et al.Chimeric antigen receptor T cellspersist and induce sustained remissions in relapsed refractory chroniclymphocytic leukemia.Sci Transl Med ,2015,7(303): 303ra139.Maude SL,etal.Managing cytokine release syndrome associated with novel T cell-engaging therapies.Cancer J,2014,20(2):119-122.Sadelain M.Chimeric antigen receptors: a paradigm shift in immunotherapy. Annu Rev Canc Biol, 2017, 1:447-466.)

Method used

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  • Synergistic and depletion-resistant chimeric antigen receptor T cell and application thereof in preparation of medicine for treating tumors
  • Synergistic and depletion-resistant chimeric antigen receptor T cell and application thereof in preparation of medicine for treating tumors
  • Synergistic and depletion-resistant chimeric antigen receptor T cell and application thereof in preparation of medicine for treating tumors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1. Construction of a CAR lentivirus containing five elements of DAP12-4-1BB-T2A-CD19(scFv)-KIRS2

[0053] In order to prove that CAR-T cells containing DAP12-4-1BB-KIRS2 intracellular signaling domain contain 4-1BB-CD3ζ, DAP12-KIRS2, DAP12-4-1BB-CD3ζ-KIRS2-4-1BB, DAP12 -41BB-KIRS2-4-1BB, DAP12-KIRS2-4-1BB, and DAP12-CD3ζ-KIRS2-4-1BB stimulated CAR-T cells have more advantages, so it is necessary to construct viral vectors containing different combinations of stimulating signals. In this example, the single-chain antibody targeting B-cell malignancies (CD19) is used as a unified extracellular antigen recognition structure, and the following eight chimeric antigen receptors need to be constructed respectively ( figure 1 and figure 2 ):

[0054] CD19(scFv)-CD8α-4-1BB-CD3ζ (KT1)

[0055] DAP12-T2A-CD19(scFv)-KIRS2 (KT2)

[0056] DAP12-4-1BB-CD3ζ-T2A-CD19(scFv)-KIRS2 (KT3)

[0057] DAP12-4-1BB-T2A-CD19(scFv)-KIRS2 (KT4)

[0058] DAP12-4-1BB-CD3ζ-T2A-CD19(scFv)...

Embodiment 2

[0103] Embodiment 2, virus infection T cell

[0104] 1. Isolation and activation of T cells and virus infection

[0105] (1) Isolation of human peripheral blood mononuclear cells

[0106] About 10 ml of peripheral blood was collected with a blood collection tube containing an anticoagulant, centrifuged at 3000 rpm for 30 min, and the upper layer plasma was collected, and the collected upper layer plasma was centrifuged at 5000 rpm for 10 min. Add it to the lymphocyte separation medium (purchased from Tianjin Haoyang Biological Products Technology Co., Ltd.) at a volume ratio of 1:1, gradient centrifugation, 3000rpm, and centrifugation for 30min. After centrifugation, the centrifuge tubes are layered from top to bottom: the first The first layer is the plasma layer; the second layer is the buffy coat layer of lymphocytes; the third layer is the transparent separation liquid layer; the fourth layer is the red blood cell layer. Aspirate the buffy coat of lymphocytes, wash twice...

Embodiment 3

[0111] Example 3. In vitro killing effect evaluation of virus-infected CAR-T cells

[0112] (1) Culture target cells MCF7-CD19 cells (CD19 positive cell line), 293T (CD19 negative cell line) and effector cells KT1, KT2, KT3, KT4, KT5, KT6, KT7, KT8, a total of 8 groups of CAR-T cells ;

[0113] (2) Collect target cells and effector cells, centrifuge at 1500rpm for 5min, and discard the supernatant;

[0114] (3) Resuspend target cells and effector cells with 10% FBS+1640 complete medium;

[0115] (4) Using the real-time cell analysis system (RTCA), add 50 μL of 1640 medium to the well of E-Plate16

[0116] (5) Use RTCA to detect the baseline, and confirm that the contact of the selected well is normal;

[0117] (6) Set the effect-target ratio to 0:1, 1:1, 5:1, 10:1;

[0118] (7) Take out E-Plate16, add 50 μL of uniformly mixed target cell suspension into each well according to the effect-to-target ratio, so that the number of cells in each well is 10 4 cells / 50μL;

[0119...

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Abstract

The invention discloses a synergistic and depletion-resistant chimeric antigen receptor T cell and application thereof in preparation of a medicine for treating tumors. The synergistic and depletion-resistant chimeric antigen receptor T cell comprises an antigen binding structural domain (scFv) and a signal transduction structural domain, wherein the signal transduction structural domain comprises a first transduction structural domain and a second transduction structural domain, and the antigen binding structural domain is connected in series between the first transduction structural domain and the second transduction structural domain; the first conduction structural domain comprises a receptor signal structural domain and / or a costimulatory structural domain; the second conductive domain comprises a transmembrane structural domain DAP12, and the transmembrane structural domain DAP12 is or is not linked to the costimulatory structural domain and / or an elementary signal domain. According to the T cell expressing the chimeric antigen receptor, the chimeric antigen receptor can be more efficiently and stably expressed in the T cell, so that a killing effect on a target cell can be better played, the durability of CAR-T is improved, the expression of a depletion marker is lower, and the memory type T cell is richer.

Description

technical field [0001] The invention belongs to the field of biology, and in particular relates to a chimeric antigen receptor T cell with synergistic and anti-exhaustion and its use in the preparation of drugs for treating tumors. Background technique [0002] In recent years, although conventional tumor treatment methods such as radiotherapy, chemotherapy, and surgery can achieve certain therapeutic effects, the problems of tumor metastasis, recurrence, and low survival rate of patients have not been properly resolved. With the development of tumor immunology theory and technology, the role of immune cells in tumor treatment has been paid more and more attention. Among them, chimeric antigen receptor T cell technology is a cell therapy technology that has developed very rapidly in recent years. [0003] Chimeric antigen receptor (CAR) is the core component of CAR-T. Using the ligand-binding domain properties, CAR can redirect the specificity and reactivity of selected imm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K39/00A61P35/00A61P35/02A61P35/04
CPCC07K16/2803C07K14/70578C07K14/70503C12N15/86C12N5/0636A61K39/001112A61P35/00A61P35/02A61P35/04C07K2317/622C07K2319/03C07K2319/33C07K2319/74C12N2740/15043C12N2800/107C12N2510/00A61K2039/5156A61K2039/812A61K2039/892A61K2039/82A61K2039/844A61K2039/868A61K2039/804A61K2039/86A61K2039/876A61K2039/884A61K2039/852Y02A50/30
Inventor 王恩秀汪晨陈颖
Owner NANJING CART MEDICAL TECH LTD
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