CAR-T cell capable of efficiently and stably expressing inhibiting antibody and application thereof

Abstract

The invention provides a CAR-T cell capable of efficiently and stably expressing an inhibiting antibody and application thereof. Specifically, the invention provides a transgenic T cell, and the genome of the transgenic T cell is stably integrated with expression cassettes containing nucleic acid sequences coding chimeric antigen receptors and immune checkpoint inhibiting antibodies, wherein two ends of the expression cassettes contain inverted terminal repeats of transposons. In the genome of the pluripotent CAR-T cell, the expression cassettes of the inhibiting antibodies are stably integrated via a transposon system, so the CAR-T cell has the activity of stably and efficiently expressing the inhibiting antibodies on the premise that original killing activity of the T cell is maintained, transfer-back CAR-T cells are prevented from inhibition by a tumor microenvironment, and residual tumor-specific T cells can be activated in situ. Meanwhile, a molecular brake system is introduced to ensure the security of CAR-T cell therapy, and transfer-back pluripotent CAR-T cells can be timely removed if necessary.

Description

technical field [0001] The invention belongs to the field of cell biology and oncology, and relates to a CAR-T (Chimeric antigen receptor, CAR) cell capable of stably expressing an inhibitory antibody at a high level. Background technique [0002] Immunotherapy against malignant tumors has developed rapidly in recent years and has achieved remarkable clinical efficacy. Among them, the immune checkpoint (such as CTLA4, PD1 / PDL1) monoclonal antibody therapy plays a role by in situ activation of the patient's residual tumor-specific T cells, and the overall effective rate for a variety of malignant tumors reaches 30%. Efficacy and long-term survival; transgenic CAR-T / TCR-T cell therapy is to quickly obtain tumor-specific T cells through in vitro genetic modification, and then adoptive reinfusion for treatment to play a role in the treatment of relapsed and refractory B-cell leukemia. The remission rate was over 90%. [0003] A chimeric antigen receptor (Chimeric Antigen Recep...

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Problems solved by technology

[0008] 2. Lack of suitable CAR-T therapeutic targets

[0011] Although there have been reports of transfection of exogenous genes into, for example, T cells, the commonly used gene transfection vector systems have a low transfection rate of effector immune cells with cell killing toxicity, or it is difficult to make exogenous genes in their cells at a high level. Express

The use of adenovirus vectors (non-integrated) can mediate the efficient short-term expression of exogenous genes in T cells, but the proliferation of activated T cells is very fast, and the expression cassettes of exogenous genes carried will be quickly lost during cell passage , the expression is difficult to last; the integration of exogenous genes in the T cell genome can be mediated by retrovirus or lentivirus, and stable expression can be achieved in theory, but the antibody contains light chain and heavy chain, and the coding sequence is long and the molecular weight is large, resulting in Retroviruses or lentiviruses carrying full-length antibody expression cassettes are difficult to package and prepare, and it is difficult to express antibodies efficiently (for example, using CAR-T cells to express antibodies, the concentration is only 200ng / ml, Oncotarget.2016Apr 29.doi:10.18632 / oncotarget.9114), generally used to express single-chain antibodies with simple structure (lack of constant region fragments, incomplete function and short half-life)

Therefore, before this, there are no reports of transgenic cells that have cytotoxicity and can stably and high-level express antibodies containing human constant regions

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