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

Abstract

The invention provides a CAR-T cell capable of efficiently and stably expressing an activated antibody and application thereof. Specifically, the genome of the transgenic T cell in the invention is stably integrated with expression cassettes containing nucleic acid sequences coding activated antibodies of chimeric antigen receptors and immune costimulatory molecules or receptors of the immune costimulatory molecules, 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 activated antibodies are stably integrated via a transposon system, so the CAR-T cell has the activity of stably and efficiently expressing the activated antibodies on the premise that original killing activity of the CAR-T cell is maintained, and transfer-back cells are allowed to rapidly propagate at a tumor site. 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 cell that efficiently and stably expresses an activated antibody and its use. 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 Receptor, CAR) generally consists ...

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Problems solved by technology

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

[0010] 3. It is difficult to reach an effective amount in the body

[0013] 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 Packaging and preparation of retroviruses or lentiviruses carrying full-length antibody expression cassettes is difficult, 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 is no report of CAR-T cells highly expressing antibodies

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