Hypoxia-inducible chimeric antigen receptor capable of achieving specific activation of tumor microenvironment

A chimeric antigen receptor and tumor microenvironment technology, applied in the field of biomedicine, can solve the problems of inflammatory factors storming the central nervous system, toxicity, side effects, etc., and achieve the effect of delaying the progress of the disease

Active Publication Date: 2019-01-29
SHANGHAI PUBLIC HEALTH CLINICAL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reason is that the antigens targeted by CAR-T cells are almost all tumor-associated antigens (TAAs), and most of them are still expressed at low levels in normal tissues, resulting in "toxic and side effects targeting non-tumor cells" (On- target off tumor), and can lead to inflammatory factor storm and central nervous system toxicity

Method used

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  • Hypoxia-inducible chimeric antigen receptor capable of achieving specific activation of tumor microenvironment
  • Hypoxia-inducible chimeric antigen receptor capable of achieving specific activation of tumor microenvironment
  • Hypoxia-inducible chimeric antigen receptor capable of achieving specific activation of tumor microenvironment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Hypoxia-inducible chimeric antigen receptor construct specifically activated by tumor microenvironment and its mode of action

[0053] The present invention provides a hypoxia-inducible chimeric antigen receptor (Hi-CAR) specifically activated by the tumor microenvironment, which includes two components, the first component sequentially includes a signal peptide, an extracellular antigen-specific binding domain, Extracellular spacer domain, transmembrane domain, co-stimulatory signaling domain and intracellular signaling domain, the second component is the oxygen-dependent degradation domain (ODD), wherein the oxygen-dependent degradation domain is taken from Hypoxia-inducible factor 1α (HIF-1α), its amino acid sequence is SEQ ID No: 1, or its sequence homology is at least 80% of SEQ ID No: 1, and its 23rd position is proline and 185th position is proline.

[0054] The Hi-CAR specifically activated by the tumor microenvironment, under normal tissue aerobic en...

Embodiment 2

[0067] Example 2 Screening and Identification of Oxygen-Dependent Degradation Domain (ODD)

[0068] 1. Synthesis of mCherry, mCherry-ODD (N-ter, 1-112), mCherry-ODD (C-ter, 113-224) and mCherry-ODD (1-224) genes by Shanghai Jierui Bioengineering Co., Ltd. , the nucleotide sequences of which are respectively SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14 and SEQ ID No: 15. The synthesized gene was cloned into the lentiviral expression plasmid pHAGE-EF-1α-MCS-IRES-ZsGreen to obtain the following four recombinant lentiviral expression plasmids, pHAGE-mCherry, pHAGE-mCherry-ODD (N-ter , 1-112), pHAGE-mCherry-ODD (C-ter, 113-224) and pHAGE-mCherry-ODD (1-224);

[0069] 2. Transfect the prepared recombinant lentiviral plasmid together with the lentiviral backbone plasmid psPAX2 and the envelope plasmid PMD2.G into human embryonic kidney HEK293T cells for lentiviral packaging, collect the viral supernatant after 48 hours, and filter with a 0.45 μM filter. Obtain lentiviral vectors LV-...

Embodiment 3

[0075] Example 3 Changes in CAR of T lymphocyte line CD19 Hi-CAR-Jurkat cells under hypoxic environment

[0076] 1. The CD19 CAR and CD19 Hi-CAR genes were synthesized by Shanghai Jierui Bioengineering Co., Ltd. The nucleotide sequences are SEQ ID No: 17 and SEQ ID No: 18 respectively, and the synthesized genes were cloned into lentivirus Expression plasmid pHAGE-EF-1α-MCS-IRES-ZsGreen to obtain the following two recombinant lentiviral plasmids, namely pHAGE-CD19 CAR and pHAGE-CD19 Hi-CAR;

[0077] 2. Transfect the prepared recombinant lentiviral plasmid, lentiviral backbone plasmid psPAX2, and envelope plasmid PMD2.G into human embryonic kidney HEK293T cells for lentiviral packaging, collect the viral supernatant after 48 hours, and filter with a 0.45 μM filter. Obtain lentiviral vectors LV-CD19 CAR and LV-CD19 Hi-CAR, and freeze them at -80°C for future use

[0078] 3. Spread the T lymphocyte line Jurkat E6-1 in a 48-well flat bottom plate, 2×10 per well 5 cells;

[0079]...

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Abstract

The invention belongs to the technical field of biomedical science, and relates to a hypoxia-inducible chimeric antigen receptor capable of achieving specific activation of a tumor microenvironment. The structural domain of the hypoxia-inducible chimeric antigen receptor comprises: (1) one or more structural domains with specific combination of target antigens; (2) a structural domain in an extracellular spacer region; (3) a transmembrane structural domain; (4) one or more costimulatory signal structural domain and a cytokine receptor signal-transduction structural domain; (5) an intracellularsignal-transduction structural domain; and (6) an oxygen-dependent degradation structural domain. Once the hypoxia-inducible chimeric antigen receptor is exposed to an anoxic environment, efficient expression of the Hi-CAR on effect cell membranes is achieved, and specific recognition of target antigens is achieved, so that the specific activation property of the anoxic microenvironment is achieved; and since the anoxic situation of the tumor microenvironment is more significant than that of normal tissue, damage to the normal tissue can be reduced significantly, and meanwhile the activity ofspecific killing of tumors is improved.

Description

technology field [0001] The invention belongs to the technical field of biomedicine, and relates to a hypoxia-inducible chimeric antigen receptor specifically activated by the tumor microenvironment and genetic engineering of the hypoxia-inducible chimeric antigen receptor capable of expressing the specifically activated tumor microenvironment Cell. Background technique [0002] Genetically engineered T cells include T cell receptor T cells (TCR-T) and chimeric antigen receptor T cells (CAR-T). The adoptive transfer of genetically engineered T cells has become a promising approach in cancer immunotherapy. A means and method, especially the CAR-T cell immunotherapy that has been approved by the US FDA in recent years. A chimeric antigen receptor (CAR) is an artificially synthesized receptor consisting of the following five key domains, which are single-chain variable antibodies that specifically recognize tumor-specific antigens (TSA) or tumor-associated antigens (TAA) outsi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/867C12N5/10
CPCC07K14/7051C07K16/2803C07K16/30C07K2319/02C07K2319/33C12N15/86C12N2501/51C12N2501/515C12N2510/00C12N2740/15043
Inventor 徐建青张晓燕廖启彬
Owner SHANGHAI PUBLIC HEALTH CLINICAL CENT
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