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Muc1-specific CAR-T cells capable of stably expressing PD-1 antibodies and usage thereof

A PD-1, cell technology, applied in the direction of targeting specific cell fusion, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, DNA/RNA fragments, etc., can solve the problem of expensive treatment, toxic Side effects, high cost of drugs

Active Publication Date: 2019-07-05
SHANGHAI CELL THERAPY RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the one hand, since PD-1 monoclonal antibody is administered intravenously, most patients who receive PD-1 antibody blocking therapy will experience varying degrees of drug toxicity
On the other hand, the production of PD-1 monoclonal antibody involves complex production, preparation and purification processes, which are costly and result in high treatment costs.
[0006] In summary, CAR-T cells have the ability to kill tumor cells and can effectively enter tumor tissues, but their activity is easily inhibited in the tumor microenvironment; while PD-1 antibodies can reactivate the anti-tumor activity of T cells , but the penetration of macromolecular antibodies to solid tumors is insufficient, systemic administration has relatively large toxic and side effects, and the drug cost is high

Method used

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  • Muc1-specific CAR-T cells capable of stably expressing PD-1 antibodies and usage thereof
  • Muc1-specific CAR-T cells capable of stably expressing PD-1 antibodies and usage thereof
  • Muc1-specific CAR-T cells capable of stably expressing PD-1 antibodies and usage thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Example 1: Recombinant plasmids pNB328-Muc1CAR, pS328-Muc1CAR, pNB328-m279V, pS328- Construction of m279V-wt, pS328-m279V, pNB328-Muc1CAR-2A-m279V, pNB328-m279V-IRES-Muc1CAR and the acquisition of various types of pluripotent T cells.

[0104] 1. Construction of recombinant plasmids

[0105] Entrusted Shanghai Jierui Biological Company to synthesize Muc1CAR exogenous gene (including CD8 signal peptide, antigen recognition single-chain antibody, IgG4CH2CH3 hinge region, CD28 transmembrane region, CD28 intracellular domain and CD3ζ tyrosine activation motif, its nucleotide The sequence is shown in SEQ ID NO: 14, the encoded amino acid sequence is shown in SEQ ID NO: 13), and a multiple cloning restriction site (BglII-XbaI-EcoRI-BamHI) is introduced upstream, and the enzyme is inserted downstream Cutting site (SalI-NheI-HindIII-SpeI), which is loaded into the pNB328 vector or pS328 vector cut with EcoR1+SalI double enzymes (for the structure and sequence of pNB328, p...

Embodiment 2

[0113] Example 2: Expression of PBMCs modified and activated by different combinations of Muc1CAR gene and PD-1 antibody gene The positive rate of Muc1CAR gene and the expression of PD-1 antibody were determined.

[0114] The activated T cells of different types and origins from different patients constructed above were cultured on the 12th day after electroporation according to 1×10 6 Cell pellet, wash cell pellet 2 times with PBS, add 2.5ul Biotin-Muc1 antibody, incubate at 4°C for 30min, wash cell pellet 2 times with PBS, add 2ul PE-streptomycin secondary antibody, wash cell pellet 2 times with PBS, add 400ul PBS was used to transfer the cells to flow tubes for detection on the machine. Similarly, collect 2×10 6 Cell number and press 2×10 6 Cells / well were spread in a 6-well plate with 3ml of AIM-V culture medium, cultured in a 5% CO2 incubator at 37°C, and the cell supernatant was collected after 24 hours of culture, and stored at -20°C for future use. Double-antibo...

Embodiment 3

[0117] Example 3: Expression of PBMCs modified with different mass ratios of pNB328-Muc1CAR and pS328-m279V plasmids Determination of Muc1CAR gene positive rate and PD-1 antibody expression

[0118]As described in Example 1, the recombinant plasmids pNB328-Muc1CAR and pS328-m279V were electrotransfected into PBMCs cells at different mass ratios (1:1, 3:5, 1:3, 1:7), and multiple simultaneous expression of Muc1CAR gene and PD -1 antibody T cells. On the 12th day after electroporation, these T cells were divided into 2×10 6 Cell number The cells were collected and divided into 2×10 6 Cells / well were spread in a 6-well plate with 3ml of AIM-V culture medium, cultured in a 5% CO2 incubator at 37°C, and the cell supernatant was collected after 24 hours of culture, and stored at -20°C for future use. Double-antibody sandwich ELISA method (use PD-1 recombinant protein of human origin to coat the microtiter plate, detect with HRP-labeled mouse anti-human IgG4 mAb, use commercial...

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Abstract

The invention relates to CAR-T cells with specifically targeted Muc1 antigens capable of stably expressing PD-1 antibodies at high levels, as well as usage thereof. Specifically, the CAR-T cells provided by the invention have a coding sequence of a chimeric antigen receptor expressing and recognizing Muc1 antigens and a coding sequence of PD1 antibodies; and / or the CAR-T cells comprise chimeric antigen receptors expressing and recognizing the Muc1 antigens, as well as PD-1 antibodies. The chimeric antigen receptors provided by the invention sequentially comprise, from N-terminals to C-terminals, membrane protein signal peptides, anti-Muc1 membrane proximal single-chain antibodies, hinge regions of which the lengths are 50 amino acid residues or above, transmembrane regions, intracellular domains of costimulatory signaling molecules and immunoreceptor tyrosine-based activation motifs. The CAR-T cells provided by the invention are capable of overcoming inhibition of immune micro-environment, promoting apoptosis of tumor cells, and exerting anti-tumor immune response; and thus, the CAR-T cells provided by the invention can be used for treating a multiple kinds of Muc1-positive malignant tumors.

Description

technical field [0001] The present invention relates to Mucl-specific CAR-T cells stably expressing PD-1 antibodies and uses thereof. Background technique [0002] Chimeric antigen receptor T cell (chimeric antigen receptor T cell, CAR-T) therapy technology is undoubtedly a rising star in the field of tumor immune cell therapy. CAR-T technology splices the variable region gene sequence of an antibody that recognizes an antigen molecule with the intracellular region sequence of T lymphocyte immune receptors through genetic engineering technology, and uses retrovirus or lentiviral vectors, transposons or transposons to The seat enzyme system or direct mRNA transduction into lymphocytes, and express fusion protein on the cell surface, so that T lymphocytes can recognize specific antigens in a non-MHC-restricted manner, and enhance their ability to recognize and kill tumors. [0003] Since the structure of CAR was first proposed in 1989 by the Eshhar research group in Israel, a...

Claims

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Application Information

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IPC IPC(8): C12N5/10C07K16/28C12N15/13C12N15/62A61K39/395A61P35/00
CPCC07K16/2818C07K14/7051A61K2039/505C07K2319/02C07K2319/33C07K2319/30A61K39/46447A61K2239/31A61K39/4611A61K39/4631A61K2239/38A61K39/395A61P35/00C07K16/28C12N5/10C12N15/62
Inventor 钱其军金华君何周李林芳刘祥箴王超崔连振
Owner SHANGHAI CELL THERAPY RES INST
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