CAR-T cell targeting ErbB receptor family and self-expressing PD-1 antibody and use thereof

A cell and antibody technology, applied in the fields of genetic engineering and immunology, can solve the problems of low cost, high production cost and response rate.

Pending 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

PD-1 antibody blocking therapy has a certain degree of effect on advanced or refractory melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cel

Method used

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  • CAR-T cell targeting ErbB receptor family and self-expressing PD-1 antibody and use thereof
  • CAR-T cell targeting ErbB receptor family and self-expressing PD-1 antibody and use thereof
  • CAR-T cell targeting ErbB receptor family and self-expressing PD-1 antibody and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Example 1: Construction of recombinant plasmids pS328-antiPD1, pS328-antiPD1-wt and pNB328-EHCAR-EK-28TIZ

[0112] Commissioned Shanghai Jierui Biological Co., Ltd. to synthesize EHCAR-EK-28TIZ gene, anti-PD1 gene and anti-PD1-wt gene, the structural model is as follows figure 1 shown. Each gene was loaded into the pNB328 and pS328 vectors which were double digested with EcoR1+SalI (see CN 201510638974.7 for the structure and sequence of pNB328, which is incorporated herein by reference in its entirety; compared with pNB328, pS328 lacks PB transposition subsequence, and other elements are the same as the pNB328 vector), construct plasmids, respectively named as pNB328-EHCAR-EK-28TIZ, pS328-antiPD1 and pS328-antiPD1-wt.

[0113] The nucleotide sequence of the light chain signal peptide in the structural model diagram is shown in the 1-60 base sequence of SEQ ID NO:2; the encoding of Anti-PD1-wt is shown in SEQ ID NO:4 (the amino acid sequence is shown in SEQ ID NO: 3 s...

Embodiment 2

[0114] Example 2: Determination of positive rate and antibody expression of chimeric antigen receptor-modified T cells constructed by pNB328-EHCAR-EK-28TIZ and pS328-antiPD1 plasmids with different mass ratios

[0115] Set the amount of pNB328-EHCAR-EK-28TIZ and pS328-antiPD1 plasmids to 7 ratios of 1ug+7ug, 2ug+6ug, 3ug+5ug, 4ug+4ug, 5ug+3ug, 6ug+2ug, 7ug+1ug , Carry out CART cell construction. The build method is as follows:

[0116] Peripheral blood mononuclear cells (PBMCs) were isolated from Shanghai Cell Therapy Production Center. Cultivate PBMCs for 2-4 hours. The unattached suspension cells are initial T cells. Collect the suspension cells into a 15ml centrifuge tube, centrifuge at 1200rmp for 3min, discard the supernatant, add physiological saline, centrifuge at 1200rmp for 3min, discard the physiological saline, and repeat this step; take eight 1.5ml centrifuge tubes and add 5×10 6 Cells, numbered a, b, c, d, e, f, g and h, centrifuged at 1200rmp for 3min, discard...

Embodiment 3

[0134] Example 3: Construction of EHCAR-EK-28TIZ T cells and EHCAR-EK-28TIZ-antiPD1T cells and determination of positive rate and antibody expression

[0135]Take 6ug pNB328-EHCAR-EK-28TIZ plasmid for the construction of EHCAR-EK-28TIZ T cells, respectively take 4ugpNB328-EHCAR-EK-28TIZ and 4ug pS328-antiPD1 plasmids for the construction of EHCAR-EK-28TIZ-antiPD1T cells, construction method With embodiment 2.

[0136] The positive rate of EHCAR-EK-28TIZ T cells and EHCAR-EK-28TIZ-antiPD1T cells was detected by flow cytometry, and the method was the same as in Example 2. see results Figure 3A , Self-expression of PD1 antibody does not reduce the positive rate of CART cells.

[0137] ELISA detection EHCAR-EK-28TIZ-antiPD1 T cell antibody expression, the method is the same as in Example 2, see the results Figure 3B .

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Abstract

The present invention provides a CAR-T cell self-expressing a PD-1 immunological checkpoint inhibitory antibody and targeting a chimeric antigen receptor of ErbB receptor family and a use thereof. TheT cell comprises a coding sequence expressing the chimeric antigen receptor targeting the ErbB receptor family and a coding sequence of the PD1 antibody; and/or expressing the chimeric antigen receptor targeting the ErbB receptor family and the PD1 antibody. The T cell has better proliferation and activation ability than those of a T cell singly targeting the chimeric antigen receptor of the ErbBreceptor family, can overcome inhibition of an immune microenvironment, promotes apoptosis of tumor cells, and exerts an anti-tumor immune response and a tumor cell killing function.

Description

technical field [0001] The invention belongs to genetic engineering and immunology, and relates to CAR-T cells targeting ErbB receptor family and self-expressing PD-1 antibody and its use PD1 antibody PD1 antibody. Background technique [0002] Cancer has now become the number one killer of human health. The fast pace of life, huge work pressure, unhealthy eating habits, and poor environment are all accomplices to the occurrence of cancer, making the high incidence and younger trend of cancer more and more obvious. The current commonly used treatment methods are very limited in effect, and a more effective treatment method still needs to be explored to improve the survival rate and quality of life of cancer patients. [0003] As one of the important branches of tumor immunotherapy, chimeric antigen receptor T cell (CAR-T) therapy has achieved very good results in malignant hematological tumors, and the complete remission rate for relapsed and refractory B-cell leukemia excee...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/13C12N15/62C12N15/63C07K19/00C07K16/28A61K39/395A61P35/00A61P35/02
CPCC07K16/2863C07K16/2818C07K14/7051A61K35/17C07K2319/33C07K2319/02C07K2317/622C07K2317/73A61K2039/505A61K39/395A61P35/00A61P35/02C07K14/705C07K16/28C07K19/00C12N5/10C12N15/62C12N15/63
Inventor 钱其军金华君游术梅江芏青刘祥箴李林芳王超
Owner SHANGHAI CELL THERAPY RES INST
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