T cell comprising CD40 antibody and muc1-specific chimeric antigen receptor gene and use thereof

A technology of chimeric antigen receptors and cells, applied in the fields of genetic engineering and immunology

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

Although these new sugar chains and peptide epitopes can be ideal targets for CAR-T cell therapy, since different sugar chains and peptide epitopes are displayed on the surface of different tumor cells, specific targeting of a certain A glycopeptide CAR-T cell may not be used in many types of tumors

Method used

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  • T cell comprising CD40 antibody and muc1-specific chimeric antigen receptor gene and use thereof
  • T cell comprising CD40 antibody and muc1-specific chimeric antigen receptor gene and use thereof
  • T cell comprising CD40 antibody and muc1-specific chimeric antigen receptor gene and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1: Construction of recombinant plasmids pS328-αCD40-wt, pS328-αCD40 and pNB328-Muc1CAR

[0107] Entrusted Shanghai Jierui Biological Co., Ltd. to synthesize Muc1CAR gene, anti-CD40 gene and anti-CD40-wt gene, the structural model is as follows figure 1 shown. They were respectively loaded into pNB328 and pS328 vectors cut with EcoR1+SalI (for the structure and sequence of pNB328, please refer to CN 201510638974.7, the entire contents of which are incorporated herein by reference; compared with pNB328, pS328 lacks the PB transposon sequence, and other elements are the same as the pNB328 vector), construct plasmids, named pNB328-Muc1CAR, pS328-αCD40 and pS328-αCD40-wt, respectively.

[0108] The nucleotide sequence of the light chain signal peptide in the structural model diagram is shown in the 1st-60th base sequence of SEQ ID NO: 1; the nucleotide sequence of Anti-CD40-wt is shown in SEQ ID NO: 3; The nucleotide sequence of Anti-CD40 is shown in the 61-1491 ba...

Embodiment 2

[0109] Example 2: Determination of positive rate and antibody expression of chimeric antigen receptor-modified T cells constructed by pNB328-Muc1CAR and pS328-αCD40 plasmids with different mass ratios

[0110] Set the amount of pNB328-Muc1CAR and pS328-αCD40 plasmids to 7 ratios of 1ug+7ug, 2ug+6ug, 3ug+5ug, 4ug+4ug, 5ug+3ug, 6ug+2ug, 7ug+1ug, and carry out CAR T cell building. The build method is as follows:

[0111] 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, discarded the supernatant...

Embodiment 3

[0129] Example 3: Construction of Muc1CAR T cells and Muc1CAR-αCD40 T cells and determination of positive rate and antibody expression

[0130] 6ug pNB328-Muc1CAR plasmid was used to construct Muc1CAR T cells, and 4ug pNB328-Muc1CAR and 4ug pS328-αCD40 plasmids were used to construct Muc1CAR-αCD40 T cells respectively. The construction method was the same as in Example 2.

[0131] The positive rate of the Mock-T cells constructed in Example 2 and the Muc1CAR T cells and Muc1CAR-αCD40 T cells constructed in this example were detected by flow cytometry, and the method was the same as in Example 2.

[0132] see results Figure 3A , showing that self-expression of CD40 antibody does not reduce the positive rate of CART cells.

[0133] ELISA detection of Mock-T cells, Muc1CAR T cells and Muc1CAR-αCD40 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 T cell self-expressing a CD40 activating antibody and targeting a Muc1 antigen chimeric antigen receptor, and a use thereof. Specifically, the provided chimeric antigen receptor successively comprises a membrane protein signal peptide, an anti-Muc1 single-chain antibody, a hinge region having a length of 50 amino acid residues or more, a transmembrane region, a costimulatory signaling molecule intracellular structural domain and an immunoreceptor tyrosine activated sequence motif from an N-terminus to a C-terminus. The present invention also comprises the T cell which self-expresses the CD40 activating antibody chimeric antigen receptor. The T cell has better proliferation and activation ability and tumor cell killing function than those of a T cell singlytargeting the Muc1 antigen chimeric antigen receptor.

Description

technical field [0001] The invention belongs to genetic engineering and immunology, and relates to T cells containing CD40 antibody and mucl specific chimeric antigen receptor gene and application thereof. Background technique [0002] Immunotherapy against malignant tumors has developed rapidly in recent years and has achieved remarkable clinical efficacy. Since 2011, Nature and JCO, the top journal of clinical oncology, have published review articles with the same title "The era of tumor immunotherapy has come" (Nature.2011; 480(7378):480; J Clin Oncol.2011; 29(36) :4828), tumor immune cell therapy ushered in a new round of research upsurge. [0003] As one of the important branches of tumor immunotherapy, chimeric antigen receptor T cell therapy has achieved very good results in malignant hematological tumors, and the complete remission rate for relapsed and refractory B-cell leukemia exceeds 90%. In August 2017, the U.S. FDA approved Novartis's tisagenlecleucel chimeri...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/63C12N15/13C07K19/00C07K16/28A61K39/395A61P35/00
CPCC07K16/2878C07K14/7051C07K16/3092A61K35/17A61K39/3955C07K2319/02C07K2319/33C07K2319/30C07K2317/73A61K2300/00A61K39/395A61K48/00A61P35/00C07K16/28C07K19/00C12N5/10C12N15/63
Inventor 钱其军金华君游术梅何周唐熙李林芳王超
Owner SHANGHAI CELL THERAPY RES INST
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