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Construction and application of chimeric antigen receptor-T (CAR-T) cells capable of targeting mesothelin and carrying PD-L1 blocking agent

A PD-L1, blocker technology, applied in the field of CAR-T cell construction, can solve the problems of low survival rate, low activity, and inability to achieve therapeutic effect of immune effector cells, and achieve the effect of enhancing the killing effect of tumor cells. Effect

Inactive Publication Date: 2018-11-23
SUZHOU MAXIMUM BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although CAR-T immunotherapy has achieved ideal curative effect in some tumors, in many solid tumors, due to the protective effect of the tumor microenvironment on the tumor and the inhibition of PD-L1 on the killing ability of immune effector cells, the immune effect The survival rate and activity of cells in tumor tissue are low, and the expected therapeutic effect cannot be achieved

Method used

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  • Construction and application of chimeric antigen receptor-T (CAR-T) cells capable of targeting mesothelin and carrying PD-L1 blocking agent
  • Construction and application of chimeric antigen receptor-T (CAR-T) cells capable of targeting mesothelin and carrying PD-L1 blocking agent
  • Construction and application of chimeric antigen receptor-T (CAR-T) cells capable of targeting mesothelin and carrying PD-L1 blocking agent

Examples

Experimental program
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Effect test

preparation example Construction

[0044] The method for preparing CAR-T cells targeting mesothelin and carrying a PD-L1 blocking agent comprises the following steps:

[0045] (1) Synthesizing a chimeric antigen receptor targeting mesothelin and carrying a PD-L1 blocker, the specific method is:

[0046] a. Synthesize the leader peptide of the CD8 antigen, the gene fragment of the anti-mesothelin SS1P single-chain antibody, the gene fragment of the hinge region, the gene fragment of the transmembrane region, the gene fragment of the intracellular signal region and the gene fragment of the anti-PD-L1 antibody secretion region. The gene fragment of CD28, the gene fragment of CD3δ and the gene fragment of anti-PD-L1 antibody scFv;

[0047] b. genetically fusing the leader peptide of the CD8 antigen with the anti-mesothelin SS1P single-chain antibody gene fragment, thereby forming a guide chain-mesothelin SS1P scFv;

[0048] c. Gene fusion of the gene fragment of the hinge region, the gene fragment of the transmemb...

Embodiment 1

[0054] Example 1: Targeting Mesothelin and combining CAR-T cells secreting anti-PD-L1

[0055] In this example, the hinge region constituting the chimeric antigen receptor (CAR) is the hinge region of human CD8α, the transmembrane region is the gene fragment of the transmembrane region of human CD28, and the intracellular signal domain is the intracellular signal region of human CD3δ.

[0056] The amino acid sequence of the anti-Mesothelin antibody was searched and optimized according to the codons preferred by humans to synthesize the scfv sequence of Mesothelin.

[0057] The amino acid sequence of the anti-PD-L1 antibody was searched and optimized according to human preferred codons to synthesize the anti-PD-L1 scfv sequence.

[0058] 1. Prepare the cDNA of PBMCs as a template

[0059] PBMCs were isolated from peripheral blood, RNA was extracted, reversed into cDNA, and the cDNA obtained after reverse transcription was stored in a -80°C refrigerator for later use.

[0060]...

Embodiment 2

[0101] Example 2: Expression detection of mesoCAR in CAR-T cells

[0102] 1. Western Blot detection

[0103] By detecting the CD3 chain in mesoCAR, we can see the expression of CAR in CAR-T cells. The test group is the CAR-T cells obtained by adding CAR plasmid and T cell transfection in Example 1, and the control group is the T cells obtained by transfection without adding CAR plasmid in Example 1.

[0104] (1) Extraction and preparation of total cell protein samples

[0105] ① Collect the CAR-T cells in Example 1 and wash them twice with saline;

[0106] ② Add 160 μl cell lysate and place on ice for 10 minutes;

[0107] ③ After the cells are fully lysed, centrifuge at 12000rpm, 4°C for 10min;

[0108] ④ Collect the supernatant and store it at -20°C.

[0109] (2) Western Blot detection

[0110] The total proteins of mesoCAR-T and control T cells were extracted, and the expression of CAR in mesoCAR-T cells was detected by CD3δ chain-specific antibody and western blot. F...

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Abstract

The invention relates to the field of tumor immunology, in particular to construction and application of chimeric antigen receptor-T (CAR-T) cells capable of targeting mesothelin and carrying a PD-L1blocking agent. The CAR-T cells are constructed by transfecting a CAR capable of targeting the mesothelin and carrying the PD-L1 blocking agent, wherein the CAR capable of targeting the mesothelin andcarrying the PD-L1 blocking agent is formed by a leader peptide of a CD8 antigen, an anti-mesothelin single-chain antibody, a hinge region, a transmembrane region, an intracellular signal domain andan anti-PD-L1 antibody secretion region which are sequentially connected with one another. The mesothelin SS1P scFv of the CAR-T cells provided by the invention can specifically bind to tumor cells expressing mesothelin glycoprotein and promote the secretion of anti-tumor-related cytokines, thereby achieving a killing effect on the tumor cells; furthermore, the CAR-T cells provided by invention can also secrete a PD-L1 antibody, specifically bind to PD-L1 produced by tumor cell expression, and block the inhibitory effect of a PD-L1 / PD-1 signal on T cell activity, thus facilitating the long-term effective inhibition of the CAR-T on tumor cell growth.

Description

technical field [0001] The present invention relates to the field of tumor immunity, in particular to the construction and application of a CAR-T cell targeting mesothelin and carrying a PD-L1 blocking agent. Background technique [0002] Mesothelin (MSLN) is a glycoprotein anchored on the plasma membrane through the phosphatidylinositol region (GPI), which is hardly expressed in parenchymal organs such as the heart, kidney, and liver. Under normal circumstances, It is mainly expressed in the mesothelial cells of the peritoneum, thoracoperitoneal cavity, and pericardium, and lowly expressed on the surface of epithelial cells such as the upper trachea, ovary, tonsil, and fallopian tube. Recent studies on solid tumors have found that mesothelin is overexpressed in lung cancer, gastric cancer, esophageal cancer and thymus cancer, thereby changing the biological characteristics of cells. Mesothelin plays a role in regulating cell function mainly through two pathways in cells, o...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N5/10A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/7051C07K16/18C07K16/2827C07K2317/622C07K2319/33C12N2510/00
Inventor 尚小云
Owner SUZHOU MAXIMUM BIO TECH CO LTD
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