A chimeric antigen receptor T-lymphocyte and its application in the preparation of products for treating solid tumors

A chimeric antigen receptor and cell technology, applied in the field of biomedicine, can solve the problems of no clinical efficacy, off-target toxicity, isolation of immune microenvironment, etc. Effects of tumor immune responses

Active Publication Date: 2021-05-04
CARBIOGENE THERAPEUTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing targets at this stage, such as Glypican 3 (GPC-3), Claudin18.2, EpCAM, Mesothelin (MSLN), etc., have achieved certain results in preclinical research, but they have not made further progress in clinical efficacy
Although some abnormally high-expressed proteins on the surface of tumor cells can be screened only through omics detection and big data analysis, when immunotherapy products (such as antibody therapy, CAR-T therapy) targeting these protein antigens are released in human When working in vivo, it is often isolated by the immune microenvironment of tumor cells, and cannot really exert its anti-tumor effect
One of the reasons is that in the process of interaction between tumor cells and immune effector cells (such as T cells, macrophages, etc.), the immune checkpoint pathway (such as B7 family protein) of immune cells is often activated, resulting in a decrease in the viability of effector cells. , resulting in immune escape
In addition, the continuous proliferation and insufficient residence time of CAR-T cells in the body also limits the response rate of CAR-T therapy and increases the recurrence rate of CAR-T therapy
More importantly, some solid tumor targets such as HER2, VEGFR, etc. are prone to off-target toxicity due to non-specific expression, and the safety in the actual clinical application process is questionable

Method used

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  • A chimeric antigen receptor T-lymphocyte and its application in the preparation of products for treating solid tumors
  • A chimeric antigen receptor T-lymphocyte and its application in the preparation of products for treating solid tumors
  • A chimeric antigen receptor T-lymphocyte and its application in the preparation of products for treating solid tumors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0139] Example 1. Preparation of CAR-T cells

[0140] 1. Construction of retroviral vector

[0141] 1. Optimization of the full-length cDNA sequence of wild-type human CD27 gene

[0142] The full-length sequence of wild-type human CD27 gene cDNA is called nCD27. In order to make nCD27 more suitable for expression in human cells, while ensuring that the amino acid sequence encoded by nCD27 remains unchanged, the nCD27 sequence was codon optimized on the website http: / / sg.idtdna.com / site to obtain oCD27, The nucleotide sequence of oCD27 is shown in positions 2719-3501 of SEQ ID NO.1.

[0143] 2. Design and synthesis of S15-CAR-CD27 gene sequence

[0144] The S15-CAR-CD27 gene sequence sequentially includes the coding gene sequence of human CD8 leader peptide, the coding gene sequence of S15 scFv, the coding gene sequence of human CD8 hinge transmembrane region, the coding gene sequence of human 4-1BB intracellular region, the coding gene sequence of human CD3ζ The coding gen...

Embodiment 2

[0184] Example 2, CFSE labeling method to detect the specific killing effect of CAR-T cells on tumor cells

[0185] CFSE (CFDA-SE) is a cell staining reagent that can fluorescently label living cells. It can easily penetrate the cell membrane, covalently bind to intracellular proteins in living cells, and release green fluorescence after hydrolysis. The principle of CFSE labeling living cells can be used to label and quantify tumor cells, so as to detect the killing efficiency of CAR-T cells on tumor target cells. The specific method is: the target cells are equally divided into two groups and adjusted to the same cell density. Stained with low concentration and high concentration of CFSE respectively, wherein high concentration stained target cells and non-stained immune cells were co-cultured according to a certain ratio. After a period of incubation, the high-concentration stained tube of target cells (along with immune cells) was mixed in equal volume with the low-concent...

Embodiment 3

[0200] Example 3. Tumor transplantation model to detect the tumor killing effect of CAR-T cells in animals

[0201] Experimental materials: B-NDG severe combined immunodeficiency mice aged 5-6 weeks and weighing 18-22 g (Biocytogen Biotechnology Co., Ltd.).

[0202] Experimental grouping: The experimental materials were randomly divided into 3 experimental groups, with 5 mice in each group. The specific treatment methods for each group are as follows:

[0203] S15 CAR-CD27 T: B-NDG severe combined immunodeficiency mice were inoculated with U87-MG tumor cell solution (solvent: PBS) in the tail vein, the inoculation volume was 0.3ml (containing 2×10 6 tumor cells). Five days after tumor cell inoculation, the S15 CAR-CD27 T cell solution prepared in Example 1 (solvent is PBS) was injected into the tail vein of the mouse, and the injection volume of S15 CAR-CD27 T cells was 0.2ml (containing 5×10 6 S15 CAR-CD27 T cells).

[0204] S15 CAR T: B-NDG severe combined immunodeficien...

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Abstract

The invention discloses a chimeric antigen receptor T lymphocyte and its application in preparing products for treating solid tumors. The chimeric antigen receptors in the chimeric antigen receptor T lymphocytes sequentially include human CD8 leader peptide, anti-Siglec-15 single-chain antibody, human CD8 hinge transmembrane region, human 4-1BB intracellular region, human CD3ζ cell Inner region, self-cleaving peptide, CSF2Ra signal peptide, EGFRt protein, self-cleaving peptide and human CD27. It is proved by experiments that the chimeric antigen receptor T lymphocytes of the present invention highly express IFNγ and CD107a, have a strong killing function on Siglec-15 positive tumor cells, and the killing efficiency exceeds 1 when the effect-target ratio is 1:1. 80%. Tumor transplantation model experiments show that the chimeric antigen receptor T lymphocytes of the present invention also have a strong killing function against Siglec‑15 positive tumor cells in animals.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a chimeric antigen receptor T lymphocyte and its application in the preparation of products for treating solid tumors, in particular to a Siglec-15-CAR-CD27 chimeric antigen receptor gene Modified T lymphocytes and their application in the preparation of products for treating solid tumors. Background technique [0002] As the research on the mechanism of T lymphocyte tumor immune response has been paid more and more attention, chimeric antigen receptor T (CAR-T) cell therapy is becoming a new immunotherapy strategy in the field of tumor immunotherapy. Since the recognition of target cells by T cells depends specifically on the T cell receptor (TCR), the single-chain antibody fragment (scFv) targeting tumor cell-associated antigens and CD3ζ or FcεRIγ of the T cell receptor Intracellular signal activation motifs are fused into a chimeric antigen receptor (Chimeric antigen rece...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N15/867A61K39/00A61P35/00
CPCA61P35/00A61K39/001129A61K2039/80A61K2039/836A61K2039/844A61K2039/86A61K2039/868A61K2039/876A61K2039/892C07K14/7051C07K14/70578C07K16/2803C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N15/86C12N2510/00C12N2740/10043C12N2740/15043
Inventor 朱建高杨文君
Owner CARBIOGENE THERAPEUTICS CO LTD
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