A chimeric antigen receptor against human caix antigen and its application

A chimeric antigen receptor and antigen technology, applied in the field of biomedicine, can solve the problem of no clinical objective curative effect observed in renal cancer patients, and achieve the effect of good killing renal cancer cells and high specificity.

Active Publication Date: 2022-02-22
XUZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In 2013, Lamers et al. reported the phase I / II clinical trial of CAR-T cells in the treatment of kidney cancer. Unfortunately, no objective clinical efficacy was observed in the 12 patients with kidney cancer.

Method used

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  • A chimeric antigen receptor against human caix antigen and its application
  • A chimeric antigen receptor against human caix antigen and its application
  • A chimeric antigen receptor against human caix antigen and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1 Cloning of Encoding Full-length Human CAIX and CAIX Extracellular DNA

[0076] According to the experimental requirements for the preparation of anti-human CAIX monoclonal antibodies, two regions of human CAIX were selected and connected with two vectors for cloning: CAIX extracellular segment (38-414AA) expression plasmid PET-28a-CAIX EX , full-length (1-459AA) expression plasmid pEGFP-CAIX full ; Amplified by PCR, identified by agarose gel electrophoresis, the length of the target fragment is consistent with the expected, the full-length CAIX is shown in Figure 1 (A), and the extracellular segment of CAIX is shown in Figure 1 (B), and then recovered with a DNA purification kit spare.

Embodiment 2

[0077] Example 2 full-length human CAIX expression plasmid pEGFP-CAIX full and human CAIX extracellular segment expression plasmid PET-28a-CAIX EX build

[0078] The DNA fragment encoding the full-length human CAIX recovered from the gel and the expression vector pEGFP were subjected to double enzyme digestion, and the DNA fragment encoding the extracellular segment of human CAIX and the double enzyme digestion product of the expression vector PET-28a were run on the gel respectively, and the gel was carried out. Recovery and purification; after ligation reaction, then transforming competent E. coli cells, after the clones grow out, pick clones and carry out PCR identification;

[0079] As shown in Figure 2(A), the 8 pEGFP-CAIX selected full All are positive clones; as shown in Figure 2(B), the eight PET-28a-CAIX picked out EX are also positive clones.

Embodiment 3

[0080] Example 3 Expression and purification of human CAIX extracellular segment recombinant protein

[0081] The recombinant expression plasmid PET-28a-CAIX EX Transform Escherichia coli Rosetta competent cells for inducible expression of recombinant proteins, such as image 3 As shown, the small amount of expression detection results show that the recombinant protein CAIX EX - GST expression is correct;

[0082] The expanded culture expressed and purified protein was detected by SDS-PAGE gel electrophoresis, such as image 3 As shown, the recombinant protein CAIX EX - The purity of GST is above 90%, which can meet the requirements of immunized mice.

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Abstract

The present invention provides a chimeric antigen receptor against human CAIX antigen and its application; the chimeric antigen receptor consists of a scFv recognizing human CAIX antigen, a hinge region, a transmembrane region and an intracellular signal domain in sequence. A new anti-human CAIX scFv was screened by a hybridoma antibody screening system, and the scFv was concatenated with the Hinge and transmembrane region of human CD8, the intracellular signaling domain of 4‑1BB and CD3ζ to construct a new chimeric antigen receptor scFv‑CD8‑4‑1BB‑CD3ζ, with higher specificity and better tumor killing effect.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a chimeric antigen receptor against human CAIX antigen and its application. Background technique [0002] Hybridoma technology, that is, lymphocyte hybridoma technology, also known as monoclonal antibody technology. It is developed on the basis of somatic cell fusion technology. In 1975, Kohler and Milstein proved that the fusion of mouse myeloma cells and immunized mouse splenocytes could secrete a homogeneous and highly specific antibody against the antigen—monoclonal antibody. This technology is called hybridoma technology. The basis of this technology is cell fusion technology. Myeloma cells can be continuously passaged in vitro, while spleen cells are terminal cells that cannot be propagated in vitro. If mouse myeloma cells are fused with lymphocytes that secrete certain antibodies, the fused cells not only have the characteristics of unlimited proliferation of tumor c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/85C12N15/867A61K35/17A61P35/00
Inventor 张青郑骏年芦萌萌李慧忠
Owner XUZHOU MEDICAL UNIV
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