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Folr1-msln dual targeting CAR-T cells, chimeric antigen receptors and vectors for the treatment of ovarian cancer

A chimeric antigen receptor and single-chain antibody technology, applied in the fields of immunology and molecular biology, can solve the problems of insufficient T cell persistence, difficult T cell infiltration, easy recurrence, etc.

Active Publication Date: 2022-03-18
THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, CAR-T therapy encounters difficulties in solid tumors. The main bottlenecks are: 1) the heterogeneity of solid tumors, and the single-target CAR-T treatment is prone to relapse (antigen escape); Immunosuppressive microenvironment (hypoxia, low pH, Treg cells, indoleamine 2,3-dioxygenase) leading to insufficient T cell activation and / or T cell suppression; 3) Insufficient persistence and expansion of T cells ; 4) Solid tumors have capsules, making it difficult for T cells to infiltrate and homing

Method used

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  • Folr1-msln dual targeting CAR-T cells, chimeric antigen receptors and vectors for the treatment of ovarian cancer
  • Folr1-msln dual targeting CAR-T cells, chimeric antigen receptors and vectors for the treatment of ovarian cancer
  • Folr1-msln dual targeting CAR-T cells, chimeric antigen receptors and vectors for the treatment of ovarian cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1 lentiviral vector construction

[0052] 1. Construction and verification of plasmid vectors

[0053] 1. The construction process of the recombinant plasmids FOLR1-CAR, MSLN-CAR and Tandem-CAR: use the gene synthesis method of PCR bridging to artificially synthesize CD8 signal peptide, single-chain antibody (FOLR1-scFv or MSLN-scFv or Tandem-scFv ), CD8a hinge region, CD28-41BB-CD3z CAR structure, IL-12 gene sequence, introduce XbaI enzyme cutting site in its upstream, and insert NotI enzyme cutting site in its downstream, entrust Beijing Qingke Biological Company to synthesize and load it into In the pCDH vector (purchased from Addgene) double-digested with XbaI and NotI,

[0054] PCDH.

[0055] 2. Extract a large amount of plasmid pCDH

[0056] 1) Take a small amount of frozen glycerol bacteria and inoculate them on LB solid culture plates containing Amp resistance, and place them in a 37°C incubator for overnight culture.

[0057] 2) The bacteria were...

Embodiment 2

[0119] Embodiment 2 separates PBMC and CD3

[0120] 1. From the PBMC

[0121] 1) Collect 10ml of peripheral blood from healthy volunteers, put it in an ice box immediately, bring it back to the cell room in the laboratory, wash it with PBS three times, and add 10ml of PBS+2%FBS buffer.

[0122] 2) Density gradient centrifugation: add 15ml of Stemcell Ficoll solution, centrifuge at 1500rpm, 19°C for 30 minutes, after centrifugation, it can be seen that the tube is divided into 4 layers: the uppermost layer is plasma, containing some platelets; the second layer is a thin white film layer, mainly mononuclear cells, mixed with a small amount of platelets; the third layer is the separation liquid layer; the fourth layer is granulocytes and red blood cells. A thin white film.

[0123] 3) Carefully absorb the buffy coat cells, add 5 times of PBS solution to the obtained PBMCs, gently pipette evenly with a capillary pipette to avoid the production of small air bubbles, and the heigh...

Embodiment 3

[0135] Example 3 lentivirus infection of CD3 and determination of infection results

[0136] 1. Infect CD3 cells per well with a multiplicity of infection (MOI) of 10, add polybrene 8ug / ml, and infect.

[0137] 2. APC-F(ab') after 72 hours of infection 2 Staining, detection of infection rate by flow cytometry.

[0138] 3. Experimental results

[0139] The 4th generation FOLR1-CAR plasmid, MSLN-CAR plasmid and Tandem-CAR plasmid, the structure diagram is shown in figure 1 , the constructed plasmid was digested, and the nucleic acid gel electrophoresis analysis conformed to the size of the target gene fragment. At the same time, we sequenced and verified the three plasmids, and the sequences were correct, indicating that three lentiviral plasmids containing two co-stimulatory molecules CD28 and 4-1BB and IL-12 gene were successfully constructed.

[0140] CD3 cells were infected with MOI=10 for 5 days, and the results of flow cytometry showed that the expression of CAR was 40%,...

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Abstract

The present invention provides a chimeric antigen receptor, comprising sequentially connected single-chain antibody segments, an extracellular hinge region, a transmembrane region, an intracellular immunoreceptor tyrosine activation motif, and an IL-12 gene region; wherein, The single-chain antibody segment is selected from one of folate receptor α (FOLR1) single-chain antibody, mesothelin (MSLN) single-chain antibody or folate receptor α single-chain antibody-mesothelin single-chain antibody tandem complex. The present invention also provides a plasmid vector, a lentiviral vector, and three kinds of CAR-T cells comprising the above-mentioned chimeric antigen receptor. All three CAR-Ts based on the present invention, FOLR1-CAR, MSLN-CAR and Tandem-CAR, can obviously secrete IL-12 and inhibit tumor growth, among which Tandem-CAR has the most obvious effect.

Description

technical field [0001] The invention belongs to the field of immunology and molecular biology, and specifically relates to a FOLR1-MSLN dual-targeting CAR-T cell, a chimeric antigen receptor and a carrier for treating ovarian cancer. Background technique [0002] The mortality rate of ovarian cancer ranks first among the three major malignant tumors of the female reproductive system, 90% of which are epithelial ovarian cancer (epithelial ovarian cancer, EOC). transfer. 55% to 75% of patients who are in remission after first-line treatment still relapse within 2 years, resulting in a five-year survival rate of less than 40%. Therefore, it is of great significance to explore new treatment methods. [0003] In 1989, Gross first proposed the concept of "chimeric receptor", that is, the variable region of the T cell receptor was replaced with a single-chain antibody, so that the modified T cells had antigen-targeting functions. The design of chimeric antigen receptor modified T...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/867C12N15/62C12N5/10A61P35/00A61P15/08
CPCC07K16/28C07K16/18C07K14/70521C07K14/70578C07K14/7051C07K14/5434C12N15/86C12N5/0636A61P35/00A61P15/08C07K2317/622C07K2317/31C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N2740/15043C12N2800/107C12N2510/00
Inventor 梁振梁志清蔡雄伟王延洲
Owner THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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