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Bispecific chimeric antigen receptor combining two single chain antibodies and expression vector

A chimeric antigen receptor and single-chain antibody technology, applied in the field of biomedicine, can solve the problems of low effective rate of ALL, small number of clinical studies, and low effective rate, so as to reduce high recurrence rate, increase long-term survival rate, increase Effect on Complete Response Rate

Active Publication Date: 2018-12-18
WUHAN BIO RAID BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0028] The number of clinical studies of Anti-CD19 CAR-T cells in the treatment of CLL is less than that of ALL, and its effective rate is also lower than that of ALL
In 2013, the MD Anderson team reported 4 cases of relapsed and refractory CLL patients treated with Anti-CD19 CAR-T cells after allogeneic transplantation. Stable (SD), 2 cases of PD, the low efficiency of this treatment may be related to the lack of effective chemotherapy pretreatment
For the treatment of B-cell tumors, CD19 is an ideal target, but Anti-CD19 CAR-T cells cannot meet the requirements of CD19 target loss in recurrent patients and patients with plasma cell tumors

Method used

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  • Bispecific chimeric antigen receptor combining two single chain antibodies and expression vector
  • Bispecific chimeric antigen receptor combining two single chain antibodies and expression vector
  • Bispecific chimeric antigen receptor combining two single chain antibodies and expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Implementation example 1, construction of recombinant lentiviral vector pLVX-CD19-CD22

[0076] The nucleotide sequence of the light chain+heavy chain of the first single-chain antibody can be called the CAR19 (CD19scFv) sequence, and the nucleotide sequence of the light chain+heavy chain of the second single-chain antibody can be called the CAR22 (CD22scFv) sequence, The sequence of CAR19 (CD19scFv) can be shown in SEQ ID NO.1, and the sequence of CAR22 (CD22scFv) can be shown in SEQ ID NO.2.

[0077] In order to further improve the design of CAR, CD28 and 4-1BB, the transmembrane region of leukocyte antigen differentiation group molecules of the third generation CAR, were used as transmembrane co-stimulatory signal molecules. The CAR sequence is as follows:

[0078] SEQ ID NO.3 is CAR19 (CAR19+CD8hinge+CD28+4-1BB+CD3ζ).

[0079] SEQ ID NO.3 is CAR22 (CAR22+CD8hinge+CD28+4-1BB+CD3ζ).

[0080] By sequence 1 (CAR19, see Figure 13 , marked as CAR19, SEQ ID NO.3) and ...

Embodiment 2

[0091] Implementation example 2, the preparation of pLVX-CD19-CD22 plasmid

[0092] Inoculate the DH5alpha strain containing the pLVX-CD19-CD22 plasmid into 250 mL of LB culture solution containing 100 μg / mL ampicillin, and culture overnight at 37° C. and 220 rpm. The culture solution was centrifuged at 6000g for 20min at 4°C, and the supernatant was discarded.

[0093] Take out Buffers P1 from the EndoFree plasma mega kit (Qiagen), add 120mL pre-cooled Buffers P1 to the centrifuged E. coli pellet, cover the cap of the centrifuge bottle, shake the centrifuge bottle vigorously to completely disperse the E. coli pellet in Buffers P1 .

[0094] Add 120mL Buffers P2 to the centrifuge bottle, put the cap on the roller mixer, slowly increase the speed to 50rpm, mix thoroughly and place at room temperature for 5min.

[0095] Add 120mL Buffers P3 to the centrifuge bottle, put the cap on the roller mixer, slowly increase the speed to the maximum speed of the roller mixer 70rpm, and m...

Embodiment 3

[0117] Implementation example 3, preparation of recombinant lentivirus LV Anti-CD19-CD22 CAR

[0118] Insert 130.0~140.0×10 in multilayer cell culture bottle (Hyperflask) (Corning) 6 Number of 293T cells (Takara), a total of 560 mL DMEM complete medium (50 mL fetal bovine serum, 5 mL Antibiotic-Antimycotic (100×)), at 37 °C with 5% CO 2 Incubate for 24 hours in the incubator. Add DMEM complete medium mixed with 320 μg plasmid (pLVX-CD19-CD22: gag plasmid: vsvg plasmid=6:3:2) into a 960 μg PEI tube, vortex, and equilibrate at room temperature for 10 minutes. Mix the above-mentioned 35mL PEI-plasmid mixture with 525mL DMEM complete medium, and replace it into the above-mentioned multi-layer cell culture flask. Place the multilayer cell culture flask at 37 °C with 5% CO 2 After 3 days in the incubator, the cell culture supernatant was collected.

[0119] After the supernatant was centrifuged at 4000 rpm (or 3000 g) for 30 min, cryonase (Takara) was added to the centrifuged su...

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Abstract

The present invention provides a bispecific chimeric antigen receptor combining two single chain antibodies, a coding gene, a combined expression vector, a method for preparing a recombinant lentivirus, a method for preparing CD3+T genetically modified by the bispecific chimeric antigen receptor combining the two single chain antibodies, and use of the genetically-modified CD3+T, and the bispecific chimeric antigen receptor combining the two single chain antibodies includes a CD8 signal peptide, an antigen binding domain composed of the two different single chain antibodies, a transmembrane domain, and an intracellular signaling domain. The treatment of patients with CD19 target loss recurrence and plasma cell tumor can be achieved, and a more effective treatment route is provided for tumor diseases.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to a bispecific chimeric antigen receptor combined with two kinds of single-chain antibodies, an encoding gene, a combined expression vector, a method for preparing recombinant lentivirus, and a method for preparing a bispecific chimeric antigen receptor combined with two kinds of single-chain antibodies. A method for specific chimeric antigen receptor genetically modified CD3+T cells and use of genetically modified CD3+T cells. Background technique [0002] Tumor Immunotherapy [0003] The theoretical basis of tumor immunotherapy is that the immune system has the ability to recognize tumor-associated antigens and regulate the body's ability to attack tumor cells (highly specific cytolysis). This biological process is complex and is still under investigation. In the 1990s, several scientific research groups have discovered tumor antigens, and T lymphocytes can recognize these tu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N7/01C12N5/10A61K35/17A61P35/00A61P35/02
CPCA61K35/17A61P35/00A61P35/02C07K14/7051C07K16/2803C07K2319/02C07K2319/33C12N7/00C12N15/86C12N2510/00C12N2740/15021C12N2740/15043
Inventor 张同存顾潮江吴寒许婧李结珍范博文舒冲
Owner WUHAN BIO RAID BIOTECH CO LTD
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