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Combined chimerism antigen receptor, expression vector, lentivirus and T-cell

A technology of chimeric antigen receptors and cells, applied in the field of biomedicine, can solve the problems of CD19, low effective rate of ALL, and small number of clinical studies, etc., and achieve the goal of increasing complete remission rate, preventing relapse, and increasing long-term survival rate Effect

Inactive Publication Date: 2019-07-09
WUHAN BIO RAID BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0027] The number of clinical studies of CART-19 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 CART-19 treatment of 4 relapsed and refractory CLL patients after allogeneic transplantation, 1 patient achieved partial remission (PR) at 8 weeks, and 1 patient achieved stable disease (SD) for more than 15 months. 2 cases of PD, the low effective rate 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 CART-19 cannot meet the requirements of CD19 target loss and recurrence patients and patients with plasma cell tumors

Method used

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  • Combined chimerism antigen receptor, expression vector, lentivirus and T-cell
  • Combined chimerism antigen receptor, expression vector, lentivirus and T-cell
  • Combined chimerism antigen receptor, expression vector, lentivirus and T-cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Implementation example 1, construction of recombinant lentiviral vector pCAR19, pCAR22

[0074] By sequence 1 (CAR19, see Figure 13 , marked as CAR19, SEQ ID NO.1) and sequence 2 (CAR22, see Figure 14 , marked as CAR22, SEQ ID NO.2), was synthesized by Nanjing GenScript Biotechnology Co., Ltd., and the synthesized sequence was cloned into the T vector.

[0075] 1. The length of the CAR19 sequence fragment is 1596bp, with EcoRI and MluI restriction sites designed at both ends, and cloned into the multiple cloning site of the lentiviral backbone plasmid pLVX-EF1α-IRES-Puro to complete the construction of the vector. To build a complete graph see figure 1 .

[0076] The part structure of CAR19 is figure 2 As shown, CD19 is a third-generation CAR that uses CD28 and 4-1BB as co-stimulatory signals.

[0077] Comparison chart of CAR19 sequence sequencing image 3 Shown: The black line at the bottom of the figure indicates the standard sequence, and the gray line indic...

Embodiment 2

[0084] Plasmid preparation of implementation example 2, pCAR19, pCAR22

[0085] Inoculate the DH5alpha strain of the pCAR19 or pCAR22 plasmid into 250 mL of LB culture medium 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.

[0086] 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 .

[0087] 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.

[0088] 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 mix thoroughly...

Embodiment 3

[0109] Implementation example 3, preparation of recombinant lentivirus LVCAR19, LVCAR22

[0110] 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 (pCAR19 or pCAR22: gag plasmid: vsvg plasmid = 6:3:2) into a 960 μg PEI tube, vortex, and equilibrate at room temperature for 10 minutes. Mix 35mL of the mixture of PEI and plasmid with 525mL of 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.

[0111] After the supernatant was centrifuged at 4000 rpm (or 3000 g) for 30 min, cryonase (Takara) was added to the centrifuged supern...

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Abstract

The invention discloses a combined chimerism antigen receptor, an expression vector, lentivirus and a T-cell. The combined chimerism antigen receptor comprises chimeric protein CAR19 and chimeric protein CAR22, and the chimeric protein CAR19 is formed by coupling a fragment comprising at least a single chain antibody that specifically identifies CD19 and a T-cell activation motif; the CAR22 is formed by coupling a fragment comprising at least a single chain antibody that specifically identifies CD22 and a T-cell activation motif. In addition, the expression vector, lentivirus and T-cell are based on the principle of CART, combined with CAR19T and CAR22T to effectively reduce high recurrence of CAR19T and to prevent severe CR of the CAR19T. The combined chimerism antigen receptor, an expression vector, lentivirus and a T-cell provide more effective treatment for tumor diseases.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to two kinds of ligands combined with CD19 and CD22, specifically a combined chimeric antigen receptor related to CD19 and CD22, an expression vector, lentivirus and 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 tumor antigens in a major histocompatibility complex (MHC)-dependent manner. [0004] Tumor immunotherapy is generally divided into two categories, nonspecific immunity and specific immunity. Non-specific immunotherapy mainly includes interleukin-2 (interleukin-2, IL-2)...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61P35/00
CPCC07K14/7051C07K16/2803C12N15/86C12N5/0636A61K35/17C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N2740/15043C12N2800/107C12N2510/00
Inventor 张同存顾潮江廖兴华
Owner WUHAN BIO RAID BIOTECH CO LTD
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