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Lentiviral vector of CAR and PD1-CD28 chimeric receptor for simultaneous expression of targeted CD19 and CD20

A PD1-CD28, lentiviral vector technology, applied in the field of molecular biology, can solve problems such as unoptimistic survival rate and heavy medical burden, and achieve the effects of optimized selection and connection sequence, strong killing, and high-efficiency expression

Active Publication Date: 2018-05-29
BEIJING CANCER HOSPITAL PEKING UNIV CANCER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The 5-year survival rate is not optimistic
At present, the China Food and Drug Administration (CFDA) has not approved the clinical use of Nivolumab and Pembrolizumab in China
Moreover, Nivolumab and Pembrolizumab need to be used multiple times, and for most patients, the medical burden is too heavy

Method used

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  • Lentiviral vector of CAR and PD1-CD28 chimeric receptor for simultaneous expression of targeted CD19 and CD20
  • Lentiviral vector of CAR and PD1-CD28 chimeric receptor for simultaneous expression of targeted CD19 and CD20
  • Lentiviral vector of CAR and PD1-CD28 chimeric receptor for simultaneous expression of targeted CD19 and CD20

Examples

Experimental program
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Effect test

Embodiment 1

[0041] This example is used to illustrate the construction method of the lentiviral vector of the present invention.

[0042] 1. Raw materials

[0043] The original vector pCDH-EF1-Luc2-T2A-tdTomato: purchased from Wuhan Miaoling Biotechnology Co., Ltd.

[0044] XbaI and SalI endonucleases: purchased from New England Biolabs (Beijing) LTD.

[0045] 2. Construction of the carrier

[0046] (1) Link the nucleotide sequence of the CAR targeting CD19&CD20 and the PD1-CD28 chimeric receptor using Furin-linker-spacer-F2A to synthesize a fusion gene, which contains XbaI and SalI restriction site, and loaded on the pUC57 vector;

[0047] (2) XbaI and SalI double-digest the pUC57 vector containing the target gene, and cut the gel to recover the target gene fragment;

[0048] (3) The original vector pCDH-EF1-Luc2-T2A-tdTomato was digested with XbaI and SalI, and the vector fragment of about 6.5kb was recovered by gel cutting;

[0049] (4) Ligate the recovered target gene fragment an...

Embodiment 2

[0051] This example is used to illustrate the selection of key elements in the vector and the optimization of the ligation sequence during the process of constructing the vector.

[0052] In this example, a combination of various promoters highly expressed in T cells and the main connection methods of two target genes was attempted (see figure 1 ).

[0053] synthesis figure 1 The 18 target gene sequences were constructed in the pCDH-EF1-Luc2-T2A-tdTomato vector by XbaI and SalI double digestion, and the 18 constructed lentiviral vectors were packaged with lentivirus, and then infected peripheral blood T cells, targeting Flow cytometric staining was performed on CD19&CD20 CAR and PD-1 antibodies, and analyzed by flow cytometry. The results are shown in Table 1 and figure 2 shown.

[0054] It should be noted that since the pCDH-EF1-Luc2-T2A-tdTomato vector has its own promoter EF1α, when using other promoters, it is necessary to first introduce restriction site sequences ...

Embodiment 3

[0061] The difference between this example and Example 1 is that the gene positions of CD19&CD20CAR and PD1-CD28 in the vector were exchanged, and the experiment found that after the position exchange, the expression efficiency of the target protein was not affected ( Figure 4 ).

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Abstract

The invention relates to the field of molecular biology, in particular to a lentiviral vector of a CAR and PD1-CD28 chimeric receptor for simultaneous expression of targeted CD19 and CD20. Through theCAR and PD1-CD28 chimeric receptor which simultaneously efficiently expresses targeted CD19 and CD20, the lentiviral vector ensures that a carrier-infected T cell can not only realize targeted identification of a tumor cell which expresses CD19 and CD20 but also remove PD-L1 / PD1 mediated immunosuppression, so that a stronger tumor cell killing capability is achieved. Furthermore, selection and connection sequences of key elements in the carrier are optimized, and the expression efficiency of the CAR and PD1-CD28 chimeric receptor of the targeted CD19 and CD20.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular to a lentiviral vector that simultaneously expresses a CAR targeting CD19 and CD20 and a chimeric receptor of PD1-CD28. Background technique [0002] In recent years, anti-CD19 CAR-T has achieved world-renowned achievements in the treatment of relapsed, drug-resistant, and refractory B-cell leukemia and lymphoma, which has aroused great interest from cancer clinicians around the world. However, the treatment of B cell-derived tumors with CAR-T targeting only CD19 will lead to low or no expression of CD19 in tumor cells, which will lead to tumor cell immune escape and relapse and drug resistance. Therefore, CARs targeting both CD19 and CD20 can simultaneously target CD19 and CD20 in B-cell-derived tumors. Therefore, for B-cell-derived tumors, especially for patients who fail CD19 CAR-T therapy, dual-target CAR-T will achieve a better therapeutic effect. CD19 and CD20 are ce...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/66C12N15/62C12N5/10A61K35/17A61P35/00
CPCA61K35/17C07K14/70521C07K16/2803C07K16/2887C07K2319/00C07K2319/33C07K2319/74C12N5/0636C12N15/66C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 陆哲明张超亭
Owner BEIJING CANCER HOSPITAL PEKING UNIV CANCER HOSPITAL
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