Three-target composition capable of regulating and controlling CAR immune cells and application thereof

A technology of immune cells and compositions, applied in the field of immunity, can solve the problems of low density, poor efficacy of CAR-T cells, and difficulty in ensuring the safety of CAR-T treatment.

Pending Publication Date: 2022-08-09
PEKING UNIV SHENZHEN GRADUATE SCHOOL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in solid tumors, due to the heterogeneity of tumor antigens and the complex microenvironment, it is difficult for immune cells such as CAR-T cells to fully function, so the curative effect of CAR-T cells in solid tumors is not good at present.
In addition, most of the specific receptors that are highly expressed on the surface of tumors are also widely expressed (lower density) in normal tissue cells. Therefore, traditional CAR-T cells continue to expand in vivo and attack cells with target targets, namely The targeting effect on normal tissues will bring serious side effects, making it difficult to guarantee the safety of CAR-T therapy

Method used

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  • Three-target composition capable of regulating and controlling CAR immune cells and application thereof
  • Three-target composition capable of regulating and controlling CAR immune cells and application thereof
  • Three-target composition capable of regulating and controlling CAR immune cells and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0065] Further, the preparation method of the three-target CAR immune cells targeting vascular endothelial cells VEGFR1 / 2 and tumor antigens includes: cloning the sequence encoding its chimeric antigen receptor into a lentivirus or retrovirus vector, preparing reverse record virus. After infecting human T cells with the virus, three targets targeting vascular endothelial cells VEGFR1 / 2 and tumor antigens can be prepared to recognize CAR immune cells.

[0066] Optionally, the preparation method of human T cells includes the following steps: collecting peripheral blood mononuclear cells; sorting T cells from peripheral blood lymphocytes; activating and culturing the T cells.

[0067] In a third aspect, an embodiment of the present application also provides a three-target recognition regulatory molecule targeting vascular endothelial cells VEGFR1 / 2 and tumor antigens. Specifically, the three-target recognition regulatory molecules targeting vascular endothelial cells VEGFR1 / 2 an...

Embodiment 1

[0076] Example 1: Construction of CAR molecules

[0077] Based on the lentiviral vector pELPS, a CAR that can recognize vascular endothelial cell receptor VEGFR1 / 2 and tumor antigen Her2 was constructed as an example, VEGF121 and 4D5 scFv were oriented in different molecules, and G4S short peptide was used for tandem, and introduced into the second generation In the structure of CAR, the conventional second-generation CAR structure includes CD8 signal peptide, targeting molecule (4D5 scFv and / or VEGF121), CD8 hinge region, CD8 transmembrane region, 4-1BB costimulatory domain and CD3ζ domain. After the constructed sequence was digested with BamHI+SalI double enzyme, it was cloned into the pELPS vector. The schematic diagram of the CAR structure of the present embodiment is as follows. figure 1 shown.

[0078] A representative CAR structure is as follows:

[0079] Amino acid sequence of CAR-121-4D5-BBZ:

[0080] MALPVTALLLPLALLLHAARPAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEI...

Embodiment 2

[0083] Example 2: Construction of regulatory molecules

[0084] We designed a three-target regulatory molecule for the design of controllable CAR-T cells, in which each component of the three-target regulatory molecule encodes the sequence molecular structure and schematic diagram as shown in figure 2 As indicated, a biofusion expression strategy was employed. The target genes of 4D5 / Myc / 121, 4D5 / 121 / Myc and Myc / 4D5 / 121 were introduced into the N-terminal His tag, the thrombin cleavage site and the homology arms on both sides of MscI and XhoI by PCR. After double digestion with XhoI, it was cloned into pET32a(+) vector using homologous recombinase, transformed into DH5α competent cells and plated, and single clones were picked for sequencing and identification.

[0085] Amino acid sequence of 4D5 / Myc / 121 (SEQ ID NO: 1):

[0086] GSGAMDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL...

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Abstract

The invention relates to a three-target-point composition capable of regulating and controlling CAR immune cells and application of the three-target-point composition. The composition of the CAR immune cells comprises three-target-point regulation and control molecules and the CAR immune cells, the three-target regulation molecule comprises a tag molecule and a target binding domain, the target binding domain can specifically bind to three different targets, and the targets are related to solid tumor treatment; the CAR immune cell can express a chimeric antigen receptor, and the chimeric antigen receptor comprises a tag molecule binding domain capable of specifically binding to the tag molecule. According to the invention, a strategy of combining Her2 / VEGFR1 / VEGFR2 targeted recognition and an adjustable switch is adopted for the first time, so that the composition capable of adjusting and controlling the CAR immune cells aiming at the Her2 / VEGFR1 / VEGFR2 three targets is constructed, the influence of factors such as tumor heterogeneity and microenvironment influence on immunotherapy can be better overcome, and the safety of a CAR-T therapy is improved.

Description

technical field [0001] The invention belongs to the field of immunity technology, and particularly relates to a composition with three targets that can regulate CAR immune cells and its application. Background technique [0002] Cancer is currently the biggest threat that seriously affects human health. According to data released by the World Health Organization in 2017, there were about 14 million new cancer cases in 2012, and new cases are expected to increase by about 70% in the next two decades. Cancer has become a very important public health problem. Standard treatments for cancer include surgery, chemotherapy, radiation, and targeted therapy. With the development of science and technology, the clinical efficacy of these therapies has been continuously improved, but the toxic and side effects have also increased. In recent years, cancer immunotherapy represented by chimeric antigen receptor T cells (CAR-T) has attracted much attention and is the focus of cancer trea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N5/10A61K39/00A61P35/00
CPCC07K16/32C07K14/49C12N5/0636C07K14/70517C07K14/70578C07K14/7051A61K39/001106A61K39/001109A61P35/00C07K2317/622C07K2319/41C07K2319/00C12N2510/00C07K2319/02C07K2319/03C07K2319/33A61K2039/5156
Inventor 曹宇王雪纯郭建师利采李书宏
Owner PEKING UNIV SHENZHEN GRADUATE SCHOOL
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