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TM4SF1-specific chimeric antigen receptor and application thereof

A chimeric antigen receptor, -TM4SF1 technology, used in NGF-receptor/TNF-receptor superfamily, antibody mimics/scaffolds, for targeting specific cell fusions, etc.

Active Publication Date: 2017-09-29
上海科棋药业科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, CAR-T has not made a breakthrough in the field of solid tumors, so there is an urgent need for new tumor targets and new CAR molecular designs to promote its application in the field of solid tumors

Method used

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  • TM4SF1-specific chimeric antigen receptor and application thereof
  • TM4SF1-specific chimeric antigen receptor and application thereof
  • TM4SF1-specific chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] This example provides a method for constructing the above-mentioned CAR molecule into the lentiviral vector pCDH.

[0048] According to the construction of the TM4SF1 CAR molecule in this example, according to the different signaling regions of the CAR molecule, it can be divided into three types: L6-28Z, L6-BBZ, and L6-28BBZ: the intracellular signaling region used in L6-28Z is CD28-CD3ζ; The intracellular signal region used in L6-BBZ is 4-1BB-CD3ζ; while the intracellular signal region used in L6-28BBZ is CD28-4-1BB-CD3ζ, and its molecular structure pattern is shown in figure 1 shown. The above CAR molecule was constructed into the lentiviral vector pCDH by conventional molecular cloning methods. The structure of the lentiviral vector pCDH is as follows: figure 2 shown.

[0049] Construction of L6-28BBZ vector: First, the L6-28BBZ nucleic acid fragment was chemically synthesized into the pUC57 vector (Shanghai Aoji Biological Co., Ltd.), and the L6-28BBZ fragment w...

Embodiment 2

[0053] This example is the construction of TM4SF1 CAR-T cells.

[0054] Packaging of CAR lentivirus: First, use the QIAGEN endotoxin-free large-scale plasmid extraction kit to extract the lentiviral plasmids pCDH-L6-28Z, pCDH-L6-BBZ, pCDH-L6-28BBZ, and pCDH-19-28BBZ constructed in Example 1 (non-targeting TM4SF1 control CAR molecule plasmid) and lentiviral system auxiliary packaging plasmids pMD2.G and pSPAX2. Spread 2.5×10E7 293T into T75 culture flasks one day before transfection. 1 hour before transfection, the medium of 293T cells was replaced with 10 ml of serum-free medium. The plasmid was co-transfected into 293T cells using the calcium phosphate precipitation method, and the cell culture medium was replaced with 15 ml of complete medium DMEM+10% FBS 24 hours after transfection. Cell supernatant was collected 48 hours after transfection, and 15 ml of fresh complete medium was added. After 72 hours, the cell supernatant was collected again, and the cells were discarde...

Embodiment 3

[0057] This example is the determination of the activity of TM4SF1 CAR-T cells.

[0058] Identification and construction of TM4SF1 CAR-T target cells: qPCR was used to detect the expression of TM4SF1 on Calu-6, Calu-3 and other lung cancer cell lines, such as Figure 5 As shown, the Calu-3 cell line highly expresses the TM4SF1 gene, while Calu-6 hardly expresses the TM4SF1 gene. A lentiviral vector encoding the TM4SF1 gene was used to screen with puromycin to construct a Calu-6 cell line highly expressing the TM4SF1 gene. TM4SF1 expression levels in Calu-6-TM4SF1 cell line see Figure 5 . according to Figure 5 The results showed that the Calu-3 and Calu-6-TM4SF1 cell lines were positive for TM4SF1 expression, while the Calu-6 cell line was negative for TM4SF1.

[0059] IFN-γ secretion experiment: L6-28Z, L6-BBZ, L6-28BBZ, 19-28BBZ four kinds of CAR-T cells (effector cells) were mixed with target cells at a 5:1 effect-to-target ratio in a 96-well plate. After culturing at 3...

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Abstract

The invention discloses a TM4SF1-specific chimeric antigen receptor. The TM4SF1-specific chimeric antigen receptor comprises an extracellular identification region, a hinge region, a transmembrane region and an intracellular signal region, wherein the extracellular identification region is an scFv sequence, the hinge region is a CD8 region, the transmembrane region is one of a CD8 transmembrane region and a CD28 transmembrane region, and the intracellular signal region comprises at least one of CD28, 4-1BB and CD3zeta. The chimeric antigen receptor concretely is L6-28Z, L6-BBZ and L6-28BBZ which are nucleic acid codes of sequences represented by SEQ ID NO.12 to SEQ ID NO.14. The invention also provides a recombinant expression vector comprising the above nucleic acids, and a host cell, a construction method and a use in the treatment of mammalian tumors thereof. The TM4SF1-specific chimeric antigen receptor can be used to modify T cells in order to make specific CAR-T cells highly-efficiently and specifically treat solid tumors.

Description

technical field [0001] The present invention relates to the field of biological immunity, in particular, the present invention relates to a TM4SF1 specific chimeric antigen receptor and its application. Background technique [0002] A chimeric antigen receptor (CAR) is composed of an antigen recognition region, a transmembrane region, and an intracellular signal region. Generally, through viral transduction or electroporation, CAR molecules are expressed on the surface of T cells to form CAR-T cells. CAR-T cells recognize tumor antigens through the extracellular antigen recognition region, recruit T cells to the surface of tumor cells, activate T cells through the intracellular signaling region, and enable T cells to perform killing functions to kill tumor cells, thereby achieving the purpose of treating tumors . The design of CAR molecules has undergone four generations of development according to the difference in the intracellular signaling region. The first-generation C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K16/2896C07K16/30C07K2317/622C07K2319/00C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N2510/00
Inventor 郝瑞栋刘根桃吴国祥
Owner 上海科棋药业科技有限公司