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A preparation method and application of fully human CAR-T cells targeting CD276

A CD276, VH-CDR1 technology, applied in the field of CAR-T cell preparation, can solve problems such as short survival, and achieve the effect of high killing activity

Active Publication Date: 2022-01-28
江苏集萃崛创生物科技研究所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reason is that murine scFv has strong immunogenicity, which induces the body to produce human anti-mouse antibody response, which leads to the transient survival of murine CAR-T cells in the human body.

Method used

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  • A preparation method and application of fully human CAR-T cells targeting CD276
  • A preparation method and application of fully human CAR-T cells targeting CD276
  • A preparation method and application of fully human CAR-T cells targeting CD276

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] Example 1 Screening of scFv targeting CD276

[0117] 1. Experimental condition setting

[0118] Experimental group: CD276 antigen + CD276-Phage

[0119] Control group 1: other non-biotin antigen (PRPS1)+CD276-Phage

[0120] Experimental group 2: no antigen + CD276-Phage

[0121] 2. Experimental method

[0122] After four rounds of screening to enrich specific binding antibody sequences, the total amount of phage input, the amount of added antigen, and reaction time were changed in each round.

[0123] The final result is to judge the enrichment by calculating the number of phages with infectious ability contained in every 100 μl of 0.1M HCl (PH=2.0) eluate in the experimental group and the control group.

[0124] 3. Screening result analysis

[0125] Enrichment occurred in the third round of screening, and the ratio of the number of phage eluted in the experimental group (antigen-antibody specific binding) to the control group (antigen-antibody non-specific binding...

Embodiment 3

[0166] Example 3 Preparation of fully human CD276 CAR-T cells

[0167] 1. Steps

[0168] (1) Prepare PBMC cells

[0169] Take peripheral blood from healthy people, centrifuge and save autologous plasma for later use, dilute remaining blood cells with an equal volume of normal saline, add to the upper layer of lymphocyte separation medium, centrifuge, absorb middle buffy coat cells, add normal saline to wash, centrifuge and discard supernatant, Instantly.

[0170] (2) Construction of the shuttle plasmid MSCV-M13B702 containing the CAR structure

[0171] a. Synthesize the CAR encoding nucleotide sequence targeting human CD276, the CAR encoding nucleotide sequence is shown in SEQ ID NO.17, wherein the nucleotide sequence of the heavy chain VH of the scFv targeting human CD276 is shown in SEQ ID NO .9, the nucleotide sequence of the light chain VL is shown in SEQ ID NO.10, the nucleotide sequence of the G4S short peptide is shown in SEQ ID NO.12, and the nucleotide sequence of ...

Embodiment 4

[0195] Example 4 In vivo functional verification of fully human CD276 CAR-T cells

[0196] 1. Steps

[0197] 5*10 of the fluorescent signal will be carried 5 SKOV3-luc-GFP was injected intraperitoneally into NCG mice, and the tumor formation in the peritoneal cavity of the mice was monitored by taking pictures every week by using small animal in vivo imaging. After the formation of abdominal tumors, fully human fhCD276-02CAR-T cells were injected intraperitoneally, and the control group was used Humanized CD276 CAR-T cells. Thereafter, tumor regression in the peritoneal cavity of the mice was observed by intravital imaging every week.

[0198] 2. Results

[0199] The result is as Figure 10 As shown, the CAR-T cells of the present invention can clear the xenograft tumor of mouse peritoneal ovarian cancer.

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Abstract

The present invention provides a preparation method and application of fully human CAR-T cells targeting CD276. The CD276-targeted CAR-T cells prepared by the present invention have a killing effect on various solid tumor cells, have high killing activity, are safe and effective, and can be used for renal cancer, lung cancer, liver cancer, glioma, ovarian cancer, breast cancer, etc. immune cell therapy.

Description

technical field [0001] The invention belongs to the field of immunotherapy, and relates to a preparation method and application of fully human CAR-T cells targeting CD276. Background technique [0002] Chimeric Antigen Receptor modified T cell (CAR-T) is a genetically modified T cell, which uses gene transduction technology to combine tumor antigen-specific recognition single-chain antibody (scFv) and T cell The CAR that activates the motif is introduced into the patient's T cells, so that these CAR-transduced T cells can directly recognize and activate the surface antigens of cancer cells, and then kill cancer cells. It is precisely because CAR-T cells do not need antigen presentation to kill cancer cells, which greatly improves the killing efficiency of cancer cells. In 2012, Professor Carl June from the University of Pennsylvania used chimeric antigen receptor-modified T cells targeting CD19 to cure leukemia child Emily Whitehead. In 2017, the US FDA made a breakthrough ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00G01N33/574
CPCC07K16/2827C07K14/70578C07K14/70517C07K14/7051C07K14/71C12N15/86C12N5/0636A61K39/001111A61K39/001104A61P35/00G01N33/574G01N33/57492C07K2317/565C07K2317/56C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N2740/10043C12N2800/107C12N2510/00A61K2039/5156G01N2333/70532
Inventor 王刚郑骏年李慧忠曹培育王蒙
Owner 江苏集萃崛创生物科技研究所有限公司
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