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Double-target chimeric antigen receptor targeting CD19 and CD123 and application of double-target chimeric antigen receptor

A chimeric antigen receptor and dual-antigen technology, which is applied in the field of bispecific chimeric antigen receptor and CAR-T cells, can solve the problems that CAR vectors are difficult to transduce T cells, CAR transduction ability is different, recurrence, etc. Achieve the effects of reducing the risk of tumor immune escape, high CAR transduction efficiency, and easy virus preparation

Active Publication Date: 2021-10-12
CHONGQING PRECISION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the heterogeneity of tumors, especially solid tumors, more than one target antigen is often expressed on the surface of tumor cells, and with the progress of research, researchers have also found that a small number of patients treated with CAR-T cells have down-regulated or mutated tumor cells. The expressed CAR-T target antigen escapes (Robbie G. Majzner et al. (2018) Tumor AntigenEscape from CAR T-cell Therapy), resulting in poor therapeutic effect and recurrence
Therefore, it is necessary to design a CAR structure targeting multiple targets. Simply expressing two CARs with different targets at the same time involves different CAR structures with different infectivity, and will generate a variety of CAR-T cell subsets, which is not conducive to the clinical application of the product. ; If only two simple CAR structures with different targets are connected in series in one carrier, such a large CAR carrier is not easy to transduce T cells. Different structures involve different CAR carrier virus preparation capabilities and different CAR transduction capabilities. There are many factors such as different stimuli and different curative effects

Method used

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  • Double-target chimeric antigen receptor targeting CD19 and CD123 and application of double-target chimeric antigen receptor
  • Double-target chimeric antigen receptor targeting CD19 and CD123 and application of double-target chimeric antigen receptor
  • Double-target chimeric antigen receptor targeting CD19 and CD123 and application of double-target chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Plasmid Construction

[0045] designed as figure 1 The CAR structure shown, the nucleic acid sequence is shown in SEQ ID NO: 1 or SEQ ID NO: 2, the fragments were cut and recovered by double enzyme digestion, the gene fragments were connected, transformed and single cloned, and the vector numbers obtained were respectively 10 , 11. The CAR structural element comprises: a nucleotide sequence such as SEQ ID NO:3, an amino acid sequence such as SEQ ID NO:10 targeting CD19 ScFv; a nucleotide sequence such as SEQ ID NO:4, and an amino acid sequence such as SEQ ID NO:11 ScFv targeting CD123; nucleotide sequence such as SEQ ID NO:5, amino acid sequence such as the hinge hinge structure of SEQ ID NO:12; nucleotide sequence such as SEQ ID NO:6, amino acid sequence such as SEQ ID NO:13 Transmembrane structure; nucleotide sequence such as SEQ ID NO:7, amino acid sequence such as the intracellular costimulatory domain of SEQ ID NO:14, nucleotide sequence such as SEQ ID...

Embodiment 2

[0047] Example 2: Preparation of lentivirus and infection of T lymphocytes

[0048] In this example, the calcium phosphate method was used to package the lentivirus, specifically: 293T cells were cultured to a better state with DMEM medium containing 10% FBS (w / v), and the packaging plasmid (RRE:REV:2G) and expression plasmid Add the scale to a 1.5 centrifuge tube, add CaCl2 and 2× HBS, mix well, let it stand at room temperature, then add it to the treated 293T cell culture medium, and change the medium again to 10mL DMEM containing 10% FBS after 3-5h Culture medium, after 48h or 72h, the cell supernatant was collected, the virus was purified, and the titer was determined.

[0049] Titer table:

[0050]

[0051] The prepared lentivirus was used to infect CHO cells, and the CD19CART, CD123CAR-T, double CART10 and double CART11 infected CHO cells were labeled with CD19-FC, CD123-His (Acro, 1741d-971F1-NZ) flow cytometry labeling reagents and detected The expression of CD19-...

Embodiment 3

[0054] Example 3: Target cell preparation and target antigen detection

[0055] The cultured target cells Raji, Thp-1, and Nalm-6 were prepared as Luc-labeled target cells by lentivirus infection with Luc-GFP virus, and anti-CD19-FC, CD123-His (Acro, 1741d-971F1-NZ) Detection of target cell surface antigen expression. see results image 3 Shown: Raji is only CD19 positive cells, Thp-1 is only CD123 positive cells, Nalm-6 is CD19 and CD123 double positive cells.

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Abstract

The invention belongs to the technical field of immunotherapy, and particularly relates to a bispecific chimeric antigen receptor capable of recognizing CD19 and CD123 tumor associated antigens, an expression vector of the chimeric antigen receptor, a CAR-T cell and application of the CAR-T cell. The bispecific chimeric antigen receptor can recognize double targets of CD19 and CD123, not only can be effectively expressed in T lymphocytes, but also can reduce the probability of tumor immune escape and reduce the tumor recurrence rate after CAR-T treatment.

Description

technical field [0001] The invention belongs to the technical field of immunotherapy, and specifically relates to a bispecific chimeric antigen receptor, a carrier comprising a bispecific chimeric antigen receptor, a CAR-T cell expressing a bispecific chimeric antigen receptor and its application. Background technique [0002] CAR-T (Chimeric Antigen Receptor T) cell therapy has achieved remarkable results in the treatment of hematological malignancies. However, due to the heterogeneity of tumors, especially solid tumors, more than one target antigen is often expressed on the surface of tumor cells, and with the progress of research, researchers have also found that a small number of patients treated with CAR-T cells have down-regulated or mutated tumor cells. The expressed CAR-T target antigen escapes (Robbie G. Majzner et al. (2018) Tumor AntigenEscape from CAR T-cell Therapy), resulting in poor therapeutic effect and recurrence. Therefore, it is necessary to design a CA...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K39/00A61P35/00
CPCC07K16/2803C07K16/2866C07K14/7051C12N5/0636C12N15/86A61K39/0011A61P35/00C07K2319/03C07K2319/33C12N2510/00C12N2740/15043
Inventor 陈雪娇赵永春黄霞沈俊杰陈军徐艳敏齐亚男赵文旭张巍单娟娟
Owner CHONGQING PRECISION BIOTECH CO LTD
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