Dual CAR (chimeric antigen receptor) structure targeting bispecific sites and application thereof

A bispecific, targeted technology, applied in the direction of peptides containing localization/targeting motifs, medical preparations containing active ingredients, specific peptides, etc., can solve the problem that CAR vectors are not easy to transduce T cells and CAR transduction ability. Different problems, recurrence, etc., to reduce the risk of tumor immune escape, high CAR transduction efficiency, and reduce tumor recurrence.

Pending 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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  • Dual CAR (chimeric antigen receptor) structure targeting bispecific sites and application thereof
  • Dual CAR (chimeric antigen receptor) structure targeting bispecific sites and application thereof
  • Dual CAR (chimeric antigen receptor) structure targeting bispecific sites and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Plasmid construction

[0054] designed as figure 1 The CAR structure shown, the nucleic acid sequence is shown as SEQ ID NO: 1 or SEQ ID NO: 2, the fragments were respectively cut and recovered by double enzyme digestion, the gene fragments were connected, transformed and single cloned, and the vector numbers obtained were respectively 15 , 16. The CAR structural element comprises: a nucleotide sequence such as SEQ ID NO: 3, an amino acid sequence such as SEQ ID NO: 13 targeting CD19 ScFv; a nucleotide sequence such as SEQ ID NO: 4, and an amino acid sequence such as SEQ ID NO: 14 The ScFv targeting CD22; the Linker connecting the ScFv heavy chain VH and light chain VL, the nucleotide sequence is shown in SEQ ID NO:5; the nucleotide sequence is like SEQ ID NO:7, and the amino acid sequence is like SEQ ID NO: 15 hinge hinge structure; nucleotide sequence such as SEQ ID NO:8, amino acid sequence such as the transmembrane structure of SEQ ID NO:16; nucleotide ...

Embodiment 2

[0055] Example 2: Preparation of lentivirus and infection of T lymphocytes

[0056] In this example, the calcium phosphate method is used to package the lentivirus, specifically: 293T cells are 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.

[0057] The prepared lentivirus was used to infect CHO cells, and the CD19CART, CD22CAR-T, double CART19 and double CART20 infected CHO cells were respectively labeled with CD19-FC, CD22-His (Acro, 2028b-81XF1-KY) flow cytometry labeling reagents and detected The expression of CD19-targeted CAR and CD22-targe...

Embodiment 3

[0062] Example 3: Target cell preparation and target antigen detection

[0063] Using K562-Luc as a model cell, construct target cells with high exogenous expression of CD19, CD22, and co-expression of CD19 and CD22, and use anti-CD19 antibody and anti-CD22 antibody (Acro, 2028b-81XF1-KY) to detect the expression of antigens on the surface of target cells . see results Figure 4 Shown: K562-CD19 is only CD19 positive cells, K562-CD22 is only CD22 positive cells, K562-CD19-CD22 is CD19 and CD22 double positive cells.

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PUM

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Abstract

The invention belongs to the technical field of immunotherapy, and particularly relates to a dual-CAR (chimeric antigen receptor) structure targeting bispecific sites, an expression vector containing the dual-CAR structure of the bispecific sites, an immune cell and an application. The dual-CAR structure is a ScFv1-hinge-transmembrane-41BB intracellular signal-CD3 [zeta]-self-cleavage polypeptide-ScFv2-hinge-transmembrane-CD28 intracellular signal-CD3 [zeta] or a ScFv1-hinge- transmembrane-CD28 intracellular signal-CD3 [zeta]-self-cleavage polypeptide-ScFv2-hinge-transmembrane-41BB intracellular signal-CD3 [zeta]. The construction of the dual-CAR structure overcomes the technical effect defect of dual-target CAR in the prior art, two different tumor antigens can be targeted at the same time, the killing rate of the tumor cells is increased, and the probability of immune escape of the tumor cells is reduced.

Description

technical field [0001] The invention belongs to the technical field of immunotherapy, and specifically relates to a dual CAR structure targeting a bispecific site, and an expression vector, immune cells and applications of the dual CAR structure containing the dual specific site. 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 CAR structure targeti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N5/10C12N15/63A61K39/00A61P35/00
CPCC07K14/7051C12N5/0636A61K39/001112A61K39/001113A61P35/00A61K2039/5156C07K2319/03C12N2510/00
Inventor 陈雪娇赵文旭黄霞齐亚男徐艳敏单娟娟陈军张巍赵永春
Owner CHONGQING PRECISION BIOTECH CO LTD
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