CLL1 and CD33 double-target chimeric antigen receptor and application thereof

A chimeric antigen receptor, dual-target technology, applied in the field of biomedicine, can solve the problems of recurrent antigen and escape, and achieve the effects of high targeting efficiency, efficient targeting activity and strong tumor killing effect.

Active Publication Date: 2021-08-13
GUANGZHOU BIO GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] It is worth noting that CD19 CAR-T cells have shown good performance in early clinical trials of hematological malignancies such as AML, but recurrence after treatment and antigen escape are still problems to be overcome

Method used

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  • CLL1 and CD33 double-target chimeric antigen receptor and application thereof
  • CLL1 and CD33 double-target chimeric antigen receptor and application thereof
  • CLL1 and CD33 double-target chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Example 1 Source of Antibody

[0106] In this example, anti-CD33 antibody Gemtuzumab and anti-CLL1 antibody 1075.7 or anti-CLL1 antibody M26 were used as antigen-binding domains for the construction of CLL1 and CD33 dual-target CAR molecules, wherein Gemtuzumab included SEQ ID NO: 1-2. The variable region, 1075.7 includes the variable region shown in SEQ ID NO:3-4, and M26 includes the variable region shown in SEQ ID NO:5-6.

Embodiment 2

[0107] Example 2 Expression of CLL1 and CD33 by tumor cells

[0108] In this example, first, FITC anti-human CD371 (CLL1) antibody (biolegend brand) and PEanti-CD33 antibody (biolegend brand) were incubated with U937 and Raji, and the expression of CLL-1 and CD33 by target cells was detected by flow cytometry. Such as figure 1 As shown, U937 is a CLL-1 and CD33 double-positive cell, and Raji is a CLL-1 and CD33 double-negative cell.

[0109] In this example, a stable lentiviral vector was further constructed according to the gene sequences of CLL1 and CD33, infected with Raji cells, and screened with puromycin (5 μg / mL) to obtain stable transfected cell lines Raji-CLL1 and Raji- CD33. Such as figure 1 As shown, Raji-CLL1 stably expresses CLL1, and Raji-CD33 stably expresses CD33.

Embodiment 3

[0110] Example 3 Design of Chimeric Antigen Receptor

[0111] In this example, anti-CD33 antibody Gemtuzumab and anti-CLL1 antibody 1075.7 or anti-CLL1 antibody M26 are used as antigen-binding domains to bind CD8α signal peptide (SEQ ID NO: 18-19) and CD8α hinge region (SEQ ID NO: 20-21) and transmembrane region (SEQ ID NO:22~23), 4-1BB co-stimulatory domain (SEQ ID NO:24~25) and CD3ζ signaling domain (SEQ ID NO:26~27), construct anti-CLL1 and The CD33 dual-target CAR molecule (SEQ ID NO: 9-16), the scFv of the CAR molecule is shown in Table 1, and the structural diagram is as follows Figure 2A As shown, wherein, the structural representation of 75-33-75-CAR (SEQ ID NO: 12) is shown in Figure 2B shown;

[0112] In this example, anti-CLL1 single-target 1075.7-CAR (SEQ ID NO: 28-29) / M26-CAR (SEQ ID NO: 30-31) was also constructed as a control, and the structural diagram is as follows Figure 2A shown.

[0113] Table 1 scFv structure of CLL1-CD33 bispecific CAR

[0114] ...

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Abstract

The invention provides a CLL1 and CD33 double-target chimeric antigen receptor and application thereof. The chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain and a signal transduction domain, and the antigen binding domain comprises an anti-CLL1 single-chain antibody and an anti-CD33 single-chain antibody. According to the constructed CAR molecule simultaneously targeting CLL1 and CD33, the effect of comprehensively targeting acute myelogenous leukemia cells is achieved, the CAR-T cells for expressing CLL1 and CD33 double-target CAR are remarkable in tumor removal effect, the antigen escape phenomenon is avoided, and the CAR molecule has important significance in the field of tumor treatment.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a chimeric antigen receptor with dual targets of CLL1 and CD33 and an application thereof. Background technique [0002] In the field of tumor immunotherapy, CAR-T is the most popular tumor immunotherapy method, and it has shown amazing effects in the treatment of leukemia, lymphoma, and multiple myeloma. At present, research on CAR-T products targeting CD19 is relatively in-depth. Kymriah and Yescarta approved in the United States are both CAR-T products targeting CD19 for the treatment of hematological tumors. From a global perspective, the R&D pipeline of CAR-T is rapidly expanding, including the exploration of new targets, such as BCMA, CD123, CD33, etc., and the expansion of new indications, such as the advancement from hematological tumors to solid tumors. The projects of many companies around the world have advanced to the clinical stage, and it is expected that CAR-T p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N7/01C12N5/10A61K39/00A61P35/02C12R1/93
CPCC07K16/2851C07K16/2803C07K14/7051C12N15/86C12N7/00C12N5/0636A61K39/001102A61K39/001111A61P35/02C07K2319/02C07K2319/03C07K2319/33C07K2317/622C07K2317/56C12N2740/15043C12N2740/15021C12N2510/00C12N2800/107A61K2039/5158
Inventor 李光超罗敏周兆丁雯王学俊
Owner GUANGZHOU BIO GENE TECH CO LTD
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