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CD19 and BCMA dual target chimeric antigen receptors and applications

A chimeric antigen receptor and single-chain antibody technology, applied in the field of biomedicine, can solve problems such as tumor recurrence, achieve efficient targeting, avoid immune escape, and avoid target escape.

Active Publication Date: 2022-05-17
GUANGDONG ZHAOTAI INVIVO BIOMEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the new generation of CAR-T therapy targeting B Cell Maturation Antigen (BCMA, CD269) has achieved great success in the treatment of multiple myeloma. However, similar to CD19-CAR therapy, it faces BCMA The Problem of Recurrence of Positive and BCMA-Negative Malignancies

Method used

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  • CD19 and BCMA dual target chimeric antigen receptors and applications
  • CD19 and BCMA dual target chimeric antigen receptors and applications
  • CD19 and BCMA dual target chimeric antigen receptors and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Construction of CAR Molecular Carrier

[0057] In this example, the anti-CD19 and anti-BCMA dual-target chimeric antigen receptor 19-BCMA-CAR was constructed, and the structural diagram is as follows figure 1 Shown, the amino acid sequence is shown in SEQ ID NO: 3, and the coding gene is shown in SEQ ID NO: 6;

[0058] First, the whole gene is synthesized as SEQ ID NO: 6, and EcoRI and BamHI restriction sites and their protective bases are added at both ends;

[0059] Double-digest the coding gene with restriction endonucleases EcoRI and BamHI, incubate in a water bath at 37°C for 30 minutes, and use 1.5% agarose gel electrophoresis to recover the digested product containing sticky ends;

[0060] The digested product was ligated into the linearized pLVX-EF1-MCS plasmid (containing cohesive ends) digested with EcoRI and BamHI. The ligation system was shown in Table 1, and the CAR containing the dual targets of CD19 and BCMA was obtained. Lentiviral vector enc...

Embodiment 2

[0064] Example 2 lentiviral packaging

[0065] In this example, the lentiviral vector constructed in Example 1 is used for lentiviral packaging, using a four-plasmid system, and the steps are as follows:

[0066] Mix the helper plasmids gag / pol, Rev and VSV-G with the recombinant vector in proportion, add it to a certain volume of serum-free DMEM, mix it and let it stand for 15 minutes; add the above mixture to the cell culture flask lined with 293T cells , mixed gently, at 37°C, 5% CO 2 Cultivate in the cell incubator for 6 hours; after 6 hours, replace the fresh medium, continue to cultivate, and add 10mM sodium butyrate solution; after 72 hours, collect the lentivirus culture supernatant for purification and detection.

[0067] Recombinant vectors include lentiviral vectors containing genes encoding CARs targeting CD19 and BCMA dual targets, lentiviral vectors containing genes encoding CARs targeting CD19 single targets, lentiviral vectors containing genes encoding CARs ta...

Embodiment 3

[0068] Example 3 T cell activation and lentiviral transfection

[0069] Peripheral blood mononuclear cells (PBMC) were separated from whole blood using Ficoll density gradient centrifugation kit (GE Company), and after red blood cells were removed, T cells were sorted out using MACS Pan-T magnetic beads;

[0070] The sorted T cells were diluted with medium (AIM-V medium + 5% FBS + penicillin 100 U / mL + streptomycin 0.1 mg / mL) to a cell concentration of 2.5×10 6 pcs / mL for use;

[0071] CD2 / CD3 / CD28 T cell activation expansion kit (Miltenyi Company) was used to activate T cells, that is, the coated magnetic beads were mixed with T cells at a ratio of 1:2, and the final density of T cells was 5×10 6 piece / mL / cm 2 , after mixing, place at 37°C, 5% CO 2 The incubator was stimulated for 48 hours;

[0072] After T cells were activated for 48 hours, the beads were demagnetic, centrifuged at 300 g for 5 min, and the supernatant was removed. T cells were resuspended in fresh medium...

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Abstract

The present invention provides the CD19 and BCMA dual -target church antigen receptors and its applications, which include the antigen -binding domain, the cross -membrane domain, and the signal conversion domain;Anti -CD19 single -chain antibody and anti -BCMA single -chain antibody.The invention of anti -CD19 and BCMA dual -target chimeric antigen receptor has small side effects and high safety. The constructed CAR‑T cells have a killing effect on B -cell tumors and can significantly improve the therapeutic effect of physical tumors.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a CD19 and BCMA dual-target chimeric antigen receptor and an application thereof. Background technique [0002] In recent years, with the development of tumor immunology theory and clinical technology, chimeric antigen receptor T-cell immunotherapy (CAR-T) has become one of the most promising tumor immunotherapies. At present, the new generation of CAR-T therapy targeting B Cell Maturation Antigen (BCMA, CD269) has achieved great success in the treatment of multiple myeloma. However, similar to CD19-CAR therapy, it faces BCMA The problem of recurrence in positive and BCMA-negative malignancies. [0003] Multiple myeloma, as a typical B-cell tumor, usually does not express CD19, however, some reports indicate that in multiple myeloma cases, although 99.5% of malignant hyperplastic plasma cells lack CD19, CD19-targeted CAR-T After treatment, the patient was completely cured. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N7/01C12N5/10A61K39/00A61P35/00
CPCC07K16/2803C07K16/2878C07K14/7051C12N15/86C12N7/00C12N5/0636A61K39/0011A61P35/00C07K2319/02C07K2319/03C07K2319/33C12N2510/00C12N2740/15021C12N2740/15043
Inventor 汤朝阳秦乐吴迪邓殷健吴海鹏王翠花冯世忠冯嘉昆其他发明人请求不公开姓名
Owner GUANGDONG ZHAOTAI INVIVO BIOMEDICINE CO LTD
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