Optimized connecting peptide combination and application thereof

A single-chain antibody, bispecific technology, applied in the field of immune cells and applications, CAR structure, can solve the problems that CAR vectors are not easy to transduce T cells, different efficacy, different virus preparation capabilities, etc., to reduce the probability of immune escape, The effect of reducing tumor recurrence rate and improving anti-tumor effect

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

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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 tumor cells are down-regulated or mutated in a small number of patients treated with CAR-T cells and The CAR-T target antigen expressed on its surface escapes (Robbie G.Majzner et al. (2018) TumorAntigen Escape from CAR T-cell Therapy), which eventually leads to poor therapeutic effect and recurrence
Therefore, it is necessary to design a CAR structure targeting multiple targets. If two CARs with dif

Method used

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  • Optimized connecting peptide combination and application thereof
  • Optimized connecting peptide combination and application thereof
  • Optimized connecting peptide combination and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0057] Example 1 Plasmid construction

[0058] Design Figure 1A with 1b The CAR structure shown (the intracellular signal in the structure contains intracellular co-stimulation domains and intracellular activation signals), VL (SCFV1) -Linker1-VL (SCFv2) -Linker1-VH (SCFv1) Or VH (SCFV1) -Linker1-VL (SCFV2) -Linker-VH (SCFv2) -Linker1-VL (SCFv1) or VL (SCFv1) -Linker1-VH (SCFv2) -Linker-VL (SCFv2) -Linker1-VH ( Structure. Using targeted CD19, CD123 and CD19, CD22 dual target CARs, designation encoding 12: anti-CD19SCFV light chain - LINKER1-anti-CD123SCFV light chain-Linker2-anti-CD123SCFV heavy chain-Linker1-anti-CD19SCFV heavy chain, nucleoside Acid sequences such as SEQ ID NO: 3, amino acid sequence, such as SEQ ID NO: 25; encoding 13: anti-CD123SCFV light chain - LINKER1-anti-CD19SCFV light chain-Linker2-anti-CD19SCFV heavy chain-Linker1-anti-CD123SCFV heavy chain, core The antimony acid sequence such as SEQ ID NO: 4, amino acid sequence, such as SEQ ID NO: 26; No. 19: CD19 SC...

Example Embodiment

[0061] Example 2 Preparation of a slow virus and infection T lymphocytes

[0062] In this example, the packed lepid virus is calcium phosphate method. Specifically, a 293T cells containing 10% FBS (w / v) culture 293T cells to a preferred state, and the packaging plasmid (RRE: REV: 2G) and expression plasmid are pressed. Add to the 1.5 centrifuge tube, add CaCl 2 And 2 × HBs, after mixing after mixing, it was added to the treatment of a well-treated 293T cell culture medium, and then evolved to 10 ml of DMEM medium containing 10% FBS after 3-5 h, 48h or 72h was collected, and the cells were collected. Purification of viruses and measured by titer.

[0063] Prepaired Double Car (12 and 13) lentiviral infection with CHO cells, respectively, CD19CART, CD123CAR-T, Double CART12 and dual CART 13, which were infected with CHO cells, and Double CART13, respectively, after labeled double The expression of CD19 CAR and targeted CD123 Car structure is targeted, and the total CAR expression ...

Example Embodiment

[0070] Example 3 Target cell preparation and target antigen detection

[0071] (1) CD19 and CD123 target antigen detection

[0072] Cultured target cells Raji (human lymphoma cells), THP-1 (mononuclear macrophages), NALM-6 (human B lymphocyte cells) were prepared by lentiviral infection with Luc-GFP virus as a target cell with LUC labeling The antibody expression of the target cell surface antigen was detected by anti-CD19 antibody and anti-CD123 antibody. Such as Figure 4A As shown: Raji is only CD19 positive cells, THP-1 is only CD123 positive cells, NALM-6 is CD19 and CD123 duplex positive cells.

[0073] (2) CD19 and CD22 target antigen detection

[0074] A target cells expressing CD19 and CD22 were constructed with K562-LUC, and a target cell having a co-expression of CD19 and CD22 was co-expressing CD19 and CD22, respectively, and an anti-CD19 antibody and antigen expression were used to detect target cell surface antigen expression. See Figure 4B As shown: K562-CD19 is CD19...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to an optimized connecting peptide, a bispecific single-chain antibody connected with the optimized connecting peptide, a CAR (Chimeric Antigen Receptor) structure containing the bispecific single-chain antibody, an expression vector, an immune cell and application. In the bispecific single-chain antibody, two linkers are used for connecting heavy chains or light chains of two single-chain antibodies; the construction of the CAR structure overcomes the technical effect defect of a double-target CAR in the prior art; two different tumour antigens can be targeted at the same time; killing of tumour cells is improved; and the probability of immune escape of the tumour cells is reduced.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to an optimized connecting peptide and its linked bispecific single-chain antibody, as well as a CAR structure comprising the bispecific single-chain antibody, an expression vector, immune cells and applications. 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 and The CAR-T target antigen expressed on its surface escapes (Robbie G. Majzner et al. (2018) TumorAntigen Escape from CAR T-cell Therapy), which eventually leads to poor therapeutic effe...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/867C12N5/10A61K39/395A61P35/00A61P35/02
CPCC07K16/2803C07K16/2866C12N5/0636A61P35/00A61P35/02C07K2317/622C07K2317/31C12N2510/00A61K2039/505
Inventor 黄霞赵永春陈雪娇赵文旭陈军徐艳敏齐亚男单娟娟张巍
Owner CHONGQING PRECISION BIOTECH CO LTD
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