T lymphocyte modified by double specific chimeric antigen receptors as well as preparation method and application thereof

A chimeric antigen receptor and bispecific technology, applied in the field of bispecific chimeric antigen receptor modified T lymphocytes and its preparation, to achieve the effect of overcoming off-target toxicity, specific recognition and killing

Active Publication Date: 2017-11-07
SHILI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the antigen receptor EGFRvIII-CD3ζ and the antigen receptor PD-L1-4-1BB were integrated into the CAR structure at the same time, so th

Method used

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  • T lymphocyte modified by double specific chimeric antigen receptors as well as preparation method and application thereof
  • T lymphocyte modified by double specific chimeric antigen receptors as well as preparation method and application thereof
  • T lymphocyte modified by double specific chimeric antigen receptors as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0058] Example 1 Determination of EGFRvIII-CD3ζ / PD-L1-4-1BB gene sequence

[0059] 1.1 Obtain the human CD8α signal peptide gene (SEQ ID NO.9), Linker region (Gly4-Ser) 3() from the GenBank database of the National Library of Medicine website (http: / / www.ncbi.nlm.nih.gov / entrez) 10), human CD8α hinge region (CD8αHinge) (SEQ ID NO.11), human CD8α transmembrane region (CD8αTM) (SEQ IDNO.12), immune receptor tyrosine activation motif CD3ζ (SEQ ID NO .13), human 4-1BB hinge region (4-1BB Hinge) (SEQ ID NO.14), human 4-1BB transmembrane region (4-1BB TM) (SEQ ID NO.15) and 4-1BB intracellular The sequence of the signal region (SEQ ID NO.16), combined with the EGFRvIII antibody light chain variable region (EGFR vIIIscfv-VL) (SEQ ID NO.17), EGFRvIII antibody heavy chain variable region (EGFRvIIIscfv-VH) (SEQ ID NO.18) ), PD-L1 antibody light chain variable region (PD-L1scfv-VL) (SEQ ID NO.19), PD-L1 antibody heavy chain variable region (PD-L1scfv-VH) (SEQ ID NO.20) sequence , Combined...

Example Embodiment

[0060] Example 2 Construction of pCDH-EGFRvIII-CD3ζ, pCDH-PD-L1-4-1BB, pCDH-EGFRvIII-CAR and pCDH-PD-L1-CAR plasmids

[0061] 2.1 Whole gene synthesis:

[0062] The complete EGFRvIII-CD3ζ (SEQ ID NO.5), PD-L1-4-1BB (SEQ ID NO.6), EGFRvIII-CAR (SEQ ID NO.7), PD- L1-CAR (SEQ ID NO.8) sequence, and added Xba I restriction site at the 5'end and EcoRI restriction site at the 3'end.

[0063] 2.2 Clone EGFRvIII-CD3ζ, PD-L1-4-1BB, EGFRvIII-CAR and PD-L1-CAR into pCDH-EF1α-GFP or pCDH-EF1α-RFP lentiviral expression vector, respectively. The details are as follows: pCDH-EF1α-GFP vector, pCDH-EF1α-RFP vector, EGFRvIII-CD3ζ fragment, PD-L1-4-1BB fragment, EGFRvIII-CAR fragment, PD-L1-CAR fragment were used Xba I / EcoR I Enzyme digestion, 8192bp, 8111bp, 1297bp, 1258bp, 1483bp, 1477bp fragments were recovered. Use T4DNA ligase to ligate the digested pCDH-EF1α-GFP vector with EGFRvIII-CD3ζ, EGFRvIII-CAR, PD-L1-CAR, pCDH-EF1α-RFP vector and PD-L1-4-1BB, and transform with the ligation product Tr...

Example Embodiment

[0064] Example 3 Packaging, concentration and titer determination of lentivirus

[0065] 3.1 Packaging of lentivirus:

[0066] 3.1.1 Cell processing: Collect the 3-10th generation 293T cells in the logarithmic growth phase 24 hours before transfection, and inoculate the 293T cells in a 10cm cell culture dish, the inoculation volume is 1×10^7, and the cells contain 10ml 10 Grow in DMEM medium containing %FBS and place it in a 37°C 5%CO2 cell incubator for 18 hours, and the cell density can reach 60-80% before transfection.

[0067] 3.1.2 Co-transfection of lentiviral expression vector plasmids (pCDH-EGFRvIII-CD3ζ, pCDH-PD-L1-4-1BB, pCDH-EGFRvIII-CAR, pCDH-PD-L1-CAR) and its packaging plasmids into 293T cells ( pLP1, pLP2, pLP / VSVG);

[0068] 3.1.3 24 hours after transfection, observe the GFP / RFP fluorescence expression of 293T cells after transfection under a fluorescence microscope. Collect the 293T culture supernatant at 48 hours and 72 hours after transfection, centrifuge at 3000 ...

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Abstract

The invention discloses T lymphocyte modified by double specific chimeric antigen receptors and a preparation method and application thereof. The surface of the T lymphocyte can express a specific chimeric antigen receptor EGFRvIII-CD3zeta and a specific chimeric antigen receptor PD-L1-4-1BB. An amino acid sequence of the specific chimeric antigen receptor EGFRvIII-CD3zeta is shown in SEQ ID NO.1; an amino acid sequence of the specific chimeric antigen receptor PD-L1-4-1BB is shown in SEQ ID NO.2. The T lymphocyte modified by the double specific chimeric antigen receptors can be used for specifically identifying and killing and simultaneously expressing the tumor cells of EGFRvIII-CD3zeta and PD-L1 antigens, and the specificity is very strong.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a bispecific chimeric antigen receptor-modified T lymphocyte and its preparation method and application. Background technique [0002] With the development of tumor immunology theory and clinical technology, chimeric antigen receptor T-cell immunotherapy (CAR-T) (June CH, Blazar BR, Riley JL. Engineering lymphocyte subsets: tools, trials and tribulations .NatRevImmunol.2009; 9:704-16) has become one of the most promising tumor immunotherapy. CAR-T cell therapy uses exogenous gene transfection technology to express fusion proteins of single chain fragment variable (scFv) that recognizes tumor-associated antigens and T cell activation sequences on the surface of T cells, enabling specific recognition of tumor-associated antigens The scFv is coupled to the activation and proliferation signaling domain in T cells through the transmembrane region. T cells expressing CAR bind to tumor an...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867A61K35/17A61P35/00
Inventor 樊克兴高同同
Owner SHILI BIOTECH CO LTD
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