A kind of T-lymphocyte modified by bispecific chimeric antigen receptor and its preparation method and application

A technology of chimeric antigen receptors and lymphocytes, applied in the biological field, to achieve the effect of overcoming off-target toxicity, specific recognition and killing

Active Publication Date: 2019-09-24
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 that they could be expressed in T cells, and EGFRvIII-CD3ζ / PD-L1-4-1BB-CAR-T cells were constructed. CAR-T cell immunotherapy has not yet been reported

Method used

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  • A kind of T-lymphocyte modified by bispecific chimeric antigen receptor and its preparation method and application
  • A kind of T-lymphocyte modified by bispecific chimeric antigen receptor and its preparation method and application
  • A kind of T-lymphocyte modified by bispecific chimeric antigen receptor and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0059] 1.1 Obtain human CD8α signal peptide gene (SEQ ID NO.9), Linker region (Gly4-Ser) 3 ( SEQ ID NO.10), human CD8α hinge region (CD8α Hinge) (SEQ ID NO.11), human CD8α transmembrane region (CD8α TM) (SEQ ID NO.12), immunoreceptor tyrosine activation motif CD3ζ (SEQ ID NO.11) 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.1) and 4-1BB Intracellular signal region (SEQ ID NO.16) sequence, combined with EGFRvIII antibody light chain variable region (EGFRvIIIscfv-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 to form complete EGFRvIII-CD3ζ (SEQ ID NO.5), PD-L1-4-1BB (SEQ ID NO.6), EGFRvIII-CAR (SEQ ID NO.7), PD-L...

Embodiment 2

[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]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 add Xba I restriction site at its 5' end, and add EcoR I restriction site at its 3' end.

[0063] 2.2 Cloning EGFRvIII-CD3ζ, PD-L1-4-1BB, EGFRvIII-CAR, and PD-L1-CAR into pCDH-EF1α-GFP or pCDH-EF1α-RFP lentiviral expression vectors, respectively. The details are as follows: the pCDH-EF1α-GFP vector, pCDH-EF1α-RFP vector, EGFRvIII-CD3ζ fragment, PD-L1-4-1BB fragment, EGFRvIII-CAR fragment, and PD-L1-CAR fragment were respectively treated with Xba I / EcoR I Digest with enzymes, and recover 8192bp, 8111bp, 1297bp, 1258bp, 1483bp, 1477bp fragments respectively. Use T4 DNA ligase to ligate pCDH-EF1α-GFP vector and EGFRvIII-CD3ζ, EGFRvIII-CAR, PD-L1-CAR, pCDH-EF1α-RFP vector and PD-L1-4-1BB res...

Embodiment 3

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

[0065] 3.1 Packaging of lentivirus:

[0066] 3.1.1 Cell treatment: 24 hours before transfection, collect 293T cells of passage 3-10 in logarithmic growth phase, inoculate 293T cells in a 10cm cell culture dish, the inoculum size is 1×10^7, and the cells are contained in 10ml 10 Grow in DMEM medium with %FBS, place in a 5% CO2 cell incubator at 37°C for 18 hours, and transfect when the cell density reaches 60-80%.

[0067] 3.1.2 Co-transfect lentiviral expression vector plasmids (pCDH-EGFRvIII-CD3ζ, pCDH-PD-L1-4-1BB, pCDH-EGFRvIII-CAR, pCDH-PD-L1-CAR) and their packaging plasmids ( pLP1, pLP2, pLP / VSVG);

[0068] 3.1.3 24 hours after transfection, the expression of GFP / RFP fluorescence in 293T cells after transfection was observed under a fluorescent microscope. Collect the 293T culture supernatant at 48 hours and 72 hours after transfection, centrifuge at 3000rpm for 15 minutes, collect the...

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