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Chimeric antigen receptor of targeted GPC3 (Glypican 3) and application thereof

An antigen, single-chain antibody technology, applied in applications, hybrid peptides, anti-tumor drugs, etc., can solve problems such as not having a good effect

Active Publication Date: 2016-09-21
HRAIN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The GPC3-GC33 antibody in 15 patients was observed to have a significant therapeutic effect in the phase I clinical trial for the treatment of HCC. It is speculated that the naked antibody will not have a good effect against human liver cancer

Method used

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  • Chimeric antigen receptor of targeted GPC3 (Glypican 3) and application thereof
  • Chimeric antigen receptor of targeted GPC3 (Glypican 3) and application thereof
  • Chimeric antigen receptor of targeted GPC3 (Glypican 3) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0122] Example 1: Determination of mGPC3scFv-CD8α-41BB-CD3ζ gene sequence

[0123] The gene sequence information of human CD8α hinge region, human CD8α transmembrane region, human 41BB intracellular region and human CD3ζ intracellular region was searched from the NCBI website database. The anti-GPC3 single chain antibody clone number is GC33. These sequences are available on the website Codon optimization is performed on http: / / sg.idtdna.com / site to ensure that it is more suitable for human cell expression without changing the encoded amino acid sequence.

[0124] Using overlapping PCR, the above sequences were sequentially connected according to anti-GPC3 scFv, human CD8α hinge region gene, human CD8α transmembrane region gene, human 41BB intracellular region gene, and human CD3ζ intracellular region gene sequence, and different restriction enzymes were introduced at the junction of each sequence site, forming the complete mGPC3-CAR gene sequence information, and obtaining th...

Embodiment 2

[0131] Example 2: Determination of mGPC3CAR-tEGFR gene sequence

[0132] The human EGFR extracellular region gene sequence information was searched from the NCBI website database, and the sequence was codon-optimized on the website http: / / sg.idtdna.com / site to ensure that it is more suitable for human cells without changing the encoded amino acid sequence Express.

[0133] Using overlapping PCR, the above sequences were sequentially connected according to the GPC3CAR, GMCSFR leader sequence, and tEGFR in Example 1, and different restriction sites were introduced at the junctions of each sequence to form complete mGPC3CAR-tEGFR gene sequence information, and the CAR molecule was obtained. As shown in SEQ ID NO:1, the encoded amino acid sequence is shown in SEQ ID NO:2.

[0134] The nucleotide sequence of the CAR molecule was double-digested with NotI (NEB) and EcoRI (NEB), connected and inserted into the NotI-EcoRI site of retrovirus MSCV (Addgene) by T4 ligase (NEB), and tran...

Embodiment 3

[0140] Example 3: Retroviral Packaging

[0141] 1. Day 1: 293T cells should be less than 20 passages, not overgrown. Plate at 0.6*10^6 cells / ml, add 10ml of DMEM medium to a 10cm dish, mix the cells well, and culture overnight at 37 degrees.

[0142] 2. Day 2: 293T cell confluency reaches about 90% for transfection (usually about 14-18h after plating); prepare plasmid complexes, the amount of various plasmids is 12.5ug of the MSCV backbone vector of Example 1 or 2, Gag-pol10ug, VSVg 6.25ug, CaCl 2 250ul,H 2 O 1ml, the total volume is 1.25ml; add HBS equal to the volume of the plasmid complex in another tube, and vortex for 20 seconds while adding the plasmid complex. Gently add the mixture to the 293T dish along the side, incubate at 37°C for 4 hours, remove the medium, wash with PBS, and add pre-warmed fresh medium again.

[0143] 3. Day 4: 48 hours after transfection, collect the supernatant and filter it with a 0.45um filter, store in -80°C, and continue to add preheat...

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Abstract

The invention relates to a chimeric antigen receptor of a targeted GPC3 (Glypican 3) and an application thereof. Specifically, the invention provides a polynucleotide sequence, wherein the polynucleotide sequence is selected from: polynucleotide sequences (1) containing a coding sequence of a CD8 antigen leading peptide, a coding sequence of an anti-GPC3 single-chain antibody, a coding sequence of a human CD8 alpha hinge region, a coding sequence of a human CD8 transmembrane region, a coding sequence of a human 41BB intracellular region, and a coding sequence of a human CD3 zeta intracellular region, and a coding sequence of an optional fragment of an EGFR (Epidermal Growth Factor Receptor) sequence containing an extracellular domain III and an extracellular domain IV, wherein the coding sequences are sequentially connected; and complementary sequences (2) of the polynucleotide sequences (1). The invention provides a CAR (chimeric antigen receptor) coded by the polynucleotide sequences and a T cell expressing the CAR. The prepared CAR-T cell has a function of strongly killing a specific tumor cell, and the kill effectiveness is more than 90% under the condition that the effector-target ratio is 20:1.

Description

technical field [0001] The invention belongs to the field of chimeric antigen receptors, and in particular relates to chimeric antigen receptors targeting GPC3 and uses thereof. Background technique [0002] Liver cancer is the fifth most prevalent tumor and the third most common cause of cancer-related death in the world (Bosch et al., Gastroenterology 127:S5-S16, 2004; El–Serag et al., Gastroenterology 132:2557 76, 2007). According to the American Cancer Society, hepatocellular carcinoma (HCC) accounts for approximately 75 percent of liver cancer cases, and symptoms are often not present until advanced stages of liver cancer. Surgery is the standard treatment for liver cancer because this type of cancer does not respond well to most chemotherapy drugs. Therefore, there is an urgent need to develop new drugs with different mechanisms of action. [0003] Glypican 3 (GPC3) is a cell surface protein belonging to the heparan sulfate proteoglycan family. The GPC3 gene codes ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
Inventor 黄飞王海鹰金涛何凤史子啸
Owner HRAIN BIOTECHNOLOGY CO LTD
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