Mesothelin-targeting chimeric antigen receptor and uses thereof

A technology of mesothelin and receptors, applied in the field of chimeric antigen receptors targeting mesothelin, which can solve the problems of exceeding the treatment requirements, T cell attack, and high amplification

Active Publication Date: 2018-03-27
HRAIN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this treatment has not been perfected, and T cells will go off target and attack other tissues, or expand too much, beyond the treatment needs

Method used

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  • Mesothelin-targeting chimeric antigen receptor and uses thereof
  • Mesothelin-targeting chimeric antigen receptor and uses thereof
  • Mesothelin-targeting chimeric antigen receptor and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1: Determination of mMesothelin-scFv-CD8α-CD28-41BB-CD3ζ gene sequence

[0093] The gene sequence information of human CD8α hinge region, human CD8α transmembrane region, human CD28, 41BB intracellular region and human CD3ζ intracellular region was searched from the NCBI website database. The anti-mesothelin single-chain antibody clone number is SS1. 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.

[0094] Using overlapping PCR, the above sequences were sequentially linked according to the anti-mesothelin scFv, human CD8α hinge region gene, human CD8α transmembrane region gene, human CD28, 41BB intracellular region gene, and human CD3ζ intracellular region gene sequence. Different enzyme cutting sites were introduced to form the complete mMesothelin-CAR gene sequence information.

[0095] The nu...

Embodiment 2

[0101] Example 2: Determination of Mesothelin-CAR-GMCSFR leader sequence-tEGFR gene sequence

[0102] The gene sequence information of the extracellular region of human EGFR 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 cell expression under the condition that the encoded amino acid sequence remains unchanged .

[0103] Using overlapping PCR, the above sequences were sequentially connected according to the Mesothelin-CAR, GMCSFR leader sequence, and tEGFR in Example 1, and different enzyme cutting sites were introduced at the junctions of each sequence to form a complete mMesothelin-CAR-GMCSFR leader sequence-tEGFR gene sequence information.

[0104] 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 transformed...

Embodiment 3

[0110] Example 3: Retroviral Packaging

[0111] 1. Day 1: 293T cells should be less than 20 passages, not overgrown. Take 0.6×10 6For cell / ml plating, add 10ml of DMEM medium to a 10cm dish, mix the cells well, and culture overnight at 37 degrees;

[0112] 2. Day 2: 293T cell confluency reaches about 90% for transfection (usually about 14-18 hours after plating); prepare plasmid complexes, the amount of various plasmids is MSCV backbone 12.5ug, Gag-pol 10ug, VSVg 6.25 Ug, 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.

[0113] 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 preheated fresh DMEM mediu...

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Abstract

The present invention relates to a mesothelin-targeting chimeric antigen receptor and uses thereof, and specifically provides a polynucleotide sequence, which is selected from (1) a polynucleotide sequence containing the coding sequence of an anti-mesothelin single chain antibody, the coding sequence of a human CD8[alpha] hinge region, the coding sequence of a human CD8 transmembrane region, the coding sequence of a human CD28 intracellular region, the coding sequence of a human 41BB intracellular region, the coding sequence of a human CD3[zeta] intracellular region, and the coding sequence ofan optionally EGFR fragment containing an extracellular domain III and an extracellular domain IV, wherein the coding sequences are sequentially linked; and (2) the complementary sequence of the polynucleotide sequence (1). The invention further provides a related fusion protein, a vector containing the coding sequence, and uses of the fusion protein, the coding sequence and the vector.

Description

technical field [0001] The invention belongs to the field of chimeric antigen receptors, and in particular relates to chimeric antigen receptors targeting mesothelin and uses thereof. Background technique [0002] Pancreatic cancer is a common malignant tumor of the digestive system in clinical practice, and it is more common in people over 50 years old. Its incidence rate has obvious regional differences, and its incidence rate has been gradually increasing in recent years. It has become the fourth most common malignant tumor in European and American countries, ranking second in the cause of death of digestive tract cancer, second only to colorectal cancer. The onset of pancreatic cancer is occult, the early symptoms are non-specific, and the surgical resection rate is low. In the process of tumorigenesis and development, the activation of many genes and the expression of their products play an important role, and the exact molecular mechanism is not completely clear. With...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/867C12N7/01C12N5/10C07K19/00A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K14/71C07K16/18C07K2317/56C07K2317/622C07K2319/02C07K2319/33C07K2319/74C12N7/00C12N15/86C12N2510/00C12N2740/10021C12N2740/10043
Inventor 黄飞金涛王海鹰何凤史子啸
Owner HRAIN BIOTECHNOLOGY CO LTD
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