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Nano switch molecules and activation control solid tumor targeted CAR-T cells thereof

A CAR-T and nano-switch technology, applied in the field of tumor immunology, can solve the problems of complex CAR-T design, production and preparation, and large-scale clinical application, and the failure to find specific surface antigens for solid tumors, etc., to solve off-target effects , the effect of prolonging the half-life of the drug

Active Publication Date: 2020-07-03
NANJING DRUM TOWER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The main ideas for controlling the off-target effect of CAR-T in solid tumors include: 1. Find new targets or design multi-target activation of CAR-T cells: the ideal CAR-T target should satisfy the following requirements: ① located on the surface of tumor cells; ② located on the surface of specific tumor cells High surface expression ③ It is not expressed in normal tissues, but no solid tumor-specific surface antigen that fully meets the above requirements has been found; 2. Control CAR-T inactivation, including introducing herpes simplex virus thymidine kinase (HSV-tk), Fas intracellular domain (ΔFAS), iCasp9 and other artificially synthesized death control switches, but none of the above methods can fundamentally solve the problem of non-specific activation of CAR-T, and the complex design of CAR-T leads to production and large-scale Scale clinical application poses difficulties

Method used

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  • Nano switch molecules and activation control solid tumor targeted CAR-T cells thereof
  • Nano switch molecules and activation control solid tumor targeted CAR-T cells thereof
  • Nano switch molecules and activation control solid tumor targeted CAR-T cells thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The preparation of gelatinase-targeted nanoparticles includes the following four steps:

[0041] Step A) Synthesis of PCL-NH2: Using ε-CL (ε-caprolactone, ε-caprolactone) as raw material, adopting free radical polymerization method to synthesize PCL (polycaprolactone) containing carboxyl group, denoted as PCL-COOH , and then use EDC to aminate the end of PCL-COOH to form PCL with an active amino group, which is denoted as PCL-NH2. The specific reaction is as follows:

[0042]

[0043] Step B) Synthesis of mPEG-PEP: The activated mPEG-NHS reacts with the amino terminal of polyethylene glycol monomethyl ether (mPEG) to synthesize mPEG-PEP. The specific reaction is as follows:

[0044]

[0045] Step C) synthesis of enzyme targeting carrier: in the presence of EDC, DMAP, NHS, the carboxyl group at the end of mPEG-PEP and the amino group at the end of PCL-NH2 react to form mPEG-PEP-PCL diblock copolymer, the Compared with the traditional two-block copolymer mPEG-PCL (...

Embodiment 2

[0048] The preparation of nano-switch molecules, the nano-switch molecules are tumor-targeted nanoparticles loaded with switch molecules, which are constructed by double emulsification-volatility method, specifically including the following steps:

[0049] Step 1) dissolving the hydrophobic switch molecule and the tumor-targeting nanocarrier in a dichloromethane solution to form an organic phase, a mixed solution A;

[0050] Step 2) Add mixed solution A to the first external aqueous phase containing 3% polyvinyl alcohol and use ultrasonic emulsification technology to fully mix to form mixed solution B, wherein the mPEG-PEP-PCL diblock copolymer is self-contained in the aqueous phase Assembled into polymer nanoparticles with the hydrophilic end outward and the hydrophobic end inward;

[0051] Step 3) adding the mixed solution B into the second external aqueous phase containing 1% polyvinyl alcohol and fully mixing it with ultrasonic emulsification technology to form the mixed s...

Embodiment 3

[0057] The preparation of solid tumor-targeted CAR-T cells includes the following steps:

[0058] 1) Preparation of nano-switch molecules: using the nano-switch molecules prepared in Example 2;

[0059] 2) Construct and amplify the assembled CAR-T plasmid: T2A gene (T2A gene sequence is shown in SEQ ID NO.), FRB sequence (FRB sequence is shown in SEQ ID NO.2), FKBP sequence using gene recombination technology (FKBP sequence shown in SEQ ID NO.3) and the ScFv segment targeting solid tumors (ScFv segment sequence shown in SEQ ID NO.4) were introduced into the CAR-T plasmid to construct an assembled CAR-T plasmid (pDonor-SB- HER2-4-1BB-FKBP-DPA10-FRB-EGFP plasmid, such as figure 1 shown), using Escherichia coli DH5α as the carrier to amplify the assembled CAR-T plasmid;

[0060] Among them, T2A gene sequence: 5'-CGGAAGCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGACCTA-3'(SEQ ID NO.1);

[0061] FKBP序列:5’-GGAGTGCAGGTGGAAACCATCTCCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTG...

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Abstract

The present invention discloses nano switch molecules and activation control solid tumor targeted CAR-T cells thereof. The solid tumor targeted CAR-T cells are prepared by the following steps of 1) preparing the nano switch molecules, wherein switch molecules are loaded on tumor targeted nanoparticles by adopting a double emulsification-volatilization method; 2) constructing assembled CAR-T plasmids, wherein T2A elements, FRB and FKBP sequences and solid tumor targeted ScFv fragments are introduced in CAR-T plasmids by using a genetic recombination method; 3) preparing assembled CAR-T cells, wherein the assembled CAR-T plasmids are transfected with T lymphocytes; and 4) preparing the solid tumor targeted CAR-T cells, wherein the assembled CAR-T cells endocytose the nano switch molecules. The switch molecules are loaded on the tumor targeted nanoparticles, so that the switch molecules can be specifically gathered on the tumor tissues; and FKBP and FRAP of CAR-T are connected by the switch molecules, so that two parts of CAR-T are combined to have the attack activity, and the problem of CAR-T cell off-target effect can be solved fundamentally.

Description

technical field [0001] The invention belongs to the technical field of tumor immunology, and in particular relates to a nano-switch molecule and solid tumor-targeted CAR-T cells controlled and activated by the same. Background technique [0002] CAR-T therapy (Chimeric Antigen Receptor T-Cell Immunotherapy, Chimeric Antigen Receptor T-cell Immunotherapy) has made a major breakthrough in the treatment of leukemia, bringing new hope to patients with certain blood cancers. However, CAR-T therapy faces obstacles when it is applied to solid tumors. The most critical reason is that the targets of CAR-T in solid tumors are expressed in a small amount in normal tissues, so CAR-T can non-specifically attack normal tissues. Serious off-target effects have been caused, and related deaths have been reported. [0003] The preparation method of CAR-T cells is to transfect CAR gene plasmids into T lymphocytes to make them into CAR-T cells. The transfection methods include virus transfecti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/62A61K47/60A61K47/59A61K9/14A61K31/365A61K31/436A61K39/00A61P35/00C12N5/10C12N15/867
CPCA61K9/146A61K31/365A61K31/436A61K47/593A61K47/60A61K47/62A61P35/00A61K39/001111C07K14/7051C07K16/30C12N5/0636C12N15/86C12N2510/00C12N2740/15043A61K2300/00
Inventor 李茹恬刘宝瑞
Owner NANJING DRUM TOWER HOSPITAL
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