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Simple and efficient lentivirus cryopreservation liquid and application thereof

A technology of lentivirus and cryopreservation solution, which is applied in the field of chimeric antigen receptor lentivirus cryopreservation solution, which can solve the problems of loss of lentivirus infection efficiency and other issues

Pending Publication Date: 2021-05-25
东莞清实生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, CAR-T therapy mainly adopts lentiviruses carrying CARs specific for tumor antigens as gene delivery vectors, but a current bottleneck is that lentiviruses that have been concentrated or purified by ultracentrifugation can be used in PBS or After reconstitution in normal saline, fresh lentivirus can infect T cells with a high efficiency, but if the lentivirus is stored at -80 degrees, the infection efficiency of the lentivirus after freezing and thawing will be lost by about 40%-60%. In the industry, there is an urgent need for a high-efficiency lentivirus cryopreservation solution that complies with the Pharmacopoeia. Correspondingly, the cryopreservation solution of the present invention is not limited to CAR-T treatment, and can be used in all biotechnologies that use lentivirus as a gene therapy method

Method used

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  • Simple and efficient lentivirus cryopreservation liquid and application thereof
  • Simple and efficient lentivirus cryopreservation liquid and application thereof
  • Simple and efficient lentivirus cryopreservation liquid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: CD19 CAR lentiviral vector construction

[0021] FMC63 scFv fragment SEQ ID NO: 1, and the second-generation CAR hinge region, transmembrane region, and intracellular region sequence SEQ ID NO: 2 corresponding to the signal peptide, were synthesized by BGI. After obtaining the two synthetic genes, construct the vector of CD19 CAR. First, amplify by PCR and add restriction sites at the head and tail of the two sequences, and then digest and ligate to obtain SEQ ID NO: 3, which will be slow The viral vector pCDH-EF1a-T2A-puro and SEQ ID NO: 3 were double-enzymatically digested and ligated with T4 DNA ligase respectively, transformed into competent cells, single clones were picked, and after the bacterial liquid was sequenced correctly, the plasmid was extracted to obtain the correct sequence Lentiviral vector pCDH-EF1a-CD19 CAR plasmid.

Embodiment 2

[0022] Example 2: Preparation of CD19 CAR lentivirus

[0023] After mixing the lentiviral packaging plasmid mixture (including PSPAX2 and VSVG) and the pCDH-EF1a-CD19 CAR plasmid in a pre-optimized ratio, add transfection-assisting reagents and incubate at room temperature for 30 minutes. The transfection mixture was then slowly added to 293T cells (purchased from ATCC). After 1-3 days, the medium supernatant was collected to obtain the crude lentivirus solution. A total of 180 ml of the supernatant was collected after centrifugation at 500 g for 10 min at 4°C. Divide 180 ml into 6 tubes on average, and place each 30 ml tube in an ultra-high-speed centrifuge tube. After balancing, ultra-centrifuge at 20,000 g for 2 hours to concentrate the lentivirus.

Embodiment 3

[0024] Example 3: Pre-experiment of lentiviral cryopreservation solution

[0025] In this example, the lentivirus cryopreservation solution of the present invention (hereinafter referred to as cryoS001) and two commercial lentivirus cryopreservation solutions (brand 1, Yumeibo, article number UR40202, hereinafter code-named UB; brand 2, Andy FUNO, Cat. No. D022 hereinafter code-named ABP) compared the protection efficiency of CD19 CAR lentivirus to clarify the effectiveness of the lentivirus cryopreservation solution.

[0026] After the ultracentrifugation of the aliquoted lentivirus in the above Example 2, carefully discard the supernatant, and resuspend the lentiviral particles with the pre-cooled lentiviral freezing solution: put 60 ml of lentivirus in two ultracentrifugation tubes After concentrating the lentivirus stock solution, discard the supernatant, resuspend each tube with 0.5 ml of UB or ABP, or cryoS001, that is, divide the ultra-concentrated 180 ml lentivirus sto...

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Abstract

The invention provides a simple and efficient lentivirus cryopreservation liquid and an application thereof, and belongs to the field of immune cell therapy. The formula of the lentivirus cryopreservation liquid comprises human plasma albumin (HSA) injection and 0.9% normal saline injection; wherein the concentrations of the HAS comprise 10%, 20%, 30% and 40%.

Description

technical field [0001] The present invention relates to the field of immune cell therapy, in particular to a chimeric antigen receptor (CAR-T) lentivirus cryopreservation solution and its application. Background technique [0002] Chimeric Antigen Receptor modified T cells (CART) express an artificially synthesized CAR molecule (Chimeric Antigen Receptor) on the T cell membrane through genetic modification, allowing T cells to bind with antigens and antibodies. way to recognize and kill tumor cells. The CAR molecule includes an extracellular binding region (a single-chain antibody (scFv) derived from a monoclonal antibody that can specifically recognize a target antigen), a hinge region, a transmembrane region, and an intracellular signal segment. This technology has several advantages. For example, CAR protein recognizes TAA (tumor associated antigen, tumorassociated antigen) on the tumor surface in an MHC (major histocompatibility complex)-independent manner, so CAR T cel...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/62C12N5/10A61K39/00A61P35/00
CPCC12N15/86C07K16/2803C12N5/0636A61K39/001112A61P35/00C12N2740/15043C12N2800/107C12N2740/15011C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N2510/00A61K2039/5156
Inventor 王冰李治寰李艳群彭方理卢佳豪
Owner 东莞清实生物科技有限公司
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