Method for transduction of cells by viral vectors

A virus carrier and cell technology, applied in the field of genetic engineering, can solve the problems of delaying the timing of tumor treatment, affecting the clinical treatment effect, and taking a long time for T cells

Pending Publication Date: 2022-08-05
CARSGEN THERAPEUTICS
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  • Claims
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Problems solved by technology

However, since viral vectors carrying recombinant nucleic acids usually take a long time to infect transduced cells, such as the preparation of CAR T cells, in the conventional process of transducing exogenous nucleic acids into T cells with viral vectors, the T cells must be activated for at least It takes one day (sometimes up to 3 days or more), and the viral transduction also takes a long time, which makes it time-consuming for T cells to specifically recognize tumor-associated antigens, which not only increases the time cost and reagent cost of cell product preparation, but also It may increase the risk of cell mutation, and due to the time course of preparation, some patients have already experienced tumor progression when they are given cell therapy products, thus delaying the timing of tumor treatment and affecting the effect of clinical treatment

Method used

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  • Method for transduction of cells by viral vectors
  • Method for transduction of cells by viral vectors
  • Method for transduction of cells by viral vectors

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preparation example Construction

[0132] In some embodiments, the method of preparation includes the step of freezing (eg, cryopreserving) the cells before or after isolation, selection and / or enrichment and / or incubation for transduction and engineering. In some embodiments, the freezing and subsequent thawing steps remove granulocytes, and to some extent monocytes, in the cell population. In some embodiments, the cells are suspended in a freezing solution to remove plasma and platelets, such as after a washing step. In some aspects, any of a variety of known freezing solutions and parameters can be used. An example involves the use of PBS containing 20% ​​DMSO and 8% human serum albumin (HSA), or other suitable cell freezing medium. It was then diluted 1:1 with medium so that the final concentrations of DMSO and HSA were 10% and 4%, respectively. Cells are then typically frozen to -80°C at a rate of 1° / min and stored in the gas phase of a liquid nitrogen storage tank.

[0133] In some embodiments, isolati...

Embodiment 1

[0512] Example 1. Evaluation of Lentiviral Transduction Efficiency

[0513] Leukocyte-rich samples were collected from subjects using leukapheresis, and buffy coat was collected using Ficoll density gradient centrifugation to obtain peripheral blood mononuclear cells (PBMCs) with high purity.

[0514] PBMCs were washed and resuspended in buffer containing phosphate buffered saline (PBS), EDTA and human serum albumin, washed cells in sorting buffer were mixed with commercially available magnetic beads coupled to monoclonal antibodies The reagents were incubated together for 30 minutes at room temperature and sorted using a magnetic separation column to obtain an enriched population of T cells. The resulting enriched T cell population was resuspended in X-VIVO 15 medium (purchased from Lonza), and T cell activator was added (conjugated to anti-CD3 and / or lentiviral vector particles) Solid supports for anti-CD28 and / or anti-41-BB monoclonal antibodies (e.g., beads, including mag...

Embodiment 2

[0529] Example 2: Evaluation of Transduction Efficiency of Primary T Cell Activated Transduction Culture for 24-48 Hours

[0530] Leukocyte-rich samples were collected from subjects using leukapheresis, and buffy coat was collected using Ficoll density gradient centrifugation to obtain peripheral blood mononuclear cells (PBMCs) with high purity.

[0531] Aseptically transfer the obtained PBMCs into transfer bags. PBMCs were washed and resuspended in selection buffer containing PBS, EDTA and human serum albumin for affinity-based selection. Washing was performed in a sterile single-use disposable kit for regenerative medicine sold by Biosafe SA, which includes a centrifuge chamber (A-200F). The transfer bag containing the cells and the bag containing the buffer were aseptically attached to the kit, which was placed in connection with the Sepax2 processing unit. Two (2) wash cycles were performed, each with approximately 200 g of RCF at the inner wall of the cavity for 180 sec...

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Abstract

The present invention provides methods of making genetically engineered cells, as well as the obtained cells and compositions thereof transduced with recombinant or heterologous genes, and methods of use in adoptive immunotherapy.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a method for entering recombinant nucleic acid through virus transduction. Background technique [0002] The role of immune effector cells (such as T cells, NK cells, NK T cells, etc.) in tumor immunotherapy has been paid more and more attention. In recent years, people have modified immune effector cells with exogenous receptors to obtain T cells that specifically recognize tumor-related antigens, and then carry out tumor therapy, such as chimeric antigen receptor-modified CAR T cells and chimeric TCR receptor-modified T cells. TCR T cells, etc. [0003] Generally, cells recognizing tumor-associated antigens are obtained by introducing a recombinant nucleic acid encoding a foreign receptor that recognizes tumor-associated antigens into a viral vector, and then infecting the transduced cells with the viral vector carrying the recombinant nucleic acid. However, beca...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10C12N15/62A61K39/00A61K39/395A61P31/00A61P35/00A61P37/00
CPCC12N15/86C07K16/303C07K16/2803C07K16/2878C07K14/7051C12N5/0636A61K39/001102A61K39/00114A61K39/3955A61K39/0011A61P35/00A61P37/00A61P31/00C12N2740/15043C12N2510/00C12N2800/107C07K2319/33A61K2039/5158A61K2039/5154
Inventor 王华茂高慧萍童潇姚晔风朱寅玉李宗海
Owner CARSGEN THERAPEUTICS
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