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Immortalized car-t cells genetically modified to eliminate t-cell receptor and beta2-microglobulin expression

A cell receptor and immortalization technology, applied in the field of immunotherapy, can solve problems such as death, tissue damage, and limit the efficacy of transferred cells

Pending Publication Date: 2020-07-31
JANSSEN BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current use of allogeneic cells has many disadvantages
In immunocompetent hosts, allogeneic cells are rapidly rejected, a process known as host versus graft rejection (HvG), and this substantially limits the efficacy of transferred cells
In immunocompromised hosts, allogeneic cells are capable of engraftment, but their endogenous T-cell receptor (TCR) specificity may recognize host tissue as foreign, leading to graft-versus-host disease (GvHD), which May cause serious tissue damage and death

Method used

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  • Immortalized car-t cells genetically modified to eliminate t-cell receptor and beta2-microglobulin expression
  • Immortalized car-t cells genetically modified to eliminate t-cell receptor and beta2-microglobulin expression
  • Immortalized car-t cells genetically modified to eliminate t-cell receptor and beta2-microglobulin expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0074] The chimeric antigen receptor (CAR) is designed for adoptive immunotherapy by connecting the extracellular antigen binding domain with the transmembrane domain and the intracellular signal domain (internal domain). It is a useful anti-tumor method for eradicating tumor cells by adoptively transferring T cells expressing chimeric antigen receptors to recognize specific antigens present on tumor cells and activate T cells to specifically lyse these tumor cells. The key aspect of this CAR strategy is the selection of target epitopes that are specifically or selectively expressed on tumors, are present on all tumor cells, and are membrane epitopes that are not easily detached from the cell surface or regulated. . However, ideally, CAR-T cells will be able to be used as general reagents or drugs suitable for any mammalian (such as human) recipient. In order to use cells in this way, one must prevent them from being rejected in the graft-versus-host response without compromis...

Embodiment 1

[0246] Example 1: Preparation of TALL-104 cells for electroporation

[0247] Divide exponentially growing TALL-104 cells to 0.7×10 6 Cells / mL were seeded in complete TALL-104 cell culture medium [Myelocult H5100 medium (StemCell Technologies 05150); 1% sodium pyruvate (Invitrogen 11360-070); 1% non-essential amino acids (Invitrogen 11140-050); 4uM Hydrocortisone (StemCell Technologies 07904); 100IU / ml recombinant human IL-2 (R&D Systems 202-IL, 2.1E4 IU / ug)] and incubate at 37°C. On the next day, collect the required number of cells (1×10 6 / Electroporation). The cells were washed twice with 10 mL of cold Opti-MEM (ThermoFisher Scientific, 31985062), centrifuged at 100×g for 10 min and resuspended in 0.1 mL×(total number of electroporation experiments+1) previously equilibrated to room temperature OPTI-MEM.

Embodiment 2

[0248] Example 2: Preparation of ribonucleoprotein complex

[0249] Guide RNA

[0250] A gRNA is designed to target the first exon of the constant chain of the TCRα gene (TRAC). The targeted sequence located upstream of the TCRα transmembrane domain is necessary for TCRα and β to assemble and address the cell surface. After Cas9 endonuclease-mediated DNA cleavage, non-homologous end joining (NHEJ) or the integration of CAR through homology-directed repair (HDR) will cause the ablation of the TRAC gene. In order to disrupt the B2M locus, a gRNA targeting the first exon was designed. For the gene KIR3DL2, which is responsible for the production of transmembrane glycoproteins on natural killer cells and T cell subsets, gRNA targeting the third exon was designed.

[0251] Table 1: Sequence of guide RNA used for gene editing .

[0252]

[0253] gRNA: tracrRNA duplex formation

[0254] Customized synthesis of target specific Alt-R by Integrated DNA Technologies TM CRISPR-Cas9 guide R...

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Abstract

The present invention pertains to engineered immortalized T-cell lines, method for their preparation and their use as medicament, particularly for immunotherapy. The engineered immortalized T-cell lines of the invention are characterized in that the expression of endogenous T-cell receptors (TCRs) and beta 2-microglobulin (B2M) is inhibited, e.g., by using an endonuclease able to selectively inactivate the TCR and B2M genes in order to render the immortalized T-cells non-alloreactive. In addition, expression of immunosuppressive polypeptide can be performed on those engineered immortalized T-cells in order to prolong the survival of these T-cells in host organisms. Such engineered immortalized T-cells are particularly suitable for allogeneic transplantations, especially because it reducesboth the risk of rejection by the host's immune system and the risk of developing graft versus host disease. The invention opens the way to standard and affordable adoptive immunotherapy strategies using immortalized T-cells for treating cancer, infections and auto-immune diseases.

Description

[0001] Sequence Listing [0002] This application contains a sequence table that has been submitted electronically in ASCII format, and is incorporated by reference in its entirety accordingly. The ASCII copy was created on November 20, 2018, named JBI5146WOPCT1_SL.txt, and has a size of 67,258 bytes. Technical field [0003] The present invention relates to an engineered immortalized T cell line expressing chimeric antigen receptor (CAR); its preparation method; and its use as a medicine, especially for immunotherapy. The engineered immortalized CAR T cell of the present invention is characterized by inhibiting the expression of endogenous T cell receptor (TCR) and β2-microglobulin (B2M), for example, by using an endonuclease. The TCR and B2M genes can be selectively inactivated so that the immortalized CAR T cells are non-aloreactive. The engineered immortalized CAR T cell line is particularly suitable for allogeneic transplantation, especially because it not only reduces the r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C07K16/28C07K14/725C07K14/74A61K35/17A61P35/00
CPCC07K14/7051C07K14/70539C07K2319/03A61P35/00C07K2317/622C07K16/2878C07K16/18C12N15/90A61K48/00A61K39/4631C12N5/0636A61K39/4611A61K39/464457A61K39/464417C07K16/28C12N2510/00C07K2319/02A61K35/17C07K14/78C07K14/4748C12N15/902C07K14/7151C12N15/8509C07K14/70578C07K14/705C07K2317/53C12N15/86C12N15/87C12N2740/15043
Inventor J.李J.莫尼M.纳松
Owner JANSSEN BIOTECH INC
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