Method for generating T-cells compatible for allogenic transplantation

A cell and lymphocyte technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problems of invisible host CTL, damage to MHCI, etc.

Active Publication Date: 2016-11-09
CELLECTIS SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, abrogation of β2-M expression in CAR T cells impairs MHC class I expression and renders them invisible to host CTLs

Method used

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  • Method for generating T-cells compatible for allogenic transplantation
  • Method for generating T-cells compatible for allogenic transplantation
  • Method for generating T-cells compatible for allogenic transplantation

Examples

Experimental program
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Embodiment approach

[0188] As a preferred embodiment of the present invention, for example, by electroporation, the nucleic acid molecule encoding the endonuclease of the present invention is transfected in the form of mRNA to obtain transient expression and avoid chromosomal integration of foreign DNA. The present inventors have identified different optimal conditions for mRNA electroporation in T cells presented in Table 1. The present inventors have used CytoPulse technology, which allows the delivery of substances into living cells by transiently permeating living cells using pulsed electric fields (US patents US6010613 and WO 2004 / 083379). Pulse duration, intensity, and interval between pulses can be modified to achieve optimal conditions for high transfection efficiency with minimal mortality. Essentially, the first high electric field pulse allows pore formation, while subsequent low electric field pulses allow the migration of the polynucleotide into the cell. In one aspect of the invent...

Embodiment

[0264] TALE nuclease that cleaves human CIITA

[0265] The mRNA encoding the TALE nuclease targeting the exons of the human CIITA gene was ordered from Cellectis Bioresearch (8, rue de la Croix Jarry, 75013 PARIS). Table 3 below indicates the target sequences cleaved by each of two independent entities (termed half-TALE nucleases), each containing a target sequence engineered to bind and cleaved by two separated by a 15bp spacer. Repeated sequences between target sequences consisting of open 17bp long sequences (called half-targets). Because exon 2 and exon 3 are shared by all transcript variants of CIITA, two TALEN pairs were designed for exon 2 and exon 3. The human genome has been predicted without significant dislocation targeting using TALE nucleases targeting these sequences.

[0266]

[0267]

[0268] Table 3: Description of CIITA TALE nucleases and their associated target sequences

[0269] TALE nuclease that cleaves human β2m

[0270] The mRNA encoding t...

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Abstract

The present invention pertains to engineered T-cells, method for their preparation and their use as medicament, particularly for immunotherapy. The engineered T-cells of the invention are characterized in that the expression of beta 2-microglobulin (B2M) and / or class I I major histocompatibility complex transactivator (CIITA) is inhibited, e.g., by using rare-cutting endonucleases able to selectively inactivating by DNA cleavage the gene encoding B2M and / or CIITA, or by using nucleic acid molecules which inhibit the expression of B2M and / or CIITA. In order to further render the T-cell non-alloreactive, at least one gene encoding a component of the T-cell receptor is inactivated, e.g., by using a rare-cutting endonucleases able to selectively inactivating by DNA cleavage the gene encoding said TCR component. In addition, expression of immunosuppressive polypeptide can be performed on those modified T-cells in order to prolong the survival of these modified T cells in host organism. Such modified T-cell is particularly suitable for allogeneic transplantations, especially because it reduces both 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 T-Cells for treating cancer, infections and auto-immune diseases.

Description

technical field [0001] The present invention relates to engineered T cells, methods for their preparation and their use as medicines, especially for immunotherapy. The engineered T cells of the invention are characterized by suppressed expression of β2-microglobulin (B2M) and / or class II major histocompatibility complex transactivator (CIITA), e.g., by using DNA cleavage-encoded A rare-cutting endonuclease capable of selectively inactivating the genes of B2M and / or CIITA, or by using a nucleic acid molecule that inhibits the expression of B2M and / or CIITA. To further render T cells non-alloreactive, at least one gene encoding a T cell receptor module is inactivated, for example, selective inactivation can be achieved by using DNA cleavage of the gene encoding said TCR module rare-cutting endonuclease. In addition, steps of expression of immunosuppressive polypeptides such as viral MHC I homologues or NKG2D ligands may be performed on these modified T cells in order to prolon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/74C12N5/0783
CPCC07K14/70539C12N5/0636A61P31/12A61P35/00A61P35/02C07K2319/03C07K14/7051A61K39/0011A61K2039/5156A61K2039/5158C07K14/70503C07K2317/24C07K2317/622C12N15/1138C12N15/85C12N15/907A61K35/28A61K35/17C07K14/705
Inventor 劳伦特·普瓦罗戴维·苏尔迪夫菲利普·迪沙泰奥让-皮埃尔·卡巴尼奥尔
Owner CELLECTIS SA
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