Methods for engineering allogeneic and highly active t cell for immunotherapy

a technology of immunotherapy and t cells, applied in the direction of peptide/protein ingredients, cell culture active agents, fusion polypeptides, etc., can solve problems such as interfering with their function

Inactive Publication Date: 2015-01-15
CELLECTIS SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0021]In another aspect, the present invention relates to the polypeptides and the polynucleotides, which encode the rare-cutting endonucleases, to precisely target the above genes of interest, in particular TCRalpha, TCRbeta, immune checkpoi

Problems solved by technology

Moving signaling domains from their natural juxtam

Method used

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  • Methods for engineering allogeneic and highly active t cell for immunotherapy
  • Methods for engineering allogeneic and highly active t cell for immunotherapy
  • Methods for engineering allogeneic and highly active t cell for immunotherapy

Examples

Experimental program
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Effect test

example 1

TALE-Nucleases Cleaving the Human GR Gene

[0296]6 heterodimeric TALE-nucleases targeting exons of the human GR gene were designed and produced. Table 1 below indicates the target sequences cleaved by each TALE-nuclease. GR TALE-nuclease was composed of two independent entities (called half TALE-nucleases) each containing a repeat sequence engineered to bind and cleave GR target sequences consisting of two 17-bp long sequences (called half targets) separated by a 15-bp spacer.

TABLE 1Description of the GR TALE-nucleases and sequences of the TALE-nucleases targetsites in the human GR gene.Half TALE-nucleaseTarget nameTarget sequenceRepeat sequencesequenceGRex2TATTCACTGATGGACTCRepeat GRex2-LPT9-L1GRex2-L TALENcaaagaatcattaac(SEQ ID NO: 7)(SEQ ID NO: 19)TCCTGGTAGAGAAGAAARepeat-GRex2-LPT9-R1GRex2-R TALEN(SEQ ID NO: 1)(SEQ ID NO: 8)(SEQ ID NO: 20)GRex3T2TGCCTGGTGTGCTCTGARepeat-GRex3T2-L1GRex3T2-L TALENtgaagcttcaggatg(SEQ ID NO: 9)(SEQ ID NO: 21)TCATTATGGAGTCTTAARepeat-GRex3T2-R1GRex3T2-R TA...

example 2

TALE-Nucleases Cleaving the Human CD52 Gene, the Human T-Cell Receptor Alpha Constant Chain (TRAC) and the Human T-Cell Receptor Beta Constant Chains 1 and 2 (TRBC)

[0308]As described in example 1, heterodimeric TALE-nucleases targeting respectively CD52, TRAC and TRBC genes were designed and produced. The targeted genomic sequences consist of two 17-bp long sequences (called half targets) separated by an 11 or 15-bp spacer. Each half-target is recognized by repeats of half TALE-nucleases listed in table 5. The human genome contains two functional T-cell receptor beta chains (TRBC1 and TRBC2). During the development of alpha / beta T lymphocytes, one of these two constant chains is selected in each cell to be spliced to the variable region of TCR-beta and form a functional full length beta chain. The 2 TRBC targets were chosen in sequences conserved between TRBC1 and TRBC2 so that the corresponding TALE-nuclease would cleave both TRBC1 and TRBC2 at the same time.

TABLE 5Description of t...

example 3

TALE-Nucleases Cleaving the Human CTLA4 Gene and the Human PDCD1 Gene

[0317]As described in example 1, heterodimeric TALE-nucleases targeting respectively PDCD1 and CTLA4 genes were designed and produced. The targeted genomic sequences consist of two 17-bp long sequences (called half targets) separated by an 11 or 15-bp spacer. Each half-target is recognized by repeats of half TALE-nucleases listed in table 10.

TABLE 10Description of the CTLA4 and PDCD1 TALE-nucleases and sequences of the TALE-nucleases target sites in the human corresponding genes.TargetTarget sequenceRepeat sequenceHalf TALE-nucleaseCTLA4_T01TGGCCCTGCACTCTCCTRepeat CTLA4_T01-LCTLA4_T01-L TALENgttttttcttctctt(SEQ ID NO: 79)(SEQ ID NO: 89)CATCCCTGTCTTCTGCARepeat CTLA4_T01-RCTLA4_T01-R TALEN(SEQ ID NO: 74)(SEQ ID NO: 80)(SEQ ID NO: 90)CTLA4_T03TTTTCCATGCTAGCAATRepeat CTLA4_T03-LCTLA4_T03-L TALENgcacgtggcccagcc(SEQ ID NO: 81)(SEQ ID NO: 91)TGCTGTGGTACTGGCCARepeat CTLA4_T03-RCTLA4_T03-R TALEN(SEQ ID NO: 75)(SEQ ID NO: 82...

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Abstract

The present invention relates to methods for developing engineered T-cells for immunotherapy that are non-alloreactive. The present invention relates to methods for modifying T-cells by inactivating both genes encoding T-cell receptor and an immune checkpoint gene to unleash the potential of the immune response. This method involves the use of specific rare cutting endonucleases, in particular TALE-nucleases (TAL effector endonuclease) and polynucleotides encoding such polypeptides, to precisely target a selection of key genes in T-cells, which are available from donors or from culture of primary cells. The invention opens the way to standard and affordable adoptive immunotherapy strategies for treating cancer and viral infections.

Description

FIELD OF THE INVENTION[0001]The present invention relates to methods for developing engineered non-alloreactive T-cells for immunotherapy and more specifically to methods for modifying T-cells by inactivating both genes encoding T-cell receptor and an immune checkpoint gene to unleash the potential of immune response. This method involves the use of specific rare cutting endonucleases, in particular TALE-nucleases (TAL effector endonuclease) and polynucleotides encoding such polypeptides, to precisely target a selection of key genes in T-cells, which are available from donors or from culture of primary cells. The invention also relates to preTCRα (“pTalpha”) and functional derivatives thereof, Chimeric Antigen Receptor (CAR), multichain CAR and the use thereof to enhance the efficiency of the immunotherapy. The invention opens the way to standard and affordable adoptive immunotherapy strategies for treating cancer and viral infections.BACKGROUND OF THE INVENTION[0002]Adoptive immuno...

Claims

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Application Information

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IPC IPC(8): A61K35/14A61K35/17
CPCA61K35/17C07K16/2803C12N5/0636C07K2317/622C07K2319/00C12N2501/39C12N2501/51C12N2501/515C12N2501/599C12N2502/99C07K14/7051A61P31/12A61P35/00A61P35/02A61P37/00A61P37/06C07K2319/03A61K39/0011A61K2039/5156A61K2039/5158A61K38/00A61P5/38A61P21/00A61P31/00A61P37/02A61P37/04A61P43/00C07K14/70517C07K14/70521C07K14/70578C07K16/28C07K2317/569C12N2510/00C07K14/70535A61K39/00C07K2317/14C07K2317/24C07K2319/74C12N15/85
Inventor GALETTO, ROMANGOUBLE, AGN SGROSSE, STEPHANIEMANNIOUI, CECILEPOIROT, LAURENTSCHARENBERG, ANDREWSMITH, JULIANNE
Owner CELLECTIS SA
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