Multi-chain chimeric antigen receptor and uses thereof

A technology of chimeric antigen receptors and ligands, applied in antibody mimics/stents, anti-inflammatory agents, drug combinations, etc., can solve problems such as limiting the success of immunotherapy

Active Publication Date: 2015-07-08
CELLECTIS SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ability of tumors to take up (co-opt) these inhibitory pathways represents an important mechanism in immune resistance and limits the success of immunotherapy

Method used

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  • Multi-chain chimeric antigen receptor and uses thereof
  • Multi-chain chimeric antigen receptor and uses thereof
  • Multi-chain chimeric antigen receptor and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0296] Embodiment 1: TALE nuclease cleaves human GR gene

[0297] Design and production of six heterodimeric TALE-nucleases targeting human GR gene exons. Table 1 below shows the target sequence cleaved by each TALE nuclease. GR TALE nucleases consist of two separate entities (termed half-TALE nucleases), each containing two 17-bp long sequences (termed half-targets) engineered to bind and cleave two 17-bp long sequences separated by a 15-bp spacer. ) consisting of repeats of the GR target sequence.

[0298]

[0299] Table 1: Description of GR TALE nucleases and TALE nuclease target site sequences in the human GR gene

[0300] Amino acid sequences and repeats of the N-terminal, C-terminal domains are based on AvrBs3 TALE (ref: GenBank: X16130.1). The C-terminal and N-terminal domains are separated by two BsmBI restriction sites. Repeat arrays (SEQ ID NOs: 7 to 18) targeting desired sequences (SEQ ID NOs: 1 to 6) were synthesized using a solid support method consisting o...

Embodiment 2

[0321] Example 2: TALE nucleases that cleave 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)

[0322] Heterodimeric TALE nucleases targeting CD52, TRAC and TRBC genes, respectively, were designed and produced as described in Example 1. The target genomic sequence consists 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 the half-TALE nucleases listed in Table 5. The human genome includes two functional T cell receptor beta chains (TRBC1 and TRBC2). During the development of α / β T lymphocytes, one of these two constant chains is selected in each cell to splice into the variable region of TCR-β and form a functional full-length β chain. Two TRBC targets were selected in sequences conserved between TRBC1 and TRBC2 such that the corresponding TALE nucleases would cleave both TRBC1 and TRBC2.

[0323]

[0...

Embodiment 3

[0349] Example 3: TALE nucleases that cleave human CTLA4 gene and human PDCD1 gene.

[0350]Heterodimeric TALE nucleases targeting PDCD1 and CTLA4 genes, respectively, were designed and produced as described in Example 1. The target genomic sequence consists 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 the half-TALE nucleases listed in Table 10.

[0351]

[0352] Table 10: Description of CTLA4 and PDCD1 TALE nucleases and TALE nuclease target site sequences in human corresponding genes

[0353] Activities of CTLA4-TALE nuclease and PDCD1-TALE nuclease in HEK293 cells

[0354] Each TALE-nuclease construct was subcloned under the control of the pEF1α long promoter using restriction enzyme digestion in a mammalian expression vector. One million HEK293 cells were seeded 1 day before transfection. Two half-targets in the genomic sequence of interest in the PDCD1 and CTLA-4 genes were iden...

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Abstract

The present invention relates to the generation of chimeric antigen receptors (CAR) referred to as multi-chain CARs. Such CARs, which aim to redirect immune cell specificity and reactivity toward a selected target exploiting the ligand-binding domain properties, comprise separate extracellular ligand binding and signaling domains in different transmembrane polypeptides. The signaling domains are designed to assemble in juxtamembrane position, which forms flexible architecture closer to natural receptors, that confers optimal signal transduction. The invention encompasses the polynucleotides, vectors encoding said multi- chain CAR and the isolated cells expressing them at their surface, in particularly for their use in immunotherapy. The invention opens the way to efficient adoptive immunotherapy strategies for treating cancer and viral infections.

Description

technical field [0001] The present invention relates to chimeric antigen receptors (CARs). Taking advantage of ligand-binding domain properties, CARs are able to redirect immune cell specificity and reactivity against a chosen target. In particular, the invention relates to multi-chain chimeric antigen receptors in which the extracellular ligand-binding and signaling domains are separated with different transmembrane polypeptides to improve their function. The different transmembrane polypeptides that make up the multi-chain CAR of the present invention, once assembled together, can specifically bind to one or several ligands in the target and induce the activation of immune cells, where they are expressed and an immune response. The present invention also relates to polynucleotides encoding such transmembrane polypeptides, vectors, and isolated cells expressing the multi-chain CAR on their surface for immunotherapy. The invention also relates to methods for engineering immu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C07K16/28C07K14/735
CPCC12N2501/39C07K16/2803C12N2502/99C12N5/0636C07K2319/00C12N2501/51C12N2501/599C07K14/70535C12N2501/515C07K2317/622A61P29/00A61P35/00A61P37/02A61P37/04A61P37/06
Inventor 朱莉安娜·史密斯安德鲁·沙伦贝格塞西尔·曼尼维贾斯廷·埃康
Owner CELLECTIS SA
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