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mAb-DRIVEN CHIMERIC ANTIGEN RECEPTOR SYSTEMS FOR SORTING/DEPLETING ENGINEERED IMMUNE CELLS

a technology of engineered immune cells and chimeric antigen receptors, which is applied in the field of improved chimeric antigen receptors, can solve the problems of inability to provide prolonged expansion and anti-tumor activity in vivo, car t cells can promote acute adverse events after being transferred into patients, and discrimination, so as to enhance the cytotoxicity of engineered immune cells and promote cdc cytotoxicity

Pending Publication Date: 2018-01-04
CELLECTIS SA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text discusses the possibility of enhancing the ability of immune cells to kill cancer cells by combining an epitope-specific antibody with a cytotoxic drug. The text also mentions that using engineered antibodies that have parts of the complement system can increase the cytotoxicity of the immune cells. These technical effects may improve the effectiveness of immune cell-based therapies for cancer treatment.

Problems solved by technology

First generation CARs have been shown to successfully redirect T cell cytotoxicity, however, they failed to provide prolonged expansion and anti-tumor activity in vivo.
However, despite their unprecedent efficacy for tumor eradication in vivo, CAR T cells can promote acute adverse events after being transferred into patients.
This system presents some limitations from the industrial perspective, as first, it requires the cloning large retroviral inserts, and second, to ensure that the transformed cells express both RQR8 and CAR polypeptides, to eliminate possible “false-positive” i.e. T-cells that would not express both polypeptides, in particular the RQR8 suicide gene allowing the depletion of the engineered immune cells in the event of undesirable effects.
However, despite their immunosuppressive efficacy, these drugs are not discriminative as they affect the proliferation of all T and B cells.
As such, the use of such antibodies would not allow the selective elimination of the engineered immune cells endowed with CARs.

Method used

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  • mAb-DRIVEN CHIMERIC ANTIGEN RECEPTOR SYSTEMS FOR SORTING/DEPLETING ENGINEERED IMMUNE CELLS
  • mAb-DRIVEN CHIMERIC ANTIGEN RECEPTOR SYSTEMS FOR SORTING/DEPLETING ENGINEERED IMMUNE CELLS
  • mAb-DRIVEN CHIMERIC ANTIGEN RECEPTOR SYSTEMS FOR SORTING/DEPLETING ENGINEERED IMMUNE CELLS

Examples

Experimental program
Comparison scheme
Effect test

example 1

n of Rituximab-Driven Depletion Systems Embedded in an Anti-CD123 CAR

[0254]All 10 CARs having different conformations in terms of chimeric scFv (anti-CD123 scFv with CD20 mimotope(s)) are depicted in FIG. 4: their resulting polypeptide sequences are shown in SEQ ID NO 1 to 10.

[0255]The DNA construct of the 10 CARs are transcribed into their corresponding mRNA via in vitro transcription and used to transfect by electroporation primary T cells freshly isolated from buffy coat via a standard ficoll procedure. One day post transfection, T cells were recovered and used to performed a flow based cytotoxicity assay as described as follows.

Generation of Anti CD123 CAR T Cells.

[0256]To generate primary T cells expressing anti-CD123 CAR, primary T cells are first purified from buffy-coat samples and activated using Dynabeads human T activator CD3 / CD28. 3 days post activation, 1 million of activated T cells are transduced with lentiviral vectors harboring an anti-CD123 CAR expression cassette ...

example 2

ty of the mAb-Driven Depletion Systems in the Anti-CD123 Chimeric Antigen Receptor

[0261]To further demonstrate the flexibility of the mAb-driven depletion system, different epitopes or mimotopes (SEQ ID NO 35-42) specific for cetuximab, palivizumab and nivolumab mAbs are inserted within the anti-CD123 CAR constructions using the same procedure and architecture as the one used for the CD20 mimotope described in Example 1. The results aim to show that transfected T cells retain their cytolytic activity and degranulation capacity toward CD123 positive tumor cells. In addition, the experiments are designed also to indicate that transfected T cells are depleted by some of the aforementioned mAbs

example 3

-Dependent Depletion of Anti-CD123 CAR Containing an mAb-Driven Depletion System

[0262]To explore the ability of the mAb-driven depletion system to allow depletion of anti-CD123 CAR T cells, transduced T cell expressing a CAR of SEQ ID NO 1, 2, 3 or 4 or an unmodified anti-CD123 CAR (SEQ ID NO 142), were subjected to a complement dependant cytotoxicity assay (CDC).

[0263]CDC Assay

[0264]The CDC assay consisted in incubating 0.2 106 transduced T cells either alone, or in the presence of Rituximab (RTX, ROCHE, 400 ng) and Babby Rabbit Complement (BRC, AbD Serotec, ref# C12CA, 100 μL of the solution diluted according to the manufacturer protocol) for 3 hours at 37° C. in a final volume of 400 μL of Xvivo 10% FBS. At the end of incubation, anti CD123-CAR T cells were recovered and labeled with recombinant CD123 protein fused to an FC fragment (SEQ ID 143) and a PE labeled anti-FC secondary monoclonal antibody (Jackson ImmunoResearch, ref#115-115-164, diluted 1 / 200). Cells were then recover...

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Abstract

A polypeptide encoding a chimeric antigen receptor (CAR) comprising at least one extracellular binding domain that comprises a scFv formed by at least a VH chain and a VL chain specific to an antigen, wherein said extracellular binding domain comprises at least one mAb-specific epitope.

Description

FIELD OF THE INVENTION[0001]The present invention relates to improved chimeric antigen receptors (CAR) to be used in immunotherapy, the extracellular binding domains (scFv) of which have been modified by insertion of a mAb-specific epitope to allow both sorting and / or depletion of the immune cells endowed with said CARs. The present invention relates also to the immune cells expressing said CARs, to the methods of in vivo depleting and / or in vitro sorting said CAR-expressing immune cells, and is drawn to the their therapeutic use.BACKGROUND[0002]Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated ex vivo, is a promising strategy to treat viral infections and cancer. The T cells used for adoptive immunotherapy can be generated either by expansion of antigen-specific T cells or redirection of T cells through genetic engineering (Park, Rosenberg et al. 2011). Transfer of viral antigen specific T cells is a well-established procedure used...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K14/705G01N15/10G01N15/14C12N5/0783C07K14/725G01N15/00
CPCC07K16/2887C07K14/70517C07K14/7051C07K14/70578C12N5/0636G01N15/14G01N15/1031C07K2317/622C07K2317/56C07K2319/03C07K2317/53C12N2510/00G01N2015/149G01N2015/1081G01N2015/1006G01N2015/008C07K16/2866C07K16/3061C07K16/2803C07K2317/24C07K14/70596A61P35/00C07K2319/02C07K2319/33C12N5/0638A61K39/4611A61K39/4631A61K39/4644C07K2319/00C12N5/0093A61K2039/6056A61K2039/64A61P35/02A61P43/00C07K16/28C07K14/70535A61K35/17A61K39/0011A61K39/39558A61K2039/505A61K2039/5156A61K2039/5158G01N2015/016G01N2015/1028G01N15/149A61K39/395C07K19/00C12N5/10C12N15/63A61K39/001176
Inventor SASU, BARBARA JOHNSONRAJPAL, ARVINDDUCHATEAU, PHILIPPEJUILLERAT, ALEXANDREVALTON, JULIEN
Owner CELLECTIS SA
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