Method for improving production of car T cells
Inventors
BOYNE, Alex • Poirot, Laurent • Duchateau, Philippe • Juillerat, Alexandre
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12365733-B2
Publication Date
2025-07-22
Expiration Date
Abstract
A method for engineering less alloreactive immune cells, including T-cells that express chimeric antigen receptors (CARs), using a nucleotide sequence in form of an RNA encoding a anti-TCR CAR to achieve the transient expression of anti-TCR CAR at the cell surface. The transient expression of the anti-TCR CAR recognized by the alpha beta TCR on the cell surface unexpectedly enabled the a purification of the TCR-negative CAR expressing cells. The TCR-negative CAR expressing immune cells can be used in adoptive therapy to treat diseases associated with cell surface antigens, such as cancer with less side effects, in particular less GVHD.
Core Innovation
The invention relates to a method for enriching for T Cell Receptor (TCR)-negative chimeric antigen receptor (CAR)-T cells during manufacturing of engineered CAR-T cells. The method includes a first disruption step that inactivates endogenous TCR component genes in donor T cells by introducing mRNA encoding a rare cutting endonuclease specific for the endogenous TCR component gene, where the rare cutting endonuclease is a TAL-effector protein or a CRISPR Cas9. The method then follows with a first transformation step that introduces a recombinant CAR into the genome of the T cells either before, after, or concomitantly with the disruption step.
After the first transformation step, the method includes a second transformation step that transiently expresses an anti-TCR complex CAR. This second transformation step is performed by introducing into the T cells a synthetic mRNA encoding the anti-TCR complex CAR, thereby producing a population of engineered CAR-T cells enriched for TCR-negative CAR-T cells. The disclosure describes transient surface expression of the anti-TCR CAR as self-limited via mRNA degradation, enabling lysis of TCR-positive targets and enrichment of TCR-negative CAR+ cells.
The problem addressed is the manufacturing need to enrich engineered CAR-T cells for TCR-negative cells, i.e., to produce a population that is depleted of alpha beta TCR-positive cells and enriched for TCR-negative CAR-T cells. The approach combines gene disruption of endogenous TCR components using rare cutting endonuclease mRNA delivery with recombinant CAR introduction into the genome and transient anti-TCR complex CAR expression using synthetic mRNA.
Claims Coverage
The independent claim is clm-00001. It describes a manufacturing method with three main inventive features: disruption of endogenous TCR components using TAL-effector or CRISPR Cas9 delivered as mRNA, introduction of a recombinant CAR into the T-cell genome, and transient expression of an anti-TCR complex CAR using synthetic mRNA to enrich TCR-negative CAR-T cells.
Enriching TCR-negative CAR-T cells via endonuclease mRNA disruption
A first disruption step comprising inactivating at least one gene encoding an endogenous TCR component in T cells from a donor by introducing into the T cells at least one mRNA encoding a rare cutting endonuclease specific for the at least one gene encoding the endogenous TCR component, wherein the rare cutting endonuclease is a TAL-effector protein or a CRISPR Cas9.
Genomic recombinant CAR introduction before, after, or concomitantly with disruption
A first transformation step comprising modifying the T cells by introducing at least one exogenous polynucleotide encoding a recombinant CAR into the genome of the T cells, wherein the first transformation step is performed before, after, or concomitantly with the first disruption step.
Transient anti-TCR complex CAR expression by synthetic mRNA
A second transformation step comprising transiently expressing an anti-TCR complex CAR by introducing into the T cells a synthetic mRNA encoding the anti-TCR complex CAR.
Across the independent claim, TCR-negative CAR-T enrichment is achieved by combining TAL-effector or CRISPR Cas9 rare cutting endonuclease mRNA disruption of endogenous TCR component genes, introducing a recombinant CAR exogenous polynucleotide into the T-cell genome, and transient synthetic mRNA expression of an anti-TCR complex CAR.
Stated Advantages
Produces a population of engineered CAR-T cells enriched for TCR-negative CAR-T cells.
Reduces alpha beta TCR-positive contamination as defined by explicit thresholds.
Enables lysis of TCR-positive targets and enrichment of TCR-negative CAR+ cells.
Uses a self-limited transient anti-TCR CAR expression enabled by mRNA degradation.
Improves antitumor activity in vitro.
Claims a reduced GVHD/side effects compared with purification-based approaches that require removing TCR-positive cells before dosing.
Documented Applications
Manufacturing of engineered CAR-T cells in an allogeneic context, to generate an enriched TCR-negative CAR-T cell population.
Antitumor activity testing in vitro.
In vivo evaluation described in the provided disclosure summary using a Raji-bearing mouse model for tumor burden/survival.
Interested in licensing this patent?