Abstract
The remissions achieved using autologous T-cells expressing chimeric antigen receptors (CARs) in patients with advanced B cell leukemia and lymphomas have encouraged the use of CAR technology to treat different types of cancers by targeting distinct tumor-specific antigens. Since the current autologous approach utilizes CAR T-cells manufactured on a "per patient" basis, we propose an alternative approach based on the use of a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic "off-the-shelf" CAR T-cell-based frozen products.
In the present work we have adapted this allogeneic platform to the production of T-cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed on tumor cells from the majority of patients with Acute Myeloid Leukemia (AML).
Multiple antigen recognition domains were screened in the context of different CAR architectures to identify candidates displaying activity against cells expressing variable levels of the CD123 antigen. The three lead candidates were tested in an orthotopic human AML cell line xenograft mouse model. From the three candidates that displayed comparable activity in vitro, we found two candidates capable of eradicating tumor cells in vivo with high efficiency.
Subsequently, Transcription Activator-Like Effector Nuclease (TALEN) gene editing technology was used to inactivate the TCRα constant (TRAC) gene, eliminating the potential for engineered T-cells to mediate Graft versus Host Disease (GvHD). Editing of the TRAC gene can be achieved at high frequencies, and allows efficient amplification of TCR-deficient T-cells that no longer mediate alloreactivity in a xeno-GvHD mouse model. In addition, we show that TCR-deficient T-cells display equivalent in vitro and in vivo activity to non-edited T-cells expressing the same CAR.
We have performed an initial evaluation of the expression of CD123 in AML patients and found an average cell surface expression of CD123 was of 67% in leukemic blasts (95% CI 48-82), 71% in CD34+CD38+ cells (95% CI 56-86), and 64% in CD34+CD38- (95% CI 41-87). Importantly, we have found that CD123 surface expression persists in CD34+CD38-CD90- cells after therapy in at least 20% of patients in remission (n=25), thus emphasizing the relevance of the target. Currently, the sensitivity of primary AML cells to CAR T-cells is being tested.
Finally, we will also present our large scale manufacturing process of allogeneic CD123 specific T-cells from healthy donors, showing the feasibility for this off-the-shelf T-cell product that could be available for administration to a large number of AML patients.
Disclosures
Galetto: Cellectis SA: Employment. Lebuhotel:Cellectis SA: Employment. Gouble:Cellectis SA: Employment. Smith:Cellectis: Employment, Patents & Royalties.
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