Conditioning Intensity and T Cell Dose Determine Efficacy of CD19-Targeted T Cell-Mediated Tumor Eradication in an Immunocompetent Mouse Model of B-ALL.
Authors: Davila Marco L , Kloss Christopher , Brentjens Renier J , Sadelain Michel 
Published: October 2019

Abstract
Abstract 2613
Recent work by our group and others demonstrates the therapeutic potential of CD19-targeted T cells to treat patients with indolent B cell malignancies. These studies make use of T cells that are genetically engineered with chimeric antigen receptors (CARs) comprising an scFv fused to various T cell activating elements. Whereas firs-generation CARs only direct T cell activation, second-generation CARs include two signal elements, such as CD3z and CD28 signaling domains (19–28z). We and our colleagues at MSKCC are currently evaluating the safety of 19–28z-transduced T cells in patients with acute leukemia (B-ALL) in a Phase I protocol (NCT01044069). Pre-clinical studies performed to date have mostly relied on xenogeneic models utilizing immunodeficient animals, which enable the evaluation of human engineered T cells but do not recapitulate all the interactions that may affect tumor eradication by CAR-modified T cells. We have therefore developed a pre-clinical immunocompetent mouse model of B-ALL, and addressed therein the impact of conditioning and T cell dose on the eradication of leukemia by syngeneic, CAR-targeted T cells.
To establish an immunocompetent mouse model of B cell leukemia, we generated a clone from the lymph node of an Eμ-myc B6 transgenic mouse. The immunophenotype and gene-expression profile of clone Eμ-ALL01 is consistent with a progenitor B cell origin. Syngeneic B6 mice inoculated with this clone develop florid acute leukemia and die approximately 2–4 weeks after injection from progressive bone marrow infiltration. We created an anti-mouse CD19 CAR comprising all murine elements, including the CD8 signal peptide, a CD19-specific single chain variable fragment, the CD8 transmembrane region, and the CD28 and CD3z signaling domains. Transduction of the murine 19–28z CAR into mouse T cells was robust and successfully retargeted the T cells to B cells. In vitro assays demonstrated that m19–28 z transduced T cells mediated effective killing of CD19-expressing target cells and the production of effector cytokines such as IFNγ and TNFα.
Cyclophosphamide either alone or in combination with control syngeneic T cells is insufficient to eradicate established Eμ-ALL01 in B6 mice. However, treatment with cyclophosphamide and m19–28z-transduced T cells cured nearly all mice. Mice sacrificed six months after treatment exhibited a dramatic reduction of B cells in the bone marrow (BM), blood, and spleen. The few remaining B lineage cells found in the BM had a phenotype consistent with early pro-B cells, suggesting that endogenous reconstitution of the B cell compartment was thwarted by persisting, functional m19–28z+ T cells. Thus, T cells are retained at the site of antigen expression, which is maintained through regeneration of progenitor B cells. The persisting CD19-targeted T cells in the BM exhibited a cell surface phenotype consistent with effector and central memory cells.
Using B cell aplasia as a surrogate endpoint for assessing in vivo T cell function and persistence, we evaluated how conditioning chemotherapy and T cell dose determine the level of B cell depletion induced by adoptively transferred CD19-targeted T cells. Overall, increasing the cyclophosphamide or T cell dose, increased the degree and duration of B cell depletion and the number of persisting CAR-modified T cells. Significantly, increasing the T cell dose at a set cyclophosphamide level had a lesser impact than increasing the conditioning intensity for a given T cell dose. In summary, the new Eμ-ALL01 syngeneic, immunocompetent B-ALL model we describe here is a valuable tool for modeling CD19 CAR therapies. Our results indicate that m19–28z transduced T cells are effective at eradicating B-ALL tumor cells and persist long-term, preferentially in bone marrow. Our findings further establish that conditioning intensity and T cell dose directly determine B cell elimination and long-term T cell persistence. These studies in mice will serve as an important framework to further model and perfect our studies in patients with B-ALL.

Disclosures:
No relevant conflicts of interest to declare.