716. Durable Acute Myeloid Leukemia Remission Without Myeloablation in an Innovative Xenotolerant Mouse Model of CD44v6 CAR-T Cell Immunotherapy
Authors: Margherita Norelli, Monica Casucci, Barbara Camisa, Laura Falcone, Aurore Saudemont, Pietro Genovese, Luigi Naldini, Chiara Bonini, Attilio Bondanza
Journal: Molecular Therapy (2015)

Despite the remarkable clinical results of chimeric antigen receptor (CAR) T-cell immunotherapy in B-cell tumors, its long-term success is limited by the emergence of CD19-loss tumor escape variants. We have developed a CD28-endocostimulated CD44v6 CAR for the targeting of multiple tumors, including acute myeloid leukemia (AML), multiple myeloma and epithelial cancers. Besides its widespread tumor expression, the appeal of CD44v6 as a CAR target stems from its non-redundant role in tumor progression and absent expression on hematopoietic stem cells (HSCs). Unfortunately, currently available immunocompromised mouse models are not suited for studying the antitumor efficacy of CAR T cells in the long term, because of the occurrence of xenograft-versus-host disease (X-GVHD) by 3-4 weeks. For the same reasons, evaluating the kinetics of the potential hematological toxicities due to off-tumor antigen recognition becomes a challenge. To develop an innovative xenotolerant mouse model to validate the antitumor efficacy and the safety of CD44v6 CAR-T cells in the long term. NSG mice transgenic for human IL-3, GM-CSF and SCF(NSG-3GS) were injected intra-liver with human HSCs soon after birth, enabling an accelerated and better balanced lympho-hematopoietic reconstitution, compared with NSG mice. Reconstituting human T cells were single CD4 or CD8 T cells, representing all memory (TNA, TSCM, TCM, TEM and TEMRA) populations. After ex vivo isolation, they could be transduced with the CD44v6 CAR using CD3/CD28-beads IL-7/IL-15 stimulation, retaining an early-differentiated (TSCM/TCM) phenotype and full antitumor functionality against AML. Importantly, these T cells did not respond in vitro to NSG-derived dendritic cells (DCs), but did so against genetically unrelated mouse DCs or human allogeneic DCs and, when transferred into irradiated secondary recipients, persisted at low levels up to 24 weeks, without causing X-GVHD. Accordingly, AML-bearing recipients infused with xenotolerant CD44v6 CAR-T cells were cured in the long-term (P<0.001 vs CD19 CAR-T cells), without the emergence of antigen-loss variants. Moreover, when transferred into mice previously reconstituted with human HSCs, these T cells caused cytokine-release storms characterized by fever and elevated IL-6 levels, but not myeloablation. Hematological toxicity was indeed limited to selective and prolonged elimination of CD44v6-expressing monocytes. Co-expressing a suicide gene (TK or inducible caspase-9) enabled fast and efficient CD44v6 CAR-T cell ablation in vivo, potentially rescuing any toxicity. CD44v6 CAR-T cells induce durable AML remission, without myeloblation in an innovative xenotolerant mouse model enabling long-term evaluations. This model could be a valuable tool for the preclinical validation of other CAR-based strategies against hematological tumors.