Overcoming CAR T Resistance with Non-Covalent BTK Inhibitor Loxo-305 in Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is a distinctive B-cell non-Hodgkin's lymphoma characterized by poor prognosis. Despite clinical success of the covalent Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, a subset of patients need to discontinue ibrutinib therapy due to treatment related adverse events, which are primarily caused by off-target effects. Furthermore, primary or acquired resistance to ibrutinib continues to emerge and often leads to dismal clinical outcomes. Therefore, exploration of more target-specific BTK inhibitors is crucial to minimize the adverse events and provide clinical benefit. CAR T therapy has achieved unprecedented response in patients with relapsed or refractory MCL. However, the development of resistant phenotypes is a new emerging medical challenge in MCL patients with unknown mechanisms. Here, we characterize the therapeutic efficacy of LOXO-305, a next generation non-covalent small molecule inhibitor with high selectivity for BTK. Preclinical efficacy of LOXO-305 alone or in combination with venetoclax (ABT199), a selective Bcl-2 inhibitor, was evaluated in MCL using in vitro and in vivo CAR T-resistant PDX models.

Methods:In vitro cell viability was measured after 72 hour treatment with LOXO-305 alone and in combination with ABT-199 in MCL cell lines using Cell Titer Glo luminescent cell viability assay (Promega). To determine whether LOXO-305 induces cell death through cell apoptosis, we used annexin V/PI staining followed by flow cytometry analysis. To evaulate in vivo drug efficacy we used patient-derived xenograft (PDX) models established from primary patient samples.

Results: LOXO-305 treatment, as a single agent, resulted in effective MCL cell growth inhibition in a panel of MCL cell lines including ibrutinib and/or ABT-199-resistant cell lines (IC₅₀=6.6-24.4μM), except for JeKo BTK KD cells with BTK knockdown (KD) via CRISPR/Cas9 technology (IC₅₀>30 μM). To improve the efficacy, we decided to investigate the potential of LOXO-305 in combination with ABT199, since the combo of ibrutinib and ABT199 is clinically beneficial in MCL patients. Indeed, LOXO-305 significantly improved the inhibitory effect of ABT-199 in the ABT-199 resistant Mino-R and JeKo BTK KD cells, suggesting that this combination could be further explored in overcoming ABT-199 resistance in MCL. The compelling synergistic effect was further confirmed by annexin V/PI apoptosis assay. Next, we assessed the in vivo efficacy of LOXO-305 in an ibrutinib-CAR T dual-resistant PDX model. LOXO-305 effectively reduced tumor size after 40 days of treatment as a single agent. Moreover, LOXO-305 treatment showed significant anti-tumor effects in an ibrutinib-ABT199-CAR T triple-resistant PDX model that recapitulates the most aggressive human MCL variants invivo. In this model, LOXO-305 treatment effectively decreased the tumor load in mice spleen and liver (p<0.05) as well as in bone marrow and peripheral blood, compared to vehicle-treated mice (p<0.001).

Conclusions: By using various in vitro and in vivo multiple resistant MCL models we determined that LOXO-305 holds great promise for an effective single agent or combined treatment of the most eggressive forms of MCL, and that a continued investigation of the rationale for a combined therapy with ABT-199 is imperative to understand its role in overcoming ibrutinib-ABT199-CAR T triple resistance.