Cox, Michelle J

Abstract The receptor tyrosine kinase AXL is a member of the TYRO3, AXL, and proto-oncogene tyrosine-protein kinase MER family and plays pleiotropic roles in cancer progression. AXL is expressed in immunosuppressive cells, which contributes to decreased efficacy of immunotherapy. Therefore, we hypothesized that AXL inhibition could serve as a strategy to overcome resistance to chimeric antigen receptor T (CAR T)–cell therapy. To test this, we determined the impact of AXL inhibition on CD19-targeted CAR T (CART19)–cell functions. Our results demonstrate that T cells and CAR T cells express high levels of AXL. Specifically, higher levels of AXL on activated Th2 CAR T cells and M2-polarized macrophages were observed. AXL inhibition with small molecules or via genetic disruption in T cells demonstrated selective inhibition of Th2 CAR T cells, reduction of Th2 cytokines, reversal of CAR T-cell inhibition, and promotion of CAR T-cell effector functions. AXL inhibition is a novel strategy to enhance CAR T-cell functions through two independent, but complementary, mechanisms: targeting Th2 cells and reversing myeloid-induced CAR T-cell inhibition through selective targeting of M2-polarized macrophages.

Abstract Pivotal clinical trials of B-cell maturation antigen-targeted chimeric antigen receptor T (CART)-cell therapy in patients with relapsed/refractory multiple myeloma (MM) resulted in remarkable initial responses, which led to a recent US Food and Drug Administration approval. Despite the success of this therapy, durable remissions continue to be low, and the predominant mechanism of resistance is loss of CART cells and inhibition by the tumor microenvironment (TME). MM is characterized by an immunosuppressive TME with an abundance of cancer-associated fibroblasts (CAFs). Using MM models, we studied the impact of CAFs on CART-cell efficacy and developed strategies to overcome CART-cell inhibition. We showed that CAFs inhibit CART-cell antitumor activity and promote MM progression. CAFs express molecules such as fibroblast activation protein and signaling lymphocyte activation molecule family-7, which are attractive immunotherapy targets. To overcome CAF-induced CART-cell inhibition, CART cells were generated targeting both MM cells and CAFs. This dual-targeting CART-cell strategy significantly improved the effector functions of CART cells. We show for the first time that dual targeting of both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART-cell therapy in MM.

Abstract Although chimeric antigen receptor T (CART)–cell therapy has been successful in treating certain hematologic malignancies, wider adoption of CART-cell therapy is limited because of minimal activity in solid tumors and development of life-threatening toxicities, including cytokine release syndrome (CRS). There is a lack of a robust, clinically relevant imaging platform to monitor in vivo expansion and trafficking to tumor sites. To address this, we utilized the sodium iodide symporter (NIS) as a platform to image and track CART cells. We engineered CD19-directed and B-cell maturation antigen (BCMA)–directed CART cells to express NIS (NIS+CART19 and NIS+BCMA-CART, respectively) and tested the sensitivity of 18F-TFB-PET to detect trafficking and expansion in systemic and localized tumor models and in a CART-cell toxicity model. NIS+CART19 and NIS+BCMA-CART cells were generated through dual transduction with two vectors and demonstrated exclusive 125I uptake in vitro. 18F-TFB-PET detected NIS+CART cells in vivo to a sensitivity level of 40,000 cells. 18F-TFB-PET confirmed NIS+BCMA-CART-cell trafficking to the tumor sites in localized and systemic tumor models. In a xenograft model for CART-cell toxicity, 18F-TFB-PET revealed significant systemic uptake, correlating with CART-cell in vivo expansion, cytokine production, and development of CRS-associated clinical symptoms. NIS provides a sensitive, clinically applicable platform for CART-cell imaging with PET scan. 18F-TFB-PET detected CART-cell trafficking to tumor sites and in vivo expansion, correlating with the development of clinical and laboratory markers of CRS. These studies demonstrate a noninvasive, clinically relevant method to assess CART-cell functions in vivo.

Abstract Chimeric antigen receptor T (CAR-T) cell therapy is a new pillar in cancer therapeutics; however, its application is limited by the associated toxicities. These include cytokine release syndrome (CRS) and neurotoxicity. Although the IL-6R antagonist tocilizumab is approved for treatment of CRS, there is no approved treatment of neurotoxicity associated with CD19-targeted CAR-T (CART19) cell therapy. Recent data suggest that monocytes and macrophages contribute to the development of CRS and neurotoxicity after CAR-T cell therapy. Therefore, we investigated neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential strategy to manage CART19 cell–associated toxicities. In this study, we show that GM-CSF neutralization with lenzilumab does not inhibit CART19 cell function in vitro or in vivo. Moreover, CART19 cell proliferation was enhanced and durable control of leukemic disease was maintained better in patient-derived xenografts after GM-CSF neutralization with lenzilumab. In a patient acute lymphoblastic leukemia xenograft model of CRS and neuroinflammation (NI), GM-CSF neutralization resulted in a reduction of myeloid and T cell infiltration in the central nervous system and a significant reduction in NI and prevention of CRS. Finally, we generated GM-CSF–deficient CART19 cells through CRISPR/Cas9 disruption of GM-CSF during CAR-T cell manufacturing. These GM-CSFk/o CAR-T cells maintained normal functions and had enhanced antitumor activity in vivo, as well as improved overall survival, compared with CART19 cells. Together, these studies illuminate a novel approach to abrogate NI and CRS through GM-CSF neutralization, which may potentially enhance CAR-T cell function. Phase 2 studies with lenzilumab in combination with CART19 cell therapy are planned.