Citations

Abstract Despite the remarkable outcomes and recent FDA approval of CD19 directed chimeric antigen receptor T (CART19) cell therapy in B cell malignancies, the durable responses in diffuse large B cell lymphoma are less than 40% and CART activity in chronic lymphocytic leukemia (CLL) is further limited. This is thought to be related to loss of CART persistence, poor trafficking to lymph nodes and inhibition by the leukemic microenvironment. Therefore, strategies to enhance CART cell function to overcome these limitations are needed. Recent studies have shown that abnormal expression of the receptor tyrosine kinase (RTK) AXL is associated with poor prognosis in human cancers. AXL signaling is associated with tumor proliferation, survival, metastasis, and drug resistance. Inhibition of AXL RTK with TP-0903, a high affinity AXL inhibitor has been found to induce robust apoptosis of CLL B cells. Based on the significant modulation of T cell functions observed with BTK inhibitor, we examined the role of AXL RTK inhibition with TP-0903 on T cell function in CLL and other B cell malignancies. First, we investigated the effect of AXL inhibition on T cell phenotype in normal donors. When naïve T cells were stimulated with PMA/Ionomycin and cultured with low dose TP0903, cytokine production was favorably altered through the promotion of Th1 and reduction of Th2 cytokines. This was associated with a significant reduction of inhibitory receptors (Fig 1a). Western blot of T cell lysates suggests low dose TP-0903 results in inhibition of LCK. When effector T cells and regulatory T cells (Treg) were treated with TP-0903 for 3 days, there was a preferential reduction of Treg (Fig 1b). Next, we investigated the influence of TP-0903 on CART19 cell phenotype and functions. Here, we used 41BB costimulated, lentiviral-transduced CART cells. Similar to our findings on naïve T cells, TP-0903 treatment led to polarization of CART cells into a Th1 phenotype when T cells were stimulated with the CD19+ mantle cell lymphoma (MCL) cell line JeKo or with leukemic B cells isolated from CLL patients (Fig 1c). TP-0903 treatment also significantly downregulated inhibitory receptors on activated CART cells, including a reduction of canonical cytokines known to be associated with the development of cytokine release syndrome (CRS) (Fig 1c). The combination of CART19 cells and TP-0903 yielded a synergistic antitumor activity against JeKo in vitro, at low E:T ratios (Fig 1d). Western blot of T cell lysates revealed phosphorylation of LCK was remarkably reduced in the presence of TP-0903, suggesting a mechanism for the observed Th1 polarization. We compared the transcriptome of activated CART cells treated with TP-0903 and more than 100 genes were differentially expressed compared to non-treated cells. Among these genes, immune synapse related genes such as cell junction and cell migration related genes were significantly increased in activated CART cells treated with TP-0903. To investigate the effect of AXL RTK inhibition of CART cells with TP-0903 in vivo, we established MCL xenografts through the injection of 1.0x106 of JeKo into NSG mice. A week after the injection of JeKo, mice were treated with either vehicle alone, TP-0903 (20mg/kg/day) alone, 0.5x106 of CART19 alone, or TP-0903 (20mg/kg/day)+0.5x106 of CART19. Three weeks after the treatment, mice were rechallenged with 1.0x106 of JeKo. Mice treated with CART19 and TP-0903 rejected the JeKo tumor challenge while mice previously treated with CART19 alone redeveloped JeKo, suggesting that AXL inhibition enhanced CART cell persistence (Fig 1e). Finally, we validated our preclinical findings in a correlative analyses of Phase I clinical trial of TP-0903 for patients with solid tumors (NCT02729298). Blood T cells from 3 patients were isolated and analyzed before and a week after treatment with TP-0903. Similar to our findings, there was a significant reduction in Tregs, reduction of inhibitory receptors and polarization to a Th1 phenotype. These findings will be further investigated in a planned Phase I clinical trial of TP-0903 in relapsed/refractory CLL (NCT03572634). In summary, we demonstrated for the first time that AXL inhibitior is capable of polarizing T cells into a Th1 phenotype, downregulates inhibitory receptors, reduces CRS associated cytokines and synergizes with CART cells in B cell malignancies. These findings encourage further study of TP-0903 as an enhancer of T cell immunotherapies. Disclosures Mouritsen: Tolero Pharmaceuticals: Employment. Foulks:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment. Parikh:Janssen: Research Funding; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; MorphoSys: Research Funding; Pharmacyclics: Honoraria, Research Funding; Gilead: Honoraria. Ding:Merck: Research Funding. Kay:Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees. Kenderian:Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding; Novartis: Patents & Royalties.

Abstract Introduction: Unprecedented clinical outcomes were reported after CD19 chimeric antigen receptor T cell (CART19) therapy and led to their FDA approval in diffuse large B cell lymphoma and in acute lymphoblastic leukemia. However, the complete response rate in chronic lymphocytic leukemia (CLL) after CART19 therapy is much lower, at approximately 20-30%, and the mechanism(s) for this relative lack of success is unclear. The dominant known mechanism(s) that prevent successful CART cell therapy in CLL have been limited to CART expansion and poor persistence. However, potential mechanisms are not limited to the CLL T-cell. Several immune defects have been identified in CLL that result from the complex bi-directional interaction between B-CLL cells and their microenvironment. In CLL the leukemic microenvironment is rich with extracellular vesicles (EVs) secreted by B-CLL cells. There is growing evidence that these vesicles play an important role in intracellular communication by the delivery of growth factors, genetic material and microenvironmentally relevant molecules. Therefore, we aimed to investigate the role and interactions of EVs in the diminished or absent CART response seen in some CLL patients. Methods: EVs were isolated from peripheral blood of 16 patients with untreated CLL at different Rai stages (8 patients had early and 8 had advanced stage disease) and risk profile by FISH (8 patients had low risk and 8 patients had high risk disease, based on the presence of 17p deletion). Cytometry was used to determine size, number of particles per µl, Annexin V and CD19 expression. These variables were correlated to the Rai stage and risk category of the disease. To investigate the impact of EVs on CART cell functions, CART19 cells were stimulated with either CLL EVs alone or in combination with the CD19 positive cell line JeKo1. After coincubation different effector functions were analysed. Results: Two patterns of EVs in CLL patients were identified; a single versus two distinct EV size populations (small [EVssmall]; 50-240nm, median=110nm) and large [EVslarge]; 180-560nm, median = 360nm Fig 1.A). In 25% of patients, EVs were CD19 positive (EVCD19+). CD19 positivity was detected only in patients with the EVslarge (Fig 1.B). The EVs concentration, CD19 expression (EVsCD19+ vs EVsCD19-), or the size (EVssmall vs EVslarge) did not correlate with disease stage (early vs advanced Rai stage) or risk profile of CLL (low vs high risk) although some variation could be seen (Fig 1.C). To investigate our hypothesis that EVs could modulate CART19 function, CART19 cell effector functions were examined in the presence of EVsCD19+, EVsCD19-, EVssmall, or EVslarge. EVs, 1.5x10e5 particles, alone were insufficient to stimulate CART19 cells. However when CART19 cells were stimulated with the CD19 positive cell line JeKo1, their effector functions were reduced only in the presence of EVsCD19+, 50,000 particles, 2.5 x 10e3/ µl, but not EVsCD19- at the same concentration. This included a significant reduction in CART specific killing (Fig 1.D) and a reduction in cytokine production. The impairment of CART cell functions was independent of the size of EVs, i.e. there was no impairment of CART functions with large or small size EVCD19- in co-culture. Summary: We identify CD19 positive large size EVs from patients with CLL and demonstrate that these EVs play a role in the leukemic microenvironment by reducing CART cell activity. Studies are ongoing to define the mechanism(s). Disclosures Parikh: Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Gilead: Honoraria; MorphoSys: Research Funding; Abbvie: Honoraria, Research Funding. Kay:Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees. Kenderian:Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding; Novartis: Patents & Royalties.