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Despite the remarkable outcomes 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 strategies to enhance this response are desperately needed. Inhibition of AXL RTK with TP0903, a high-affinity AXL inhibitor has been found to induce robust apoptosis of malignant B cells. Here, we aimed to examine the role of AXL RTK inhibition with TP0903 on T-cell function in B-cell malignancies. First, we investigated the influence of TP0903 on CART19 cell phenotype and functions. Here, we used 41BB costimulated, lentiviral-transduced CART cells. AXL inhibition 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 patients with chronic lymphocytic leukemia (CLL), in the presence of TP0903 (Fig 1A). Exposure of activated CART cells to TP0903 also resulted in significant downregulation of inhibitory receptors on activated CART cells (Fig 1B), a reduction of conical cytokines known to be associated with the development of cytokine release syndrome (CRS) (Fig 1C). To investigate the effect of AXL RTK inhibition of CART cells with TP0903 in vivo, we established MCL xenografts through the injection of 1.0x106 of Jeko into NSG mice. A week later, mice were treated with either vehicle alone, TP0903 (20 mg/kg/day) alone, 0.5x106 of CART19 alone, or TP0903 (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 TP0903 rejected the tumor challenge while mice previously treated with CART19 alone redeveloped MCL, suggesting that AXL inhibition enhanced CART cell persistence (Fig 1D). Finally, we validated our preclinical findings in correlative analyses of phase 1 clinical trial of TP0903 in patients with solid tumors (NCT02729298). 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 1 clinical trial of TP0903 in relapsed/refractory CLL (NCT03572634) In summary, we demonstrated for the first time that AXL inhibition polarizes T cells into a Th1 phenotype, downregulates inhibitory receptors, and synergizes with CART cells in B-cell malignancies. These findings encourage further study of TP0903 as an enhancer of T-cell immunotherapies.

Background: Unprecedented clinical outcomes were reported after CD19 chimeric antigen receptor T-cell (CART19) therapy. However, the complete response rate in chronic lymphocytic leukemia (CLL) is much lower, at approximately 20%–30%. Several immune defects have been identified in CLL that result from the complex interaction between CLL cells and the microenvironment. This leukemic microenvironment is rich with extracellular vesicles (EVs) secreted by B-CLL cells. Here, we aimed to investigate the role of EVs play in the diminished CART response seen in some CLL patients. Methods: EVs were isolated from peripheral blood of 16 patients with untreated CLL at different Rai stages and risk profile by FISH. Cytometry was used to determine size, number of particles per μl, and CD19 expression. To investigate the impact of EVs on CAR T-cell functions, CART19 cells were stimulated with either CLL B cells or the CD19-positive cell line JEKO and different effector functions were analyzed. Results: Two patterns of EVs in CLL patients were identified; a single versus 2 distinct EV populations, characterized by size (small [EVssmall] and large [EVslarge], Fig 1A). In 25% of patients, EVs were CD19 positive (EVCD19+). CD19 positivity was detected only in patients with the EVslarge (Fig 1B). 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). 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 alone were insufficient to stimulate CART19 cells. However when CART19 cells were stimulated with the CD19-positive cell line JEKO, their effector functions were reduced only in the presence of EVsCD19+ but not EVsCD19-. This included a significant reduction in CART-specific killing (Fig 1C) and a reduction in cytokine production. The impairment of CART cell functions was independent of the size of EVs, ie, there was no impairment of CART functions with large or small size EVCD19- in co-culture. Conclusion: 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 CAR T-cell activity. Studies are ongoing to define the mechanism(s).

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.

AbstractBackgroundScreening for vancomycin‐resistant Enterococcus (VRE) is performed at many transplant centers, but data on the impact of VRE colonization and bloodstream infection (BSI) on hematopoietic cell transplantation (HCT) outcomes remain conflicting.MethodsConsecutive adults with acute myeloid leukemia who underwent allogeneic HCT between 2004 and 2014 were retrospectively reviewed. Patients were screened by perirectal PCR swabs targeting vanA and vanB twice weekly while inpatient.ResultsOf a total of 203 patients (median age 54 years), 73 (36%) were VRE colonized prior to HCT, 23 (11%) became colonized within the first 100 days, and 107 (53%) remained non‐colonized through day 100 post HCT. A landmark analysis on HCT day 0 revealed no significant difference in overall survival according to pre‐transplant colonization status (P=.20). However, patients with subsequent VRE colonization within the first 100 days of HCT had a significantly worse survival on both univariable (P=.04) and multivariable (P=.03) analyses. During the first 30 days post HCT, 11 (5% of total and 11% of the VRE colonized) patients developed VRE BSI. Ten (91%) of these had screened positive for VRE colonization before the bacteremia. Age ≥60 years, HCT‐comorbidity index ≥3, and VRE colonization were independent risk factors for VRE BSI on multivariable analysis (P=.04, .03, .003, respectively). Only 1 (9%) patient with VRE BSI died within the first 100 days post HCT.ConclusionVRE colonization is a surrogate marker and not an independent predictor of worse outcomes post HCT. VRE BSI is associated with increased morbidity, but does not impact post‐HCT survival.

BACKGROUNDABO incompatibility is not a contraindication to hematopoietic cell transplantation (HCT), but it has been associated with additional risks including delayed engraftment, pure red cell aplasia (PRCA), and higher transfusion needs. Data on these events and on patient survival after reduced‐intensity conditioning (RIC) HCT are limited.STUDY DESIGN AND METHODSA total of 127 consecutive patients, 86 with acute myeloid leukemia and 41 with myelodysplastic syndromes, who underwent HLA‐matched peripheral blood RIC allogenic HCT between 2005 and 2014 were retrospectively analyzed.RESULTSEighty ABO‐compatible, 26 major/bidirectional, and 21 minor‐ABO‐mismatch HCT were identified. Compared to the ABO‐compatible group, major/bidirectional mismatches had increased red blood cell (RBC) transfusion requirement during the first 100 days (p = 0.009), delayed RBC and PLT engraftment (p = 0.0011 and p = 0.005, respectively), and higher incidence of grade II to IV acute graft‐versus‐host disease (aGVHD; p = 0.037). In multivariable analysis, major/bidirectional mismatches had significantly higher non‐relapse mortality (NRM) and inferior disease‐free survival (DFS) and overall survival (OS) compared with ABO‐compatible patients (p = 0.01, p = 0.04, and p = 0.035, respectively). Minor ABO mismatch had no impact on survival (p = 0.99). Four (15%) of 26 major/bidirectional mismatches developed PRCA. There was a significant association between fludarabine plus busulfan conditioning and PRCA (p = 0.0046).CONCLUSIONMajor/bidirectional ABO mismatch is associated with higher NRM and shortened DFS and OS in the setting of RIC HCT. Increased transfusion need, delayed RBC and platelet engraftment, PRCA, and increased severity of aGVHD are additional complications contributing to the morbidity.