Sakemura, Reona

Abstract BACKGROUND Efficacy of chimeric antigen receptor T cell (CART) therapy remains limited in solid tumors. Given the heterogeneity of surface receptor expression and immunosuppressive stromal microenvironment, strategies to target tumor neovasculature and tumor stromal cells are needed to help overcome CART inhibition by GBM. Eph receptors are the largest family of receptor tyrosine kinases and are integral to cell adhesion, migration, and axon guidance, during development and homeostasis. EphA3 is a receptor tyrosine kinase which is lowly expressed in adult tissues but is highly expressed in tumor neovasculature and tumor stromal cells in GBM and other solid tumors. EphA3 is over-expressed in up to 40% of GBM samples. We aimed to develop CART cells directed against EphA3 to use in targeting tumor neovasculature and tumor stromal cells in GBM. METHODS we developed a second generation CD28 co-stimulated CAR construct in a third generation lentivirus backbone to generate EphA3 CART cells using the single chain variable fragment of ifabotuzumab, a monoclonal antibody directed against EphA3. Patient derived GBM xenograft cell lines were used in these experiments. RESULTS EphA3 directed CART cells exhibited specific and potent antitumor activity against EphA3+ GBM cell lines with variable transcriptome EphA3 expression indicating its broader applicability in patients with GBM. Killing over 24-hour incubation was significant at low effector: target ratio: 52.5% killing at 1.25:1 against cell lines with 25.05% EphA3 expression and 37.1% killing at 1.25:1and 90% killing at 5:1 ratio against a cell line with 19.28% expression. Conversely, when co-cultured with UTD controls there was significantly lower killing, or growth of tumor cells. CONCLUSION We demonstrate for the first time that targeting EphA3 with CART cells is feasible, efficacious and represents a novel therapeutics strategy to target GBM. Data from in vivo and combinatorial CART experiments will be reported at the meeting.

Abstract Despite its efficacy, chimeric antigen receptor T-cell therapy (CART) is limited by the development of cytokine release syndrome (CRS) and neurotoxicity (NT). While CRS is related to extreme elevation of cytokines and massive T cell expansion, the exact mechanisms for NT have not yet been elucidated. Preliminary studies suggest that NT might be mediated by myeloid cells that cross the blood brain barrier. This is supported by correlative analysis from CART19 pivotal trials where CD14+ cell numbers were increased in the cerebrospinal fluid of patients that developed severe NT (Locke et al, ASH 2017). Therefore, we aimed to investigate the role of GM-CSF neutralization in preventing CRS and NT after CART cell therapy via monocyte control. First, we investigated the effect of GM-CSF blockade on CART cell effector functions. Here, we used the human GM-CSF neutralizing antibody (lenzilumab, Humanigen, Burlingame, California) that has been shown to be safe in phase II clinical trials. Lenzilumab (10 ug/kg) neutralizes GM-CSF when CART19 cells are stimulated with the CD19+ Luciferase+ acute lymphoblastic leukemia (ALL) cell line NALM6, but does not impair CART cell function in vitro. We have found that malignancy associated macrophages reduce CART proliferation. GM-CSF neutralization with lenzilumab results in enhanced CART cell antigen specific proliferation in the presence of monocytes. To confirm this in vivo, NOD-SCID-g-/- mice were engrafted with high disease burdens of NALM6 and treated with low doses of CART19 or control T cells (to induce tumor relapse), in combination with lenzilumab or isotype control antibody. The combination of CART19 and lenzilumab resulted in significant anti-tumor activity and overall survival benefit compared to control T cells (Fig 1A), similar to mice treated with CART19 combined with isotype control antibody, indicating that GM-CSF neutralization does not impair CART cell activity in vivo. This anti-tumor activity was validated in an ALL patient derived xenograft model. Next, we explored the impact of GM-CSF neutralization on CART cell related toxicities in a novel patient derived xenograft model. Here, NOD-SCID-g-/- mice were engrafted with leukemic blasts (1-3x106 cells) derived from patients with high risk ALL. Mice were then treated with high doses of CART19 cells (2-5x106 intravenously). Five days after CART19 treatment, mice began to develop progressive motor weakness, hunched bodies, and weight loss that correlated with massive elevation of circulating human cytokine levels. Magnetic Resonance Imaging (MRI) of the brain during this syndrome showed diffuse enhancement and edema, associated with central nervous system (CNS) infiltration of CART cells and murine activated myeloid cells. This is similar to what has been reported in CART19 clinical trials in patients with severe NT. The combination of CART19, lenzilumab (to neutralize human GM-CSF) and murine GM-CSF blocking antibody (to neutralize mouse GM-CSF) resulted in prevention of weight loss (Fig 1B), decrease in critical myeloid cytokines (Fig 1C-D), reduction of cerebral edema (Fig 1E), enhanced leukemic disease control in the brain (Fig 1F), and reduction in brain macrophages (Fig 1G). Finally, we hypothesized that disrupting GM-CSF through CRISPR/Cas9 gene editing during the process of CART cell manufacturing would result in functional CART cells with reduced secretion of GM-CSF. We designed guide RNA targeting exon 3 of the GM-CSF gene and generated GM-CSFk/o CART19 cells. Our preliminary data suggest that these CARTs produce significantly less GM-CSF upon activation but continue to exhibit similar production of other cytokines and exhibit normal effector functions in vitro (Fig 1H). Using the NALM6 high tumor burden relapse xenograft model as described above, GM-CSFk/o CART19 cells resulted in slightly enhanced disease control compared to CART19 cells (Fig 1I). Thus, modulating myeloid cell behavior through GM-CSF blockade can help control CART mediated toxicities and may reduce their immunosuppressive features to improve leukemic control. These studies illuminate a novel approach to abrogate NT and CRS through GM-CSF neutralization that also potentially enhances CART cell functions. Based on these results, we have designed a phase II clinical trial using lenzilumab as a modality to prevent CART related toxicities in patients with diffuse large B cell lymphoma. Disclosures Ahmed: Humanigen: Employment. Sahmoud:Humanigen: Employment. Durrant:Humanigen: Employment. Russell:Vyriad: Equity Ownership. Kay:Morpho-sys: 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; 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; 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; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding. Kenderian:Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding.

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 BACKGROUND Respiratory failure syndromes are common after allogeneic hematopoietic cell transplantation (allo-HCT), and are associated with substantial morbidity and mortality. These syndromes are incompletely understood, and may be associated with histopathologic evidence of acute lung injury (ALI). The role of biopsy in this setting is controversial, with uncertain benefits and considerable procedural risk. We report the clinical, radiologic and histopathologic features of patients with biopsy-proven ALI following allo-HCT in a large multi-site retrospective series. METHODS The medical records of 1,864 consecutive adults who underwent allo-HCT at Mayo Clinic, Minnesota (n = 916), Arizona (n = 694), and Florida (n = 254) (Jan 2005-Dec 2017) were reviewed. A total of 152 patients underwent a lung biopsy: 126 bronchoscopic, 22 surgical, and 4 CT-guided biopsies (Figure 1). Patients with a histopathologic diagnosis of ALI were included in the study, and were classified into clinicopathologic subgroups per published American Thoracic Society guidelines (Am J Resp Crit Care Med, 2011). Patients whose diagnosis remained uncertain after a detailed review of the medical records were included in a separate subgroup as undifferentiated ALI. Transplant characteristics, radiologic features, pulmonary function test (PFT) results, treatments, and outcomes were compared between groups. RESULTS Clinicopathologic diagnoses in 62 patients with biopsy-proven ALI consisted of an infectious etiology in 15 (24%), cryptogenic organizing pneumonia (COP) in 15 (24%), idiopathic pneumonia syndrome (IPS) in 9 (15%), bronchiolitis obliterans syndrome (BOS) in 9 (15%), diffuse alveolar hemorrhage in 3 (5%), periengraftment respiratory distress syndrome in 2 (3%), and undifferentiated ALI in 9 (15%) patients (Figure 2). Transplant characteristics were comparable between groups. Infectious pneumonia and IPS were the most common diagnoses during the first 100 days post HCT (50% and 36%, respectively). Between 100 and 365 days, a range of diagnoses were seen including infectious pneumonia (20%), COP (23%), IPS (14%), BOS (23%), and undifferentiated ALI (20%) (Table 1). In 14/15 (93%) infectious pneumonia cases, biopsy did not change management and diagnosis was concurrently established by bronchoalveolar lavage (BAL) or non-invasive testing. Patients with IPS, BOS, and undifferentiated ALI more commonly had a pleural effusion (66%, 44%, and 44%, respectively). Nodular infiltrates were most commonly seen in patients with infectious pneumonia (55%). Other radiographic findings were not clearly discriminative. BOS and infectious pneumonia were more commonly associated with an obstructive PFT (78% and 33%, respectively), whereas restrictive pattern was more frequently seen in COP and IPS (50% and 57%, respectively) (Table 2). Despite a histopathologic diagnosis of ALI, 9 (15%) patients had clear clinical and radiographic evidence of BOS. Steroids were used commonly in noninfectious syndromes: 93% of patients with COP and in 100% of IPS patients. Of 4 patients with IPS who received Etanercept, 2 (50%) showed complete recovery, whereas the remaining 5 patients who did not receive Etanercept died from fulminant respiratory failure. Most common treatment response was complete recovery in COP (73%), fulminant respiratory failure leading to death in IPS (78%), and chronic respiratory failure in BOS (78%). Estimated OS was shortest in the group with IPS and undifferentiated ALI (median 5 and 17 mons, respectively), whereas patients with COP had the best prognosis (mOS not reached) followed by the infectious pneumonia and the BOS groups (mOS of 59 and 55 mons, respectively). Pulmonary complications were the most common cause of death in patients with non-infectious ALI. Lung biopsy was associated with post-procedural pneumothorax in 17 (27%) patients; 12 of whom required a chest tube. CONCLUSIONS In this largest case series to date of histopathologic ALI after allo-HCT, biopsy does not appear to meaningfully change management in the majority of patients, and is associated with substantial procedural risk. Histopathologic findings do not clearly correlate with the clinical and radiographic picture in a sizeable proportion of the patients. PFT results, BAL findings, the timing of the onset of respiratory failure after HCT, and the response to steroids seem to be of greater diagnostic and/or prognostic value. Disclosures No relevant conflicts of interest to declare.

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.

Background:The expression of the receptor tyrosine kinase (RTK) Axl on cancer cells has been associated with poor prognosis and implicated in disease progression. Inhibition of Axl‐RTK with TP‐0903, a high affinity Axl‐RTK inhibitor induces robust apoptosis of several tumors, including chronic lymphocytic leukemia (CLL). Axl‐RTK inhibition has also been found to induce nonspecific immunomodulatory effects in syngeneic tumor models.Aims:We aimed to investigate the impact and downstream effects of Axl‐RTK inhibition on naïve T cells and chimeric antigen receptor T cells (CART), using CD19 directed CART cells (CART19) as a model.Methods:We utilized either PMA/Ionomycin or the CD19+ mantle cell lymphoma cell line, JeKo as a stimulant for naïve T cells and CART19, respectively. Cells were analyzed for cytokine profile, T cell phenotype, and kinetics after the stimulation in the presence or the absence of the Axl‐RTK inhibitor TP‐0903. Regarding in vivo experiment, a relapse JeKo xenograft model was used. Mice were treated with vehicle alone, TP‐0903 alone, CART19 alone, or TP‐0903 + CART19. Three weeks after the treatment, mice were re‐challenged with JeKo as a CART19 relapse model.Results:Stimulation of naïve T cells in the presence of the Axl‐RTK inhibitor TP‐0903 resulted in a significant reduction of Th2 cytokines and downregulation of inhibitory receptors. The differential expansion of either effector T cells or Tregs in the presence of TP‐0903 resulted in a preferential reduction of Tregs (Fig 1A). Axl‐RTK inhibition of CART19 led to Th1 polarization upon stimulation with the JeKo, as evident by the selective reduction of Th2 cytokine. Additionally, this resulted in a reduction of myeloid attracting cytokines, such as CXCL1, IL‐8, IL‐6, and IL‐10, cytokines implicated in the development cytokine release syndrome and neurotoxicity after CART cell therapy and in adaptive resistance to immunotherapy (Fig 1B). TP‐0903 also significantly downregulated inhibitory receptors on activated CARTs (Fig 1C). In our relapsed in vivo model, mice treated with CART19 + TP‐0903 rejected the JeKo challenge while mice received CART19 alone redeveloped disease, suggesting that Axl‐RTK inhibition enhanced CART cell persistence. Peripheral blood analysis of mice after the rechallenge showed a higher number of circulating T cells and lower level of MCP‐1. Western blotting was performed to investigate the downstream effect of Axl‐RTK inhibition on CART19 and it revealed a relatively selective inhibition of LCK on stimulated CART19. Transcriptome analysis of activated CARTs treated with TP‐0903 revealed >100 genes that were differentially expressed compared to untreated cells. Among these, Th cell and Treg polarization genes were significantly altered in activated CARTs treated with TP‐0903.imageFinally, we validated our findings in phase I clinical trial of TP‐0903 (NCT02729298). T cells isolated from 3 cancer patients showed a significant reduction of Tregs and Th1 polarization. This will be further investigated in a planned phase I clinical trial of TP‐0903 in relapsed/refractory CLL (NCT03572634).Summary/Conclusion:We showed for the first time that Axl‐RTK inhibition polarizes T cells into a Th1 functional phenotype, downregulates inhibitory receptors, and reduces myeloid associated cytokine production by CART19. The combination therapy yielded synergistic cytotoxicity against JeKo in vitro and in a CART19 relapse model. These findings encourage further study of Axl‐RTK inhibition with TP‐0903 as an enhancer of T cell immunotherapies.

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.