Background Blinatumomab is a bispecific T cell antibody construct, that binds and allows CD-3 positive cytotoxic T cells to recognize and eradicate CD19-positive acute lymphocytic leukemia (ALL) blasts. The drug is approved for patient (pts) with relapsed/ refractory (RR) B-cell ALL and those with minimal residual disease (MRD) based on efficacy in clinical trials. We sought to evaluate the safety and efficacy of blinatumomab in the "real world" setting. Methods We conducted a retrospective multicenter study in collaboration with 11 U.S. academic institutions, and evaluated the outcome of RR B-cell ALL pts, who received blinatumomab outside of clinical trials. These pts were evaluated for response, duration of response (DOR), overall survival (OS) from the time of blinatumomab initiation and toxicity. DOR was estimated among pts who had achieved complete remission (CR)/CR with incomplete count recovery (CRi). Survival curves were estimated using the Kaplan-Meier method and compared via log-rank test. Results From December 2014 to May 2019, 239 pts with B-cell ALL were identified. Baseline characteristics are summarized in Table 1. The median age of pts at blinatumomab initiation was 48 years (yrs) (range, 18-85). Sixty-one (26%) pts had Philadelphia chromosome [t(9;22)] positive (Ph+ve) disease. Sixty-one (26%) pts had ≥ 3 prior therapies and 46 (19%) pts had allogeneic stem cell transplantation (allo-HCT) prior to blinatumomab. Twenty-two (9%) pts received inotuzumab ozagamicin prior to blinatumomab. Twenty-nine (12%) pts received blinatumomab in combination with tyrosine kinase inhibitor (TKI). Twelve (5%) pts received blinatumomab for MRD. Response rate (CR/CRi) in pts with RR disease was 61%; 44% had CR with MRD negativity by flow cytometry. Rate of CR/CRi in Ph+ve B-cell ALL was 74.5%. Among 12 pts who received Blinatumomab for MRD, 9 (75%) pts achieved MRD negativity. Overall 113 (48%) pts were successfully bridged to allo-HCT. Median DOR in pts with RR disease was 32.1 months (95% CI; 9.5-not reached [NR]) (Fig. 1a) and when censored at allo-HCT was 14.8 months (95% CI; 5.2-NR) (Fig. 1b). Median OS in pts with RR disease after blinatumomab was 12.7 months (95% CI; 9.2 -17.9) (Fig. 1c) and when censored at allo-HCT was 8.6 months (95% CI; 6.4-11.6) (Fig.1d). Median OS after blinatumomab in RR B-cell ALL Ph+ve pts was NR (59% alive at 2 yr). Among pts who received blinatumomab for MRD, median DOR was NR and 54% sustained MRD negativity at 2 yrs. Median OS was 34.7 months (95% CI; 8.8-34.7) in group of pts who received blinatumomab for MRD. Seven (3%) pts discontinued blinatumomab due to adverse events and 40 (21%) pts had dose interruptions. Cytokine release syndrome (CRS) of any grade (G) was reported in higher number of pts (93 [39%]) compared to clinical trials, though G3-4 CRS appears to be similar (G1-2 [36%], G3-4 [3%]). Forty-four (44%) pts were treated with steroids and 8 (8%) pts required tocilizumab with steroids for CRS. Ninety-three percent of pts had complete resolution of CRS. CNS toxicities were observed in 31 (13%) pts, among them 60% (n= 19) had G3-4 toxicities. Hepatic toxicities were observed in 84 (35%) pts as outlined in Table 1. Six (2.5%) pts died due to blinatumomab induced toxicities. Conclusion: Our real-world data with multicenter collaboration suggest that overall blinatumomab was well tolerated and had led to significant response in pts with RR B-cell ALL. Disclosures Advani: Amgen: Research Funding; Abbvie: Research Funding; Pfizer: Honoraria, Research Funding; Macrogenics: Research Funding; Kite Pharmaceuticals: Consultancy; Glycomimetics: Consultancy, Research Funding. Aldoss:AUTO1: Consultancy; Helocyte: Consultancy, Honoraria, Other: travel/accommodation/expenses; Agios: Consultancy, Honoraria; Jazz Pharmaceuticals: Honoraria, Other: travel/accommodation/expenses, Speakers Bureau. Podoltsev:Agios Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Research Funding; Astellas Pharma: Research Funding; Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Research Funding; Sunesis Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Research Funding; Pfizer: Research Funding; Astex Pharmaceuticals: Research Funding; CTI Biopharma: Research Funding; Celgene: Other: Grant funding, Research Funding; Genentech: Research Funding; AI Therapeutics: Research Funding; Samus Therapeutics: Research Funding; Arog Pharmaceuticals: Research Funding; Kartos Therapeutics: Research Funding. Curran:Incyte: Research Funding; Merck: Research Funding; Gilead: Research Funding. Yang:Agios: Consultancy; AstraZeneca: Research Funding. Mattison:Pfizer: Membership on an entity's Board of Directors or advisory committees. Liedtke:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; IQVIA/Jazz: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech/Roche: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celator: Research Funding; Caelum: Membership on an entity's Board of Directors or advisory committees; BlueBirdBio: Research Funding; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees; Agios: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Atallah:Helsinn: Consultancy; Takeda: Consultancy, Research Funding; Jazz: Consultancy; Pfizer: Consultancy; Helsinn: Consultancy; Novartis: Consultancy; Jazz: Consultancy.
Improved Anti-Tumor Response of Chimeric Antigen Receptor T Cell (CART) Therapy after GM-CSF Inhibition Is Mechanistically Supported By a Novel Direct Interaction of GM-CSF with Activated Carts
Despite the remarkable activity of CD19 directed chimeric antigen receptor T cell (CART19) therapy in the treatment of B cell malignancies, the therapy is limited by the development of severe life-threatening complications such as neurotoxicity (NT) and cytokine release syndrome (CRS). Additionally, durable efficacy following CART19 therapy is not optimal. Emerging literature suggests that inhibitory myeloid cells and their cytokines play an important role in inducing CAR-T cell toxicities and also contribute to the inhibition of their effector functions. Specifically, GM-CSF was identified as a critical cytokine in the development of NT and CRS after CART19 therapy. Neutralization of GM-CSF in preclinical models has been shown to prevent CRS and enhance CART anti-tumor activity through modulation of myeloid cell behavior, resulting in reduction of tumor associated macrophages. In addition to the predominant effect of GM-CSF on myeloid cells, there appears to be a direct effect on CART19 cells. In this study, we aimed to evaluate the direct effect of GM-CSF on CART cells. Our initial finding of enhanced anti-tumor activity of CART19 cells after GM-CSF inhibition suggested a direct effect of GM-CSF on CART cells (Sterner et al. 2019, Blood). In these experiments, a guide RNA (gRNA) targeting exon 3 of GM-CSF in a CRISPR-Cas9 lentiviral vector was used to knock out GM-CSF during CART cell manufacturing. This resulted in a disruption efficiency of approximately 70% of the GM-CSF gene. Using a high tumor burden xenograft model for relapsed acute lymphoblastic leukemia established through the engraftment of the CD19+ luciferase+ NALM6 cell line (1x106 cells intravenously) in immunocompromised NOD-SCID-γ-/- mice, treatment with low doses of GM-CSFk/o CART19 resulted in improved anti-tumor activity and overall survival compared to GM-CSFwt CART19. The lack of myeloid cells in this model pointed to an intrinsic effect of GM-CSF on CAR-T cells. To ensure that this was not related to an off-target effect of the gRNA, whole exome sequencing (WES) of the modified cells was performed. There was no difference in the single nucleotide variants or indel counts between GM-CSFk/o CART19 and GM-CSFwt CART19 (Figure 1A). WES was significant for only two alterations in the exon 3 targeted by the gRNA (Figure 1B). The high efficiency and accuracy of targeting exon 3 of GM-CSF indicated that the improvement in CART function is unlikely related to an off-target effect of the gRNA and suggested the possibility of a direct interaction between GM-CSF and CART cells as a potential mechanism behind the improved anti-tumor activity. To investigate this interaction, we first assessed the expression of GM-CSF receptors on CART cells. While resting CART cells do not express any GM-CSF receptors, our analysis robustly indicates that activated CART cells significantly upregulate both α and β subunits of the GM-CSF receptor. This finding was significant both when CART cells are activated through their T cell receptor with CD3/CD28 beads (Figure 1C) or through the CAR with irradiated NALM6 cells (Figure 1D). Additionally, activated GM-CSFk/o CART19 cells also upregulated GM-CSF receptors, indicating this upregulation is induced by T cell stimulation. These results suggest a direct interaction between GM-CSF and upregulated GM-CSFR on activated CART cells. Having demonstrated that 1) GM-CSF depletion enhances CART19 efficacy in xenograft models in the absence of monocytes and 2) T cell activation increases GM-CSF receptor expression, we sought to uncover the downstream changes resulting from this effect. Transcriptome interrogation of GM-CSFk/o CART19 revealed a distinct signature including a significant inhibition of the Fas death pathway, a known critical pathway in inducing CART cell apoptosis. This suggests a potential mechanism for enhanced CART19 activity following GM-CSF depletion (Figure 1E). In summary, our results strongly indicate that CART cells increase expression of GM-CSF receptor subunits when activated, resulting in modulation of CART cell functions. Furthermore, GM-CSFk/o CART19 revealed a distinct transcriptome signature compared to GM-CSFwt CART19. These results illuminate a novel mechanism for a direct modulatory effect of GM-CSF on activated CART cells. Disclosures Cox: Humanigen: Patents & Royalties. Sterner:Humanigen: Patents & Royalties. Sakemura:Humanigen: Patents & Royalties. Ahmed:Humanigen: Employment. Chappell:Humanigen: Employment. Durrant:Humanigen: Employment. Parikh:Acerta Pharma: Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Genentech: Honoraria; Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Ascentage Pharma: Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Kenderian:Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Lentigen: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Kite/Gilead: Research Funding; Morphosys: Research Funding.
Targeting of CD19 By Tafasitamab Does Not Impair CD19 Directed Chimeric Antigen Receptor T Cell Activity in Vitro
CD19 directed chimeric antigen receptor T cell (CART) therapy has shown remarkable activity in B cell lymphoma and acute lymphoblastic leukemia leading to the approval of two CART therapies. With the emergence of therapeutic anti-CD19 antibodies for the treatment of B cell malignancies, it remains to be elucidated whether such antibodies would interfere with the ability of CD19 targeting CARTs to exert their anti-tumor effect in a subsequent therapy. To address a part of this question, we investigated the potential for functional interference between the monoclonal anti-CD19 antibody tafasitamab (MOR208) and CD19 directed CART cells (CART19). CART19 cells were generated through lentiviral transduction of healthy donor T cells with a second generation CD19 CAR construct (FMC63-CD8h-CD8TM-41BBζ) which is similar to the construct used for the FDA-approved CART tisagenlecleucel. Tafasitamab, is an Fc-enhanced humanized monoclonal antibody which mediates antibody-dependent cellular toxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and direct cytotoxicity. It is currently being studied in phase 2 and 3 clinical trials in diffuse large B-cell lymphoma (DLBCL) in combination with the immunomodulatory agent lenalidomide (L-MIND) and the chemotherapeutic drug bendamustine (B-MIND). As a first step we confirmed the relevance of the tested CD19-positive target cell lines, JEKO (mantel cell lymphoma), Ly7 (DLBCL) and NALM-6 (ALL) based on functional activity of tafasitamab and CART19. In a 24 hours ADCC (tafasitamab titration plus natural killer (NK) cells; Figure 1A) and T cell cytotoxicity assays (CART19, E:T titrations; data not shown) distinct activity was observed for both therapies on all tested cell lines. Secondly, we studied whether the observed CART19 activity may be influenced by tafasitamab in case of a direct CD19 binding competition between tafasitamab and the CAR. To test for such binding competition we incubated the CD19+ cell lines NALM6 or JEKO with up to 100 µg/ml tafasitamab, to saturate the receptors. Subsequent flow cytometry analysis using the FMC63 antibody (carrying the same CD19 binding domain as contained in CART19) failed to detect CD19 expression, indicating a direct binding competition between FMC63 and tafasitamab (Figure 1B). Next, to investigate the potential impact of such binding competition on CART19 cell effector functions, we co-cultured tafasitamab CD19+ JEKO cell line at increasing concentrations of up to 100µg/ml, and then added CART19 cells at different effector to target ratios to the cell culture. The presence of tafasitamab, binding to the CD19 antigen, did not affect important CART cell effector functions such as antigen specific killing (Figure 1C), degranulation (Figure 1D), cytokine production or proliferation of CART19 (Figure 1E). In summary, our studies indicate that CART19 continue to exhibit potent antigen specific effector functions despite presence of tafasitamab and the related competition for CD19 binding. Besides the presented in vitro work the questions of therapeutic sequencing of tafasitamab and CART19 is being studied in xenograft models and will be presented at the meeting. Disclosures Sakemura: Humanigen: Patents & Royalties. Cox:Humanigen: Patents & Royalties. Schanzer:MorphoSys AG: Employment. Endell:MorphoSys AG: Employment, Patents & Royalties. Nowakowski:Selvita: Membership on an entity's Board of Directors or advisory committees; NanoString: Research Funding; MorphoSys: Consultancy, Research Funding; Genentech, Inc.: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Curis: Research Funding; Bayer: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Kenderian:Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding.
EXTH-32. DEVELOPMENT OF EPHA3 DIRECTED CHIMERIC ANTIGEN RECEPTOR T CELL THERAPY FOR THE TREATMENT OF GLIOBLASTOMA MULTIFORME
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.
GM-CSF Blockade during Chimeric Antigen Receptor T Cell Therapy Reduces Cytokine Release Syndrome and Neurotoxicity and May Enhance Their Effector Functions
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.
Axl-RTK Inhibition Modulates T Cell Functions and Synergizes with Chimeric Antigen Receptor T Cell Therapy in B Cell Malignancies
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.
Histopathologic Acute Lung Injury after Allogeneic Hematopoietic Cell Transplantation: Clinical Findings, Radiologic Features, Treatments and Outcomes
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.
Size Matters: Identification of Larger Size CD19 Positive Extracellular Vesicles in Chronic Lymphocytic Leukemia That Inhibit Chimeric Antigen Receptor T Cell Functions
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.
Relapses after Achieving EFS24 in Patients with Diffuse Large B-Cell Lymphoma in the Rituximab Era
Abstract Introduction: The addition of Rituximab to chemotherapy has significantly improved the outcome of patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL). Patients treated with immunochemotherapy for DLBCL who achieve EFS24 (event-free for 2 years after diagnosis) have an overall survival equivalent to that of the age- and sex-matched general population. Relapses after achieving EFS24 have been considered to be unusual but have been understudied. We sought to define the rate, clinical characteristics, treatment pattern, and outcomes of such relapses. Methods: 1448 patients with newly diagnosed DLBCL from March 2002 to June 2015 were included. Patients were enrolled in the Molecular Epidemiology Resource (MER) of the University of Iowa/Mayo Clinic Lymphoma SPORE, treated per physician choice (predominantly R-CHOP immunochemotherapy) and followed prospectively. An event was defined as progression or relapse, unplanned re-treatment after initial therapy, or death from any cause. Cumulative incidence of relapse and non-relapse mortality after achieving EFS24 were analyzed as competing events using Gray's test in the EZR software. Post-relapse survival was defined as time from relapse to death from any cause and analyzed using Kaplan-Meier method in SPSS (V22). Results: Among the 1448 patients, 1260 (87%) had DLBCL alone at diagnosis, and 188 (13%) had concurrent indolent lymphoma (follicular lymphoma 115, marginal zone lymphoma 18, chronic lymphocytic leukemia 14, lymphoplasmacytic lymphoma 4, unspecified 37) at diagnosis. After a median follow-up of 83.9 months, 896 patients achieved EFS24. For all 896 patients who achieved EFS24, the cumulative incidence of relapse (CIR) was 5.7%, 9.3% and 13.2%, respectively, at 2, 5 and 10 years after achieving EFS24. Patients with concurrent indolent lymphoma at diagnosis had a higher CIR compared to those with DLBCL alone at diagnosis (10.2 vs 4.8% at 2 years, 15.7 vs 8.0% at 5 years, 28.8 vs 9.7% at 10 years, P<0.001; Figure 1). There were a total of 84 patients who relapsed after achieving EFS24. The median age at initial diagnosis was 66 years (range 35-92), and 48 (57%) were male. At diagnosis, 11 (13%) had ECOG PS >1, 37 (50%) had LDH elevation, 62 (74%) were stage III-IV, 14 (17%) had more than 1 extranodal site, and 26 (31%) were poor risk by R-IPI score. There were 58 patients with DLBCL alone at diagnosis who relapsed after achieving EFS24, and 38 (75%) relapsed with DLBCL, 13 (25%) relapsed with indolent lymphoma (predominantly follicular lymphoma), and pathology was unknown in 7 patients. In contrast, there were 26 patients with concurrent indolent lymphoma at diagnosis who relapsed after achieving EFS24, and 9 (41%) relapsed with DLBCL, 13 (59%) relapsed with indolent lymphoma, and pathology was unknown in 4 patients. In the 47 patients who relapsed with DLBCL after achieving EFS24, 45% received intensive salvage chemotherapy, 19% received regular intensity chemotherapy, 9% received CNS directed chemotherapy, and 36% went on to receive autologous stem cell transplant (ASCT). In the 26 patients who relapsed with indolent lymphoma after achieving EFS24, 27% were initially observed, 54% received regular intensity chemotherapy, 4% received intensive salvage chemotherapy, and 19% received ASCT after subsequent progression. The median post-relapse survival (PRS) for all patients with a relapse after achieving EFS24 was 38.0 months (95% CI 27.5-48.5). The median PRS for patients who relapsed with DLBCL and indolent lymphoma after achieving EFS24 were 29.9 (19.9-39.9) and 89.9 (NR-NR) months, respectively (P=0.002; Figure 2). Conclusions: Relapses after achieving EFS24 in patients with DLBCL were uncommon in the rituximab era. Patient with DLBCL alone at diagnosis can relapse with either DLBCL or indolent lymphoma (3:1 ratio). Patients with concurrent DLBCL and indolent lymphoma at diagnosis had a significantly higher CIR, and relapses with DLBCL and indolent lymphoma were similar (2:3 ratio). Even with high intensity salvage chemotherapy and consolidative ASCT, patients who relapsed with DLBCL had a significantly worse survival compared to those who relapsed with indolent lymphoma. Late relapses with DLBCL remain clinically challenging, with a median survival of 2.5 years after relapse. Figure 1. Figure 1. Disclosures Maurer: Celgene: Research Funding; Nanostring: Research Funding; Morphosys: Research Funding. Witzig:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ansell:Takeda: Research Funding; Pfizer: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; Seattle Genetics: Research Funding; Celldex: Research Funding; LAM Therapeutics: Research Funding; Trillium: Research Funding; Merck & Co: Research Funding; Bristol-Myers Squibb: Research Funding. Cerhan:Celgene: Research Funding; Jannsen: Other: Scientific Advisory Board; Nanostring: Research Funding.
Fludarabine Busulfan Compared to Fludarabine Melphalan Is Associated with Increased Relapse Risk in Reduced Intensity Conditioning Transplant Despite Pharmacokinetic Dosing
Abstract Background: Fludarabine plus busulfan (FB) and fludarabine plus melphalan (FM) are two commonly used reduced intensity conditioning (RIC) regimens for allogeneic hematopoietic stem cell transplantation (SCT). A recent analysis showed that both regimens had similar overall survival (OS) but relapse incidence (RI) was significantly higher with FB with a trend towards higher non-relapse mortality (NRM) in FM (Baron et al., Cancer 2015). However, that cohort included patients treated with oral and intravenous busulfan without pharmakokinetic targeting of intravenous which may impact anti-leukemic activity. Aim: Compare transplant related outcomes of FB vs. FM using intravenous (IV) busulfan targeted to the area under the curve (AUC). Methods: After due IRB approval, patients with acute myelogenous leukemia (AML) and myelodysplastic syndromes (MDS) receiving RIC-SCT from 2008-2014 were identified. All baseline and laboratory features were retrospectively extracted. Busulfan dose in FB was 0.8 mg/kg IV for 10 doses with therapeutic AUC target of 900-1500 mcmol/L (min). All patients received T-cell replete grafts. Categorical and continuous variables were compared using Pearson's chi-squared and Wilcoxon/Kruskal-Wallis, respectively. Survival estimates were calculated using the Kaplan-Meier method and curves were compared using the log-rank test. Cumulative incidence was computed as competing events using Grey's model. Univariate and multivariate analyses were performed using Cox regression modeling. Results: A. Baseline characteristics and AUC monitoring: 134 pts were identified (47 FB and 87 FM). Median follow up of the entire cohort was 40 months (0-63.3) and at last follow up, 60% and 29% have died or relapsed, respectively. Baseline characteristics stratified according to conditioning regimen are shown in table 1. Younger age (60 yrs FB vs. 61 yrs FM, p = 0.015) and a trend towards a higher CMV R-/D- status (28% FB vs. 14% FM p = 0.064) were the only significant differences identified. All patients in the FB group received intravenous busulfan and 46/47 patients had evaluable AUC data with a range of 732-1354 mcmol/L (min). A total of 19 patients were outside of range, all <900 mcmol/L (min) and required a median dose increase of 28.1% (3.3-50.2%) B. Engraftment and toxicity data: The median time for platelet engraftment was 19 days (0-48) for FB vs. 16 days (14-183) for FM (p = 0.0023) whereas the median time to absolute neutrophil count (ANC) engraftment was 18 days (7-30) for FB and 15 days (11-40) for FM (p = 0.077). Cumulative incidence of grade II-IV, III-IV acute GVHD and chronic GVHD at 2-yr was 57.3%, 11% and 52.8% for FB and 48.6%, 17% and 63.4% for FM (p = 0.73, 0.4 and 0.21, respectively). Two patients in the FM group died of cardiac causes (heart failure and sudden cardiac arrest). No cases of sinusoidal obstructive syndrome were observed in either arm C. Transplant outcomes: A significantly higher 2-yr relapse incidence (RI) was associated with FB vs. FM at 35.6% vs 17.3%, respectively (p = 0.0058). 2-yr progression free survival (PFS) was also significantly lower in the FB vs. FM at 51.2% vs. 65.1%, respectively (p 0.031). However, 2-yr OS and NRM was similar for FB vs. FM (53.1% and 22.9% vs 63.9% and 21.9%, respectively p = 0.26 and 0.89). Necessitating a dose adjustment based on AUC did not increase the risk for relapse or affect NRM. In multivariate analysis, FB was associated with increased RI with hazard ratio (HR) 2.29 (1.07-4.88; p = 0.033). Other factors significantly associated with RI on multivariate analysis were secondary/therapy related disease with HR 2.84 (1.34-6.02; p = 0.0067) and CR1 vs. other with HR 0.39 (0.17-5.32; p = 0.019). The significance of the results remained unchanged after exclusion of MDS patients (data not shown) Conclusion: Despite AUC dose adjustment, FB compared to FM was associated with increased RI with a similar OS and NRM. AUC dose adjustment did not impact transplant outcomes and its routine use in RIC should be further evaluated. Given the wide use of FB as a conditioning regimen, these important observations should be prospectively studied in a randomized fashion. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Al-Kali: Novartis: Research Funding. Wolf:Janssen Scientific Affairs, LLC: Consultancy.