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Increased number of MDSCs present in sarcoma tumor-bearing mice, but not in neuroblastoma tumor-bearing mice.

Genetically engineered T cells expressing CD19-specific chimeric antigen receptors (CAR) have shown impressive activity against B-cell malignancies, and preliminary results suggest that T cells expressing a first-generation disialoganglioside (GD2)-specific CAR can also provide clinical benefit in patients with neuroblastoma. We sought to assess the potential of GD2-CAR therapies to treat pediatric sarcomas. We observed that 18 of 18 (100%) of osteosarcomas, 2 of 15 (13%) of rhabdomyosarcomas, and 7 of 35 (20%) of Ewing sarcomas expressed GD2. T cells engineered to express a third-generation GD2-CAR incorporating the 14g2a-scFv with the CD28, OX40, and CD3ζ signaling domains (14g2a.CD28.OX40.ζ) mediated efficient and comparable lysis of both GD2+ sarcoma and neuroblastoma cell lines in vitro. However, in xenograft models, GD2-CAR T cells had no antitumor effect against GD2+ sarcoma, despite effectively controlling GD2+ neuroblastoma. We observed that pediatric sarcoma xenografts, but not neuroblastoma xenografts, induced large populations of monocytic and granulocytic murine myeloid-derived suppressor cells (MDSC) that inhibited human CAR T-cell responses in vitro. Treatment of sarcoma-bearing mice with all-trans retinoic acid (ATRA) largely eradicated monocytic MDSCs and diminished the suppressive capacity of granulocytic MDSCs. Combined therapy using GD2-CAR T cells plus ATRA significantly improved antitumor efficacy against sarcoma xenografts. We conclude that retinoids provide a clinically accessible class of agents capable of diminishing the suppressive effects of MDSCs, and that co-administration of retinoids may enhance the efficacy of CAR therapies targeting solid tumors. Cancer Immunol Res; 4(10); 869–80. ©2016 AACR.

GD2 expression on pediatric sarcomas

Fig S1: Site density cell lines with cytokine analyses of normal healthy donors and patient-derived CD22 CART. Fig S2: Development and evaluation of high-affinity CD22V1 CAR. Fig S3: PKCB2 effects of Bryostatin1 and CD22 modulation with PKC-inhibitors. Fig S4: RNA-seq analysis of Bryostatin1 effects on KOPN8 and SEM Fig S5: Exhaustion markers in Bryostatin1 and CD19 CAR with Bryostatin1 Fig S6: Bioluminescence for Bryostatin1 in vivo experiments Table S1: Enrichment analysis of RNA-seq Bryostatin1-treated cells specific to intrinsic components of the plasma membrane

Supplementary methods to paper

Purpose:Chimeric antigen receptor T-cell (CART) therapy targeting CD22 induces remission in 70% of patients with relapsed/refractory acute lymphoblastic leukemia (ALL). However, the majority of post-CD22 CART remissions are short and associated with reduction in CD22 expression. We evaluate the implications of low antigen density on the activity of CD22 CART and propose mechanisms to overcome antigen escape.Experimental Design:Using ALL cell lines with variable CD22 expression, we evaluate the cytokine profile, cytotoxicity, and in vivo CART functionality in the setting of low CD22 expression. We develop a high-affinity CD22 chimeric antigen receptor (CAR) as an approach to improve CAR sensitivity. We also assess Bryostatin1, a therapeutically relevant agent, to upregulate CD22 and improve CAR functionality.Results:We demonstrate that low CD22 expression negatively impacts in vitro and in vivo CD22 CART functionality and impairs in vivo CART persistence. Moreover, low antigen expression on leukemic cells increases naïve phenotype of persisting CART. Increasing CAR affinity does not improve response to low-antigen leukemia. Bryostatin1 upregulates CD22 on leukemia and lymphoma cell lines for 1 week following single-dose exposure, and improves CART functionality and in vivo persistence. While Bryostatin1 attenuates IFNγ production by CART, overall in vitro and in vivo CART cytotoxicity is not adversely affected. Finally, administration of Bryostain1 with CD22 CAR results in longer duration of in vivo response.Conclusions:We demonstrate that target antigen modulation is a promising strategy to improve CD22 CAR efficacy and remission durability in patients with leukemia and lymphoma.See related commentary by Guedan and Delgado, p. 5188

The prognosis of patients with large B-cell lymphoma (LBCL) that progresses after treatment with chimeric antigen receptor (CAR) T-cell therapy targeting CD19 (CAR19) is poor. We report on the first 3 consecutive patients with autologous CAR19-refractory LBCL who were treated with a single infusion of autologous 1 × 106 CAR+ T cells per kilogram targeting CD22 (CAR22) as part of a phase 1 dose-escalation study. CAR22 therapy was relatively well tolerated, without any observed nonhematologic adverse events higher than grade 2. After infusion, all 3 patients achieved complete remission, with all responses continuing at the time of last follow-up (mean, 7.8 months; range, 6-9.3). Circulating CAR22 cells demonstrated robust expansion (peak range, 85.4-350 cells per microliter), and persisted beyond 3 months in all patients with continued radiographic responses and corresponding decreases in circulating tumor DNA beyond 6 months after infusion. Further accrual at a higher dose level in this phase 1 dose-escalation study is ongoing and will explore the role of this therapy in patients in whom prior CAR T-cell therapies have failed. This trial is registered on clinicaltrials.gov as #NCT04088890.

Chimeric antigen receptor T cells (CAR-Ts) have demonstrated remarkable efficacy in leukemia and lymphomas but limited responses in solid tumors. We conducted a phase I trial (NCT02107963) of GD2 CAR-Ts (GD2-CAR.OX40.28.z.ICD9), demonstrating feasibility and safety of administering GD2 CAR-Ts in children and young adults with neuroblastoma and, for the first time, osteosarcoma. 15 patients aged 8-28 years were enrolled on four dose levels, of which 13 patients were infused. No dose-limiting toxicities were observed, and administration of up to 1x107 GD2-CAR-T/kg was feasible and safe for children and young adults with neuroblastoma and osteosarcoma. At Day 28 following GD2 CAR-T infusion, 23.1% (3/13) of evaluable patients had progressive disease and 76.9% (10/13) had stable disease (SD). 3/10 SD patients remained stable at 60 days post-infusion of GD2 CAR-T, but all patients eventually progressed. Since a major barrier to CAR-T efficacy is inadequate CAR-T expansion, we evaluated CAR-T levels and found that patients stratified into good and poor expander groups, observed across dose levels and associated with pro-inflammatory cytokine signatures in patients. To understand the immune cell contributors to CAR-T expansion, patient pre-treatment apheresis, CAR-T product, and post-infusion samples were evaluated by high-dimensional proteomic (CyTOF), transcriptomic (RNAseq), and epigenetic (ATACseq) analyses. In patient apheresis, good CAR-T expansion associated with more open chromatin and with both proteomic and transcriptomic enrichment of naïve T cells, while poor CAR-T expansion associated with increased levels of T effector memory (TEMRA) cells and enrichment of myeloid derived suppressor cell (MDSC) transcriptomic signatures. CAR-T products across patients, regardless of CAR-T expansion, demonstrated increased T cell activation proteomic signatures, with enhanced exhaustion transcriptomic signatures in poor expanders compared to good. The most robust cellular correlate to good CAR-T expansion was a population of CXCR3-expressing monocytes in pre-treatment apheresis. Interestingly, this CXCR3+ monocyte population reduced in post-infusion timepoints of good expanders, resembling levels found in poor expanders. Our findings were validated in TARGET-OS patient data in The Cancer Genome Atlas, where high CXCR3 expression was found to be associated with survival benefit in osteosarcoma patients. CXCR3 has been extensively studied on T cells, but its function on myeloid populations is yet to be fully explored. These results are the first to demonstrate that the peripheral immune environment prior to CAR-T administration may effectively predict and modulate CAR-T expansion in patients. Citation Format: Tara Murty, Sabina Kaczanowska, Ahmad Alimadadi, Cristina Contreras, Caroline Duault, Priyanka Balasubrahmanyan, Warren Reynolds, Norma Gutierrez, Reema Baskar, Catherine Wu, Franziska Michor, Jennifer Altreuter, Yang Liu, Aashna Jhaveri, Vandon Duong, Hima Anbunathan, Radim Moravec, Joyce Hong, Roshni Biswas, Stephen Van Nostrand, James Lindsay, Mina Pichavant, Elena Sotillo, Bita Sahaf, Sean Bendall, Holden Maecker, Steven Highfill, David Stroncek, Melinda Merchant, John Glod, Catherine Hedrick, Crystal Mackall, Sneha Ramakrishna, Rosandra Kaplan. Immune determinants of CAR-T expansion in solid tumor patients receiving GD2 CAR-T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2142.

Introduction: H3K27M-mutated diffuse intrinsic pontine glioma (DIPG) and spinal cord diffuse midline glioma (DMG) are universally lethal central nervous system (CNS) tumors in children and young adults. We previously demonstrated safety and activity of GD2.41BB.z chimeric antigen receptor T cells (CAR-Ts) at dose level 1, 1x106 GD2 CAR-T/kg (Majzner/Ramakrishna et al. Nature 2022) and reported results of dose level 2, 3x106 GD2 CAR-T/kg (Majzner et al. AACR 2022). Here, we present in depth high-dimensional analyses to define the immune states that contribute to CAR-T activity in patients. Methods: Thirteen patients (10 DIPG/3 spinal DMG; 4-30 years old; 7F/6M) were enrolled in this GD2 CAR-T phase 1 clinical trial (NCT04196413). GD2 CAR-Ts were administered to 12/13 enrolled patients. In the first cohort, CAR-Ts were administered initially intravenously (IV), followed by serial intracerebroventricular infusions (ICV; range 0-11 infusions/patient). Patient GD2 CAR-T product, peripheral blood, and cerebrospinal fluid (CSF) samples were evaluated for CAR-T expansion (qPCR; flow cytometry), cytokine signatures (Multiplex Luminex), and immune cell profiles (single cell RNA-sequencing). Data were analyzed in the context of clinical trajectory and patient response. Results: 10/12 infused subjects demonstrated clinical and/or radiographic benefit, with less systemic toxicity following ICV compared to IV infusion. CAR-T expansion was noted in the periphery and CSF of all treated patients and following serial ICV infusions. In peripheral blood, cytokine concentrations, including IFN-gamma, IL6, and CXCL9, were higher after IV compared to ICV CAR-T infusions, correlating with increased systemic inflammation. Conversely in CSF, cytokine concentrations, such as CCL2 and CXCL9, were higher following ICV compared to IV CAR-T infusions. Transcriptomic analysis was conducted on 576,199 single cells from 91 samples, including GD2 CAR-T products and patient CSF. This is the largest CAR-T dataset in CNS tumors. Patient CSF samples were dominated by T cell and myeloid populations. After IV CAR-T infusion, patient CSF exhibited an increased fraction of regulatory T cells and suppressive myeloid populations from baseline. These immune suppressive cells reduced after ICV infusion. Ongoing analyses are underway to explore the relation of these immune populations to patient response. Conclusions: H3K27M-mutated DIPG/DMG patients demonstrate continued clinical response with serial ICV GD2 CAR-T infusions, with heterogeneity in the durability of response across patients. In-depth correlative analyses profile distinct immune populations and demonstrate population shifts depending on route of administration and over the course of treatment. Key findings from these data will allow for iterative improvement in CAR-T therapies for H3K27M+ DIPG/DMG patients, providing hope to shift the paradigm of this fatal disease. Citation Format: Sneha Ramakrishna, Zinaida Good, Moksha Desai, Daniel Zamler, Rebecca Mancusi, Jasia Mahdi, Robbie Majzner, Liora Schultz, Rebecca Richards, Jennifer Kamens, Valentin Barsan, Cynthia Campen, Sonia Partap, Zachary Ehlinger, Warren Reynolds, Yiyun Chen, Mark P. Hamilton, Jennifer Moon, Christina Baggott, Michael Kunicki, Michelle Fujimoto, Amy Li, Sneha Jariwala, Sharon Mavroukakis, Emily Egeler, Ashley Jacobs, Courtney Erickson, Karen Yamabe-Kwong, Snehit Prabhu, Kara Davis, Steve Feldman, Bita Sahaf, Crystal L. Mackall, Michelle Monje. Immune signatures of GD2 CAR T cell activity in H3K27M+ diffuse midline glioma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 959.

Chimeric antigen receptor (CAR) T cell therapy is an effective salvage therapy for pediatric relapsed B-cell acute lymphoblastic leukemia (B-ALL), yet is challenged by high rates of post-CAR relapse. Literature describing specific relapse patterns and extramedullary (EM) sites of involvement in the post-CAR setting remains limited, and a clinical standard for post-CAR disease surveillance has yet to be established. We highlight the importance of integrating peripheral blood minimal residual disease (MRD) testing and radiologic imaging into surveillance strategies, to effectively characterize and capture post-CAR relapse. Main body: Here, we describe the case of a child with multiply relapsed B-ALL who relapsed in the post-CAR setting with gross non-contiguous medullary and EM disease. Interestingly, her relapse was identified first from peripheral blood flow cytometry MRD surveillance, in context of a negative bone marrow aspirate (MRD

BACKGROUND: H3K27M-mutated DMGs express high levels of the disialoganglioside GD2 and GD2-CAR T-cells (GD2-CART) regress DMG in preclinical models. METHODS: NCT04196413 is a 3 + 3 Phase I dose escalation trial testing GD2-CART in patients with biopsy-proved H3K27M DMG, with dose-limiting toxicities (DLT) considered independently for DIPG and spinal DMG (sDMG). Arm A tested escalating doses of IV GD2-CART (DL1=1e6 GD2-CART/kg; DL2=3e6 GD2-CART/kg) following lymphodepletion (LD). After the DLT period, patients with clinical and/or radiographic benefit were eligible for subsequent ICV GD2-CART infusions (10-30e6 GD2-CART) administered via Ommaya without LD. RESULTS: Twelve subjects were treated after standard radiotherapy, 7 of whom began treatment at the time of progression [n=4 DL1 (3 DIPG/1 sDMG); n=8 DL2 (6 DIPG/2 sDMG)]. No DLTs were observed on DL1. Three subjects experienced DLT on DL2 (2 DIPG/1 sDMG) due to grade-4 cytokine release syndrome (CRS). On both dose levels, all subjects exhibited transient symptoms related to on-tumor inflammation, termed Tumor Inflammation-Associated Neurotoxicity (TIAN); no DLT due to TIAN has occurred. Ten subjects experienced radiographic and/or clinical benefit after IV infusion and received subsequent ICV infusions (median=4 ICV infusions/pt, range=1-7). ICV infusions were not associated with high-grade CRS. Four patients continue to receive ICV infusions on study and have experienced continued clinical and radiographic benefit, currently 7-11 months following enrollment. Two patients (one sDMG, one DIPG) have experienced near-complete (>95%) tumor volume reduction. CONCLUSIONS: IV treatment of DIPG and sDMG with GD2-CART is safe at a dose of 1e6/kg, but associated with frequent high-grade CRS at 3e6/kg. ICV GD2-CART has been well tolerated and has mediated impressive sustained clinical benefit in some patients with DIPG/sDMG. Given these findings, we are launching a new arm to assess safety and activity and to define the recommended phase 2 dose for ICV delivery of GD2-CART without LD.

Purpose: Chimeric antigen receptor T-cell (CART) therapy targeting CD22 induces remission in 70% of patients with relapsed/refractory acute lymphoblastic leukemia (ALL). However, the majority of post-CD22 CART remissions are short and associated with reduction in CD22 expression. We evaluate the implications of low antigen density on the activity of CD22 CART and propose mechanisms to overcome antigen escape. Experimental Design: Using ALL cell lines with variable CD22 expression, we evaluate the cytokine profile, cytotoxicity, and in vivo CART functionality in the setting of low CD22 expression. We develop a high-affinity CD22 chimeric antigen receptor (CAR) as an approach to improve CAR sensitivity. We also assess Bryostatin1, a therapeutically relevant agent, to upregulate CD22 and improve CAR functionality. Results: We demonstrate that low CD22 expression negatively impacts in vitro and in vivo CD22 CART functionality and impairs in vivo CART persistence. Moreover, low antigen expression on leukemic cells increases naïve phenotype of persisting CART. Increasing CAR affinity does not improve response to low-antigen leukemia. Bryostatin1 upregulates CD22 on leukemia and lymphoma cell lines for 1 week following single-dose exposure, and improves CART functionality and in vivo persistence. While Bryostatin1 attenuates IFNγ production by CART, overall in vitro and in vivo CART cytotoxicity is not adversely affected. Finally, administration of Bryostain1 with CD22 CAR results in longer duration of in vivo response. Conclusions: We demonstrate that target antigen modulation is a promising strategy to improve CD22 CAR efficacy and remission durability in patients with leukemia and lymphoma. See related commentary by Guedan and Delgado, p. 5188

Background: H3K27M-mutated DMGs are universally lethal central nervous system tumors that express high levels of the disialoganglioside GD2. IV administered GD2-CAR T cells (GD2-CART) regress DMG in preclinical models, and locoregionally delivered CARs demonstrate enhanced activity in xenograft models of brain tumors. Methods: NCT04196413 is a 3+3 Phase I dose escalation trial testing GD2-CART in patients with H3K27M DMG, with dose-limiting toxicities (DLT) considered independently for DIPG and spinal DMG (sDMG). Arm A tested escalating doses of IV GD2-CART (DL1: 1e6 GD2-CART/kg; DL2=3e6 GD2-CART/kg) following lymphodepletion (LD). After the DLT period, patients with clinical and/or radiographic benefit were eligible for subsequent ICV GD2-CART (10-30e6 GD2-CART) administered via Ommaya catheter without LD every 4-8 weeks for a maximum of 12 doses. We previously reported early results from 4 patients treated on DL1, which demonstrated clinical activity and manageable toxicity. Here we provide updated results for DL1 and DL2. Results: Thirteen subjects were enrolled and 11 treated [n=4 DL1 (3 DIPG/1 sDMG); n=9 DL2 (7 DIPG/2 sDMG)]. Two subjects were removed prior to treatment due to rapid progression. No DLTs were observed on DL1. Three subjects experienced DLT on DL2 (2 DIPG/1 sDMG) due to grade 4 cytokine release syndrome (CRS), successfully managed with tocilizumab, anakinra, and corticosteroids. CRS occurred earlier on DL2 vs. DL1 (Day 3 vs 7). On both dose levels, all subjects exhibited transient symptoms related to on-tumor inflammation, termed Tumor Inflammation-Associated Neurotoxicity (TIAN), which was successfully managed with anakinra and, in some cases, CSF drainage and dexamethasone. No DLT due to TIAN has occurred. Ten patients have had adequate follow-up to assess benefit. Nine experienced radiographic and/or clinical benefit after IV infusion, and they received subsequent ICV GD2-CART infusions (median= 4 ICV infusions/pt, range 1-6). ICV infusions were not associated with high-grade CRS, although some subjects developed transient fever, headache, meningismus, nausea, and/or vomiting, and several subjects developed TIAN. Four patients continue to receive ICV infusions on study and have experienced continued clinical and radiographic benefit at 11+, 9.5+, 8+ and 7+ months following enrollment. A 31-year-old with sDMG has experienced a near-complete (>95%) reduction in tumor volume and a 17-year-old with DIPG experienced a near-complete (>98%) reduction in volume of a pontine tumor. Conclusions: IV treatment of DIPG and sDMG with GD2-CART is safe at a dose of 1e6/kg, but associated with unacceptable rates of high-grade CRS at 3e6/kg. ICV GD2-CART without LD, administered following a previous course of IV GD2-CART with LD, has been well tolerated and has mediated impressive sustained clinical benefit in some patients with DIPG/sDMG. Given these findings, we are launching a new arm to assess safety and activity and to define the recommended phase 2 dose for ICV delivery of GD2-CART without LD. Patients are eligible for up to 12 ICV infusions of GD2-CART administered every 4-6 weeks. Clinical benefit will be formally assessed using patient-reported outcomes. GD2-CART has the potential to transform therapy for patients with H3K27M+ DIPG/sDMG. Citation Format: Robbie G. Majzner, Jasia Mahdi, Sneha Ramakrishna, Shabnum Patel, Harshini Chinnasamy, Kristen Yeom, Liora Schultz, Valentin Barsan, Rebecca Richards, Cynthia Campen, Agnes Reschke, Angus Martin Shaw Toland, Christina Baggott, Sharon Mavroukakis, Emily Egeler, Jennifer Moon, Ashley Jacobs, Karen Yamabe-Kwong, Lindsey Rasmussen, Esther Nie, Sean Green, Michael Kunicki, Michelle Fujimoto, Zach Ehlinger, Warren Reynolds, Snehit Prabhu, Katherine E. Warren, Tim Cornell, Sonia Partap, Paul Fisher, Gerald Grant, Hannes Vogel, Bita Sahaf, Kara Davis, Steven Feldman, Michelle Monje, Crystal L. Mackall. Major tumor regressions in H3K27M-mutated diffuse midline glioma (DMG) following sequential intravenous (IV) and intracerebroventricular (ICV) delivery of GD2-CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT001.

Despite high remission rates following CAR-T cell therapy in B-ALL, relapse due to loss of the targeted antigen is increasingly recognized as a mechanism of immune escape. We hypothesized that simultaneous targeting of CD19 and CD22 may reduce the likelihood of antigen loss, thus improving sustained remission rates. A systematic approach to the generation of CAR constructs incorporating two target-binding domains led to several novel CD19/CD22 bivalent CAR constructs. Importantly, we demonstrate the challenges associated with the construction of a bivalent CAR format that preserves bifunctionality against both CD19 and CD22. Using the most active bivalent CAR constructs, we found similar transduction efficiency compared to that of either CD19 or CD22 single CARs alone. When expressed on human T cells, the optimized CD19/CD22 CAR construct induced comparable interferon γ and interleukin-2 in vitro compared to single CARs against dual-antigen-expressing as well as single-antigen-expressing cell lines. Finally, the T cells expressing CD19/CD22 CAR eradicated ALL cell line xenografts and patient-derived xenografts (PDX), including a PDX generated from a patient with CD19− relapse following CD19-directed CAR therapy. The CD19/CD22 bivalent CAR provides an opportunity to test whether simultaneous targeting may reduce risk of antigen loss. Keywords: chimeric antigen receptor, adoptive cell therapy, leukemia

Background We previously discovered high expression of the disialoganglioside GD2 on H3K27M+ gliomas and demonstrated preclinical efficacy of intravenous (IV) GD2-targeted chimeric antigen receptor (CAR) T-cells in preclinical models of H3K27M-mutated diffuse intrinsic pontine glioma (DIPG) and diffuse midline gliomas (DMGs). We are now conducting a Phase I clinical trial (NCT04196413) of autologous GD2-targeting CAR T-cells for H3K27M+ DIPG and spinal cord DMG. Here we present the results of subjects treated at dose level 1 (DL1; 1 million GD2-CAR T-cells/kg IV). Methods Four patients (3 DIPG, 1 spinal DMG; ages 4–25; 1M/3F) were enrolled at DL1. Three subjects with H3K27M+ DIPG received 1e6 GD2-CAR T-cells/kg IV on study. One patient with spinal DMG enrolled but became ineligible after manufacturing and was treated on an eIND at DL1. An Ommaya reservoir was placed in all subjects for therapeutic monitoring of intracranial pressure. Subjects underwent lymphodepletion with fludarabine/cyclophosphamide and remained inpatient for at least two weeks post-infusion. Results All subjects developed cytokine release syndrome (Grade 1–3) manifested by fever, tachycardia and hypotension. Other toxicities included ICANS (Grade 1–2) and neurological symptoms/signs mediated by intratumoral inflammation which we have termed Tumor Inflammation-Associated Neurotoxicity (TIAN). No evidence of on-target, off-tumor toxicity was observed in any patients. No dose-limiting toxicities occurred. CAR T cells trafficked to the CNS and were detected in CSF and blood. 3/4 patients exhibited marked improvement or resolution of neurological deficits and radiographic improvement. The patient treated on an eIND exhibited >90% reduction in spinal DMG volume but progressed by month 3. Re-treatment of this subject via intracerebroventricular administration resulted in a second reduction in spinal DMG volume by ~80%. Conclusions GD2-CAR T-cells at DL1 demonstrate a tolerable safety profile in patients with H3K27M+ DIPG/DMG with clear signs of T-cell expansion and activity including clinical responses.

The use of radiotherapy as bridging therapy to chimeric antigen receptor T-cell therapy (CAR-T) in pre-B acute lymphoblastic leukemia (B-ALL) has been minimally explored. Here, we present a boy with B-ALL who relapsed after allogeneic bone marrow transplant with disseminated disease, including significant symptomatic cardiovascular and gastrointestinal (GI) involvement. The cardiac and GI leukemic infiltrates were successfully treated with bridging radiation therapy (BRT) prior to CAR-T infusion. Using this approach, he successfully tolerated CAR-T with no evidence of disease or sequelae on 3-month follow-up. This is the first reported case of safe and effective delivery of cardiac BRT in B-ALL.

Despite impressive progress, more than 50% of patients treated with CD19-targeting chimeric antigen receptor T cells (CAR19) experience progressive disease. Ten of 16 patients with large B cell lymphoma (LBCL) with progressive disease after CAR19 treatment had absent or low CD19. Lower surface CD19 density pretreatment was associated with progressive disease. To prevent relapse with CD19− or CD19lo disease, we tested a bispecific CAR targeting CD19 and/or CD22 (CD19-22.BB.z-CAR) in a phase I clinical trial (NCT03233854) of adults with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL) and LBCL. The primary end points were manufacturing feasibility and safety with a secondary efficacy end point. Primary end points were met; 97% of products met protocol-specified dose and no dose-limiting toxicities occurred during dose escalation. In B-ALL (n = 17), 100% of patients responded with 88% minimal residual disease-negative complete remission (CR); in LBCL (n = 21), 62% of patients responded with 29% CR. Relapses were CD19−/lo in 50% (5 out of 10) of patients with B-ALL and 29% (4 out of 14) of patients with LBCL but were not associated with CD22−/lo disease. CD19/22-CAR products demonstrated reduced cytokine production when stimulated with CD22 versus CD19. Our results further implicate antigen loss as a major cause of CAR T cell resistance, highlight the challenge of engineering multi-specific CAR T cells with equivalent potency across targets and identify cytokine production as an important quality indicator for CAR T cell potency., Bispecific CAR T cells targeting CD19 and CD22 exhibit clinical activity and low toxicity in patients with large B cell lymphoma and B cell acute lymphoblastic leukemia, with relapses associated with loss of CD19 but not CD22.

BackgroundChimeric antigen receptor (CAR) therapy and hematopoietic stem cell transplantation (HSCT) are therapeutics for relapsed acute lymphocytic leukemia (ALL) that are increasingly being used in tandem. We identified a non-physiologic CD19+/CD3+ T-cell population in the leukapheresis product of a patient undergoing CAR T-cell manufacturing who previously received a haploidentical HSCT, followed by infusion of a genetically engineered T-cell addback product. We confirm and report the origin of these CD19+/CD3+ T cells that have not previously been described in context of CAR T-cell manufacturing. We additionally interrogate the fate of these CD19-expressing cells as they undergo transduction to express CD19-specific CARs.Main bodyWe describe the case of a preteen male with multiply relapsed B-ALL who was treated with sequential cellular therapies. He received an αβ T-cell depleted haploidentical HSCT followed by addback of donor-derived T cells genetically modified with a suicide gene for iCaspase9 and truncated CD19 for cell tracking (RivoCel). He relapsed 6 months following HSCT and underwent leukapheresis and CAR T-cell manufacturing. During manufacturing, we identified an aberrant T-cell population dually expressing CD19 and CD3. We hypothesized that these cells were RivoCel cells and confirmed using flow cytometry and PCR that the identified cells were in fact RivoCel cells and were eliminated with iCaspase9 activation. We additionally tracked these cells through CD19-specific CAR transduction and notably did not detect T cells dually positive for CD19 and CD19-directed CARs. The most likely rationale for this is in vitro fratricide of the CD19+ ‘artificial’ T-cell population by the CD19-specific CAR+ T cells in culture.ConclusionsWe report the identification of CD19+/CD3+ cells in an apheresis product undergoing CAR transduction derived from a patient previously treated with a haploidentical transplant followed by RivoCel addback. We aim to bring attention to this cell phenotype that may be recognized with greater frequency as CAR therapy and engineered αβhaplo-HSCT are increasingly coupled. We additionally suggest consideration towards using alternative markers to CD19 as a synthetic identifier for post-transplant addback products, as CD19-expression on effector T cells may complicate subsequent treatment using CD19-directed therapy.

Introduction: Chimeric antigen receptor (CAR) T cell therapy has provided patients with relapsed/refractory B cell malignancies a new therapeutic option, but this class of therapeutics has not demo...

Immunotherapy for the treatment of acute lymphoblastic leukemia (ALL) broadens therapeutic options beyond chemotherapy and targeted therapy. Here, we review the use of monoclonal antibody-based drugs and cellular therapies to treat ALL. We discuss the challenges facing the field regarding the optimal timing and sequencing of these therapies in relation to other treatment options as well as considerations of cost effectiveness. By early identification of patients at risk for leukemic relapse, monoclonal antibody and cellular immunotherapies can be brought to the forefront of treatment options. Novel CAR design and manufacturing approaches may enhance durable patient response. Multiple clinical trials are now underway to evaluate the sequence and timing of monoclonal antibody, cellular therapy, and/or stem cell transplantation. The biologic and clinical contexts in which immunotherapies have advanced the treatment of ALL confer optimism that more patients will achieve durable remissions. Immunotherapy treatments in ALL will expand through rationally targeted approaches alongside advances in CAR T cell therapy design and clinical experience.

BACKGROUND: Patients with relapsed/refractory (R/R) large B-cell lymphoma (LBCL) after failure of CD19-directed CAR T-cell therapy (CAR19) have a dire prognosis, with an overall response rate (ORR) of 29% to conventional salvage therapies, and a median overall survival (OS) of 6 months. CD22 is expressed on the majority of B-cell malignancies. Autologous CAR T-cells targeting CD22 (CAR22) have yielded an ORR of 70-90% in pediatric patients with R/R B-cell acute lymphoblastic leukemia (B-ALL), including those who had previously failed CAR19 therapy. Based on these encouraging results, we evaluated CAR22 in adult patients with R/R LBCL, focusing on those with CAR19-refractory disease. METHODS: This ongoing single-institution phase I dose escalation clinical trial (NCT04088890) is evaluating a CAR construct incorporating the m971 CD22 single chain variable fragments and 41BB/CD3z endodomains integrated within autologous T-cells via lentiviral transduction. After lymphodepletion (LD) with fludarabine and cyclophosphamide, patients are infused with cryopreserved CAR T-cells after a 7- to 11-day closed manufacturing process utilizing the CliniMACS Prodigy device (Miltenyi). Primary objectives assess the ability to successfully manufacture CAR22 and safety. Secondary objectives include efficacy and durability of responses. RESULTS: Twenty-one patients with LBCL [n=12 at dose level 1 (DL1), 1x10 6 CAR+ cells/kg; n=9 at dose level 2 (DL2), 3x10 6 CAR+ cells/kg] have been enrolled with a median age of 64 years (range, 36-79) and a median of 4 (range, 3-8) prior lines of therapy. All patients had at least one high risk feature, including failure of prior CAR19 therapy (n=20); refractory disease to second-line or later therapy (n=17); elevated lactate dehydrogenase (LDH) pre-LD (n=17); high tumor burden (n=9); a history of primary refractory disease (n=7); failure of prior autologous hematopoietic stem cell transplantation (HSCT) (n=6); never achieving CR to any therapy (n=5); or LBCL with MYC gene rearrangements (n=5). Successful manufacturing of cells was achieved in all patients. All patients reached day 28 post-infusion and are included in the safety and efficacy analysis presented here; updated results will be presented at the meeting. Every patient experienced cytokine release syndrome (CRS); 20/21 (95%) were Grade 1-2, 1/21 (5%) were Grade 3. Four patients (19%) experienced immune effector cell-associated neurotoxicity syndrome (ICANS); all cases were Grade 1-2 and resolved within 2 days. Five patients (24%) experienced a hyperinflammatory macrophage activation syndrome (MAS), manifested in all cases by pancytopenia and consumptive coagulopathy (DIC) requiring transfusion and/or growth factor support. One patient who received DL2 had a Grade 5 infectious event in the setting of ongoing MAS and pancytopenia. Relative to DL1, higher prevalence of Grade ≥3 cytopenias beyond D28 (89% vs. 50%) and MAS (33% vs. 17%) were observed at DL2; thus, DL1 was selected as the maximally tolerated dose (MTD). ORR at D28 was 86% (CR, n=11; PR, n=7), and was similar between DL1 and DL2 (92% vs. 78%; p=ns). 3/7 (43%) initial PR improved to CR at a median of 3 months post-infusion. All 14 patients (67% of cohort) who achieved CR remain in remission, with a mean follow-up of 7.3 months (range, 1.2-21.3); median progression free survival (PFS) and OS have not yet been reached. Five patients died from disease progression, and one patient died from septic shock in CR. CD22 expression by flow was downregulated or absent in 1/3 (33%) patients evaluated after relapse. Peak CAR-T expansion as detected by peripheral blood flow cytometry occurred at a median of 14 days, with a trend towards earlier and higher peak levels in DL2 patients. Significantly higher mean CAR-T levels occurred at peak expansion in patients who developed MAS (1070±915 vs. 196±209 CAR+ cells/μL; p=0.001). CONCLUSIONS: Infusion of CAR22 in R/R LBCL is safe and well tolerated at DL1. Manufacturing of CAR22 was uniformly successful. With a mean follow-up of 7.3 months, the ORR and CR rates are 18/21 (86%) and 14/21 (67%), respectively. These data demonstrate CAR22 to be an effective salvage therapy for CAR19-refractory or CD19-negative LBCL. Figure 1 Figure 1. Disclosures Frank: Allogene Therapeutics: Research Funding; Adaptive Biotechnologies: Research Funding; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees. Oak: Kite Pharma-Gilead: Research Funding. Arai: Magenta Therapeutics: Research Funding. Rezvani: Kaleido: Other: One-time scientific advisory board; Nohla Therapeutics: Other: One-time scientific advisory board; Pharmacyclics-Abbvie: Research Funding; US Department of Justice: Consultancy. Shiraz: Kite Pharma-Gilead: Research Funding. Sidana: Janssen: Consultancy, Research Funding; Allogene: Research Funding; Magenta Therapeutics: Consultancy, Research Funding; BMS: Consultancy. Weng: Kite Pharma: Research Funding. Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding. Feldman: Samsara Biocapital: Consultancy; Obsidian: Consultancy; Lonza PerMed: Consultancy; Gradalis: Consultancy. Mackall: Lyell: Consultancy, Current equity holder in publicly-traded company, Other: Founder; Syncopation Life Sciences: Consultancy, Current holder of individual stocks in a privately-held company, Other: Founder; Apricity: Consultancy, Current equity holder in publicly-traded company; Neoimmune Tech: Consultancy; Nektar: Consultancy, Research Funding. Miklos: Kite, a Gilead Company, Amgen, Atara, Wugen, Celgene, Novartis, Juno-Celgene-Bristol Myers Squibb, Allogene, Precision Bioscience, Adicet, Pharmacyclics, Janssen, Takeda, Adaptive Biotechnologies and Miltenyi Biotechnologies: Consultancy; Pharmacyclics: Patents & Royalties; Pharmacyclics, Amgen, Kite, a Gilead Company, Novartis, Roche, Genentech, Becton Dickinson, Isoplexis, Miltenyi, Juno-Celgene-Bristol Myers Squibb, Allogene, Precision Biosciences, Adicet, Adaptive Biotechnologies: Research Funding; Adaptive Biotechnologies, Novartis, Juno/Celgene-BMS, Kite, a Gilead Company, Pharmacyclics-AbbVie, Janssen, Pharmacyclics, AlloGene, Precision Bioscience, Miltenyi Biotech, Adicet, Takeda: Membership on an entity's Board of Directors or advisory committees. Muffly: Astellas, Jasper, Adaptive, Baxalta: Research Funding; Adaptive: Honoraria, Other: fees for non-CME/CE services: , Research Funding; Pfizer, Amgen, Jazz, Medexus, Pfizer: Consultancy. OffLabel Disclosure: CD22-directed CAR-T therapy for the treatment of adults with relapsed/refractory large B-cell lymphoma

Background: Diffuse intrinsic pontine glioma (DIPG) and other H3K27M-mutated diffuse midline gliomas (DMGs) are universally lethal central nervous system tumors. We previously discovered that the disialoganglioside GD2 is highly and homogenously expressed on H3K27M+ gliomas and demonstrated that GD2 CAR T cells are effective in preclinical models (Mount/Majzner et al., Nat Med, 2018). Methods: Four subjects (3 DIPG, 1 spinal cord DMG; 4-25 yr; 1M/3F) were enrolled at DL1. Three subjects with H3K27M+ DIPG received 1e6 autologous GD2 CAR T cells/kg intravenously (IV) on study. One patient, a 25 y/o with spinal cord DMG, developed rapidly progressive disease after enrollment, resulting in complete paraparesis that led to removal from the study prior to cell infusion; she was treated on a single patient eIND with the same treatment regimen as DL1. We utilized a retroviral vector expressing a 14g2a.4-1BB.z CAR construct and an inducible iCasp9 safety switch. Manufacturing was performed in the Miltenyi Prodigy on CD4/CD8 enriched apheresis product. CAR T cells were cultured in the presence of dasatinib to improve T cell fitness (Weber et al., Science, 2021). An Ommaya reservoir was placed in all patients for monitoring of intracranial pressure (ICP). Results: We generated GD2 CAR T cell products meeting release criteria for all four patients. All subjects received lymphodepletion with cyclophosphamide and fludarabine and remained inpatient for 14+ days after infusion. All patients developed cytokine release syndrome (Grade 1-3) manifested by fever, tachycardia and hypotension, beginning 6-7 days after infusion. Due to concern for tumoral edema and increased ICP, patients were managed with conservative fluid resuscitation, and early intervention with tocilizumab and anakinra +/- corticosteroids. Other toxicities included ICANS (Grade 1-2) and neurotoxicity mediated by inflammation in sites of disease which we have termed Tumor Inflammation-Associated Neurotoxicity (TIAN). TIAN most often manifested as worsening of existing deficits, but one patient developed symptoms of increased ICP which quickly resolved upon removal of CSF via the Ommaya. No evidence of on-target, off-tumor toxicity was observed in any patients. No dose-limiting toxicities occurred.CAR T cells trafficked to the CNS and were detected in both the CSF and peripheral blood. Inflammatory cytokines including IL-6 were elevated in the CSF and blood. 3/4 patients exhibited marked improvement or resolution of neurological deficits and some radiographic improvement. The patient treated on a single patient eIND exhibited a >90% reduction in her spinal cord DMG tumor volume at two months post-infusion. Durability of the therapeutic benefit remains to be determined. Conclusions: This is the first report of GD2 CAR T cell therapy for DIPG and spinal cord DMG. Toxicities are similar to other CAR T cells with additional, manageable complications due to inflammation at CNS sites of tumor. Treatment at DL1 demonstrated a tolerable safety profile and clear signs of T cell expansion and activity including clinical responses. This approach has the potential to transform therapy for patients with H3K27M+ DIPG/DMG. Further correlative studies, including single-cell RNAseq, longer-term outcomes and results from patients on subsequent dose levels will also be presented. Citation Format: Robbie G. Majzner, Sneha Ramakrishna, Aaron Mochizuki, Shabnum Patel, Harshini Chinnasamy, Kristen Yeom, Liora Schultz, Rebecca Richards, Cynthia Campen, Agnes Reschke, Jasia Mahdi, Angus Martin Shaw Toland, Christina Baggott, Sharon Mavroukakis, Emily Egeler, Jennifer Moon, Kayla Landrum, Courtney Erickson, Lindsey Rasmussen, Valentin Barsan, John S. Tamaresis, Anne Cunniffe Marcy, Michael Kunicki, Michelle Fujimoto, Zach Ehlinger, Sreevidya Kurra, Timothy Cornell, Sonia Partap, Paul Fisher, Gerald Grant, Hannes Vogel, Bita Sahaf, Kara Davis, Steven Feldman, Crystal L. Mackall, Michelle Monje. GD2 CAR T cells mediate clinical activity and manageable toxicity in children and young adults with DIPG and H3K27M-mutated diffuse midline gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT031.

Introduction We are conducting a Phase I clinical trial utilizing chimeric antigen receptor (CAR) T-cells targeting GD2 (NCT04196413) for H3K27M-mutant diffuse intrinsic pontine glioma (DIPG) and spinal cord diffuse midline glioma (DMG). Cerebrospinal fluid (CSF) is collected for correlative studies at the time of routine intracranial pressure monitoring via Ommaya catheter. Here we present single cell RNA-sequencing results from the first 3 subjects. Methods Single cell RNA-sequencing was performed utilizing 10X Genomics on cells isolated from CSF at various time points before and after CAR T-cell administration and on the CAR T-cell product. Output was aligned with Cell Ranger and analyzed in R. Results As detailed in the Majzner et al. abstract presented at this meeting, three of four subjects treated at dose-level one exhibited clear radiographic and/or clinical benefit. We have to date completed single cell RNA-sequencing for three of these four subjects (two with benefit, one without). After filtering out low-quality signals and doublets, 89,604 cells across 3 subjects were analyzed. Of these, 4,122 cells represent cells isolated from CSF and 85,482 cells represent CAR T-cell product. Two subjects who demonstrated clear clinical and radiographic improvement exhibited fewer S100A8+S100A9+ myeloid suppressor-cells and CD25+FOXP3+ regulatory T-cells in the CSF pre-infusion compared to the subject who did not derive a therapeutic response. In one subject with DIPG who demonstrated improvement, polyclonal CAR T-cells detectable in CSF at Day +14 demonstrated enrichment of CD8A, GZMA, GNLY and PDCD1 compared to the pre-infusion CAR T-cells by trajectory analysis, suggesting differentiation toward a cytotoxic phenotype; the same subject exhibited increasing numbers of S100A8+S100A9+ myeloid cells and CX3CR1+P2RY12+ microglia over time. Further analyses will be presented as data become available. Conclusions The presence of immunosuppressive myeloid populations, detectable in CSF, may correlate to clinical response in CAR T cell therapy for DIPG/DMG.

Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent effects in relapsed and/or refractory pre-B cell acute lymphoblastic leukemia (B-ALL), but antigen loss is a frequent cause of resistance to CD19-targeted immunotherapy. CD22 is also expressed in most cases of B-ALL and is usually retained following CD19 loss. We report results from a phase 1 trial testing a new CD22-targeted CAR (CD22-CAR) in 21 children and adults, including 17 who were previously treated with CD19-directed immunotherapy. Dose-dependent antileukemic activity was observed, with complete remission obtained in 73% (11/15) of patients receiving ≥1 × 106 CD22-CAR T cells per kg body weight, including 5 of 5 patients with CD19dim or CD19- B-ALL. Median remission duration was 6 months. Relapses were associated with diminished CD22 site density that likely permitted CD22+ cell escape from killing by CD22-CAR T cells. These results are the first to establish the clinical activity of a CD22-CAR in B-ALL, including leukemia resistant to anti-CD19 immunotherapy, demonstrating potency against B-ALL comparable to that of CD19-CAR at biologically active doses. Our results also highlight the critical role played by antigen density in regulating CAR function.

Single-cell analysis provides novel insights into elements of CAR T cell toxicities and response.

BACKGROUND: CD22 is expressed on the majority of B-cell malignancies. Autologous CAR T-cells targeting CD22 (CAR22) have yielded objective response rates (ORR) of 70-90% in pediatric patients with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (ALL), including those who had previously failed CD19-directed CAR T-cell (CAR19) therapy. Based on these encouraging results, we evaluated CAR22 in adult patients with R/R ALL and for the first time in patients with R/R large B-cell lymphoma (LBCL), including those who had failed prior autologous CAR19 therapy. METHODS: This single-institution phase I dose escalation clinical trial (NCT04088890) is evaluating a CAR construct incorporating the m971 CD22 single chain variable fragments and 41BB/CD3z endodomains integrated within autologous T-cells via lentiviral transduction. After lymphodepletion with fludarabine and cyclophosphamide, patients were infused with fresh or cryopreserved CAR T-cells after a 7- to 11-day closed manufacturing process utilizing the CliniMACS Prodigy device (Miltenyi). The current cohort includes patients treated at dose level 1 (DL1), which was 1x106 CAR+ cells/kg. Primary objectives assessed the ability to successfully manufacture CAR22 and safety. Overall response rate (ORR) at 28 days post-infusion (D28) was a secondary objective. RESULTS: Three patients with LBCL have been enrolled with a median age of 53 years (range, 51-57) and a median of 6 (range, 5-8) prior lines of therapy. All three patients received prior CAR19 and had refractory disease to second-line or later therapy (n=3); had not undergone autologous hematopoietic stem cell transplantation (HSCT) (n=3); had MYC and BCL2 gene rearrangements (double-hit lymphoma; n=2); had high tumor burden (SPD >50 cm2; n=2); had a history of primary refractory disease (n=1); or had never achieved CR to any therapy (n=1). Six patients with ALL have been enrolled with a median age of 43.5 years (range, 23-62) and a median of 6 (range, 4-8) prior lines of therapy. All six patients received prior allogeneic HSCT and had Ph-positive disease (n=3); had central nervous system (CNS) involvement (n=3); had extramedullary disease (n=2); had high disease burden (BM blasts >5%; n=2); had received prior CD19-directed therapy (n=5); or had received prior CD22-directed therapy (n=3). Successful manufacturing of cells at DL1 was achieved in all patients. All patients (LBCL n=3, ALL n=6) reached day 28 and are included in the safety and response analysis presented here; updated results will be presented at the meeting. Eight patients (88.9%) experienced cytokine release syndrome (CRS); all were Grade 1-2. There were no cases of immune effector cell-associated neurotoxicity syndrome (ICANS). No differences in toxicities were seen across the patient age spectrum and no Grade 5 toxicities occurred following CAR22 infusion. In LBCL, all patients achieved a response at D28 (ORR=100%; CR, n=1, PR, n=2). Both patients with a D28 PR improved to CR by day 90 and 180. All patients remain in CR, with a median follow-up of 8.4 months (range, 6-9.3). In ALL, all patients achieved a CR at D28 (ORR=100%; MRD-, n=5, MRD+, n=1). After a median follow up of 5.1 months (range, 1-8.2), three patients relapsed at 2.5, 4, and 5.5 months after infusion; one patient died while undergoing subsequent therapy 7.3 months post-infusion. CD22 expression by flow cytometry was downregulated or absent in two patients after relapse. Peak CAR expansion as detected by peripheral blood flow cytometry reached a median level of 90.1 (LBCL; range, 85.4-350) and 43.4 (ALL; range, 0.9-399.6) CAR+ cells/µL between D14 and D21. In two LBCL patients with progression following CAR19, CAR22 levels were 11.7 and 55.9 fold higher than prior CAR19 levels at peak expansion. CONCLUSIONS: Infusion of CD22-targeting CAR T-cells in R/R LBCL and ALL is safe and well tolerated. Manufacturing of CAR22 was uniformly successful. To date, 3 of 3 heavily treated adult patients with LBCL whose disease relapsed after prior CAR19 have each achieved CR durable to at least 6 months. All adult ALL patients have achieved CR following CAR22, with some early relapses observed. Accrual is ongoing. Disclosures Negrin: Amgen: Consultancy; Biosource: Current equity holder in private company; UpToDate: Honoraria; KUUR Therapeutics: Consultancy; Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company; BioEclipse Therapeutics: Current equity holder in private company. Rezvani:Pharmacyclics: Research Funding. Shiraz:ORCA BioSystems: Research Funding; Kite, a Gilead Company: Research Funding. Sidana:Janssen: Consultancy. Mackall:BMS: Consultancy; Allogene: Current equity holder in publicly-traded company; Apricity Health: Consultancy, Current equity holder in private company; Nektar Therapeutics: Consultancy; NeoImmune Tech: Consultancy; Lyell Immunopharma: Consultancy, Current equity holder in private company. Miklos:Kite-Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Adaptive Biotech: Consultancy, Other: Travel support, Research Funding; Allogene Therapeutics Inc.: Research Funding; Juno-Celgene-Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Novartis: Consultancy, Other: Travel support, Research Funding; Pharmacyclics: Consultancy, Other: Travel support, Patents & Royalties, Research Funding; Janssen: Consultancy, Other: Travel support; Miltenyi Biotec: Research Funding. Muffly:Amgen: Consultancy; Adaptive: Research Funding; Servier: Research Funding. OffLabel Disclosure: CD22-directed CAR T-cell Therapy for the treatment of adults with relapsed/refractory LBCL and B-ALL

Chimeric antigen receptor (CAR) T-cell therapy has proven effective in relapsed and refractory B-cell malignancies, but resistance and relapses still occur. Better understanding of mechanisms influencing CAR T-cell cytotoxicity and the potential for modulation using small-molecule drugs could improve current immunotherapies. Here, we systematically investigated druggable mechanisms of CAR T-cell cytotoxicity using >500 small-molecule drugs and genome-scale CRISPR-Cas9 loss-of-function screens. We identified several tyrosine kinase inhibitors that inhibit CAR T-cell cytotoxicity by impairing T-cell signaling transcriptional activity. In contrast, the apoptotic modulator drugs SMAC mimetics sensitized B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma cells to anti-CD19 CAR T cells. CRISPR screens identified death receptor signaling through FADD and TNFRSF10B (TRAIL-R2) as a key mediator of CAR T-cell cytotoxicity and elucidated the RIPK1-dependent mechanism of sensitization by SMAC mimetics. Death receptor expression varied across genetic subtypes of B-cell malignancies, suggesting a link between mechanisms of CAR T-cell cytotoxicity and cancer genetics. These results implicate death receptor signaling as an important mediator of cancer cell sensitivity to CAR T-cell cytotoxicity, with potential for pharmacological targeting to enhance cancer immunotherapy. The screening data provide a resource of immunomodulatory properties of cancer drugs and genetic mechanisms influencing CAR T-cell cytotoxicity.

Genetically engineered T cells expressing CD19-specific chimeric antigen receptors (CAR) have shown impressive activity against B-cell malignancies, and preliminary results suggest that T cells expressing a first-generation disialoganglioside (GD2)-specific CAR can also provide clinical benefit in patients with neuroblastoma. We sought to assess the potential of GD2-CAR therapies to treat pediatric sarcomas. We observed that 18 of 18 (100%) of osteosarcomas, 2 of 15 (13%) of rhabdomyosarcomas, and 7 of 35 (20%) of Ewing sarcomas expressed GD2. T cells engineered to express a third-generation GD2-CAR incorporating the 14g2a-scFv with the CD28, OX40, and CD3ζ signaling domains (14g2a.CD28.OX40.ζ) mediated efficient and comparable lysis of both GD2+ sarcoma and neuroblastoma cell lines in vitro. However, in xenograft models, GD2-CAR T cells had no antitumor effect against GD2+ sarcoma, despite effectively controlling GD2+ neuroblastoma. We observed that pediatric sarcoma xenografts, but not neuroblastoma xenografts, induced large populations of monocytic and granulocytic murine myeloid-derived suppressor cells (MDSC) that inhibited human CAR T-cell responses in vitro. Treatment of sarcoma-bearing mice with all-trans retinoic acid (ATRA) largely eradicated monocytic MDSCs and diminished the suppressive capacity of granulocytic MDSCs. Combined therapy using GD2-CAR T cells plus ATRA significantly improved antitumor efficacy against sarcoma xenografts. We conclude that retinoids provide a clinically accessible class of agents capable of diminishing the suppressive effects of MDSCs, and that co-administration of retinoids may enhance the efficacy of CAR therapies targeting solid tumors. Cancer Immunol Res; 4(10); 869–80. ©2016 AACR.

Following CAR T cell therapy, many patients receive consolidative hematopoietic stem cell transplantation (HSCT), with the available donor pool recently expanding to include genetically engineered cell products. One such example with striking early promise is the α/Β T-cell depleted haploidentical HSCT (α/Β haplo-HSCT) given with bystander α/Β genetically modified T cells, termed BPX-501 cells. These cells are engineered to express an inducible caspase 9 (iCas9) suicide vector that can be activated by the AP1903 (Rimiducid) dimerizing agent in the event of graft-versus-host disease (GVHD) (US NCT03301168). Here, we report the identification of an unexpected dual expressing CD19+/CD3+ T cell subset in a 10 year old patient who underwent apheresis collection for CAR T cell manufacturing upon relapse six months after α/Β haplo-HSCT and BPX-501 cell addback. A leukapheresis product was collected and CD4 and CD8 T cells were selected from this product for CAR transduction using the Miltenyi CliniMACS Prodigy. T cell purity was assessed using flow cytometry. Characterization of his T cell product at this stage revealed an aberrant cell population expressing both surface CD3 and CD19 (Fig 1a) and lacking additional B cell surface markers, excluding leukemic origin of these cells. This patient was the recipient of the described BPX-501 cell product. BPX-501 cells are also engineered to express a truncated version of CD19 to permit tracking of these modified T cells in patients, post-infusion. We hypothesized that these dual positive CD19+/CD3+ cells were the BPX-501 cells derived from his paternal haploidentical donor and still circulating despite leukemia relapse. We utilized the patient's leukapheresis sample to better characterize these cells. Flow cytometry confirmed the presence of this dual positive CD19+/CD3+ population in the apheresis product. We subsequently treated the apheresis product with Rimiducid in vitro and observed elimination of the CD19+/CD3+ cell subset in a dose dependent manner, thus confirming that these cells were BPX-501 cells (Fig 1b). We additionally investigated the fate of the BPX-501 cells following CAR transduction and observed an absence of this subset post-CAR transduction (Fig 1a). Cells co-expressing CD19-targeting CARs and surface CD19 were not observed in the final manufactured product. The most likely explanation for this phenomenon is in vitro fratricide of the CD19+ T cell population by the CD19-specific CAR+ T cells in culture. We aim to bring attention to this cell phenotype that may be recognized with greater frequency as CAR T therapy and engineered α/Β haplo-HSCT are increasingly coupled. We additionally suggest consideration towards using alternative markers to CD19 as a synthetic identifier for post-transplant add-back products, as CD19-expression on effector T cells may complicate subsequent treatment using CD19-directed therapy. Disclosures Majzner: Xyphos Inc.: Consultancy; Lyell Immunopharma: Consultancy; Xyphos Inc.: Consultancy. Mackall:Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board; Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Feldman:Octane Biotech, Inc.: Employment; Personalized Medicine Initiative Science: Membership on an entity's Board of Directors or advisory committees.

Introduction: Chimeric antigen receptor (CAR) T cells targeting either CD19 or CD22 have yielded striking complete remission (CR) rates of 70%-90% in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (ALL), but CD19 negative and CD22 low relapse limits the curative potential of these single-antigen CAR T cell approaches. We hypothesized that a bivalent CAR-T construct that can target CD19 and/or CD22 would prevent antigen negative/low relapse. Here we present the combined single institution experience to date of pediatric and adult patients with R/R ALL treated with this novel bispecific CAR. Methods: We conducted parallel Phase I clinical trials of CD19/CD22 bispecific CAR T cells in pediatric and adult patients with relapsed/refractory ALL. We utilized lentiviral transduction of a bivalent CAR construct incorporating the fmc63 CD19 and m971 CD22 single chain variable fragments (scFvs) and a 41BB costimulatory endodomain. After lymphodepletion with fludarabine and cyclophosphamide, patients were infused with fresh or cryopreserved CAR T cells manufactured using a 7-11 day process. Two dose levels were tested during dose escalation: Dose level 1 was 1x106 CAR T cells/kg and dose level 2 was 3x106 cells/kg. Primary objectives assessed the ability to successfully manufacture CAR19/22 CAR T cells and safety while response at Day 28 post-infusion was a secondary objective. Blood, bone marrow and cerebrospinal fluid samples were obtained at protocol defined intervals for correlative biology studies. Results: Nineteen patients have been enrolled (10 pediatric; 9 adult) with a median age of 23 years (range, 2-68) and median of 4 (range, 2-11) prior lines of leukemia-directed therapy. Ten patients received prior HCT, 9 were treated with prior Blinatumomab, 3 with prior CD19 directed CAR T cells and 4 with prior Inotuzumab. Fourteen patients (8 pediatric, 6 adult) have been infused to date with CD19/CD22 bispecific CAR T cells; 7 were treated at dose level 1 (DL1) and 7 at dose level 2 (DL2). Successful manufacturing of cells at target dose levels was achieved in all patients. Twelve patients have reached day 28 and are included in the safety and response analysis presented here. Nine of 12 (75%) experienced cytokine release syndrome (CRS) and 2/12 (17%) developed immune-effector cell neurotoxicity syndrome (ICANS). The CRS and ICANS were all grade 1 or 2 across both dose levels and across pediatric and adult patients except for one adult with high disease burden who experienced grade 4 CRS and grade 4 ICANS, both of which were reversible. No differences in toxicities were seen across the patient age spectrum and there were no cases of treatment-related mortality within 28 days following CAR T infusion. Eleven of 12 (92%) patients achieved a CR, 10 of whom achieved CR at day 28 and one with a PR of extramedullary disease at day 28 which improved to CR by day 180 without further leukemia-directed intervention. One patient had primary progressive disease prior to day 28. Peak CAR expansion as detected by peripheral blood flow cytometry reached a median level of 11.13% (DL1) and 29.1% (DL2) CAR T of CD3+ cells with a range of 0.7-22.54% and 3.8-86.96%, respectively. To date, 3 patients (1 pediatric and 2 adult patients) have relapsed, all with retention of CD19. Post-remission practice differed across pediatric and adult patients; Six pediatric patients reaching day 28 underwent consolidative hematopoietic cell transplantation (HCT) whereas no adult patients received subsequent HCT. One patient died from complications post HCT while in remission. Therefore, the overall survival for all infused patients was 92% with a median follow-up of 9.5 months from time of infusion (range, 1-20). Conclusion: The combined pediatric and adult phase I trials of bispecific CD19/CD22 targeting CAR T cells in relapsed/refractory ALL demonstrates safety and tolerability at two dose levels. Expanded accrual at dose level 2 is ongoing and clinical outcomes will be updated. This work additionally demonstrates feasibility of delivering unified B-ALL CAR T cell therapy across age boundaries. Multi-parametric CyTOF studies permitting CAR T cell phenotyping in conjunction with single cell TCR tracking, proteomics, epigenomics and cytokine profiling are ongoing and will be used to further characterize persisting CAR T cells and define inter-product and inter-patient variability. Disclosures Muffly: Pfizer: Consultancy; KITE: Consultancy; Adaptive: Research Funding. Majzner:Xyphos Inc.: Consultancy; Lyell Immunopharma: Consultancy. Feldman:Octane Biotech, Inc.: Employment; Personalized Medicine Initiative Science: Membership on an entity's Board of Directors or advisory committees. Miklos:Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Juno: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Becton Dickinson: Research Funding; Miltenyi Biotech: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Membership on an entity's Board of Directors or advisory committees; AlloGene: Membership on an entity's Board of Directors or advisory committees. Mackall:Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board; Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

The proliferation-specific Forkhead Box m1 (Foxm1 or Foxm1b) transcription factor (previously called HFH-11B, Trident, Win, or MPP2) regulates expression of cell cycle genes essential for progression into DNA replication and mitosis. Expression of Foxm1 is found in a variety of distinct human cancers including hepatocellular carcinomas, intrahepatic cholangiocarcinomas, basal cell carcinomas, ductal breast carcinomas, and anaplastic astrocytomas and glioblastomas. In this study, we show that human Foxm1 protein is abundantly expressed in highly proliferative human non–small cell lung cancers (NSCLC) as well as in mouse lung tumors induced by urethane. To determine the role of Foxm1 during the development of mouse lung tumors, we used IFN-inducible Mx-Cre recombinase transgene to delete mouse Foxm1 fl/fl–targeted allele before inducing lung tumors with urethane. We show that Mx-Cre Foxm1−/− mice exhibit diminished proliferation of lung tumor cells causing a significant reduction in number and size of lung adenomas. Transient transfection experiments with A549 lung adenocarcinoma cells show that depletion of Foxm1 levels by short interfering RNA caused diminished DNA replication and mitosis and reduced anchorage-independent growth of cell colonies on soft agar. Foxm1-depleted A549 cells exhibit reduced expression of cell cycle–promoting cyclin A2 and cyclin B1 genes. These data show that Foxm1 stimulates the proliferation of tumor cells during progression of NSCLC. (Cancer Res 2006; 66(4): 2153-61)

The Forkhead Box f1 (Foxf1) transcription factor (previously known as HFH-8 or Freac-1) is expressed in the septum transversum and splanchnic (visceral) mesoderm and is required for proper development of gut-derived organs. Sequence comparisons of mouse and human Foxf1 genes have revealed highly conserved DNA sequences located within the -5.3-kb Foxf1 promoter region and the 400-nucleotide regulatory element located 1 kb 3' to the Foxf1 gene (3'RE). To examine their transcriptional activity during mouse embryonic development, we generated transgenic mice in which the expression of the beta-galactosidase transgene was controlled by the -2.7-kb Foxf1 promoter region, the -5.3-kb Foxf1 promoter region, or the -5.3-kb Foxf1 promoter region fused to the 3'RE. The -5.3-kb Foxf1 promoter sequences induced appropriate transgene expression in the midgut and developing intestine, whereas the -2.7-kb Foxf1 promoter region was transcriptionally inactive. Addition of 3'RE to the -5.3-kb Foxf1 promoter restored proper transgene expression in the foregut, liver, and lung mesenchyme and prevented ectopic transgene expression in the developing nervous system. Cotransfection studies demonstrated that FoxA2 protein bound to the 3'RE region (+4506/+4529 bp) and was sufficient to inhibit expression of the -5.3-kb Foxf1 promoter. Furthermore, C/EBPbeta and HNF-6 proteins bound to the 3'RE region (+4647/+4694 bp) and provided synergistic transcriptional activation of the -5.3-kb Foxf1 promoter in cotransfection assays. These studies demonstrated that the conserved Foxf1 3'RE region is essential for proper tissue-specific regulation of the Foxf1 promoter region during mouse embryogenesis.

The Forkhead Box m1 (Foxm1) transcription factor is expressed in cardiomyocytes and cardiac endothelial cells during heart development. In this study, we used a novel Foxm1 −/− mouse line to demonstrate that Foxm1-deletion causes ventricular hypoplasia and diminished DNA replication and mitosis in developing cardiomyocytes. Proliferation defects in Foxm1 −/− hearts were associated with a reduced expression of Cdk1-activator Cdc25B phosphatase and NFATc3 transcription factor, and with abnormal nuclear accumulation of the Cdk-inhibitor p21Cip1 protein. Depletion of Foxm1 levels by siRNA caused altered expression of these genes in cultured HL-1 cardiomyocytes. Endothelial-specific deletion of the Foxm1 fl/fl allele in Tie2-Cre Foxm1 fl/fl embryos did not influence heart development and cardiomyocyte proliferation. Foxm1 protein binds to the −9,259/−9,288-bp region of the endogenous mouse NFATc3 promoter, indicating that Foxm1 is a transcriptional activator of the NFATc3 gene. Foxm1 regulates expression of genes essential for the proliferation of cardiomyocytes during heart development. Developmental Dynamics 236:1000–1013, 2007. © 2007 Wiley-Liss, Inc.

The Forkhead box f1 (Foxf1) transcription factor is expressed in mesenchymal cells of the lung, liver, and gallbladder. Although Foxf1 deficiency causes severe abnormalities in the development of these organs, the molecular mechanisms underlying Foxf1 function remain uncharacterized. In this study we inactivated Foxf1 function in lung mesenchymal cells and mouse embryonic fibroblasts (MEFs) by use of either short interfering RNA duplexes or a membrane-transducing Foxf1 dominant negative (DN) mutant protein (Foxf1 DN), the latter of which is fused to the human immunodeficiency virus TAT protein transduction domain. Although Foxf1 did not influence DNA replication or cell survival, Foxf1 depletion severely diminished mesenchyme migration. Foxf1 deficiency in mesenchymal cells was associated with reduced expression of the integrin-beta3 (Itgbeta3) subunit. Furthermore, we generated transgenic mice containing a tetracycline-inducible Foxf1 DN transgene. Adenovirus-mediated activation of Foxf1 DN in transgenic MEFs caused diminished cell migration and reduced Itgbeta3 expression. A chromatin immunoprecipitation assay demonstrated that Foxf1 protein binds to the bp -871 to -815 region of the mouse Itgbeta3 promoter. Deletion of the -871 to -815 Itgbeta3 promoter region completely abolished the ability of Foxf1 to activate transcription of the Itgbeta3 promoter in cotransfection experiments, indicating that the mouse Itgbeta3 is a direct transcriptional target of Foxf1 protein. Foxf1 plays an essential role in mesenchyme migration by transcriptionally regulating Itgbeta3.

Transgenic and gene knock-out studies demonstrated that the mouse Forkhead Box m1 (Foxm1 or Foxm1b) transcription factor (previously called HFH-11B, Trident, Win, or MPP2) is essential for hepatocyte entry into mitosis during liver development, regeneration, and liver cancer. Targeted deletion of Foxm1 gene in mice produces an embryonic lethal phenotype due to severe abnormalities in the development of liver and heart. In this study, we show for the first time that Foxm1(-/-) lungs exhibit severe hypertrophy of arteriolar smooth muscle cells and defects in the formation of peripheral pulmonary capillaries as evidenced by significant reduction in platelet endothelial cell adhesion molecule 1 staining of the distal lung. Consistent with these findings, significant reduction in proliferation of the embryonic Foxm1(-/-) lung mesenchyme was found, yet proliferation levels were normal in the Foxm1-deficient epithelial cells. Severe abnormalities of the lung vasculature in Foxm1(-/-) embryos were associated with diminished expression of the transforming growth factor beta receptor II, a disintegrin and metalloprotease domain 17 (ADAM-17), vascular endothelial growth factor receptors, Polo-like kinase 1, Aurora B kinase, laminin alpha4 (Lama4), and the Forkhead Box f1 transcription factor. Cotransfection studies demonstrated that Foxm1 stimulates transcription of the Lama4 promoter, and this stimulation requires the Foxm1 binding sites located between -1174 and -1145 bp of the mouse Lama4 promoter. In summary, development of mouse lungs depends on the Foxm1 transcription factor, which regulates expression of genes essential for mesenchyme proliferation, extracellular matrix remodeling, and vasculogenesis.

9531 Background: Genetic manipulation of T cells may provide promising new therapies for pediatric solid tumors. T cells can be modified to express chimeric antigen receptors (CAR), which link the ...