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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.

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 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.

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.

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.