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PURPOSE Nonresponse and relapse after CD19-chimeric antigen receptor (CAR) T-cell therapy continue to challenge survival outcomes. Phase II landmark data from the ELIANA trial demonstrated nonresponse and relapse rates of 14.5% and 28%, respectively, whereas use in the real-world setting showed nonresponse and relapse rates of 15% and 37%. Outcome analyses describing fate after post-CAR nonresponse and relapse remain limited. Here, we aim to establish survival outcomes after nonresponse and both CD19+ and CD19– relapses and explore treatment variables associated with inferior survival. METHODS We conducted a retrospective multi-institutional study of 80 children and young adults with B-cell acute lymphoblastic leukemia experiencing nonresponse (n = 23) or relapse (n = 57) after tisagenlecleucel. We analyze associations between baseline characteristics and these outcomes and establish survival rates and salvage approaches. RESULTS The overall survival (OS) at 12 months was 19% across nonresponders (n = 23; 95% CI, 7 to 50). Ninety-five percent of patients with nonresponse had high preinfusion disease burden. Among 156 morphologic responders, the cumulative incidence of relapse was 37% (95% CI, 30 to 47) at 12 months (CD19+; 21% [15 to 29], CD19–; 16% [11 to 24], median follow-up; 380 days). Across 57 patients experiencing relapse, the OS was 52% (95% CI, 38 to 71) at 12 months after time of relapse. Notably, CD19– relapse was associated with significantly decreased OS as compared with patients who relapsed with conserved CD19 expression (CD19– 12-month OS; 30% [14 to 66], CD19+ 12-month OS; 68% [49 to 92], P = .0068). Inotuzumab, CAR reinfusion, and chemotherapy were used as postrelapse salvage therapy with greatest frequency, yet high variability in treatment sequencing and responses limits efficacy analysis across salvage approaches. CONCLUSION We describe poor survival across patients experiencing nonresponse to tisagenlecleucel. In the post-tisagenlecleucel relapse setting, patients can be salvaged; however, CD19– relapse is distinctly associated with decreased survival outcomes.

Chimeric antigen-receptor (CAR)-associated hemophagocytic lymphohistiocytosis (HLH)-like toxicities involving hyperferritinemia, multi-organ dysfunction, coagulopathy, and/or hemophagocytosis are described as occurring in a subset of patients with cytokine release syndrome (CRS). Case series report poor outcomes for those with B-acute lymphoblastic leukemia (B-ALL) who develop HLH-like toxicities, although larger outcomes analyses of children and young adults (CAYA) with B-ALL who develop these toxicities following commercial tisagenlecleucel are not described. Using a multi-institutional database of 185 CAYA with B-ALL, we conducted a retrospective cohort study including groups that developed HLH-like toxicities, high grade CRS without HLH-like toxicities, or no to low grade CRS without HLH-like toxicities. Primary objectives included characterizing the incidence, outcomes, and pre-infusion factors associated with HLH-like toxicities. Among 185 CAYA infused with tisagenlecleucel, 26 (14.1%) met criteria for HLH-like toxicities. One-year overall survival and relapse-free survival were 25.7% and 4.7% in those with HLH-like toxicities, compared with 80.1% and 57.6% in those without. In multivariable analysis for death, meeting criteria for HLH-like toxicities carried a hazard ratio of 4.61 (95% confidence interval: 2.41-8.83), controlling for disease burden, age, and sex. Patients who developed HLH-like toxicities had higher pre-tisagenlecleucel disease burden, ferritin, C-reactive protein levels, and lower platelet and absolute neutrophil counts than patients with high grade or no/low grade CRS without HLH-like toxicities. Overall, CAYA with B-ALL who developed HLH-like toxicities following tisagenlecleucel experienced high rates of relapse and non-relapse mortality, indicating the urgent need for further investigations into prevention and optimal management of patients who develop HLH-like toxicities following tisagenlecleucel.

Remarkable complete response rates have been shown with tisagenlecleucel, a chimeric antigen receptor (CAR) T-cell therapy targeting CD19, in patients up to age 26 years with refractory/relapsed B-cell acute lymphoblastic leukemia; it is US Food and Drug Administration approved forthis indication. Currently, patients receive a single dose of tisagenlecleucel across a wide doserange of 0.2 to 5.0× 106 and 0.1 to 2.5× 108 CAR T cells per kg for patients ≤50 and >50 kg, respectively. The effect of cell dose on survival and remission is not yet well established. Our primary goal was to determine if CAR T-cell dose affects overall survival (OS), event-free survival (EFS), or relapse-free-survival (RFS) in tisagenlecleucel recipients. Retrospective data were collected from Pediatric Real World CAR Consortium member institutions and included 185 patients infused with commercial tisagenlecleucel. The median dose of viable transduced CAR T cells was 1.7× 106 CAR T cells per kg. To assess the impact of cell dose, we divided responders into dose quartiles: 0.134 to 1.300× 106 (n= 48 [27%]), 1.301 to 1.700× 106 (n= 46 [26%]), 1.701 to 2.400×106 (n= 43 [24%]), and 2.401 to 5.100× 106 (n= 43 [24%]). OS, EFS, and RFS were improved in patients who received higher doses of tisagenlecleucel (P= .031, .0079, and .0045, respectively). Higher doses of tisagenlecleucel were not associated with increased toxicity. Because the current tisagenlecleucel package insert dose range remains broad, this work has implications in regard totargeting higher cell doses, within the approved dose range, to optimize patients' potential for long-standing remission.

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 cells provide a therapeutic option in hematologic malignancies. However, treatment failure after initial response approaches 50%. In allogeneic hematopoietic cell transplantation, optimal fludarabine exposure improves immune reconstitution, resulting in lower nonrelapse mortality and increased survival. We hypothesized that optimal fludarabine exposure in lymphodepleting chemotherapy before CAR T-cell therapy would improve outcomes. In a retrospective analysis of patients with relapsed/refractory B-cell acute lymphoblastic leukemia undergoing CAR T-cell (tisagenlecleucel) infusion after cyclophosphamide/fludarabine lymphodepleting chemotherapy, we estimated fludarabine exposure as area under the curve (AUC; mg × h/L) using a validated population pharmacokinetic (PK) model. Fludarabine exposure was related to overall survival (OS), cumulative incidence of relapse (CIR), and a composite end point (loss of B-cell aplasia [BCA] or relapse). Eligible patients (n = 152) had a median age of 12.5 years (range

Purpose: Patients with metastatic colorectal cancer refractory to chemotherapy have limited treatment options. Ensituximab (NEO-102) is a novel chimeric mAb targeting a variant of MUC5AC with specificity to colorectal cancer. Patients and Methods: Single-arm, phase II trial assessed the efficacy and safety of ensituximab in patients with advanced, refractory cancer who expressed MUC5AC antigen in tumor tissue. Ensituximab was administered intravenously every 2 weeks with 3 mg/kg as recommended phase II dose (RP2D). A minimum sample size of 43 patients was required on the basis of the assumption that ensituximab would improve median overall survival (OS) by 7 months using a one-sided significance level of 10% and 80% power. Written informed consent was obtained from all patients. Results: Sixty-three patients with advanced, refractory colorectal cancer were enrolled and 53 subjects were treated in phase II arm. Median age was 58 years and 46% of the patients were female. Among 57 evaluable patients, median OS was 6.8 months. No responses were observed, and stable disease was achieved in 21% of the patients. The most common treatment-related adverse events (AE) at RP2D included fatigue (38%), anemia (30%), nausea (15%), vomiting (11%), increased bilirubin (9%), constipation (8%), decreased appetite (6%), and diarrhea (6%). Serious AEs at least possibly related to ensituximab occurred in 4 patients and included anemia, nausea, increased bilirubin, and hypoxia. No patients discontinued treatment due to drug-related AEs. Conclusions: Ensituximab was well tolerated and demonstrated modest antitumor activity in patients with heavily pretreated refractory colorectal cancer.

Key Points CD19 CAR therapy for R/R EM disease, particularly CNS, offers a beneficial option with similar toxicity and survival to BM disease.There was no increased cytokine release syndrome or neurotoxicity in patients with R/R EM disease, including active CNS disease at infusion., Visual Abstract, Chimeric antigen receptor (CAR) T cells have transformed the therapeutic options for relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia. Data for CAR therapy in extramedullary (EM) involvement are limited. Retrospective data were abstracted from the Pediatric Real World CAR Consortium (PRWCC) of 184 infused patients from 15 US institutions. Response (complete response) rate, overall survival (OS), relapse-free survival (RFS), and duration of B-cell aplasia (BCA) in patients referred for tisagenlecleucel with EM disease (both central nervous system (CNS)3 and non-CNS EM) were compared with bone marrow (BM) only. Patients with CNS disease were further stratified for comparison. Outcomes are reported on 55 patients with EM disease before CAR therapy (CNS3, n = 40; non-CNS EM, n = 15). The median age at infusion in the CNS cohort was 10 years (range

ImportanceTreatment options are limited for patients with advanced pancreatic ductal adenocarcinoma (PDAC) beyond first-line 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX), with such individuals commonly being treated with gemcitabine and nab-paclitaxel.ObjectiveTo determine whether NPC-1C, an antibody directed against MUC5AC, might increase the efficacy of second-line gemcitabine and nab-paclitaxel in patients with advanced PDAC.Design, Setting, and ParticipantsThis multicenter, randomized phase II clinical trial enrolled patients with advanced PDAC between April 2014 and March 2017 whose disease had progressed on first-line FOLFIRINOX. Eligible patients had tumors with at least 20 MUC5AC staining by centralized immunohistochemistry review. Statistical analysis was performed from April to May 2022.InterventionsPatients were randomly assigned to receive gemcitabine (1000 mg/m2) and nab-paclitaxel (125 mg/m2) administered intravenously on days 1, 8, and 15 of every 4-week cycle, with or without intravenous NPC-1C 1.5 mg/kg every 2 weeks.Main Outcomes and MeasuresThe primary end point was overall survival (OS). Secondary end points were progression-free survival (PFS), objective response rate (ORR), and safety. Pretreatment clinical variables were explored with Cox proportional hazards analysis.ResultsA total of 78 patients (median [range] age, 62 [36-78] years; 32 [41%] women; 9 [12%] Black; 66 [85%] White) received second-line treatment with gemcitabine plus nab-paclitaxel (n = 40) or gemcitabine plus nab-paclitaxel and NPC-1C (n = 38). Median OS was 6.6 months (95% CI, 4.7-8.4 months) with gemcitabine plus nab-paclitaxel vs 5.0 months (95% CI, 3.3-6.5 months; P = .22) with gemcitabine plus nab-paclitaxel and NPC-1C. Median PFS was 2.7 months (95% CI, 1.9-4.1 months) with gemcitabine plus nab-paclitaxel vs 3.4 months (95% CI, 1.9-5.3 months; P = .80) with gemcitabine plus nab-paclitaxel and NPC-1C. The ORR was 3.1% (95% CI, 0.4%-19.7%) in the gemcitabine plus nab-paclitaxel and NPC-1C group and 2.9% (95% CI, 0.4%-18.7%) in the gemcitabine plus nab-paclitaxel group. No differences in toxicity were observed between groups, except that grade 3 or greater anemia occurred more frequently in patients treated with gemcitabine plus nab-paclitaxel and NPC-1C than gemcitabine plus nab-paclitaxel (39% [15 of 38] vs 10% [4 of 40]; P = .003). The frequency of chemotherapy dose reductions was similar in both groups (65% vs 74%; P = .47). Lower performance status, hypoalbuminemia, PDAC diagnosis less than or equal to 18 months before trial enrollment, lymphocyte-to-monocyte ratio less than 2.8, and CA19-9 greater than 2000 IU/mL were independently associated with poorer survival.Conclusions and RelevanceIn this randomized clinical trial of advanced PDAC, NPC-1C did not enhance the efficacy of gemcitabine/nab-paclitaxel. These data provide a benchmark for future trials investigating second-line treatment of PDAC.Trial RegistrationClinicalTrials.gov Identifier: NCT01834235

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.

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.

NEO-102 is a novel chimeric IgG1 monoclonal antibody which recognizes a variant form of MUC5AC expressed specifically by human pancreatic and colorectal tumors. Preclinical models have demonstrated encouraging signs of anti-tumor activity through antibody-dependent cell-mediated cytotoxicity. This is a phase 1, dose-escalation trial of NEO-102 (Ensituximab) for patients with refractory pancreatic and colorectal cancer. The primary objective was to determine safety and tolerability of escalating doses of NEO-102. Secondary objectives were to assess pharmacokinetics, anti-tumor activity and biologic correlates. Patients whose tumors express NPC-1 antigen were eligible. Dose-escalation was performed in a 3 + 3 design at doses of 1.5, 2, 3 and 4 mg/kg. A total of 19 patients (4 pancreatic and 15 colon cancer) were enrolled at participating institutions in the treatment phase. Most common treatment-related adverse events included anemia, fatigue, fevers, chills and flushing. There was no detectable hemolysis. Of twelve patients evaluable for disease response, the response rate at week 8 included 5 patients with stable disease and 8 patients with progressive disease (PD). Treatment-related grade 3/4 hyperbilirubinemia and anemia were observed at 4 mg/m2. Reversible hypoxia at 3 mg/kg was a dose-limiting toxicity. The maximum tolerated dose was established at 3 mg/kg. Of 74 patients who underwent tissue screening, positive NPC-1 expression was 47 % in colon and 59 % in pancreatic cancer. Treatment with the NEO-102, in this first-in-human study, is well tolerated with a manageable safety profile. A maximum tolerated dose of 3 mg/kg has been established. Toxicity profile is typical for this therapeutic class and allows for combination with conventional cytotoxic therapies.

NEO-201 is a novel humanized IgG1 monoclonal antibody that was derived from an immunogenic preparation of tumor-associated antigens from pooled allogeneic colon tumor tissue extracts. It was found to react against a variety of cultured human carcinoma cell lines and was highly reactive against the majority of tumor tissues from many different carcinomas, including colon, pancreatic, stomach, lung, and breast cancers. NEO-201 also exhibited tumor specificity, as the majority of normal tissues were not recognized by this antibody. Functional assays revealed that treatment with NEO-201 is capable of mediating both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against tumor cells. Furthermore, the growth of human pancreatic xenograft tumors in vivo was largely attenuated by treatment with NEO-201 both alone and in combination with human peripheral blood mononuclear cells as an effector cell source for ADCC. In vivo biodistribution studies in human tumor xenograft-bearing mice revealed that NEO-201 preferentially accumulates in the tumor but not organ tissue. Finally, a single-dose toxicity study in non-human primates demonstrated safety and tolerability of NEO-201, as a transient decrease in circulating neutrophils was the only related adverse effect observed. These findings indicate that NEO-201 warrants clinical testing as both a novel diagnostic and therapeutic agent for the treatment of a broad variety of carcinomas.

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.

Chimeric Antigen Receptor (CAR) therapy targeting CD19 achieves complete remission (CR) rates of 70%-90% in relapsed/refractory B-ALL. Relapse due to loss of the CD19 targeted epitope presents a therapeutic challenge as evidenced by the largest global pediatric CD19-CAR experience which showed 15 of 16 relapses to be explained by CD19 downregulation (Maude et al, NEJM 2018). Alternatively targeting CD22 using CD22-CAR therapy has demonstrated a CR rate of approximately 70% in both CD19+ and CD19- B-ALL, however relapse due to CD22 downregulation limits the curative potential of singularly-targeting CD22 (Fry et al, Nat Med. 2018). We hypothesized that simultaneous targeting of CD19 and CD22 via a bispecific CAR-T cell would be a safe and tolerable treatment strategy in relapsed/refractory B-cell ALL and address immune evasion. Here, we report the first clinical experience in pediatric patients using bispecific CD19-CD22 CAR T cells. We describe a single institution phase I dose escalation study in pediatric patients with relapsed or refractory B cell ALL. We utilized lentiviral transduction of a bivalent CAR construct incorporating the fmc63 CD19 and m971 CD22 single chain variable fragments (scFvs) used in clinically tested CAR constructs and a 41BB costimulatory endodomain (Fry et al, Nat Med. 2018). Our primary objectives are feasibility of production of this bivalent CAR and safety at 3 dose escalation levels (1x106, 3x106 and 1x107 CAR T cells/kg). Clinical response assessment is evaluated as a secondary aim. All patients described received lymphodepletion with fludarabine (25mg/m2 x 3 days) and cyclophosphamide (900mg/m2 x 1) followed by fresh or cryopreserved CAR T cell infusion after a 7-9 day production time. Patients were prospectively monitored at predefined intervals for disease response and correlative assessments. Four pediatric patients with precursor-B ALL, age 2-17, have been enrolled and treated with CD19/CD22 bispecific CAR T cells at dose level 1 (1x106) [Table 1]. Three patients entered CAR therapy with low disease burden detected by minimal residual disease (MRD) alone and 1 patient initiated therapy with 12% bone marrow blasts. All patients were CNS1 at time of treatment. The toxicity profile mirrored that of the singular CD19 and CD22 CAR experience with 3 patients experiencing reversible CRS (2 Grade I, 1 Grade II), onset day 3-8, and 2 patients experiencing grade I neurotoxicity, onset day 3-9. In our cohort, we experienced lower grade toxicities than previously reported, likely due to a mean lower disease burden. Only 1 patient with CRS met criteria for tocilizumab and this patient was the singular study patient treated with higher burden disease. Neurotoxicity was managed with supportive care and fully reversible. Peripheral blood flow cytometry analysis detects circulating CAR by day 6 in all patients and demonstrates peak CAR expansion between day 6-10. Peak CAR T expansion reached levels of 10-25% of total T cells with inter-patient variability in CD4 and CD8 predominance, favoring CD8 expansion in 3 of 4 patients. Clinical symptoms and inflammatory markers expectedly correlate with peak CAR expansion. Four of 4 patients achieved complete remission (CR) at day 28 post-CD19/CD22 bispecific CAR therapy. Three of 4 patients demonstrated MRD- remissions by flow cytometry and of these, next generation sequencing (NGS) was negative where available (N=2). 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. In this phase I study, we demonstrate safety and tolerability of this bispecific CD19/CD22 CAR at a dose of 1x106 CAR T cells/kg in pediatric patients with relapsed/refractory B cell ALL. The CD19/22-bispecific CAR mediated antileukemic activity in 100% of patients studied thus far. Long-term follow up and further accrual will be required to inform the effect of bispecific CAR targeting on surface antigen remodeling. Disclosures Muffly: Adaptive Biotechnologies: Research Funding; Shire Pharmaceuticals: Research Funding. Miklos:Genentech: Research Funding; Kite - Gilead: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics - Abbot: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

Autologous CD19 directed CAR T-cell therapy has response rates of >70% in adult acute lymphoblastic leukemia (ALL) and >40% in adult diffuse large B cell lymphoma (DLBCL). Large trials (ZUMA-1/JULIET/TRANSCEND) have highlighted that many patients fail to achieve durable responses. Several groups have reported on the phenomenon of CD19 immune escape as a cause (Grupp et al, NEJM 2013, Neelapu et al, NEJM 2017) and the NIH Pediatric Oncology Branch has shown CD22 as an alternative target (Fry et al, Nat Med. 2018). We developed a bi-specific CAR construct targeting CD19 & CD22 with intracellular signaling domains incorporating 4-1BB and CD3ζ (CD19/CD22.BB.z) to overcome CD19 immune escape. Here, we present our Phase I experience with this bi-specific CAR in adults. This is a single institution phase I dose escalation study enrolling patients Age ≥ 18 years with relapsed/refractory B-ALL or DLBCL after standard therapies. Primary aim is to determine feasibility of manufacturing the bi-specific CAR and safety at three dose levels (1 x 106 CAR T cells/kg, 3 x 106 CAR T cells/kg, 1 x 107 CAR T cells/kg). Clinical response was evaluated as a secondary endpoint utilizing standard response criteria for ALL and DLBCL. All patients underwent lymphodepletion with cyclophosphamide (500mg/m2 daily x3 doses) and fludarabine (30mg/m2 daily x 3 doses) followed by CAR infusion two days later. Patients were assessed at pre-defined time-points (Day 28, Month 3, 6, 9, 12 then every 6-12 months) with correlative assessments including immunophenotyping, single cell RNAseq, CAR qtPCR, serum and single cell cytokine analysis. Seven adult patients (5 DLBCL, 2 ALL), aged 35 - 75 years have been enrolled and 6 treated, all at dose Level 1 [Table 1]. The first 3 patients received freshly harvested cells and the remaining received cryopreserved cells (1 patient treated twice received initial fresh then cryopreserved product). None received systemic bridging therapy before CAR T infusion. Six patients developed reversible cytokine release syndrome (CRS,4 with Grade 1, 2 with Grade 2), onset between Day 1 to 13, and 2 patients received tocilizumab & systemic steroids. Three patients developed neurotoxicity (1 with grade 2, Day 8-11 and the others grade 1) with grade 2 neurotoxicity managed with steroids. Four patients required growth factor support beyond Day 28 and all treated patients show persistent B-cell aplasia. Two patients achieved CR: an ALL patient with disease in bone marrow/blood/CNS was MRD negative at day 28 & 60; a 75yo DLBCL patient achieved PR at day 28 and CR at month 3. Three others have ongoing PR and one died of progressive disease after initial PR at Day 28. A patient with PD at Day 28 subsequently treated with radiation and 2-months of revlimid/rituximab, now has no detectable disease 6 months post CAR-T. One patient with initial 6-month PR received a second infusion due to PD, did not develop CRS or CRES with 2nd infusion and has SD at Day 28 Notably, the patient experienced a lack of CAR-T expansion with the second infusion, raising the possibility of an immunogenic response to the CAR-T cell infusion. Flow analysis of all patients' peripheral blood showed CAR expansion peaked at median Day 13 (range Day 10-20) and CARs remained detectable [Figure 1]. Multi-parametric CyTOF phenotyping of the CAR19-22 products showed significant numbers of transduced CAR-T memory stem cells (phenotype: CD3+CD8+CD45RA+CD127+CD27+CCR7+). Single cell cytokine secretion analysis (Isoplexis,Rossi et al Blood 2018) revealed high polyfunctional strength index (PSI) in both CD4+ and CD8+ cell subsets in each patient's pre-infusion CAR product that reflected phenotypic expansion in patients. Additional correlative studies, including cytokine analysis, qtPCR based CAR quantification and CyTOF phenotypic analysis of the CAR-T cells will be presented. This first adult phase I trial of bi-specific CAR targeting CD19 & CD22 shows low toxicity with promising efficacy including achievement of CR in adult DLBCL and ALL patients. We have escalated dose to 3x 106 CAR T cells/kg and an expansion study of 60 patients will follow. CAR-T cells expanded within the first 20 days and continue to be detectable through 6 months. Disclosures Muffly: Shire Pharmaceuticals: Research Funding; Adaptive Biotechnologies: Research Funding. Miklos:Janssen: Consultancy, Research Funding; Genentech: Research Funding; Pharmacyclics - Abbot: Consultancy, Research Funding; Kite - Gilead: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

The inhibitory receptor CTLA4 has a key role in peripheral tolerance of T cells for both normal and tumor-associated antigens. Murine experiments suggested that blockade of CTLA4 might have antitumor activity and a clinical experience with the blocking antibody ipilimumab in patients with metastatic melanoma did show durable tumor regressions in some patients. Therefore, a phase II study of ipilimumab was conducted in patients with metastatic renal cell cancer with a primary end point of response by Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Two sequential cohorts received either 3 mg/kg followed by 1 mg/kg or all doses at 3 mg/kg every 3 weeks (with no intention of comparing cohort response rates). Major toxicities were enteritis and endocrine deficiencies of presumed autoimmune origin. One of 21 patients receiving the lower dose had a partial response. Five of 40 patients at the higher dose had partial responses (95% confidence interval for cohort response rate 4% to 27%) and responses were seen in patients who had previously not responded to IL-2. Thirty-three percent of patients experienced a grade III or IV immune-mediated toxicity. There was a highly significant association between autoimmune events (AEs) and tumor regression (response rate = 30% with AE, 0% without AE). CTLA4 blockade with ipilimumab induces cancer regression in some patients with metastatic clear cell renal cancer, even if they have not responded to other immunotherapies. These regressions are highly associated with other immune-mediated events of presumed autoimmune origin by mechanisms as yet undefined.

CD25+CD4+ T regulatory (Treg) cells regulate peripheral self tolerance and possess the ability to suppress antitumor responses, which may in part explain the poor clinical response of cancer patients undergoing active immunization protocols. We have previously shown that in vitro incubation of human PBMC with LMB-2, a CD25-directed immunotoxin, significantly reduced CD25+FOXP3+CD4+ Treg cells without impairing the function of the remaining lymphocytes. In the current study, eight patients with metastatic melanoma were treated with LMB-2 followed by MART-1 and gp100-specific peptide vaccination. LMB-2 administration resulted in a preferential, transient reduction in mean circulating CD25+CD4+ T cell number, from 83 ± 16 cells/μl to a nadir of 17 ± 5 cells/μl, a 79.1% reduction. FOXP3 analysis revealed a less robust depletion with mean FOXP3+CD4+ Treg cell number decreasing from 74 ± 15 cells/μl to 36 ± 8 cells/μl, a 51.4% reduction. FOXP3+CD4+ Treg cells that survived LMB-2-mediated cytotoxicity expressed little or no CD25. Similar to the peripheral blood, immunohistochemical analysis showed a 68.9% mean reduction in FOXP3+CD4+ Treg cell frequency in evaluable lesions. Despite inducing a reduction in Treg cell numbers in vivo, LMB-2 therapy did not augment the immune response to cancer vaccination and no patient experienced an objective response or autoimmunity. These data demonstrate the capacity of a CD25-directed immunotoxin to selectively mediate a transient partial reduction in circulating and tumor-infiltrating Treg cells in vivo, and suggest that more comprehensive Treg cell elimination may be required to bolster antitumor responses in patients with metastatic melanoma.

Purpose: A phase I study was conducted to assess the safety of adoptive immunotherapy using gene-modified autologous T cells for the treatment of metastatic ovarian cancer. Experimental Design: T cells with reactivity against the ovarian cancer–associated antigen α-folate receptor (FR) were generated by genetic modification of autologous T cells with a chimeric gene incorporating an anti-FR single-chain antibody linked to the signaling domain of the Fc receptor γ chain. Patients were assigned to one of two cohorts in the study. Eight patients in cohort 1 received a dose escalation of T cells in combination with high-dose interleukin-2, and six patients in cohort 2 received dual-specific T cells (reactive with both FR and allogeneic cells) followed by immunization with allogeneic peripheral blood mononuclear cells. Results: Five patients in cohort 1 experienced some grade 3 to 4 treatment-related toxicity that was probably due to interleukin-2 administration, which could be managed using standard measures. Patients in cohort 2 experienced relatively mild side effects with grade 1 to 2 symptoms. No reduction in tumor burden was seen in any patient. Tracking 111In-labeled adoptively transferred T cells in cohort 1 revealed a lack of specific localization of T cells to tumor except in one patient where some signal was detected in a peritoneal deposit. PCR analysis showed that gene-modified T cells were present in the circulation in large numbers for the first 2 days after transfer, but these quickly declined to be barely detectable 1 month later in most patients. An inhibitory factor developed in the serum of three of six patients tested over the period of treatment, which significantly reduced the ability of gene-modified T cells to respond against FR+ tumor cells. Conclusions: Large numbers of gene-modified tumor-reactive T cells can be safely given to patients, but these cells do not persist in large numbers long term. Future studies need to employ strategies to extend T cell persistence. This report is the first to document the use of genetically redirected T cells for the treatment of ovarian cancer.

We previously reported our experience in treating 56 patients with metastatic melanoma using a human anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) antibody. Durable tumor regressions were seen that correlated with the induction of autoimmune toxicities. In this study, we treated 46 additional patients using an intrapatient dose escalation schema to test whether higher doses of anti–CTLA-4 antibody would induce increased autoimmunity and concomitant tumor regression. Twenty-three patients started anti–CTLA-4 antibody administration at 3 mg/kg and 23 patients started treatment at 5 mg/kg, receiving doses every 3 weeks. Patients were dose-escalated every other dose to a maximum of 9 mg/kg or until objective clinical responses or grade III/IV autoimmune toxicity were seen. Escalating doses of antibody resulted in proportionally higher plasma concentrations. Sixteen patients (35%) experienced a grade III/IV autoimmune toxicity. Five patients (11%) achieved an objective clinical response. Two of the responses are ongoing at 13 and 16 months, respectively. Flow cytometric analysis of peripheral blood revealed significant increases in both T-cell surface markers of activation and memory phenotype. Thus, higher serum levels and prolonged administration of anti–CTLA-4 antibody resulted in a trend toward a greater incidence of grade III/IV autoimmune toxicity than previously reported, but did not seem to increase objective response rates.

The identification of many tumor-associated epitopes as nonmutated “self” Ags led to the hypothesis that the induction of large numbers of self/tumor Ag-specific T cells would be prevented because of central and peripheral tolerance. We report in this study on vaccination efforts in 95 HLA-A*0201 patients at high risk for recurrence of malignant melanoma who received prolonged immunization with the “anchor-modified” synthetic peptide, gp100209–217(210M). Vaccination using this altered peptide immunogen was highly effective at inducing large numbers of self/tumor-Ag reactive T cells in virtually every patient tested, with levels as high as 42% of all CD8+ T cells assessed by tetramer analysis. From 1 to 10% of all CD8+ cells were tumor-Ag reactive in 44% of patients and levels >10% were generated in 17% of patients. These studies were substantiated using the ELISPOT assay and a bulk cytokine release assay. Although our data regarding “tumor escape” were inconclusive, some patients had growing tumors that expressed Ag and HLA-A*0201 in the presence of high levels of antitumor T cells. There was no difference in the levels of antitumor Ag-specific T cells in patients who recurred compared with those that remained disease-free. Thus, the mere presence of profoundly expanded numbers of vaccine-induced, self/tumor Ag-specific T cells cannot by themselves be used as a “surrogate marker” for vaccine efficacy. Further, the induction of even high levels of antitumor T cells may be insufficient to alter tumor progression.

Cytotoxic T lymphocyte-associated antigen (CTLA)-4 can inhibit T-cell responses and is involved in tolerance against self antigens. We previously reported autoimmune manifestations and objective cancer regressions in patients with metastatic melanoma treated with CTLA-4 blockade. The possibility of activating tumor-reactive T cells while removing inhibitory activity with CTLA-4 blockade has stimulated interest in using anti-CTLA-4 antibodies in combination with other cancer immunotherapies to improve clinical outcomes. In this study, we assessed the antitumor activity and autoimmune toxicity of CTLA-4 blockade in combination with an immune-activating stimulus, interleukin (IL)-2, in patients with metastatic melanoma.Thirty-six patients received anti-CTLA-4 antibody every 3 weeks. Three patients per cohort received doses of .1, .3, 1.0, and 2.0 mg/kg. Twenty-four patients received 3.0 mg/kg. All patients received IL-2 therapy (720,000 IU/kg every 8 hours to a maximum of 15 doses).Eight patients (22%) experienced objective tumor responses (three complete and five partial), including metastases in the lungs, lymph nodes, mediastinum, and subcutaneous tissues. Six of the eight patients have ongoing objective responses at 11 to 19 months. Five patients (14%) developed grade III/IV autoimmune toxicities secondary to anti-CTLA-4 administration, including four patients with enterocolitis and one with arthritis and uveitis.There is not evidence to support a synergistic effect of CTLA-4 blockade plus IL-2 administration, because the 22% objective response rate is that expected from the sum of these two agents administered alone. Durable cancer regressions were seen in patients treated with this combination.

PurposePreviously, we reported our experience treating 14 patients with metastatic melanoma using a fully human antibody to cytotoxic T-lymphocyte antigen-4 (anti–CTLA-4) in conjunction with peptide vaccination. We have now treated 56 patients to evaluate two different dose schedules of anti–CTLA-4 and to explore the relationship between autoimmunity and tumor regression.Patients and MethodsA total of 56 patients with progressive stage IV melanoma were enrolled onto the study. All had Karnofsky performance status ≥ 60% with no prior history of autoimmunity. Twenty-nine patients received 3 mg/kg anti–CTLA-4 every 3 weeks, whereas 27 received 3 mg/kg as their initial dose with subsequent doses reduced to 1 mg/kg every 3 weeks. In both cohorts patients received concomitant vaccination with two modified HLA-A*0201-restricted peptides from the gp100 melanoma-associated antigen, gp100:209-217(210M) and gp100:280-288(288V).ResultsTwo patients achieved a complete response (ongoing at 30 and 31 months, respectively) and five patients achieved a partial response (durations of 4, 6, 25+, 26+, and 34+ months, respectively), for an overall objective response rate of 13%. Tumor regression was seen in lung, liver, brain, lymph nodes, and subcutaneous sites. Of 14 patients with grade 3/4 autoimmune toxicity, five (36%) experienced a clinical response compared with only two responses in the 42 patients (5%) with no autoimmune toxicity (P = .008). There were no significant differences in response rate or toxicity between the two dose schedules.ConclusionAdministration of anti–CTLA-4 monoclonal antibody plus peptide vaccination can cause durable objective responses, which correlate with the induction of autoimmunity, in patients with metastatic melanoma.

3081 Background: E is an investigational, novel, chimeric monoclonal IgG1 antibody derived from an immunogenic neoantigen with sequence homology to MUC5AC that is preferentially expressed with exquisite specificity to pancreatic cancer and CRC. Its mechanism of action is via antibody-dependent cellular cytotoxicity (ADCC). The efficacy and safety of E was evaluated in a single-arm, open-label, phase 1/2 clinical trial of adult pts with refractory mCRC. Methods: Pts were selected based on > 20% expression of tumor antigen, as measured by immunohistochemistry. Based on phase 1 results, E was administered 3 mg/kg IV every 2 weeks until unacceptable toxicity or disease progression. Primary endpoint was overall survival (OS). Serum cytokine levels were analyzed at baseline, day 4, and day 15. E-mediated ADCC of CD16 genotype V/V, V/F, and F/F pt PBMCs was measured with an IN-111 release assay using the E target-expressing ASPC-1 pancreatic cancer cell line. Results: Fifty-seven and 63 pts were evaluable for OS and safety, respectively. Median OS was significantly longer than historical control: 6.8 vs 5.0 months (mo); p = 0.007; 95% CI: 5.39,8.02. Three pts were alive at end of study (21, 21, and 24 mo); 21 pts survived ≥ 12 mo. Pts had a median of 4 prior therapies (range 2-9); 25% had received regorafenib. Forty-seven pts were evaluable by RECIST, and 20 (43%) had stable disease of target lesions at end of first course (day 57). E was well tolerated, with < 2% grade 3 and no grade 4 toxicities. There were no trends in serum cytokine and chemokine levels. Analysis of 56 samples (8 V/V, 26 V/F, 17 F/F, and 5 undetermined) showed that V/V PBMCs had significantly higher E-mediated ADCC than PBMCs harboring other genotypes. No correlation between CD16 polymorphism and pt outcome was observed. Conclusions: E demonstrated excellent tolerability and encouraging OS in this heavily pretreated population. Correlative in vitro data suggest that E can mediate higher levels of ADCC activity in individuals with a V/V versus other genotypes. The lack of correlation between CD16 polymorphism and pt outcomes in this study suggests that other immune-related factors (under investigation) may impact the efficacy of E in vivo. Clinical trial information: NCT01040000.

Interleukin-2 (IL-2) has been used to treat patients with metastatic melanoma and renal cell cancer for nearly two decades, and much progress has been made in ameliorating its adverse effects. One bothersome adverse effect, oral pain or oral irritation, is usually treated with an oral antifungal antibiotic, nystatin. The authors performed a prospective, randomized, double-blind, placebo-controlled trial involving 64 patients to evaluate the effect of prophylactic administration of nystatin or placebo on the development of oral irritation in patients receiving high-dose intravenous IL-2. No difference was found between patients randomized to receive nystatin or placebo in their rates of development of oral irritation, the severity of IL-2 adverse effects, the duration of their treatment, the rate of development of positive studies for oral yeast, or their pattern of experiencing other adverse effects. Thus, patients who receive high-dose intravenous IL-2 should not be treated prophylactically with nystatin to prevent oral irritation, and clinicians should seek evidence of the presence of oral thrush before using antifungal agents to treat oral pain in these patients.

3080Background: NEO-102 is an investigational, novel, chimeric monoclonal IgG1 antibody raised against an immunogenic membrane fraction of an allogeneic CRC vaccine. It's target has sequence homolo...

500 Background: NEO-102 is a novel chimeric mAb targeting a variant of MUC5AC with specificity to colorectal cancer. Its mechanism of action is through antibody dependent cellular cytotoxicity (ADCC). An earlier, phase I study, established the maximum tolerated dose at 3.0 mg/kg IV every 2 weeks with encouraging early signs of clinical activity. We report initial results of the subsequent phase II study. Methods: This is a single arm, open label multi-center clinical trial of NEO-102 in adults with mCRC pts who failed at least two lines of standard chemotherapy (C). An immunohistochemistry (IHC) based companion diagnostic assay was used to select eligible pts whose tumors express the target in > 20% of tumor cells. NEO-102 at 3.0 mg/kg IV was administered q 2 weeks until disease progression. The primary endpoint was OS. A minimum of 43 pts were needed assuming that treatment with NEO-102 will improve OS by 40% (7.0 months) using a one-sided significance level of 10% and 80% power for this study compared to historical control of 5 months. Additional objectives were to evaluate response rate as measured by RECIST criteria and analyze patient PBMCs for ADCC and immune cytokine profiling. Results: A total of 47 pts enrolled were evaluable. 26 pts were male and 35 pts were white. Twenty-four out of 47 pts (51%) remain alive as of September 2015 with an ongoing median OS of 7.0 months (Range 2-22 months). Of these heavily pre-treated pts, 42 were evaluable for response, 13 (31%) demonstrated stable disease by RECIST. Seven pts had more than 4 doses of treatment, maximum 13 doses. Grade 3 adverse events were anemia 1/47 (2%), hyperbilirubinemia 1/47 (2%), diarrhea 1/47 (2%), fatigue 1/47 (2%), headache 1/47 (2%), nausea 1/47 (2%) and vomiting 1/47 (2%). No grade 4 toxicities were reported. Conclusions: In the monotherapy phase 2 study of NEO 102 in patients with refractory mCRC preliminary results demonstrate excellent tolerability and encouraging OS. Updated OS and Progression Free Survival data will be presented at the ASCO 2016 GI Cancers symposium. Additional combination trials with NEO-102 and C are underway. Clinical trial information: NCT01040000.

e14013 Background: NEO-102 (N) is a novel chimeric monoclonal antibody that recognizes a variant form of MUC5AC expressed by colorectal and pancreatic tumors with minimal expression on normal tissu...

TPS499 Background: PC carries a poor prognosis. F has improved overall survival (OS) compared to G leading to its use as a 1st line treatment for patients (pts) with advanced PC. Similarly, the combination of G and A has demonstrated OS benefit. No data exists for G/A in the 2nd line setting. Many oncologists continue to sequence F treatment followed upon disease progression by G/A for pts with advanced PC with good performance status. N is a chimeric monoclonal IgG1 antibody recognizing an aberrantly glycosylated MUC5AC specific to GI tumors. Its mechanism of action is through ADCC. An IHC based companion diagnostic assay is utilized to select pts whose tumors express the target (approx. 65-70%). A prior study administering N to pts with advanced PC refractory to chemotherapy was well tolerated with disease stabilization and prolongation of OS. Furthermore 2 of 5 pts receiving the combination of G+N following progression on F remain alive at 10 and 12 months respectively. Methods: The primary objective of this phase 2.5 study is to determine if the combination of N and G plus A can improve the OS for pts who have PC over G plus A alone in the post F setting. The trial is a 2-arm randomized 1:1 multi-institution trial of N plus G and A vs. G and A. Eligible pts would be expected to have an estimated 4.5 to 4.8 month median OS with G alone, but this may be enhanced somewhat with the addition of A. The goal is to determine if the use of N along with G and A will result in pts having an increased median OS of 8 months vs. an estimated 5 months without N. Kaplan-Meier curves and a two-tailed log-rank test will be the primary analysis methods. All pts must have tissue positive for the MUC5AC variant (≥20% by IHC). Because N has been established to be safe when given with G alone but not with the combination of G and A, stopping rules for safety will be adhered to for the first 6 pts randomized to G/A plus N. Enrollment to the study has commenced. Correlative studies will utilize both PBMCs and serum for immune monitoring. Frequency, phenotype and function of different immune cells (including CD8+ T cells, CD4+ T cells, Tregs and MDSCs) will be analyzed. Clinical trial information: NCT01834235.

240 Background: NPC-1C (NEO102) is a novel chimeric monoclonal antibody that recognizes a variant form of MUC5AC expressed specifically by human pancreatic (P) and colorectal (CC) tumors. The mechanism of action is through Antibody-dependent cell-mediated cytotoxicity (ADCC). We are presenting results of dose escalation and preplanned expansion cohort of NEO-102. Methods: This is a Phase Ib/IIa, open label, multicenter clinical trial and expansion cohort of NEO102. Patients are preselected based on an Immunuhistochemistry (IHC) companion diagnostic assay. Antigen expression cut off was set at 20% expression. NEO102 was administered every 2 weeks IV with tumor assessments every 8 weeks. The primary objective was to determine safety and tolerability of NEO102. Secondary objectives include overall survival, clinical benefit, pharmacokinetics and explore the immunologic correlates. Results: Twenty-six patients (3 pancreatic and 23 colon cancer) are evaluable for toxicity and response. Median age is 55 (32 - 68) years, 14 are male. Two dose limiting toxicities were experienced at 4.0 mg/kg: grade 3 hyperbilirubinemia (n=4) and grade 3 anemia (n=1). The maximum tolerated dose (MTD) and recommended phase-2 dose (RP2D) was established at 3.0 mg/kg. Of 12 patients evaluated for response, 5 demonstrated stable disease by RECIST (42%; 4 CC, 1 P) after the first course (Day 57) and 1 PC had SD after the second course (Day 114). OS is 4.5+ months (1+ - 13.5+), median duration of treatment of 74+ days (36-133). Of those screened, 60-70% tumors were IHC positive. Immune correlates (including cytokine profile, HAMA) will be presented. Conclusions: Treatment with NEO102 is well tolerated with a manageable safety profile. RP2D is 3.0 mg/kg. Preliminary results with NEO102 have demonstrated signs of clinical activity based on stabilization of disease in heavily pretreated patients with PC and CC. A Phase II study of NEO102 monotherapy in pancreatic and colon cancer is now enrolling patients. In addition, a combination study using NEO102 with cytotoxic chemotherapy in metastatic pancreatic cancer is underway. Clinical trial information: NCT01040000.

Through the adoptive transfer of lymphocytes after host immunodepletion, it is possible to mediate objective cancer regression in human patients with metastatic melanoma. However, the generation of tumor-specific T cells in this mode of immunotherapy is often limiting. Here we report the ability to specifically confer tumor recognition by autologous lymphocytes from peripheral blood by using a retrovirus that encodes a T cell receptor. Adoptive transfer of these transduced cells in 15 patients resulted in durable engraftment at levels exceeding 10% of peripheral blood lymphocytes for at least 2 months after the infusion. We observed high sustained levels of circulating, engineered cells at 1 year after infusion in two patients who both demonstrated objective regression of metastatic melanoma lesions. This study suggests the therapeutic potential of genetically engineered cells for the biologic therapy of cancer.

Lymphopenia is a serious consequence of HIV infection and the administration of cancer chemotherapeutic agents. Although growth factors can be administered to patients to increase circulating neutrophils, there is no effective method to stimulate CD8+ lymphocyte production in humans, in vivo. This report is the first to describe the administration of recombinant interleukin-7 to humans and demonstrates the ability of this cytokine to mediate selective increases in CD4+ and CD8+ lymphocytes along with a decrease in the percentage of CD4+ T-regulatory cells. These studies suggest an important role for interleukin-7 in the treatment of patients with lymphopenia.

Purpose: Two clinical trials were conducted to evaluate the clinical efficacy and immunologic impact of vaccination against the tyrosinase protein plus systemic interleukin 2 (IL-2) administration in patients with advanced metastatic melanoma.Experimental Design: Full-length tyrosinase was employed as an immunogen to induce diverse immunologic responses against a commonly expressed melanoma antigen. Heterologous prime/boost vaccination with recombinant vaccinia and fowlpox vectors encoding tyrosinase was first explored in a randomized three-arm phase II trial, in which vaccines were administered alone or concurrently with low-dose or high-dose IL-2. In a subsequent single cohort phase II trial, all patients received the same vaccines and high-dose IL-2 sequentially rather than concurrently.Results: Among a total of 64 patients treated on these trials, 8 objective partial responses (12.5%) were observed, all in patients receiving high-dose IL-2. Additional patients showed evidence of lesional regression (mixed tumor response) or overall regression that did not achieve partial response status (minor response). In vitro evidence of enhanced immunity against tyrosinase following protocol treatments was documented in 3 of 49 (6%) patients tested serologically, 3 of 23 (13%) patients tested for T-cell recognition of individual tyrosinase peptides, and 4 of 16 (25%) patients tested for T-cell recognition of full-length tyrosinase protein with real-time reverse transcription-PCR techniques.Conclusions: Whereas prime/boost immunization with recombinant vaccinia and fowlpox viruses enhanced antityrosinase immunity in some patients with metastatic melanoma, it was ineffective alone in mediating clinical benefit, and in combination with IL-2 did not mediate clinical benefit significantly different from that expected from treatment with IL-2 alone.

Efforts to develop effective cancer vaccines often use combinations of immunogenic peptides to increase the applicability and effectiveness of the immunizations. The immunologic consequences of combining more than 1 self/tumor antigen in a single vaccine emulsion remain unclear, however. We performed 2 sequential clinical trials in patients at high risk for melanoma recurrence. Patients were given the highly immunogenic gp100:209–217(210M) peptide and the less immunogenic tyrosinase:368–376(370D) peptide once every 3 weeks for 4 weeks. This vaccination course was 12 weeks long, and patients were vaccinated for up to 4 courses (16 total vaccinations). In the first trial in 31 patients, the peptides were emulsified separately in incomplete Freund adjuvant and injected at 2 different sites. In the second trial in 33 patients, the peptides were emulsified together and injected at the same site. Cryopreserved lymphocytes were obtained by apheresis after each course and were evaluated for antipeptide activity using tetramer, enzyme-linked immunospot, and in vitro sensitization boost assays. When the peptides were injected at separate sites, robust specific reactivity to the native gp100:209–217 peptide was measured by each of the assays, whereas immunization with the tyrosinase:368–376(370D) peptide was far less effective. When the peptides were emulsified and injected together at the same site, immunization to the gp100:209–217(210M) epitope dropped precipitously, whereas reactivity to the tyrosinase: 368–376(370D) peptide was enhanced. These cautionary data indicate that mixing peptides in the same emulsion can alter reactivity compared with peptides injected separately by mechanisms that may include the induction of localized nonspecific inflammation or competitive binding of peptides to major histocompatibility complex molecules.

PurposeWe investigated the combination of lymphodepleting chemotherapy followed by the adoptive transfer of autologous tumor reactive lymphocytes for the treatment of patients with refractory metastatic melanoma.Patients and MethodsThirty-five patients with metastatic melanoma, all but one with disease refractory to treatment with high-dose interleukin (IL) -2 and many with progressive disease after chemotherapy, underwent lymphodepleting conditioning with two days of cyclophosphamide (60 mg/kg) followed by five days of fludarabine (25 mg/m2). On the day following the final dose of fludarabine, all patients received cell infusion with autologous tumor-reactive, rapidly expanded tumor infiltrating lymphocyte cultures and high-dose IL-2 therapy.ResultsEighteen (51%) of 35 treated patients experienced objective clinical responses including three ongoing complete responses and 15 partial responses with a mean duration of 11.5 ± 2.2 months. Sites of regression included metastases to lung, liver, lymph nodes, brain, and cutaneous and subcutaneous tissues. Toxicities of treatment included the expected hematologic toxicities of chemotherapy including neutropenia, thrombocytopenia, and lymphopenia, the transient toxicities of high-dose IL-2 therapy, two patients who developed Pneumocystis pneumonia and one patient who developed an Epstein-Barr virus–related lymphoproliferation.ConclusionLymphodepleting chemotherapy followed by the transfer of highly avid antitumor lymphocytes can mediate significant tumor regression in heavily pretreated patients with IL-2 refractory metastatic melanoma.

During the past decade, the identification of tumor-associated antigens has opened new opportunities for the development of cancer immunotherapies.1 Cancer vaccines using immunogenic peptide epitopes from tumor antigens have been used in clinical trials to treat patients with metastatic melanoma, with limited success. Many of these trials used immunization with human leukocyte antigen (HLA) class I-restricted peptides. Even though many of these peptides were efficient in generating high levels of peptide and/or tumor-reactive CD8+ T cells (CTLs) in vivo, positive immunologic response has not correlated with objective clinical responses.2 In the present trial, we administered HLA class I- and class II-restricted peptides derived from the tumor-associated antigen NY-ESO-1 to patients with metastatic melanoma. We hypothesized that providing specific helper CD4+ T cells by immunization with a class II-restricted epitope would lead to enhanced CTL activation and a sustained CTL response against tumor. Helper CD4+ T cells have been shown to play a central role in the initiation and maintenance of an effective CTL anti-tumor response3,4 by a variety of mechanisms, including the elaboration of appropriate cytokines and activation of antigen-presenting cells.5,6 NY-ESO-1 is an immunogenic cancer/testis antigen expressed in a variety of tumors, including melanoma, breast, ovary, lung cancers, and sarcomas.7,8 Its expression in normal tissues is limited to testis. Antibodies and/or CTL reactivity directed against NY-ESO-1 were detected in approximately 50% of patients whose tumors expressed NY-ESO-1. Both humoral and cellular immune responses to ESO-1 have been reported in patients with a variety of cancers.9,10 We recently identified two patients with strong pre-existing immunity to NY-ESO-1, both in peripheral blood and tumor-infiltrating lymphocytes (11 and unpublished data). Both patients had dramatic responses to immunotherapy. Whether NY-ESO-1 immunity played any role in their responses remains to be defined. The NY-ESO-1:157-165 was previously found to be the minimal epitope recognized by specific CD8+ T cells in an HLA-A2-restricted fashion.9 This peptide was modified in an attempt to improve peptide binding and stability in solution. The cysteine residue at P9 was replaced with valine to prevent dimerization of the peptide. The modified peptide was designated NY-ESO-1:157-165(9V) or ESO-1:165V. Stability studies using mass spectrometry revealed that the native peptide rapidly dimerized and lost activity while the modified ESO-1:165V peptide was stable in solution.12 In addition, our group recently identified a naturally processed helper epitope from NY-ESO-1 that was recognized by CD4+ T cells in the context of HLA-DPB1*0401 and *0402.13 DP4 is the most prevalent class II HLA allele in Caucasians (43–70%) and is also found to be associated with the development of antibody against ESO-1.13 Therefore, the modified HLA-A2-restricted epitope ESO-1:165V and the HLA-DP4 restricted epitope (ESO-1:161-180) were used in our ESO-1 vaccine study of patients with metastatic melanoma. In this article we report on the clinical and immunologic responses from patients who received the class I-restricted peptide alone, the class II-restricted peptide alone, or a combination of both class I- and class II-restricted peptides. The primary objective of this study was to determine whether the administration of a class I- and/or class II-restricted NY-ESO-1 peptide in patients with metastatic melanoma could elicit an immunologic response against the vaccinated peptides as measured by an in vitro sensitization (IVS) assay, and tumor regression. The secondary objective of this study was to determine toxicity and to gain information for the possible conduct of future protocols.

Summary: Cancer vaccines targeting CD8 + T cells have been successful in eliciting immunologic responses but disappointing in inducing clinical responses. Strong evidence supports the importance of CD4 + T cells in “helping” cytotoxic CD8 + cells in antitumor immunity. We report here on two consecutive clinical trials evaluating the impact of immunization with both human leukocyte antigen class I- and class II-restricted peptides from the gp100 melanoma antigen. In Protocol 1, 22 patients with metastatic melanoma were immunized with two modified class I A * 0201-restricted peptides, gp100:209-217(210M) and MART-1:26-35(27L). In Protocol 2, 19 patients received the same class I-restricted peptides in combination with a class II DRB1 * 0401-restricted peptide, gp100:44-59. As assessed by in vitro sensitization assays using peripheral blood mononuclear cells (PBMC) against the native gp100:209-217 peptide, 95% of patients in Protocol 1 were successfully immunized after two vaccinations in contrast to 50% of patients in Protocol 2 (P2 < 0.005). Furthermore, the degree of sensitization was significantly lower in patients in Protocol 2(P = 0.01). Clinically, one patient in Protocol 2 had an objective response, and none did in Protocol 1. Thus, the addition of the class IIrestricted peptide gp100:44-59 did not improve clinical response but might have diminished the immunologic response of circulating PBMC to the class I-restricted peptide gp100:209-217. The reasons for this decreased immune reactivity are unclear but may involve increased CD4 + CD25 + regulatory T-cell activity, increased apoptosis of activated CD8 + T cells, or the trafficking of sensitized CD8 + reactive cells out of the peripheral blood. Moreover, the sequential, nonrandomized nature of patient enrollment for the two trials may account for the differences in immunologic response.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a critical immunoregulatory molecule (expressed on activated T cells and a subset of regulatory T cells) capable of down-regulating T cell activation. Blockade of CTLA-4 has been shown in animal models to improve the effectiveness of cancer immunotherapy. We thus treated 14 patients with metastatic melanoma by using serial i.v. administration of a fully human anti-CTLA-4 antibody (MDX-010) in conjunction with s.c. vaccination with two modified HLA-A*0201-restricted peptides from the gp100 melanoma-associated antigen, gp100:209–217(210M) and gp100:280–288(288V). This blockade of CTLA-4 induced grade III/IV autoimmune manifestations in six patients (43%), including dermatitis, enterocolitis, hepatitis, and hypophysitis, and mediated objective cancer regression in three patients (21%; two complete and one partial responses). This study establishes CTLA-4 as an important molecule regulating tolerance to “self” antigens in humans and suggests a role for CTLA-4 blockade in breaking tolerance to human cancer antigens for cancer immunotherapy.

Immunization with plasmid DNA represents a theoretically attractive method for increasing T cell responses against cancer antigens. We administered plasmid DNA encoding the gp100 melanoma-melanocyte differentiation antigen to 22 patients with metastatic melanoma and evaluated immunologic and clinical responses. Patients were randomized to receive plasmid DNA either intradermally (n = 10) or intramuscularly (n = 12). One patient (4.5%) exhibited a partial response of several subcentimeter cutaneous nodules. All other patients had progressive disease. Of 13 patients with cells available before and after immunization, no patient exhibited evidence of the development of anti-gp100 cell responses using in vitro boost assays. The same assays were capable of demonstrating immunologic precursors after immunization with fowl poxvirus encoding gp100 or with gp100 peptides. We were thus unable to demonstrate significant clinical or immunologic responses to plasmid DNA encoding the "self" nonmutated gp100 tumor antigen.

We report here the adoptive transfer, to patients with metastatic melanoma, of highly selected tumor-reactive T cells directed against overexpressed self-derived differentiation antigens after a nonmyeloablative conditioning regimen. This approach resulted in the persistent clonal repopulation of T cells in those cancer patients, with the transferred cells proliferating in vivo, displaying functional activity, and trafficking to tumor sites. This led to regression of the patients’ metastatic melanoma as well as to the onset of autoimmune melanocyte destruction. This approach presents new possibilities for the treatment of patients with cancer as well as patients with human immunodeficiency virus-related acquired immunodeficiency syndrome and other infectious diseases.

PURPOSE: A strain of Salmonella typhimurium (VNP20009), attenuated by chromosomal deletion of the purI and msbB genes, was found to target to tumor and inhibit tumor growth in mice. These findings led to the present phase I study of the intravenous infusion of VNP20009 to patients with metastatic cancer. PATIENTS AND METHODS: In cohorts consisting of three to six patients, 24 patients with metastatic melanoma and one patient with metastatic renal cell carcinoma received 30-minute intravenous bolus infusions containing 106 to 109 cfu/m2 of VNP20009. Patients were evaluated for dose-related toxicities, selective replication within tumors, and antitumor effects. RESULTS: The maximum-tolerated dose was 3 × 108 cfu/m2. Dose-limiting toxicity was observed in patients receiving 1 × 109 cfu/m2, which included thrombocytopenia, anemia, persistent bacteremia, hyperbilirubinemia, diarrhea, vomiting, nausea, elevated alkaline phosphatase, and hypophosphatemia. VNP20009 induced a dose-related increase in the circulation of proinflammatory cytokines, such as interleukin (IL)-1β, tumor necrosis factor alpha, IL-6, and IL-12. Focal tumor colonization was observed in two patients receiving 1 × 109 cfu/m2 and in one patient receiving 3 × 108 cfu/m2. None of the patients experienced objective tumor regression, including those patients with colonized tumors. CONCLUSION: The VNP20009 strain of Salmonella typhimurium can be safely administered to patients, and at the highest tolerated dose, some tumor colonization was observed. No antitumor effects were seen, and additional studies are required to reduce dose-related toxicity and improve tumor localization.