Abstract: Parasites and pathogens are increasingly recognized as significant drivers of ecological and evolutionary change in natural ecosystems. Concurrently, transmission of infectious agents among human, livestock, and wildlife populations represents a growing threat to veterinary and human health. In light of these trends and the scarcity of long‐term time series data on infection rates among vectors and reservoirs, the National Ecological Observatory Network ( NEON ) will collect measurements and samples of a suite of tick‐, mosquito‐, and rodent‐borne parasites through a continental‐scale surveillance program. Here, we describe the sampling designs for these efforts, highlighting sampling priorities, field and analytical methods, and the data as well as archived samples to be made available to the research community. Insights generated by this sampling will advance current understanding of and ability to predict changes in infection and disease dynamics in novel, interdisciplinary, and collaborative ways.

Abstract: Nonstructural protein 5A (NS5A) represents a novel target for the treatment of hepatitis C virus (HCV). Daclatasvir, recently reported by Bristol–Myers–Squibb, is a potent NS5A inhibitor currently under investigation in phase 3 clinical trials. While the performance of daclatasvir has been impressive, the emergence of resistance could prove problematic and as such, improved analogues are being sought. By varying the biphenyl‐imidazole unit of daclatasvir, novel inhibitors of HCV NS5A were identified with an improved resistance profile against mutant strains of the virus while retaining the picomolar potency of daclatasvir. One compound in particular, methyl (( S )‐1‐(( S )‐2‐(4‐(4‐(6‐(2‐(( S )‐1‐((methoxycarbonyl)‐ L ‐valyl)pyrrolidin‐2‐yl)‐1 H ‐imidazol‐5‐yl)quinoxalin‐2‐yl)phenyl)‐1 H ‐imidazol‐2‐yl)pyrrolidin‐1‐yl)‐3‐methyl‐1‐oxobutan‐2‐yl)carbamate ( 17 ), exhibited very promising activity and showed good absorption and a long predicted human pharmacokinetic half‐life. This compound represents a promising lead that warrants further evaluation.

Abstract: Background: B7-H3 expression on central nervous system (CNS) tumors offers a potential therapeutic target for adoptive cellular therapy. Methods: A 3+3 statistical design was employed to assess the safety of repeated B7-H3 chimeric antigen receptor (CAR) T cells administered into the intracranial ventricular system (ICV) using an intrapatient dose escalation without lymphodepleting chemotherapy in pts 1-26 years of age with recurrent/refractory CNS tumors including (non-pontine) diffuse midline glioma (DMG) (NCT04185038, Arm B). Primary endpoints were feasibility to manufacture sufficient CARs and safety of weekly infusions for 3 weeks of 4-week cycles for 2 cycles. Secondary endpoints were best overall response (BOR) and correlatives of CAR activity. Results: CAR manufacturing was achieved in 20/22 (91%) enrolled pts. Eight pts were inevaluable (rapidly progressive exam precluding initial infusion (n=5), removal from protocol therapy due to progressive disease (n=2), and lack of evaluable disease prior to initial infusion (n=1)). No dose limiting toxicities were observed, identifying the maximally tolerated dose regimen (MTDR) as DR3 (maximum dose: 10 × 107 B7-H3CARs). Subjects received 98 ICV doses (median: 6 doses/patient, range: 3-13). For evaluable patients, the most common adverse events were headache (12/12, 100%), fatigue (9/12, 75%), nausea/vomiting (7/12, 58%), and fever (6/12, 50%). There was no cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). Imaging analysis is ongoing with current BOR being stable disease. CAR T cells were detected in the CSF in 9/12 evaluable pts (75%). Serial CSF/serum cytokine analysis and mass spectrometry are in process. Conclusion: Repeated ICV administration of B7-H3 CAR T cells is tolerable with circulating CAR T cells detectable in the CSF post infusion. Further investigation is warranted, along with future studies interrogating iterative enhancements such as modifications for enhanced potency and multi-antigen targeting. Citation Format: Nicholas A. Vitanza, Juliane Gust, Rebecca Ronsley, Ashley L. Wilson, Wenjun Huang, Kristy Seidel, Catherine M. Albert, Navin Pinto, Rimas J. Orentas, Rebecca A. Gardner, Michael C. Jensen, Julie R. Park. BrainChild-03: Intraventricular B7-H3 CAR T cells for recurrent/refractory central nervous system tumors and non-pontine diffuse midline glioma in children and young adults [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB121.

Abstract: Diffuse intrinsic pontine glioma (DIPG) remains a fatal brainstem tumor demanding innovative therapies. As B7-H3 (CD276) is expressed on central nervous system (CNS) tumors, we designed B7-H3–specific chimeric antigen receptor (CAR) T cells, confirmed their preclinical efficacy, and opened BrainChild-03 (NCT04185038), a first-in-human phase I trial administering repeated locoregional B7-H3 CAR T cells to children with recurrent/refractory CNS tumors and DIPG. Here, we report the results of the first three evaluable patients with DIPG (including two who enrolled after progression), who received 40 infusions with no dose-limiting toxicities. One patient had sustained clinical and radiographic improvement through 12 months on study. Patients exhibited correlative evidence of local immune activation and persistent cerebrospinal fluid (CSF) B7-H3 CAR T cells. Targeted mass spectrometry of CSF biospecimens revealed modulation of B7-H3 and critical immune analytes (CD14, CD163, CSF-1, CXCL13, and VCAM-1). Our data suggest the feasibility of repeated intracranial B7-H3 CAR T-cell dosing and that intracranial delivery may induce local immune activation. Significance: This is the first report of repeatedly dosed intracranial B7-H3 CAR T cells for patients with DIPG and includes preliminary tolerability, the detection of CAR T cells in the CSF, CSF cytokine elevations supporting locoregional immune activation, and the feasibility of serial mass spectrometry from both serum and CSF. This article is highlighted in the In This Issue feature, p. 1

Abstract: Patients with H3K27M-mutant diffuse midline glioma (DMG) have no proven effective therapies. ONC201 has recently demonstrated efficacy in these patients, but the mechanism behind this finding remains unknown. We assessed clinical outcomes, tumor sequencing, and tissue/cerebrospinal fluid (CSF) correlate samples from patients treated in two completed multisite clinical studies. Patients treated with ONC201 following initial radiation but prior to recurrence demonstrated a median overall survival of 21.7 months, whereas those treated after recurrence had a median overall survival of 9.3 months. Radiographic response was associated with increased expression of key tricarboxylic acid cycle–related genes in baseline tumor sequencing. ONC201 treatment increased 2-hydroxyglutarate levels in cultured H3K27M-DMG cells and patient CSF samples. This corresponded with increases in repressive H3K27me3 in vitro and in human tumors accompanied by epigenetic downregulation of cell cycle regulation and neuroglial differentiation genes. Overall, ONC201 demonstrates efficacy in H3K27M-DMG by disrupting integrated metabolic and epigenetic pathways and reversing pathognomonic H3K27me3 reduction. Significance: The clinical, radiographic, and molecular analyses included in this study demonstrate the efficacy of ONC201 in H3K27M-mutant DMG and support ONC201 as the first monotherapy to improve outcomes in H3K27M-mutant DMG beyond radiation. Mechanistically, ONC201 disrupts integrated metabolic and epigenetic pathways and reverses pathognomonic H3K27me3 reduction.

Abstract: Relapse following chimeric antigen receptor (CAR) T-cell therapy directed against CD19 for relapsed/refractory B-acute lymphoblastic leukemia (r/r B-ALL) remains a significant challenge. Three main patterns of relapse predominate: CD19 positive (CD19pos) relapse, CD19 negative (CD19neg) relapse, and lineage switch (LS). Development and validation of risk factors that predict relapse phenotype could help define potential pre- or post-CAR T-cell infusion interventions aimed at decreasing relapse. Our group sought to extensively characterize preinfusion risk factors associated with the development of each relapse pattern via a multicenter, retrospective review of children and young adults with r/r B-ALL treated with a murine-based CD19-CAR construct. Of 420 patients treated with CAR, 166 (39.5%) relapsed, including 83 (50%) CD19pos, 68 (41%) CD19neg, and 12 (7.2%) LS relapses. A greater cumulative number of prior complete remissions was associated with CD19pos relapses, whereas high preinfusion disease burden, prior blinatumomab nonresponse, older age, and 4-1BB CAR construct were associated with CD19neg relapses. The presence of a KMT2A rearrangement was the only preinfusion risk factor associated with LS. The median overall survival following a post-CAR relapse was 11.9 months (95% CI, 9-17) and was particularly dismal in patients experiencing an LS, with no long-term survivors following this pattern of relapse. Given the poor outcomes for those with post-CAR relapse, study of relapse prevention strategies, such as consolidative hematopoietic stem cell transplantation, is critical and warrants further investigation on prospective clinical trials.

Abstract: CD19-targeted chimeric antigen receptor T cells (CD19-CAR) and blinatumomab effectively induce remission in relapsed or refractory B-cell acute lymphoblastic leukemia (ALL) but are also associated with CD19 antigen modulation. There are limited data regarding the impact of prior blinatumomab exposure on subsequent CD19-CAR outcomes. PATIENTS AND METHODS We conducted a multicenter, retrospective review of children and young adults with relapsed or refractory ALL who received CD19-CAR between 2012 and 2019. Primary objectives addressed 6-month relapse-free survival (RFS) and event-free survival (EFS), stratified by blinatumomab use. Secondary objectives included comparison of longer-term survival outcomes, complete remission rates, CD19 modulation, and identification of factors associated with EFS. RESULTS Of 420 patients (median age, 12.7 years; interquartile range, 7.1-17.5) treated with commercial tisagenlecleucel or one of three investigational CD19-CAR constructs, 77 (18.3%) received prior blinatumomab. Blinatumomab-exposed patients more frequently harbored KMT2A rearrangements and underwent a prior stem-cell transplant than blinatumomab-naïve patients. Among patients evaluable for CD19-CAR response (n = 412), blinatumomab nonresponders had lower complete remission rates to CD19-CAR (20 of 31, 64.5%) than blinatumomab responders (39 of 42, 92.9%) or blinatumomab-naive patients (317 of 339, 93.5%), P 〈 .0001. Following CD19-CAR, blinatumomab nonresponders had worse 6-month EFS (27.3%; 95% CI, 13.6 to 43.0) compared with blinatumomab responders (66.9%; 95% CI, 50.6 to 78.9; P 〈 .0001) or blinatumomab-naïve patients (72.6%; 95% CI, 67.5 to 77; P 〈 .0001) and worse RFS. High-disease burden independently associated with inferior EFS. CD19-dim or partial expression (preinfusion) was more frequently seen in blinatumomab-exposed patients (13.3% v 6.5%; P = .06) and associated with lower EFS and RFS. CONCLUSION With the largest series to date in pediatric CD19-CAR, and, to our knowledge, the first to study the impact of sequential CD19 targeting, we demonstrate that blinatumomab nonresponse and high-disease burden were independently associated with worse RFS and EFS, identifying important indicators of long-term outcomes following CD19-CAR.

Abstract: Introduction: Chimeric antigen receptor (CAR) T-cells redirected against CD19 have demonstrated remarkable clinical activity in children and adults with relapsed/refractory (r/r) B-cell malignancies. The risk of lineage switch (LS) following CD19-directed therapies has been well documented but has been primarily limited to case reports. Additionally, the risk of subsequent malignant neoplasms (SMN) following CAR T-cells has not yet been described. Distinguishing LS (B-ALL to myeloid malignancy) from a therapy-related myeloid neoplasm is both clinically and biologically relevant. The former emerges from a highly refractory leukemic clone, likely resistant to salvage therapy, whereas the latter represents a new malignancy that can be associated with long-term survival. Methods: We conducted a multicenter, retrospective review of children and young adults with r/r B-acute lymphoblastic leukemia (B-ALL) who received either commercial tisagenlecleucel or 1 of 3 investigational murine-based CD19-CAR constructs on clinical trials at 7 US centers between 2012-2019. Patients diagnosed with B-ALL before age 25 years were included and patients who had received any prior CAR product were excluded. Results: Of 420 CAR-treated patients, with a median follow-up of 30.1 months, 12 (2.9%) experienced LS and 6 (1.4%) developed a SMN (Table). The median time to diagnosis of LS following CAR T-cell infusion was significantly shorter compared to diagnosis of SMN (65.5 days vs. 883.5 days; p=0.005). Eleven of 12 patients (91.7%) with LS converted to acute myeloid leukemia (AML). One patient converted to mixed phenotype acute leukemia, B/myeloid type. The leukemia of 10 of 12 patients with LS harbored cytogenetics similar to those at initial diagnosis. For the remaining 2 patients with LS, cytogenetics were unavailable, but the leukemias were considered LS by the treating institution. KMT2Ar rearrangement (KMT2Ar) was a predominant cytogenetic abnormality seen in patients with LS. Overall, 38 of 420 patients (9%) had a KMT2Ar. KMT2Ar was present in 9 of 12 (75%) patients with LS compared to 20 of 408 (7.1%) non-LS patients (p & lt;0.001). Patients with LS were younger at initial diagnosis compared to the remaining cohort (median age, 1.6 years vs. 7.7 years; p=0.001), reflecting the inherent association between KMT2Ar and infant ALL. Otherwise, there were no significant differences in gender, prior hematopoietic stem cell transplant (HSCT), prior blinatumomab exposure, or CAR response. Within the KMT2Ar cohort, 31 (81.6%) patients achieved a complete remission post-CAR. Eight of these patients received a consolidative HSCT (representing 4 first and 4 second HSCTs). No KMT2Ar patient experienced a post-HSCT LS and 3 are alive with a median follow-up of 1164 days post-CAR. In contrast, of the 23 KTM2Ar patients who did not receive HSCT post-CAR, 7 developed LS and 14 are alive with a median follow-up of 864 days post-CAR. Relative contraindications to post-CAR HSCT included a prior HSCT (n=11) or early LS (n=5). Of the 7 CAR non-responding patients with KMT2Ar, 2 (28.6%) had rapid emergence of LS by the first restaging timepoint. There are no long-term survivors following LS, regardless of KMT2A status, dying a median of 123 days (range, 36-594 days) after diagnosis of LS. The 6 SMNs were cholangiocarcinoma, synovial sarcoma, malignant melanoma and 3 therapy-related myeloid neoplasms (MDS/AML), distinguished from LS based on loss of original cytogenetics. Notably, 4/6 (67%) patients that developed a SMN had received an allogeneic HSCT prior to development of SMN. Four patients (67%) remain alive and in remission with a median follow-up of 304 days after diagnosis of SMN, including 2 patients with MDS/AML. Conclusions: In the largest series of pediatric patients treated with CAR T-cell therapy, we show that LS occurs in 2.9% of children. The presence of a KMT2Ar was the biggest risk factor, with 23.7% of these patients experiencing LS. We found that LS can occur very early in a patient's post CAR T-cell course, and despite a variety of treatment approaches, the outcomes for these patients are dismal. Given the predisposition to LS, the role for consolidative HSCT in KMT2Ar patients warrants further study. Limited by a short follow-up period, we saw SMNs in only 1.4% of our patients. Causality is unknown and likely unrelated to CAR-T, but this further supports the long-term safety of CAR T-cells in children with B-ALL. Figure 1 Figure 1. Disclosures Borowitz: Amgen, Blueprint Medicines: Honoraria. Lee: Harpoon Therapeutics: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Other: trial funding; Gilead: Other: trial funding. Grupp: Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Kite, Vertex, and Servier: Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Verneris: jazz: Other: advisory board; Novartis: Other: advisory board; Fate Therapeutics: Consultancy. Gore: Mirati: Current equity holder in publicly-traded company; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Honoraria; Clovis: Current equity holder in publicly-traded company; Celgene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Sanofi Paris: Current equity holder in publicly-traded company; Anchiano: Current equity holder in publicly-traded company; Blueprint Medicines: Current equity holder in publicly-traded company. Brown: Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Kura: Membership on an entity's Board of Directors or advisory committees; KIte: Membership on an entity's Board of Directors or advisory committees. Pulsipher: Equillium: Membership on an entity's Board of Directors or advisory committees; Adaptive: Research Funding; Jasper Therapeutics: Honoraria. Rheingold: Pfizer: Research Funding; Optinose: Other: Spouse's current employment. Gardner: BMS: Patents & Royalties; Novartis: Consultancy.