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PURPOSE: WNK3 kinase (PRKWNK3) has been implicated in the development and function of the brain via its regulation of the cation-chloride cotransporters, but the role of WNK3 in human development is unknown. METHOD: We ascertained exome or genome sequences of individuals with rare familial or sporadic forms of intellectual disability (ID). RESULTS: We identified a total of 6 different maternally-inherited, hemizygous, 3 loss-of-function or 3 pathogenic missense variants (p.Pro204Arg, p.Leu300Ser, p.Glu607Val) in WNK3 in 14 male individuals from 6 unrelated families. Affected individuals had identifier with variable presence of epilepsy and structural brain defects. WNK3 variants cosegregated with the disease in 3 different families with multiple affected individuals. This included 1 large family previously diagnosed with X-linked Prieto syndrome. WNK3 pathogenic missense variants localize to the catalytic domain and impede the inhibitory phosphorylation of the neuronal-specific chloride cotransporter KCC2 at threonine 1007, a site critically regulated during the development of synaptic inhibition. CONCLUSION: Pathogenic WNK3 variants cause a rare form of human X-linked identifier with variable epilepsy and structural brain abnormalities and implicate impaired phospho-regulation of KCC2 as a pathogenic mechanism.

Immune effector cell (IEC) therapies have revolutionized our approach to relapsed B-cell malignancies, and interest in the investigational use of IECs is rapidly expanding into other diseases. Current challenges in the analysis of IEC therapies include small sample sizes, limited access to clinical trials and a paucity of predictive biomarkers of efficacy and toxicity associated with IEC therapies. Retrospective and prospective multi-center cell therapy trials can assist in overcoming these barriers through harmonization of clinical endpoints and correlative assays for immune monitoring, allowing additional cross-trial analysis to identify biomarkers of failure and success. The Consortium for Pediatric Cellular Immunotherapy (CPCI) offers a unique platform to address the aforementioned challenges by delivering cutting-edge cell and gene therapies for children through multi-center clinical trials. Here the authors discuss some of the important pre-analytic variables, such as biospecimen collection and initial processing procedures, that affect biomarker assays commonly used in IEC trials across participating CPCI sites. The authors review the recent literature and provide data to support recommendations for alignment and standardization of practices that can affect flow cytometry assays measuring immune effector function as well as interpretation of cytokine/chemokine data. The authors also identify critical gaps that often make parallel comparisons between trials difficult or impossible.

BACKGROUND: Thoracic endovascular repair (TEVAR) is considered the first-line therapy in the repair of acute complicated type B aortic dissection (AC-BAD). Given the difficulty of designing randomized trials in this surgical cohort, long-term outcome data is limited. This systematic review and meta-analysis provide a complete aggregation of reported long-term survival and freedom from reintervention of AC-BAD patients based on the existing literature. METHODS: Three databases were searched from date of database inception to January 2021. The relevant references were identified and baseline cohort characteristics, survival and freedom from reintervention were extracted. The primary endpoints were survival and freedom from reintervention, whilst secondary endpoints were post-operative outcomes such as cord ischemia and endoleak. Kaplan-Meier curves were digitized and aggregated as per established procedure. RESULTS: A total of 2,812 references were identified in the literature search for review, with 46 selected for inclusion. A total of 2,565 patients were identified, of which 1,920 (75%) were male. The mean age of the cohort was 59.8±5.8. Actuarial survival at 2, 4, 6 and 10 years was 87.5%, 83.2%, 78.5% and 69.7%, respectively. Freedom from all secondary reintervention at 2, 4, 6, 8 and 10 years was 74.7%, 69.1%, 65.7%, 63.9% and 60.9%, respectively. When accounting for study quality, actuarial survival at 2, 4, 6 and 8 years was 85.4%, 79.1%, 69.8% and 63.1%, respectively. Freedom from all secondary reintervention at 2, 4, 6 and 8 years was 73.2%, 67.6%, 63.7% (maintained), respectively. CONCLUSIONS: TEVAR is associated with promising long-term survival extended to 10 years, though rates of freedom from reintervention remain an ongoing point for improvement. Randomized controlled trials comparing endovascular with open repair in the setting of acute, complicated type B aortic dissection are needed.

The benefits of cancer information-seeking may be particularly salient to individuals impacted by childhood cancer, including patients, caregivers, health professionals, and advocates. The purpose of this study was to explore information-seeking patterns for childhood cancer through the National Cancer Institute's Cancer Information Service (CIS), a multi-channel, bilingual resource for cancer information. The study team conducted descriptive analyses on secondary data characterizing 1820 caregivers, health professionals, organizations, and members of the general public who contacted the CIS about childhood cancer between September 2018 and June 2022. Almost 80% of inquiries about childhood cancer were initiated by caregivers, followed by the public, health professionals, and organizations. Although English was the primary language used by individuals to reach the CIS when discussing childhood cancer, there were variations in points of access (i.e., telephone, instant messaging, email, social media) across the four user groups. Most childhood cancer inquiries were about staging and treatment, and the primary cancer sites discussed by CIS users were neurologic or brain, hematologic, and musculoskeletal cancers. Discussion topics included managing and coping with cancer, clinical trials, and treatment side effects. Just over half (54%) of CIS contacts about childhood cancer resulted in a health professional referral. Findings provide direction for the CIS and other public health organizations to deliver, prioritize, and tailor their services to support the information needs of childhood cancer survivors and their families-as well as those who care and advocate for them-who may have a significant need for credible cancer information.

Locoregional delivery of chimeric antigen receptor (CAR) T cells has resulted in objective responses in adults with glioblastoma, but the feasibility and tolerability of this approach is yet to be evaluated for pediatric central nervous system (CNS) tumors. Here we show that engineering of a medium-length CAR spacer enhances the therapeutic efficacy of human erb-b2 receptor tyrosine kinase 2 (HER2)-specific CAR T cells in an orthotopic xenograft medulloblastoma model. We translated these findings into BrainChild-01 ( NCT03500991 ), an ongoing phase 1 clinical trial at Seattle Children’s evaluating repetitive locoregional dosing of these HER2-specific CAR T cells to children and young adults with recurrent/refractory CNS tumors, including diffuse midline glioma. Primary objectives are assessing feasibility, safety and tolerability; secondary objectives include assessing CAR T cell distribution and disease response. In the outpatient setting, patients receive infusions via CNS catheter into either the tumor cavity or the ventricular system. The initial three patients experienced no dose-limiting toxicity and exhibited clinical, as well as correlative laboratory, evidence of local CNS immune activation, including high concentrations of CXCL10 and CCL2 in the cerebrospinal fluid. This interim report supports the feasibility of generating HER2-specific CAR T cells for repeated dosing regimens and suggests that their repeated intra-CNS delivery might be well tolerated and activate a localized immune response in pediatric and young adult patients. In an interim analysis of a phase 1 trial, repeated intracranial infusions of HER2-specific CAR T cells were well tolerated with no observed dose-limiting toxicities in three young adult patients with CNS tumors.

Over the past two decades surgical approaches for mitral valve (MV) disease have evolved with the advent of minimally invasive techniques. Robotic mitral valve repair (RMVr) safety and efficacy has been well documented, however, mid- to long-term data are limited. The aim of this review was to provide a comprehensive analysis of the available mid- to long-term data for RMVr.Electronic searches of five databases were performed to identify all relevant studies reporting minimum five-year data on RMVr. Pre-defined primary outcomes of interest were overall survival, freedom from MV reoperation and from moderate or worse mitral regurgitation (MR) at five years or more post-RMVr. A meta-analysis of proportions or means was performed, utilizing a random effects model, to present the data. Kaplan-Meier curves were aggregated using reconstructed individual patient data.Nine studies totaling 3,300 patients undergoing RMVr were identified. Rates of overall survival at 1-, 5- and 10-year were 99.2%, 97.4% and 92.3%, respectively. Freedom from MV reoperation at eight-years post RMVr was 95.0%. Freedom from moderate or worse MR at seven years was 86.0%. Rates of early post-operative complications were low with only 0.2% all-cause mortality and 1.0% cerebrovascular accident. Reoperation for bleeding was low at 2.2% and successful RMVr was 99.8%. Mean intensive care unit and hospital stay were 22.4 hours and 5.2 days, respectively.RMVr is a safe procedure with low rates of early mortality and other complications. It can be performed with low complication rates in high volume, experienced centers. Evaluation of available mid-term data post-RMVr suggests favorable rates of overall survival, freedom from MV reoperation and from moderate or worse MR recurrence.

Robotic-assisted mitral valve surgery (RMVS) is becoming an increasingly performed procedure in cardiac surgery, however, its true safety and efficacy compared to the gold standard conventional sternotomy approach [conventional sternotomy mitral valve surgery (CSMVS)] remains debated. The aim of this meta-analysis was to provide a comprehensive analysis of all available literature comparing RMVS to CSMVS.An electronic search of five databases was performed to identify all relevant studies comparing RMVS to CSMVS. Pre-defined primary outcomes of interest included all-cause mortality, cerebrovascular accidents (CVA) and re-operation for bleeding. Secondary outcomes of interest included cross clamp time, cardiopulmonary bypass (CPB) time, intensive care unit (ICU) and hospital length of stay (LOS), post-operative atrial fibrillation (POAF) and red blood cell (RBC) transfusion.The search strategy identified fourteen studies qualifying for inclusion in this meta-analysis comparing RMVS to CSMVS. The outcomes of 6,341 patients (2,804 RMVS and 3,537 CSMVS) were included. RMVS had significantly lower mortality when compared to CSMVS group in both the unmatched [odds ratio (OR) 0.33; 95% confidence interval (CI): 0.19-0.57; P0.001] and matched cohorts (OR 0.35; 95% CI: 0.15-0.80; P=0.01). There was no significant difference in rates of CVA or re-operation for bleeding between the two groups in either the entire included cohort or matched patients. CSMVS had significantly shorter cross clamp time by 28 minutes (95% CI: 19.30-37.32; P0.001) and CPB time by 49 minutes (95% CI: 36.16-61.01; P0.001) which remained significantly shorter in the matched cohorts. RMVS had shorter ICU [mean difference (MD) 26 hours; 95% CI: -34.31 to -18.52; P0.001] and hospital LOS (MD 2 days; 95% CI: -2.66 to -1.37; P0.001), which were again both significantly shorter in the matched cohort. RMVS group also had fewer RBC transfusions (OR 0.44; 95% CI: 0.28-0.70; P0.001).Current evidence on comparative outcomes of RMVS and CSMVS is limited with only low-quality studies currently available. This present meta-analysis suggests that RMVS may have lower mortality and shorter ICU and hospital LOS, however CSMVS may be associated with significantly shorter cross clamp and CPB times. Further analysis of high-quality studies with randomized data is required to verify these results.

Cells rely on activity-dependent protein-protein interactions to convey biological signals, but the state-dependent interactome is notoriously cell-specific and undercharacterized1. In the case of chimeric antigen receptor (CAR) T cells containing a 4-1BB costimulatory domain, receptor engagement is thought to trigger the formation of protein complexes similar to those triggered by T cell receptor (TCR)-mediated signaling, but the number and type of protein-interaction-mediating binding domains differ between CARs and TCRs. Here, we performed co-immunoprecipitation mass spectrometry of a 2nd generation CD19-directed 4-1BB:zeta CAR (referred to as bbζCAR) and identified 67 proteins that increased their co-association after target engagement. We compared activity-induced TCR and CAR signalosomes using quantitative multiplex co-immunoprecipitation and showed that bbζCAR engagement leads to activation of two modules of protein interactions, one similar to TCR signaling that is more weakly engaged in bbζCAR vs. TCR, and one composed of TRAF signaling complexes that is not engaged by the TCR. Batch-to-batch and inter-individual variations in IL2 production correlated with differences in the magnitude of protein network activation. Future CAR T cell manufacturing protocols could measure, and eventually control, biological variation by monitoring these signalosome activation markers.One Sentence SummaryWe define a network of protein interactions engaged by chimeric antigen receptors following target binding, and show that the magnitude of network activation correlates with IL-2 secretion, a proxy measure for CAR T cell function.

Background: Outcomes for relapsed osteosarcoma (OS) remain poor and there are no systemic therapies that have been shown to provide a survival benefit. Chimeric Antigen Receptor (CAR) T cell immunotherapy involves the adoptive transfer of T-lymphocytes that have been engineered to recognize tumor-specific antigens, resulting in targeted lysis of malignant cells. Folate receptors (FR) are membrane-bound surface proteins that bind folates with high affinity. FRs are overexpressed in OS and have very limited expression in normal tissue. UB-TT170 (Umoja Biopharma) is a small molecule bispecific “adapter” consisting of folate conjugated to fluorescein (FL). It penetrates tumors in minutes and is retained for long periods of time due to high affinity for the FR, while unbound UB-TT170 rapidly clears from the blood and from FR- tissues. We hypothesize that administration of fixed dosing anti-FL(FITC-E2) CAR T cells followed by escalating doses of UB-TT170 will deliver personalized immunotherapy to OS patients. In addition, this approach offers an attractive safety mechanism since administration of NaFL should reverse CAR T cell reactivity. Methods: We designed a Phase I study for young adult (15-30 yr) patients with recurrent/refractory OS to examine the safety and feasibility of administering autologous, peripheral blood-derived T cells that have been genetically modified to express a 2nd generation FL-specific CAR in combination with intra-subject dose escalation of UB-TT170. The primary objective is to identify a recommended dose escalation sequence of UB-TT170 to move forward in clinical development. A 3+3 design will be used to investigate 3 possible dosing sequences. The trial opened in July 2022. As of January 5, 2023, 5 subjects have enrolled and one subject has been treated on dose regimen 1. T cell products have been successfully manufactured for all enrolled subjects. The trial remains open to enrollment. Clinical trial registration numbers: NCT05312411 Citation Format: Catherine M. Albert, Navin R. Pinto, Mallory Taylor, Ashley Wilson, Stephanie Rawlings-Rhea, Prabha Narayanaswamy, Wenjun Huang, Kristy Seidel, Stephanie Mgebroff, Christopher Brown, Jacob Garcia, Jeffrey Teoh, Rimas J. Orentas, Rebecca A. Gardner, Michael C. Jensen, Julie R. Park. ENLIGHTen-01: a phase 1 study of fluorescein-specific (FITC-E2)-car T cells in combination with folate-fluorescein (UB-TT170) for osteosarcoma [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 CT137.

Survey length and monetary incentives are design features that have been found to affect survey participation in opposing ways. Declining response rates make these key design features even more important. However, survey length has received surprisingly little attention, possibly being viewed as a static survey attribute rather than an alterable design feature. In addition, experimental tests of the interaction between survey length and monetary incentives could not be found, despite calls for the need for such research and theories that support such an interaction. Leverage-salience theory leads to opposing expectations, depending on the relative importance people give to different factors affecting survey participation. Making the survey request in parts, to reduce burden, may increase participation. Yet, it may instead decrease participation relative to the status quo of offering the full incentive for the entire survey if incentives play a larger role than survey length. When factoring both burden and motivation, the results for survey length could likely depend on the structure of monetary incentives. We designed an experiment to test a modularized survey, ending one part of the survey with a request to complete another part. Respondents in the modular survey design were also assigned to proportionate or disproportionate incentives—offering a larger proportion of the incentive for the initial survey request. Surprisingly, the two-part survey request led to not lower but greater nonresponse. There was increased potential for nonresponse bias attributable to nonresponse to the second request, without reduction in item nonresponse and measurement error. Disproportionate allocation of incentives across modules seemed to increase overall participation compared with proportionate allocation. The lack of a beneficial effect of a request for a shorter survey and the results on incentive allocation have important implications for survey practitioners. Furthermore, the inconsistency in findings on survey length requires further research to identify mediating factors.

Background Cardiac surgery is associated with a high rate of blood use. The aim of this study is to identify preoperative patient factors associated with allogeneic Red Blood Cell (RBC) or non-Red Blood Cell (NRBC) use in cardiac surgery. Methods All adult cardiac surgical procedures conducted at a single Western Australian institution were retrospectively analysed. Data was collected from the Australia and New Zealand Cardiac Surgery Database from 2015 to 2018. A number of preoperative factors were identified, relating to past medical history or preoperative cardiac status. Outcome 1 was defined as the use of one or more RBC products intra or post-operatively. Outcome 2 was defined as the use of one or more NRBC products intra or post-operatively. Multivariate logistical regression analysis was done to assess for the association between preoperative factors and allogeneic blood product use. Results A total of 1595 patients were included in this study, of which 1488 underwent a Coronary Artery Bypass Graft, Valve or a combined procedure. Patients on dialysis preoperatively and those who had preoperative cardiogenic shock demonstrated the greatest risk of requiring RBC transfusion with an odds ratio of 5.643 (95% CI 1.305–24.40) and 3.257 (95% 1.801–5.882) respectively. Patients who had preoperative cardiogenic shock demonstrated the greatest risk of requiring NRBC transfusion with an odds ratio of 3.473 (95% CI 1.970–6.135). Patients who have had a previous cardiothoracic intervention are at increased risk of both RBC and NRBC transfusion, with adjusted odds ratios of 1.774 (95% CI 1.353–2.325) and 2.370 (95% CI 1.748–3.215) respectively. Conclusion A number of factors relating to past medical history or preoperative cardiac status are implicated with increased allogeneic blood product use in cardiac surgery. Identifying high-risk patients in a preoperative setting can enable us enrol them in a blood conservation program, therefore minimizing the risk of exposure to blood transfusion.

Because of the COVID-19 pandemic, in-person services for individuals with neurodevelopmental disabilities were disrupted globally, resulting in a transition to remote delivery of services and therapies. For individuals with neurogenetic conditions, reliance on nonclinical caregivers to facilitate all therapies and care was unprecedented. The study aimed to (1) describe caregivers’ reported impact on their dependent’s services, therapies, medical needs, and impact on themselves as a result of the COVID-19 pandemic and (2) assess the relationship between the extent of disruption of services and the degree of self-reported caregiver burden. Two online questionnaires were completed by caregivers participating in Simons Searchlight in April and May 2020. Surveys were completed by caregivers of children or dependent adults with neurodevelopmental genetic conditions in Simons Searchlight. Caregivers reported that the impact of the COVID-19 pandemic moderately or severely disrupted services, therapies, or medical supports. The majority of caregivers were responsible for providing some aspect of therapy. Caregivers reported “feeling stressed but able to deal with problems as they arise,” and reported lower anxiety at follow-up. Caregivers reported that telehealth services were not meeting the needs of those with complex medical needs. Future surveys will assess if and how medical systems, educational programs, therapists, and caregivers adapt to the challenges arising during the COVID-19 pandemic.

Bullous pemphigoid (BP) is the most common autoimmune blistering disorder of the skin. It is typified by tense blisters with a subepidermal split and mixed dermal inflammatory infiltrate on histology. Biopsy of the perilesional skin for direct immunofluorescence (DIF) has become the gold standard in the diagnosis of BP. Currently there is a pervasive clinical opinion that the lower extremity is a site with a high false-negative rate (FNR) for DIF in the diagnosis of BP. This notion is primarily based on 2 early studies from the 1980s without more recent confirmatory studies. To readdress this question regarding the lower extremities, a retrospective study from 2012 to 2018 was performed in our institution that evaluated the FNR of DIF by an anatomical site in the diagnosis of BP. Cases of BP were identified using standard criteria (clinical and histological data reviewed in cases with negative DIF), and overall, 79 patients were included in the study. A total of 4 false-negative DIF biopsies were verified. Two negative DIF were from the lower extremity yielding a FNR of 10% compared with 4% on the trunk and 3% from the upper extremity, with no statistically significant difference by anatomical sites. Our study fails to demonstrate a high FNR of DIF from the lower extremity in the diagnosis of BP.

CD19 CAR T cell therapy has emerged as a promising therapy in the treatment of B cell malignancies, such as B cell acute lymphoblastic leukemia (B-ALL). However, questions remain regarding the heterogeneity of the CAR T cell infusion products that lead to differential clinical outcomes and side effects, including cytokine release syndrome and neurotoxicity. The purpose of this study is to identify biomarkers and T cell subpopulations within 1) leukapheresis samples of patients receiving CD19 CAR T cell therapy and 2) pre-infusion manufactured CAR T cell products to identify correlates with clinical outcomes. We hypothesize that different manufacturing practices used to generate CAR T cells from the patient’s leukapheresis T cells drive heterogeneity within the infusion products that drive differential patient outcomes, and that cell intrinsic properties within the leukapheresis T cells drive phenotypic dispositions observed in the infusion products. Leukapheresis samples and pre-infusion CAR T cells were obtained from pediatric and adult B-ALL patients enrolled in CD19 CAR T cell clinical trials. Briefly, leukapheresis T cells from pediatric patients were CD4/8 sorted and then stimulated with CD3/CD28 beads followed by CAR transduction. Pediatric CD4 CAR T cells were cultured with IL-7 + IL-15 while CD8 CAR T cells were cultured with IL-2 + IL-15. CAR T cells from adults were stimulated with LCLs expressing CD19. To identify T cell subpopulations and biomarkers leukapheresis T cells and CAR T cells from each patient were stained with two mass cytometry panels: 1) a phenotype panel of antibodies to characterize surface markers, transcription factors and chemokine receptors and 2) a functional panel of antibodies specific for cytokines to broadly characterize the secretion profile of leukapheresis and CAR T cells upon stimulation with PMA and Ionomycin. Additional multi-omic profiling was performed with CITE-seq and 10X Chromium on leukapheresis samples and CAR T cells of select adult patients to provide in depth analysis on transcriptomic drivers of phenotypes observed within CAR T cell infusion products. We hierarchically clustered leukapheresis T cells and CAR T cells and correlated the abundance of T cell subclusters within each patient to their neurotoxicity grade (0-5). We determined that CD4 CAR T cells that secrete elevated levels of IL-3 upon stimulation correlate with an increased risk of severe neurotoxicity (designated as grades 3-5) within our pediatric cohort. Identification of IL-3 as a driver of neurotoxicity within the CD4 infusion products may inform strategies to improve manufacturing practices used to generate safer CAR T cell products. Citation Format: Tony Chour, Alexandre Hirayama, Ye Zheng, Alyssa Sheih, Summer Zhuang, Ashley Wilson, Vicky Wu, Raphael Gottardo, Cameron Turtle, Rebecca Gardner, Evan Newell. Cellular profiling of leukapheresis and infusion products of patients enrolled in CD19 CAR T cell therapy identifies biomarkers associated with clinical outcomes [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 3910.

Background: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Despite the survival rate of 90% for newly diagnosed children with ALL, the outcome for relapsed patients is historically poor with a less than 30% survival. CD19 CAR T-cell therapy (CART19) has shown remarkable response rates, between 80-90% in relapsed/refractory disease. Little is known about antigen-independent factors that predict initial resistance to CART19. We hypothesized that leukemias that are resistant to CART19 are distinct from sensitive leukemias and that these differences can be detected prior to therapy. Methods: To interrogate differences between resistant and sensitive leukemias, we obtained pre-treatment bone marrow aspirates (BMAs) from patients enrolled in a clinical trial at Seattle Children’s Hospital (PLAT-02). Samples were categorized based on patient response, with non-response defined as not achieving and maintaining minimal residual disease negativity at Day +63. Our study included 7 resistant and 7 sensitive leukemias as controls. We performed whole exome sequencing, bulk RNA-seq, PacBio-seq of the CD19 locus, array-based methylation, ATAC-seq, scRNA-seq, and CyTOF. Results: We found that non-response to CART19 is independent of leukemic subtype. Despite blasts being CD19+ in all patients by flow cytometry, we identified alternative splicing of CD19 in one non-responder, while the remaining non-responders expressed high levels of wildtype CD19. We discovered a distinctive DNA methylation pattern in the non-responders characterized by hypermethylation of PRC2 targets in embryonic and cancer stem cells (p = 8.15E-25) Furthermore, using gene set enrichment analysis of ATAC-seq data, we found increased accessibility of chromatin at regions associated with stem cell proliferation (NES = 2.31; p < 0.0001) and cell cycling (NES = 2.27; p < 0.0001). We found a greater similarity between accessibility patterns of non-responders to hematopoietic progenitors, including hematopoietic stem cells (p = 0.037) and common myeloid progenitors (p = 0.047). These findings were supported by an increased frequency of cell subpopulations expressing a multi-lineage phenotype (CD19, CD20, CD33, CD34; p = 0.009). Moreover, we find decreased expression of antigen presentation and processing pathways across all leukemic cells relative to responders (p = 0.0001). Conclusions: This study, one of the most comprehensive multi-omic analyses of samples from patients treated with CAR T-cells, identified resistance mechanisms that can be detected prior to treatment. We report the novel association of a stem cell phenotype, lineage plasticity, and decreased antigen presentation with resistance. These results support further refinement of eligibility for CART19 for children with leukemia and highlights the need for alternative of complimentary approaches for these patients. Citation Format: Katherine E. Masih, Rebecca Gardner, Hsien-Chao Chou, Abdalla Abdelmaksoud, Young K. Song, Luca Mariani, Vineela Gangalapudi, Berkley E. Gryder, Ashley Wilson, Serifat O. Adebola, Benjamin Z. Stanton, Chaoyu Wang, Xinyu Wen, Gregoire Altan-Bonnet, Michael C. Kelly, Jun S. Wei, Martha L. Bulyk, Michael C. Jensen, Rimas J. Orentas, Javed Khan. Multi-omic analysis identifies mechanisms of resistance to CD19 CAR T-cell therapy in children with acute lymphoblastic leukemia [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 3581.

TPS7078 Background: The acute and late effects of acute myeloid leukemia (AML) therapy in children can be devastating, and relapse rates remain high. Targeted therapies are needed to decrease toxicity and improve cure rates. Most AML patients express CD33 on their leukemic blasts. CD33 is a viable target with CD33 targeted therapies approved for AML. CD33 targeted CAR T cell therapies are being developed, but safety and efficacy remain a challenge. Safety concerns include cytokine release syndrome, neurotoxicity and aplasia. Efficacy is limited, with antigen escape and T cell exhaustion proposed as mechanisms of treatment failure. Dimerizing Agent Regulated ImmunoReceptor Complex (DARIC) is a next-generation technology that utilizes a split receptor design separating the antigen binding and intracellular signaling subunits (Leung, 2019). These subunits remain inactive until the administration of rapamycin which allows for dimerization of the subunits, enabling T cell activation when antigen is present. Modulating CAR T cell activation may aid hematopoietic recovery, enhance CAR T cell function and persistence, and mitigate toxicity. We developed a DARIC T cell product, SC-DARIC33, that couples the DARIC technology with a lentiviral construct that incorporates the use of a novel humanized camelid nanobody for targeting the C2 splice isoform within the membrane proximal domain of CD33. Methods: Up to 18 patients will be enrolled in this phase 1 study (NCT05105152). Patients must have early first relapse of AML (≤ 6 months from diagnosis), first relapse refractory to at least one reinduction attempt, second or greater relapse, or refractory de novo AML. The first 3 patients must be ≥18 years, all patients who meet eligibility criteria must be ≤28 years. The primary objectives of this Phase I study (NCT05105152) are assessment of safety/toxicity and feasibility of manufacturing of SC-DARIC33. Secondary objectives include assessment of efficacy, on/off rate of DARIC expression, engraftment, expansion and persistence of SC-DARIC33. Also, the effect of cessation of rapamycin on activation state of SC-DARIC 33 and on hematopoietic recovery will be evaluated. Subjects receive lymphodepletion prior to a single dose of SC-DARIC33 T cells on Day 0 followed by initiation of rapamycin on Day +2 with the purpose of activating the T cells. Rapamycin continues until Day 21 and bone marrow evaluation occurs on Day 28. Subsequent cycles of rapamycin are allowed for patients who respond. The trial will implement a BOIN design with three possible dose levels (1 x 106, 5 x 106, and 10 x 106 DARIC+ cells/kg) to determine the maximum tolerated dose (MTD). The trial is open for enrollment at Seattle Children’s Hospital. Clinical trial information: NCT05105152.

BrainChild-03 is a phase 1 clinical trial delivering repeated locoregional 2nd generation B7-H3 CAR T cells with 4-1BB co-stimulation to children with central nervous system (CNS) tumors without lymphodepleting chemotherapy. The primary endpoints are feasibility and safety, with secondary endpoints of disease response and correlatives of CAR T cell activity. There are 3 arms: (A) – weekly delivery into the tumor cavity, (B) – weekly delivery into the lateral ventricle for metastatic disease, (C) – biweekly delivery into the lateral ventricle for diffuse intrinsic pontine glioma (DIPG). In total, 23/24 (96%) enrolled patients have had successful CAR T manufacturing. 16/24 patients are evaluable and have received a total of 141 intracranial CAR T cell doses. Unevaluable patients include 5 never treated and 3 who progressed prior to receiving the minimum doses to become evaluable. The most common adverse events have been headache (16/16, 100%), nausea/vomiting (12/16, 75%), and fever (10/16, 63%). There has been 1 DLT for an intratumoral hemorrhage and no cytokine release syndrome (CRS). 7 evaluable patients with DIPG (Arm C) have received a cumulative 50 infusions. 5/7 DIPG patients enrolled after progression and have a median survival of 246.5 days post-initial CAR T cell infusion, with 4/5 still alive. The 2 DIPG patients enrolled prior to progression had radiographic improvement, including 1 with improvement of a cranial nerve 6 palsy who self-withdrew from protocol therapy after 18 infusions over 12 months and 1 still on protocol therapy after 11 infusions over 6 months. DIPG patients have had increased CSF levels of proinflammatory mediators (e.g. CXCL10, CCL2, IFNg, GM-CSF, IL-12) without systemic cytokine changes. 5/7 DIPG patients had detectable CAR T cells in their CSF post-infusion. Ultimately, the preliminary experience suggests locoregional delivery of B7-H3 CAR T cells may be feasible and tolerable in children with CNS tumors, including DIPG.

Considering the high expression of B7-H3 (CD276) on diffuse intrinsic pontine glioma (DIPG) and preliminary evidence that repeated locoregional delivery of CAR T cells to patients with CNS tumors was feasible and tolerable, we open the phase 1 trial BrainChild-03 (NCT04185038). Children with DIPG in Dose Regimen 1 received 40 intraventricular B7H3CAR doses, with the two patients enrolling post-progression surviving >400 days from initial CAR T infusion. To evaluate for immune and tumor responses that may correlate with clinical and radiographic evaluations, we performed targeted proteomic analysis on serial CSF and serum biospecimens. MRM-MS is a targeted mass spectrometry that provides sensitive measurement of proteins in cancer tissues and fluids. In 2 patients with longitudinal biospecimens, we identified 50 CSF and 59 serum proteins above level of detection. In general, there were fewer serum fluctuations compared to the CSF, supporting that intracranial delivery provides local immune activation. Sharp fluctuations of several immunoregulatory peptides were measured in the CSF at pre and post infusion timepoints, including BCL10, CXCL13, TIM-3, ICOSLG, and PD-L2. Notably, several analytes tracked consistently in the CSF of both patients, including markers of macrophage maturation and immune cell recruitment, including CD14, CD163, CD44, CSF-1, CXCL13 and VCAM-1. B7-H3 was detected in the CSF and serum of both patients. S005, who progressed on protocol therapy, had a sharp increase in B7-H3 in the CSF during Course 2, while S008, who had clinical improvement on protocol therapy, had consistently lower B7-H3 present. Notably, serum B7-H3 steadily declined in both patients over time, with the exception of a transient increase in S005 between Courses 4 and 5. Future work for all enrolled patients with DIPG will explore the correlation of these targeted proteomic measurements with clinical end points for potential use as markers of efficacy of therapy or adverse events.

10011 Background: B7H3 (CD276) has limited expression in normal tissues and high cell-surface expression in pediatric solid malignancies providing rationale for immunologic therapeutic targeting. We present a first-in-human experience of B7H3 chimeric antigen receptor T cells (CAR-T) for children and young adults (CYA) with relapsed or refractory solid tumors (R/RST). Methods: CYA patients with R/RST were enrolled onto a Phase 1 trial (NCT04483778) to examine the safety of autologous T cells genetically modified to express scFV-IgG4hinge-CD28tm-4-1BB-zeta B7H3-specific CAR with the methotrexate resistance/selection cassette DHFRdm and the tracking/suicide construct EGFRt. All patients received lymphodepleting fludarabine and cyclophosphamide prior to infusion of cryopreserved CAR-T at the prescribed dose level. The maximal tolerated dose or biologically effective dose (BED) was determined based upon observed toxicity through day 28 from initial CAR-T infusion and using a 3+3 statistical design. Results: Sixteen subjects (age 11-24, median 17 years) enrolled and received dose level (DL) 1 (0.5 x 106 CAR-T/kg, n = 3) or DL2 (1 x 106 CAR-T cells/kg, n = 6). No dose limiting toxicity was observed following first infusion, most common toxicities were fatigue and cytokine release syndrome (CRS) (n = 2, maximum CTCAE grade 2). Maximum circulating CAR-T expansion on first infusion was 4.98 cells/uL (range 0.23-4.98 cells/uL) with median persistence of 28 days (range 14-90). Best overall response of Stable Disease was observed in 3 of the 9 subjects infused. Given observed expansion and persistence, DL2 was determined to be the BED. A second infusion at DL2 in one subject demonstrated CAR T expansion to 1590 cells/uL (86% of circulating CD3 cells) with CTCAE grade 2 CRS and transient dose limiting CTCAE grade 4 liver enzyme elevation. A partial metabolic response on FDG-PET by PERCIST criteria was observed in this subject at Day 28. Conclusions: B7H3 CAR T cells are safe and demonstrate anti-tumor activity in CYA with R/RST. CAR-T cell expansion and persistence may be necessary to achieve objective responses. STRIvE-02 Arm B will explore dual expression of CD19 CAR with B7H3 CAR, using lymphocytic CD19 expression to drive CAR expansion and persistence. Clinical trial information: NCT04483778.

2541 Background: The epidermal growth factor receptor (EGFR) is a cell surface tyrosine kinase receptor associated with cell proliferation and differentiation. EGFR expression and activating mutations are associated with aggressive neoplastic disease, chemotherapy resistance, and increased metastatic potential. Published data and EGFR immunohistochemistry (IHC) performed on tissue microarrays indicate that 15-40% of pediatric solid tumors (ST) express EGFR. The unique EGFR monoclonal antibody (mAb) 806 selectively binds to an epitope that is conformationally hidden when EGFR is tethered but revealed when tethering is perturbed as occurs with EGFR overexpression, truncation, or through extra-cellular domain missense mutations. Methods: Children and young adults (CYA) with EGFR-expressing recurrent/refractory (R/R) ST were enrolled on a Phase 1 trial to examine the safety and feasibility of administering autologous chimeric antigen receptor (CAR) T cells derived from autologous T cells genetically modified to express a second generation EGFR806-specific scFV-IgG4hinge-CD28tm/cyto-4-1-BB-zeta and EGFRt tracking/suicide contract. All subjects received lymphodepleting chemotherapy with fludarabine and cyclophosphamide prior to the administration of cryopreserved CAR T cells a the prescribed dose level. The biologically effective dose (BED) or maximum tolerated dose was determined based upon observed toxicity through day 28 from initial CAR-T infusion and using a 3+3 statistical design. Results: Eleven subjects (n=10 evaluable, age range 9-25, median 18) were enrolled and received either dose level (DL) 1 (0.5 x 106 CAR-T/kg, n=4) or DL2 (1 x 106 CAR-T cells/kg, n=7). CAR T were manufactured successfully in all subjects. Most common toxicities were fatigue, tumor-related pain and cytokine release syndrome (n=2, maximum CTCAE grade 1). Dose limiting toxicity of CTCAE grade 4 transaminase level and hyperbilirubinemia occurred at DL2 (n=1). Maximum circulating CAR-T expansion was 29.66 cells/uL (range 0.05-29.66 cells/uL) with median persistence of 28 days (range 0-90). Two subjects on DL1 and one subject on DL2 demonstrated mixed response on day 28 and tolerated additional CAR T infusion without dose limiting toxicity. Conclusions: EGFR806 directed CAR-T cells have an acceptable toxicity profile in CYA with R/RST and demonstrate anti-tumor activity in some patients. Additional analyses are ongoing to identify biomarkers of response and toxicity. Clinical trial information: NCT03618381.

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Background: CD19 targeting chimeric antigen receptor (CAR) T cells have induced unprecedented remission rates in high-risk precursor B Acute Lymphoblastic Leukemia (ALL); however recurrent disease with CD19 antigen escape variants is not uncommon. Therefore, we developed a novel CD22 targeting CAR, and following preclinical validation, tested it in a first-in-human pediatric and young adult phase 1 clinical trial, PLAT-04 (NCT03244306). Four subjects were treated at 2 dose levels (DL) (1x10 6/kg (DL1) and 3x10 6/kg (DL2)). The CD22 CAR T cell product (SCRI-CAR22v1) was successfully manufactured (n=4) and no dose limiting toxicity (DLTs), cytokine release syndrome (CRS) or neurotoxicity was observed. However, all subjects had minimal CAR T cell expansion, with 3 of 4 subjects demonstrating persistent or progressive disease at day 21 evaluation despite continued CD22 expression on leukemic blasts. Based on the poor in vivo expansion and lack of activity, enrollment was voluntarily halted to interrogate and optimize the CAR construct for enhanced performance. Methods: Human T cells were transduced to express one of two CD22 CAR constructs. We designed SCRI-CAR22v2, a CD22 CAR that utilizes the same scFv as SCRI-CAR22v1 but with a shorter linker between M971 VH and VL and a shorter hinge with differing transmembrane region, and both using CD8 alpha (Figure A). This construct maintained the truncated EGFR extracellular tag (EGFRt) for tracking and potential in vivo suicide mechanism. The two transduced CAR T cell products were compared preclinically by flow cytometry, chromium release assay and in an in vivo murine model to understand differing T cell activity between the CAR constructs. Additionally, SCRI-CAR22v2 is currently under investigation in a dose finding phase 1 clinical trial, PLAT-07 (NCT04571138). Results: Following use of cetuximab-APC and biotinylated anti-human Fab antibody for surface EGFRt and CAR detection, the SCRI-CAR22v1 expresses lower levels of EGFRt but similar CAR levels on the cell surface demonstrated by MFI (Figure B). Biotinylated, soluble CD22 antigen was also used to evaluate CD22 CAR receptor activity and, as measured by MFI, a higher affinity is suggested via SCRI-CAR22v2 as compared to SCRI-CAR22v1 (Figure B). K562 cells expressing low, medium or high CD22 were used to evaluate the impact of surface antigen expression on the CAR activity level. SCRI-CAR22v2 demonstrates improved targeted cell lysis at all 3 antigen quantity levels by chromium release assay (Figure C). In NSG mice inoculated with Raji tumor cells expressing ffluc, SCRI-CAR22v2 demonstrated improved survival compared to SCRI-CAR22v1 (Figure D) and clearance of Raji tumor cells (Figure E). Based on this promising preclinical data, we initiated enrollment onto PLAT-07, a phase 1 dose finding trial (2x10 5cells/kg (DL1), 5x10 5cells/kg (DL2) and 1x10 6cells/kg (DL3)) of SCRI-CAR22v2. To date, 3 subjects have been enrolled and successfully infused at DL1. All had prior CD19-CAR therapy and 2 lacked CD19 leukemic expression at the time of SCRI-CAR22v2 infusion. At the time of cell infusion, one subject had only extramedullary disease, one had MRD of Conclusions: Despite encouraging preclinical data, SCRI-CAR22v1 demonstrated poor expansion and engraftment in a Phase 1 trial. Notably, minor CAR alterations lead to encouraging in-human activity in early clinical findings. Our experience suggests a shorter linker and hinge as well as incorporation of an CD8 alpha transmembrane region improves the clinical activity of CD22 targeted CAR T cells in subjects with recurrent disease following CD19 CAR T cells. Further evaluation is needed to elucidate the critical CAR components and/or assays at the preclinical level that can best predict which CAR should be brought to the clinic for further evaluation. Figure 1 Figure 1. Disclosures Orentas: Lentigen: Patents & Royalties. Jensen: BMS: Patents & Royalties; Umoja Biopharma: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bluebird Bio: Research Funding. Gardner: Novartis: Consultancy; BMS: Patents & Royalties.

Introduction: CAR T cells in B-ALL have recently focused on the dual targeting of CD19 and CD22 to enhance long term remissions and prevent antigen negative recurrence that is frequently encountered with single antigen targeting. However, a barrier to this approach has been the retention of dual specificity killing and ongoing persistence. PLAT-05 is a multisite phase 1 trial (NCT03330691) that was undertaken to evaluate the safety and feasibility of SCRI-CAR19x22v1, a dual transduced patient-derived product with lentiviral vectors encoding for either a CD19- or CD22-specific CAR, both with 4-1BB co-stimulation. Early results of the first 27 subjects infused demonstrated feasibility and a favorable safety profile with encouraging CR rates. Products were fractionated evenly between CD19 CAR, CD19+CD22 CAR and CD22 CAR. However, engraftment was predominated by the single CD19 CAR population, leading to unsuccessful eradication of CD19-CD22+ leukemia. This finding led to re-engineering the CD22 CAR construct for enhanced CD22 targeting, and re-initiation of dose finding with the new product, SCRI-CAR19v2. Methods: After enrollment, subjects undergo apheresis followed by a combined CD4/CD8 positive immunomagnetic selection and seeded at a prescribed ratio for co-culture in a closed-system G-Rex bioreactor. Following anti-CD3xCD28 bead stimulation, T cells are transduced with two lentiviral vectors that encode for either a CD19- or CD22-specific CAR. After flu/cy lymphodepletion, CAR T cells are infused at one of three dose levels: 0.5, 1 or 3 X 10 6 CAR T cells/kg. Toxicity is graded according to CTCAEv5 except for CRS and ICANS which are graded per ASTCT criteria. Leukemic response and CAR T cell persistence are evaluated by flow cytometry. Results: 14 subjects enrolled onto PLAT-05 for the SCRI-CAR19x22v2 dose escalation and products were successfully manufactured in all subjects with an average of 8.9 days in culture (range 7-12 days). In contrast to v1 products, the CAR composition of v2 products was skewed in favor of CD22 CAR expression, with median expression of each population as follows: 42% CD22 only, 33% CD19 and CD22, 3.2% CD19 only. Twelve subjects were infused (0.5x10 6/kg n=3, 1x10 6/kg n=3, 3x10 6/kg n=6), 11 of whom had prior exposure to CD19 or CD22 targeted therapies with diverse expression of CD19 and CD22 on the leukemic blasts. No dose limiting toxicities occurred in the 11 fully evaluable subjects (1 subject is pending) and the recommended phase 2 dose was determined as 3x10 6 CAR + cells/kg. CRS was present in 45% of subjects, all grade 1. Neurotoxicity occurred in 45% of subjects, all grade 1 except a single self-limited grade 3 ICANS event (due to a single time point CAPD score). 91% of infused subjects obtained a CR, of which 100% were MRD negative. The non-responder had persistent disease that was CD19-CD22-. The in vivo engraftment of CAR T cells peaked most frequently between day +7 and +14 and was predominated by the CD22 CAR T cells, with some minimal contribution of dual and CD19 CAR T cells. Of the 4 subjects who had previously received an FMC63 based CD19 CAR, expansion was due to solely to the CD22 CARs in all 4 subjects, with apparent rejection of the T cells expressing CD19 CAR. Conclusions: We demonstrate enhanced activity of SCRI-CAR19x22v2 compared to v1, now with dual activity against both CD19 and CD22 demonstrated by elimination of ALL with single antigen expression. We maintained encouraging CR rates with a favorable toxicity profile. Interestingly, the product is predominated by CD22 CAR and CD19/CD22 CAR populations, while in vivo engraftment is predominated by single CD22 CAR expressing T cells. Subjects exposed to prior CD19 murine based CAR rejected the CD19 CAR but engrafted the CD22 CAR with demonstratable activity, a potential advantage of a dual transduced product. The impact of lower CD19 CAR engraftment on durable remissions is unknown. While limited expansion of the CD19 CAR population could be protective against exhaustion, the uneven engraftment of the CAR populations may ultimately lead to single antigen targeting. Optimization of transduction may be required for a more balanced product to maintain dual targeting and give further insight into the behavior of dual-expressing CAR T cells. An expansion cohort is currently underway to further characterize engraftment kinetics and in vivo performance to best inform future development of this product. Figure 1 Figure 1. Disclosures Pulsipher: Jasper Therapeutics: Honoraria; Adaptive: Research Funding; Equillium: Membership on an entity's Board of Directors or advisory committees. Li: Novartis Canada: Membership on an entity's Board of Directors or advisory committees. Jensen: Bluebird Bio: Research Funding; Umoja Biopharma: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Patents & Royalties. Gardner: Novartis: Consultancy; BMS: Patents & Royalties. OffLabel Disclosure: investigational use of SCRI-CAR19x22 will be discussed

Following preclinical optimization of B7-H3-specific CAR T cells against pediatric brain tumor models, we opened BrainChild-03 (NCT04185038), a phase 1 clinical trial of repeatedly dosed, outpatient, locoregional B7-H3-specific CAR T cells for children with recurrent/refractory central nervous system (CNS) tumors or diffuse intrinsic pontine glioma (DIPG). Here, we report the interim findings from patients enrolled on Arm C, dedicated to DIPG. The primary endpoints are feasibility and safety, with secondary endpoints of disease response. We utilize second-generation CAR T cells with a 4-1BB costimulatory domain and a methotrexate-resistant human DHFR mutein (huDHFRFS; L22F,F31S), allowing for methotrexate selection. We do not deliver conditioning chemotherapy. The first three evaluable patients with DIPG all met feasibility for generating a balanced CD4:CD8 CAR T cell product, with 3.85x109 CAR T cells generated for S005, 4.29x109 for S008, and 2.45x109 for S012, allowing for greater than 6 months of biweekly dosing for each patient. All subjects were treated at Dose Level 1 (1x107 CAR T cells). S005 received 10 doses before clinical progression greater than 2 years from diagnosis, S008 has received 10 doses and continues on therapy with decreased tumor volume, and S012 has received 5 doses and continues on study with stable disease. There have been no dose limiting toxicities (DLT). 3/3 patients exhibited post infusion fever, headache, and elevated serum CRP but no evidence of cytokine release syndrome (CRS) or systemic CAR T cells. 0/3 patients required PICU admissions. In the cerebrospinal fluid (CSF), 2/3 patients have had elevations of cytokines such as CXCL10 and CCL2, as well as circulating CSF CAR T cells. Advanced serial patient CSF proteomic and transcriptomic profiling are underway. Ultimately, this report provides preliminary evidence that outpatient locoregional B7-H3 CAR T cells for children with DIPG may be feasible and tolerable.