Cell surface proteins are valuable for generation of targeted and immune-based cancer therapies. While large molecular datasets are available for multiple central nervous system (CNS) tumors, identifying such targets on a tumor-level scale to generate novel therapeutics for patients is limited by a lack of analytical pipelines. Here, we present CARTarT (CAR-T Target identification Tool), a no-code, web application that utilizes user-inputted bulk transcriptomic data to identify putative surface targets for pediatric tumors. CARTarT was built on R 4.3.1 and performs differential gene expression between tumors and normal tissue (GTEX) utilizing an empirical bayes method. Data is then filtered based on predicted-surfaceome expression, non-immune cell expression, and limited normal tissue expression to mitigate possible off-target toxicities. Targets are finally scored using a weighted scoring metric based on RNA-level log fold change, t-value, average tumoral expression, and prevalence in oncogene databases to prioritize putative targets. Targets with a score less than 0.20 were removed from our final table. A total of 919 samples from OpenPedCan were utilized to validate the tool. Amongst diffuse midline glioma specimens (n=196), we identified 100 putative surface targets. 60 (60%) of surface targets were previously identified oncogenic or cancer-associated genes. 17 surface targets (15%) are shown to have previously described immune or targeted therapeutics across cancer types, most notably identifying B7-H3, IL13RA2, EGFR, and GPC2. In addition, we identified 45 targets with potential across multiple pediatric neuro-oncologic malignancies (high grade glioma, medulloblastoma, ependymoma, atypical teratoid/rhabdoid tumor) including CHRNA5, GLP1R, B7-H3 and GPC2. These findings highlight the potential utility of CARTarT as a no-code solution for surface target identification and describe established and novel surface targets across numerous pediatric CNS tumors. We present CARTarT to the scientific community as an open-access opportunity to advance immune-based and targeted therapies for tumors.

Abstract BACKGROUND GD2-directed Chimeric Antigen Receptor (CAR) T cell therapy is a promising immunotherapeutic modality for diffuse midline glioma (DMG). We developed mRNA CAR T cells as a safe alternative to virally transduced CAR T cells and have shown that intra-tumoral (IT) infusions of mRNA GD2-directed CAR T cells result in significant tumor regression with improved toxicity in murine DMG models. For clinical translation, we sought to determine the optimal route and trafficking patterns of CAR T cells administered into the cerebrospinal fluid (CSF). METHODS NSG mice engrafted with DMG xenograft SU-DIPG13P* in the pons were treated with 5x106 mRNA CAR T cells either into the lateral ventricle (LV) or into the cisterna magna (CM), with brains harvested after 24 hours for analysis using confocal microscopy. RESULTS Both LV and CM treated mice showed higher GD2-directed CAR T cell migration to tumors compared to CD19-directed controls, with higher accumulation of GD2-directed CAR T cells in the parenchyma for LV treated mice compared to CM (p<0.001). To evaluate efficacy of delivery in the CSF, NSG mice engrafted with DMG xenograft 7316-6349 in the pons were treated with 5x106 mRNA CAR T cells IT, LV, or CM twice a week for 3 weeks totaling 6 doses. Mice were imaged weekly to monitor bioluminescent tumor signal, showing that mice treated with GD2-directed CAR T cells IT had significantly decreased tumor burdens compared to LV and CM GD2 CAR treated groups (p < 0.05 and p < 0.01 respectively), as well as compared to CD19 controls (p < 0.01). CONCLUSIONS Despite local infiltration, mRNA CAR T cells delivered into the CSF did not fully reduce tumor burden, and thus, future work is aimed at evaluating potential routes of priming tumors to attract subsequent cellular therapy doses delivered intra-ventricularly.

Abstract BACKGROUND Pediatric diffuse hemispheric glioma (DHG) is a histone-mutated (H3.3G34R/V) high-grade glioma with poor prognosis. Clinical observation and previous reports have identified that a subset of patients present with tumor-associated hemorrhage. Here, we present our findings from radiological review of these patients and determine genomic risk factors predictive for hemorrhage risk in this cohort. METHODS Data was abstracted through the Children’s Brain Tumor Network (CBTN) and EGAS00001004301. Transcriptomic and genomic analyses were completed in R 4.3.1 using edgeR, msigdbr, and xCell. Presence of blood products was determined on preoperative MRI by a board certified pediatric neuroradiologist. RESULTS 48 samples were available across cohorts with transcriptomic or genomic data. 10 samples (21%) had pre-operative imaging and transcriptomic data available. Initial analysis determined that 6 samples (60%) had acute/chronic hemorrhage based on radiological review. Samples with hemorrhage had increased levels of VEGFA (LFC: 3.45, p=2E-07) and CA9 (LFC: 5.97, p=8E-05) expression on the transcriptomic level suggesting that patients with high expression of these markers (AngioHi) had increased risk of hemorrhage compared to patients with low expression of these markers (AngioLo). AngioHi patients had notably higher levels of IL8 (LFC: 3.56, p=0.01) and increased macrophage populations. Genomic information was available for 15 samples (31%, 7 AngioHi, 8 AngioLo). PTEN alterations (n=4, 57%), FBXW7 alterations (n= 2, 29%), or PDGFRa driver alterations (n= 3, 43%) were found in the AngioHi cohort while none of these alterations were identified in the AngioLo cohort. Notably, at least one of these mutations was seen in all cases in the AngioHi cohort. CONCLUSIONS These findings identify potential mutational alterations predictive for tumor-associated hemorrhage in pediatric DHG. Current work is being completed to further characterize how these mutations impact vascular remodeling for patients with DHG and may offer targeted therapeutic opportunities.

Abstract BACKGROUND GD2-directed chimeric antigen receptor (CAR) T cells have shown promise as a potential therapeutic for diffuse midline glioma. We have previously shown intra-tumoral infusions of mRNA CAR T cells result in tumor regression with improved safety and toxicity profile in murine models. For human trials, the optimal route of CAR T cell administration into the central nervous system should maximize in vivo potency while minimizing procedural morbidity. Prior mRNA CAR T literature has reported decreased solid tumor infiltration with systemic delivery, so we sought to determine mRNA CAR T cell trafficking into pontine tumors from the cerebral spinal fluid to inform clinical translation of this therapy. METHODS Mice engrafted with SU-DIPG13P* tumor cells in the pons were treated with 5x106 mRNA CAR T cells into the lateral ventricle (LV) and brains were harvested 24 hours later. Tumor and T cells were analyzed using confocal microscopy to determine the migration of CAR T cells. RESULTS LV injection of GD2-directed CAR T cells resulted in T cell trafficking to the subarachnoid spaces, tumor site, and surrounding parenchyma. The highest concentration of T cells was in the subarachnoid space directly adjacent to the tumor (p< 0.0001) and decreased on a gradient to subcortical tumor. Even at 24 hours after injection, T cells were identified within the center of the tumor mass. CD19-directed control CAR T cells showed similar distribution within the subarachnoid with significantly decreased T cells at the tumor site. CONCLUSION Ventricular infusions of mRNA CAR T cells showed effective migration to pontine tumors within 24 hours. While CAR T cells did not fully penetrate the parenchyma within that timeframe, GD2-directed mRNA CAR T cells were detected within the center of the tumor mass, indicating ventricular administration may be sufficient for clinical delivery. Additional routes are being explored in ongoing work.