Abstract 685: GPC2 is an oncogene and immunotherapeutic target in high-risk neuroblastoma
Bibliographic record
Abstract
Abstract Background: GD2-directed immunotherapeutic strategies have improved outcomes in neuroblastoma; however, the majority of patients treated suffer relapse and GD2 expression on pain fibers causes dose-limiting toxicities. Methods: To identify alternative cell surface immunotherapeutic targets, we compared high-risk neuroblastoma (n=126 tumors) and normal tissue RNA sequencing data (GTEx; n=7859 samples from 31 normal tissues) and prioritized genes by differential and absolute expression and cell surface prediction. Genes were further surveyed for somatic copy number gain and correlative expression with MYCN amplification. Differential protein expression and localization were confirmed in neuroblastoma primary tumors (n=98), patient-derived xenografts (n=32; PDXs), cell lines (n=23), and normal pediatric tissues (n=36). Cell lines were subjected to candidate gene gain and loss of function studies (n=11). Additional pediatric tumor RNA sequencing data was surveyed followed by confirmatory immunohistochemistry (IHC). Finally, candidate specific antibodies were isolated from a human Fab phage library and utilized for antibody-drug conjugate (ADC) engineering followed by cytotoxicity studies. Results: We identified 33 differentially expressed cell surface molecules from which we prioritized glypican-2 (GPC2) for validation given GPC2’s robust differential expression (log-fold change tumor vs. normal tissue = 1.71-9.22; p=1.99 x 10-9-1.88 x10-300), high-level absolute RNA expression (median FPKM=60), and frequent DNA copy number gain associated with higher GPC2 expression (35%, n=182 tumors; p<0.005). GPC2 expression was also higher in MYCN amplified neuroblastomas (p<0.05), MYCN binds the GPC2 promoter shown by chromatin immunoprecipitation (ChIP) sequencing and reporter assays, and MYCN depletion resulted in decreased GPC2 expression. Immunoblot, flow cytometry, immunofluorescence, and IHC analysis of primary tumors, PDXs, and cell lines confirmed dense cell surface GPC2 expression. Medulloblastomas (n=62) were also found to have high GPC2 expression that positively correlated with MYC, MYCN, and GPC2 loci gain (p<0.0001). Pediatric normal tissues had very restricted cell surface GPC2 expression, with only low levels found in the esophagus and skin. GPC2 depletion in neuroblastoma cell lines resulted in apoptosis and growth inhibition and GPC2 forced over-expression increased neuroblastoma cell proliferation (p<0.001 for all assays). Finally, a human GPC2 antibody, D3-GPC2-Fab, was developed and shown to bind GPC2 with high affinity and specificity. D3-GPC2-IgG1 induced internalization of GPC2 and was conjugated to pyrrolobenzodiazepine (PBD) dimers to form an ADC which induced potent and specific cytotoxicity to GPC2 expressing neuroblastoma cells (IC50 = 1.7-11 pM). Conclusions: GPC2 is an oncogene and immunotherapeutic target in neuroblastoma and potentially other cancers. Citation Format: Kristopher R. Bosse, Pichai Raman, Maria Lane, Robyn T. Sussman, Jo Lynne Harenza, Daniel Martinez, Sabine Heitzeneder, Zhongyu Zhu, Komal Rathi, Michael Randall, Laura Donovan, Sorana Morrissy, Doncho V. Zhelev, Yang Feng, Jennifer Hwang, Yanping Wang, Bruce Pawel, Tricia Bhatti, Mariarita Santi, Javed Khan, Michael Taylor, Dimiter S. Dimitrov, Crystal Mackall, John M. Maris. GPC2 is an oncogene and immunotherapeutic target in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 685. doi:10.1158/1538-7445.AM2017-685
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".