Purpose We undertook a multi-dimensional clinical genomics study of children and adolescent young adults with relapsed and refractory cancers to determine the feasibility of genome guided precision therapy. Experimental Design Patients with non-central nervous system solid tumors underwent a combination of whole exome sequencing (WES), whole transcriptome sequencing (WTS), and high-density single nucleotide polymorphism array analysis of the tumor, with WES of matched germline DNA. Clinically actionable alterations were identified as a reportable germline mutation, a diagnosis change, or a somatic event (including a single nucleotide variant, an indel, an amplification, a deletion, or a fusion gene), which could be targeted with drugs in existing clinical trials or with Food and Drug Administration approved drugs. Results Fifty-nine patients in 20 diagnostic categories were enrolled from 2010 to 2014. Ages ranged from 7-months-old to 25-years-old. Seventy-three percent of the patients had prior chemotherapy, and the tumors from these patients with relapsed or refractory cancers had a higher mutational burden than that reported in the literature. Thirty patients (51% of total) had clinically actionable mutations, of which 24 (41%) had a mutation that was currently targetable in a clinical trial setting, 4 patients (7%) had a change in diagnosis, and 7 patients (12%) had a reportable germline mutation. Conclusions We found a remarkably high number of clinically actionable mutations in 51% of the patients, and 12% with significant germline mutations. We demonstrated the clinical feasibility of next generation sequencing in a diverse population of relapsed and refractory pediatric solid tumors.

Figure 1. CD4CARNK cells kill both peripheral T cell lymphoma cell line and primary patient malignant cells at effector to target ratios of 2 to 1 and 5 to 1. Peripheral T cell lymphoma cell lines and primary patient T cell leukemia lymphoma samples were co-cultured for 24 hours with CD4CAR NK cells. The percent of malignant cell killing was determined by comparison to vector control transduced NK cells via flow cytometry analysis of cell survival.

Background: Rhabdomyosarcoma (RMS) is most common soft tissue sarcoma in childhood and adolescence. There are two major subtypes of RMS - alveolar RMS (ARMS) and embryonal RMS (ERMS). With current treatment modalities the 5 year survival rate of patients with metastatic disease is only about 30%, thus necessitating development of novel targeted therapeutic strategies. Fibroblast Growth Factor Receptor 4 (FGFR4) is highly differentially expressed gene and activating mutations in FGFR4 is associated with increase in RMS metastasis. We hypothesize that FGFR4 can be exploited as a potential therapeutic target in RMS. Monoclonal antibodies (mAbs) against specific cancer cell surface antigens, have gained importance as therapeutic agents in other cancer types. Therefore, mAbs against FGFR4 may be developed either alone or conjugated with a chemotherapeutic agent for treatment of RMS. Methods: We have developed 15 mAbs that are of mouse, rabbit and human origin. The immunogen for rabbit antibodies is human FGFR4 extracellular domain (ECD), while human FGFR4-Fc chimeric protein or hFGFR4 transfected cells were used for mouse antibodies and were produced using hybridoma technology. Using recombinant DNA technology, human immunoglobulin library was selected using FGFR4 ECD to derive human anti-FGFR4 mAbs. All mAbs were purified using affinity chromatography and reactivity was confirmed by ELISA. These mAbs were tested for their ability to bind cell surface FGFR4 and internalization of mAb bound FGFR4 (upon incubation at 37°C) by flow cytometry using RMS cell lines. To further characterize these antibodies we have performed immunohistochemistry (IHC) on tissue microarrays (TMA) of normal tissue, xenografts of RMS cell lines, and primary tumor from RMS patients. Finally, to select a potential mAb candidate as therapeutic agent, we tested appropriate secondary antibody-drug conjugate (ADC) using cell based cytotoxic assay. Results: We observed that tested mAbs bind to both ARMS and ERMS cell lines. The level of FGFR4 expression is variable in both subtypes. Internalization assays demonstrated mAbs bound FGFR4 was internalized in RMS cell lines. Analysis by IHC on TMAs suggests that FGFR4 protein is expressed at a considerably higher level in RMS tumor tissue and xenografts compared to normal tissue. Based on these findings, we are currently screening the mAbs for their ability to elicit a cytotoxic response either alone or in conjunction with secondary ADC. Conclusion: These results suggest anti-FGFR4 mAbs can be used as therapeutic intervention for RMS. High expression of FGFR4 in other cancers such as prostate, melanoma, lung, breast, colorectal, and gastric cancers suggests the potential use of anti-FGFR4 mAbs and their derivatives in these cancers. Citation Format: Sivasubramanian Baskar, Nityashree Shivaprasad, Zhongyu Zhu, Dimiter Dimitrov, Mhairi Sigrist, Poul Sorensen, Marielle Yohe, John Shern, John Maris, Crystal Mackall, Javed Khan. FGFR4 as a potential therapeutic target for monoclonal antibody based intervention in rhabdomyosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2488. doi:10.1158/1538-7445.AM2015-2488

Background: Despite decades of multimodule therapies, RMS remains incurable once it has metastasized; thus, new therapeutic strategies are warranted. FGFR4 is a developmentally regulated cell surface receptor tyrosine kinase, overexpressed in virtually all, mutationally activated in about 7.5% of RMS, and directly activated by PAX3-FOXO1 fusion protein, which makes it a tractable target for immunotherapy. Material and Methods: Using monoclonal antibody technologies and a yeast display B-cell library, we generated 15 human or mouse binders specific to human FGFR4 and engineered into human scFvFc. All binders were successfully produced in vitro, and we further characterized them using FACS and ELISA for their specificity. Octet was used to measure the binding affinity against human FGFR4. For those lead hits, they were made into different formats of therapeutic including CAR and BiTE. We then performed in vitro killing assays and/or in vivo xenograft model to determine the efficacy of those therapeutics in killing RMS cells. Results: m410 and m412 were two lead hits and scFvFcs of these two binders were successfully produced in vitro and showed FGFR4 specificity with a binding affinity at nanomolar concentration. By ELISA, these binders showed dose-dependent binding to FGFR4 protein but not to other FGFR family members. We then made m410 and m412 into CAR and BiTE format, respectively. T cells transduced with m410 CAR construct were found highly potent in inducing gamma interferon, TNF alpha, and cytotoxicity when the FGFR4-CART are cocultured with RMS cells. Our in vivo testing found them to be effective in eliminating RMS cells in murine xenograft models. When T cells were cocultured with RMS cells in the presence of m412 BiTE in vitro, potent selective antitumor effect was observed, suggesting this can be another promising strategy for RMS immunotherapy. Conclusions: Here our data demonstrated that we had successfully generated binders specific to human FGFR4. The CAR and BiTE developed from these binders were able to kill FGFR4-positive target cells. Our data suggest that these FGFR4 CARs and FGFR4 BiTEs could provide effective immune therapies for rhabdomyosarcoma and other FGFR4-expressing cancers. Citation Format: Adam Cheuk, Nityashree Shivaprasad, Dina Schneider, Marielle Yohe, Meijie Tan, Peter Azorsa, Ronald Sams, Silvia Pomella, Berkley Gryder, Rossella Rota, Ben Stanton, Jun Wei, Young Song, Xinyu Wen, Sivasish Sindiri, Jeetendra Kumar, Robert Hawley, Joon-Yong Chung, Doncho Zhelev, Zhongyu Zhu, Dimiter Dimitrov, Stephen Hewitt, Boro Dropulic, Rimas Orentas, Javed Khan. Development of FGFR4-specific chimeric antibody receptor (CAR) T cell and bispecific T cell engager (BiTE) for rhabdomyosarcoma (RMS) immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A08.