Analysis and establishment of new 'nanobody-drug-conjugates' as a therapeutic option for late stage and cisplatin-resistant germ cell tumors as well as an alternative to standard chemotherapy - revised version.
Final Report Abstract
With steadily increasing incidences, testicular germ cell tumors (GCT) represent the most common tumor type in young men aged 14 - 45 years. All type II GCT, including seminoma and non-seminoma, arise from a common precursor lesion, the so-called 'germ cell neoplasia in situ' (GCNIS), which originates from a misguided germ cell development. The therapeutic approach for GCT generally comprises orchiectomy followed by chemo- or radiotherapy. Even though most GCT respond well towards cisplatin-based chemotherapy, the disease can have a lethal progression due to the development of metastases and resistance mechanisms. Moreover, chemotherapy is often accompanied by strong side effects and diminished fertility. For this reason, there is an urgent need to identify novel therapeutic concepts presenting with fewer side effects for the treatment of therapy-resistant GCT. This study aimed at identifying alternative therapeutic options for the treatment of GCT based on immunotherapeutic approaches, such as nanobody- / antibody-drug-conjugates (NDC / ADC) or natural killer cells with a chimeric antigen receptor (NK-CAR). More specifically for the treatment of yolk-sac tumors (YST), multi-kinase inhibitors have been further evaluated. Initially, putative therapeutic targets have been identified. For this purpose, based on previous work, factors, whose mRNA expression was significantly increased in GCT cells compared to non-cancerous fibroblast control cells, were identified as suitable therapeutic targets. Further, using flow cytometry- and immunofluorescence analyses, the membranous location of these targets was validated in 16 different (cisplatin-resistant) GCT cell lines as well as in 11 non-cancerous (immune) cells, such as fibroblasts or macrophages. Based on this screen, an ADC targeting the protein Claudin6 (CLDN6) has been successfully established, where a CLDN6-binding antibody was conjugated to the microtubule-disrupting agent MMAE. In GCT, high levels of CLDN6 were detected, accordingly, treatment with CLDN6-ADC was very efficient in GCT cells in vitro. However, aggressive and therapy-resistant YST harbored a CLDN6-negative fraction of about 40 %, eventually leading to the conclusion that the efficacy of the CLDN6-ADC approach might be reduced in YST. Further molecular characterization of (resistant) YST identified and verified various druggable signaling cascades, which could be targeted using multi-kinase inhibitors. Moreover, during this study, a CXCR4 targeting NDC was evaluated. Also, the CXCR4-NDC was able to induce apoptosis specifically in CXCR4+ seminoma and YST cells. Beyond that, an uncoupled nanobody against CXCR7 (a CXCR4- related chemokine receptor) has been tested. As such, blockage of the CXCR7 receptor resulted in diminished migratory and proliferative potential of GCT cells. Finally, an immunotherapy against CD24 has been established. Here, high levels of CD24 were observed in embryonal carcinoma (a non-seminomatous GCT subtype), as well as other urologic tumor malignancies, such as urothelial-, prostate-, and renal cell carcinoma. As such, the application of NK-CD24-CAR cells enhanced apoptosis induction specifically in CD24+ urological tumor cells. In summary, in this study, we could generate an atlas of novel therapeutic targets and establish promising therapeutic strategies for the treatment of GCT as well as urologic malignancies, which could pave the road for prospective continuative studies.
Publications
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CD24 targeting with NK‐CAR immunotherapy in testis, prostate, renal and (luminal‐type) bladder cancer and identification of direct CD24 interaction partners. The FEBS Journal, 290(20), 4864-4876.
Söhngen, Christian; Thomas, David J.; Skowron, Margaretha A.; Bremmer, Felix; Eckstein, Markus; Stefanski, Anja; Driessen, Marc D.; Wakileh, Gamal A.; Stühler, Kai; Altevogt, Peter; Theodorescu, Dan; Klapdor, Rüdiger; Schambach, Axel & Nettersheim, Daniel
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Molecular characterization of the CXCR4 / CXCR7 axis in germ cell tumors and its targetability using nanobody-drug-conjugates. Experimental Hematology & Oncology, 12(1).
Wakileh, Gamal A.; Bierholz, Philipp; Kotthoff, Mara; Skowron, Margaretha A.; Bremmer, Felix; Stephan, Alexa; Anbuhl, Stephanie M.; Heukers, Raimond; Smit, Martine J.; Ströbel, Philipp & Nettersheim, Daniel
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Targeting CLDN6 in germ cell tumors by an antibody-drug-conjugate and studying therapy resistance of yolk-sac tumors to identify and screen specific therapeutic options. Molecular Medicine, 29(1).
Skowron, Margaretha A.; Kotthoff, Mara; Bremmer, Felix; Ruhnke, Katja; Parmaksiz, Fatma; Richter, Annika; Küffer, Stefan; Reuter-Jessen, Kirsten; Pauls, Stella; Stefanski, Anja; Ströbel, Philipp; Stühler, Kai & Nettersheim, Daniel
