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.

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Testicular germ cell tumors represent the most common tumor type of young men age 17-40 and incidence rates are steadily rising, especially in Western industrial countries. In general, germ cell tumor patients are treated by removal of the testis and a cisplatinum-based chemotherapy, leading to high cure rates (> 90%). Thus, research focusses on development of novel therapeutic options that maintain the high cure rates but reduce strong side effects in parallel. Nevertheless, germ cell tumors can still be a lethal threat if the tumor metastasized or a cisplatin resistance was acquired. The prognosis for these young patients is very bad, demonstrating the need for novel therapies to improve standard therapy and treat those late stage patients.So called antibody-drug-conjugates (ADC), i. e. antibodies coupled to a cytotoxin, are nowadays in the focus of research and are already used in clinical routine (e. g. Brentuximab Vedotin, a CD30-antibody coupled to a spindle toxin). After binding of the antibody to its target protein on the cell surface followed by internalization, the ADC is cleaved in the cytoplasm and the toxin is released.Further progress in the field of antibody-based therapies led to the development of nanobodies, which consist of only the variable part of heavy-chain-antibodies as found in camelids (e.g. camels and alpacas). Nanobodies are much smaller than conventional antibodies (15 vs. 150 kDa) allowing for an increased tissue penetration. Additionally, due to the lack of the Fc-part nanobodies are taken up by cells more antigen-specific than antibodies, increasing their efficacy. Specificity and affinity to their target protein are comparable to conventional antibodies. Nanobodies can be modified chemically in many ways, also allowing for coupling to toxins. Thus, nanobody-drug-conjugates (NDC) are a versatile alternative and advancement to ADCs.In this study, three NDCs each to ten different promising target molecules, which were identified in previous experiments, should be developed. All target molecules are expressed with very high intensities in germ cell tumors compared to normal testis tissue and fibroblasts, making them ideal candidates for nanobody-based therapies. We will demonstrate the cytotoxic effect of each NDC in (cisplatin-resistant) germ cell tumor cell lines, followed by analyses of apoptosis-induction and changes in the cell cycle. Finally, we will confirm efficacy of the NDCs in vivo after xenografting of germ cell tumor cell lines into nude mice.In summary, we will analyze and establish novel targeted therapy options, which are based on state of the art antibody-related techniques. These new therapies might not only improve standard therapy but also be beneficial for hard-to-treat patients showing metastases and cisplatin-resistance.