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.

睾丸生殖细胞肿瘤是17-40岁年轻男性中最常见的肿瘤类型，发病率正在稳步上升，特别是在西方工业国家。一般来说，生殖细胞肿瘤患者通过切除睾丸和以顺铂为基础的化疗进行治疗，从而获得高治愈率(&gt；90%)。因此，研究的重点是开发新的治疗方案，在保持高治愈率的同时减少强烈的副作用。然而，如果肿瘤转移或获得顺铂耐药，生殖细胞肿瘤仍然是一个致命的威胁。这些年轻患者的预后非常差，这表明需要新的治疗方法来改进标准治疗和治疗晚期患者。所谓的抗体-药物结合物(ADC)，即与细胞毒素偶联的抗体，目前是研究的重点，并已用于临床常规治疗(例如，Brentuximab Vedotin，一种CD30抗体与纺锤体毒素偶联)。抗体与细胞表面的靶蛋白结合后内化，ADC在细胞质中被裂解，毒素被释放。基于抗体的治疗领域的进一步进展导致了纳米体的发展，纳米体只由骆驼(如骆驼和羊驼)中发现的重链抗体的可变部分组成。纳米抗体比传统抗体(15 kDa比150 kDa)小得多，从而增加了组织穿透能力。此外，由于缺少Fc部分，纳米抗体被比抗体更具抗原特异性的细胞所摄取，从而提高了它们的疗效。其特异性和对目标蛋白的亲和力可与常规抗体相媲美。纳米体内可以通过许多方式进行化学修饰，还可以与毒素偶联。因此，纳米体-药物结合物(NDC)是ADCs的一种通用的替代品和进展。在本研究中，需要开发三个NDCs，每个到十个不同的有希望的靶分子，这些都是在先前的实验中被确定的。与正常睾丸组织和成纤维细胞相比，所有靶分子在生殖细胞肿瘤中的表达强度都非常高，使它们成为基于纳米体的治疗的理想候选者。我们将展示每个NDC在(顺铂耐药)生殖细胞肿瘤细胞系中的细胞毒作用，然后分析细胞凋亡的诱导和细胞周期的变化。最后，我们将在将生殖细胞肿瘤细胞系移植到裸鼠体内后证实其体内的有效性。综上所述，我们将基于最先进的抗体相关技术来分析和建立新的靶向治疗方案。这些新疗法不仅可能改进标准疗法，还可能对表现出转移和顺铂耐药的难以治疗的患者有利。

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.