Tri-functional antibody- fusion proteins for cancer immunotherapy

调节免疫反应的细胞因子/配体-受体相互作用的抗肿瘤潜力已成为癌症治疗策略的焦点。在这里，我们提出了一种由肿瘤导向的重组抗体和两种不同的细胞因子/趋化因子组成的三功能抗体融合蛋白的方法。根据这一概念，靶向地将适当的细胞因子/趋化因子组合输送到肿瘤部位将支持有效的局部抗肿瘤免疫反应。因此，靶向应该减少全身毒性和自身免疫效应的风险，而不同的免疫调节分子的联合使用应该能够克服肿瘤逃逸机制。在前面的项目中，我们已经成功地合成了与IL-15连接的抗体融合蛋白，并通过额外掺入共刺激配体4-1BBL产生了第一个三功能融合蛋白。因此，在体外通过靶向形式实现了更好的免疫刺激活性，并在体内报道了抗肿瘤作用。鉴于这些令人鼓舞的结果，我们的目标是通过改进和扩大三功能抗体融合蛋白的生成来继续发展这一概念，从而满足结构简单、尺寸最小以及所有化合物以可溶和靶向形式存在的良好可及性的标准。此外，我们将集中于IL-15与不同的配体的结合，无论是肿瘤坏死因子超家族(OX40L，GITRL，LIGHT)还是趋化因子CCL-家族(CCL16，CCL21)。我们将分析这些分子的构型对以可溶性和靶向形式显示的活性的影响，以及它们对体外T细胞和NK细胞免疫反应的影响。随后，将产生最有希望的三功能融合蛋白的小鼠版本，并在免疫活性肿瘤小鼠模型中评估它们的抗肿瘤潜力。因此，可望深入了解这一创新方法的实施情况和前景。

The antitumor potential of cytokine/ligand-receptor interactions that modulate an immune response has moved into the spotlight of cancer treatment strategies. Here, we propose an approach with tri-functional antibody-fusion proteins composed of a tumor-directed recombinant antibody and two different cytokines/chemokines. According to the concept, target-directed delivery of appropriate combinations of cytokine/ chemokine to the tumor site will support an efficient local antitumor immune response. Thereby, targeting should reduce the risk of systemic toxicity and autoimmune effects, while the combinatorial use of different immune-modulatory molecules should allow overcoming tumor evasion mechanisms. In the preceding project we have already successfully generated antibody-fusion proteins with IL-15 linked to part of the IL-15Ralpha chain and generated first tri-functional fusion proteins by the additional incorporation of the costimulatory ligand 4-1BBL. Thus, improved immune-stimulatory activity was achieved by the targeted form in vitro and antitumoral effects were reported in vivo. In light of these encouraging results, we aim to continue developing this concept by improving and expanding the generation of tri-functional antibody-fusion proteins, thereby meeting criteria of simple configuration, minimal size and good accessibility of all compounds in soluble and targeted form. Furthermore, we will focus on the combination of IL-15 with different ligands either of the TNF-superfamily (OX40L, GITRL, LIGHT) or the chemokine CCL-family (CCL16, CCL21). We will analyze the influence of the configuration of these molecules on the activity displayed in soluble and targeted form and their impact on the immune response of T cells and NK cells in vitro. Subsequently, murine versions of the most promising tri-functional fusion proteins will be generated and their antitumor potential evaluated in an immune competent tumor mouse model. Thus, insight into the implementation and prospect of this innovative approach is expected to be gained.