Bi- und tri-functional IL-15 based cytokine fusion proteins for targeted 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，我们提出了一种利用多功能抗体融合蛋白将不同的细胞因子和配体靶向输送到肿瘤部位的方法，以支持有效的抗肿瘤免疫反应。靶向应减少全身毒性和自身免疫影响的风险。此外，不同免疫调节分子的组合使用应该可以克服肿瘤逃逸机制。在前面的项目中，我们已经成功地制备了双功能抗体融合蛋白，将细胞因子IL-15连接到IL-15Rpha链的一部分，在体外和体内获得了更好的活性。另外，与共刺激配体4-1BBL结合形成三功能融合蛋白，在体外以靶向形式增强免疫刺激能力。鉴于这些令人鼓舞的结果，我们的目标是开发第二代三功能抗体融合蛋白，满足以下标准：结构简单、尺寸最小、所有化合物都以可溶和靶向形式存在。因此，我们将重点研究IL-15与4-螺旋束细胞因子家族(IL-7、IL-21)、共刺激配体(OX40L、GITRL、LIGHT)或趋化因子CCL家族(CCL16、CCL21)的不同配体的结合。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，最有希望的三功能融合蛋白的抗肿瘤潜力将在体内肺转移瘤小鼠模型中进行评估。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 multifunctional antibody-fusion proteins for target-directed delivery of different cytokines and ligands to the tumor site in order to support an efficient antitumor immune response. Targeting should reduce the risk of systemic toxicity and autoimmune effects. Furthermore, the combinatorial use of different immune-modulatory molecules should allow to overcome tumor evasion mechanisms. In the preceding project we have already successfully generated bi-functional antibody-fusion proteins with the cytokine IL-15 linked to part of the IL-15Ralpha chain, obtaining improved activity in vitro and in vivo. Additional combination with the costimulatory ligand 4-1BBL into a tri-functional fusion protein rendered enhanced immune-stimulatory capacity in targeted form in vitro. In light of these encouraging results, we aim to develop second generation tri-functional antibody-fusion proteins, meeting criteria of simple configuration, minimal size and good accessibility of all compounds in soluble and targeted form. Therefore we will focus on the combination of IL-15 with different ligands either of the 4-helix-bundle cytokine family (IL-7, IL-21), costimulatory ligands of the TNF-family (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 the antitumor potential of the most promising tri-functional fusion proteins will be evaluated in a lung metastasis tumor mouse model in vivo. Thus, insight into the implementation and prospect of this innovative approach is expected to be gained.