Role and therapeutic modulation of the RANK-RANKL molecule system in the NK cell immune surveillance of malignant hematopoietic diseases

NK细胞在肿瘤的免疫调节中发挥着重要作用，并且由于其能够有效地介导抗体依赖的细胞毒作用(ADCC)，在很大程度上决定了治疗性抗体如利妥昔单抗的临床疗效。因此，目前有多种策略旨在提高抗体诱导NK细胞对肿瘤细胞的ADCC的潜力。NK细胞的直接抗肿瘤活性和ADCC均受NK细胞表达的受体的不同配体的影响，因此中和抑制分子有助于提高结构性和治疗性诱导的NK细胞抗肿瘤免疫。在上一个资助阶段，我们证明了TNFR家族成员RANK(主要以其在骨代谢中的作用而闻名)是由NK细胞表达的，而其对应的RANKL是由恶性造血细胞表达的。我们发现RANKL能够将信号转导到配体表达的肿瘤细胞(反向信号)，从而导致细胞因子的释放，这些细胞因子在恶性肿瘤中作为自分泌/旁分泌生长和生存因子。此外，RANKL信号通路释放的因子可削弱NK细胞的抗肿瘤活性并诱导NK细胞RANK的表达，后者进一步被发现可将信号转导至NK细胞，从而削弱其抗肿瘤活性，从而促进表达RANKL的恶性细胞的免疫逃逸。基于这些发现，我们开发了Fc工程RANK-Ig融合蛋白，能够中和RANKL在抗肿瘤免疫中的不利功能，同时通过靶向肿瘤细胞来有效刺激NK细胞ADCC。在第二个资助期，我们旨在阐明RANKL信号后介导细胞因子释放和代谢活性的信号通路。此外，我们的目标是确定RANK介导对NK细胞杀伤和细胞因子产生的不同抑制的分子机制。基于我们在同基因小鼠肿瘤模型中观察到的人和小鼠NK细胞对ADCC诱导的不同敏感性的发现，即将到来的研究的重点是建立合适的人源化小鼠模型，以进一步在临床前表征我们的Fc工程RANK-Ig融合蛋白。这些模型有望最终验证我们的策略，即通过中和RANKL的抑制效应，同时针对表达RANKL的恶性肿瘤细胞的NK细胞ADCC，双重增强NK细胞对恶性造血细胞的反应性。在临床前验证后，我们设想我们的结构将在大学拥有的生产单位中根据GMP指南生产，目的是使癌症患者能够进行快速临床测试。

NK cells play an important role in the immunosurveillane of tumors and, due to their ability to potently mediate antibody-dependent cellular cytotoxicity (ADCC), largely contribute to the clinical efficacy of therapeutic antibodies like Rituximab. Accordingly, multiple strategies presently aim to increase the potential of antibodies to induce ADCC of NK cells against tumor cells. Both the direct anti-tumor reactivity and ADCC of NK cells are influenced by the various ligands for receptors expressed by NK cells, and neutralization of inhibitory molecules thus serves well to increase constitutive and therapeutically-induced NK cell anti-tumor immunity. In the last funding period we demonstrated that the TNFR family member RANK (mainly known for its role in bone metabolism) is expressed by NK cells while its counterpart RANKL is expressed by malignant hematopoietic cells. We found that RANKL is capable to transduce signals into the ligand-expressing malignant cells (reverse signaling) which lead to release of cytokines that act as autocrine/paracrine growth and survival factors in the malignant diseases. In addition, the factors released upon RANKL signaling impaired the anti-tumor reactivity of NK cells and induced RANK expression on NK cells, The latter was further found to transduce signals into NK cells that impair their anti-tumor reactivity thereby facilitating immune escape of the RANKL-expressing malignant cells. Based on these findings we developed Fc-engineered RANK-Ig fusion proteins that are capable to neutralize the detrimental function of RANKL on anti-tumor immunity while at the same time potently stimulating NK cell ADCC by targeting of the malignant cells.In the second funding period we aim to elucidate the signaling pathways that mediate the induction of cytokine release and metabolic activity in malignant hematopoietic cells following RANKL signaling. Moreover, we aim to determine the molecular mechanisms by which RANK mediates the observed differential inhibition of NK cell cytotoxicity and cytokine production. Based on our findings regarding the differential susceptibility of human and murine NK cells for ADCC-induction observed in syngeneic mouse tumor models, the focus of the upcoming studies is to establish suitable humanized mouse models to further preclinically characterize our Fc-engineered RANK-Ig fusion proteins. Such models are envisaged to finally validate our strategy to dually reinforce NK reactivity against malignant hematopoietic cells by neutralizing the inhibitory effects of RANKL and simultaneously targeting the RANKL-expressing malignant cells for NK cells ADCC. After the preclinical validation, we envisage our constructs to be produced in an University-owned production unit according to GMP guidelines with the aim to enable rapid clinical testing in cancer patients.