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

NK细胞在肿瘤的免疫监视中起重要作用，并且由于其有效介导抗体依赖性细胞毒性（ADCC）的能力，在很大程度上有助于治疗性抗体如利妥昔单抗的临床功效。因此，多种策略目前旨在增加抗体诱导NK细胞针对肿瘤细胞的ADCC的潜力。NK细胞的直接抗肿瘤反应性和ADCC均受NK细胞表达的受体的各种配体的影响，因此抑制性分子的中和很好地用于增加组成性和治疗诱导的NK细胞抗肿瘤免疫。在上一个资助期间，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）由NK细胞表达，而其对应物RANKL由恶性造血细胞表达。我们发现RANKL能够将信号传递到表达配体的恶性细胞中（反向信号传导），导致细胞因子的释放，这些细胞因子在恶性疾病中充当自分泌/旁分泌生长和存活因子。此外，RANKL信号传导后释放的因子损害NK细胞的抗肿瘤反应性并诱导NK细胞上的RANK表达，后者进一步发现将信号传递到NK细胞中，损害其抗肿瘤反应性，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工程化的RANK-Ig融合蛋白，其能够中和RANKL对抗肿瘤免疫的有害功能，同时通过靶向恶性细胞而有效地刺激NK细胞ADCC。在第二个资助期，我们的目标是阐明介导RANKL信号传导后恶性造血细胞中细胞因子释放和代谢活性诱导的信号传导途径。此外，我们的目的是确定RANK介导观察到的NK细胞细胞毒性和细胞因子产生的差异抑制的分子机制。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诱导的差异敏感性的发现，即将进行的研究的重点是建立合适的人源化小鼠模型，以进一步临床前表征我们的Fc工程化RANK-Ig融合蛋白。设想此类模型最终验证我们的策略，即通过中和RANKL的抑制作用并同时靶向表达RANKL的恶性细胞的NK细胞ADCC来双重增强NK对恶性造血细胞的反应性。在临床前验证之后，我们设想我们的构建体将根据GMP指导方针在大学拥有的生产单位中生产，目的是在癌症患者中进行快速临床测试。

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.