A Fc-Engineered RANK-Fc Fusion Protein Neutralizes Soluble RANK Ligand and Targets Malignant Hematopoietic Cells for NK Cell Reactivity.
Authors: Schmiedel Benjamin J , Baessler Tina , Scheible Carolin , Wende Constantin M , Azuma Miyuki , Schneider Pascal , Kanz Lothar , Grosse-Hovest Ludger , Salih Helmut R 
Published: October 2019

Abstract
Abstract 411
Bone resorption is commonly associated with aging, but also with certain cancers. Recent studies identified Receptor Activator of NFκB (RANK) ligand (RANKL) and its receptors RANK and osteoprotegerin (OPG) as key regulators of bone resorption. Multiple myeloma (MM) disrupts the balance within this molecule system, and severe bone destruction due to inappropriate osteoclastogenesis is a prominent feature of this disease. Besides MM cells, other malignant hematopoietic cells have also been found to express RANKL at the cell surface and to release this molecule in soluble form (sRANKL). Neutralization of RANKL using RANK-Fc fusion protein or monoclonal antibody (Denosumab/AMG162), which mimics the RANKL-neutralizing endogenous effects of osteoprotegerin, decreases osteolysis in multiple in vivo models and is presently being evaluated as a means to treat both non-malignant and malignant osteolysis. We here confirmed and extended previously published data and report that all investigated MM cell lines (n = 5) as well as primary leukemic cells of CLL patients (n = 12) displayed substantial levels of RANKL mRNA and surface expression. Moreover, we report that substantial levels of sRANKL can be detected in culture supernatants of MM and primary CLL cells, but not in supernatants of healthy PBMC. Next we engineered RANK-Fc fusion proteins with modified affinity to FcγR by mutating amino acids in the Fc portion as previously described (Lazar et al., PNAS 2006; Armour et al., Eur J Immunol 1999). Compared to wildtype RANK-Fc (RANK-Fc-wt), our mutants (S239D/I332E and E233P/L234V/L235A/DeltaG236/A327G/A330S) displayed highly enhanced and abrogated (RANK-Fc-ADCC+ and RANK-Fc-KO, respectively) affinity to FcγRIIIa expressed on NK cells, which play an important role in anti-tumor immunity due to their ability to lyse target cells directly and to mediate antibody-dependent cellular cytotoxicity (ADCC) upon application of therapeutic antibodies. The RANK-Fc-ADCC+ displayed similar capacitiy to neutralize sRANKL compared to the RANK-Fc-KO and the RANK-Fc-wt as revealed by binding competition assays. Next we cultured NK cells with L cells or P815 cells transfected to express RANKL and the parental, RANKL-negative controls in the presence or absence of the different RANK-Fc constructs. Addition of RANK-Fc-KO or RANK-Fc-wt did not substantially alter NK cell reactivity against the target cells. However, presence of the RANK-Fc-ADCC+ dramatically enhanced NK cell cytotoxicity and cytokine production in cultures with the RANKL-expressing target cells (increase from 20% to 89%, E:T ratio 30:1 and 12 pg/ml to 290 pg/ml respectively; both p<0.01, Student's t-test). Neither of the three RANK-Fc proteins altered NK cell cytotoxicity and cytokine production in cultures with the RANKL negative controls demonstrating that the RANK-Fc-ADCC+ specifically induced NK cell reactivity against RANKL-expressing malignant cells. Moreover, treatment with RANK-Fc-ADCC+ also significantly augmented NK cell anti-tumor reactivity in cultures with RANKL-expressing primary CLL cells of patients, and this was observed both in settings using allogenic NK cells and analyzing autologous NK cells among PBMC of the leukemia patients (both p<0.01, Student's t-test). Taken together, our Fc-engineered RANK-Fc-ADCC+ fusion protein may neutralize detrimental effects of sRANKL, can target RANKL-expressing malignant cells for NK cell anti-tumor reactivity and may thus constitute an attractive immunotherapeutic means for treatment of hematopoietic malignancies.

Disclosures:
No relevant conflicts of interest to declare.