Post-Doc University Medical Center Schleswig-Holstein Kiel, Schleswig-Holstein, Germany
Introduction: T cell-based therapies have transformed the treatment landscape of multiple myeloma (MM), yet their efficacy can be hampered by cytokine release syndrome and T cell exhaustion. The engagement of other effector cell populations, such as Natural Killer (NK) cells, represents a promising complementary therapeutic strategy. NK cells distinguish healthy from stressed (virus-infected or malignantly transformed) cells by activating and inhibitory receptors. In this way, they contribute to immune surveillance by exerting cytotoxic activity and producing immunoregulatory cytokines. However, to evade immune recognition, tumor cells downregulate or shed activating ligands. To counteract this, bispecific NK cell engager (NKCE) have been developed to restore NK cell activation by simultaneously targeting an activating receptor, such as NKp30, and a tumor-associated antigen (TAA), and thereby promoting NK cell recruitment and activation at the tumor site.
Methods: NKp30-specific nanobodies were processed into IgG-like NKCE harboring a Fab-fragment derived either from CD38 antibody daratumumab or G protein-coupled receptor family C group 5 member D (GPRC5D) antibody talquetamab. Both NKCE were produced in CHO-S cells and carry either a silent or an active Fc domain. Simultaneous binding of both, NKp30 and the TAA, was analyzed via flow cytometry using a NKp30-Fc fusion protein. NK cell-mediated tumor cell killing was measured in standard 51Cr release assays using different MM cell lines as targets. Antibody-mediated phagocytosis was analyzed with pHrodo-labeled tumor cells andmonocyte-derived macrophages using live cell imaging. NK cell-induced cytokine release was investigated via ELISA.
Results: A camelid-derived NKp30 nanobody was incorporated into NKCE targeting either CD38 [CD38´NKp30] or GPRC5D [GPRC5D´NKp30] on MM cells. The NKCE demonstrated binding comparable to daratumumab and talquetamab, respectively. In addition, they were capable of simultaneously engaging NKp30 and the corresponding TAA. Furthermore, both, [GPRC5D´NKp30] and [CD38´NKp30], induced effective NK cell-mediated tumor cell killing in the picomolar range. Of note, [GPRC5D´NKp30] also mediated potent tumor cell killing of MM cells with low levels of CD38 that could not be lysed by daratumumab or [CD38´NKp30]. Incorporation of an active Fc domain further enhanced these anti-myeloma effects and additionally induced robust phagocytosis of MM cells. Moreover, while our NKCE triggered some cytokine release by NK cells, the levels were significantly lower than those observed with talquetamab induced cytokine release of T cells.
Conclusions: Our novel NKp30-targeting NKCE exhibit strong anti-myeloma activity, which can be further enhanced by co-engagement of Fc γ receptors. As a perspective, these NKCE may represent a strategy to further improve MM therapy, with potential application both as monotherapy and in combination with adoptively transferred NK cells.
