Impact of antibody architecture and paratope valency on effector functions of bispecific NKp30 x EGFR natural killer cell engagers

ABSTRACT

Natural killer (NK) cells emerged as a promising effector population that can be harnessed for anti-tumor therapy. In this work, we constructed NK cell engagers (NKCEs) based on NKp30-targeting single domain antibodies (sdAbs) that redirect the cytotoxic potential of NK cells toward epidermal growth factor receptor (EGFR)-expressing tumor cells. We investigated the impact of crucial parameters such as sdAb location, binding valencies, the targeted epitope on NKp30, and the overall antibody architecture on the redirection capacity. Our study exploited two NKp30-specific sdAbs, one of which binds a similar epitope on NKp30 as its natural ligand B7-H6, while the other sdAb addresses a non-competing epitope. For EGFR-positive tumor targeting, humanized antigen-binding domains of therapeutic antibody cetuximab were used. We demonstrate that NKCEs bivalently targeting EGFR and bivalently engaging NKp30 are superior to monovalent NKCEs in promoting NK cell-mediated tumor cell lysis and that the architecture of the NKCE can substantially influence killing capacities depending on the NKp30-targeting sdAb utilized. While having a pronounced impact on NK cell killing efficacy, the capabilities of triggering antibody-dependent cellular phagocytosis or complement-dependent cytotoxicity were not significantly affected comparing the bivalent IgG-like NKCEs with cetuximab. However, the fusion of sdAbs can have a slight impact on the NK cell release of immunomodulatory cytokines, as well as on the pharmacokinetic profile of the NKCE due to unfavorable spatial orientation within the molecule architecture. Ultimately, our findings reveal novel insights for the engineering of potent NKCEs triggering the NKp30 axis.

KEYWORDS:

• ADCC
• antibody engineering
• bispecific antibody
• EGFR
• NK cell engager
• NKp30
• paratope
• protein engineering
• single domain antibody
• valency
• VHH

Acknowledgments

We gratefully acknowledge Britta von Below and Anja Muskulus for excellent technical assistance. We further thank Kerstin Hallstein, Laura Unmuth, Sigrid Auth, Vanessa Siegmund, and Dirk Mueller-Pompalla for experimental support. Furthermore, we would like to thank Mantas Malisauskas (DMPK-NBE), Luca Marchiando (DMPK-NBE), Elisa Bertotti (DMPK-NBE), Patrizia Tavano (DMPK-NBE), Andrea Paoletti (DMPK-NBE) and Klaus-Georg Gerth (DMPK-NCE) for the support regarding the PK study.

Disclosure statement

ASB, CLG, SK, MP and KK received research funding from Merck KGaA. LP, KK, BL, BR, AE, YX, SK, RZ, LT, SP and SZ are or were employees at either Merck Healthcare KGaA or EMD Serono. Besides, this work was conducted in the absence of any further commercial interest.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2024.2315640

Additional information

Funding

The work was supported by research funds within the clinical research unit CATCH-ALL funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – 444949889 to MP.