Multifunctional NK Cell–Engaging Antibodies Targeting EGFR and NKp30 Elicit Efficient Tumor Cell Killing and Proinflammatory Cytokine Release

Abstract

In this work, we have generated novel Fc-comprising NK cell engagers (NKCEs) that bridge human NKp30 on NK cells to human epidermal growth factor receptor (EGFR) on tumor cells. Camelid-derived VHH single-domain Abs specific for human NKp30 and a humanized Fab derived from the EGFR-specific therapeutic Ab cetuximab were used as binding arms. By combining camelid immunization with yeast surface display, we were able to isolate a diverse panel of NKp30-specific VHHs against different epitopes on NKp30. Intriguingly, NKCEs built with VHHs that compete for binding to NKp30 with B7-H6, the natural ligand of NKp30, were significantly more potent in eliciting tumor cell lysis of EGFR-positive tumor cells than NKCEs harboring VHHs that target different epitopes on NKp30 from B7-H6. We demonstrate that the NKCEs can be further improved with respect to killing capabilities by concomitant engagement of FcγRIIIa and that soluble B7-H6 does not impede cytolytic capacities of all scrutinized NKCEs at significantly higher B7-H6 concentrations than observed in cancer patients. Moreover, we show that physiological processes requiring interactions between membrane-bound B7-H6 and NKp30 on NK cells are unaffected by noncompeting NKCEs still eliciting tumor cell killing at low picomolar concentrations. Ultimately, the NKCEs generated in this study were significantly more potent in eliciting NK cell–mediated tumor cell lysis than cetuximab and elicited a robust release of proinflammatory cytokines, both features which might be beneficial for antitumor therapy.

NKp46‐specific single domain antibodies enable facile engineering of various potent NK cell engager formats

“…For this, we focused on VHHs that address distinct epitopes and elicit a robust activation of NK cells. Similar to what has been shown by Vivier for NKp46 (Gauthier et al, 2019) and our group for NKp30 (Klausz et al, 2022; Pekar et al, 2021), co‐engagement of FcγRIIIa by utilizing an effector functional Fc portion enhanced killing capacities of VHH‐based NKCEs.…”

“…Our group recently described that killing capacities of NKp30‐directed EGFR‐targeting bispecific NKCEs can be further enhanced by co‐triggering FcγRIIIa (Klausz et al, 2022; Pekar et al, 2021). This was also demonstrated by Vivier and colleagues for NKp46‐specific Fab‐derived paratopes incorporated into multifunctional NKCEs (Gauthier et al, 2019).…”

“…The authors generated NKp46‐directed paratopes based on canonical VH as well as VL comprising antigen binding sites and furthermore characterized several different sophisticated NKCE formats. Our group has recently described an efficient route for generating NKCEs based on NKp30‐directed camelid‐derived sdAbs (Klausz et al, 2022). This strategy involved immunization of camelids followed by YSD‐based antibody selection.…”

“…For instance, bifunctional immunoligands have been described in which a tumor‐associated antigen (TAA)‐directed paratope was fused to the extracellular region of a ligand for an activating receptor or to affinity‐optimized versions thereof (Peipp et al, 2015; Pekar et al, 2021; von Strandmann et al, 2006). Moreover, bispecific or multifunctional NKCEs were constructed that bridge a TAA on tumor cells with an activating receptor on NK cells such as NKG2D or NKp30 (Colomar‐Carando et al, 2022; Klausz et al, 2022; Klewinghaus et al, 2022; Raynaud et al, 2021). In one of the most prominent examples, Vivier and colleagues engineered trifunctional NKCEs based on the engagement of two NK cell activating receptors, that is, NKp46 as well as FcγRIIIa, for very potent effector cell redirection (Gauthier et al, 2019).…”

Role of B7 family members in glioma: Promising new targets for tumor immunotherapy

“…The intracytoplasmic domain has many signaling motifs, e.g. an inhibition motif based on immunoreceptor tyrosine (SaYtpL), a SH2 (Src homology 2)-binding domain (YqlQ), and a SH3-binding motif (PdaPilPvsP) ( 95 ). B7-H6 also presents a selective expression on several tumor cell types (melanoma, neuroblastoma, primary blood or bone marrow cells from various hematological malignancies, etc.…”