The combination of CD16A/EGFR bispecific innate cell engager AFM24 with SNK01 NK cells promotes efficacious targeting and killing of EGFR(+) tumor cells

The epidermal growth factor receptor (EGFR) is an overexpressed antigen on several solid cancers resulting in uncontrolled cell proliferation. Current EGFR-targeting agents such as tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAbs) mainly rely on EGFR signaling inhibition which is challenged by limited efficacy due to dose limiting toxicity, and/or intrinsic or acquired resistance of the tumor by e.g., KRAS or BRAF mutations. To overcome these limitations and to address the high medical need across EGFR-positive cancers, we investigate a unique approach of combining AFM24, a tetravalent bispecific EGFR- and CD16A-binding innate cell engager (ICE), with the patient-specific NK cell product SNK01. AFM24 mediates high affinity targeting and ADCC of EGFR+ tumor cells by NK cells, independent of EGFR signaling inhibition and irrespective of the mutational status of the tumor. AFM24 is currently being tested as monotherapy in a Phase 1/2a study in patients with EGFR-positive tumors. SNK01 is a first-in-kind, autologous non genetically modified NK cell product with significant anti-tumor cytotoxicity and high and homogeneous expression of CD16A, NKG2D, NKp46 and DNAM-1 , that can be consistently produced even from heavily pre-treated cancer patients. SNK01 showed activity against numerous solid tumors in preclinical studies and in the phase I trials. In order to investigate the synergistic potential of this unique combination, SNK01 cells were generated from PBMCs of healthy individuals and their functional activation by AFM24 in response to EGFR-positive A-431 cells was assessed in cytotoxicity assays and flow cytometry-based CD107a (marker for NK cell degranulation) and IFN-γ mobilization assays. AFM24 binding to and CD16A expression on SNK01 cells was measured by flow cytometry. Both, binding of AFM24 and expression of CD16A were detected on nearly 100% of SNK01 cells, indicating saturation of CD16A-positive SNK01 cells with AFM24. In the presence of AFM24, lysis of EGFR-positive A-431 cells by SNK01 cells was significantly enhanced. Likewise, in response to AFM24, upregulation of CD107a and IFN-γ expression by SNK01 cells was significantly increased upon cross-linking to A-431 cells. By contrast, AFM24 alone did not lead to significant NK cell activation in the absence of target cells or vice versa. Hence, activation of SNK01 cells was strictly dependent on AFM24-mediated cross-linking of SNK01 cells with target cells. In conclusion, this combination holds promise to direct the AFM24-augmented anti-tumor activity of SNK01 cells to EGFR-positive tumor cells independent of EGFR signaling pathway mutations thereby providing a potential new treatment option for underserved patient populations. An IND for a Phase I/II combination trial of AFM24 and SNK01 has been cleared and first-patient-in is on track for the second half of 2021. Citation Format: Jens Pahl, Ting-Ying (Hazel) Cheng, Uwe Reusch, Arndt Schottelius, Wolfgang Fischer, Yong Man Kim, Stephen Chen, Joachim Koch. The combination of CD16A/EGFR bispecific innate cell engager AFM24 with SNK01 NK cells promotes efficacious targeting and killing of EGFR⁺ tumor cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P191.