Abstract 1546: The CD30/CD16A bispecific innate immune cell engager AFM13 elicits heterogeneous single-cell NK cell responses and effectively triggers memory-like (ML) NK cells
Authors: Marin Nancy , Becker-Hapak Michelle , Koch Joachim , Berrien-Elliott Melissa M. , Foster Mark , Neal Carly , McClain Ethan , Desai Sweta , Wagner Julia A. , Schappe Timothy , Marsala Lynne , Wong Pamela , Treder Martin , Fehniger Todd A. 
Published: August 2019

Abstract
Natural killer (NK) cells are crucial effector cells of the innate immune system capable of rapidly recognizing and eliminating infected, stressed and malignant cells. NK cells discriminate tumor targets from healthy cells via integration of activating and inhibitory receptor signals. One barrier to the broad application of NK cells across many cancer types is inconsistent cancer cell recognition, which may be overcome by immune cell engagers. AFM13 is a tetravalent bispecific antibody based on the ROCK platform characterized by bivalent binding to CD30 and CD16A with clinical efficacy in CD30+ malignancies. However, our understanding of NK cell functional responses triggered via AFM13 remain incomplete. Moreover, adoptively transferred memory-like (ML) NK cells have demonstrated enhanced anti-tumor activity (Romee R et al, Sci Transl Med, 2016) that may be receptive to AFM13-based targeting to enhance target cell recognition. To address these questions, we analyzed single-cell conventional (cNK) and ML (IL-12/15/18-induced) NK cell functional responses to NK-resistant CD30+ lymphoma cells +/- AFM13. Primary cNK cells co-incubated with AFM13-treated Hut-78 cells demonstrated increased IFN-γ, TNF, and degranulation, compared to Hut-78 cells or Raji (CD30-) targets + AFM13 as a negative control (p&lt;0.05). To define the single-cell specificity of NK cell responses to AFM13, similar assays were performed using mass cytometry analysis of 39 lineage, maturation, activating and inhibitory receptors, and function-relevant NK cell markers. tSNE-based multidimensional analyses revealed marked distinctions between Hut-78 and AFM13-Hut-78 stimulated cNK cells, due in part to IFN-γ, MIP-1α, CD107a, and CD16. To define the impact of specific NK receptors, SPADE was used to define highly activated IFN-γ+ NK cell sub-populations. In a KIR3DL1+ donor, activation was primarily within the KIR3DL1+ subset, consistent with the lack of its inhibitory ligand (HLA-Bw4). In KIR3DL1 negative donors, responding NK cells were enriched in mature KIR2DL2/L3+CD57+ NK cells that lacked NKG2A. Additional experiments revealed that both control and ML NK cells exhibited increased IFN-γ, degranulation, and cytotoxicity with AFM13 (P&lt;0.01), and AFM13-stimulated ML NK cells exhibited the highest IFN-γ response and killing. Collectively, these data indicate that target cell recognition of NK cells can be significantly enhanced by AFM13, yet influenced by inhibitory receptor expression, maturation state, and memory-like differentiation. Thus, these data suggest that the status and repertoire of NK cells in a patient may offer diagnostic potential for therapeutic response, and the combination of ML NK cells with AFM13 appears to be a promising therapeutic approach.
Citation Format: Nancy Marin, Michelle Becker-Hapak, Joachim Koch, Melissa M. Berrien-Elliott, Mark Foster, Carly Neal, Ethan McClain, Sweta Desai, Julia A. Wagner, Timothy Schappe, Lynne Marsala, Pamela Wong, Martin Treder, Todd A. Fehniger. The CD30/CD16A bispecific innate immune cell engager AFM13 elicits heterogeneous single-cell NK cell responses and effectively triggers memory-like (ML) NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1546.