Abstract 2950: CD16A shedding facilitates repetitive targeting of tumor cells by AFM13-armed NK cells

Abstract

Abstract Antibody-Dependent Cellular Cytotoxicity (ADCC) is a powerful mechanism of Natural Killer (NK) cells to kill antibody-opsonized target cells. However, ADCC mediated by conventional antibodies has its limitations in killing of tumor cells commonly being characterized by low tumor antigen expression. Several strategies have been implemented to boost ADCC including shedding inhibition of CD16A, a key ADCC-mediating Fc receptor on the plasma membrane of NK cells. However, previous results have shown that genetic engineering of the cleavage site of CD16A leads to decreased serial engagement of target cells. Another approach to boost ADCC is provided by tetravalent-bispecific innate cell engagers (ICE®) cross-linking CD16A and tumor antigens triggering anti-tumoral cytotoxicity. In the current study, we have investigated whether ICE molecules can induce effective ADCC while maintaining the natural function of CD16A with a particular focus on preserved CD16A shedding. To study cytotoxicity and contact dynamics, we have used an in-house developed live-cell microchip screening with single cell resolution together with a microcontact printing-based assay combined with an ICE®. Single cell analysis using microchip screening revealed that the CD16A/CD30 targeting ICE® AFM13 induces stronger ADCC of NK cells towards CD30+ target cells when compared to anti-CD30 antibodies. This stronger response was reached through increasing both the overall number of cytotoxic NK cells and the fraction of NK serial killers i.e., NK cells performing three or more kills in sequence. Interestingly, the differences were most prominent towards target cells expressing low levels of CD30. Combination of AFM13 and inhibition of CD16 shedding increased NK cell conjugation time with target cells, which could potentially limit targeting of additional tumor cells located at a distance from the primary target. To investigate this phenomenon further, we used a microcontact printing assay. Here, a grid of spatially distributed protein patches consisting of AFM13 and anti-LFA-1 was printed on glass surfaces enabling the formation of “artificial immune synapses” when probed by NK cells. On these “artificial immune synapses” we investigated the influence of shedding inhibition on NK cell migration and interaction dynamics. In conclusion, we show that AFM13 increases both the fraction of tumor-target responsive NK cells and the fraction of serial killing NK cells compared to conventional monoclonal antibodies. Based on our data we hypothesize that CD16A shedding facilitates AFM13 induced ADCC potential of NK cells by allowing potent migration to distantly located tumor cells and serial killing. Especially in the context of AFM13 primary indications, Hodgkin and peripheral T cell lymphoma, migration of NK cells might have a particularly strong impact when treating cancer patients due to the disseminated nature of the disease. Citation Format: Chiara Zambarda, Karolin Guldevall, Damien Toullec, Susanne Wingert, Christian Breunig, Sheena Pinto, Jacopo Fontana, Joachim Koch, Björn Önfelt. CD16A shedding facilitates repetitive targeting of tumor cells by AFM13-armed NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2950.