Abstract B54: Antibody therapy prototype leverages NK cells: A “TCR-like” antibody specific for a model neoepitope-MHC complex induces ADCC activity by mouse NK cells


 Background: Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action by which many anticancer monoclonal antibodies lead to the destruction of cancer cells. Most of these antibodies are specific for cell-surface molecules that, while overexpressed in cancer cells, are not cancer-specific. In contrast, a truly cancer-specific approach is to target neoepitope peptides presented on the MHC molecules of cancer cells. In C57BL/6 mice, the ovalbumin-derived peptide SIINFEKL constitutes a model neoepitope because it is a non-self sequence presented by the mouse MHC molecule H2Kb. Antibodies specific for peptide-MHC complexes are called “TCR-like” antibodies. We tested the ability of a mouse “TCR-like” antibody specific for the SIINFEKL-H2Kb complex to induce ADCC activity by mouse NK cells.Methods: Sequences of the heavy chain and light chain of anti-SIINFEKL-H2Kb antibody clone 25-D1.16 were retrieved from NCBI. To change the isotype from IgG1 to IgG2a, the variable region sequences of 25-D1.16 were inserted into the pTRIOZ-mIgG2a plasmid (Invivogen). The IgG2a form of this anti-SIINFEKL-H2Kb antibody was then expressed in CHO cells, and purified using protein A agarose. To test the induction of ADCC, 4-hour cocultures were performed at 3 different Effector:Target (E:T) ratios (0.5:1, 1.5:1, and 5:1) using primary mouse NK cells and CFSE-stained, SIINFEKL-pulsed EL4 target cells (C57BL/6 mouse cell line expressing H2Kb). The purified anti-SIINFEKL-H2Kb IgG2a antibody was added at 3.5 µg/mL to induce ADCC, or not added to test “natural” cytotoxicity. Cytotoxic activity was measured by (1) Annexin V and SYTOX staining of apoptotic and dead target cells, respectively, using flow cytometry; (2) a colorimetric assay for LDH activity used to corroborate the flow cytometry cytotoxicity results; and (3) CD107a staining to assess degranulation by NK cells, also using flow cytometry.Results: NK cell cytotoxic activity was increased in the presence of the antibody at all three E:T ratios. This boost in killing of target cells represents ADCC activity mediated by the added “TCR-like” antibody. At the low E:T ratio of 0.5:1, ADCC contributed a 10.4% increase in target cell killing over the background “natural” cytotoxic activity of the NK cells when the antibody was absent. At the higher ratios of 1.5:1 and 5:1, the increase in cytotoxic activity attributable to ADCC was 17.7% and 38.1%, respectively. Release of LDH in the cell culture supernatants correlated strongly with the target cell death measured by flow cytometry. At all E:T ratios, degranulation by NK cells was significantly higher in the presence of antibody.Conclusions: The “TCR-like” anti-SIINFEKL-H2Kb antibody, with mouse isotype IgG2a, induced mouse NK cells to kill SIINFEKL-pulsed EL4 cells via the ADCC mechanism. This result supports the concept that neoepitope-specific “TCR-like” antibodies could leverage ADCC by NK cells, hinting at potential new approaches to cancer therapy that would target cancer cells with high specificity and may even be personalizable.
 Citation Format: Marc A Gillig, Nicholas S Ingala, Rachit Ohri. Antibody therapy prototype leverages NK cells: A “TCR-like” antibody specific for a model neoepitope-MHC complex induces ADCC activity by mouse NK cells [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B54.