Knockout of the inhibitory receptor TIGIT enhances the antitumor response of ex vivo expanded NK cells and prevents fratricide with therapeutic Fc-active TIGIT antibodies

Abstract

BackgroundInhibitory receptor T-cell Immunoreceptor with Ig and ITIM domains (TIGIT) expressed by Natural Killer (NK) and T cells regulates cancer immunity and has been touted as the next frontier in the development of cancer immunotherapeutics. Although early results of anti-TIGIT and its combinations with antiprogrammed death-ligand 1 were highly exciting, results from an interim analysis of phase III trials are disappointing. With mixed results, there is a need to understand the effects of therapeutic anti-TIGIT on the TIGIT+immune cells to support its clinical use. Most of the TIGIT antibodies in development have an Fc-active domain, which binds to Fc receptors on effector cells. In mouse models, Fc-active anti-TIGIT induced superior immunity, while Fc receptor engagement was required for its efficacy. NK-cell depletion compromised the antitumor immunity of anti-TIGIT indicating the essential role of NK cells in the efficacy of anti-TIGIT. Since NK cells express TIGIT and Fc-receptor CD16, Fc-active anti-TIGIT may deplete NK cells via fratricide, which has not been studied.MethodsCRISPR-Cas9-based TIGIT knockout (KO) was performed in expanded NK cells. Phenotypic and transcriptomic properties of TIGIT KO and wild-type (WT) NK cells were compared with flow cytometry, CyTOF, and RNA sequencing. The effect of TIGIT KO on NK-cell cytotoxicity was determined by calcein-AM release and live cell imaging-based cytotoxicity assays. The metabolic properties of TIGIT KO and WT NK cells were compared with a Seahorse analyzer. The effect of the Fc-component of anti-TIGIT on NK-cell fratricide was determined by co-culturing WT and TIGIT KO NK cells with Fc-active and Fc-inactive anti-TIGIT.ResultsTIGIT KO increased the cytotoxicity of NK cells against multiple cancer cell lines including spheroids. TIGIT KO NK cells upregulated mTOR complex 1 (mTORC1) signaling and had better metabolic fitness with an increased basal glycolytic rate when co-cultured with cancer cells compared with WT NK cells. Importantly, TIGIT KO prevented NK-cell fratricide when combined with Fc-active anti-TIGIT.ConclusionsTIGIT KO in ex vivo expanded NK cells increased their cytotoxicity and metabolic fitness and prevented NK-cell fratricide when combined with Fc-active anti-TIGIT antibodies. These fratricide-resistant TIGIT KO NK cells have therapeutic potential alone or in combination with Fc-active anti-TIGIT antibodies to enhance their efficacy.

PM21-particle stimulation augmented with cytokines enhances NK cell expansion and confers memory-like characteristics with enhanced survival

“…A common strategy to enhance NK cells is to genetically knock out the expression of inhibitory receptors. To determine if CAP-expanded NK cells are amenable to this approach, a triple inhibitory receptor knockout was performed using CRISPR/Cas9 targeting as previously described ( 41 ). Simultaneous knockout of TIGIT, CD96, and PVRIG could be achieved ( Figure 9A ) with less than 10% of CAP-NK cells expressing each receptor ( Figure 9B ).…”

“…Donor-matched PM21-NK cells and CAP-NK cells were used as a source for total RNA extractions. Preparation of RNA, sequencing, and analysis were performed as previously described ( 41 ). ClusterProfiler was used for preranked Gene Set Enrichment Analysis to determine enriched hallmark-gene sets in CAP-NK cells compared to PM21 NK cells and enrichplot ( 42 ) was used to generate dot plots of enriched gene sets.…”