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Fcγ receptor–mediated cross-linking codefines the immunostimulatory activity of anti-human CD96 antibodies
Anne Rogel, … , Emma V. King, Aymen Al-Shamkhani
Anne Rogel, … , Emma V. King, Aymen Al-Shamkhani
Published August 23, 2022
Citation Information: JCI Insight. 2022;7(19):e158444. https://doi.org/10.1172/jci.insight.158444.
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Research Article Immunology

Fcγ receptor–mediated cross-linking codefines the immunostimulatory activity of anti-human CD96 antibodies

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Abstract

New strategies that augment T cell responses are required to broaden the therapeutic arsenal against cancer. CD96, TIGIT, and CD226 are receptors that bind to a communal ligand, CD155, and transduce either inhibitory or activating signals. The function of TIGIT and CD226 is established, whereas the role of CD96 remains ambiguous. Using a panel of engineered antibodies, we discovered that the T cell stimulatory activity of anti-CD96 antibodies requires antibody cross-linking and is potentiated by Fcγ receptors. Thus, soluble “Fc silent” anti-CD96 antibodies failed to stimulate human T cells, whereas the same antibodies were stimulatory after coating onto plastic surfaces. Remarkably, the activity of soluble anti-CD96 antibodies was reinstated by engineering the Fc domain to a human IgG1 isotype, and it was dependent on antibody trans-cross-linking by FcγRI. In contrast, neither human IgG2 nor variants with increased Fcγ receptor IIB binding possessed stimulatory activity. Anti-CD96 antibodies acted directly on T cells and augmented gene expression networks associated with T cell activation, leading to proliferation, cytokine secretion, and resistance to Treg suppression. Furthermore, CD96 expression correlated with survival in HPV+ head and neck squamous cell carcinoma, and its cross-linking activated tumor-infiltrating T cells, thus highlighting the potential of anti-CD96 antibodies in cancer immunotherapy.

Authors

Anne Rogel, Fathima M. Ibrahim, Stephen M. Thirdborough, Florence Renart-Depontieu, Charles N. Birts, Sarah L. Buchan, Xavier Preville, Emma V. King, Aymen Al-Shamkhani

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Figure 7

Anti-CD96 mAb stimulates the proliferation of tumor-infiltrating lymphocytes from head and neck squamous cell carcinomas.

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Anti-CD96 mAb stimulates the proliferation of tumor-infiltrating lymphoc...
(A) Using data from The Cancer Genome Atlas, Kaplan-Meier curves of cumulative survival were generated for HPV+ and HPV– patients with head and neck squamous cell carcinoma (HNSCC), based on CD96 expression levels. (B) The expression of CD96 on tumor-infiltrating CD8+ T cells, conventional CD4+ T cells (CD4+Foxp3–), and Tregs (CD4+Foxp3+) isolated from n = 10 HNSCC biopsies was analyzed by flow cytometry. Data show (left) representative histogram overlays of CD96 expression and (right) the frequency of CD96hi cells in each T cell subset. (C) Coexpression of CD96 and PD-1 on tumor-infiltrating CD8+ T cells from n = 11 HNSCC biopsies was analyzed by flow cytometry. Data show dot plots of marker coexpression. FMO, fluorescence minus one. (D) Tumor-infiltrating T cells from n = 13 HPV+ tumors were stimulated with plate-bound OKT3 and anti-CD96 huG1 mAb (clone 19-134) or a matching isotype control for 5 days. Cells were pulsed with tritiated thymidine in the last 16 hours of culture. Each symbol represents the mean of the counts per minute of triplicate wells for each individual patient. Data are combined from n = 12 independent experiments. *P ≤ 0.05. (B) Friedman’s test and Dunn’s multiple comparisons test; (D) 2-tailed Wilcoxon’s paired test.

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