Understanding the role of CD96 in human T-cell activation and its potential as a target for cancer immunotherapy

Abstract

CD96 (TACTILE) is a type I transmembrane receptor and a member of the immunoglobulin superfamily (IgSF). It is commonly expressed on hematopoietic cells, including CD4⁺ and CD8⁺ T-cells, as well as Natural Killer (NK) cells. CD96 interacts with CD155 on tumour and antigen-presenting cells (APCs), competing with the TIGIT and CD226 receptors. Since its isolation in 1992, CD96 has been implicated in cell-cell adhesion, immune regulation, and cytotoxicity mediation. While blockade of CD96 in murine tumour models suggests it inhibits NK cytotoxicity, contrasting findings in human T-cells indicate that antibody targeting of CD96 in fact enhances T-cell activation and pro-inflammatory cytokine release and therefore may represent a suitable target of clinical immunostimulatory antibodies.

Antibody targeting of immunoreceptors such as 4-1BB and OX40 has shown promising results in enhancing anti-tumour immune responses. However, patient-specific factors often limit therapeutic efficacy, leading to treatment resistance or relapse. Expanding the current repertoire of immunotherapeutics is therefore essential.
To address the limited availability of CD96-targeting antibodies, we employed a phage-display panning strategy to generate novel antibodies against human and mouse CD96, as well as clones with cross-species specificity. We identified 77 antibody clones, including several that do not interfere with CD155 binding. Notably, pan-species clones were found to enhance T-cell activation in the presence of anti-CD3 antibodies, highlighting CD96’s potential role in T-cell activation and its viability as an immunotherapeutic target.

CD96 contains key intracellular signalling domains, including a proline-rich domain (PRD), an ITIM, and a YxxM motif in human CD96 - common features of co-stimulatory receptors such as CD28 and ICOS. Using a chimeric antigen receptor (CAR) model incorporating the ICD of CD96 and subsequent site-directed mutagenesis, we demonstrated that CD96 facilitates T-cell activation through its PRD. Kinase profiling further revealed that the CD96 PRD mediates activation of key protein-tyrosine and serine/threonine kinases, including ITK, PDK1, and the MEK/ERK pathway.

Finally, by employing a T-cell activation model using bacterial superantigens as MHC-II presented peptides, we have shown that CD96 augments T-cell activation via physiological interactions with CD155. However, this activity is significantly influenced by the co-expression of TIGIT and CD226.

These findings establish CD96 as a critical modulator of T-cell activation and a promising target for immunotherapy.

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Identifiers

ORCID for Michael John Spurway: orcid.org/0000-0002-3259-4064

ORCID for Aymen Al-Shamkhani: orcid.org/0000-0003-0727-4189

ORCID for Edd James: orcid.org/0000-0001-8638-7928

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