TIGIT, PVRIG, and CD96 As Potential Immune Checkpoint Targets

TIGIT Binds to CD112 or CD155 and Inhibits T Cell and Natural Killer Cell Activity

T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), also known as VSTM3, VSIG9, and WUCAM, is an inhibitory immunoglobulin superfamily (IgSF) receptor, with a gene structure that resembles members of the CD28 family.¹The TIGIT protein contains an extracellular region with a single IgV-like domain, followed by a transmembrane segment, and a cytoplasmic domain with an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoglobulin tail tyrosine-like (ITT) motif that both contribute to inhibitory signaling. TIGIT is expressed on activated and memory T cells, regulatory T cells, follicular helper T (Tfh) cells, and natural killer (NK) cells.^1-5 Similar to PD-1 and TIM-3, it is also highly expressed on exhausted T cells and exhausted natural killer (NK) cells in chronic infections and in cancer.^{6, 7}

The TIGIT protein binds to three different Nectin or Nectin-like molecules, CD155/PVR, CD112/Nectin-2, and CD113/Nectin-3, but it binds with highest affinity to CD155/PVR.^3,4,8 CD155/PVR and CD112 are primarily expressed on dendritic cells (DCs), T cells, and non-hematopoietic cell types including epithelial cells, endothelial cells, and tumor cells.^1-4 Ligation of TIGIT by CD155/PVR was originally shown to inhibit T cell proliferation and cytokine production by inducing tolerogenic DCs, but was also later found to directly inhibit TCR-mediated T cell activation, proliferation, and cytokine production.^1,4,5,9 On regulatory T cells, high level expression of TIGIT is associated with lineage stability, enhanced suppressive activity, and induction of IL-10 expression.^10-12 Significantly, ligation of TIGIT on regulatory T cells promotes the secretion of Fgl2, which inhibits both Th1 and Th17 cell differentiation, but is unable to inhibit Th2 differentiation, resulting in a shift in the immune response away from a pro-inflammatory Th1 or Th17-like response towards a Th2-type response.^{9, 13} On NK cells, binding of TIGIT by either CD155/PVR or CD112 inhibits NK cell cytotoxicity.^{3, 14} Therefore, TIGIT may suppress immune responses by acting on multiple different immune cell types.

In cancer, CD155 and CD112 are overexpressed on a broad range of tumors, and TIGIT is highly expressed on mouse and human tumor-infiltrating lymphocytes (TILs).^{6, 11, 15-20} On CD8⁺ TILs, TIGIT expression correlates with high level expression of PD-1 and TIM-3, and is associated with a dysfunctional phenotype.^{11, 20} On tumor-infiltrating regulatory T cells, TIGIT expression is associated with increased suppressive capacity and IL-10 production.¹¹ In TIGIT knockout mice bearing either B16F10 melanoma or MC38 colon carcinoma tumors, tumor growth was delayed due to enhanced CD8⁺ T cell activity.¹¹ Subsequent experiments utilizing either TIGIT deficient mice or TIGIT blockade in other mouse tumor models demonstrated similar results, although the delay in tumor progression in some studies was also attributed to increased NK cell activity.^{7, 21-23} Additionally, it was reported that while blockade of TIGIT or PD-L1 alone had a minimal effect on tumor growth and survival in a colorectal carcinoma mouse tumor model, blockade of both TIGIT and PD-L1 or TIGIT and PD-1 significantly improved anti-tumor immune responses.⁶ Similar results were obtained using dual TIGIT/PD-1 blockade in a mouse glioblastoma model.²⁴ A significant reduction in tumor growth and metastasis, coupled with prolonged survival was also observed in TIGIT deficient tumor-bearing mice treated with anti-TIM-3 antibodies compared with control mice, suggesting that dual blockade of TIGIT and TIM-3 could also synergistically improve anti-tumor immune responses.¹¹ As a result of these studies, a monoclonal anti-TIGIT blocking antibody is currently being evaluated in clinical trials for treating advanced and metastatic tumors.²⁵

In addition to TIGIT/VSTM3/VSIG9, there are a number of related VSTM and VSIG proteins whose effects on different immune cells have yet to be fully explored. R&D Systems researchers have screened several VSTM and VSIG family proteins and have unpublished data that suggests that many members of these families may regulate T cell activity. Bio-Techne exclusively offers R&D Systems^TM bioactive recombinant VSTM and VSIG family proteins to further our current understanding of the effects of these proteins on different immune cell types and determine whether they may represent next generation immune checkpoint targets.

Binding of CD112 or CD155 to DNAM-1/CD226 Promotes T Cell and Natural Killer Cell Activity

Besides binding to TIGIT, CD155/PVR and CD112 also bind with lower affinity to the co-stimulatory receptor, DNAM-1/CD226, which is primarily expressed on natural killer (NK) cells, T cells, B cells, and monocytes.^26-28 In contrast to TIGIT ligation, activation of DNAM-1/CD226 by CD155/PVR or CD112 promotes CD8⁺ T cell and NK cell cytotoxicity and cytokine production, and suppresses tumor growth.^{26, 29} As a result, DNAM-1/CD226 and TIGIT represent paired receptors with opposing effects on T cell and NK cell activity, similar to the CD28 and CTLA-4 paired receptor system.³⁰

CD96 Binds to CD155 and Functions as an Inhibitory Receptor on Mouse T Cells and NK Cells

Further adding to the complexity of the TIGIT-DNAM-1/CD226 signaling network, CD155/PVR and CD112 have both been reported to bind to additional receptors. In addition to binding to TIGIT and DNAM-1/CD226, CD155/PVR also binds to the IgSF receptor, CD96/TACTILE, which is expressed almost exclusively on T cells and natural killer (NK) cells.^31-33 Assessment of the binding affinity of CD155/PVR for CD96/TACTILE revealed that it binds to CD96/TACTILE with higher affinity than it binds to DNAM-1/CD226, but with lower affinity than it binds to TIGIT.³⁴ Notably, recent studies also identified CD111/Nectin-1 as a second CD96 ligand.^{33, 35} Although there are some discrepancies in the existing data regarding the effects of the human CD96 protein, most studies in mice suggest that similar to TIGIT, CD96 competes with DNAM-1/CD226 for CD155/PVR binding and acts as an inhibitory receptor on NK cells and CD8⁺ T cells.^{30, 36-39} Ligation of CD96 by CD155/PVR was shown to inhibit IFN-gamma production by mouse NK cells, while blockade of CD96 was found to enhance CD8⁺ T cell activity and suppress tumor growth in mouse tumor models.^34,37,38 Although this data is intriguing, further research is needed to fully understand whether human CD96 also functions as a T cell and NK cell inhibitory receptor.

PVRIG is an Inhibitory Receptor that Competes with DNAM-1 for Binding to CD112

In addition to binding to TIGIT and DNAM-1/CD226, CD112 also binds with high affinity to the PVRIG protein, a poliovirus receptor-like protein, also known as CD112 R, which is expressed on T cells, natural killer (NK) cells, and NKT cells.^40-42 While DNAM-1/CD226 competes with PVRIG for binding to CD112, TIGIT has little effect on the CD112-PVRIG interaction.⁴⁰ Similar to TIGIT, PVRIG functions as a co-inhibitory receptor on both T cells and NK cells.^40-43 Binding of CD112 to the PVRIG receptor inhibits T cell proliferation and function, and disruption of this interaction using either PVRIG deficient mice or antagonistic anti-PVRIG antibodies, enhances CD8⁺ T cell functions and reduces tumor growth in syngeneic mouse tumor models.^{40, 42} Furthermore, combined PVRIG and PD-L1 blockade was shown to synergistically improve anti-tumor immune responses in mouse tumor models.⁴² In humans, PVRIG was found to be co-expressed with PD-1 and TIGIT on tumor-infiltrating lymphocytes (TILs), and antagonistic anti-PVRIG antibodies combined with anti-TIGIT or anti-PD-1 antibodies synergistically improved the effector function of CD3⁺ TILs isolated from patients with multiple different types of cancers.⁴³ The results of these studies indicate that the PVRIG protein is an immune checkpoint target and suggest that the therapeutic potential of antagonistic anti-PVRIG antibodies alone, or in combination with anti-TIGIT or anti-PD-1 antibodies, should be investigated in cancer patients.