TIGIT Antibody

What is TIGIT and why is it a significant target for immunotherapy research?

TIGIT (T cell immunoglobulin and ITIM domain) functions as an inhibitory checkpoint molecule implicated in tumor immunosurveillance. It is specifically expressed on immune cells including natural killer (NK) cells and various T cell subsets. TIGIT is a significant target because it binds to CD155 (PVR) and CD112 (PVRL2, nectin-2), which are expressed on antigen-presenting cells, T cells, and various tumor cells . Upon binding to its ligands, TIGIT inhibits multiple signaling pathways including NF-κB, P13K, and MAPK cascades, resulting in reduced NK cell cytotoxicity and inhibition of T cell activation, proliferation, and effector functions . Research significance stems from TIGIT's characteristics including low expression in peripheral lymphoid organs but high expression in tumor-infiltrating lymphocytes (TILs), established synergy with other co-inhibitory immune checkpoints, and the widespread expression of its ligands on tumor cells .

How do TIGIT antibodies function in experimental immunology?

TIGIT antibodies function by blocking the interaction between TIGIT and its ligands (primarily CD155/PVR), preventing immunosuppressive signaling. Methodologically, these antibodies can be designed to have varying capabilities: some function purely as blocking agents that disrupt TIGIT-ligand interactions while others may incorporate Fc-dependent effector functions that can induce antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) . In experimental systems, the functionality of anti-TIGIT antibodies can be assessed through various methods including flow cytometry to measure binding to TIGIT-expressing cells, ELISA-based competition assays to evaluate blocking efficiency against ligand binding, and functional assays measuring restoration of immune cell activity following antibody treatment . When designing experiments, researchers should consider whether they require antibodies with cross-species reactivity (e.g., recognizing both human and mouse TIGIT) to facilitate translational research across model systems .

What are the key considerations for selecting a TIGIT antibody for flow cytometry applications?

When selecting TIGIT antibodies for flow cytometry, researchers should evaluate several methodological aspects. First, consider the clone's epitope specificity and whether it might be masked by ligand binding or affected by common fixation procedures. The antibody's format is also crucial—determine whether native, conjugated, or biotinylated formats are most appropriate for your multi-color panel design . Validation is essential: examine whether the antibody has been validated specifically for flow cytometry on your target cell populations (NK cells, CD4+ T cells, CD8+ T cells, or Tregs). For optimal results, titrate the antibody on your specific samples to determine the appropriate concentration that maximizes signal-to-noise ratio . When analyzing TIGIT expression patterns, include appropriate controls such as isotype controls and FMO (fluorescence minus one) controls. For co-expression studies, consider including markers like CD96, CD226, PD-1, or TIM-3 to fully characterize the inhibitory receptor landscape on your target cells . Flow cytometry gating strategies should account for the typically heterogeneous expression of TIGIT across lymphocyte subpopulations.

What are the molecular mechanisms behind the synergistic effects observed when combining TIGIT blockade with other immune checkpoint inhibitors?

The synergistic effects observed when combining TIGIT blockade with other immune checkpoint inhibitors derive from several complementary molecular mechanisms. TIGIT functions within a complex network of interactions involving CD155, CD112, CD226 (DNAM-1), and CD96, creating a balance between activating and inhibitory signals . When designing combination studies, researchers should consider that TIGIT blockade not only removes inhibitory signaling but can also indirectly enhance CD226-mediated activation by reducing competition for shared ligands . Methodologically, phosphoproteomic analysis reveals that dual blockade of TIGIT and PD-1/PD-L1 leads to enhanced TCR signaling with increased phosphorylation of ZAP70, LAT, and downstream MAPK pathway components that are not fully restored by single-agent blockade . At the transcriptional level, RNA-seq analysis shows unique gene expression signatures with dual blockade, including upregulation of genes associated with T cell effector function, proliferation, and memory formation . In tumor microenvironment studies, multiplex immunohistochemistry and flow cytometry demonstrate that combination therapy increases the ratio of effector T cells to regulatory T cells and enhances production of effector cytokines including IFN-γ, TNF-α, and IL-2 . Experimentally, these synergistic effects are most evident in research models with high expression of multiple immune checkpoints, suggesting that patient stratification based on checkpoint co-expression profiles may be valuable in translational research .

How do Fc modifications of anti-TIGIT antibodies influence their in vitro and in vivo activity profiles?

Fc modifications of anti-TIGIT antibodies significantly alter their functional profiles through modulation of Fc receptor engagement. When designing anti-TIGIT antibodies, researchers must consider whether Fc-mediated effector functions contribute to or detract from desired therapeutic outcomes . Non-glycosylated variants (e.g., IgG1NQ isotype) demonstrate reduced Fc receptor binding and minimal antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), making them suitable for pure blocking applications without depletion of TIGIT-expressing cells . Conversely, engineered Fc regions with enhanced binding to activating FcγRs can augment ADCC activity against TIGIT-high regulatory T cells or exhausted effector cells, potentially reshaping the immune landscape .

Methodologically, researchers can characterize Fc variant properties through surface plasmon resonance to measure binding kinetics to different FcγRs, in vitro ADCC/CDC assays with NK cells or complement, and flow cytometry-based assessment of target cell depletion . In vivo models comparing different Fc variants of the same anti-TIGIT clone reveal distinct mechanisms of action: pure blockers primarily relieve T cell inhibition while ADCC-enhanced variants additionally deplete immunosuppressive cell populations . When Fc-engineered antibodies are combined with other checkpoint inhibitors in preclinical models, researchers should carefully analyze whether observed synergies depend on blocking activity, Fc-mediated functions, or both mechanisms by including appropriate isotype and F(ab')2 controls .

What are the optimal protocols for evaluating anti-TIGIT antibody blocking efficiency in vitro?

For robust evaluation of anti-TIGIT antibody blocking efficiency, researchers should implement a multi-assay approach. ELISA-based competition assays provide a quantitative foundation: biotinylated TIGIT protein is captured on streptavidin-coated plates, followed by addition of test antibodies in serial dilutions along with recombinant CD155-Fc fusion protein . Blocking efficiency is calculated by measuring the reduction in CD155 binding using HRP-conjugated secondary antibodies against the Fc tag . Flow cytometry-based competition assays offer cellular context: cells expressing TIGIT (either transfected cell lines or primary T/NK cells) are pre-incubated with test antibodies before adding fluorescently-labeled CD155, with blocking efficiency determined by the reduction in CD155 binding .

For functional validation, researchers should assess the ability of anti-TIGIT antibodies to restore immune cell functions using:

• T cell activation assays measuring proliferation (CFSE dilution), cytokine production (ELISA/intracellular staining), and activation marker upregulation (CD25, CD69)

• NK cell cytotoxicity assays against target cells expressing CD155

• Mixed lymphocyte reactions with TIGIT-expressing T cells and allogeneic dendritic cells

Controls should include isotype-matched antibodies, known blocking antibodies, and F(ab')2 fragments to distinguish Fc-dependent and independent effects . Titration experiments determine IC50 values for blocking activity, allowing quantitative comparison between different antibody candidates. The most informative protocols incorporate both recombinant protein systems for mechanistic clarity and cell-based assays for physiological relevance .

What strategies can resolve contradictory findings when testing TIGIT antibodies across different experimental models?

When confronting contradictory findings across experimental models, researchers should systematically address several methodological variables. First, closely examine antibody characteristics: different clones may recognize distinct epitopes with varying abilities to block TIGIT-ligand interactions . Perform side-by-side comparisons using identical concentrations and formats (whole IgG vs. F(ab')2) to isolate antibody-specific effects from experimental variation .

Second, evaluate model-specific factors: cell line-based systems often show cleaner responses than primary cells due to homogeneous TIGIT expression and simplified microenvironments. Primary cell contradictions may stem from donor variability in TIGIT expression levels, polymorphisms in TIGIT or its ligands, or differences in immune cell activation status . For cross-species variations, consider the 25-32% sequence divergence between human and mouse TIGIT, which may affect antibody binding and function .

Third, analyze contextual influences on TIGIT biology: the presence of competing receptors (CD226, CD96) varies across models and may counter TIGIT blockade effects . The ratio of CD226:TIGIT expression particularly influences outcomes of TIGIT blockade . Additionally, examine cytokine milieu differences between models, as certain cytokines modulate TIGIT expression and signaling strength .

Methodologically, resolve contradictions by:

• Performing parallel experiments with standardized protocols across models

• Quantifying TIGIT, CD226, and CD96 expression by flow cytometry or qRT-PCR

• Controlling for activation status using resting versus pre-activated cells

• Including combination approaches with other checkpoint inhibitors to reveal context-dependent synergies

• Using genetic approaches (CRISPR/siRNA) alongside antibody blockade to validate target specificity

This systematic approach helps distinguish true biological differences from technical artifacts when reconciling contradictory findings .

How can researchers design experiments to distinguish between the direct effects of TIGIT blockade and indirect effects mediated through the TIGIT-CD226-CD155 axis?

Designing experiments that distinguish direct TIGIT blockade effects from indirect effects within the TIGIT-CD226-CD155 axis requires sophisticated methodological approaches. Researchers should implement a comprehensive experimental framework including genetic manipulation, selective blockade, and detailed phenotypic analysis .

First, create cellular systems with defined receptor expression through CRISPR/Cas9 knockout or overexpression of TIGIT, CD226, or both in relevant cell types. This allows direct comparison of anti-TIGIT antibody effects in the presence or absence of CD226 . For example, compare T cell activation following TIGIT blockade in CD226-sufficient versus CD226-deficient cells to determine CD226-dependency.

Second, employ selective blockade strategies:

• Use anti-TIGIT antibodies in combination with anti-CD226 blocking antibodies

• Compare effects of anti-TIGIT antibodies with direct CD155 blockade

• Include F(ab')2 fragments of anti-TIGIT antibodies to eliminate Fc-dependent effects

• Test anti-TIGIT antibodies that block TIGIT-CD155 interaction but do not affect TIGIT-CD112 binding

Third, perform detailed molecular analyses to track signaling pathway activation:

• Assess phosphorylation of TIGIT ITIM motifs and downstream signaling proteins (SHP1/2, SHIP1)

• Monitor CD226 phosphorylation status and association with LFA-1

• Measure recruitment of activating adapters (e.g., Grb2) to CD226

• Quantify calcium flux as an immediate readout of T cell receptor signaling modulation

For in vivo studies, use genetic models with selective deletion of TIGIT or CD226 in specific immune cell populations, followed by treatment with anti-TIGIT antibodies and comprehensive immune phenotyping . This controlled experimental approach allows differentiation between direct TIGIT blocking effects and indirect effects mediated through altered CD226 availability or signaling .

How should researchers interpret TIGIT expression heterogeneity across different immune cell subsets in their experimental readouts?

TIGIT expression heterogeneity across immune cell populations necessitates nuanced data interpretation and specialized experimental design. When analyzing TIGIT expression, researchers should implement multi-parameter flow cytometry with comprehensive gating strategies that first identify major lymphocyte populations (CD4+ T cells, CD8+ T cells, NK cells, Tregs) and then analyze TIGIT expression within each subset . This approach reveals that TIGIT is differentially expressed on effector memory CD4+ T cells (30-40%), regulatory T cells (40-50%), exhausted CD8+ T cells (50-70%), and mature NK cells (20-90%) .

For accurate interpretation, normalization to appropriate control populations is essential—researchers should compare TIGIT expression on tumor-infiltrating lymphocytes to matched circulating lymphocytes or lymphocytes from adjacent non-tumor tissue . When examining functional outcomes following anti-TIGIT treatment, stratify analyses by both cell type and TIGIT expression level (negative, low, intermediate, high) since response magnitude often correlates with baseline TIGIT expression .

Experimental design should account for this heterogeneity through:

• Cell sorting of specific populations before functional assays

• Correlation analyses linking TIGIT expression levels with functional readouts at single-cell level

• Time-course studies tracking TIGIT expression changes following activation

• Co-expression analysis with other checkpoints (PD-1, TIM-3, LAG-3) to identify multi-inhibitory receptor+ cells

The biological significance of TIGIT heterogeneity extends to therapeutic implications—in models of combination immunotherapy, TIGIT+PD-1+ double-positive T cells often show greater functional restoration than single-positive populations, suggesting particular importance for targeting these multifaceted exhausted cells .

What are the critical controls needed when evaluating the specificity and efficacy of novel anti-TIGIT antibodies?

Rigorous evaluation of novel anti-TIGIT antibodies requires a comprehensive panel of controls addressing specificity, functionality, and experimental validity. For specificity validation, researchers must include:

• Isotype-matched control antibodies with identical Fc regions to distinguish specific TIGIT binding from non-specific Fc-mediated effects

• TIGIT-knockout or knockdown cells alongside wild-type cells to confirm binding specificity

• Pre-absorption controls where anti-TIGIT antibodies are pre-incubated with recombinant TIGIT protein before cell staining

• Cross-reactivity assessment against structurally related proteins (CD96, CD112R) using overexpression systems

For functional validation, critical controls include:

• F(ab')2 fragments of the test antibody to differentiate Fc-independent blocking activity from Fc-dependent effector functions

• Known blocking antibodies (commercial standards) tested in parallel for comparative efficacy

• Recombinant TIGIT-Fc fusion proteins as alternative blocking agents

• Cells expressing mutant TIGIT with disrupted ligand-binding capacity

Experimental validity controls should incorporate:

• Dose-response relationships across a wide concentration range (1 ng/mL to 100 μg/mL)

• Multiple readout systems measuring the same biological effect

• Evaluation in both cell lines and primary cells

• Assessment under different activation conditions (resting, suboptimal, and optimal stimulation)

When reporting results, researchers should provide complete information about antibody characteristics including clone name, isotype, format (whole IgG vs. F(ab')2), concentration, and duration of treatment . Additionally, detail the specific experimental conditions where efficacy was observed, as anti-TIGIT antibody effects often vary across activation states, cell types, and model systems .

How can researchers effectively measure changes in the TIGIT-CD155 axis within the tumor microenvironment following antibody treatment?

Effectively measuring changes in the TIGIT-CD155 axis within the tumor microenvironment following antibody treatment requires a multi-modal approach that captures both cellular and molecular alterations. Researchers should implement an integrated strategy combining ex vivo tissue analysis with in situ imaging techniques .

For ex vivo analysis, fresh tumor samples should be enzymatically digested while preserving surface epitopes, followed by multi-parameter flow cytometry that simultaneously assesses:

• Receptor occupancy by therapeutic anti-TIGIT antibodies using secondary antibodies against the treatment antibody

• Available (unbound) TIGIT using antibodies recognizing non-overlapping epitopes

• CD155 and CD112 expression levels on tumor cells and myeloid populations

• CD226 expression and activation status on lymphocytes

• Functional readouts including cytokine production (IFN-γ, TNF-α) and proliferation markers (Ki-67)

For in situ assessment, multiplex immunofluorescence or immunohistochemistry allows spatial analysis of:

• TIGIT+ immune cell infiltration patterns and distribution relative to tumor nests

• Co-localization of TIGIT+ cells with CD155-expressing cells

• Changes in TIL density and composition following treatment

• Alterations in immune synapse formation between T cells and target cells

Molecular analyses should include:

• Phospho-flow cytometry measuring TIGIT-associated signaling pathway activation (SHP1/2 phosphorylation)

• RNA-seq of sorted immune populations to identify transcriptional changes following TIGIT blockade

• Spatial transcriptomics to map gene expression changes within distinct tumor microenvironment regions

• ELISA-based measurement of soluble TIGIT and CD155 in tumor interstitial fluid

To establish causality, parallel in vitro studies using autologous tumor-immune cell co-cultures treated with the same antibodies can confirm direct effects on the TIGIT-CD155 axis . This comprehensive approach enables researchers to connect antibody-mediated molecular changes with alterations in immune cell function and distribution within the tumor microenvironment .