PVR Recombinant Monoclonal Antibody

What is the molecular structure of PVR/CD155 and what epitopes are targeted by recombinant monoclonal antibodies?

PVR/CD155 is a transmembrane glycoprotein belonging to the nectin family with multiple isoforms. The predominant isoforms include membrane-bound variants (α) and soluble or secreted variants (β and γ), which are present in various body fluids including blood, cerebrospinal fluid, and urine . The protein has a molecular weight ranging from 60-80 kDa when detected by Western blotting .

Most recombinant monoclonal antibodies target the extracellular domain of human PVR/CD155. For example, some antibodies are developed against a synthetic peptide corresponding to residues surrounding Asn188 of human CD155 protein , while others target the extracellular domain spanning Met1-Asn343 (UniProt #P15151-1) . This region contains the immunoglobulin-like domains that mediate PVR's interactions with its binding partners.

How does PVR/CD155 contribute to normal immune function versus pathological conditions?

In normal immune homeostasis, PVR/CD155 plays crucial roles in:

• Mediating NK cell adhesion and triggering NK cell effector functions

• Binding to receptors CD96 and CD226 (DNAM-1), accumulating at cell-cell contact sites and forming mature immunological synapses

• Triggering adhesion, secretion of lytic granules, IFN-gamma production, and activating cytotoxicity in NK cells

• Maintaining immune tolerance and preventing autoimmunity through T-cell inhibitory signaling

In pathological conditions, particularly cancer:

• PVR is dramatically overexpressed in multiple malignancies while maintaining low or absent expression in most healthy tissues

• Overexpression promotes tumor cell invasion, migration, and proliferation

• Associated with poor prognosis and enhanced tumor progression

• Contributes to immune escape mechanisms through immunoregulatory functions

The dual role of PVR in immune activation (via DNAM-1) and inhibition (via TIGIT and CD96) creates a complex regulatory network that cancer cells exploit through PVR overexpression.

What are the optimal validated applications for PVR recombinant monoclonal antibodies and how should researchers select the appropriate clone?

PVR recombinant monoclonal antibodies have been validated for multiple applications, each with specific optimization considerations:

When selecting the appropriate clone, researchers should consider:

• Target epitope - different clones recognize distinct regions which may affect functionality detection

• Species cross-reactivity - most validated for human samples only

• Conjugation needs - whether unconjugated or directly labeled (e.g., HRP-conjugated) antibodies are required

• Validation status - preference for clones with published validation for your specific application

What are the critical methodological considerations for detecting tumor-associated PVR/CD155 in patient samples?

Detection of PVR/CD155 in patient samples requires careful optimization:

For immunohistochemistry:

• Antigen retrieval is critical - most protocols use citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Background reduction techniques are essential as PVR can be expressed at varying levels on stromal cells

• Comparison with healthy tissue controls is necessary due to the differential expression pattern

• Quantification systems should be established (H-score or percentage of positive cells) for consistent assessment

For liquid biopsies detecting soluble PVR:

• Soluble PVR isoforms (β and γ) are present in patient sera and represent potential biomarkers of cancer development and progression

• ELISA-based detection should include calibration standards

• Pre-analytical variables (collection, processing, storage) must be standardized

• Validation against tissue expression is recommended for correlation studies

When analyzing patient samples, researchers should consider that PVR is expressed at low levels on specific healthy cells including vascular endothelial cells, spinal cord motor neurons, and some immune cell subsets, which may serve as internal controls for antibody specificity .

How does PVR/CD155 modulate the tumor microenvironment through interactions with immune checkpoints?

PVR/CD155 orchestrates complex immunoregulatory functions through its interactions with multiple receptors on immune cells:

• DNAM-1 (CD226) Interaction - Activating pathway:

  • Promotes NK and T cell activation against tumor cells

  • Enhances cytotoxic functions and cytokine production

  • Facilitates immune surveillance

• TIGIT Interaction - Inhibitory pathway:

  • Functions as an immune checkpoint receptor with an ITIM domain

  • Suppresses NK and T cell functions upon binding to PVR

  • Counterbalances DNAM-1-mediated activation

  • Often upregulated in the tumor microenvironment

• CD96 Interaction - Context-dependent inhibitory role:

  • Initially characterized with inconsistent functions across species

  • Recent evidence confirms inhibitory functions on Th9 and NK cells

  • Contributes to immune suppression in the tumor context

Tumors exploit this system by:

• Overexpressing PVR to engage inhibitory receptors (TIGIT, CD96)

• Shifting the balance from immune activation toward suppression

• Promoting NK cell fratricide through PVR transfer mechanisms

• Utilizing soluble PVR to potentially compete for receptor binding

These interactions represent the molecular basis for current immunotherapeutic approaches targeting the PVR-receptor axis.

What experimental models are most appropriate for investigating PVR-targeted immunotherapies?

Researchers investigating PVR-targeted immunotherapies should consider these experimental models:

In vitro models:

• Co-culture systems using PVR-expressing tumor cells with immune effectors (NK cells, T cells)

• CRISPR/Cas9-mediated PVR knockout or overexpression cell lines

• Reporter assays measuring activation/inhibition through PVR-receptor interactions

• 3D organoid models incorporating immune components

In vivo models:

• Syngeneic mouse models with orthotopic tumors expressing human PVR

• Humanized mouse models for evaluating human-specific PVR interactions

• Genetically engineered mouse models with tissue-specific PVR expression

• Patient-derived xenograft models for testing PVR-targeting approaches

Readouts and endpoints to assess:

• Tumor volume and weight measurements (demonstrated in colon cancer models)

• Metastatic burden quantification (validated in multiple mouse tumor models)

• Immune infiltration analysis by flow cytometry and multiplex IHC

• RNA sequencing to assess transcriptional changes

Results from these models have validated several therapeutic approaches currently in clinical trials, including checkpoint inhibitors targeting TIGIT and adoptive cell therapies recognizing PVR-overexpressing tumors.

How can researchers troubleshoot specificity issues when using PVR antibodies in complex tissue samples?

Ensuring antibody specificity in complex tissue samples requires systematic validation:

• Positive and negative controls:

  • Use cell lines with known high PVR expression (many tumor cell lines) as positive controls

  • Include PVR-knockout cell lines generated by CRISPR/Cas9 as negative controls

  • Compare with tissues known to express low levels of PVR (most healthy tissues)

• Epitope validation techniques:

  • Peptide competition assays to confirm epitope specificity

  • Western blotting to verify molecular weight (60-80 kDa)

  • Cross-validation with multiple antibody clones targeting different epitopes

• Signal-to-noise optimization:

  • Titrate antibody concentrations (typical starting dilutions 1:100-1:400 for IHC)

  • Compare signal patterns with known PVR distribution (e.g., vascular endothelial cells serve as internal positive controls)

  • Use isotype controls to identify non-specific binding

• Technical considerations:

  • For FFPE tissues, optimize antigen retrieval methods

  • For flow cytometry, optimize cell preparation to preserve surface epitopes

  • For Western blotting, adjust lysis conditions to maintain protein integrity

If inconsistencies persist, consider epitope masking by protein-protein interactions or post-translational modifications affecting antibody recognition.

What strategies can optimize the detection of PVR in the context of its dynamic expression and multiple isoforms?

PVR detection requires strategies that account for its complexity:

For transmembrane isoform (α) detection:

• Use antibodies targeting extracellular domains when studying surface expression

• Employ membrane extraction protocols for Western blotting

• Consider fixation conditions that preserve membrane integrity

• Account for potential internalization during procedures

For soluble isoform (β/γ) detection:

• Optimize sample preparation techniques for bodily fluids

• Use sandwich ELISA approaches with capture/detection antibody pairs

• Consider concentration steps for dilute samples

• Recognize that soluble PVR levels are increased in cancer patient sera

For dynamic expression analysis:

• Implement time-course experiments following relevant stimuli

• Consider stress conditions that induce PVR (PVR is a stress-induced ligand)

• Analyze sorted cell populations when examining heterogeneous tissues

• Use single-cell techniques to capture expression heterogeneity

For multiparametric analysis:

• Combine PVR detection with its binding partners (DNAM-1, TIGIT, CD96)

• Correlate with functional assays (cytotoxicity, cytokine production)

• Integrate with analysis of downstream signaling pathways

• Consider context-dependent regulation of expression

These strategies will provide a more comprehensive understanding of PVR biology in experimental systems.

How might researchers develop and validate novel therapeutic approaches targeting the PVR/CD155 axis?

Several promising therapeutic approaches targeting the PVR/CD155 axis warrant investigation:

• Direct PVR targeting:

  • Development of neutralizing antibodies against PVR

  • Small molecule inhibitors disrupting PVR interactions

  • Evaluation of soluble receptor decoys competing for PVR binding

  • Validation methods should include receptor-binding competition assays and functional immune readouts

• Checkpoint inhibition strategies:

  • Anti-TIGIT antibodies blocking the PVR-TIGIT inhibitory axis

  • Dual targeting of TIGIT and other checkpoints (PD-1, CTLA-4)

  • Bispecific antibodies engaging both inhibitory pathways

  • Validation requires immune activation assessment in PVR-expressing tumor models

• Adoptive cell therapy approaches:

  • Engineering NK or T cells with enhanced DNAM-1 signaling

  • CAR-T cells targeting PVR-overexpressing tumors

  • Genetic modification to render effector cells resistant to PVR-mediated inhibition

  • Validation through cytotoxicity assays against PVR-positive tumor panels

• Oncolytic virotherapy:

  • Recombinant polioviruses targeting PVR-overexpressing tumors

  • Modified viral vectors with enhanced tumor selectivity

  • Combination with immunomodulatory agents

  • Validation through viral entry, replication, and oncolysis assessment

These approaches require rigorous preclinical validation before clinical translation, with particular attention to potential on-target/off-tumor effects given PVR expression on some healthy tissues.

What are the emerging areas in understanding the biology of PVR beyond its canonical functions?

Emerging research areas exploring non-canonical PVR functions include:

• Metabolic regulation:

  • Investigating PVR's influence on cellular metabolism in tumors

  • Examining potential interactions with metabolic checkpoints

  • Exploring metabolic consequences of PVR signaling in immune cells

  • Experimental approaches include metabolomics and bioenergetic analyses

• Extracellular vesicle biology:

  • Analyzing PVR incorporation into exosomes and microvesicles

  • Studying intercellular transfer of PVR through vesicular mechanisms

  • Investigating the immunomodulatory roles of PVR-containing vesicles

  • Methods include vesicle isolation, characterization, and functional studies

• PVR in tissue homeostasis:

  • Exploring functions beyond immune regulation and viral entry

  • Investigating roles in tissue repair and regeneration

  • Examining developmental functions in organogenesis

  • Approaches include tissue-specific knockout models and developmental studies

• Post-translational regulation:

  • Characterizing glycosylation patterns affecting PVR function

  • Investigating proteolytic processing generating soluble forms

  • Studying intracellular trafficking and recycling

  • Techniques include glycoproteomics and protein turnover analyses

Understanding these non-canonical functions may reveal new therapeutic vulnerabilities and explain context-dependent PVR activities observed in different pathological states.