CD96 Recombinant Monoclonal Antibody

What is CD96 and what is its biological significance in immune function?

CD96, also known as TACTILE (T cell-activated increased late expression), is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. It is expressed on various immune cells including natural killer (NK) cells, T cells, and dendritic cells. CD96 plays critical roles in immune regulation through several mechanisms:

CD96 functions primarily in adhesive interactions between activated T and NK cells during the late phase of immune responses. It promotes NK cell-target adhesion by interacting with PVR (CD155) present on target cells, including tumor cells. This interaction contributes to the regulation of immune cell activation and plays a role in recognition and elimination of target cells .

Functionally, CD96 appears to operate after T and NK cells have penetrated the endothelium using integrins and selectins, when they are actively engaging diseased cells and moving within areas of inflammation . It belongs to the same immunoglobulin superfamily as CD226 (DNAM-1) and TIGIT, with CD96 and TIGIT functioning as inhibitory receptors and CD226 as a co-stimulatory receptor .

Recent research has revealed that CD96 inhibits NK cell cytokine release and plays an inhibitory role in Th9 cells. In certain cancers, such as lung adenocarcinoma (LUAD), CD96 levels in CD8+ T cells are elevated compared to healthy individuals, potentially promoting a hypo-immune response .

How are CD96 recombinant monoclonal antibodies produced for research applications?

The production of CD96 recombinant monoclonal antibodies involves a sophisticated multi-stage process that combines molecular biology and protein engineering techniques:

• Initial isolation: The CD96 monoclonal antibody is collected and its gene sequence is thoroughly analyzed to identify the variable regions responsible for antigen binding.

• Vector construction: A vector containing the CD96 monoclonal antibody gene is constructed, incorporating the necessary regulatory elements for expression.

• Host cell transfection: The vector is transfected into a suitable host cell line (typically mammalian cells) for culturing and expression.

• Immunogen preparation: During production, recombinant human CD96 protein is used as an immunogen to stimulate specific antibody production.

• Purification: The expressed CD96 recombinant monoclonal antibody is purified using affinity chromatography to isolate the target antibody from cellular components and other proteins.

• Quality assessment: The purified antibody is assessed for specificity using ELISA applications to confirm binding properties .

This recombinant approach allows for more consistent production with higher reproducibility compared to traditional hybridoma methods while maintaining the specificity of monoclonal antibodies.

What are the key methodological considerations when using CD96 antibodies in flow cytometry?

When utilizing CD96 antibodies for flow cytometry applications, researchers should consider several methodological factors to ensure optimal results:

• Antibody clone selection: Different clones recognize different epitopes of CD96, which can significantly impact results. For example, clone NK92.39 has been validated for flow cytometry of human samples .

• Fluorophore selection: CD96 antibodies are available conjugated with various fluorophores including PE, which should be selected based on your panel design and potential spectral overlap with other markers .

• Sample preparation: CD96 is expressed predominantly on NK cells and T cells, requiring proper lymphocyte isolation and potential activation protocols depending on the research question.

• Titration optimization: Each antibody lot should be titrated to determine optimal concentration for staining, as over-staining can increase background while under-staining may miss positive populations.

• Appropriate controls: Include both isotype controls and FMO (fluorescence minus one) controls to accurately set gates, particularly when analyzing cells with variable CD96 expression levels.

• Consideration of CD96 regulation: CD96 expression increases with activation ("increased late expression"), so timing of analysis after stimulation should be carefully controlled across experimental conditions.

• Co-staining strategies: Consider co-staining with antibodies against CD155 (PVR), CD226 (DNAM-1), and TIGIT to provide context for CD96 expression and binding competition studies .

These methodological considerations ensure more reliable and reproducible flow cytometry results when investigating CD96 expression and function.

How does the binding domain of anti-CD96 antibodies affect their functional properties?

The binding domain of anti-CD96 antibodies significantly influences their functional properties and therapeutic potential, as evidenced by comparative studies:

Research comparing different anti-mouse CD96 monoclonal antibodies has revealed domain-specific effects on function. Antibodies that bind to the first Ig domain of mouse CD96 (such as clones 3.3 and 6A6) compete with CD155 binding and block CD96-CD155 interactions. In contrast, antibodies binding to the second Ig domain (such as clone 8B10) do not block this interaction .

Interestingly, comparative studies in multiple metastasis models showed that while all three antibody clones demonstrated anti-metastatic activity, their relative potency followed the order: 6A6 > 3.3 > 8B10. This suggests that the specific epitope binding properties significantly influence therapeutic efficacy beyond simply blocking the CD96-CD155 interaction .

The most remarkable finding was that anti-CD96 antibodies can promote NK cell anti-metastatic activity even without blocking CD96-CD155 interactions. This challenges the conventional immune checkpoint blockade paradigm and suggests alternative mechanisms through which these antibodies might function, potentially involving antibody-dependent cellular cytotoxicity or other signaling effects .

These domain-specific functional differences highlight the importance of epitope selection in antibody development and suggest that comprehensive epitope mapping should be conducted when developing anti-CD96 therapeutic antibodies.

What pharmacokinetic parameters are critical when designing in vivo studies with CD96 antibodies?

Several critical pharmacokinetic parameters must be considered when designing in vivo studies with CD96 antibodies to ensure reliable interpretation of results:

• Dosing regimen: Studies have employed doses around 10 mg/kg administered intraperitoneally in mouse models, though optimal dosing may vary between antibody clones. The timing and frequency of administration significantly impact efficacy .

• Half-life determination: Monitoring antibody concentrations at multiple timepoints (0.5, 1, 2, 4, 8, 24, 72, 144, 216, 240 and 336 hours post-administration) is essential to establish the pharmacokinetic profile .

• Anti-drug antibody (ADA) formation: One of the most critical factors affecting CD96 antibody pharmacokinetics is the development of anti-drug antibodies. Research has shown that anti-CD96 concentrations can drop dramatically after approximately 10 days due to ADA formation that increases antibody clearance .

• Correlation with efficacy: Higher antibody concentrations in plasma have been shown to correlate with better therapeutic efficacy. Therefore, maintaining adequate plasma concentrations is essential for accurate assessment of treatment potential .

• Analytical methodology: Combined approaches using ligand binding assays and mass spectrometry (requiring as little as 10 microliters of plasma) provide comprehensive pharmacokinetic profiling .

When designing studies, researchers should plan for regular sampling to monitor both antibody concentrations and ADA formation throughout the experiment. Without such measurements, pharmacodynamic data cannot be properly interpreted and could lead to underestimation of therapeutic efficacy .

How do CD96 antibodies function in combination with other immune checkpoint inhibitors?

CD96 antibodies demonstrate significant potential when used in combination with other immune checkpoint inhibitors, particularly through complementary mechanisms of action:

The mechanistic basis for this synergy appears to involve enhanced CD8+ T cell infiltration into tumors and an increased CD8/Treg ratio in the tumor microenvironment. This suggests that while anti-PD-1 primarily relieves T cell exhaustion, anti-CD96 may contribute through distinct mechanisms involving NK cell activation and altered immune cell trafficking .

It's worth noting that pharmacokinetic interactions between combined antibodies may occur. Studies have observed that anti-PD-1 concentrations were lower at later time points in animals receiving combination treatment compared to anti-PD-1 monotherapy, highlighting the importance of monitoring drug concentrations during combination studies .

The synergistic effect between anti-CD96 and other therapies extends beyond immune checkpoint inhibitors. Research suggests that CD96 blockade combined with other approaches, such as doxorubicin chemotherapy, has shown promise in enhancing anti-tumor responses .

These findings support the development of combination strategies targeting CD96 alongside established immune checkpoint pathways, potentially addressing resistance mechanisms and expanding the population of responsive patients.

What experimental models are most informative for evaluating CD96 antibody efficacy?

Several experimental models have proven particularly informative for evaluating CD96 antibody efficacy, each offering unique insights into different aspects of anti-tumor activity:

• Experimental metastasis models: Multiple models have demonstrated the anti-metastatic activity of CD96 antibodies:

  • B16F10 melanoma lung metastasis model

  • LWT1 BRAFV600E mutant melanoma model

  • RM-1 prostate carcinoma model

  • CT26 colon adenocarcinoma model

  These models allow assessment of metastatic burden following intravenous tumor cell injection and have consistently shown that anti-CD96 antibodies can reduce metastatic colonization .

• Spontaneous metastasis models: The orthotopic 4T1.2 mammary carcinoma model with primary tumor surgical resection provides a clinically relevant system for evaluating anti-CD96 efficacy against spontaneous distant metastases. This model has demonstrated the neoadjuvant efficacy of anti-CD96 antibodies, showing significant survival benefits .

• Combination therapy models: Models evaluating CD96 antibodies alongside other therapeutic modalities (such as anti-PD-1 or chemotherapy) provide insights into potential synergistic effects. These have shown enhanced CD8+ T cell infiltration and improved tumor control with combination approaches .

• Mechanistic models: Studies focusing on NK cell and IFN-γ dependency have revealed that the anti-metastatic activity of anti-CD96 antibodies is highly dependent on these factors, informing our understanding of their mechanism of action .

When designing studies to evaluate CD96 antibody efficacy, researchers should select models aligned with their specific research questions, considering factors such as immune cell composition, metastatic potential, and relevance to clinical scenarios.

What methodological approaches can mitigate anti-drug antibody formation in CD96 antibody studies?

Anti-drug antibody (ADA) formation presents a significant challenge in CD96 antibody studies, potentially leading to accelerated clearance and reduced efficacy. Several methodological approaches can help mitigate this issue:

• Antibody engineering strategies:

  • Humanization of antibody sequences to reduce immunogenicity

  • Framework modification to eliminate potential T-cell epitopes

  • Fc engineering to reduce immunogenicity while maintaining effector functions

• Administration protocols:

  • Optimization of dosing schedules to minimize ADA development

  • Intermittent dosing with drug holidays to reduce chronic immune stimulation

  • Consideration of alternative administration routes

• Immunomodulation approaches:

  • Co-administration of immunosuppressive agents during initial antibody exposure

  • Induction of tolerance through specific immunomodulatory regimens

  • Use of immunologically privileged delivery methods

• Monitoring and analytical strategies:

  • Implementation of sensitive ADA detection methods combining ligand binding assays and mass spectrometry

  • Regular monitoring throughout study duration (not just at endpoint)

  • Correlation of ADA levels with antibody concentrations and efficacy endpoints

• Consideration of genetic backgrounds:

  • Selection of animal strains less prone to ADA development

  • Genetic modification to humanize relevant immune components

Research has demonstrated that without proper monitoring of both plasma concentration and anti-drug antibody formation throughout in vivo studies, pharmacodynamic data cannot be properly interpreted. This could lead to an underestimation of therapeutic efficacy when rapid clearance occurs due to ADA formation .

How do the findings from CD96 antibody research inform cancer immunotherapy development?

Findings from CD96 antibody research have provided several key insights informing cancer immunotherapy development:

CD96 has emerged as a promising target in cancer immunotherapy due to its role as an immune checkpoint receptor that inhibits NK cell function and cytokine release. The identification of CD96 as a cancer stem cell marker in certain malignancies further highlights its potential relevance for targeting therapy-resistant tumor populations .

Mechanistic studies have demonstrated that CD96 blockade can enhance anti-tumor immunity through multiple mechanisms:

• Enhanced NK cell activity: Anti-CD96 antibodies promote NK cell anti-metastatic activity, which appears to be dependent on both NK cells and IFN-γ .

• Increased T cell infiltration: Combination strategies involving anti-CD96 and anti-PD-1 have shown enhanced CD8+ T cell infiltration into tumors and improved CD8/Treg ratios .

• Disruption of inhibitory signaling: By blocking CD96-CD155 interactions or through other mechanisms, these antibodies may release inhibitory signals that normally suppress immune responses against tumors .

Importantly, research has revealed that anti-CD96 antibodies need not necessarily block CD96-CD155 interactions to promote NK cell anti-metastatic activity. This finding challenges conventional immune checkpoint blockade paradigms and suggests alternative mechanisms through which these antibodies might function .

In specific cancer contexts such as lung adenocarcinoma, where CD96 levels in CD8+ T cells are elevated compared to healthy individuals, targeting this pathway may be particularly relevant for restoring anti-tumor immunity .

The continuing development of strategies to block CD96 inhibitory immune responses represents an active area of research, potentially opening new avenues of treatment for cancer patients, particularly in combination with established immunotherapies .

What are the key experimental variables that influence reproducibility in CD96 antibody research?

Reproducibility in CD96 antibody research is influenced by several key experimental variables that should be carefully controlled and reported:

• Antibody characteristics:

  • Clone selection: Different clones (e.g., 3.3, 6A6, 8B10, NK92.39) recognize different epitopes and domains of CD96, significantly affecting function

  • Isotype selection: While Fc isotype may be irrelevant for some anti-metastatic activities, it could affect other functions through Fc receptor engagement

  • Concentration: Optimal concentrations (ranging from 250-400 μg/dose in mouse models) significantly impact efficacy

• Experimental model selection:

  • Species specificity: Some antibodies only recognize human CD96, while others are specific for mouse CD96

  • Model system: Different tumor models (B16F10, LWT1, RM-1, CT26, 4T1.2) show variable responses to the same antibody

  • Route of administration: Intraperitoneal injection is commonly used, but other routes may affect pharmacokinetics

• Timing considerations:

  • Treatment schedule: Early vs. late intervention can dramatically alter outcomes

  • Duration of experiments: Studies should extend beyond 10 days to account for potential anti-drug antibody development

  • Sampling timepoints: Multiple timepoints are necessary for accurate pharmacokinetic profiling

• Analytical methods:

  • Detection techniques: Combined ligand binding assays and mass spectrometry provide comprehensive monitoring

  • Minimum sample requirements: Methods requiring as little as 10 microliters of plasma enable frequent sampling

  • Anti-drug antibody monitoring: Essential for proper interpretation of pharmacodynamic data

• Readout parameters:

  • Primary endpoints: Metastatic burden, survival, immune cell infiltration

  • Mechanistic assessments: NK cell dependency, IFN-γ production, CD8/Treg ratios

What are the current knowledge gaps and future research directions for CD96 antibody applications?

Despite significant advances in CD96 antibody research, several important knowledge gaps remain that represent promising future research directions:

• Mechanism of action clarification:

  • The finding that antibodies not blocking CD96-CD155 can still promote anti-metastatic activity raises questions about alternative mechanisms beyond classical immune checkpoint blockade

  • Further research into how antibody binding to different domains affects downstream signaling is needed

• Optimizing combination strategies:

  • While combinations with anti-PD-1 show promise, the optimal sequencing, dosing, and additional combination partners remain to be determined

  • Understanding potential antagonistic interactions between CD96 targeting and other immunotherapies

• Biomarker development:

  • Identification of predictive biomarkers for CD96 antibody response

  • Development of companion diagnostics to select patients most likely to benefit

• Addressing anti-drug antibody challenges:

  • More effective strategies to mitigate ADA formation without compromising efficacy

  • Understanding the immunological factors predisposing to ADA development

• Translational considerations:

  • Bridging preclinical findings to early clinical studies

  • Species differences in CD96 expression, structure, and function that may affect translation

• Engineering next-generation antibodies:

  • Development of bispecific antibodies targeting CD96 and complementary pathways

  • Fc engineering to enhance specific effector functions while reducing immunogenicity

• Expanding therapeutic applications:

  • Beyond cancer: potential roles in infectious disease, autoimmunity

  • Tissue-specific targeting approaches for localized immune modulation

• Long-term efficacy and resistance mechanisms:

  • Understanding acquired resistance to CD96-targeted therapies

  • Strategies to overcome resistance through rational combinations