CHR27 Antibody

What is CD27 and what role does it play in immunological research?

CD27 (also known as TNFRSF7) is a type I glycoprotein and member of the tumor necrosis factor (TNF) receptor family. It is expressed on specific B cell populations and the majority of T cells, playing a crucial role in the generation and long-term maintenance of T cell immunity . Functionally, CD27 serves as a receptor for CD70 (CD27L), and this interaction triggers strong ubiquitination of TRAF and activates both canonical and non-canonical NF-kappaB pathways, as well as the JNK pathway .

CD27 has become a significant target in immunology research due to its involvement in:

• T cell activation and proliferation

• Long-term T cell memory formation

• Potential role in apoptotic pathways through association with SIVA1

• Promising therapeutic target in cancer immunotherapy

Understanding CD27 biology is essential for researchers studying adaptive immunity, T cell responses, and potential immunotherapeutic interventions.

How can researchers select the appropriate CD27 antibody for their experimental needs?

Selecting the appropriate CD27 antibody requires consideration of multiple experimental factors:

For example, the CD27 antibody AB-65132 has been specifically validated for flow cytometric analysis of human samples, with recommended usage of 5 μl per 10^6 cells in a 100 μl suspension . Similarly, antibody A01271-2 shows reactivity with human, mouse, and rat samples and is validated for multiple applications including Western blot (1:500-2000), immunohistochemistry (1:50-400), flow cytometry, and ELISA (1:100-1000) .

What experimental controls are essential when working with CD27 antibodies?

Rigorous experimental controls are essential for generating reliable data with CD27 antibodies:

• Positive controls: Include samples known to express CD27 (such as activated T cells or specific lymphoma cell lines).

• Negative controls: Include samples lacking CD27 expression or use blocking peptides when available.

• Isotype controls: Use antibodies with the same isotype, host species, and conjugate but with irrelevant specificity. For example, with mouse IgG1 kappa CD27 antibodies, use a matched isotype control .

• Fluorescence Minus One (FMO) controls: For multicolor flow cytometry, include all antibodies except anti-CD27 to assess spectral overlap contribution.

• Titration controls: Optimize antibody concentration by testing a range of dilutions to identify the optimal signal-to-noise ratio.

• Secondary antibody-only controls: For indirect detection methods, include samples with only secondary antibody to assess non-specific binding.

Implementing these controls helps distinguish specific from non-specific signals and ensures accurate interpretation of experimental results.

What applications are CD27 antibodies commonly used for in research settings?

CD27 antibodies are utilized across multiple research applications, each requiring specific optimization:

Beyond these applications, CD27 antibodies are increasingly employed in:

• Immunoprecipitation for protein complex analysis

• ChIP assays for studying transcriptional regulation

• Immunofluorescence microscopy for cellular localization studies

• Functional blocking experiments to elucidate CD27-CD70 signaling pathways

How does epitope specificity influence the functional activity of CD27 agonistic antibodies?

Epitope specificity critically determines the functional activity of CD27 agonistic antibodies, with significant implications for both research applications and therapeutic development:

Research has demonstrated that antibodies binding to membrane-distal and external-facing residues of CD27 typically function as stronger agonists . This phenomenon relates to how antibody binding affects receptor clustering and subsequent signal transduction.

Epitope mapping studies using truncation mutants of human CD27 expressing or lacking various Cysteine-Rich Domains (CRDs) have revealed distinct binding profiles among different antibodies:

• Antibodies binding to CRD1-containing domains (e.g., AT133-2, AT133-5, AT133-11) show different functional properties compared to those binding CRD3-containing mutants (e.g., AT133-14)

• Varlilumab (a clinically relevant CD27 antibody) requires both CRD2 and CRD3 for binding

• Some antibodies (e.g., hCD27.15) and the natural ligand CD70 only recognize the complete wild-type molecule containing all three CRDs

These binding differences correlate with varying capacities to:

• Induce receptor multimerization

• Activate downstream signaling pathways

• Stimulate T cell proliferation and effector functions

• Modulate interactions with the natural ligand CD70

Understanding these epitope-function relationships is crucial for developing antibodies with desired agonistic or antagonistic properties for specific experimental or therapeutic applications.

What role does antibody isotype play in determining CD27 antibody efficacy in immunotherapy models?

Antibody isotype profoundly influences the therapeutic efficacy of CD27-targeting antibodies through several mechanisms:

Studies have demonstrated that suboptimal agonism due to poor epitope selection can be partially overcome through strategic Fc-engineering, specifically by employing isotypes that promote receptor clustering . The relationship between isotype and efficacy involves complex interactions with Fc gamma receptors (FcγRs):

This pattern of isotype-dependent response closely mirrors observations with anti-CD40 antibodies, where both epitope specificity and isotype contribute to agonistic potential . The mechanistic explanation involves how different isotypes engage FcγRs on antigen-presenting cells, which can create a scaffold that facilitates CD27 receptor cross-linking on T cells, thereby enhancing agonistic signaling.

These findings highlight the importance of strategic isotype selection or engineering when developing CD27 antibodies for both research and therapeutic applications, particularly when optimal epitope binding is challenging to achieve.

How can CD27 agonistic antibodies complement checkpoint inhibition in cancer immunotherapy research?

CD27 agonistic antibodies offer complementary mechanisms to checkpoint inhibitors, creating synergistic anti-tumor immunity through distinct but converging pathways:

MK-5890, a novel CD27 agonistic antibody, exemplifies this complementary approach and has demonstrated potential to enhance PD-1 checkpoint inhibition in cancer immunotherapy . The mechanistic basis for this complementarity stems from fundamental differences in how these agents modulate immune responses:

The complementary actions create a more comprehensive immune activation strategy:

• CD27 agonism enhances initial T cell priming, expansion, and effector differentiation

• PD-1 blockade prevents subsequent exhaustion and maintains T cell functionality

• Combined, they address both insufficient activation and premature termination of anti-tumor responses

This approach has particular relevance for:

• Overcoming resistance mechanisms to single-agent immunotherapies

• Addressing tumor microenvironments with multiple immunosuppressive mechanisms

• Potentially reducing required doses of individual agents, minimizing toxicity

Research using these complementary approaches requires careful consideration of timing, dosing, and sequence of administration to optimize therapeutic synergy while managing potential immune-related adverse events.

What mechanisms determine broad neutralizing capacity in therapeutic antibodies?

While the search results primarily discuss SC27 in the context of coronavirus neutralization rather than CD27, the principles governing broad neutralizing capacity are highly relevant to antibody research across disciplines:

SC27 demonstrates exceptional neutralizing breadth through several mechanisms that inform antibody engineering approaches:

• Multiple binding sites engagement: SC27 targets multiple regions of the SARS-CoV-2 spike protein, including both the ACE2 binding site and a "cryptic" conserved site . This multi-epitope targeting minimizes escape through single mutations.

• Conservation-focused targeting: By binding to highly conserved regions that experience evolutionary constraint, SC27 maintains effectiveness against diverse viral variants and even related coronaviruses .

• Structural impediments to mutation: SC27 targets regions where mutations would likely compromise viral fitness, creating a higher barrier to resistance development .

These principles translate to antibody development across research domains:

Understanding these mechanisms can guide the rational design of CD27 antibodies with enhanced functional properties, cross-species reactivity, and therapeutic potential.

What techniques are most effective for epitope mapping of CD27 antibodies?

Epitope mapping is critical for understanding antibody function and guiding therapeutic development. Several complementary approaches are particularly effective for CD27 antibodies:

For comprehensive epitope characterization, researchers typically employ multiple complementary techniques. For example, domain mapping through truncation mutants might identify CRD1 as the binding region, followed by alanine scanning to identify specific critical residues within CRD1, and finally structural confirmation through X-ray crystallography.

What protocols ensure proper validation of CD27 antibodies for research applications?

Rigorous validation is essential for ensuring CD27 antibody specificity and functionality. A comprehensive validation protocol includes:

• Multi-application testing: Validate antibodies across relevant applications (WB, IHC, Flow cytometry, ELISA) using standardized protocols. For example, Boster validates all antibodies on multiple platforms with positive and negative controls to ensure specificity and high affinity .

• Binding kinetics characterization:

  • Employ surface plasmon resonance (SPR) to determine association/dissociation rates (ka/kd) and affinity constants (KD)

  • Compare binding to target species (human, mouse, rhesus) when cross-reactivity is claimed

  • Document concentration-dependent binding curves

• Functional validation:

  • For agonistic antibodies: Confirm activity in NF-κB reporter assays and T-cell proliferation assays

  • For blocking antibodies: Verify inhibition of CD27-CD70 interaction using competitive binding assays

  • Assess downstream signaling effects (phosphorylation, gene expression)

• Specificity testing:

  • Knockout/knockdown validation: Test on CD27-deficient cells/tissues

  • Peptide competition: Pre-incubate with immunizing peptide to confirm specific binding

  • Cross-reactivity: Test against related TNFR family members

• Reproducibility assessment:

  • Lot-to-lot consistency testing

  • Inter-laboratory validation

  • Multiple detection methods to confirm specificity

Implementing this comprehensive validation approach ensures reliable antibody performance and minimizes the risk of non-specific or artifact-based results.

What are the optimal conditions for using CD27 antibodies in flow cytometry?

Optimizing flow cytometry protocols for CD27 antibodies requires careful consideration of several parameters:

When analyzing specific cell populations, consider these additional recommendations:

• For T cells: Include markers like CD3, CD4/CD8, and activation markers (CD69, HLA-DR)

• For B cells: Include markers like CD19, CD20, and differentiation markers (IgD, CD27 itself)

• For NK cells: Include CD56, CD16, and exclude lineage markers

The Atlantic Blue™ fluorescent dye-conjugated CD27 antibody (AB-65132) has excitation/emission maxima of 404 nm / 458 nm , making it compatible with violet laser excitation and requiring appropriate filter sets for detection.

How can researchers optimize CD27 antibody-based immunohistochemistry protocols?

Optimizing immunohistochemistry (IHC) protocols for CD27 antibodies requires systematic approach to several critical parameters:

Additional optimization strategies:

• Consider dual IHC with lineage markers (CD3, CD20) to identify specific CD27+ populations

• For formalin-fixed paraffin-embedded (FFPE) tissues, extend antigen retrieval times compared to frozen sections

• For multiplexed staining, use antibodies raised in different host species or directly conjugated primaries

• When quantifying expression, standardize image acquisition parameters and use appropriate analysis software

Thorough documentation of optimized protocols ensures reproducibility and facilitates troubleshooting when working with different tissue types or experimental conditions.

What strategies can address inconsistent results when using CD27 antibodies?

Inconsistent results with CD27 antibodies can stem from multiple sources. Implementing systematic troubleshooting strategies can restore experimental reliability:

When troubleshooting CD27 antibody experiments, implement a systematic approach:

• First verify antibody quality and storage conditions

• Evaluate sample preparation and handling procedures

• Review critical protocol parameters (concentrations, incubation times, buffers)

• Confirm appropriate positive and negative controls are included

• Document all experimental variables to identify patterns in inconsistency

How do different storage and handling conditions affect CD27 antibody performance?

Proper storage and handling of CD27 antibodies is critical for maintaining their performance characteristics:

Specific recommendations for CD27 antibodies from the search results:

• Antibody A01271-2: Store at -20°C as supplied; after reconstitution, store at 2-8°C for up to 6 months; avoid repeated freezing and thawing

• Antibody AB-65132 (Atlantic Blue™ conjugate): Store at 2-8°C; avoid exposure to light; stable for one year after shipment under proper storage conditions

When preparing working dilutions:

• Use fresh, cold buffer

• Prepare immediately before use when possible

• If storage of dilutions is necessary, minimize time and verify activity before critical experiments

• Document date of reconstitution and dilution on all containers

What analytical approaches are recommended for quantifying CD27 expression in complex samples?

Quantifying CD27 expression in complex samples requires appropriate analytical approaches tailored to the experimental technique:

For Flow Cytometry:

• Standardized quantification methods:

  • Molecules of Equivalent Soluble Fluorochrome (MESF) calibration

  • Antibody Binding Capacity (ABC) determination using calibration beads

  • Relative fluorescence intensity compared to isotype control

• Population analysis strategies:

  • Clearly define positive populations using FMO controls

  • Employ biexponential scaling for optimal visualization

  • Report percentage positive and median fluorescence intensity (MFI)

  • Use dimensionality reduction techniques (tSNE, UMAP) for high-parameter analysis

For Immunohistochemistry/Immunofluorescence:

• Quantitative assessment methods:

  • Digital image analysis with standardized acquisition settings

  • Use of H-score (intensity × percentage positive cells)

  • Automated cell counting and intensity measurement software

  • Spatial analysis of CD27+ cells relative to other markers

• Standardization approaches:

  • Include reference standards on each slide

  • Normalize to internal controls

  • Blind scoring by multiple observers

  • Use positive cell density (cells/mm²) for objective comparison

For Western Blot:

• Quantification strategies:

  • Normalize CD27 signal to loading controls (β-actin, GAPDH)

  • Use standard curves with recombinant CD27 protein

  • Employ digital image analysis software with background subtraction

  • Report relative expression compared to reference sample

For qPCR/RNA Analysis:

• Data normalization approaches:

  • Use multiple reference genes for robust normalization

  • Apply the ΔΔCt method for relative quantification

  • Consider splice variant-specific analysis

  • Correlate transcript with protein levels when possible

Each of these approaches requires appropriate statistical analysis and representation of biological and technical replicates to ensure reliable quantification of CD27 expression across different experimental contexts.

How can researchers determine if their CD27 antibody is suitable for therapeutic development?

Evaluating a CD27 antibody's potential for therapeutic development requires assessment of multiple parameters beyond basic research applications:

Examples from therapeutic CD27 antibody development:

• MK-5890 underwent rigorous selection based on agonist activity in NF-κB luciferase reporter assays and was characterized for epitope binding using X-ray crystallography

• Anti-CD27 antibodies were evaluated for therapeutic efficacy in human CD27 knock-in mouse models, with pharmacodynamic properties examined in mice, rhesus monkeys, and eventually in phase 1 clinical studies

• Antibody isotype significantly affected therapeutic efficacy, with mouse IgG1 (m1) and mouse IgG2a (m2a) variants showing different activities in lymphoma models

Researchers should consider partnering with translational scientists and regulatory experts early in development to ensure appropriate characterization for potential therapeutic applications.

What future directions are emerging in CD27 antibody research?

CD27 antibody research is evolving rapidly, with several promising directions emerging at the intersection of basic science and translational medicine:

• Engineered antibody formats: Development of bispecific antibodies targeting both CD27 and tumor-associated antigens to enhance tumor-specific T cell activation while minimizing systemic immune stimulation.

• Precision epitope engineering: Rational design of antibodies targeting specific CD27 epitopes that confer optimal agonistic properties, based on structural understanding of epitope-function relationships .

• Combinatorial immunotherapy approaches: Systematic investigation of CD27 antibodies in combination with other immunomodulatory agents beyond PD-1 inhibitors, including novel checkpoint inhibitors, cytokine therapies, and cellular therapies .

• Biomarker development: Identification and validation of predictive biomarkers for response to CD27-targeted therapies, enabling patient stratification for clinical trials.

• Novel delivery approaches: Development of targeted delivery systems for CD27 antibodies to enhance tumor penetration and minimize off-target effects.

• Cross-species reactive antibodies: Engineering broadly reactive CD27 antibodies that maintain functionality across multiple species, facilitating more predictive preclinical studies .

Understanding the complex interplay between epitope specificity, isotype effects, and functional outcomes will remain central to advancing both basic research tools and therapeutic applications targeting CD27.

How can researchers contribute to improving standardization in CD27 antibody research?

Improving standardization in CD27 antibody research requires collective effort from the scientific community:

• Comprehensive reporting: Include detailed methodological information in publications, including:

  • Antibody clone, catalog number, and lot

  • Exact dilutions and incubation conditions

  • Complete buffer compositions

  • Specific epitope information when available

  • Validation data demonstrating specificity

• Reference standards establishment: Develop community-accepted positive and negative controls for CD27 detection in different applications and sample types.

• Protocol repositories: Contribute optimized protocols to public repositories with detailed troubleshooting guidance.

• Interlaboratory validation: Participate in multi-center studies to validate antibody performance across different laboratory settings.

• Data sharing: Make raw data available through appropriate repositories, enabling meta-analyses and facilitating identification of factors affecting reproducibility.