CRRSP7 Antibody

What is CCR7 and why is it a significant target for antibody development?

CCR7, also known as CD197, is a seven-transmembrane G-protein-coupled receptor specific for two CC chemokines: CCL19 (also known as MIP-3β, Exodus-3, and ELC) and CCL21 (also known as 6Ckine, Exodus-2, SLC, TCA4, and SCYA21). CCR7 is primarily expressed in lymphoid tissues including the spleen, lymph nodes, and tonsil, as well as in bone marrow and on peripheral T and B lymphocytes, cord blood CD34-positive cells, and mature dendritic cells . Its primary function is mediating the homing of leukocytes to secondary lymphoid tissues in response to its cognate chemokines . This receptor plays a crucial role in immune cell trafficking and has been implicated in various disease processes, making it an important target for antibody-based research and potential therapeutic development.

What are the common formats of anti-CCR7 antibodies available for research?

Based on the search results, researchers can access several formats of anti-CCR7 antibodies:

• Fluorescently conjugated monoclonal antibodies: These include PE/Cy7 conjugated mouse anti-human CCR7 monoclonal antibodies (clone G043H7) and APC-conjugated mouse anti-human CCR7 antibodies (clone 2-L1-A) .

• Blocking antibodies: Fully human anti-CCR7 blocking antibodies such as R707, which can block human CCR7 signaling and function in response to its natural ligands .

• Custom polyclonal antibodies: These can be developed against CCR7 through specialized services, offering different purification and conjugation options depending on specific research needs .

The choice of antibody format depends on the intended application, with conjugated antibodies being particularly useful for flow cytometry, while blocking antibodies serve better for functional studies.

How can I verify the specificity of a CCR7 antibody?

Verifying antibody specificity is crucial for reliable experimental results. For CCR7 antibodies, several approaches are recommended:

• Immunofluorescent staining with flow cytometric analysis: Each lot of quality CCR7 antibodies should be tested by this method to confirm specific binding to CCR7-expressing cells .

• Testing on positive and negative controls: Use cell lines with known CCR7 expression patterns. For instance, many CCR7 antibody developers use CCR7-transfected cells as positive controls .

• Cross-reactivity assessment: Check if the antibody recognizes CCR7 across different species as claimed. For example, some anti-human CCR7 antibodies also react with CCR7 from African Green monkey, Baboon, Cynomolgus, and Rhesus .

• Functional validation: For blocking antibodies, verify their capacity to inhibit CCR7-mediated functions such as chemotaxis toward CCL19/CCL21 in vitro .

• Signal-to-noise ratio evaluation: As demonstrated in AR-V7 antibody testing, analyzing the signal-to-noise ratio helps identify antibodies that provide clear, specific nuclear signals versus background .

How do different anti-CCR7 antibody clones compare in their epitope recognition and functional effects?

Different anti-CCR7 antibody clones target distinct epitopes, resulting in varied binding characteristics and functional effects. For instance, clone G043H7 and clone 2-L1-A are both used for human CCR7 detection but may differ in their epitope recognition and binding affinity .

The R707 antibody represents a fully human anti-CCR7 blocking antibody that specifically blocks human CCR7 signaling in response to its two natural ligands. Interestingly, this antibody showed less activity against the murine CCR7 orthologue, highlighting species-specific differences in epitope recognition . In preclinical studies, R707 significantly reduced xenogeneic acute graft-versus-host disease (aGVHD) induced by human peripheral blood mononuclear cells (PBMCs) while limiting CD4+ and particularly CD8+ T cell expansion during administration .

When selecting an anti-CCR7 antibody clone for a specific application, researchers should consider:

• The exact epitope recognized (if known)

• Whether blocking activity is desired

• Species cross-reactivity requirements

• The specific application (flow cytometry, functional studies, etc.)

What are the considerations for using anti-CCR7 antibodies in multiplex flow cytometry?

When incorporating anti-CCR7 antibodies into multiplex flow cytometry panels, researchers should consider:

• Fluorochrome selection: Choose fluorochromes with minimal spectral overlap with other markers in your panel. Options include PE/Cy7 conjugated antibodies (e.g., clone G043H7) or APC-conjugated antibodies (e.g., clone 2-L1-A) .

• Titration optimization: It is recommended to titrate the antibody for optimal performance. For instance, the suggested use of PE/Cy7 anti-human CCR7 is 5 μl per million cells or 5 μl per 100 μl of whole blood, but this should be optimized for each application .

• Temperature considerations: For best staining results, cell samples should be stained and maintained at 4-8°C before flow cytometric analysis .

• Appropriate controls: Include isotype controls at the same concentration as the CCR7 antibody to differentiate specific from non-specific binding .

• Instrument settings: Ensure appropriate laser and filter settings for the selected fluorochrome. For example, APC-conjugated reagents can be used in flow cytometers equipped with a dye, HeNe, or red diode laser .

How can CCR7 antibodies be applied in studying graft-versus-host disease (GVHD) and transplantation immunology?

CCR7 antibodies have shown significant potential in transplantation immunology research, particularly in understanding and potentially treating acute graft-versus-host disease (aGVHD). Research indicates that CCR7 is critical for aGVHD pathogenesis but dispensable for beneficial graft-versus-leukemia responses .

The fully human anti-CCR7 blocking antibody R707 has demonstrated promising results in preclinical models:

• It blocks human CCR7 signaling and function in vitro in response to its natural ligands.

• While it was less effective in standard murine allogeneic hematopoietic stem cell transplantation (HSCT) models due to lower activity against murine CCR7, it significantly reduced xenogeneic aGVHD induced by human peripheral blood mononuclear cells.

• R707 specifically limited CD4+ and particularly CD8+ T cell expansion during antibody administration, with effects being transient as T cell numbers recovered after antibody cessation.

• Importantly, the antibody did not substantially impair the antitumor potential of PBMC inoculum, with treated mice retaining their capacity to reject a human acute myeloid leukemia cell line .

These findings suggest that CCR7-targeting antibodies might represent a viable new approach for aGVHD prevention in clinical settings, offering the potential to reduce GVHD while preserving beneficial anti-tumor effects.

What are the optimal storage and handling conditions for CCR7 antibodies?

Proper storage and handling of CCR7 antibodies are crucial for maintaining their functionality and specificity. Based on the search results, the following guidelines should be followed:

• Storage temperature: CCR7 antibodies should typically be stored undiluted between 2°C and 8°C (refrigerated, not frozen) .

• Light protection: Conjugated antibodies, such as PE/Cy7 or APC-conjugated anti-CCR7 antibodies, should be protected from prolonged exposure to light to prevent photobleaching of the fluorochromes .

• Freezing precautions: It is generally recommended not to freeze antibody solutions, as this can lead to degradation and loss of activity .

• Diluent conditions: CCR7 antibodies are typically supplied in phosphate-buffered solution at pH 7.2, containing preservatives such as 0.09% sodium azide and 0.2% (w/v) BSA (origin USA) .

• Safety considerations: Note that sodium azide, a common preservative in antibody solutions, yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing .

How should titration of CCR7 antibodies be performed for flow cytometry?

Proper titration of CCR7 antibodies is essential for optimal performance in flow cytometry applications. Here is a methodological approach:

• Starting recommendation: Begin with the manufacturer's suggested concentration. For example, for PE/Cy7 anti-human CCR7, the suggested use is 5 μl per million cells or 5 μl per 100 μl of whole blood .

• Preparation of serial dilutions: Prepare a series of antibody dilutions (e.g., 1:2, 1:5, 1:10, 1:20, 1:50, 1:100) using appropriate buffer.

• Cell preparation: Use a consistent number of cells (typically 1×10^6 cells) per test tube for each dilution.

• Staining procedure:

  • Incubate cells with different antibody dilutions at 4-8°C (optimal temperature for CCR7 staining)

  • Include unstained controls and isotype controls at each concentration

  • Follow standard washing and fixation protocols

• Analysis metrics: When analyzing results, consider:

  • Signal-to-noise ratio (specific staining vs. background)

  • Separation between positive and negative populations

  • Stain index calculation: (Median positive - Median negative)/(2 × Standard deviation of negative)

• Temperature considerations: For best staining results with CCR7 antibodies, maintain cell samples at 4-8°C before flow cytometric analysis .

The optimal antibody concentration will provide maximum specific signal with minimal background staining.

What benchmarking methods can be used to compare different anti-CCR7 antibodies for specific applications?

When comparing different anti-CCR7 antibodies for specific research applications, several benchmarking approaches can be employed:

• Western blotting: Compare antibodies by western blotting using cell lines with known CCR7 expression profiles to assess specificity and sensitivity .

• Immunocytochemistry signal-to-noise ratio analysis: As demonstrated in the AR-V7 antibody comparison study, comparing signal-to-noise ratios across different antibody clones can help identify those with optimal specificity. This approach involves using a set of cell lines with known CCR7 status to evaluate each antibody's performance .

• Flow cytometry metrics:

  • Compare mean fluorescence intensity (MFI) on positive and negative populations

  • Evaluate stain index calculations across antibodies

  • Assess coefficient of variation (CV) values for positive populations

• Functional blocking assays: For blocking antibodies like R707, compare their ability to inhibit CCR7-mediated functions such as chemotaxis or signaling in response to CCL19/CCL21 .

• Deep learning prediction methods: Recent advances in antibody research have employed deep learning methods to predict antibody fitness for various properties including expression, thermostability, immunogenicity, and aggregation. These computational approaches can complement experimental validation .

What are common pitfalls in CCR7 antibody-based experiments and how can they be addressed?

Researchers working with CCR7 antibodies may encounter several common challenges. Here are potential solutions based on the search results:

• Cross-reactivity issues:

  • Problem: Unexpected binding to non-target proteins

  • Solution: Verify antibody specificity using appropriate positive and negative controls. For example, CCR7-transfected cells versus non-transfected cells .

• Weak or absent staining in flow cytometry:

  • Problem: Poor detection of CCR7-positive populations

  • Solutions:

    • Ensure proper temperature maintenance (4-8°C) during staining and analysis

    • Optimize antibody concentration through careful titration

    • Check for potential epitope masking due to cell preparation methods

• High background in imaging applications:

  • Problem: Poor signal-to-noise ratio

  • Solution: As demonstrated in the AR-V7 antibody comparison, thorough comparative testing of multiple antibody clones can identify those with optimal signal-to-noise characteristics .

• Species cross-reactivity limitations:

  • Problem: Antibody functions in one species but not another

  • Example: R707 antibody showed activity against human CCR7 but was less effective against murine CCR7

  • Solution: Carefully verify cross-reactivity claims and test on target species before designing complex experiments

• Inconsistent blocking efficiency in functional assays:

  • Problem: Variable inhibition of CCR7 signaling

  • Solution: Establish dose-response relationships and determine optimal concentrations for consistent blocking activity

How can researchers validate the specificity of CCR7 antibody staining in complex tissue samples?

Validating CCR7 antibody specificity in complex tissue samples requires rigorous controls and complementary approaches:

• Multi-parameter validation approach:

  • Use multiple antibody clones targeting different CCR7 epitopes

  • Compare staining patterns for consistency across antibodies

  • Employ an antibody with established high specificity (like clone E308L for AR-V7) as a reference standard

• Biological validation controls:

  • Include tissues with known high CCR7 expression (lymph nodes, spleen) as positive controls

  • Include tissues with minimal CCR7 expression as negative controls

  • Consider using tissues from CCR7 knockout animals (if available) as definitive negative controls

• Technical validation methods:

  • Perform blocking experiments with recombinant CCR7 protein to demonstrate staining specificity

  • Use competing unlabeled antibody to confirm epitope-specific binding

  • Implement isotype controls at matched concentrations to assess non-specific binding

• Complementary molecular validation:

  • Correlate protein detection with mRNA expression (e.g., in situ hybridization or RT-PCR)

  • Confirm specificity through correlation with known CCR7 expression patterns

  • Use western blotting of tissue lysates to verify antibody specificity by molecular weight

How might machine learning and computational approaches enhance CCR7 antibody development and application?

Recent advances in computational biology and machine learning offer promising avenues for enhancing CCR7 antibody research:

• Deep learning for antibody fitness prediction: Current research is benchmarking deep learning methods for predicting antibody properties including expression, thermostability, immunogenicity, and aggregation. These approaches could be applied to CCR7 antibodies to predict their performance characteristics before experimental validation .

• Model-based distinctions: Machine learning models have shown varying abilities to distinguish between intra-family antibody variants (multi-point mutants from the same wild type) versus inter-family antibodies (diverse antibodies from different wild type origins). For CCR7 antibodies, this could help predict which modifications might improve specificity or affinity while maintaining other desired properties .

• Training data considerations: The performance of predictive models for antibodies depends significantly on training datasets. Models trained on observed antibody spaces (OAS) containing millions of antibody sequences show promise for predicting various antibody properties .

• Model size and performance correlation: Research indicates that increasing model size typically leads to improved prediction performance for antibody characteristics. This suggests that larger computational models might provide better predictions for CCR7 antibody design and optimization .

• Integration of structural data: Combining sequence-based predictions with structural modeling could enhance the design of highly specific CCR7 antibodies with optimal binding characteristics to specific epitopes.

What emerging technologies might transform the application of CCR7 antibodies in clinical research?

Several emerging technologies show potential to revolutionize how CCR7 antibodies are used in clinical research:

• Circulating tumor cell (CTC) analysis: The methodology developed for AR-V7 detection in CTCs demonstrates how carefully validated antibodies can enable non-invasive liquid biopsy approaches . Similar techniques could be applied using CCR7 antibodies to detect and characterize CCR7-expressing cells in blood samples from patients with various diseases.

• Multiparameter single-cell analysis: Integration of CCR7 antibodies into high-dimensional cytometry panels (mass cytometry, spectral flow cytometry) can provide unprecedented insights into complex immune cell trafficking and differentiation in clinical samples.

• Therapeutic antibody development: The success of R707 in reducing xenogeneic aGVHD while preserving beneficial graft-versus-leukemia responses suggests potential clinical applications for CCR7-blocking antibodies . Future research may focus on optimizing such antibodies for therapeutic use.

• Antibody engineering: Advanced engineering approaches including bispecific antibodies targeting CCR7 and another relevant molecule could enable more precise targeting of specific cell populations in complex diseases.

• In vivo imaging: Development of imaging-compatible CCR7 antibody conjugates could enable non-invasive monitoring of CCR7-expressing cell trafficking in living organisms, potentially translating to clinical diagnostic applications.