LCR7 Antibody

What is CCR7 and why is it important in immunological research?

CCR7 (Chemokine Receptor 7, also known as CD197) is a 7 transmembrane (7TM) G protein-coupled chemokine receptor that binds to the homeostatic chemokines CCL19/MIP-3 beta and CCL21/6Ckine. It plays a crucial role in lymphoid organogenesis and the recruitment of naïve T lymphocytes and activated dendritic cells to lymph nodes, where they initiate immune responses .

CCR7 is expressed on multiple immune cell types including dendritic cells, naïve and memory T cells, regulatory T cells (Treg), NK cells, and B cells following inflammatory stimulation. Its expression enables immune cell trafficking to and retention in regional lymph nodes for expansion of adaptive immune responses .

The receptor's importance extends to pathological conditions, as CCR7 is associated with various diseases including immunological disorders, inflammatory diseases, and cancer, making it a valuable research target .

What cell types typically express CCR7 and how can antibodies help quantify this expression?

CCR7 is expressed by several immune cell populations, though at varying levels:

Flow cytometry with CCR7 antibodies can effectively quantify expression across these populations. Experimental approaches should consider that tumor cells (particularly CLL) express significantly higher levels of CCR7 than normal lymphocytes, which may require different antibody titrations for optimal detection .

What are the optimal sample preparation methods for CCR7 detection by flow cytometry?

For optimal CCR7 detection by flow cytometry:

• Sample preparation: For peripheral blood mononuclear cells (PBMCs), isolate cells using density gradient centrifugation and wash in buffer containing 2% human serum and 0.5 mM EDTA in PBS .

• Antibody concentration: Titrate antibody concentrations to determine optimal dilution. For PE-conjugated anti-CCR7, a typical starting concentration is 5 μl per million cells or 5 μl per 100 μl of whole blood .

• Staining protocol:

  • Stain cells with CCR7 antibody for 30 minutes at room temperature in the dark

  • For multicolor analysis, include appropriate markers such as CD4 (for T cells) or other lineage markers

  • Include proper isotype controls (e.g., Mouse IgG2a-PE for PE-conjugated CCR7 antibodies)

• Special considerations:

  • Protect conjugated antibodies from light

  • Do not freeze conjugated antibodies

  • Store at 2-8°C for up to 12 months from receipt date

  • When staining whole blood, be aware that PE-conjugated versions may show non-specific staining in all leukocyte populations

How do I troubleshoot weak or absent signals when using CCR7 antibodies in immunoassays?

When experiencing weak or no signal with CCR7 antibodies, systematically troubleshoot using this methodology:

• Antibody functionality:

  • Verify antibody activity using a positive control (CCR7+ cell line like transfected HEK293 cells)

  • Check antibody storage conditions (protect from light, store at 2-8°C, avoid freezing conjugated antibodies)

  • Determine optimal antibody concentration through serial dilution

• Sample-related issues:

  • Ensure sample contains sufficient CCR7+ cells (use known positive controls)

  • Check for protein degradation by adding protease inhibitors to sample buffers

  • For low-abundance targets, use enrichment steps (e.g., isolate lymphocyte populations)

• Technical protocol issues:

  • For Western blots: Verify protein transfer using Ponceau S stain; check transfer direction and membrane activation (pre-soak PVDF in methanol)

  • For flow cytometry: Ensure compensation is properly set for multi-color experiments

  • For IHC: Test different fixation methods and antigen retrieval techniques

• Detection system issues:

  • Ensure secondary antibody is compatible with primary antibody species

  • Verify detection reagents are fresh and active

  • Avoid sodium azide when using HRP-conjugated antibodies

If problems persist after these steps, consider alternative antibody clones or detection methods to confirm your findings .

What are effective strategies for reducing non-specific binding and background with CCR7 antibodies?

To minimize non-specific binding and background when using CCR7 antibodies:

• Optimize blocking conditions:

  • Increase blocking incubation period (1 hour at room temperature)

  • Test different blocking agents (3-5% non-fat dry milk, BSA, or normal serum)

  • Include mild detergents like Tween-20 in blocking and wash buffers

• Antibody concentration optimization:

  • Titrate primary antibody to determine optimal concentration

  • Consider longer incubation with more dilute antibody (e.g., overnight at 4°C)

  • Run secondary antibody controls (omitting primary antibody) to identify non-specific secondary binding

• Sample preparation improvements:

  • Filter blocking agents to remove particulates

  • Use fresh samples with protease inhibitors

  • For Western blots, ensure complete protein denaturation (boil for 10 minutes in Laemmli buffer)

• Technical adjustments:

  • For Western blots: decrease voltage during electrophoresis to prevent "smile effect"

  • For flow cytometry: include appropriate FcR blocking reagents

  • For IHC: optimize antigen retrieval methods and reduce incubation temperature

These methodological adjustments should be tested systematically to identify the optimal conditions for your specific experimental system.

How can CCR7 antibodies be used to study lymph node homing of cancer cells?

CCR7 antibodies provide valuable tools for investigating lymph node homing of cancer cells through several methodological approaches:

• Blocking migration in vitro:

  • Trans-endothelial migration (TEM) assays using CCR7 antibodies can quantify inhibition of cancer cell migration across endothelial barriers toward CCL19/CCL21 gradients

  • Calcium signaling assays can assess CCR7 antibody's ability to block ligand-induced signaling

• In vivo homing studies:

  • Pre-incubate CCR7+ cells with antibodies before tail vein transfer into mice

  • Quantify reduction in lymph node homing compared to control antibody-treated cells

  • Analyze redistribution of cells to peripheral blood, spleen, and other tissues

• Mechanism analysis:

  • Competition binding assays using fluorescently labeled CCL19 (e.g., Alexa-Fluor647 labeled CCL19) to determine if antibodies block ligand binding

  • Flow cytometry to quantify antibody binding to CCR7 on cancer cells versus healthy lymphocytes

  • Combined with functional assays to correlate receptor occupancy with migration inhibition

• Therapeutic antibody development:

  • Generation of neutralizing antibodies like CAP-100 that specifically bind CCR7 and block its ligand-binding site

  • Evaluation of both Fab-mediated activities (blocking) and Fc-mediated functions (ADCC, CDC)

These approaches have demonstrated that CCR7-blocking antibodies can significantly inhibit lymph node homing of malignant cells, particularly in chronic lymphocytic leukemia (CLL), providing both research insights and potential therapeutic applications .

What technical considerations are important when developing anti-CCR7 antibodies with therapeutic potential?

Developing anti-CCR7 antibodies with therapeutic potential presents several technical challenges that require specific methodological approaches:

• Epitope selection and immunogen design:

  • Target specific epitopes involved in ligand binding

  • Address limited immunogenicity due to high sequence homology between human and mouse CCR7 (87%)

  • Consider using synthetic peptide mimics of optimal CCR7 immunogen regions

• Functional screening methodology:

  • Implement competition binding assays with fluorescently labeled CCL19/CCL21

  • Develop calcium signaling assays to evaluate inhibition of downstream signaling

  • Establish migration inhibition assays using transwell or trans-endothelial models

• Antibody engineering considerations:

  • Optimize binding to activating Fcγ receptors over inhibitory receptors for enhanced ADCC

  • Engineer appropriate binding to FcRn for optimal plasma half-life

  • Consider humanization to reduce immunogenicity in clinical applications

• Validation strategy:

  • Quantify differential binding to CCR7 on malignant versus healthy cells

  • Assess effects on various CCR7+ immune cells to predict potential immunological side effects

  • Develop appropriate animal models despite limitations in cross-species reactivity

• Specificity testing:

  • Test binding to related chemokine receptors

  • Evaluate binding to CCR7 across different cell types and activation states

  • Confirm blocking activity extends to both CCL19 and CCL21 ligands

These technical considerations are illustrated by the development of CAP-100, a humanized IgG1 anti-CCR7 antibody that demonstrated preferential blocking of migration in CLL cells compared to healthy lymphocytes .

How should I select the appropriate CCR7 antibody clone and conjugate for my specific application?

Selecting the optimal CCR7 antibody requires systematic evaluation of several parameters:

• Clone selection based on application:

  • Clone 150503 has been validated for flow cytometry, IHC, ICC, and functional blocking assays

  • G043H7 clone is recommended for flow cytometry applications with multicolor panels

  • Consider whether you need a neutralizing antibody (e.g., for blocking studies) or just detection

• Conjugate selection methodology:

  Application	Recommended Conjugate	Considerations
  Flow cytometry (single color)	PE	Bright signal, potential non-specific staining in whole blood
  Flow cytometry (multicolor)	Alexa Fluor 750 or PE/Cy7	Minimal spectral overlap with common fluorophores
  Western blot	Unconjugated	Use with appropriate secondary antibody
  IHC/ICC	Unconjugated	Compatible with various detection systems
  Functional assays	Unconjugated	Minimal interference with receptor function

• Validation approach:

  • Test antibody on known positive controls (e.g., CCR7-transfected HEK293 cells)

  • Include appropriate negative controls (e.g., irrelevant transfectants)

  • Perform titration experiments to determine optimal concentration

  • Validate with multiple techniques if possible (e.g., flow cytometry and Western blot)

• Application-specific considerations:

  • For flow cytometry: Consider brightness, spectral compatibility with other markers

  • For microscopy: Consider fluorophore photostability

  • For functional assays: Ensure antibody has documented blocking activity

  • For quantitative studies: Select antibodies with established quantitative performance

Remember that different application needs may require different clones or conjugates for optimal results.

What are effective methods for validating the specificity of CCR7 antibodies in research applications?

To rigorously validate CCR7 antibody specificity, implement these methodological approaches:

• Positive and negative control systems:

  • Test antibody on CCR7-transfected cell lines (e.g., HEK293-CCR7) versus non-transfected controls

  • Compare staining between known CCR7+ populations (naïve/central memory T cells) and CCR7- populations (effector memory T cells)

  • Use CCR7 knockout/knockdown models where available

• Competitive binding validation:

  • Pre-incubate with unlabeled antibody to block binding of labeled antibody

  • Use CCR7 ligands (CCL19/CCL21) to compete with antibody binding

  • Employ peptides matching the antibody's epitope to confirm binding specificity

• Cross-technique validation:

  • Compare detection across multiple techniques (flow cytometry, Western blot, IHC)

  • For Western blot: Verify band molecular weight (MW) matches predicted CCR7 MW

  • For IHC/ICC: Compare with in situ hybridization for CCR7 mRNA

• Antibody isotype controls:

  • Include appropriate isotype controls (e.g., Mouse IgG2a for many CCR7 clones)

  • Match isotype control concentration to test antibody

  • Use same fluorochrome conjugate on isotype control

• Advanced validation approaches:

  • Proximity ligation assay (PLA) can confirm specific protein-protein interactions

  • For neutralizing antibodies, confirm functional inhibition in migration assays

  • Consider immunoprecipitation followed by mass spectrometry to confirm target identity

This systematic validation approach will provide strong evidence for antibody specificity and help troubleshoot any unexpected staining patterns.

How do I interpret and troubleshoot unexpected banding patterns when using CCR7 antibodies in Western blots?

When encountering unexpected bands with CCR7 antibodies in Western blot experiments, apply this systematic interpretation and troubleshooting methodology:

• Common unexpected banding patterns with CCR7 antibodies:

  Pattern	Possible Causes	Verification Approach
  Multiple bands	Post-translational modifications, multimerization, partial degradation	Treat with glycosidases, increase denaturation time, add protease inhibitors
  Higher MW than expected	Glycosylation, dimerization	Treat with glycosidases, boil samples longer (10 min) in reducing buffer
  Lower MW than expected	Proteolytic degradation, alternative splicing	Add protease inhibitors, compare with positive control lysates
  Diffuse bands	Excessive antibody concentration, sample overloading	Titrate antibody, reduce protein amount

• Distinguishing specific from non-specific bands:

  • Run lysates from CCR7-transfected cells alongside non-transfected controls

  • Use blocking peptides that should eliminate only specific bands

  • Compare banding patterns across different cell types with known CCR7 expression

• Technical optimization methodology:

  • For membrane proteins like CCR7, optimize lysis conditions (detergent type/concentration)

  • Test different reducing agent concentrations and denaturation temperatures

  • Consider native versus reducing conditions to evaluate potential multimers

  • Examine literature reports of CCR7 banding patterns for comparison

• Advanced troubleshooting approaches:

  • If multiple bands persist, consider immunoprecipitation followed by mass spectrometry

  • For suspected cross-reactivity, perform BLAST analysis of the epitope sequence

  • Consider alternative antibody clones that target different epitopes

  • Use CCR7 knockdown/knockout samples as definitive negative controls

By systematically applying these interpretation and troubleshooting strategies, you can distinguish genuine CCR7 signals from artifacts and optimize your Western blot protocol.

What factors might cause discrepancies between CCR7 protein detection methods, and how can these be reconciled?

When facing discrepancies in CCR7 detection between different methods, consider these methodological approaches to reconciliation:

• Common discrepancies between techniques:

  Discrepancy Type	Possible Causes	Reconciliation Approach
  Flow cytometry positive, Western blot negative	Conformational epitope disrupted by denaturation	Use non-denaturing conditions or alternative antibody clone
  Western blot positive, IHC negative	Fixation altering epitope accessibility	Test different fixation methods and antigen retrieval techniques
  Different expression levels between techniques	Varying sensitivity thresholds	Standardize detection using quantitative controls across methods

• Epitope-specific considerations:

  • CCR7 is a 7-transmembrane protein where epitope accessibility varies by technique

  • Flow cytometry typically detects extracellular domains

  • Western blot may detect any domain but requires denaturation

  • IHC/ICC results depend heavily on fixation and permeabilization methods

• Standardization methodology:

  • Use the same antibody clone across techniques when possible

  • Include consistent positive controls (e.g., CCR7-transfected cell lines)

  • Quantify relative expression using reference standards

  • Consider cell-specific factors that might affect detection (e.g., glycosylation patterns)

• Technical reconciliation approaches:

  • For IHC/ICC: Test multiple fixation and antigen retrieval methods

  • For Western blot: Compare reducing vs. non-reducing conditions

  • For flow cytometry: Test surface vs. intracellular staining protocols

  • Validate with orthogonal methods (e.g., RT-PCR for mRNA expression)

• Biological interpretation considerations:

  • Surface CCR7 may be internalized upon ligand binding, affecting flow cytometry results

  • Post-translational modifications may vary between cell types

  • Protein expression doesn't always correlate with functionality (test with migration assays)

By systematically addressing these factors, you can reconcile discrepancies and develop a more complete understanding of CCR7 expression in your experimental system.

How can CCR7 antibodies be used in therapeutic development research?

CCR7 antibodies have become valuable tools in therapeutic development, particularly for targeting diseases involving lymph node homing. The methodological approaches include:

• Therapeutic antibody development strategy:

  • CAP-100 represents a prototype therapeutic anti-CCR7 antibody designed to block tumor cell homing to lymph nodes

  • Development involves creating antibodies that specifically bind CCR7 and neutralize ligand-binding sites and signaling

  • Testing includes both Fab-mediated blocking activity and Fc-mediated effector functions

• Preclinical research methodology:

  • Migration inhibition assays to confirm blocking of CCR7-dependent cell movement

  • Trans-endothelial migration models to evaluate inhibition of extravasation

  • In vivo models to assess prevention of lymph node homing

  • Cell killing assays to measure ADCC and CDC potential

• Target disease considerations:

  • Chronic lymphocytic leukemia (CLL) represents a primary target due to lymph node dependence

  • CCR7 surface expression in CLL exceeds other targets like CD20

  • CCR7 antibodies can potentially overcome resistance to existing therapies like ibrutinib

  • Other potential applications include autoimmune disorders and additional lymph node-dependent cancers

• Technical development challenges:

  • Generating antibodies against complex transmembrane structures

  • Targeting specific epitopes involved in ligand binding

  • Balancing blocking activity with effector functions

  • Overcoming the high sequence homology between human and mouse CCR7

This therapeutic development research has already progressed to clinical trials, with CAP-100 entering first-in-human studies for CLL patients (NCT04704323) .

What are the specific technical considerations when using CCR7 antibodies for studying immune cell trafficking in complex tissues?

When investigating immune cell trafficking using CCR7 antibodies in complex tissues, implement these specialized methodological approaches:

• Tissue-specific sample preparation:

  • For lymph nodes: Use fresh frozen sections rather than paraffin-embedded when possible to preserve epitope accessibility

  • For high endothelial venules (HEVs): Combine CCR7 staining with endothelial markers (CD31, MECA-79)

  • For spleen: Consider region-specific analysis (white pulp vs. red pulp)

• Multiparameter imaging strategies:

  • Combine CCR7 antibodies with markers for cell subsets (CD4, CD8, CD19)

  • Include markers for tissue microenvironments (HEVs, fibroblastic reticular cells)

  • Use CCL19/CCL21 staining to correlate receptor with ligand distribution

  • Consider multiplex approaches to capture complex cellular interactions

• Dynamic trafficking analysis methods:

  • Ex vivo tissue slice models can maintain 3D architecture while allowing live imaging

  • Intravital microscopy with fluorescently labeled antibodies for in vivo tracking

  • Adoptive transfer of labeled cells pre-treated with blocking vs. non-blocking CCR7 antibodies

  • Correlation of CCR7 expression with positional data in tissue sections

• Technical optimization for tissue analysis:

  • Test multiple fixation protocols as CCR7 epitopes can be fixation-sensitive

  • Optimize antigen retrieval methods for each tissue type

  • Consider signal amplification methods for detecting low expression

  • Use proximity ligation assays to detect receptor-ligand interactions in situ

• Quantitative analysis approaches:

  • Develop algorithms for automated quantification of cell positioning relative to landmarks

  • Establish metrics for migration (distance from HEVs, penetration depth)

  • Use spatial statistics to analyze cellular distribution patterns

  • Correlate CCR7 expression levels with positioning data

These specialized approaches enable sophisticated analysis of how CCR7 governs immune cell trafficking and positioning within complex tissue microenvironments.

What approaches can be used to develop and characterize small molecule CCR7 antagonists?

Developing small molecule CCR7 antagonists requires specialized methodological approaches distinct from antibody-based strategies:

• Virtual screening methodology:

  • Ligand-based virtual screening using known CCR7 ligands and antagonists (e.g., cosalane) as templates

  • Structure-based approaches utilizing the recently solved X-ray co-crystal structure of CCR7 with cmp2105

  • Joint screening campaigns combining multiple computational approaches

  • Focused library generation based on chemokine receptor pharmacophore models

• Screening cascade design:

  • Primary screening using thermal shift assays (thermofluor) to identify stabilizing compounds

  • Secondary validation using membrane-based competition binding with radiolabeled CCL19

  • Functional confirmation with β-arrestin recruitment assays

  • Cell-based migration inhibition assays to confirm biological activity

• Validation and characterization strategy:

  • CCL19-induced calcium signaling assays for functional antagonism assessment

  • Binding affinity determination (IC50 values against both CCL19 and CCL21)

  • Selectivity profiling against related chemokine receptors

  • Assessment of allosteric versus orthosteric binding mechanisms

• Current landscape analysis:

  Compound	Discovery Method	IC50 (CCL19)	IC50 (CCL21)	Binding Site	Key Limitations
  Cosalane	Screening	0.2 μM	2.7 μM	Unknown	High lipophilicity, complex structure
  Cmp2105	Thermal-shift	35 nM (binding) 7.3 μM (function)	Not reported	Allosteric Gi binding pocket	Moderate functional potency
  Navarixin	Thermal-shift	33.9 μM	Not reported	Unknown	Low potency

• Challenges and considerations:

  • CCR7 remains underexplored in drug discovery with few potent/selective antagonists

  • Current virtual screening efforts have yielded verified decoys but limited hits

  • Potent antagonists discovered for other chemokine receptors (e.g., maraviroc for CCR5) lack activity on CCR7

  • Small molecule approach offers potential advantages in tissue penetration and oral bioavailability over antibodies

This systematic approach to small molecule antagonist development complements antibody-based strategies and may lead to novel research tools and therapeutic agents targeting CCR7.

What are the methodological considerations when studying LCR7 antibodies for plant research applications?

When working with LCR7 antibodies in plant research contexts, particularly with Arabidopsis thaliana (Mouse-ear cress), consider these specialized methodological approaches:

These methodological considerations recognize the unique challenges of plant-focused antibody applications and provide strategies to optimize LCR7 detection in Arabidopsis thaliana research .