CRR3 Antibody

What is CCR3 and why is it a significant target for therapeutic antibodies?

CCR3 (cysteine-cysteine chemokine receptor-3) is a receptor for several CC chemokines, including CCL5/RANTES, CCL7/MCP-3, and CCL11/eotaxin. It is predominantly expressed on the surface of eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells . CCR3 plays a central role in eosinophil trafficking into various tissues, particularly the gastrointestinal (GI) tract, making it an attractive therapeutic target for conditions characterized by eosinophilic inflammation .

Previous research has demonstrated that CCR3 and its ligands are significantly involved in pathological processes underlying allergic asthma, ocular allergies, and certain cancers . The receptor's critical role in mediating eosinophil recruitment to sites of inflammation makes anti-CCR3 antibodies valuable both as research tools and potential therapeutic agents for eosinophil-mediated diseases such as eosinophilic gastroenteritis (EGE) .

How do anti-CCR3 antibodies affect eosinophil trafficking in experimental models?

Anti-CCR3 antibodies directly interfere with the recruitment pathway of eosinophils from bone marrow into peripheral blood and subsequently into target tissues. In mouse models of food allergen-induced gastrointestinal eosinophilic inflammation, administration of anti-CCR3 antibodies significantly reduced the number of eosinophils in both peripheral blood and intestinal mucosa, though interestingly, it did not affect eosinophil levels in bone marrow .

This selective inhibition suggests that anti-CCR3 antibodies primarily disrupt the migration process rather than affecting bone marrow production of eosinophils. The mechanism involves blocking the interaction between CCR3 and its ligands (particularly eotaxins), which are crucial for directing eosinophils to sites of inflammation. This disruption in trafficking leads to significantly reduced eosinophilic infiltration in target tissues, which correlates with improved pathological outcomes in experimental models .

What experimental models are most appropriate for studying anti-CCR3 antibody efficacy?

For studying anti-CCR3 antibody efficacy, ovalbumin (OVA)-induced allergen models in BALB/c mice have proven particularly valuable. The standard protocol involves:

• Intraperitoneal sensitization with OVA

• Intragastric challenge with OVA to induce eosinophilic inflammation

• Administration of anti-CCR3 antibody or control IgG via intraperitoneal injection 1 hour before each OVA challenge

• Assessment of outcomes 1 hour after the final challenge

This approach allows researchers to evaluate multiple parameters including:

• Eosinophil counts in peripheral blood, bone marrow, and target tissues

• Histological assessment of tissue inflammation and damage

• Functional outcomes such as weight change and diarrhea severity

• Molecular analysis of relevant cytokines and chemokines

Alternative models include airway inflammation models for asthma research and tissue-specific models for other eosinophilic conditions. The selection should be guided by the specific disease context being investigated, with consideration for the tissue-specific patterns of eosinophil recruitment.

What are the optimal techniques for epitope mapping of anti-CCR3 antibodies?

Effective epitope mapping of anti-CCR3 antibodies employs a multi-faceted approach:

• CCR3 Extracellular Domain-Substituted Mutant Analysis: This technique involves creating mutants where domains of CCR3 are systematically substituted and then testing antibody binding through flow cytometry. This approach successfully identified that antibodies C3Mab-3, C3Mab-4, and J073E5 all recognize the N-terminal region (amino acids 1-38) of mouse CCR3 .

• Alanine Scanning: Following identification of the broader binding region, alanine scanning provides higher resolution mapping. In this approach, individual amino acids within the identified region are substituted with alanine, and changes in antibody binding affinity are measured. This method revealed specific amino acid requirements for various antibodies:

  • C3Mab-3 requires Ala2, Phe3, Asn4, and Thr5

  • C3Mab-4 requires Ala2, Phe3, and Thr5

  • J073E5 requires Ala2 and Phe3

• Flow Cytometry Validation: Results from mutation studies should be validated using flow cytometry to confirm binding patterns and specificity.

This methodical approach allows precise identification of binding epitopes, which is crucial for understanding antibody function and optimizing therapeutic potential.

How can computational approaches improve anti-CCR3 antibody design and optimization?

Computational approaches offer powerful tools for antibody design and optimization, applicable to anti-CCR3 antibodies through several advanced techniques:

• Homology Modeling with CDR Loop Prediction: Predicting antibody structure using guided homology modeling that incorporates de novo CDR loop conformation prediction provides detailed structural insights. This is particularly valuable for understanding how anti-CCR3 antibodies interact with their target epitopes .

• Batch Modeling for Variant Analysis: Accelerated model construction allows comparison of a parent sequence with multiple variants, enabling efficient screening of potential modifications to enhance antibody performance .

• Antibody-Antigen Interaction Prediction: Ensemble protein-protein docking can predict complex structures between anti-CCR3 antibodies and CCR3, helping researchers understand binding mechanisms at molecular resolution .

• Liability Prediction:

  • Identification of potential surface sites for post-translational modification

  • Detection of aggregation hotspots

  • Prediction of chemical reactivity sites

• In Silico Engineering:

  • FEP+ (Free Energy Perturbation) with lambda dynamics to rapidly identify high-quality variants

  • Protein Mutation FEP+ to accurately predict changes in binding affinity and thermostability

These computational approaches can significantly reduce experimental workload by prioritizing the most promising antibody candidates for laboratory validation.

What mechanisms explain the differential effects of anti-CCR3 antibodies on eosinophils versus mast cells?

Anti-CCR3 antibodies exhibit differential effects on eosinophils and mast cells despite both cell types expressing CCR3, which reflects complex underlying mechanisms:

• Species-Specific Receptor Function: Research indicates fundamental differences in CCR3 function between humans and mice. In vitro studies have shown that human mast cells migrate in response to CCR3 ligands like eotaxin, whereas murine mast cells fail to demonstrate this migratory response .

• Cell-Specific Signaling Pathways: In mouse models, anti-CCR3 antibody administration almost completely inhibited eosinophil recruitment to the intestinal mucosa but had no effect on mast cell accumulation in the same tissue. This suggests distinct signaling pathways govern CCR3-mediated recruitment in different cell types .

• Functional Dichotomy: Evidence indicates that while CCR3 may not mediate mouse mast cell migration, it does play a role in mast cell activation. Studies have shown that eotaxin-1/CCR3 signaling is required for IgE-mediated degranulation of murine mast cells .

• Therapeutic Implications: The partial effectiveness of anti-CCR3 antibodies in reducing diarrhea severity in mouse models (despite not affecting mast cell numbers) suggests that these antibodies may inhibit mast cell activation while not preventing their tissue recruitment. This functional separation has important implications for therapeutic applications targeting conditions where both eosinophils and mast cells contribute to pathology .

This differential impact highlights the complexity of chemokine receptor biology and underscores the importance of comprehensive functional analysis when developing targeted therapeutic approaches.

What controls and validation steps are essential when using anti-CCR3 antibodies in flow cytometry?

When using anti-CCR3 antibodies in flow cytometry, several critical controls and validation steps must be implemented:

• Isotype Controls: Include matched isotype controls (e.g., rat IgG2a for anti-mouse CCR3 antibodies like C3Mab-3 and C3Mab-4) to establish baseline non-specific binding .

• Positive and Negative Cell Controls:

  • Positive controls: Use cell lines with confirmed CCR3 expression (e.g., eosinophil cell lines)

  • Negative controls: Use cell lines known to lack CCR3 expression or CCR3-knockout cells

• Antibody Titration: Perform comprehensive titration experiments to determine optimal antibody concentration that maximizes specific signal while minimizing background.

• Epitope Validation: When studying specific anti-CCR3 antibodies, validate their binding epitopes through domain-substituted mutant analysis and alanine scanning as performed for C3Mab-3 and C3Mab-4 .

• Multi-Parameter Validation: Confirm CCR3 expression patterns using multiple antibody clones recognizing different epitopes to avoid epitope-masking artifacts.

• Functional Correlation: Correlate CCR3 detection with functional readouts such as cell migration or calcium flux in response to CCR3 ligands.

• Sample Preparation Considerations:

  • Minimize time between sample collection and analysis as CCR3 expression may change ex vivo

  • Be aware that certain activation states may alter CCR3 surface expression

  • Use appropriate buffers that preserve receptor epitopes

Implementing these validation steps ensures reliable and reproducible results when using anti-CCR3 antibodies for flow cytometric analysis.

How should researchers interpret conflicting data between anti-CCR3 antibody effects and genetic CCR3 knockout models?

Interpreting discrepancies between anti-CCR3 antibody studies and CCR3 knockout models requires careful consideration of several factors:

• Temporal Differences:

  • Antibody studies: Reflect acute inhibition of CCR3 in a fully developed immune system

  • Knockout models: Represent complete absence of CCR3 throughout development, potentially allowing compensatory mechanisms to emerge

• Epitope-Specific Effects:

  • Anti-CCR3 antibodies target specific epitopes (e.g., N-terminal region amino acids 1-38 in mouse CCR3) , which may block certain ligand interactions while sparing others

  • Knockout models eliminate all functions of the receptor regardless of epitope

• Antibody Properties:

  • Fc effector functions of antibodies may contribute additional effects beyond simple blocking

  • Different antibody isotypes (e.g., IgG2a used in C3Mab-3 and C3Mab-4) have varying capacities to engage complement or Fc receptors

• Analysis Framework:

  • Create a comparison table documenting all experimental parameters (dosage, timing, readouts)

  • Map discrepancies to specific outcomes to identify pattern-based explanations

  • Consider whether differences reflect distinct biological phenomena rather than contradiction

• Reconciliation Strategies:

  • Perform parallel experiments using both approaches under identical conditions

  • Introduce rescue experiments in knockout models using wild-type CCR3

  • Test epitope-specific antibodies targeting different regions to dissect domain-specific functions

Understanding these nuances allows researchers to extract complementary rather than contradictory insights from different experimental approaches to CCR3 inhibition.

How do findings from anti-CCR3 antibody research in gastrointestinal eosinophilic inflammation translate to other eosinophil-mediated diseases?

Research findings on anti-CCR3 antibodies in gastrointestinal eosinophilic inflammation provide valuable insights that can be cautiously translated to other eosinophil-mediated diseases:

• Shared Mechanistic Principles:

  • The fundamental mechanism of CCR3 in mediating eosinophil trafficking appears consistent across tissues

  • Anti-CCR3 antibody treatment significantly reduced eosinophil numbers in peripheral blood and intestinal tissue in food allergen-induced inflammation models

• Disease-Specific Considerations:

  • Tissue microenvironments vary significantly and may influence antibody penetration and efficacy

  • Co-expression of CCR3 on different cell populations varies between tissues (e.g., airway epithelium versus gastrointestinal mucosa)

• Translational Assessment:

• Outcome Variables:

  • Anti-CCR3 antibody treatment improved histological outcomes, reduced epithelial cell proliferation, and partially restored weight loss in EGE models

  • Similar outcome measurements should be established for each specific disease context

• Mechanistic Limitations:

  • Anti-CCR3 antibodies showed no effect on OVA-specific IgE levels or expression of eotaxin-1, IL-5, and IL-13

  • This suggests similar limits may apply across disease contexts - antibodies address recruitment but not underlying immune activation

These translational considerations provide a framework for applying insights from gastrointestinal models to broader eosinophil-mediated conditions while acknowledging tissue-specific factors that warrant targeted investigation.

What emerging technologies might enhance epitope-specific antibody development against CCR3?

Several cutting-edge technologies are poised to revolutionize epitope-specific antibody development against CCR3:

• AI-Driven Epitope Prediction:

  • Machine learning algorithms trained on existing epitope data can predict optimal binding regions

  • Neural networks can identify non-obvious epitopes that may be missed by conventional mapping approaches

  • These computational tools complement experimental methods like the alanine scanning used to identify key binding residues (Ala2, Phe3, Asn4, Thr5) in mouse CCR3

• Cryo-EM Structural Analysis:

  • High-resolution structural determination of CCR3-antibody complexes

  • Enables visualization of precise binding interfaces that can inform rational engineering

  • Particularly valuable for membrane proteins like CCR3 where crystallization has been challenging

• Single B Cell Sequencing Technologies:

  • Direct isolation and sequencing of B cells producing anti-CCR3 antibodies

  • Captures natural antibody diversity from immunized models

  • Allows paired heavy/light chain recovery for recombinant expression

• Antibody Display Technologies:

  • Yeast display systems for fine epitope mapping and affinity maturation

  • Phage display libraries with synthetic diversity in CDR regions targeting specific CCR3 epitopes

  • Mammalian display systems that maintain proper glycosylation and folding

• Structure-Guided Engineering:

  • Homology modeling workflows incorporating de novo CDR loop prediction

  • Computational docking to predict antibody-antigen interactions at atomic resolution

  • FEP+ technology to accurately predict the impact of mutations on binding affinity

• Multi-Specific Antibody Platforms:

  • Bispecific antibodies targeting CCR3 and complementary inflammatory pathways

  • Domain-specific targeting to separate signaling from trafficking functions

  • Epitope-selective inhibition to modulate rather than completely block CCR3 function

These emerging technologies will likely enable development of next-generation anti-CCR3 antibodies with enhanced specificity, improved tissue penetration, and tailored functional properties for specific disease contexts.

What factors contribute to variability in anti-CCR3 antibody effectiveness in experimental models?

Several factors can influence the consistency and effectiveness of anti-CCR3 antibodies in experimental settings:

• Antibody Characteristics:

  • Epitope specificity: Different antibodies targeting distinct regions of CCR3 show variable effects, as demonstrated by the specific amino acid requirements for binding of C3Mab-3 versus C3Mab-4

  • Antibody isotype: The Fc portion influences half-life and potential for effector functions

  • Antibody affinity: Higher affinity antibodies may achieve better receptor blockade

• Experimental Design Variables:

  • Timing of antibody administration: In the mouse model of EGE, administration occurred 1 hour before each OVA challenge

  • Dosage regimen: Insufficient dosing may lead to incomplete receptor blockade

  • Route of administration: Intraperitoneal injection was used in successful studies , but other routes may affect bioavailability

• Model-Specific Factors:

  • Mouse strain differences: BALB/c mice were used in the EGE studies , but strain-specific immune responses may vary

  • Age and sex of experimental animals: May influence CCR3 expression levels

  • Microbiome composition: Can affect baseline inflammation and response to interventions

• Technical Considerations:

  • Antibody storage and handling: Improper storage can lead to aggregation or degradation

  • Validation of blockade: Confirming effective CCR3 blockade through functional assays

  • Batch-to-batch variability in antibody production

• Readout Selection:

  • Choice of endpoints: Anti-CCR3 antibodies significantly reduced eosinophil numbers but had only partial effects on weight loss and diarrhea

  • Timing of assessment: Optimal timing may vary for different parameters (cellular, histological, functional)

Understanding these variables allows researchers to design more robust experiments and properly interpret variability between studies of anti-CCR3 antibodies.

How can researchers distinguish between CCR3 blockade effects and off-target effects of anti-CCR3 antibodies?

Distinguishing specific CCR3 blockade from potential off-target effects requires multiple complementary approaches:

• Epitope-Specific Controls:

  • Compare antibodies targeting different CCR3 epitopes (e.g., C3Mab-3 targeting Ala2/Phe3/Asn4/Thr5 versus C3Mab-4 targeting Ala2/Phe3/Thr5)

  • Effects consistent across different epitope-binding antibodies likely represent true CCR3 blockade

• Genetic Validation:

  • Compare antibody effects with CCR3 knockout or knockdown models

  • Effects present in antibody-treated wild-type mice but absent in CCR3-deficient mice confirm CCR3 specificity

• Dose-Response Relationships:

  • True on-target effects typically show dose-dependent responses that plateau at receptor saturation

  • Off-target effects often show different dose-response characteristics

• Molecular Signature Analysis:

  • Measure CCR3-dependent signaling pathways (e.g., calcium flux, ERK phosphorylation)

  • Compare with global transcriptomic or proteomic changes to identify potential off-target effects

• Cross-Reactivity Screening:

  • Test antibody binding to related chemokine receptors

  • Screen against tissue panels to identify unexpected binding targets

• Functional Dissection:

  • Evaluate CCR3-specific functions (eosinophil migration) versus broader physiological effects

  • As observed in EGE models, anti-CCR3 antibodies completely blocked eosinophil recruitment but only partially improved clinical symptoms, suggesting potential contributions from other factors

• F(ab)2 versus Whole Antibody Comparison:

  • Compare effects of whole antibodies versus F(ab)2 fragments lacking Fc regions

  • Differences suggest contribution of Fc-mediated effects beyond simple receptor blockade