LCR5 Antibody

What is CCR5 and why is it significant in immunological research?

CCR5 (C-C chemokine receptor type 5) is a beta-chemokine receptor that plays a central role in infectious disease, host defense, and cancer progression. This receptor's significance extends across multiple pathophysiological processes, making it an ideal target for therapeutic development. Notably, CCR5 functions as the major HIV entry co-receptor, where its surface density correlates with HIV plasma viremia levels. The receptor interacts with several ligands, including CCL5/RANTES (Regulated upon Activation, Normal T cell Expressed and presumably Secreted), which plays a primary role in inflammatory immune responses by attracting and activating leukocytes .

Beyond HIV research, CCR5 has emerged as a critical component in studies examining cancer progression, inflammatory responses, and host defense mechanisms. Understanding CCR5 biology requires considering its expression patterns across various cell types, its binding dynamics with multiple chemokines, and its functional significance in diverse physiological contexts .

How do CCR5 antibodies differ from other chemokine receptor antibodies in experimental applications?

CCR5 antibodies possess unique characteristics that distinguish them from antibodies targeting other chemokine receptors. Unlike many receptor antibodies, certain CCR5 antibodies (like Leronlimab) can achieve complete receptor occupancy without triggering significant receptor internalization, allowing them to stabilize cell surface CCR5 expression. This property enables detailed investigation of receptor dynamics that might otherwise be difficult to observe with antibodies that induce substantial receptor downregulation .

CCR5 antibodies also demonstrate distinctive species cross-reactivity patterns. For instance, some mouse CCR5 antibodies show partial cross-reactivity with human CCR5, while others (like the mouse CCL5/RANTES antibody) demonstrate 50% cross-reactivity with recombinant human CCL5 but no cross-reactivity with other species variants such as feline CCL5 . These cross-reactivity profiles must be carefully considered when designing experiments involving multiple species models.

What are the most reliable methods for validating CCR5 antibody specificity?

Validating CCR5 antibody specificity requires a multi-faceted approach:

• Direct ELISA validation: Test the antibody against recombinant CCR5 proteins from the target species and potential cross-reactive species. For example, the mouse CCL5/RANTES antibody (MAB478) demonstrates 50% cross-reactivity with recombinant human CCL5 in direct ELISAs, providing a quantitative measure of specificity .

• Competitive binding assays: Perform dose-dependent competition studies with known CCR5 ligands. Genuine CCR5 antibodies will show predictable patterns of competition with natural ligands like CCL5/RANTES.

• Cell-based validation: Test antibody binding on cell lines with confirmed CCR5 expression (positive controls) versus CCR5-negative cell lines (negative controls). Flow cytometry provides quantitative assessment of binding specificity.

• Functional validation: Confirm that the antibody demonstrates expected functional effects, such as chemotaxis inhibition. For instance, the mouse CCL5/RANTES antibody neutralizes chemotaxis elicited by recombinant mouse CCL5/RANTES in a dose-dependent manner with an ND50 of typically 0.1-0.5 μg/mL .

• Knockout/knockdown controls: When possible, validate using CCR5 knockout models or cells with CCR5 knockdown to confirm absence of binding in these negative controls.

How should researchers optimize CCR5 antibody concentrations for receptor occupancy (RO) studies?

Optimizing CCR5 antibody concentrations for receptor occupancy studies requires systematic titration and validation:

• Perform antibody titration: Conduct a dose-response analysis using concentrations ranging from sub-nanomolar to saturating levels (typically 1-10 μg/mL). Research with Leronlimab demonstrated that 5 μg/mL represented a saturating concentration for CCR5 receptor occupancy calculations .

• Establish saturation plateaus: Plot the percentage of CCR5+ cells versus antibody concentration to identify the concentration at which receptor binding plateaus, indicating saturation.

• Consider time-dependent effects: Monitor receptor occupancy at multiple time points after antibody addition. Complete receptor occupancy with Leronlimab was observed within eight hours following subcutaneous injection and maintained until plasma concentration fell below 5 μg/mL .

• Account for background binding: Implement appropriate controls to establish baseline signal levels. Use isotype controls and unstained controls to set accurate gating strategies.

• Validate across sample types: Test optimal concentrations across different sample types (whole blood, isolated PBMCs, tissue-derived cells) as matrix effects can influence binding kinetics.

For longitudinal monitoring of therapeutic antibody blockade efficacy, the established optimal concentration should be consistently applied throughout the study to ensure comparable results.

What are the most accurate flow cytometric methods for measuring CCR5 receptor occupancy?

Two independent flow cytometric methods have been validated for measuring CCR5 receptor occupancy, particularly using the anti-CCR5 antibody Leronlimab:

Method 1: Direct measurement of occupied receptors (Equation 1)
This approach directly measures Leronlimab-bound receptors using anti-human IgG4 antibody to detect the Leronlimab-occupied CCR5 receptors. The calculation is:

RO (%) = [% cells CCR5+ and Leronlimab+] ÷ [% cells CCR5+ and Leronlimab+ after ex vivo incubation with saturating (5 μg/mL) Leronlimab] × 100

Method 2: Measurement of unoccupied receptors (Equation 2)
This approach measures unoccupied CCR5 receptors using fluorescently labeled Leronlimab (Leronlimab-PB). The calculation is:

RO (%) = [% cells CCR5+ and Leronlimab+ (anti-IgG4+)] ÷ [% cells CCR5+ and (anti-IgG4+ plus Leronlimab-PB+)] × 100

Both methods require careful staining protocols:

• Use non-competing anti-CCR5 antibody clones (such as 3A9) that don't interfere with the therapeutic antibody binding

• Implement stringent washing steps to prevent false positives

• Include FMO (Fluorescence Minus One) controls for accurate gating

• Progressive gating on CD45+, singlets, live cells, CD3+, CD4+/CD8-, and CCR5+ populations

These methods have demonstrated high concordance in both non-human primate models and human clinical samples, with receptor occupancy measurements correlating strongly with plasma antibody concentrations.

How can researchers effectively use CCR5 antibodies to neutralize CCL5/RANTES-mediated effects in vivo?

Effective neutralization of CCL5/RANTES-mediated effects in vivo using CCR5 antibodies requires strategic experimental design:

• Establish optimal dosing regimen: Research has shown that daily administration of anti-RANTES monoclonal antibody from day 2 to 8 after primary tick-borne encephalitis virus (TBEV) infection significantly delayed mortality in mice. This illustrates the importance of timing antibody administration relative to infection or inflammatory challenge .

• Consider alternative CCR5 antagonists as controls: Include Met-RANTES (a CCR5 antagonist) as a comparative control to distinguish between effects specific to antibody-mediated neutralization versus receptor antagonism. Both Met-RANTES and anti-RANTES mAb treatments reduced inflammatory cell accumulation in cerebral cortex sections compared to vehicle or isotype antibody-treated mice .

• Monitor multiple endpoints: Track survival curves using Kaplan-Meier analysis, body weight changes, viral titers in relevant tissues, and histopathological changes to comprehensively assess neutralization efficacy. Despite extending survival, anti-RANTES mAb treatment did not significantly affect virus titers in brain tissues, indicating that protective effects may operate through modulation of inflammatory responses rather than direct antiviral activity .

• Use appropriate controls: Include isotype-matched control antibodies and vehicle controls to distinguish specific effects from non-specific responses. Statistical significance should be established between treatment groups and appropriate controls .

• Examine tissue-specific effects: Assess both systemic parameters and tissue-specific changes, as CCL5/RANTES neutralization may have compartmentalized effects that differ between circulation and tissue microenvironments.

How does CCR5 receptor occupancy by antibodies affect CCR5+CD4+ T cell populations in vivo?

Research has revealed unexpected dynamics in CCR5+CD4+ T cell populations following CCR5 antibody treatment:

• Increased CCR5+CD4+ T cell levels: Treatment with the anti-CCR5 antibody Leronlimab leads to a significant increase in the levels of circulating and tissue-resident CCR5+CD4+ T cells in vivo. This effect has been observed in both Leronlimab-treated macaques and humans receiving weekly 700 mg Leronlimab .

• Mechanism of increased detection: CCR5 antibody binding appears to stabilize cell surface CCR5 expression rather than inducing receptor internalization. This stabilization may prevent normal receptor turnover, leading to the increased detection of CCR5+CD4+ T cells .

• Protection from viral replication: In SIV-infected macaques, weekly Leronlimab treatment led to increased CCR5+CD4+ T cell levels while simultaneously suppressing plasma viremia. This occurred concomitantly with full CCR5 receptor occupancy on peripheral blood CD4+ T cells, demonstrating that CCR5+CD4+ T cells were protected from viral replication by antibody binding .

• Temporal dynamics: The increase in CCR5+CD4+ T cells appears to be temporary and correlates with antibody presence in circulation. Once antibody levels decrease below effective receptor occupancy thresholds (approximately 5 μg/mL for Leronlimab), CCR5+CD4+ T cell populations return to baseline levels .

These findings challenge previous assumptions about CCR5 antibody effects on target cell populations and highlight the importance of monitoring CCR5+CD4+ T cell dynamics in therapeutic applications of CCR5 antibodies.

What are the methodological considerations for studying tissue-resident versus circulating CCR5+CD4+ T cells?

Studying tissue-resident versus circulating CCR5+CD4+ T cells requires specialized methodological approaches:

• Tissue processing optimization: Tissue digestion protocols must be optimized to maintain CCR5 expression, as harsh enzymatic digestion can cleave surface receptors. Gentle mechanical disruption combined with selected enzymes (like collagenase P) helps preserve CCR5 expression while achieving single-cell suspensions.

• Distinguishing tissue residency: Include tissue-residency markers (CD69, CD103, CD49a) alongside CCR5 staining to differentiate true tissue-resident populations from circulating cells captured within the tissue vasculature. Intravascular staining techniques using intravenous antibody administration prior to tissue collection can further distinguish these populations.

• Accounting for tissue-specific expression levels: CCR5 expression levels vary substantially between tissues. Flow cytometric analysis must account for these differences by using tissue-matched controls and consistent gating strategies adapted to tissue-specific expression patterns.

• Receptor occupancy assessments across tissues: When measuring CCR5 receptor occupancy, parallel assessment of blood and tissue samples provides critical comparative data. Research has shown that therapeutic antibodies like Leronlimab can achieve receptor occupancy on both blood and tissue-resident CD4+ T cells, though penetration kinetics may differ .

• Functional validation: Beyond phenotypic characterization, functional assays (chemotaxis, calcium flux, signaling) should be performed on cells isolated from different anatomical compartments to determine if CCR5 function differs between tissue-resident and circulating populations.

These considerations are essential for accurate comparative analysis of CCR5+CD4+ T cells across different anatomical compartments and for understanding the tissue-specific impacts of CCR5-targeting therapeutics.

How can CCR5 antibodies be used to investigate the role of CCR5 in non-HIV pathologies?

CCR5 antibodies offer powerful tools for investigating CCR5's role in diverse pathologies beyond HIV:

• Neuroinflammatory disorders: Anti-RANTES mAb treatment has been shown to extend survival in tick-borne encephalitis virus (TBEV) infection models by reducing inflammatory cell accumulation in the cerebral cortex. This approach can be adapted to study other neuroinflammatory conditions where CCR5-CCL5 interactions may drive pathology .

• Cancer immunology research: CCR5 is upregulated in certain cancers (e.g., breast cancer) and promotes tumor progression through multiple mechanisms including attraction of pro-inflammatory macrophages, direct effects on tumor cells, modulation of stromal cells, and vascular changes. CCR5 antibodies can be used to dissect these mechanisms by selectively blocking CCR5 signaling in different tumor compartments .

• Inflammatory disease models: In conditions characterized by excessive CCL5-driven inflammation, neutralizing antibodies can help determine the contribution of CCR5-CCL5 axis to pathology. Experimental designs should include:

  • Time-course studies to determine optimal intervention windows

  • Dose-response assessments to establish minimum effective concentrations

  • Comparative studies with small molecule CCR5 antagonists to distinguish receptor blockade from ligand neutralization effects

• Mechanistic separation of CCR5 functions: Since CCR5 interacts with multiple chemokines beyond CCL5 (including CCL3/MIP-1α and CCL4/MIP-1β), careful experimental design using both anti-CCR5 antibodies and specific chemokine-neutralizing antibodies can help dissect the relative contributions of different ligand-receptor interactions to observed pathologies.

• Combined blockade strategies: For complex pathologies, combining CCR5 antibodies with blockade of other chemokine receptors or inflammatory mediators can reveal synergistic pathways and potential therapeutic combinations.

How should researchers interpret differences between CCR5 receptor occupancy and functional inhibition?

Understanding the relationship between CCR5 receptor occupancy (RO) and functional inhibition requires nuanced interpretation:

• Non-linear relationship: Complete receptor occupancy does not always translate to complete functional inhibition. The dose-response curves for RO versus functional effects often show different EC50 values, with higher antibody concentrations typically required for complete functional inhibition compared to receptor occupancy.

• Mechanistic considerations: Partial receptor occupancy may be sufficient to disrupt certain CCR5 functions while others remain intact. This phenomenon relates to concepts of receptor reserve and signaling thresholds, where different downstream pathways may require different levels of receptor availability.

• Temporal dynamics: While receptor occupancy may be achieved rapidly, functional consequences often display delayed kinetics. For example, Leronlimab achieved 100% CCR5 RO within eight hours following subcutaneous injection, but the full biological effects (like protection from viral replication) may take longer to manifest .

• Analyzing discrepancies: When discrepancies between RO and functional effects occur, researchers should consider:

  • Alternative signaling pathways that might compensate for CCR5 blockade

  • Involvement of other chemokine receptors with overlapping functions

  • Cell-specific differences in signal transduction efficiency

  • Potential for antibody-induced conformational changes that permit partial signaling despite binding

• Experimental validation: To properly interpret these differences, researchers should include complementary assays measuring different functional endpoints (chemotaxis, calcium flux, phosphorylation of downstream effectors) alongside RO measurements.

What are the common technical challenges in CCR5 antibody-based flow cytometry and how can they be addressed?

Researchers frequently encounter several technical challenges when using CCR5 antibodies in flow cytometry:

• Low receptor expression levels: CCR5 is often expressed at relatively low levels on primary cells, making detection challenging. This can be addressed by:

  • Using bright fluorochromes (PE, APC) for CCR5 detection

  • Implementing signal amplification methods

  • Careful PMT voltage optimization

  • Using specialized high-sensitivity flow cytometers like FACSymphony A5, which allows for easier detection of rare events

• Receptor internalization during processing: CCR5 can internalize during cell isolation and staining procedures. Minimize this by:

  • Keeping samples at 4°C during processing

  • Using sodium azide in staining buffers to inhibit metabolic processes

  • Reducing processing time

  • Considering fixation prior to extensive manipulation

• Antibody competition issues: When using multiple anti-CCR5 antibodies (e.g., for receptor occupancy studies), competition for binding epitopes can occur. Overcome this by:

  • Selecting non-competing antibody clones (like clone 3A9, which doesn't compete with Leronlimab)

  • Optimizing staining sequence (often staining with the lower-affinity antibody first)

  • Validating antibody combinations experimentally before critical experiments

• Background staining: High background can interfere with RO calculations. Reduce this by:

  • Implementing stringent washing steps (at least three washes with PBS following anti-IgG4 staining)

  • Using appropriate FMO controls for accurate gating

  • Blocking Fc receptors prior to staining

  • Using 10% FBS in washing buffer to reduce non-specific binding

• Cross-reactivity with non-human samples: When working with non-human primates or other animal models, antibody cross-reactivity can be inconsistent. Address this by:

  • Validating each antibody lot for cross-reactivity

  • Adjusting antibody concentrations for optimal signal-to-noise ratio

  • Adapting antibody clones for optimal species reactivity

What experimental controls are essential for validating CCR5 antibody effects in complex disease models?

Robust experimental design for studying CCR5 antibody effects in disease models requires comprehensive controls:

• Isotype-matched control antibodies: Always include isotype-matched control antibodies at equivalent concentrations to distinguish specific CCR5-mediated effects from Fc-mediated or other non-specific antibody effects. For example, studies examining anti-RANTES mAb effects in TBEV infection included isotype-matched control antibodies for comparison .

• Vehicle controls: Include appropriate vehicle controls that match the antibody formulation buffer to control for any effects of buffer components. In TBEV infection studies, both vehicle controls and isotype controls were essential for establishing the specific effects of Met-RANTES and anti-RANTES mAb .

• Dose-response relationships: Test multiple antibody doses to establish dose-response relationships. The chemotaxis neutralization assay for mouse CCL5/RANTES antibody demonstrated clear dose-dependent effects with an ND50 of 0.1-0.5 μg/mL, confirming specific activity .

• Temporal controls: Monitor effects at multiple time points to distinguish immediate from delayed consequences of CCR5 blockade. In TBEV infection studies, monitoring continued for 14 days, revealing delayed effects on survival that might have been missed with shorter observation periods .

• Multiparameter outcome assessment: Measure multiple outcomes to comprehensively characterize antibody effects. The TBEV infection studies assessed mortality, body weight changes, virus titers, and histopathological changes, revealing that anti-RANTES mAb delayed mortality and reduced inflammatory cell accumulation without significantly affecting virus titers .

• Genetic validation: When possible, include CCR5-deficient models (knockout mice or naturally CCR5-deficient subjects) to compare antibody-mediated blockade with genetic absence of the receptor.

• Competitive controls: Include experiments with CCR5 ligands (CCL5/RANTES) at saturating concentrations to compete with antibody binding and confirm specificity of observed effects.

How do findings from animal models of CCR5 antibody treatment translate to human applications?

Translating CCR5 antibody research from animal models to humans requires careful consideration of several factors:

How can CCR5 receptor occupancy measurements inform dosing strategies in clinical applications?

CCR5 receptor occupancy (RO) measurements provide critical information for optimizing clinical dosing strategies:

• Establishing minimum effective occupancy thresholds: Research indicates that plasma concentrations of approximately 5 μg/mL of Leronlimab correspond to nearly complete CCR5 RO, suggesting this as a minimum target concentration for therapeutic efficacy . By correlating RO with clinical outcomes, researchers can determine the minimum receptor occupancy required for therapeutic effects.

• Optimizing dosing intervals: The temporal relationship between antibody administration and RO duration guides dosing frequency. For Leronlimab, weekly 700 mg subcutaneous injections maintained complete CCR5 RO on peripheral blood CD4+ T cells in humans , whereas a single 50 mg/kg dose in macaques maintained high occupancy for approximately six weeks .

• Patient-specific factors: Individual variations in receptor expression, antibody clearance rates, and tissue distribution can affect RO. Monitoring RO in clinical trial participants helps identify outliers who may require dose adjustments.

• Tissue penetration considerations: While peripheral blood measurements are most accessible, they may not reflect RO in tissue compartments. The ability of CCR5 antibodies to achieve RO on both blood and tissue-resident CD4+ T cells suggests good tissue penetration, but kinetics may differ between compartments .

• Correlating RO with functional outcomes: In SIV-infected macaques, complete CCR5 RO on peripheral blood CD4+ T cells following Leronlimab treatment corresponded with full suppression of plasma viremia , establishing a functional relationship between RO and antiviral efficacy.

• Predicting drug interactions: RO measurements can help predict potential interactions with other therapies that might affect CCR5 expression or antibody binding, allowing for proactive dose adjustments.

By integrating RO measurements into clinical development programs, researchers can develop evidence-based dosing strategies that ensure optimal therapeutic exposure while minimizing unnecessary antibody administration.