CHR5 Antibody

What is CCR5 and why is it an important target for antibody research?

CCR5 (C-C chemokine receptor type 5) is a membrane protein that plays a central role in infectious disease, host defense, and cancer progression. It serves as the major HIV entry co-receptor, with its surface density correlating with HIV plasma viremia . The fundamental importance of CCR5 in HIV infection was established when researchers discovered that individuals with a homozygous 32-base pair deletion in the CCR5 gene (CCR5 Δ32/Δ32) demonstrate high resistance to HIV infection . This natural genetic variant highlighted CCR5 as an ideal target for therapeutic interventions, particularly antibody-based approaches that can block HIV entry without requiring genetic modifications .

What methodologies are available for measuring CCR5 receptor occupancy?

More recently, researchers have developed two independent flow cytometric methods specifically for calculating CCR5 RO using anti-CCR5 antibodies like Leronlimab:

• The first method uses an anti-IgG4 antibody to detect bound Leronlimab (an IgG4 antibody) and fluorescently-labeled Leronlimab to detect unoccupied CCR5 receptors.

• The second method combines both approaches in a single panel to minimize the cell numbers required for analysis .

These methods provide sensitive measurements of occupancy on both blood and tissue-resident CD4+ T cells with low background on untreated CCR5+CD4+ T cells, representing a significant improvement over previous techniques .

How can CCR5 antibodies mimic the CCR5 Δ32/Δ32 phenotype in experimental systems?

Anti-CCR5 antibodies like Leronlimab can effectively mimic the natural resistance to HIV infection observed in individuals with the CCR5 Δ32/Δ32 genotype through competitive inhibition of the CCR5-HIV Env interaction. In controlled experiments, when CD4+ T cells from CCR5 wild-type donors were treated with Leronlimab, they became fully resistant to infection with CCR5-tropic HIV while still supporting replication of CXCR4- and dual-tropic HIV . This infection pattern is identical to that observed with CD4+ T cells from CCR5 Δ32/Δ32 donors .

The ability to recreate this protective phenotype pharmacologically through antibody treatment offers significant advantages over genetic engineering approaches to knock out CCR5. It provides a reversible intervention that avoids potential off-target effects associated with genetic modifications while still conferring protection against HIV acquisition . This approach has been validated against a panel of 25 HIV isolates from multiple clades, confirming the ability of Leronlimab to protect cells from infection with CCR5-tropic isolates from diverse geographical origins .

What are the technical considerations when translating CCR5 antibody research from human to non-human primate models?

Translating CCR5 antibody research between species requires careful consideration of several factors:

• CCR5 conservation and binding affinity: While CCR5 is highly conserved between humans and rhesus macaques, subtle structural differences may affect antibody binding affinity and efficacy .

• Expression level differences: Macaque CD4+ T cells express higher numbers of CCR5 molecules per cell compared to human cells, with central memory CD4+ T cells showing the highest expression levels . This higher per-cell expression means that inhibition of SHIV infection in macaque cells may require higher concentrations of CCR5-targeted inhibitors compared to human cells .

• Dosage adjustments: Research has shown that a 10-fold higher concentration of Leronlimab was required to achieve full inhibition of SHIV SF162P3 infection in macaque cells compared to HIV in human cells in vitro .

• RO measurement adaptations: Methods for measuring CCR5 RO must be validated across species, ensuring that fluorescently-labeled antibodies have appropriate cross-reactivity and that gating strategies account for species-specific differences in CCR5 expression patterns .

• Anti-drug antibody (ADA) development: Non-human primates may develop antibodies against human or humanized therapeutic antibodies, potentially affecting long-term efficacy. Monitoring for ADA development is essential in longitudinal studies .

These considerations highlight the importance of species-specific validation when developing CCR5 antibody-based interventions.

How does Leronlimab treatment affect CCR5+CD4+ T cell dynamics in vivo?

Leronlimab treatment has been shown to have a significant impact on CCR5+CD4+ T cell dynamics in vivo, with several important observations:

• Stabilization of cell surface CCR5: Leronlimab binds to and stabilizes cell surface CCR5, preventing receptor internalization that normally occurs following ligand binding .

• Increased circulating CCR5+CD4+ T cells: Both macaque and human studies have demonstrated that Leronlimab treatment leads to a temporary increase in the levels of circulating CCR5+CD4+ T cells . This effect has been observed in both peripheral blood and tissue-resident cells.

• Protection of CCR5+CD4+ T cells from viral infection: In chronically SIV-infected macaques, weekly Leronlimab treatment led to increased CCR5+CD4+ T cell levels concurrent with fully suppressed plasma viremia . This demonstrates that despite the increase in target cells (CCR5+CD4+ T cells), these cells were protected from viral replication by Leronlimab binding.

• Tissue penetration and receptor occupancy: Studies have confirmed full CCR5 receptor occupancy on CD4+ T cells from various tissues throughout challenge phases, including lymph nodes, duodenum, and bronchoalveolar lavage samples .

These findings provide important insights into the mechanism of action of Leronlimab beyond simple receptor blocking, revealing complex immunological effects that may contribute to its therapeutic efficacy.

What are the optimal flow cytometry protocols for assessing CCR5 receptor occupancy?

Optimal flow cytometry protocols for CCR5 receptor occupancy assessment require careful consideration of multiple factors:

Protocol Design for Combined Equations Method:

• Panel design: The most efficient protocol uses four staining tubes:

  • Two tubes serving as fluorescence minus one (FMO) controls

  • One tube for directly measuring occupied CCR5 (using anti-IgG4 antibody)

  • One tube for measuring total CCR5 expression

• Sample preparation: At least 50 μL of whole blood or 3 × 10⁵ PBMCs should be washed twice with PBS before antibody staining .

• Staining procedure:

  • Add 5 μg/mL of unconjugated Leronlimab to designated tubes

  • Incubate at room temperature for 20 minutes

  • Add fluorescently-conjugated antibodies for surface markers

  • Incubate at room temperature for additional 20 minutes

  • Lyse red blood cells if using whole blood

  • Wash and fix samples before acquisition

• Calculation method: CCR5 receptor occupancy can be calculated using either of two equations:

  • Equation 1: RO = (% IgG4+ cells) ÷ (% IgG4+ cells + % Leronlimab-PB+ cells) × 100

  • Equation 2: RO = (% IgG4+ cells) ÷ (% total CCR5+ cells) × 100

Both equations yield comparable results when properly implemented, with the combined approach minimizing required cell numbers while maintaining accuracy.

How can researchers distinguish between direct antibody effects and secondary immunological responses in CCR5 antibody studies?

Distinguishing between direct antibody-mediated effects and secondary immunological responses requires careful experimental design:

• Time-course analyses: Monitor parameters at multiple time points following antibody administration to distinguish immediate (direct) effects from delayed (secondary) responses.

• Ex vivo validation: Compare in vivo observations with ex vivo antibody treatment of cells from the same subject to isolate direct antibody effects.

• Controls for CCR5 expression changes: Account for the dynamic nature of CCR5 expression, which can change in response to inflammatory and homeostatic stimuli independent of antibody effects .

• Isotype control antibodies: Include appropriate isotype-matched control antibodies that lack CCR5 specificity to identify non-specific effects of antibody administration.

• Mechanistic inhibition studies: Use inhibitors of specific signaling pathways to determine whether observed effects are dependent on active signaling or purely due to physical receptor blockade.

• Correlation analyses: Perform correlation analyses between receptor occupancy levels and observed immunological effects to establish dose-response relationships.

When investigating the increased CCR5+CD4+ T cell levels observed with Leronlimab treatment, researchers should consider both direct mechanisms (receptor stabilization preventing internalization) and indirect effects (altered chemokine gradients affecting T cell trafficking) .

What are the critical parameters for evaluating tissue penetration of CCR5 antibodies?

Evaluating tissue penetration of CCR5 antibodies requires assessment of multiple parameters:

• Tissue sampling strategy: Collect biopsies from diverse anatomical locations (lymph nodes, intestinal mucosa, lung, etc.) to assess differential penetration in various tissue compartments .

• Quantification methods:

  • Direct antibody quantification in tissue homogenates

  • Immunohistochemistry to visualize antibody distribution

  • Flow cytometry of cells isolated from tissues to measure CCR5 RO

• Pharmacokinetic considerations:

  • Timing of tissue collection relative to antibody administration

  • Correlation between plasma antibody concentrations and tissue levels

  • Half-life determination in different tissue compartments

• Functional readouts:

  • Protection from viral challenge in tissue-resident cells

  • CCR5-dependent signaling inhibition in tissues

  • Alteration of local chemokine gradients

• Anatomical barriers assessment:

  • Blood-tissue barrier penetration efficiency

  • Impact of inflammation on tissue penetration

  • Carrier systems to enhance penetration in poorly accessible tissues

Research with Leronlimab has demonstrated that tissue penetration can be effectively assessed through combined approaches, including measurement of CCR5 RO on tissue-resident CD4+ T cells and direct quantification of antibody levels in tissue samples .

How can researchers address anti-drug antibody development in longitudinal CCR5 antibody studies?

Anti-drug antibody (ADA) development is a significant challenge in longitudinal studies with therapeutic antibodies. Researchers can address this issue through:

• Regular monitoring: Implement routine screening for ADAs using validated immunoassays at predetermined timepoints throughout the study.

• Neutralization assays: Beyond detection, assess whether detected ADAs neutralize the therapeutic effect through functional assays measuring CCR5 RO in the presence of subject serum.

• Protocol modifications:

  • Consider co-administration of immunosuppressive agents in research settings where scientifically appropriate

  • Implement dosage adjustments if ADA development is detected

  • Evaluate alternative administration routes that may reduce immunogenicity

• Correlation analysis: Document the relationship between ADA development, antibody plasma concentrations, and loss of CCR5 RO in individual subjects.

• Genetic analysis: Consider host factors that may predispose to ADA development, such as certain HLA haplotypes.

In macaque studies with Leronlimab, researchers observed ADA development in some animals (e.g., animal 37032), which corresponded with loss of CCR5 RO . This highlights the importance of monitoring ADA development when interpreting variable responses to CCR5 antibody treatment.

What strategies can resolve inconsistencies between in vitro and in vivo efficacy of CCR5 antibodies?

Resolving inconsistencies between in vitro and in vivo efficacy requires systematic investigation:

• Concentration discrepancies: In vitro studies often use standardized concentrations that may not reflect tissue distribution in vivo. Measure actual tissue concentrations and adjust in vitro conditions accordingly.

• Matrix effects: Components present in vivo (proteins, lipids, etc.) may alter antibody binding characteristics. Consider supplementing in vitro systems with serum or tissue extracts.

• Dynamic expression analysis: CCR5 expression changes dynamically in vivo in response to various stimuli. Implement time-course analyses of CCR5 expression in both systems.

• Competitive binding environment: In vivo, natural ligands may compete with antibodies for CCR5 binding. Include relevant chemokines in in vitro systems at physiologically relevant concentrations.

• 3D culture systems: Traditional 2D cell cultures may not recapitulate the tissue architecture that influences antibody distribution. Consider organoid or tissue slice culture systems.

• Multi-parameter analysis: Evaluate multiple readouts (RO, functional inhibition, downstream signaling) in both systems to identify specific points of divergence.

Research with Leronlimab demonstrated that while a specific concentration was sufficient to block HIV infection in human cells in vitro, a 10-fold higher concentration was required for equivalent protection in macaque cells due to higher per-cell CCR5 expression . This highlights the importance of species-specific optimization when translating between in vitro and in vivo systems.

How should researchers interpret changes in CCR5+CD4+ T cell frequencies during antibody treatment?

Interpreting changes in CCR5+CD4+ T cell frequencies during antibody treatment requires consideration of multiple factors:

• Mechanism assessment: Distinguish between:

  • Actual increase in cell numbers (proliferation or mobilization from tissues)

  • Apparent increase due to prevented receptor internalization

  • Changes in trafficking patterns between blood and tissues

• Temporal patterns: Establish whether changes are transient or sustained, and correlate with antibody concentration over time.

• Functional status evaluation: Assess whether the phenotype and functionality of CCR5+CD4+ T cells changes during treatment through:

  • Activation marker analysis

  • Cytokine production profiling

  • Transcriptomic analysis

  • Proliferation assays

• Compartmental analysis: Compare changes across multiple anatomical compartments (blood, lymph nodes, mucosal tissues) to determine whether redistribution rather than absolute changes are occurring.

• Context-specific interpretation: In the HIV/SIV infection context, increased CCR5+CD4+ T cells despite viral suppression indicates effective protection of these target cells by the antibody .

Research with Leronlimab has shown that treatment leads to increased levels of circulating and tissue-resident CCR5+CD4+ T cells in vivo in both macaques and humans . This is attributed to Leronlimab's stabilization of cell surface CCR5, preventing natural receptor internalization processes .

How might CCR5 antibodies be applied beyond HIV in other disease models?

CCR5 antibodies have potential applications across multiple disease models beyond HIV:

• Cancer immunotherapy: CCR5 plays roles in cancer progression through:

  • Promotion of cancer cell proliferation and metastasis

  • Regulation of tumor microenvironment and immune infiltration

  • Mediation of cancer stem cell activities

  CCR5 antibodies could potentially modulate these processes, particularly in cancers where CCR5 expression correlates with poor prognosis .

• Autoimmune and inflammatory disorders: Data from CCR5 knockout models suggest advantages in decreased immune activation in certain contexts . CCR5 antibodies might provide therapeutic benefit in:

  • Rheumatoid arthritis

  • Inflammatory bowel disease

  • Multiple sclerosis

  • Transplant rejection

• Neuroinflammatory conditions: CCR5 plays roles in neuroinflammation, but knockout models also show neurological complications . Carefully titrated CCR5 antibody therapy might provide balanced modulation in:

  • Stroke recovery

  • Traumatic brain injury

  • Neurodegenerative diseases

• Infectious diseases beyond HIV: CCR5 functions in immune responses to various pathogens. While knockout models show blunted responses to some pathogens , targeted modulation through antibodies might enhance beneficial responses while limiting pathological inflammation in:

  • Viral hepatitis

  • Parasitic infections

  • Bacterial sepsis

Research should focus on defining optimal receptor occupancy levels for each application, as complete blockade may not be desirable in all disease contexts.

What novel methodologies could enhance tissue-specific delivery of CCR5 antibodies?

Several innovative approaches could enhance tissue-specific delivery of CCR5 antibodies:

• Antibody engineering strategies:

  • Bispecific antibodies incorporating tissue-targeting domains

  • Size-minimized antibody fragments with enhanced tissue penetration

  • pH-sensitive antibodies that release in specific tissue environments

  • Tissue-activated prodrug antibodies

• Delivery vehicle approaches:

  • Lipid nanoparticles with tissue-tropic formulations

  • Exosome-based delivery systems

  • Cell-mediated delivery using adoptively transferred immune cells

  • Hydrogel-based sustained release platforms for specific anatomical sites

• Physical delivery enhancement:

  • Focused ultrasound to temporarily increase blood-tissue barrier permeability

  • Electroporation-enhanced delivery to specific tissues

  • Implantable devices for localized, controlled release

• Combination approaches:

  • Co-administration with agents that modulate vascular permeability

  • Sequential administration protocols that prime tissues for enhanced antibody uptake

  • Circadian timing of administration to align with temporal patterns of tissue accessibility

These approaches could address challenges observed in studies where antibody penetration varied across tissue compartments, potentially enabling more targeted therapeutic applications with reduced systemic effects .

How can integrative data approaches enhance understanding of CCR5 antibody mechanisms?

Integrative data approaches can significantly enhance our understanding of CCR5 antibody mechanisms:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data to build comprehensive pathway maps

  • Correlate CCR5 RO with global cellular changes at multiple biological levels

  • Identify unexpected secondary effects through unbiased profiling

• Systems biology modeling:

  • Develop mathematical models of CCR5 signaling networks that predict antibody impacts

  • Simulate concentration-dependent effects across different tissue compartments

  • Model temporal dynamics of receptor occupancy and cellular responses

• Machine learning applications:

  • Train algorithms to identify patterns in complex datasets that correlate with therapeutic outcomes

  • Develop predictive models for optimal dosing regimens

  • Identify patient-specific factors that influence response to CCR5 antibodies

• Data visualization and integration platforms:

  • Create interactive platforms that integrate RO data with functional outcomes

  • Develop tissue-specific maps of antibody distribution and effects

  • Enable comparative analysis across species, tissues, and disease models

• Collaborative data repositories:

  • Establish standardized data collection protocols for CCR5 antibody research

  • Create shared repositories that allow meta-analyses across multiple studies

  • Implement common data elements to facilitate cross-study comparisons

These approaches address the need for more complex understanding of CCR5 biology beyond its role in HIV infection, as highlighted in the literature on biological complexity and data integration in CCR5 research .