LCR25 Antibody

What is leronlimab and how does it function as a CCR5 antagonist?

Leronlimab is a CCR5-binding humanized immunoglobulin G4 monoclonal antibody that functions as a competitive inhibitor of the CCR5 receptor. Unlike small molecule CCR5 antagonists, leronlimab binds directly to CCR5 on the cell surface, preventing its interaction with natural ligands and viral proteins that utilize this receptor for cell entry .

When bound to CCR5, leronlimab blocks the binding of HIV envelope proteins and natural chemokines, effectively inhibiting CCR5-mediated signaling. Interestingly, leronlimab appears to stabilize CCR5 expression on the cell surface rather than inducing internalization of the receptor, an unexpected mechanism that may contribute to its therapeutic effects .

How is receptor occupancy (RO) measured when studying CCR5 antibodies like leronlimab?

Two established methods for measuring CCR5 receptor occupancy have been validated for leronlimab:

• Competitive binding assay: This approach uses fluorescently-labeled competitor antibodies that bind to epitopes distinct from leronlimab's binding site. The reduction in binding of these reporter antibodies indicates the degree of CCR5 occupancy by leronlimab .

• Direct detection method: This utilizes labeled anti-human IgG4 antibodies that directly bind to leronlimab when it occupies CCR5 receptors on cell surfaces. The signal intensity correlates with receptor occupancy levels .

Both methods can be employed for longitudinal monitoring of anti-CCR5 therapeutic antibody blockade efficacy in both non-human primates and humans, with flow cytometry being the primary analytical platform .

What are the differences in CCR5 expression between humans and non-human primates relevant for preclinical studies?

While CCR5 is highly conserved between humans and rhesus macaques, important differences exist that affect experimental design:

These differences must be considered when designing dose-escalation studies and interpreting results from non-human primate models before translation to human clinical trials.

How should researchers design dose-response studies for CCR5 antibodies in different disease models?

When designing dose-response studies for CCR5 antibodies like leronlimab across different disease models, researchers should employ the following methodology:

• Dose selection rationale:

  • For HIV/SHIV prevention models: Evidence shows that 10 mg/kg weekly provides significant but partial protection, while 50 mg/kg biweekly achieves complete protection in macaque models .

  • For inflammatory conditions: Consider starting with lower doses (e.g., 5-10 mg/kg) based on receptor occupancy requirements.

• Treatment schedule determination:

  • Monitor receptor occupancy at different timepoints post-administration to establish optimal dosing intervals.

  • For HIV prevention, maintain >85% receptor occupancy at all times to ensure protection .

  • For inflammatory conditions like Long COVID, weekly administration may be sufficient based on clinical observations .

• Tissue-specific considerations:

  • Include analysis of target tissues beyond peripheral blood (lymph nodes, mucosal tissues, BAL) to confirm adequate drug penetration .

  • Design tissue collection timepoints strategically to capture peak and trough receptor occupancy levels.

What experimental approaches best evaluate the impact of leronlimab on immune cell populations?

Comprehensive evaluation of leronlimab's impact on immune cell populations requires:

• Flow cytometry panels should include, at minimum:

  • CCR5 expression assessment on T cell subsets (naive, central memory, effector memory)

  • Additional chemokine receptors (CXCR3, CXCR4, CCR2) to evaluate compensatory changes

  • Activation markers (CD69, CD25, HLA-DR) to monitor T cell activation status

  • Proliferation markers (Ki-67) to assess cell turnover

• Functional assays:

  • In vitro viral inhibition assays using relevant strains (R5-tropic HIV for human cells, SHIV for macaque cells)

  • Migration assays to assess chemotaxis responses to CCR5 ligands

  • Cytokine production profiles before and after treatment

• Long-term monitoring:

  • Establish baseline measurements before intervention

  • Schedule regular timepoints during treatment (days 0, 7, 14, 28, 56)

  • Include post-treatment follow-up to assess reversibility of effects

This systematic approach allows researchers to distinguish direct antibody effects from secondary immune changes and characterize the immunomodulatory profile of CCR5 blockade.

How do researchers address the paradoxical increase in CCR5+CD4+ T cells observed with leronlimab treatment?

The unexpected finding that leronlimab treatment increases CCR5+CD4+ T cell levels represents a significant paradox requiring specific experimental approaches:

• Mechanistic investigation:

  • Conduct pulse-chase experiments to determine if increased CCR5+CD4+ T cells result from proliferation, redistribution from tissues, or reduced turnover

  • Employ transcriptional analysis of CCR5+ cells before and after treatment to identify regulatory changes

  • Use cell surface half-life studies with tagged CCR5 to determine if leronlimab stabilizes receptor expression

• Correlation with clinical outcomes:

  • Track CCR5+CD4+ T cell levels longitudinally and correlate with symptom improvement

  • Stratify analysis by responders vs. non-responders to identify predictive patterns

  • Note that significant increases in cell surface CCR5 levels were observed specifically among leronlimab-treated responders but not in non-responders

• Signaling pathway analysis:

  • Investigate whether CCR5 binding by leronlimab alters canonical signaling pathways differently than natural ligands

  • Examine downstream effects on cytokines like IL-10 and CCL-2, which have shown reductions in leronlimab-treated patients

This methodological approach addresses the hypothesis that leronlimab normalizes abnormal immune downmodulation rather than simply blocking CCR5 activity.

What are the key methodological considerations for studying leronlimab in HIV prevention and treatment?

For HIV research with leronlimab, researchers should implement these methodological approaches:

• Prevention studies:

  • Use appropriate challenge models (e.g., intrarectal SHIV challenge in macaques) with well-characterized viral stocks

  • Establish receptor occupancy thresholds for prevention (target >90% occupancy)

  • Monitor protection across multiple exposures to assess durability

  • Examine tissue biopsies from protected subjects to confirm CCR5 receptor occupancy and absence of viral nucleic acids

• Treatment studies:

  • Design transition protocols from conventional ART to leronlimab monotherapy

  • Implement rigorous viral load monitoring with lower detection limits

  • Anticipate and account for transient viral blips, which occur at higher frequency (7.1%) in leronlimab monotherapy compared to combination ART (2.0%)

  • Include resistance monitoring to detect potential viral escape

• Pharmacological considerations:

  • Measure drug concentration across multiple tissue compartments, not just plasma

  • Assess anti-drug antibody (ADA) development, which has been observed in some subjects and correlates with loss of receptor occupancy

  • Consider combination approaches with other antiretrovirals for treatment studies

How should researchers design studies examining leronlimab's effects in inflammatory conditions like Long COVID?

When investigating leronlimab in inflammatory conditions:

• Patient stratification approach:

  • Screen for baseline CCR5 expression levels on immune cells

  • Categorize patients based on inflammatory profiles (e.g., cytokine signatures)

  • Consider genetic polymorphisms affecting CCR5 expression

• Outcome measurement strategy:

  • Implement validated symptom assessment tools specific to the condition

  • Include objective physiological measurements alongside subjective symptom scores

  • Monitor changes in inflammatory biomarkers (e.g., IL-6, TNF-α, CCL2) during treatment

  • Track changes in cell surface CCR5 levels as potential biomarkers of response

• Study design elements:

  • Include appropriate placebo controls and blinding

  • Consider crossover designs for heterogeneous conditions

  • Power studies adequately to detect differences in predefined subgroups

  • Include extended follow-up periods to assess durability of effects

The existing data from Long COVID studies suggest that normalization of immune downmodulation, rather than persistent immune activation, may be the target mechanism to monitor .

How can researchers reconcile contradictory findings regarding CCR5 expression changes in different disease states?

Resolving contradictory findings regarding CCR5 expression requires:

• Contextual analysis framework:

  • Distinguish between tissue-specific vs. systemic expression changes

  • Consider differential expression across cell subpopulations rather than bulk measurements

  • Account for temporal dynamics of expression patterns during disease progression

  • Compare findings with expression patterns of related chemokine receptors (CCR2) and ligands

• Standardized quantification methods:

  • Implement absolute quantification of receptor numbers per cell using standardized beads

  • Report both percentage of positive cells and mean fluorescence intensity

  • Consider single-cell approaches to identify heterogeneous expression patterns

  • Use consistent antibody clones and flow cytometry protocols across studies

• Integrated data analysis:

  • Examine correlations between CCR5 expression and corresponding ligand levels

  • Utilize multivariate analysis to identify confounding variables

  • Consider genetic factors (e.g., CCR5Δ32 heterozygosity) in human studies

  • Refer to comparative data from different disease models as shown in this example table:

ND: Not Determined; ↑: Increased; ↓: Decreased; ─: No Change

What methodological approaches can address variable responses to leronlimab observed in clinical studies?

To address variable response patterns to leronlimab:

• Predictive biomarker identification:

  • Perform comprehensive baseline immunophenotyping to identify predictor signatures

  • Monitor dynamic changes in cell surface CCR5 levels as potential response biomarkers

  • Correlate baseline CCR5 receptor density with subsequent clinical response

  • Consider pharmacogenomic analyses to identify genetic determinants of response variability

• Mechanistic differentiation protocols:

  • Compare signaling pathway activation between responders and non-responders

  • Examine ex vivo response patterns in patient-derived cells

  • Investigate potential compensatory mechanisms in non-responders

  • Assess for development of anti-drug antibodies that may neutralize therapeutic effects

• Integrated response assessment:

  • Implement composite endpoints that capture both clinical and biological responses

  • Develop standardized definitions of "response" incorporating multiple parameters

  • Consider time-to-response in statistical analyses

  • Design adaptive trials that allow dose adjustments based on early receptor occupancy data

This approach helps distinguish pharmacological failure (insufficient drug exposure or target engagement) from biological resistance to CCR5 blockade.

What are the optimal cell preparation and staining protocols for accurate assessment of CCR5 expression and receptor occupancy?

For accurate CCR5 analysis, researchers should follow these methodological guidelines:

• Sample handling protocol:

  • Process samples within 4-6 hours of collection to prevent receptor modulation

  • Avoid repeated freeze-thaw cycles of cells for longitudinal studies

  • For tissue samples, use enzymatic digestion protocols optimized to preserve CCR5 epitopes

  • Consider fixation impact on antibody binding sites when designing protocols

• Staining approach:

  • Include viability dye to exclude dead cells from analysis

  • Block Fc receptors prior to antibody staining

  • Use directly conjugated antibodies when possible to reduce background

  • Calibrate compensation carefully for multicolor panels including CCR5

  • Implement consistent gating strategies across timepoints and studies

• Controls and validation:

  • Include fluorescence-minus-one (FMO) controls for accurate gate setting

  • Use CCR5-negative cells (e.g., from CCR5Δ32 homozygous donors) as biological negative controls when available

  • Verify results using multiple anti-CCR5 antibody clones that recognize distinct epitopes

  • Establish internal quality control standards for longitudinal monitoring

These technical considerations are essential for generating reliable and reproducible data regarding CCR5 expression and occupancy measurements.

How should researchers design in vitro experiments to evaluate leronlimab's efficacy against diverse viral isolates?

When designing in vitro assessment of leronlimab against diverse viral isolates:

• Viral panel selection criteria:

  • Include isolates from multiple HIV clades to assess breadth of protection

  • Incorporate R5-tropic, X4-tropic, and dual-tropic viruses to confirm specificity

  • Select isolates with known resistance to other entry inhibitors to assess cross-resistance

  • Consider transmitted/founder viruses for prevention models versus chronic isolates for treatment models

• Assay methodology:

  • Standardize target cell preparation (e.g., activated CD4+ T cells at 1 × 10^6 cells/mL in appropriate media)

  • Pre-incubate cells with leronlimab at varying concentrations (0.1-100 μg/mL) for 1 hour at 37°C

  • Infect via spinoculation (1200 × g for 2 hours) for consistent viral entry

  • Include CCR5Δ32/Δ32 donor cells as biological controls for CCR5-independent entry

  • Measure viral replication via p24 ELISA or reporter gene expression at multiple timepoints

• Data analysis approach:

  • Calculate IC50/IC90 values for each isolate

  • Compare potency across viral subtypes

  • Assess correlation between CCR5 dependence and susceptibility to inhibition

  • Conduct parallel experiments with small molecule CCR5 antagonists for comparison

This systematic approach allows for comprehensive characterization of leronlimab's antiviral profile against diverse viral targets.