CCR5 Receptor Occupancy Analysis Reveals Increased Peripheral Blood CCR5+CD4+ T Cells Following Treatment With the Anti-CCR5 Antibody Leronlimab

CCR5 plays a central role in infectious disease, host defense, and cancer progression, thereby making it an ideal target for therapeutic development. Notably, CCR5 is the major HIV entry co-receptor, where its surface density correlates with HIV plasma viremia. The level of CCR5 receptor occupancy (RO) achieved by a CCR5-targeting therapeutic is therefore a critical predictor of its efficacy. However, current methods to measure CCR5 RO lack sensitivity, resulting in high background and overcalculation. Here, we report on two independent, flow cytometric methods of calculating CCR5 RO using the anti-CCR5 antibody, Leronlimab. We show that both methods led to comparable CCR5 RO values, with low background on untreated CCR5+CD4+ T cells and sensitive measurements of occupancy on both blood and tissue-resident CD4+ T cells that correlated longitudinally with plasma concentrations in Leronlimab-treated macaques. Using these assays, we found that Leronlimab stabilized cell surface CCR5, leading to an increase in the levels of circulating and tissue-resident CCR5+CD4+ T cells in vivo in Leronlimab-treated macaques. Weekly Leronlimab treatment in a chronically SIV-infected macaque led to increased CCR5+CD4+ T cells levels and fully suppressed plasma viremia, both concomitant with full CCR5 RO on peripheral blood CD4+ T cells, demonstrating that CCR5+CD4+ T cells were protected from viral replication by Leronlimab binding. Finally, we extended these results to Leronlimab-treated humans and found that weekly 700 mg Leronlimab led to complete CCR5 RO on peripheral blood CD4+ T cells and a statistically significant increase in CCR5+CD4+ T cells in peripheral blood. Collectively, these results establish two RO calculation methods for longitudinal monitoring of anti-CCR5 therapeutic antibody blockade efficacy in both macaques and humans, demonstrate that CCR5+CD4+ T cell levels temporarily increase with Leronlimab treatment, and facilitate future detailed investigations into the immunological impacts of CCR5 inhibition in multiple pathophysiological processes.

Tumor Budding-Derived CCL5 Recruits Fibroblasts to Promote Colorectal Cancer Progression Through CCR5-SLC25A24 Signaling

Background: Tumor buddings have been included in the routine diagnosis of colorectal cancer (CRC) and considered to be a tumor prognostic factor independent of TNM staging. This study aimed at identifying the contribution of tumor budding-derived C-C chemokine ligand 5 (CCL5) to tumor microenvironment (TME) through fibroblasts. Methods: Co-cultivation recruitment assays and human cytokine array were used to detect the main cytokine derived from CRC tumor buddings to recruit fibroblasts. siRNA transfection and inhibitor treatment were applied to investigate the effective receptor of CCL5 on fibroblasts. Transcriptome sequencing was performed to explore the mechanism inside fibroblasts when stimulated by CCL5. Stimulation with CCL5 in vitro, orthotopic xenograft mouse model and clinical specimens were designed to clarify the contribution of CCL5 to angiogenesis and collagen synthesis.Results: H&E and immunochemistry staining confirmed that CRC with tumor buddings in the invasive front was accompanied by more fibroblasts compared with CRC without tumor buddings. Further vitro study indicated that CCL5 derived from tumor buddings could recruit fibroblasts through CCR5 receptor on fibroblasts and positively regulate solute carrier family 25 member 24 (SLC25A24) expression in fibroblasts, which could activate pAkt-pmTOR signaling. Moreover, CCL5 can increase the number of α-SMA+CD90+FAP- fibroblasts to promote tumor angiogenesis through enhancing the expression of VEGFA and making fibroblasts transdifferentiate into vascular endothelial cells. Meanwhile, CCL5 also can promote collagen synthesis through fibroblasts, thus contributing to tumor progression.Conclusions: In the invasive front of CRC, tumor-budding-derived CCL5 can recruit fibroblasts through CCR5-SLC25A24 signaling, further promoting angiogenesis and collagen synthesis through fibroblasts, eventually creating a tumor-promoting microenvironment.

The Chemokine CCL5 Inhibits the Replication of Influenza A Virus Through SAMHD1 Modulation

Influenza A virus (IAV) is the main etiological agent of acute respiratory tract infections. During IAV infection, interferon triggers the overexpression of restriction factors (RFs), the intracellular antiviral branch of the innate immune system. Conversely, severe influenza is associated with an unbalanced pro-inflammatory cytokine release. It is unclear whether other cytokines and chemokines released during IAV infection modulate RFs to control virus replication. Among the molecules enhanced in the infected respiratory tract, ligands of the CCR5 receptor play a key role, as they stimulate the migration of inflammatory cells to the alveoli. We investigated here whether ligands of the CCR5 receptor could enhance RFs to levels able to inhibit IAV replication. For this purpose, the human alveolar basal epithelial cell line (A549) was treated with endogenous (CCL3, CCL4 and CCL5) or exogenous (HIV-1 gp120) ligands prior to IAV infection. The three CC-chemokines tested reduced infectious titers between 30% to 45% upon 24 hours of infection. Eploying RT-PCR, a panel of RF mRNA levels from cells treated with CCR5 agonists was evaluated, which showed that the SAMHD1 expression was up-regulated four times over control upon exposure to CCL3, CCL4 and CCL5. We also found that IAV inhibition by CCL5 was dependent on PKC and that SAMHD1 protein levels were also increased after treatment with CCL5. In functional assays, we observed that the knockdown of SAMHD1 resulted in enhanced IAV replication in A549 cells and abolished both CCL5-mediated inhibition of IAV replication and CCL5-mediated cell death inhibition. Our data show that stimuli unrelated to interferon may trigger the upregulation of SAMHD1 and that this RF may directly interfere with IAV replication in alveolar epithelial cells.

Antibody-based CCR5 blockade protects Macaques from mucosal SHIV transmission

In the absence of a prophylactic vaccine, the use of antiretroviral therapy (ART) as pre-exposure prophylaxis (PrEP) to prevent HIV acquisition by uninfected individuals is a promising approach to slowing the epidemic, but its efficacy is hampered by incomplete patient adherence and ART-resistant variants. Here, we report that competitive inhibition of HIV Env-CCR5 binding via the CCR5-specific antibody Leronlimab protects rhesus macaques against infection following repeated intrarectal challenges of CCR5-tropic SHIVSF162P3. Injection of Leronlimab weekly at 10 mg/kg provides significant but partial protection, while biweekly 50 mg/kg provides complete protection from SHIV acquisition. Tissue biopsies from protected macaques post challenge show complete CCR5 receptor occupancy and an absence of viral nucleic acids. After Leronlimab washout, protected macaques remain aviremic, and adoptive transfer of hematologic cells into naïve macaques does not transmit viral infection. These data identify CCR5 blockade with Leronlimab as a promising approach to HIV prophylaxis and support initiation of clinical trials.

Studies on the IC50 of Metabolically Stable 1-(3,3-diphenylpropyl)- piperidinyl Amides and Ureas as Human CCR5 Receptor Antagonists Based on QSAR

Background: In this study, modulators of human Chemotactic cytokine receptor 5 (CCR5) were described using a quantitative structure-activity relationship model (QSAR). This model was based on the molecule’s chemical structure. Methods:: All 56 compounds of CCR5 receptor antagonists were randomly separated into two sets, 43 were reserved for training and the other 13 for testing. In the course of this study, molecular models were drawn using ChemDraw software. By means of Hyperchem software as well as optimized with both AM1 (semi-empirical self-consistent-field molecular orbital) and MM+ (molecular mechanics plus force field), molecular models were described through numerous descriptors using CODESSA software. Results: Linear models were obtained by Heuristic Method (HM) software and nonlinear models were obtained using APS software with optimal descriptor combinations used to build linear QSAR models, involving a group of selected descriptors. As a result, values of the above two different sets were shown to result from 0.82 in testing and 0.86 in training in HM while 0.83 in testing and 0.88 in training in Gene Expression Programming (GEP). Conclusion: From this method, the activity of molecules could be predicted, and the molecular structure could be changed to alter its IC50, avoiding the testing of large numbers of compounds.

Disruption of the CCL5/RANTES-CCR5 Pathway Restores Immune Homeostasis and Reduces Plasma Viral Load in Critical COVID-19

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is now pandemic with nearly three million cases reported to date1. Although the majority of COVID-19 patients experience only mild or moderate symptoms, a subset will progress to severe disease with pneumonia and acute respiratory distress syndrome (ARDS) requiring mechanical ventilation2. Emerging results indicate a dysregulated immune response characterized by runaway inflammation, including cytokine release syndrome (CRS), as the major driver of pathology in severe COVID-193,4. With no treatments currently approved for COVID-19, therapeutics to prevent or treat the excessive inflammation in severe disease caused by SARS-CoV-2 infection are urgently needed. Here, in 10 terminally-ill, critical COVID-19 patients we report profound elevation of plasma IL-6 and CCL5 (RANTES), decreased CD8+ T cell levels, and SARS-CoV-2 plasma viremia. Following compassionate care treatment with the CCR5 blocking antibody leronlimab, we observed complete CCR5 receptor occupancy on macrophage and T cells, rapid reduction of plasma IL-6, restoration of the CD4/CD8 ratio, and a significant decrease in SARS-CoV-2 plasma viremia. Consistent with reduction of plasma IL-6, single-cell RNA-sequencing revealed declines in transcriptomic myeloid cell clusters expressing IL-6 and interferon-related genes. These results demonstrate a novel approach to resolving unchecked inflammation, restoring immunologic deficiencies, and reducing SARS-CoV-2 plasma viral load via disruption of the CCL5-CCR5 axis, and support randomized clinical trials to assess clinical efficacy of leronlimab-mediated inhibition of CCR5 for COVID-19.

Disruption of the CCL5/RANTES-CCR5 Pathway Restores Immune Homeostasis and Reduces Plasma Viral Load in Critical COVID-19

ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is now pandemic with nearly three million cases reported to date1. Although the majority of COVID-19 patients experience only mild or moderate symptoms, a subset will progress to severe disease with pneumonia and acute respiratory distress syndrome (ARDS) requiring mechanical ventilation2. Emerging results indicate a dysregulated immune response characterized by runaway inflammation, including cytokine release syndrome (CRS), as the major driver of pathology in severe COVID-193,4. With no treatments currently approved for COVID-19, therapeutics to prevent or treat the excessive inflammation in severe disease caused by SARS-CoV-2 infection are urgently needed. Here, in 10 terminally-ill, critical COVID-19 patients we report profound elevation of plasma IL-6 and CCL5 (RANTES), decreased CD8+ T cell levels, and SARS-CoV-2 plasma viremia. Following compassionate care treatment with the CCR5 blocking antibody leronlimab, we observed complete CCR5 receptor occupancy on macrophage and T cells, rapid reduction of plasma IL-6, restoration of the CD4/CD8 ratio, and a significant decrease in SARS-CoV-2 plasma viremia. Consistent with reduction of plasma IL-6, single-cell RNA-sequencing revealed declines in transcriptomic myeloid cell clusters expressing IL-6 and interferon-related genes. These results demonstrate a novel approach to resolving unchecked inflammation, restoring immunologic deficiencies, and reducing SARS-CoV-2 plasma viral load via disruption of the CCL5-CCR5 axis, and support randomized clinical trials to assess clinical efficacy of leronlimab-mediated inhibition of CCR5 for COVID-19.