Just did a GvHD search for Leronlimab/CCR5 and came up with two results. Haven’t checked number 2 yet, but this is a good reminder of RO in Leronlimab versus Maravoric. This publication only references GvVD as a key word in search, but it’s a good reminder of Sachs’s Simian work, and our 2 methods of better calculating RO. This publication has of course been posted a few years ago, but it was a fun reminder for me.
Measuring RO has been fraught with over calculation. For instance, Maravoric has been calculated at 120% Receptor Occupancy. How is this possible? Different binding method, and therefore different RO methods of verifying that are less than reliable.
“Currently, Maraviroc is the only FDA-approved CCR5 antagonist. Maraviroc inhibits CCR5 internalization following ligand binding, and thus Maraviroc CCR5 RO is indirectly measured by a MIP-1β internalization assay where CCR5 RO is defined by the percentage of cell surface CCR5 that is not down-regulated following treatment with MIP-1β (37). This indirect method of measuring CCR5 RO results in background RO of approximately 25% (38), with reports of 120% CCR5 RO in peripheral blood CD4+ T cells from both Maraviroc-treated and -untreated rhesus macaques (39). These issues of extremely high background and overcalculation of CCR5 RO are major limitations of the MIP-1β internalization assay, especially when CCR5 RO is a critical parameter in studying CCR5-blocking agents.
Directly measuring CCR5 RO with monoclonal antibodies also presents challenges as CCR5 expression is a dynamic process that must be controlled for. Indeed, the frequency of CCR5+ cells change longitudinally in response to inflammatory and homeostatic stimuli and can be impacted by the CCR5-targeting reagent itself (39–43), leading to inaccuracies in methods that use baseline CCR5 values to calculate CCR5 RO (44). Not accounting for the ability of CCR5 expression to change over time in the CCR5 RO calculation for the anti-CCR5 antibody HGS004 resulted in baseline pre-treatment CCR5 RO values of 20% in HIV-1 infected participants (45). Thus, no robust and highly sensitive method for the calculation of CCR5 RO currently exists.
Here, we report on two sensitive methods to measure CCR5 RO by the anti-CCR5 antibody Leronlimab (PRO-140; Vyrologix). We demonstrate the sensitivity of this method to longitudinally quantify CCR5 RO on blood and tissue CD4+ T cells from Leronlimab-treated macaques and describe increased levels of CCR5+CD4+ T cells in the blood of both Leronlimab-treated macaques and humans. Finally, we translate the macaque CCR5 RO method to Leronlimab-treated, HIV-naïve human participants, demonstrating the direct use for monitoring CCR5 RO by Leronlimab in human clinical trials.”
https://www.frontiersin.org/journals/immunolo...94638/full
Discussion
"Here, we created and validated two different methods of calculating CCR5 RO by the anti-CCR5 antibody Leronlimab. The methods generated comparable longitudinally CCR5 RO percentages in rhesus macaques that received a single 10 mg/kg or 50 mg/kg SC Leronlimab injection. Additionally, both methods were highly sensitive, with baseline values of 1% and fully saturated values of 99% when tested in human and non-human primates. These results are in contrast to the commonly used MIP-1β internalization assay utilized for Maraviroc, which is associated with background levels of approximately 25% in human samples (38) and yields values in excess of 100% in maraviroc-treated and -untreated rhesus macaques (39). Higher RO percentages calculated by the MIP-1β internalization assay may be due to fluctuating CCR5 frequencies or incomplete CCR5 internalization upon MIP-1β binding. In contrast, our methods did not depend on receptor internalization and all mathematical components used were gated on CCR5+ cells, compensating for any fluctuation in CCR5 frequency and allowing for precise calculation of RO. Finally, the pre-clinical Leronlimab CCR5 RO assay was extended into human participants, demonstrating the ability to longitudinally and robustly monitor CCR5 RO.
Similar to maraviroc, we found that Leronlimab stabilized surface CCR5 molecules and prevented its internalization following ligand binding. Indeed, this shared feature of both drugs likely explains their shared ability to increase frequencies of CCR5+CD4+ T cells in both humans and macaques. Because CCR5+CD4+ T cells are target cells for HIV/SIV infection, increasing the frequencies of susceptible cells could raise concerns of increased HIV/SIV replication. However, we found that weekly Leronlimab treatment in a chronically SIVmac239-infected macaque fully suppressed plasma viremia for over 20 weeks despite a rise in the CCR5+CD4+ T cell frequency immediately after the first Leronlimab injection. Both suppression of viral replication and increased CCR5+CD4+ T cell levels were temporally associated with full CCR5 RO on peripheral blood CD4+ T cells, underscoring the need to measure CCR5 RO in studies utilizing CCR5-blocking agents.
Because CCR5 is involved in multiple pathophysiologic processes, Leronlimab is being explored in clinical trials for both HIV and non-HIV indications. In HIV-positive participants, Leronlimab suppressed plasma viremia after a single 5 or 10 mg/kg SC Leronlimab injection, while Leronlimab monotherapy contributed to the maintenance of viral suppression for over six years (52–55). Moreover, the only two cases of HIV cure occurred after hematopoietic stem cell transplantation (HSCT) with donor cells homozygous for CCR5Δ32 (56, 57), while similar studies using donor cells that were wild-type CCR5 or heterozygous CCR5Δ32 led to eventual viral rebound (58). For this reason, there is intense focus on genetically disrupting ccr5 to abolish cell surface CCR5 expression in HIV-positive individuals (59, 60). However, CCR5 has many protective roles, making it difficult to predict the long-term biological effects or consequences of permanently disrupting ccr5 expression in humans. A logical approach to reproduce the phenotypic protection seen in homozygous CCR5Δ32 individuals is to instead use CCR5-blocking pharmacologic agents such as Leronlimab. Moreover, the therapeutic use of Leronlimab extends beyond HIV treatment due to its diverse roles. CCR5 is expressed in over 95% of triple-negative breast cancers (61) and influences breast cancer progression (9). In a murine model, Leronlimab prevented and reduced breast cancer metastasis suggesting a role for Leronlimab in the treatment of neoplasia (62). As CCR5 is central in inflammatory immune responses, it is currently being studied as a therapeutic for severe and critical SARS-CoV-2 infections (44, 63–65) and graft-versus-host disease (GVHD), where Leronlimab treatment reduced xeno-GVHD after HSCT of human cells to mice (66). Finally, Leronlimab is currently in phase 1 and 2 clinical studies to treat metastatic colorectal cancer, nonalcoholic Steatohepatitis, and long COVID after SARS-CoV-2 infection, demonstrating the diverse applicability of this safe and effective CCR5-targeting agent.
The appeal of monoclonal antibody-based therapeutic agents is growing due to their longer half-life and promising safety profile, and this is reflected in the dramatic rise in FDA approvals and commercial use of antibody treatments in recent years (67, 68). The CCR5 RO assays described here will be an important study measurement for any CCR5 antibody-based agent, and can be modified for any antibody-based agents that have a clearly defined cell surface protein target. In the case of the anti-CCR5 antibody Leronlimab, the ability to accurately measure CCR5 RO will advance both pre-clinical and clinical studies, furthering our understanding of the immunological impacts of CCR5 for multiple pathophysiologic processes."
Measuring RO has been fraught with over calculation. For instance, Maravoric has been calculated at 120% Receptor Occupancy. How is this possible? Different binding method, and therefore different RO methods of verifying that are less than reliable.
“Currently, Maraviroc is the only FDA-approved CCR5 antagonist. Maraviroc inhibits CCR5 internalization following ligand binding, and thus Maraviroc CCR5 RO is indirectly measured by a MIP-1β internalization assay where CCR5 RO is defined by the percentage of cell surface CCR5 that is not down-regulated following treatment with MIP-1β (37). This indirect method of measuring CCR5 RO results in background RO of approximately 25% (38), with reports of 120% CCR5 RO in peripheral blood CD4+ T cells from both Maraviroc-treated and -untreated rhesus macaques (39). These issues of extremely high background and overcalculation of CCR5 RO are major limitations of the MIP-1β internalization assay, especially when CCR5 RO is a critical parameter in studying CCR5-blocking agents.
Directly measuring CCR5 RO with monoclonal antibodies also presents challenges as CCR5 expression is a dynamic process that must be controlled for. Indeed, the frequency of CCR5+ cells change longitudinally in response to inflammatory and homeostatic stimuli and can be impacted by the CCR5-targeting reagent itself (39–43), leading to inaccuracies in methods that use baseline CCR5 values to calculate CCR5 RO (44). Not accounting for the ability of CCR5 expression to change over time in the CCR5 RO calculation for the anti-CCR5 antibody HGS004 resulted in baseline pre-treatment CCR5 RO values of 20% in HIV-1 infected participants (45). Thus, no robust and highly sensitive method for the calculation of CCR5 RO currently exists.
Here, we report on two sensitive methods to measure CCR5 RO by the anti-CCR5 antibody Leronlimab (PRO-140; Vyrologix). We demonstrate the sensitivity of this method to longitudinally quantify CCR5 RO on blood and tissue CD4+ T cells from Leronlimab-treated macaques and describe increased levels of CCR5+CD4+ T cells in the blood of both Leronlimab-treated macaques and humans. Finally, we translate the macaque CCR5 RO method to Leronlimab-treated, HIV-naïve human participants, demonstrating the direct use for monitoring CCR5 RO by Leronlimab in human clinical trials.”
https://www.frontiersin.org/journals/immunolo...94638/full
Discussion
"Here, we created and validated two different methods of calculating CCR5 RO by the anti-CCR5 antibody Leronlimab. The methods generated comparable longitudinally CCR5 RO percentages in rhesus macaques that received a single 10 mg/kg or 50 mg/kg SC Leronlimab injection. Additionally, both methods were highly sensitive, with baseline values of 1% and fully saturated values of 99% when tested in human and non-human primates. These results are in contrast to the commonly used MIP-1β internalization assay utilized for Maraviroc, which is associated with background levels of approximately 25% in human samples (38) and yields values in excess of 100% in maraviroc-treated and -untreated rhesus macaques (39). Higher RO percentages calculated by the MIP-1β internalization assay may be due to fluctuating CCR5 frequencies or incomplete CCR5 internalization upon MIP-1β binding. In contrast, our methods did not depend on receptor internalization and all mathematical components used were gated on CCR5+ cells, compensating for any fluctuation in CCR5 frequency and allowing for precise calculation of RO. Finally, the pre-clinical Leronlimab CCR5 RO assay was extended into human participants, demonstrating the ability to longitudinally and robustly monitor CCR5 RO.
Similar to maraviroc, we found that Leronlimab stabilized surface CCR5 molecules and prevented its internalization following ligand binding. Indeed, this shared feature of both drugs likely explains their shared ability to increase frequencies of CCR5+CD4+ T cells in both humans and macaques. Because CCR5+CD4+ T cells are target cells for HIV/SIV infection, increasing the frequencies of susceptible cells could raise concerns of increased HIV/SIV replication. However, we found that weekly Leronlimab treatment in a chronically SIVmac239-infected macaque fully suppressed plasma viremia for over 20 weeks despite a rise in the CCR5+CD4+ T cell frequency immediately after the first Leronlimab injection. Both suppression of viral replication and increased CCR5+CD4+ T cell levels were temporally associated with full CCR5 RO on peripheral blood CD4+ T cells, underscoring the need to measure CCR5 RO in studies utilizing CCR5-blocking agents.
Because CCR5 is involved in multiple pathophysiologic processes, Leronlimab is being explored in clinical trials for both HIV and non-HIV indications. In HIV-positive participants, Leronlimab suppressed plasma viremia after a single 5 or 10 mg/kg SC Leronlimab injection, while Leronlimab monotherapy contributed to the maintenance of viral suppression for over six years (52–55). Moreover, the only two cases of HIV cure occurred after hematopoietic stem cell transplantation (HSCT) with donor cells homozygous for CCR5Δ32 (56, 57), while similar studies using donor cells that were wild-type CCR5 or heterozygous CCR5Δ32 led to eventual viral rebound (58). For this reason, there is intense focus on genetically disrupting ccr5 to abolish cell surface CCR5 expression in HIV-positive individuals (59, 60). However, CCR5 has many protective roles, making it difficult to predict the long-term biological effects or consequences of permanently disrupting ccr5 expression in humans. A logical approach to reproduce the phenotypic protection seen in homozygous CCR5Δ32 individuals is to instead use CCR5-blocking pharmacologic agents such as Leronlimab. Moreover, the therapeutic use of Leronlimab extends beyond HIV treatment due to its diverse roles. CCR5 is expressed in over 95% of triple-negative breast cancers (61) and influences breast cancer progression (9). In a murine model, Leronlimab prevented and reduced breast cancer metastasis suggesting a role for Leronlimab in the treatment of neoplasia (62). As CCR5 is central in inflammatory immune responses, it is currently being studied as a therapeutic for severe and critical SARS-CoV-2 infections (44, 63–65) and graft-versus-host disease (GVHD), where Leronlimab treatment reduced xeno-GVHD after HSCT of human cells to mice (66). Finally, Leronlimab is currently in phase 1 and 2 clinical studies to treat metastatic colorectal cancer, nonalcoholic Steatohepatitis, and long COVID after SARS-CoV-2 infection, demonstrating the diverse applicability of this safe and effective CCR5-targeting agent.
The appeal of monoclonal antibody-based therapeutic agents is growing due to their longer half-life and promising safety profile, and this is reflected in the dramatic rise in FDA approvals and commercial use of antibody treatments in recent years (67, 68). The CCR5 RO assays described here will be an important study measurement for any CCR5 antibody-based agent, and can be modified for any antibody-based agents that have a clearly defined cell surface protein target. In the case of the anti-CCR5 antibody Leronlimab, the ability to accurately measure CCR5 RO will advance both pre-clinical and clinical studies, furthering our understanding of the immunological impacts of CCR5 for multiple pathophysiologic processes."
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