Pharamacodynamic Assessment Shows That CCR5 Surface Expression May Serve As an Indicator for Effective CCR5 Blockade in Allogeneic Stem Cell Transplant (alloSCT) Recipients Treated with Maraviroc

Background:

Brief CCR5 blockade using maraviroc (MVC) after reduced-intensity alloSCT resulted in a low incidence of acute GvHD and protection against visceral GvHD.  We observed significant variability in baseline T-cell CCR5 expression in donors and recipients.  We therefore hypothesized that effective blockade of CCR5 activation is dependent on its baseline expression.  To test this, we developed a phosphoflow assay that measures the activity of MVC in fresh blood samples. 

Methods:

The phosphoflow assay (Fig. 1) quantifies CCR5 activation by measuring phosphorylation of a C-terminal serine residue (SER349) using a phosphospecific CCR5 antibody.  To demonstrate activity, CCL4 stimulation was performed in fresh whole blood with or without excess MVC.  Change in pCCR5 levels was measured as the MFI fold-change from a baseline unstimulated control.  The surface expression of CCR5 was measured separately by flow cytometry.

Results:

Twenty-four reduced-intensity alloSCT recipients received MVC 300 mg b.i.d. orally from day -3 to day +90 on an ongoing phase II study.  Recipient blood was tested on days -6 (before MVC), 0 and 14, and donor blood was tested on day 0.  We observed significant variability in surface CCR5 expression on T-cells in donors (range 0 – 9%) and recipients (range 0 – 49%).  Recipient surface CCR5 expression increased from d-6 to d0 on both CD4 (mean 3.6 to 7.3%, p=0.005) and CD8 (mean 13.5 to 30.6%, p=0.005) T-cells, consistent with blockade of receptor internalization. 

CD8 T-cell pCCR5 levels increased in response to CCL4 stimulation on d -6 (p=0.01) and decreased in response to MVC (p=0.002), compared to unstimulated blood (Fig. 2).  A decrease in pCCR5 levels in response to MVC was observed even without CCL4 stimulation, reflecting loss of background CCR5 activity (p=0.0002).  In contrast, d0 pCCR5 levels failed to increase in response to CCL4 (p=0.32), nor did they decrease in response to additional MVC (p=0.13), mirroring effective in vivo blockade of CCR5.

The magnitude of increase in CD8 T-cell pCCR5 levels in response to CCL4 stimulation on d-6 correlated with CCR5 surface expression (r=0.55, p=0.02).  The same was true of CD4 T-cells (r=0.76, p=0.0004).  On d0, however, the ability to stimulate CCR5 inversely correlated with CCR5 surface expression for both CD8 (r=-0.46, p=0.04) and CD4 (r=-0.62, p=0.004), suggesting that greater CCR5 surface upregulation may serve as an indicator for complete receptor blockade.

Conclusions:

Intracellular phosphorylation of CCR5 in response to stimulation is efficiently blocked by MVC in patients undergoing alloSCT, as measured by a novel phosphoflow assay.  Upregulation of surface CCR5 may indicate effective CCR5 blockade and should be explored as a biomarker for drug response.

Fig. 1.  Phosphoflow assay for CCR5 activation

Fig. 2. Fold-change in pCCR5 on CD8 T-cells in response to CCL4 stimulation with or without MVC