483 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

Track: Poster Abstracts
Saturday, February 14, 2015, 6:45 PM-7:45 PM
Grand Hall CD (Manchester Grand Hyatt)
Austin P. Huffman, BA , Blood and Marrow Transplantation Program, Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA
Lee P Richman, BA , Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
David L. Porter, MD , Hematology Oncology, Blood and Marrow Transplantation Program, Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA
Robert H Vonderheide, MD DPhil , Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
Ran Reshef, MD , Blood and Marrow Transplantation Program, Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA
Presentation recording not available for download or distribution as requested by the presenting author.

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

Disclosures:
R. Reshef, Pfizer, Investigator: Research Funding
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