Abstract
The homozygous expression of phenylalanine (F/F) at codon 158 of FcγRIIIa (CD16) is associated with inferior clinical responses to the anti-CD20 monoclonal antibody rituximab in patients with indolent non-Hodgkin’s lymphoma, in contrast to higher response rates observed for Waldenstrom’s macroglobulinemia patients expressing at least one valine (V/F, V/V) and follicular NHL patients who are homozygous for valine (V/V). We attempted elucidate the potential basic mechanism(s) behind these clinical observations by analyzing all the potential implications of this polymorphism among the 3 polymorphic subgroups at FcγRIIIA-158 (F/F, V/F, and V/V). We therefore used peripheral blood isolated natural killer (NK) cells from a pool of 52 unrelated healthy donors who were genotyped for FcγRIIIA-158. We evaluated allele-specific differences for FcγRIIIa gene expression by quantitative RT-PCR and demonstrated higher transcript levels among V/V (23.2 ng/mL) versus V/F (6.7 ng/mL) and F/F (6.2 ng/mL) (p<0.0001). We then determined protein levels for CD16 in the same subgroup of donors using quantitative flow cytometry. The number of CD16 receptors per NK cell was 105,947, 94,863, and 69,130 for the V/V, V/F and F/F donors, respectively and was significantly higher among donors who expressed at least one valine (V/V and V/F) versus F/F (p=0.033). We next determined rituximab binding affinity to NK cells from V/V, V/F and F/F donors following incubation at concentrations of 10–200 ug/ml, and use of an indirect competitive assay with the anti-CD16 monoclonal antibody 3G8 (Cancer Research 64:4664). Rituximab binding to NK cells was higher among donors expressing at least one valine at all concentrations evaluated, with mean rituximab binding (defined as % of inhibition of mean fluorescence intensity for 3G8) as follow: V/V (72%); V/F (53%); and FF (37%); (p=0.017). Lastly, we assessed rituximab dependent NK cell mediated cytotoxicity (ADCC) using CD20 expressing ARH-77 B-cells and observed higher levels of ADCC killing among V/V (82%) and V/F (80%) versus F/F (23%) donors at an effector: target cell ratio of 20:1. Taken together, these studies suggest that individuals who express at least one valine at FcγRIIIA-158 might have better clinical outcomes to rituximab therapy on the basis of increased FcγRIIIA-158 receptor expression, rituximab binding and ADCC mediated killing of tumor cells.
Prenyltransferases Regulate CD20 Protein Levels and Influence Anti-CD20 Monoclonal Antibody-mediated Activation of Complement-dependent Cytotoxicity
