Abstract
Alemtuzumab is a monoclonal antibody that targets cell surface CD52 and is effective in depleting lymphocytes by cytolytic effects in vivo. Although the cytolytic effects of alemtuzumab are dependent on the density of CD52 antigen on cells, there is scant information regarding the expression levels of CD52 on different cell types. In this study, CD52 expression was assessed on phenotypically distinct subsets of lymphoid and myeloid cells in peripheral blood mononuclear cells (PBMCs) from normal donors. Results demonstrate that subsets of PBMCs express differing levels of CD52. Quantitative analysis showed that memory B cells and myeloid dendritic cells (mDCs) display the highest number while natural killer (NK) cells, plasmacytoid dendritic cells (pDCs) and basophils have the lowest number of CD52 molecules per cell amongst lymphoid and myeloid cell populations respectively. Results of complement dependent cytolysis (CDC) studies indicated that alemtuzumab mediated profound cytolytic effects on B and T cells with minimal effect on NK cells, basophils and pDCs, correlating with the density of CD52 on these cells. Interestingly, despite high CD52 levels, mDCs and monocytes were less susceptible to alemtuzumab-mediated CDC indicating that antigen density alone does not define susceptibility. Additional studies indicated that higher expression levels of complement inhibitory proteins (CIPs) on these cells partially contributes to their resistance to alemtuzumab mediated CDC. These results indicate that alemtuzumab is most effective in depleting cells of the adaptive immune system while leaving innate immune cells relatively intact.
Highlights
CD52 is a cell surface glycoprotein consisting of a short 12 aa peptide with a C terminal GPI anchor
While cytolytic effects correlated with antigen density on a majority of peripheral blood mononuclear cells (PBMCs) subsets, CD52 antigen density did not correlate with the degree of cytolysis for some myeloid cell populations and our studies indicated that they expressed higher levels of complement inhibitory proteins
Analyzing multiple cell surface markers simultaneously, we first defined phenotypically distinct cell populations corresponding to lymphocyte, myeloid and plasmacytoid cell lineages in PBMCs from 22 normal donors
Summary
CD52 is a cell surface glycoprotein consisting of a short 12 aa peptide with a C terminal GPI anchor. Alemtuzumab is a humanized monoclonal antibody to human CD52, genetically engineered by grafting rat complementarity determining regions (CDRs) into human framework regions fused to human IgG1 [4] It binds to the C-terminal part of the peptide to an epitope that includes part of the GPI anchor [5]. Alemtuzumab has been approved for the treatment of patients with advanced chronic lymphocytic leukemia (CLL) [6,7,8] This antibody has been utilized in the treatment of a wide range of diseases including rheumatoid arthritis [9,10,11], non-Hodgkin’s lymphoma [12,13] and T- cell lymphoma [14,15]. Caspase-8 dependent and independent apoptosis have been identified as other potential mechanisms of cytolytic action by alemtuzumab on cell lines and CLL cells [21,22,23]
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