Oral Presentations - Pathology and Laboratory Medicine - University ...

Graduate StudentKyluik DL and Scott MDDepartment of Pathology and Laboratory Medicine, University of British Columbia,Vancouver, Canada. Canadian Blood Services and UBC Centre for Blood ResearchAbstract # 72effect of polymer species and size onimmunocamouflage and antigenicityDana KyluikBackround/ObjectivesDespite ABO/RhD typing, alloimmunization remain significant problem in chronically transfused patients. Theseproblems arise due to the ~300 non-ABO blood group antigens present on the RBC. To address this risk, theimmunocamouflage of donor RBC, accomplished by the covalent grafting of low immunogenicity polymers to the cellmembrane, has been proposed. To date, the majority of our research has focused on methoxypoly(ethylene glycol)[mPEG]. These studies demonstrated significant immunoprotection and normal in vivo survival. However, a smallnumber of studies have reported the presence of anti-mPEG antibodies in patients receiving PEGylated proteins. Wepropose to explore the potential utility of a newly designed class of proprietary compounds (cwXXy: cw represent thenovel polymer, XX the molecular weight and y the linker chemistry). This novel compound class shares similaritieswith mPEG, but does not cross react with anti-PEG antibodies. We hypothesize that these polymers (initiallyexemplified by cw20t and cw30t) will provide similar efficacy to traditional PEGylation, while exhibiting a lower riskof immunogenicity.MethodsInitial studies were conducted to examine the differential effects of SVAmPEG and cwXXy on RBC structure andfunction. Normal human erythrocytes (12% hematocrit) were modified with either SVAmPEG or cwXXy polymers(2, 5 , 20 and 30 kDa) at concentrations ranging from 0-2 mM. Grafting reactions were carried out for 60 min. Cellmorphology was examined microscopically following fixation of control and modified RBC in 4% paraformaldehyde.To assess RBC membrane stability and spontaneous lysis, osmotic fragility assays was performed over 68 hours usingDrabkin’s reagent. Two-phase partitioning experiments (PEG8000 and dextran T500) were conducted to quantitateany differential effects of SVAmPEG and cwXXy on RBC derivatization based the effects of the grafted polymer onsurface properties and phase separation.ResultsBoth SVAmPEG and cwXXy polymer species demonstrated equivalent cellular morphology to sham treated controlRBC immediately following the derivatization process. Cellular stability, as assessed by the osmotic fragility profile,showed minimal differences between SVAmPEG and cwXXy at eqimolar concentrations and equivalent molecularweights as demonstrated by the fragility curves. However, both polymers showed elevated spontaneous lysis (0.9%saline) at 68 hours relative to unmodified cells. Of importance, previous findings demonstrated that SVAmPEG(2mM grafting concentration) modified cells exhibited normal murine in vivo survival despite this slight osmoticfragility profile variation. Grafting of cw20/30t to RBC results in comparable partitioning curves to SVAmPEG(20 and 30 kDa) in the two-phase system. However, SVAmPEG show slightly enhanced reaction kinetics due to itsdifferent linker chemistry that exhibits both greater lysine reactivity and a longer hydrolysis half-life.ConclusionOur studies suggest that the cwXXy polymer family demonstrate comparable efficacy to SVAmPEG with regards tomorphology, partitioning and osmotic fragility profiles. This data suggests that these novel polymer may exhibit similarin vivo survival and immunocamouflage efficacy.Poster Presentations * 2 0 1 083