Immunogenicity of murine mPEG-red blood cells and the risk of anti-PEG antibodies in human blood donors
2016; Elsevier BV; Volume: 47; Linguagem: Inglês
10.1016/j.exphem.2016.11.001
ISSN1873-2399
AutoresYevgeniya Le, Wendy M. Toyofuku, Mark D. Scott,
Tópico(s)Blood transfusion and management
Resumo•In vivo survival of murine methoxypoly(ethylene glycol) (mPEG)-red blood cells (RBCs) after repeated transfusions is normal.•Soluble PEG, before or after mPEG-RBC transfusions, did not induce anti-PEG immunoglobulin (Ig)G.•Findings in humans do not support reports that 25% of blood donors have anti-PEG IgG.•The high rate of anti-PEG IgG in earlier reports is due to methodologic false positives.•Methoxy-PEG-RBCs remain an option for the treatment/prevention of non-ABO alloimmunization. The immunocamouflage of non-ABO blood group antigens by membrane-grafted methoxypoly(ethylene glycol) (mPEG) may attenuate the risk of red blood cell (RBC) alloimmunization. However, concerns have been raised over the immunogenic risk of PEG and PEG-RBCs. To assess this risk, murine and human studies were performed. Mice were exposed to soluble PEG prior to, or between, multiple transfusions (∼60-day intervals) of control or mPEG-RBCs, and cell survival was determined by flow cytometry. In some studies, the control and mPEG-RBC groups were reversed after one or more transfusions. Furthermore, human blood donors and commercial intravenous immunoglobulin products were examined to detect anti-PEG antibodies and to assess the risk for false positives. Naïve mice receiving chronic mPEG-RBC transfusions had normal RBC survival curves with no evidence of anti-PEG antibodies. Similarly, challenge with soluble PEG did not elicit anti-PEG antibodies in mice. Studies in humans revealed no evidence of a high prevalence of anti-PEG antibodies in either blood donors or commercial intravenous immunoglobulin. However, by use of the methods employed by studies identifying high levels of anti-PEG antibodies, a significant level (∼15%) of "false positives" were detected in commercial antibodies of known (non-PEG) specificities. These findings suggest that methodologic problems yielded a high rate of false positives in these earlier studies. These data continue to support the clinical utility of cellular PEGylation and the low immunogenic risk of grafted mPEG. The immunocamouflage of non-ABO blood group antigens by membrane-grafted methoxypoly(ethylene glycol) (mPEG) may attenuate the risk of red blood cell (RBC) alloimmunization. However, concerns have been raised over the immunogenic risk of PEG and PEG-RBCs. To assess this risk, murine and human studies were performed. Mice were exposed to soluble PEG prior to, or between, multiple transfusions (∼60-day intervals) of control or mPEG-RBCs, and cell survival was determined by flow cytometry. In some studies, the control and mPEG-RBC groups were reversed after one or more transfusions. Furthermore, human blood donors and commercial intravenous immunoglobulin products were examined to detect anti-PEG antibodies and to assess the risk for false positives. Naïve mice receiving chronic mPEG-RBC transfusions had normal RBC survival curves with no evidence of anti-PEG antibodies. Similarly, challenge with soluble PEG did not elicit anti-PEG antibodies in mice. Studies in humans revealed no evidence of a high prevalence of anti-PEG antibodies in either blood donors or commercial intravenous immunoglobulin. However, by use of the methods employed by studies identifying high levels of anti-PEG antibodies, a significant level (∼15%) of "false positives" were detected in commercial antibodies of known (non-PEG) specificities. These findings suggest that methodologic problems yielded a high rate of false positives in these earlier studies. These data continue to support the clinical utility of cellular PEGylation and the low immunogenic risk of grafted mPEG. Immunologic recognition of foreign proteins and cells is evolutionarily important as a means of biological survival. However, modern medicine is often in conflict with this biological imperative as we attempt to treat patients with enzymopathies using exogenously sourced enzymes or to correct organ or tissue failure via transplantation of healthy cells. Indeed, consequent to these clinical needs, immunosuppressive therapy has evolved to be a key therapeutic tool. Although most immunosuppressive agents are pharmacologically active drugs addressing leukocyte activation and proliferation, one of the most clinically and commercially successful immunosuppressive approaches has been the covalent grafting of poly(ethylene glycol) (PEG) to exogenously sourced proteins (and other small molecules) for treating patients with enzymopathies. This work, pioneered in the 1970s by Abukowski et al., revealed that xenogeneic proteins could be covalently modified with PEG and methoxypoly(ethylene glycol) (mPEG; PEGylation) to block immunologic recognition of foreign proteins while increasing vascular retention and maintaining enzymatic activity [1Abuchowski A. McCoy J.R. Palczuk N.C. van Es T. Davis F.F. Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase.J Biol Chem. 1977; 252: 3582-3586PubMed Google Scholar, 2Abuchowski A. van Es T. Palczuk N.C. Davis F.F. Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol.J Biol Chem. 1977; 252: 3578-3581PubMed Google Scholar, 3Abuchowski A. Kazo G.M. Verhoest C.R.J. et al.Cancer therapy with chemically modified enzymes: I. Antitumor properties of polyethylene glycol-asparaginase conjugates.Cancer Biochem Biophys. 1984; 7: 175-186PubMed Google Scholar]. The clinical utility of protein PEGylation was rapidly proven, and there are currently a large number of Food and Drug Administration (FDA)-approved PEGylated enzymes in clinical use and trials [4Greenwald R.B. Choe Y.H. McGuire J. Conover C.D. Effective drug delivery by PEGylated drug conjugates.Adv Drug Deliv Rev. 2003; 55: 217-250Crossref PubMed Scopus (777) Google Scholar, 5Haag R. Kratz F. Polymer therapeutics: concepts and applications.Angew Chem Int Ed Engl. 2006; 45: 1198-1215Crossref PubMed Scopus (968) Google Scholar, 6Kang J.S. Deluca P.P. Lee K.C. Emerging PEGylated drugs.Expert Opin Emerg Drugs. 2009; 14: 363-380Crossref PubMed Scopus (197) Google Scholar, 7Mehvar R. Modulation of the pharmacokinetics and pharmacodynamics of proteins by polyethylene glycol conjugation.J Pharm Pharm Sci. 2000; 3: 125-136PubMed Google Scholar, 8Pasut G. Sergi M. Veronese F.M. Anti-cancer PEG-enzymes: 30 years old, but still a current approach.Adv Drug Deliv Rev. 2008; 60: 69-78Crossref PubMed Scopus (117) Google Scholar, 9Webster R. Didier E. Harris P. et al.PEGylated proteins: Evaluation of their safety in the absence of definitive metabolism studies.Drug Metab Dispos. 2007; 35: 9-16Crossref PubMed Scopus (254) Google Scholar]. These compounds, some in use for >30 years, have been found to be highly effective with limited adverse effects. The initial approach of protein PEGylation has been expanded over the last decade to encompass the immunocamouflage of intact cells by the covalent grafting of low-immunogenicity polymers such as mPEG directly to membrane surface proteins. Initial applications were focused on the red blood cell (RBC) to diminish and/or prevent alloimmunization to non-ABO blood groups [10Bradley A.J. Scott M.D. Immune complex binding by immunocamouflaged [poly(ethylene glycol)-grafted] erythrocytes.Am J Hematol. 2007; 82: 970-975Crossref PubMed Scopus (23) Google Scholar, 11Murad K.L. Mahany K.L. Brugnara C. Kuypers F.A. Eaton J.W. Scott M.D. Structural and functional consequences of antigenic modulation of red blood cells with methoxypoly(ethylene glycol).Blood. 1999; 93: 2121-2127PubMed Google Scholar, 12Scott M.D. Murad K.L. Koumpouras F. Talbot M. Eaton J.W. Chemical camouflage of antigenic determinants: Stealth erythrocytes.Proc Natl Acad Sci USA. 1997; 94: 7566-7571Crossref PubMed Scopus (186) Google Scholar, 13Scott M.D. Bradley A.J. Murad K.L. Camouflaged blood cells: Low-technology bioengineering for transfusion medicine?.Transfus Med Rev. 2000; 14: 53-63Abstract Full Text PDF PubMed Scopus (40) Google Scholar, 14Wang D. Kyluik D.L. Murad K.L. Toyofuku W.M. 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Additional studies have subsequently indicated that the immunocamouflage of cell surfaces can prevent leukocyte allorecognition (preventing graft vs. host disease in a mouse model) and also block viral invasion [18Chen A.M. Scott M.D. Immunocamouflage: prevention of transfusion-induced graft-versus-host disease via polymer grafting of donor cells.J Biomed Mater Res A. 2003; 67: 626-636Crossref PubMed Scopus (41) Google Scholar, 19Chen A.M. Scott M.D. Comparative analysis of polymer and linker chemistries on the efficacy of immunocamouflage of murine leukocytes.Artif Cells Blood Substit Immobil Biotechnol. 2006; 34: 305-322Crossref PubMed Scopus (18) Google Scholar, 20Kyluik D.L. Sutton T.C. Le Y. Scott M.D. Chapter 7: Polymer-Mediated Broad Spectrum Antiviral Prophylaxis: Utility in High Risk Environments.in: Carpi A. Progress in Molecular and Environmental Bioengineering: From Analysis and Modeling to Technology Applications. Intech, Rijeka2011: 167-190Google Scholar, 21Murad K.L. 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The immunocamouflage of cells is mediated by both steric interference and charge camouflage [14Wang D. Kyluik D.L. Murad K.L. Toyofuku W.M. Scott M.D. Polymer-mediated immunocamouflage of red blood cells: Effects of polymer size on antigenic and immunogenic recognition of allogeneic donor blood cells.Sci China Life Sci. 2011; 54: 589-598Crossref PubMed Scopus (13) Google Scholar, 15Wang D. Toyofuku W.M. Scott M.D. The potential utility of methoxypoly(ethylene glycol)-mediated prevention of rhesus blood group antigen RhD recognition in transfusion medicine.Biomaterials. 2012; 33: 3002-3012Crossref PubMed Scopus (16) Google Scholar, 25Bradley A.J. Murad K.L. Regan K.L. Scott M.D. Biophysical consequences of linker chemistry and polymer size on stealth erythrocytes: size does matter.Biochim Biophys Acta. 2002; 1561: 147-158Crossref PubMed Scopus (88) Google Scholar, 26Bradley A.J. Scott M.D. Separation and purification of methoxypoly(ethylene glycol) grafted red blood cells via two-phase partitioning.J Chromatogr B Anal Technol Biomed Life Sci. 2004; 807: 163-168Crossref PubMed Scopus (30) Google Scholar, 27Le Y. Scott M.D. Immunocamouflage: The biophysical basis of immunoprotection by grafted methoxypoly(ethylene glycol) [mpeg].Acta Biomater. 2010; 6: 2631-2641Crossref PubMed Scopus (37) Google Scholar, 28Le Y. Li L. Wang D. Scott M.D. Immunocamouflage of latex surfaces by grafted methoxypoly(ethylene glycol) (mPEG): Proteomic analysis of plasma protein adsorption.Sci China Life Sci. 2012; 55: 191-201Crossref PubMed Scopus (11) Google Scholar]. The immunocamouflage of blood cells can be induced not only by PEG, but by other polymers as well (e.g., hyperbranched polyglycerols [HPG] and polyethyloxazoline propionic acid [PEOZ]), though PEG and its derivatives (e.g., mPEG) remain the best characterized immunologically and toxicologically [29Chapanian R. Constantinescu I. Brooks D.E. Scott M.D. Kizhakkedathu J.N. In vivo circulation, clearance, and biodistribution of polyglycerol grafted functional red blood cells.Biomaterials. 2012; 33: 3047-3057Crossref PubMed Scopus (32) Google Scholar, 30Chapanian R. Constantinescu I. Rossi N.A. et al.Influence of polymer architecture on antigens camouflage, CD47 protection and complement mediated lysis of surface grafted red blood cells.Biomaterials. 2012; 33: 7871-7883Crossref PubMed Scopus (24) Google Scholar, 31Rossi N.A. Constantinescu I. Brooks D.E. Scott M.D. Kizhakkedathu J.N. Enhanced cell surface polymer grafting in concentrated and nonreactive aqueous polymer solutions.J Am Chem Soc. 2010; 132: 3423-3430Crossref PubMed Scopus (44) Google Scholar, 32Rossi N.A. Constantinescu I. Kainthan R.K. Brooks D.E. Scott M.D. Kizhakkedathu J.N. Red blood cell membrane grafting of multi-functional hyperbranched polyglycerols.Biomaterials. 2010; 31: 4167-4178Crossref PubMed Scopus (62) Google Scholar, 33Kyluik-Price D.L. Li L. Scott M.D. Comparative efficacy of blood cell immunocamouflage by membrane grafting of methoxypoly(ethylene glycol) and polyethyloxazoline.Biomaterials. 2014; 35: 412-422Crossref PubMed Scopus (22) Google Scholar]. Despite the potential of other polymers to induce the immunocamouflage of proteins and cells, PEG and PEG derivatives are used almost exclusively in pharmacologic preparations largely because of their known physical properties and toxicologic and immunologic safety profiles. Indeed, even though the polymer is grafted to potentially highly immunogenic proteins, only sporadic reports of anti-PEG antibodies have been noted in the enzyme replacement literature. In further support of the low immunogenicity of PEG, experimental generation of anti-PEG antibodies typically requires the use of very strong adjuvants (e.g., Freund's complete adjuvant) [34Richter A.W. Akerblom E. Antibodies against polyethylene glycol produced in animals by immunization with monomethoxy polyethylene glycol modified proteins.Int Arch Allergy Appl Immunol. 1983; 70: 124-131Crossref PubMed Scopus (229) Google Scholar, 35Cheng T.L. Wu P.Y. Wu M.F. Chern J.W. Roffler S.R. Accelerated clearance of polyethylene glycol-modified proteins by anti-polyethylene glycol IgM.Bioconjug Chem. 1999; 10: 520-528Crossref PubMed Scopus (129) Google Scholar, 36Cheng T.C. Chuang K.H. Chen M. et al.Sensitivity of PEGylated interferon detection by anti-polyethylene glycol (PEG) antibodies depends on PEG length.Bioconjug Chem. 2013; 24: 1408-1413Crossref PubMed Scopus (12) Google Scholar, 37Cheng T.L. Chen B.M. Chern J.W. Wu M.F. Roffler S.R. Efficient clearance of poly(ethylene glycol)-modified immunoenzyme with anti-PEG monoclonal antibody for prodrug cancer therapy.Bioconjug Chem. 2000; 11: 258-266Crossref PubMed Scopus (61) Google Scholar]. However, PEG is widely used in the food, pharmaceutical, and cosmetic industries, and this ubiquity has raised some concerns that this chronic exposure could enhance the immunogenic potential of PEG. Indeed, 2007 and 2012 reports by Armstrong et al. that more than 25% of blood donors exhibited anti-PEG antibodies (the majority being IgG) have raised significant concerns [38Armstrong J.K. Hempel G. Koling S. et al.Antibody against poly(ethylene glycol) adversely affects PEG-asparaginase therapy in acute lymphoblastic leukemia patients.Cancer. 2007; 110: 103-111Crossref PubMed Scopus (506) Google Scholar, 39Garay R.P. El-Gewely R. Armstrong J.K. Garratty G. Richette P. Antibodies against polyethylene glycol in healthy subjects and in patients treated with PEG-conjugated agents.Expert Opin Drug Deliv. 2012; 9: 1319-1323Crossref PubMed Scopus (369) Google Scholar]. Armstrong et al.'s findings were in stark contrast to those of Richter et al. [40Richter A.W. Akerblom E. Polyethylene glycol reactive antibodies in man: Titer distribution in allergic patients treated with monomethoxy polyethylene glycol modified allergens or placebo, and in healthy blood donors.Int Arch Allergy Appl Immunol. 1984; 74: 36-39Crossref PubMed Scopus (159) Google Scholar], who reported that 1 in 500 (0.2%) blood donors had "clinically insignificant" anti-PEG antibodies, as well as previous in vitro and in vivo human and murine studies examining immune recognition of mPEG-RBCs [10Bradley A.J. Scott M.D. Immune complex binding by immunocamouflaged [poly(ethylene glycol)-grafted] erythrocytes.Am J Hematol. 2007; 82: 970-975Crossref PubMed Scopus (23) Google Scholar, 11Murad K.L. Mahany K.L. Brugnara C. Kuypers F.A. Eaton J.W. Scott M.D. Structural and functional consequences of antigenic modulation of red blood cells with methoxypoly(ethylene glycol).Blood. 1999; 93: 2121-2127PubMed Google Scholar, 14Wang D. Kyluik D.L. Murad K.L. Toyofuku W.M. Scott M.D. Polymer-mediated immunocamouflage of red blood cells: Effects of polymer size on antigenic and immunogenic recognition of allogeneic donor blood cells.Sci China Life Sci. 2011; 54: 589-598Crossref PubMed Scopus (13) Google Scholar, 15Wang D. Toyofuku W.M. Scott M.D. The potential utility of methoxypoly(ethylene glycol)-mediated prevention of rhesus blood group antigen RhD recognition in transfusion medicine.Biomaterials. 2012; 33: 3002-3012Crossref PubMed Scopus (16) Google Scholar, 25Bradley A.J. Murad K.L. Regan K.L. Scott M.D. Biophysical consequences of linker chemistry and polymer size on stealth erythrocytes: size does matter.Biochim Biophys Acta. 2002; 1561: 147-158Crossref PubMed Scopus (88) Google Scholar, 41Bradley A.J. Test S.T. Murad K.L. Mitsuyoshi J. Scott M.D. Interactions of IgM ABO antibodies and complement with methoxy-PEG-modified human RBCs.Transfusion. 2001; 41: 1225-1233Crossref PubMed Scopus (45) Google Scholar]. To experimentally address the potential immunogenicity of mPEG-RBCs, a chronic murine transfusion model was used in which mice were challenged with soluble PEG polymer both prior to and subsequent to transfusion of control and mPEG-modified murine RBCs. RBC survival curves were determined and murine IgG response was measured. To assess the presence of anti-PEG antibodies in humans, flow cytometric studies using target PEGylated latex beads were done with normal blood donors and commercial intravenous immunoglobulin (IVIG, 10,000+ donors) products. Moreover, the risk of "false positives" using the method of Armstrong et al. [38Armstrong J.K. Hempel G. Koling S. et al.Antibody against poly(ethylene glycol) adversely affects PEG-asparaginase therapy in acute lymphoblastic leukemia patients.Cancer. 2007; 110: 103-111Crossref PubMed Scopus (506) Google Scholar] was also assessed using commercial antibodies of known (non-PEG) specificity. The immunogenicity of PEG and mPEG-RBC was assessed in vivo using a murine transfusion model, as previously described [11Murad K.L. Mahany K.L. Brugnara C. Kuypers F.A. Eaton J.W. Scott M.D. Structural and functional consequences of antigenic modulation of red blood cells with methoxypoly(ethylene glycol).Blood. 1999; 93: 2121-2127PubMed Google Scholar, 12Scott M.D. Murad K.L. Koumpouras F. Talbot M. Eaton J.W. Chemical camouflage of antigenic determinants: Stealth erythrocytes.Proc Natl Acad Sci USA. 1997; 94: 7566-7571Crossref PubMed Scopus (186) Google Scholar, 14Wang D. Kyluik D.L. Murad K.L. Toyofuku W.M. Scott M.D. Polymer-mediated immunocamouflage of red blood cells: Effects of polymer size on antigenic and immunogenic recognition of allogeneic donor blood cells.Sci China Life Sci. 2011; 54: 589-598Crossref PubMed Scopus (13) Google Scholar, 15Wang D. Toyofuku W.M. Scott M.D. The potential utility of methoxypoly(ethylene glycol)-mediated prevention of rhesus blood group antigen RhD recognition in transfusion medicine.Biomaterials. 2012; 33: 3002-3012Crossref PubMed Scopus (16) Google Scholar]. All experiments were done in accordance with the Canadian Council of Animal Care and the University of British Columbia (UBC) Animal Care Committee guidelines and were conducted within the Animal Resource Unit at UBC. Donor RBCs were collected from C57BL/6 mice, labeled with PKH-26, and PEGylated as described previously [11Murad K.L. Mahany K.L. Brugnara C. Kuypers F.A. Eaton J.W. Scott M.D. Structural and functional consequences of antigenic modulation of red blood cells with methoxypoly(ethylene glycol).Blood. 1999; 93: 2121-2127PubMed Google Scholar, 12Scott M.D. Murad K.L. Koumpouras F. Talbot M. Eaton J.W. Chemical camouflage of antigenic determinants: Stealth erythrocytes.Proc Natl Acad Sci USA. 1997; 94: 7566-7571Crossref PubMed Scopus (186) Google Scholar, 14Wang D. Kyluik D.L. Murad K.L. Toyofuku W.M. Scott M.D. Polymer-mediated immunocamouflage of red blood cells: Effects of polymer size on antigenic and immunogenic recognition of allogeneic donor blood cells.Sci China Life Sci. 2011; 54: 589-598Crossref PubMed Scopus (13) Google Scholar, 15Wang D. Toyofuku W.M. Scott M.D. The potential utility of methoxypoly(ethylene glycol)-mediated prevention of rhesus blood group antigen RhD recognition in transfusion medicine.Biomaterials. 2012; 33: 3002-3012Crossref PubMed Scopus (16) Google Scholar]. Importantly, previous studies have reported that C57BL/6 mice are capable of producing anti-PEG antibodies when stimulated with PEG-liposomes [42Judge A. McClintock K. Phelps J.R. Maclachlan I. Hypersensitivity and loss of disease site targeting caused by antibody responses to PEGylated liposomes.Mol Ther. 2006; 13: 328-337Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar]. PEGylated donor RBCs were grafted with 2 mmol/L of 20-kDa activated mPEG purchased from either Nektar Therapeutics (San Carlos, CA; methoxypoly(ethylene glycol)-benzotriazolyl carbonate [BTCmPEG]) or Laysan Bio (Arab, AL, USA; succinimidyl valerate [SVA]-mPEG). Of note, no differences in the in vivo circulation or immunogenicity of BTCmPEG-RBCs or SVAmPEG-RBCs were noted. Control and mPEG-modified donor RBCs were transfused (intraperitoneally) into syngeneic recipient mice, with the 24-hour circulation levels of PKH-26-labeled donor cells being defined as 100%. All transfusions were performed, on average, 60 days apart, unless specified otherwise. At defined intervals a small volume (20–40 μL) of blood was collected from the recipient mice for flow cytometric analysis (10,000 total events; BD FACSCalibur, BD Biosciences, San Jose, CA) of donor cell survival, as previously described [11Murad K.L. Mahany K.L. Brugnara C. Kuypers F.A. Eaton J.W. Scott M.D. Structural and functional consequences of antigenic modulation of red blood cells with methoxypoly(ethylene glycol).Blood. 1999; 93: 2121-2127PubMed Google Scholar, 12Scott M.D. Murad K.L. Koumpouras F. Talbot M. Eaton J.W. Chemical camouflage of antigenic determinants: Stealth erythrocytes.Proc Natl Acad Sci USA. 1997; 94: 7566-7571Crossref PubMed Scopus (186) Google Scholar, 14Wang D. Kyluik D.L. Murad K.L. Toyofuku W.M. Scott M.D. Polymer-mediated immunocamouflage of red blood cells: Effects of polymer size on antigenic and immunogenic recognition of allogeneic donor blood cells.Sci China Life Sci. 2011; 54: 589-598Crossref PubMed Scopus (13) Google Scholar, 15Wang D. Toyofuku W.M. Scott M.D. The potential utility of methoxypoly(ethylene glycol)-mediated prevention of rhesus blood group antigen RhD recognition in transfusion medicine.Biomaterials. 2012; 33: 3002-3012Crossref PubMed Scopus (16) Google Scholar]. A minimum of five mice were used per treatment group. Control groups included naïve mice and animals transfused with saline according to the same schedule used for the treatment groups. Individual treatment groups are defined in the Results, but included populations prechallenged with soluble PEG prior to control or mPEG-RBC transfusion, as well as mice challenged with soluble PEG subsequent to transfusion with control or mPEG-RBCs. As a positive clearance control, an aliquot (20% hematocrit) of donor murine RBCs was treated with 50 mmol/L phenozine methosulfate (PMS; Sigma-Aldrich, St. Louis, MO) for 60 min at 37°C to mildly oxidize the cells [43Scott M.D. Eaton J.W. Kuypers F.A. Chiu D.Y. Lubin B.H. Enhancement of erythrocyte superoxide dismutase activity: Effects on cellular oxidant defense.Blood. 1989; 74: 2542-2549PubMed Google Scholar]. Sham-treated (0 mmol/L PMS) control cells were similarly incubated. After incubation, the samples were centrifuged (1,000g for 5 min) and washed twice with isotonic saline prior to PKH-26 labeling. On completion of the in vivo murine studies, whole blood was collected from anesthetized mice into heparin sodium salt tubes via cardiac puncture. Mouse plasma was obtained by centrifugation for 5 min at 1,000g. Where specified, individual mouse plasma samples were pooled within the same control or treatment group. All plasma and serum samples were stored at −80°C for subsequent experiments. The murine samples were assessed for anti-PEG IgG antibodies via flow cytometry using fluorescently labeled anti-murine IgG antibodies. Murine anti-PEG IgG antibodies were detected using PEGylated TentaGel M OH latex particles (TentaGel, RAPP Polymere, Tubingen, Germany; 0.23 mmol PEG/g latex, PEG molecular weight ∼3 kDa). The murine samples from the described in vivo mPEG-RBC transfusion experiments were assayed for induced anti-PEG IgG antibodies by incubating murine plasma with the PEGylated TentaGel beads using the methodology described by Armstrong et al. [38Armstrong J.K. Hempel G. Koling S. et al.Antibody against poly(ethylene glycol) adversely affects PEG-asparaginase therapy in acute lymphoblastic leukemia patients.Cancer. 2007; 110: 103-111Crossref PubMed Scopus (506) Google Scholar]. Briefly, 50 μL of undiluted murine plasma was added to 100 μL phosphate-buffered saline (PBS, pH 7.2). To the plasma dilution, 25 μL of a 1% aqueous solution of PEGylated TentaGel latex particles was added, and the mixture was incubated for 30 min at room temperature, followed by three washes in PBS to remove unbound protein, prior to flow cytometric analysis. Anti-PEG antibody binding to latex particles was detected using 5 μL of secondary goat anti-mouse IgG antibody (fluorescein isothiocyanate [FITC], 1 mg/mL; Catalog No. F8264, Sigma, St. Louis, MO). All experiments in humans were done in accordance with and the approval of the University of British Columbia Clinical Research Ethics Board and in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki). The population was gender balanced and consisted of 16 females and 19 males. To experimentally re-examine the findings of Armstrong et al. that ∼25% of normal blood donors exhibited anti-PEG antibodies, serum samples were obtained from 35 healthy individuals (i.e., theoretically yielding eight or nine positive samples per Armstrong et al.) [38Armstrong J.K. Hempel G. Koling S. et al.Antibody against poly(ethylene glycol) adversely affects PEG-asparaginase therapy in acute lymphoblastic leukemia patients.Cancer. 2007; 110: 103-111Crossref PubMed Scopus (506) Google Scholar, 44Armstrong J.K. Leger R.M. Wenby R.B. Meiselman H.J. Garratty G. Fisher T.C. Occurrence of an antibody to poly(ethylene glycol) in normal donors.Blood. 2003; 102: 556aGoogle Scholar]. Serum was prepared from venous blood collected in serum separator tubes (SST Vacutainer tubes, Becton Dickinson, Fairview, NJ) subsequent to clotting for 30–60 min at room temperature and centrifugation (15 min at 1,000g). Human serum samples were stored at −80°C prior to flow cytometric analysis. To detect anti-PEG antibodies, PEGylated TentaGel particles (25 μL of a 1% aqueous solution of PEGylated TentaGel latex) beads were incubated with human serum (50 μL in 100 μL PBS) prior to addition of secondary anti-human IgG (PE mouse anti-human IgG, 0.015 mg/mL stock; Catalog No. 555787, BD Biosciences, San Jose, CA, USA) and IgM (APC mouse anti-human IgM, 0.003 mg/mL; Catalog No. 551062, BD Biosciences) antibodies. The secondary antibody levels used were reduced relative to Armstrong et al. because of concern over the exceedingly high concentration of secondary anti-human IgG antibody (stock Sigma-Aldrich antibody 10–20 mg/mL, final sample concentration of 0.1–0.2 mg/mL) used in their study. A comparison of the two methods is provided in Table 1. The binding of endogenous human anti-PEG antibodies to the beads was quantitated by flow cytometry (BD FACSCalibur, BD Biosciences), with a minimum of 10,000 total events analyzed per each sample [27Le Y. Scott M.D. Immunocamouflage: The biophysical basis of immunoprotection by grafted methoxypoly(ethylene glycol) [mpeg].Acta Biomater. 2010; 6: 2631-2641Crossref PubMed Scopus (37) Google Scholar, 38Armstrong J.K. Hempel G. Koling S. et al.Antibody against poly(ethylene glycol) adversely affects PEG-asparaginase therapy in acute lymphoblastic leukemia patients.Cancer. 2007; 110: 103-111Crossref PubMed Scopus (506) Google Scholar]. To further validate the IgG analysis, two additional secondary anti-human IgG antibodies (0.015 mg/mL) were also examined: FITC goat anti-human IgG (Catalog No. AP504F, Millipore, Billerica, MA, U
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