Pre-existing anti–polyethylene glycol antibody linked to first-exposure allergic reactions to pegnivacogin, a PEGylated RNA aptamer (2015)

Nucleic acid aptamers are a novel class of drugs that can be selected to inhibit targets of interest, including protein-protein interactions.1Nimjee S.M. Rusconi C.P. Sullenger B.A. Aptamers: an emerging class of therapeutics.Annu Rev Med. 2005; 56: 555-583Crossref PubMed Scopus (804) Google Scholar Pegnivacogin is a 2′-fluoropyrimidine–modified RNA aptamer that inhibits coagulation factor IXa, coupled to an approximately 40-kDa branched molecule of methoxypolyethylene glycol (mPEG), to increase its concentration and half-life in plasma.2Woodruff R.S. Sullenger B.A. Modulation of the coagulation cascade using aptamers.Arterioscler Thromb Vasc Biol. 2015; 35: 2083-2091Crossref PubMed Scopus (32) Google Scholar During the RADAR3Povsic T.J. Vavalle J.P. Aberle L.H. Kasprzak J.D. Cohen M.G. Mehran R. et al.A phase 2, randomized, partially blinded, active-controlled study assessing the efficacy and safety of variable anticoagulation reversal using the REG1 system in patients with acute coronary syndromes: results of the RADAR trial.Eur Heart J. 2013; 34: 2481-2489Crossref PubMed Scopus (73) Google Scholar phase 2b clinical trial in patients with acute coronary syndrome, allergic reactions occurred within minutes of a first dose of pegnivacogin in 3 of 640 patients (Table I). Two met criteria for anaphylaxis, and 1 was an isolated dermal reaction; each event was deemed serious, and 1 life-threatening, and together they led to early termination of the trial.3Povsic T.J. Vavalle J.P. Aberle L.H. Kasprzak J.D. Cohen M.G. Mehran R. et al.A phase 2, randomized, partially blinded, active-controlled study assessing the efficacy and safety of variable anticoagulation reversal using the REG1 system in patients with acute coronary syndromes: results of the RADAR trial.Eur Heart J. 2013; 34: 2481-2489Crossref PubMed Scopus (73) Google ScholarTable ISubjects experiencing SARs in RADARPatient IDOnset (min)SymptomsTreatmentResolution (h)Allergy history602-0045GI, D, P, HIVF, H1, IVV, S62 mo prior diffuse urticaria to unknown agent406-00325DS, H1, H20.4Contrast dye, hay fever418-0085P, D, HS, H1, H2, IVF, IVV, I, Inh1144Recent allergy to β-blockers and steroidsAll 3 patients were female; 602-004 was treated at a site in Poland and the other 2 patients were treated in Germany. For additional information regarding these patients, see this article's REG1-CLIN211a section in the Online Repository at www.jacionline.org. Note: As substantially more information is available on these 3 subjects than for other trial participants, inferences regarding the possible role of sex, geography, or allergic history are cautioned against.D, Dermal; GI, gastrointestinal; H, hypotension; H1, H1 blocker; H2, H2 blocker; I, intubation; Inh, inhalers; IVV, intravenous vasopressors; IVF, intravenous fluid resuscitation; P, pulmonary; S, steroids. Open table in a new tab All 3 patients were female; 602-004 was treated at a site in Poland and the other 2 patients were treated in Germany. For additional information regarding these patients, see this article's REG1-CLIN211a section in the Online Repository at www.jacionline.org. Note: As substantially more information is available on these 3 subjects than for other trial participants, inferences regarding the possible role of sex, geography, or allergic history are cautioned against. D, Dermal; GI, gastrointestinal; H, hypotension; H1, H1 blocker; H2, H2 blocker; I, intubation; Inh, inhalers; IVV, intravenous vasopressors; IVF, intravenous fluid resuscitation; P, pulmonary; S, steroids. In a broad investigation into a cause for these 3 events (detailed in Methods in this article's Online Repository at www.jacionline.org), a clinical database review found no other serious allergic reactions (SARs) to pegnivacogin; a quality analysis found no aggregation, degradation, or other deviations of the study product from specifications; and a primate pharmacology study found no evidence that pegnivacogin caused an inflammatory response, histamine release, or complement activation. However, blinded testing of more than half of all RADAR patients identified an association between high levels of antibody to polyethylene glycol (PEG) and the first-exposure allergic reactions. In addition to the immediate relevance, our findings are the first to document the potential clinical significance of pre-existing antibody to PEG, a component of numerous consumer and medicinal products. Initially, coded samples from 31 RADAR patients were tested for anti-PEG antibody (see Analytical methods and Fig E1 in this article's Online Repository at www.jacionline.org) in 2 ELISAs to detect IgG binding to pegloticase, a PEGylated urate oxidase (a protein not expressed in humans), and to the 40-kDa mPEG component of pegnivacogin. Unblinding of the data revealed that samples giving the highest signals in both ELISAs were from the 3 patients with SARs (predose from patients 418-008 and 406-003; 88-day postinfusion from patient 602-004 from whom no predose sample was available). Direct (Fig 1, A) and competition ELISAs (Fig 1, B) showed that antibody from each patient could bind to linear and branched PEGs of 5 to 40 kDa, presented as free mPEG or PEG-diol (lacking methoxy termini), or when conjugated via different linkages to 2 proteins and pegnivacogin; importantly, they did not recognize the un-PEGylated aptamer. The predominant heavy-chain isotype was IgG in all 3 patients, and for patient 418-008, who had the highest anti-PEG antibody level, IgG2 was the predominant IgG subtype (see Fig E2 in this article's Online Repository available at www.jacionline.org). To assess the overall prevalence and levels of pre-existing anti-PEG antibody, the sponsor submitted coded pretreatment plasma samples from 328 other RADAR patients who had not experienced allergic reactions (all remaining samples available). Recoded samples available from 26 of the initial 31 patients were added, to give a set of 354 unique patient samples (353 pretreatment and 1 posttreatment). In the pegloticase ELISA, 139 of these exceeded the A405 cutoff point of 0.15, with about a third having an A405 value of less than 0.2. Ninety samples with an A405 value of more than 0.2 were then retested with a validated automated competition ELISA, in which pegloticase was the plate coating and 10-kDa PEG-diol was the competitor.4Hershfield M.S. Ganson N.J. Kelly S.J. Scarlett E.L. Jaggers D.A. Sundy J.S. Induced and pre-existing anti-polyethylene glycol antibody in a trial of every 3-week dosing of pegloticase for refractory gout, including in organ transplant recipients.Arthritis Res Ther. 2014; 16: R63Crossref PubMed Scopus (31) Google Scholar Of these, 82 retested as positive with confirmed specificity for PEG (Fig 1, C). Applying this false-positive rate of 9%, we estimate that 36% of all RADAR patients tested were positive for anti-PEG antibody, with 23% having an A405 value of more than 0.2 in the pegloticase ELISA. The 3 patients with SARs were among only 8 with an A405 value of 1.0 or more (top 2.3%), with patient 418-008 having the highest A405 value of 1.9. Two of these 8 patients received heparin in the trial; of the other 6, 3 (50%) experienced allergic SARs. In further testing of 15 samples with A405 values within the 90th percentile, anti-PEG IgG titers for the 3 patients with SARs ranged from 1:120 to 1:3100 (patient 418-008), compared with approximately 1:110 to 1:950 for the 12 patients without allergies. Oligonucleotides can potentially trigger allergic-like events by activating the alternative complement pathway, or an innate immune response via Toll-like receptors.5Kwoh T.J. An Overview of the clinical safety experience of first- and second-generation antisense oligonucleotides.in: Crooke S.T. Antisense drug technology. 2nd ed. CRC Press, Boca Raton2007: 365-400Crossref Google Scholar No evidence of the former mechanism was observed with pegnivacogin in primate studies (see Fig E3, Fig E4, Fig E5 in this article's Online Repository at www.jacionline.org) or in phase 1 patients across a wide range of doses. Activation of Toll-like receptors typically elicits a mitogenic response with flu-like symptoms, which were not observed with the reactions in RADAR. Antibody-mediated mechanisms can also trigger allergy, but humans are not generally exposed to RNA oligonucleotides, and antibody to bacterial DNA and antinuclear autoantibodies do not react significantly with RNA. In contrast, free PEGs are used as additives in numerous consumer products and topical and parenteral medications, and as osmotic laxatives. Perhaps as a result of this wide exposure, anti-PEG antibodies, of no apparent clinical significance, have been detected by various methods as an incidental finding in several study populations at a prevalence of approximately 3% to more than 40%.6Yang Q. Lai S.K. Anti-PEG immunity: emergence, characteristics, and unaddressed questions.Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2015; 7: 655-677Crossref PubMed Scopus (369) Google Scholar mPEGs have also been used for more than 25 years to covalently modify (PEGylate) biologically active molecules as a means of masking epitopes and slowing elimination. Some PEGylated enzymes and liposomes have recently been found to induce anti-PEG antibodies.6Yang Q. Lai S.K. Anti-PEG immunity: emergence, characteristics, and unaddressed questions.Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2015; 7: 655-677Crossref PubMed Scopus (369) Google Scholar Findings in clinical trials of PEGylated enzymes support a role for high levels of pre-existing anti-PEG antibodies in causing first-exposure reactions to pegnivacogin. In a phase 2 trial of pegloticase for refractory gout, we found anti-PEG antibodies in pretreatment samples of 5 of 27 (19%) patients. The patient with the highest level had an adverse reaction during his first infusion of pegloticase, and patients in whom high levels of anti-PEG antibodies were induced by pegloticase had more frequent and more severe infusion reactions than did those who remained antibody negative.4Hershfield M.S. Ganson N.J. Kelly S.J. Scarlett E.L. Jaggers D.A. Sundy J.S. Induced and pre-existing anti-polyethylene glycol antibody in a trial of every 3-week dosing of pegloticase for refractory gout, including in organ transplant recipients.Arthritis Res Ther. 2014; 16: R63Crossref PubMed Scopus (31) Google Scholar Infusion reactions, including anaphylaxis-like SAEs, were also associated with higher levels of pegloticase-induced anti-PEG antibodies during phase 3 investigation.7Lipsky P.E. Calabrese L.H. Kavanaugh A. Sundy J.S. Wright D. Wolfson M. et al.Pegloticase immunogenicity: the relationship between efficacy and antibody development in patients treated for refractory chronic gout.Arthritis Res Ther. 2014; 16: R60Crossref PubMed Scopus (156) Google Scholar In a phase 1 trial for treating phenylketonuria, a PEGylated phenylalanine ammonia lyase (rAvPAL-PEG) induced anti-PEG antibodies in all 25 participants, 2 of whom had hypersensitivity reactions to an incidentally prescribed birth control medication formulated with PEG before its use was forbidden.8Longo N. Harding C.O. Burton B.K. Grange D.K. Vockley J. Wasserstein M. et al.Single-dose, subcutaneous recombinant phenylalanine ammonia lyase conjugated with polyethylene glycol in adult patients with phenylketonuria: an open-label, multicentre, phase 1 dose-escalation trial.Lancet. 2014; 384: 37-44Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar We postulate that a high level of pre-existing anti-PEG antibodies was a major, but not the sole, factor necessary for triggering first-exposure allergic reaction to pegnivacogin. A likely second factor is the relatively large amount of PEG delivered in a single 1 mg/kg bolus dose of pegnivacogin, for example, approximately 64 mg of PEG for an 80-kg patient. In contrast, with peginterferon alfa2a (PEGASYS), which also contains a single 40-kDa branched mPEG, a standard dose would deliver about 0.12 mg of PEG. Other factors must also be involved, as a few other RADAR patients with similar high levels of pre-existing anti-PEG antibodies did not experience an allergic reaction to pegnivacogin. This could reflect differences in properties of anti-PEG antibodies in individual patients, as well as differences in susceptibility to antibody-triggered allergic reactions, that is, the same factors that underlie the spectrum of responses that antidrug antibodies elicit to some antibiotics and un-PEGylated protein-based biologics.9Chung C.H. Mirakhur B. Chan E. Le Q.T. Berlin J. Morse M. et al.Cetuximab-induced anaphylaxis and IgE specific for galactose-alpha-1,3-galactose.N Engl J Med. 2008; 358: 1109-1117Crossref PubMed Scopus (1131) Google Scholar, 10Yin L. Chen X. Vicini P. Rup B. Hickling T.P. Therapeutic outcomes, assessments, risk factors and mitigation efforts of immunogenicity of therapeutic protein products.Cell Immunol. 2015; 295: 118-126Crossref PubMed Scopus (55) Google Scholar On the basis of our findings, we advise testing for pre-existing anti-PEG antibodies during clinical trials of new PEGylated therapeutic agents, particularly those containing high densities of PEG, or that are given at doses that would expose patients to relatively large amounts of PEG. Further research is needed into the prevalence of “incidental” anti-PEG antibodies, the basis for PEG immunogenicity, and the mechanism(s) by which PEG-anti-PEG immune complexes trigger allergic reactions. We appreciate helpful discussions with Susan J. Kelly during the course of this investigation, and for reviewing the manuscript. We thank former colleagues at Regado Biosciences for help in the design and execution of this investigation, and in particular Doug Brooks and the Chemistry, Manufacturing, and Controls team, the biology group for management and deidentification of plasma and serum samples, and Doug Kornbrust for help in the design and interpretation of results from the safety pharmacology study. Regado Biosciences (Basking Ridge, NJ) provided pegnivacogin and its component moieties, the 31 nucleotide un-PEGylated RNA aptamer (RB005, as noted in Fig 1, B) and a hexylamino 40-kDa branched mPEG used to produce pegnivacogin (mPEG2-NHS, Nektar, San Francisco, Calif), referred to in Fig 1 as HEXPEG. Pegloticase (PEGylated recombinant porcine urate oxidase) was obtained from Savient Pharmaceuticals (Bridgewater, NJ). The 10-kDa mPEG-NPC (10 kDa p-nitrophenylcarbonate-activated monomethoxyPEG), the activated mPEG reagent used in the manufacture of pegloticase, was obtained from NOF (Tokyo, Japan). PEGylated bovine adenosine deaminase (Adagen) was obtained from Enzon Pharmaceuticals (Piscataway, NJ). Bovine spleen adenosine deaminase and 10-kDa PEG-diol were obtained from Sigma (St Louis, Mo). A protocol for studying anti-PEG antibodies in stored, coded, and deidentified samples from RADAR participants was granted an exemption from review by the Duke University Institutional Review Board on the basis that the proposed studies met “the definition of research not involving human subjects as described in 45 CFR 46.102(f), 21 CFR 56.102(e) and 21 CFR 812.3(p) and satisfies the Privacy Rule as described in 45CFR164.514.” The studies described in the present publication were initiated in December 2010 and completed in May 2011, although additional studies of some stored samples were performed more recently in preparing this report. An initial set of 98 deidentified and coded serum and plasma samples from 31 RADAR patients was screened at 1:21 dilution in 2 ELISAs, one to detect IgG binding to pegloticase and the second to detect binding to mPEG, using the manual procedure described previously.E1Hershfield M.S. Ganson N.J. Kelly S.J. Scarlett E.L. Jaggers D.A. Sundy J.S. Induced and pre-existing anti-polyethylene glycol antibody in a trial of every 3-week dosing of pegloticase for refractory gout, including in organ transplant recipients.Arthritis Res Ther. 2014; 16: R63Crossref PubMed Scopus (165) Google Scholar However, in the second ELISA, HEXPEG, the 40-kDa branched mPEG reagent used in producing pegnivacogin, replaced the linear 10-kDa mPEG-NPC reagent used in the manufacture of pegloticase. Positive samples were retested for confirmation, and specificity for PEG was further tested by direct and competition ELISAs to assess antibody binding to 10-kDa mPEG-NPC, pegnivacogin, RB005 (un-PEGylated aptamer), and PEGylated and un-PEGylated bovine adenosine deaminase. These assays were performed essentially as previously described.E1Hershfield M.S. Ganson N.J. Kelly S.J. Scarlett E.L. Jaggers D.A. Sundy J.S. Induced and pre-existing anti-polyethylene glycol antibody in a trial of every 3-week dosing of pegloticase for refractory gout, including in organ transplant recipients.Arthritis Res Ther. 2014; 16: R63Crossref PubMed Scopus (165) Google Scholar In the competition ELISA, duplicate aliquots of samples (at final dilution 1:21) were preincubated with 0 to 20 μg of a competing antigen. The mixture was then carried through the antipegloticase ELISA. A significant decrease in A405 (compared with no-competitor control) indicated specificity for the competing antigen. Reference standard and positive quality control (QC) used in ELISAs to detect anti-PEG antibodies were prepared from immune plasma of different individuals who had developed IgG antibodies with specificity toward PEG after receiving pegloticase in a phase 1 clinical trial.E2Sundy J.S. Ganson N.J. Kelly S.J. Scarlett E. Rehrig C.D. Huang W. et al.Pharmacokinetics and pharmacodynamics of intravenous PEGylated recombinant mammalian urate oxidase in patients with refractory gout.Arthritis Rheum. 2007; 56: 1021-1028Crossref PubMed Scopus (177) Google Scholar Negative QC consisted of pooled nonimmune plasma from 3 individuals in the same trial. Fig E1 illustrates the ability of these positive QC and reference standards to recognize molecular species relevant to the RADAR trial. Following the initial 98-sample screen, further testing for anti-PEG antibody was carried out by the automated competition ELISA previously described,E1Hershfield M.S. Ganson N.J. Kelly S.J. Scarlett E.L. Jaggers D.A. Sundy J.S. Induced and pre-existing anti-polyethylene glycol antibody in a trial of every 3-week dosing of pegloticase for refractory gout, including in organ transplant recipients.Arthritis Res Ther. 2014; 16: R63Crossref PubMed Scopus (165) Google Scholar in which pegloticase was the plate coating and 20 μg of 10-kDa PEG-diol was the competitor. The cutoff points for the automated procedure were established by a statistical analysis of results obtained with 51 unique samples chosen randomly by the sponsor from among pretreatment samples from 328 patients who were not tested in the initial manual ELISA screen. From this analysis, the cutoff points established for the automated antipegloticase ELISA and competition steps were A405 values of 0.15% or more and 32% or more inhibition, respectively. Anti-PEG antibody titer was estimated by end-point dilution in the antipegloticase ELISA by analyzing duplicate serial 2-fold dilutions from 1:10 to 1:2560. IgM and IgG isotypes were determined using μ- and γ-chain–specific secondary antibodies conjugated with alkaline phosphatase, and IgG subclass was determined with biotinylated mouse antihuman IgG1-, IgG2-, IgG3-, and IgG4-specific antibodies (all from Sigma). Biotinylated antibodies were detected with the Vectastain ABC Kit AK-5000 (Vector Laboratories, Burlingame, Calif). Fig E2 shows results of the determinations of IgM and IgG isotype and IgG subclass. REG1-CLIN211a was conducted as a follow-up to REG1-CLIN211 in the 3 subjects who experienced severe, acute allergic reactions following the administration of pegnivacogin. Of the potential participants, subjects 406-003 and 418-008 agreed to participate in this study, while the third subject, 602-004, was not contacted because of refusal of the investigative site to participate. Following approval of the protocol by local ethics committee and Regado, both subjects completed informed consent and had all assessments conducted during a single visit. The visit for each subject occurred on June 28, 2011, approximately 7 months and 8 months following dosing in the RADAR study. The following assessments were included in this study: questionnaires for allergy history, family history, complete medical history and exposure (environmental and ingestion) history, chemistry, hematology and urinalysis panels, hepatitis screening, anti-PEG antibody determination, and HLA typing. No findings common to both were identified with respect to exposure, or specific allergy or medical history, except that both had experienced drug allergies with respiratory and cutaneous manifestations before the RADAR trial. Unfortunately, the frequency of drug allergies among other RADAR trial participants is not known. Both subjects remained positive for anti-PEG antibodies at follow-up, although antibody levels were lower than at dosing with pegnivacogin. Thus, when analyzed simultaneously by the automated method, A405 for pretreatment versus posttreatment samples were, respectively, 0.98 versus 0.37 for patient 406-003 and 1.83 versus 0.30 for patient 418-008. All terms defined as anaphylaxis by the National Institute of Allergy and A. R. B.A. et on the definition and management of Allergy Immunol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar and by the of on drug allergy were within the RADAR database from each individual clinical study All terms that met these criteria were then further and included was a relationship to REG1 the or those for a and they were with the properties of pegnivacogin or The of the 3 allergic-like within 5 to 25 minutes after coupled with the that plasma of pegnivacogin within 1 to minutes after intravenous led to that relevant reactions within the first after pegnivacogin on these a cutoff of 2 from administration was used to and further allergic events to pegnivacogin. the 3 SARs identified during the review of the RADAR clinical data and data from phase 1 studies did not a of hypersensitivity events associated with pegnivacogin. Thus, the estimated rate for serious allergic-like reactions to pegnivacogin in RADAR and clinical trials was with criteria for The samples tested consisted of samples from each point the study including the active the drug product and and the and clinical from at which the serious allergic-like reactions control samples analyzed for included an used in studies and 5 of pegnivacogin clinical trials testing was performed on all included to and to molecular to product and and studies of both of used in the RADAR trial provided and as well as and results from that the SARs. testing on and selected control samples included to potential or that not be observed by the and to for molecular that not be observed by The potential for pegnivacogin to was by and on and All were conducted All analyzed met and the additional testing did not or evidence of product This was a study conducted at its with conducted by the were with for dosing and and were also prepared with for of respiratory and group of was a single dose of the on 1, by a single dose of the pegnivacogin on and then a single dose of the pegnivacogin on All doses were via the at a dose of pegnivacogin was a recently of all testing pegnivacogin was an of that had with a of in PEG compared with used in the RADAR and and and the and were from at 2 before dosing at via and data in for for and were conducted at for all were conducted before at approximately 5 minutes and and after of the were and on the before each dose samples for clinical were and on and samples for of histamine level, complement factors and and were before and 5 minutes and on 1, and and were conducted by the of at the National its standard factors were in ELISA in a standard and by values as less than are as for of means and All other were performed at its standard No adverse to the administration of or pegnivacogin were observed in this study. A of data via and is presented in Fig No compared with control were observed in response to with pegnivacogin. (Fig E3, in the pegnivacogin group are to 3 of having lower before dosing on are not to the in the at approximately 5 minutes and are to sample and histamine are provided in Fig No compared with control were observed in response to with pegnivacogin. of the and were essentially at the for all points and In no the range for individual in or control are provided in Fig As with and histamine no compared with control were observed in response to with pegnivacogin, and no individual the range in or control of anti-PEG antibody isotype and IgG subclass. The samples tested are from the 3 patients who experienced first-exposure serious allergic-like reactions to pegnivacogin in response of to pegnivacogin Heart in the and are sample for all and histamine response of to pegnivacogin D, presented as for all response of to pegnivacogin D, presented as for all