Oracle or false prophet? Can we predict AAV efficacy based on preexisting antibody titers? (2019)

Recombinant adeno‐associated viral vectors (AAVs) have emerged as a vector of choice in gene therapy for hemophilia, and over the past few years there has been a proliferation of trials using AAVs introduced through the circulation to target the liver.1.Manno C.S. Pierce G.F. Arruda V.R. Glader B. Ragni M. Rasko J.J. et al.Successful transduction of liver in hemophilia by AAV‐factor IX and limitations imposed by the host immune response.Nat Med. 2006; 12: 342-7Crossref PubMed Scopus (1567) Google Scholar, 2.Nathwani A.C. Tuddenham E.G. Rangarajan S. Rosales C. McIntosh J. Linch D.C. et al.Adenovirus‐associated virus vector‐mediated gene transfer in hemophilia B.N Engl J Med. 2011; 365: 2357-65Crossref PubMed Scopus (1353) Google Scholar, 3.George L.A. Sullivan S.K. Giermasz A. Rasko J.E.J. Samelson‐Jones B.J. Ducore J. et al.Hemophilia B gene therapy with a high‐specific‐activity factor IX variant.N Engl J Med. 2017; 377: 2215-27Crossref PubMed Scopus (439) Google Scholar, 4.Rangarajan S. Walsh L. Lester W. Perry D. Madan B. Laffan M. et al.AAV5–factor VIII gene transfer in severe hemophilia A.N Engl J Med. 2017; 377: 2519-30Crossref PubMed Scopus (431) Google Scholar, 5.Miesbach W. Meijer K. Coppens M. Kampmann P. Klamroth R. Schutgens R. et al.Gene therapy with adeno‐associated virus vector 5‐human factor IX in adults with hemophilia B.Blood. 2018; 131: 1022-31Crossref PubMed Scopus (189) Google Scholar The clinical efficacy of biologics engineered from viruses, however, may be affected by the recipient's prior exposure to the wild‐type virus from which the recombinant virion is derived, and both B and T cell responses may present challenges.6.Mingozzi F. High K.A. Overcoming the host immune response to adeno‐associated virus gene delivery vectors: the race between clearance, tolerance, neutralization, and escape.Annu Rev Virol. 2017; 4: 511-34Crossref PubMed Scopus (118) Google Scholar Data from studies of diverse human populations document that a substantial proportion of adults carry circulating antibodies to AAV,7.Calcedo R. Vandenberghe L.H. Gao G. Lin J. Wilson J.M. Worldwide epidemiology of neutralizing antibodies to adeno‐associated viruses.J Infect Dis. 2009; 199: 381-90Crossref PubMed Scopus (521) Google Scholar, 8.Boutin S. Monteilhet V. Veron P. Leborgne C. Benveniste O. Montus M.F. et al.Prevalence of serum IgG and neutralizing factors against adeno‐associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors.Hum Gene Ther. 2010; 21: 704-12Crossref PubMed Scopus (626) Google Scholar and that the proportion of the population that carries these increases with age.9.Hui D.J. Edmonson S.C. Podsakoff G.M. Pien G.C. Ivanciu L. Camire R.M. et al.AAV capsid CD8+ T‐cell epitopes are highly conserved across AAV serotypes.Mol Ther Methods Clin Dev. 2015; 2: 15029Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar A role for the effects of preexisting antibody titers on clinical efficacy with AAV vectors was surmised early on, and most trials tested these as part of the clinical protocol. The pattern that emerged was that trials that targeted solid organs by direct injection (eg, intramuscular) or that delivered vector to compartments with limited access to circulating antibodies, such as the central nervous system (including the subretinal space), showed effective transduction even in the presence of detectable antibody titers,10.Manno C.S. Chew A.J. Hutchison S. Larson P.J. Herzog R.W. Arruda V.R. et al.AAV‐mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B.Blood. 2003; 101: 2963-72Crossref PubMed Scopus (598) Google Scholar, 11.Bennett J. Wellman J. Marshall K.A. McCague S. Ashtari M. DiStefano‐Pappas J. et al.Safety and durability of effect of contralateral‐eye administration of AAV2 gene therapy in patients with childhood‐onset blindness caused by RPE65 mutations: a follow‐on phase 1 trial.Lancet. 2016; 388: 661-72Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar but that delivery of vector through the circulation was sensitive to even low levels of neutralizing antibodies.1.Manno C.S. Pierce G.F. Arruda V.R. Glader B. Ragni M. Rasko J.J. et al.Successful transduction of liver in hemophilia by AAV‐factor IX and limitations imposed by the host immune response.Nat Med. 2006; 12: 342-7Crossref PubMed Scopus (1567) Google Scholar Subsequent studies in animal models further delineated this observation. In mice, the use of human intravenous immunoglobulin to model preexisting neutralizing antibodies to AAV suggested that this in vivo model may be more sensitive than the in vitro cell‐based assays,12.Scallan C.D. Jiang H. Liu T. Patarroyo‐White S. Sommer J.M. Zhou S. et al.Human immunoglobulin inhibits liver transduction by AAV vectors at low AAV2 neutralizing titers in SCID mice.Blood. 2006; 107: 1810-7Crossref PubMed Scopus (198) Google Scholar and studies in non‐human primates, which are natural hosts for AAV and thus have naturally occurring antibodies, documented that even low‐titer neutralizing antibodies (determined in a cell‐based in vitro assay) could fully block liver transduction when vector was infused intravenously.13.Jiang H. Lillicrap D. Patarroyo‐White S. Liu T. Qian X. Scallan C.D. et al.Multiyear therapeutic benefit of AAV serotypes 2, 6, and 8 delivering factor VIII to hemophilia A mice and dogs.Blood. 2006; 108: 107-15Crossref PubMed Scopus (164) Google Scholar Complicating the straightforward extrapolation of these findings to the clinical arena is the number of different AAV vectors being utilized in clinical studies; conservation of the capsid sequences at the amino acid level varies from as low as 51% up to nearly 100%, and there is some (mostly modest) variation in prevalence of neutralizing antibodies in the population depending on capsid identity. In the paper by Stanford et al14.Stanford S. Pink R. Creagh D. Clark A. Lowe G. Curry N. et al.Adenovirus‐associated antibodies in UK cohort of hemophilia patients: a seroprevalence study of the presence of adenovirus‐associated virus vector‐serotypes AAV5 and AAV8 neutralizing activity and antibodies in patients with hemophilia A.Res Pract Thromb Haemost. 2019; 3: 261-7Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar recently published in Research and Practice in Thrombosis and Haemostasis, the authors used two different assays to assess preexisting immunity to two different AAV serotypes in 100 hemophilia A patients in the UK. They reported that as many as 30%‐40% of these subjects were positive for either antibodies that bind to AAV or an inhibitor of transduction (measured using a cell‐based transduction inhibition titer assay) in one or both assays. Beyond the value of understanding seroprevalence against two commonly used capsids in a specific population cohort, the report by Stanford and colleagues highlights two important questions that remain for the most part unanswered thus far.14.Stanford S. Pink R. Creagh D. Clark A. Lowe G. Curry N. et al.Adenovirus‐associated antibodies in UK cohort of hemophilia patients: a seroprevalence study of the presence of adenovirus‐associated virus vector‐serotypes AAV5 and AAV8 neutralizing activity and antibodies in patients with hemophilia A.Res Pract Thromb Haemost. 2019; 3: 261-7Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar First, which one of the several experimental assays can predict more accurately how the presence of circulating anti‐AAV antibodies may impact in vivo transduction? And second, if such a universally accepted assay existed, should the field work together in an effort to standardize it for different capsids? On the first question, the authors suggest that, while the transduction inhibition assay is considered a standard, a positive signal in either test (binding or neutralizing activity) should trigger exclusion from trials where AAVs are delivered systemically. This notion, perhaps prudent in principle, has been recently challenged by Mingozzi and colleagues on the grounds that binding antibodies may in fact increase capsid internalization and transgene expression and thus NAb assays are better predictors of the outcome of gene transfer.15.Fitzpatrick Z. Leborgne C. Barbon E. Masat E. Ronzitti G. van Wittenberghe L. et al.Influence of pre‐existing anti‐capsid neutralizing and binding antibodies on AAV vector transduction.Mol Ther Methods Clin Dev. 2018; 9: 119-29Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar Others have suggested that in vivo neutralization assays, in which Nabs are passively transferred to mice following human serum injection to the animals, are more sensitive than those neutralization assays performed in vitro and thus better suited for inclusion/exclusion criteria.16.Wang M. Crosby A. Hastie E. Samulski J.J. McPhee S. Joshua G. et al.Prediction of adeno‐associated virus neutralizing antibody activity for clinical application.Gene Ther. 2015; 22: 984-92Crossref PubMed Scopus (37) Google Scholar However, neutralizing assays (both in vivo and in vitro) rely on the ability of a reporter vector to transduce the target cells and mediate quantifiable expression levels that decrease proportionally to the amount of circulating transduction inhibitors. This poses a number of significant limitations to their standardization, as transduction efficiency is highly serotype‐dependent and, in general, the sensitivity of the assay decreases as the AAV dose increases, compromising the comparison of NAb titers between serotypes with distinct transduction efficiencies. As an example, the assay used by the authors to measure anti‐AAV5 NAbs requires an MOI of 25 000, supplemented with etoposide, an agent that promotes transduction,17.Falese L. Sandza K. Yates B. Triffault S. Gangar S. Long B. et al.Strategy to detect pre‐existing immunity to AAV gene therapy.Gene Ther. 2017; 24: 768-78Crossref PubMed Scopus (55) Google Scholar whereas the anti‐AAV8 NAb assay uses an MOI of 200 with no requirement for agents like etoposide.18.Meliani A. Leborgne C. Triffault S. Jeanson‐Leh L. Veron P. Mingozzi F. Determination of anti‐adeno‐associated virus vector neutralizing antibody titer with an in vitro reporter system.Hum Gene Ther Methods. 2015; 26: 45-53Crossref PubMed Scopus (63) Google Scholar Other characteristics that impact NAb titers when evaluated using in vitro assays include the amount of serum used, the cell number on the plate and the reporter transgene.16.Wang M. Crosby A. Hastie E. Samulski J.J. McPhee S. Joshua G. et al.Prediction of adeno‐associated virus neutralizing antibody activity for clinical application.Gene Ther. 2015; 22: 984-92Crossref PubMed Scopus (37) Google Scholar In this regard, use of assays that do not rely on transduction performance, such as total antibody assays or the assay recently developed by Guo and colleagues, which relies on quantification of AAV binding to the target cells in vitro using a qPCR assay.19.Guo P. Zhang J. Chrzanowski M. Huang J. Chew H. Firrman J.A. et al.Rapid AAV‐neutralizing antibody determination with a cell‐binding assay.Mol Ther Methods Clin Dev. 2019; 13: 40-6Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar Further compounding the intrinsic complexity of each assay are the differences in the AAV investigational products themselves, in terms of infectivity titers and content of empty capsids, both of which influence transduction performance and thus may affect the NAb titer. Empty capsids, which contain the capsid but lack any packaged DNA, are a byproduct of all current manufacturing processes, and have the advantage of functioning to bind and neutralize circulating antibodies to AAV.20.Mingozzi F. Anguela X.M. Pavani G. Chen Y. Davidson R.J. Hui D.J. et al.Overcoming preexisting humoral immunity to AAV using capsid decoys.Sci Transl Med. 2013; 5: 194ra92Crossref PubMed Scopus (223) Google Scholar In in vivo studies in mice and non‐human primates (NHP), the presence of empty capsids has been demonstrated to result in more efficient transduction particularly at lower vector doses, by acting as a decoy to bind neutralizing antibodies.20.Mingozzi F. Anguela X.M. Pavani G. Chen Y. Davidson R.J. Hui D.J. et al.Overcoming preexisting humoral immunity to AAV using capsid decoys.Sci Transl Med. 2013; 5: 194ra92Crossref PubMed Scopus (223) Google Scholar Vectors manufactured in insect cells by introducing the DNA sequences using insect cell (baculo) viruses have demonstrated altered capsid composition and lower biological potency,21.Kondratov O. Marsic D. Crosson S.M. Mendez‐Gomez H.R. Moskalenko O. Mietzsch M. et al.Direct head‐to‐head evaluation of recombinant adeno‐associated viral vectors manufactured in human versus insect cells.Mol Ther. 2017; 25: 2661-75Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar typically owing to reduced content of one of the capsid proteins (VP1), which leads to the formation of defective particles with reduced transduction efficiency. These may function in a manner similar to empty capsids, in that they may bind anti‐AAV antibodies without driving transgene expression. These substantial differences in the AAV product from one manufacturer to another further complicate efforts to develop a standardized assay. As Stanford et al14.Stanford S. Pink R. Creagh D. Clark A. Lowe G. Curry N. et al.Adenovirus‐associated antibodies in UK cohort of hemophilia patients: a seroprevalence study of the presence of adenovirus‐associated virus vector‐serotypes AAV5 and AAV8 neutralizing activity and antibodies in patients with hemophilia A.Res Pract Thromb Haemost. 2019; 3: 261-7Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar note, the purpose of these assays is to identify accurately those potential trial participants who can be expected to exhibit some level of transduction following intravenous infusion of vector. Thus, it is difficult to judge which assays are of greatest utility without an accompanying clinical dataset. One can debate about best characteristics of the assay, ie, is it better to have a wider definition of eligible (as long as all participants exhibit an adequate level of expression), which may lead to greater variability in clinical outcomes, or is it better to set a tighter range, resulting in fewer eligible participants but greater uniformity of results at a given vector dose? Should we adjust vector doses based on pretreatment antibody titers? Differences among capsids and in final product characteristics make it difficult to extrapolate findings from one product to the next. It is safe to say that we likely have more to learn regarding this critical determinant of clinical success with AAV vectors. Dr. Anguela reports employment from Spark Therapeutics during the writing of the manuscript. In addition, Dr. Anguela is an inventor in the following patent applications pending to Spark Therapeutics: WO2013158879A1, US20140336245A1, US20150023924A1, US20160375110A1, and WO2017075619A1. Dr. High reports personal fees and other from Spark Therapeutics, outside the submitted work. Dr. High and Dr. Anguela jointly outlined the editorial, researched it, drafted it, and revised it. ErratumResearch and Practice in Thrombosis and HaemostasisVol. 3Issue 4PreviewThis erratum corrects “Oracle or false prophet? Can we predict AAV efficacy based on preexisting antibody titers?” by Xavier M. Anguela and Katherine A. High, which published in Research and Practice in Thrombosis and Haemostasis Vol. 3, No. 2, pp. 149‐151, 2019. https://doi.org/10.1002/rth2.12190 . Full-Text PDF Open Access