Abstract
Neutralizing antibodies to adeno-associated virus (AAV) vectors are highly prevalent in humans1,2, and block liver transduction3,4,5 and vector readministration6; thus, they represent a major limitation to in vivo gene therapy. Strategies aimed at overcoming anti-AAV antibodies are being studied7, which often involve immunosuppression and are not efficient in removing pre-existing antibodies. Imlifidase (IdeS) is an endopeptidase able to degrade circulating IgG that is currently being tested in transplant patients8. Here, we studied if IdeS could eliminate anti-AAV antibodies in the context of gene therapy. We showed efficient cleavage of pooled human IgG (intravenous Ig) in vitro upon endopeptidase treatment. In mice passively immunized with intravenous Ig, IdeS administration decreased anti-AAV antibodies and enabled efficient liver gene transfer. The approach was scaled up to nonhuman primates, a natural host for wild-type AAV. IdeS treatment before AAV vector infusion was safe and resulted in enhanced liver transduction, even in the setting of vector readministration. Finally, IdeS reduced anti-AAV antibody levels from human plasma samples in vitro, including plasma from prospective gene therapy trial participants. These results provide a potential solution to overcome pre-existing antibodies to AAV-based gene therapy.
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Data availability
All requests for raw and analyzed data and materials will be promptly reviewed by Généthon, Institut National de la Santé et de la Recherche Médicale or Spark Therapeutics to verify if the request is subject to any intellectual property or confidentiality obligations. Patient-related data not included in the manuscript were generated as part of a clinical trial and are not available because of patient confidentiality issues. Any data and materials that can be shared will be released via a material transfer agreement established with the relevant institution that generated the data or owns the materials. Source data for Figs. 1–4 and Extended Data Figs. 1–7 are included with this paper.
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Acknowledgements
We thank the staff of the Nantes-Atlantic National College of Veterinary Medicine, Food Science and Engineering and Virscio staff for their help in designing and performing the NHP studies. We also thank the Spark Therapeutics vector production, bioanalytical, immunology, liver, sample management and laboratory information management teams for their contribution to the NHP studies. The work was supported by the Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Sorbonne Université.
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C.L., E.B., J.M.A., S.D., H.H., F.C., S.M., D.L., J.S., K.H., L.V.W., B.M., A.M., A.F., V.D., D.M.C. and H.B. performed the experiments. C.L., G.R., S.M.A., S.L.-D. and F.M. analyzed the results and wrote the manuscript. X.M.A., S.M.A., S.L.-D. and F.M. conceived and supervised the studies.
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J.M.A., H.H., S.M., D.L., J.S., K.H., D.M.C., H.B., X.M.A., S.M.A. and F.M. are employees of Spark Therapeutics, a Roche company. F.M., S.L.D., S.M.A. and C.L. are inventors on a patent related to this work. Patent applicants: Institut National de la Santé et de la Recherche Médicale; Généthon; Sorbonne Université; Université Paris Descartes; Université Paris Diderot; Spark Therapeutics. Inventors: S. Lacroix-Desmazes, F. Mingozzi, C. Leborgne, J. D. Dimitrov, S. M. Armour. Application no. PCT/EP2019/069280 (application request filed). Specific aspects of the manuscript covered in the patent application include use of IdeS to decrease pre-existing antibody immunity to AAV and allow for vector readministration.
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Extended data
Extended Data Fig. 1 IdeS degrades anti-AAV antibodies and allows successful liver transduction in mice passively immunized with IVIg.
a, IVIg was incubated in vitro with saline (PBS), commercially sourced IdeS (IdeS-C), or lab-made IdeS (IdeS) for 24 hours prior to measurement of anti-AAV8 NAb titers (n = 1 per condition tested in triplicate, one representative out of two independent experiments shown). b, effect of IdeS on AAV8 vector transduction efficiency of HEK293 cells. An AAV8-Luc vector was incubated for 24 hours at 37 °C with PBS (- IdeS) or with increasing amounts of IdeS (+ IdeS). Control, AAV8-Luc not incubated at 37 °C. Relative Light Units (RLU) measured in duplicate at increasing multiplicity of infection (MOI). n = 1 per condition tested in duplicate, one representative out of two independent experiments shown. c, Plasma concentration of IdeS over time in non-human primates (n = 2 animals, duplicate independent measurements of each sample). The inset shows the estimation of the half-life of the enzyme. d, vector genome copy number (VGNC) in passively immunized C57BL/6 mice injected with an AAV8-GLuc vector (n = 6 mice per group, one representative out of two independent experiments shown). e, f, Effect of IdeS on anti-AAV8 IgG e, and NAb titers f, measured 24 hours after treatment in passively immunized HB mice (n = 4 mice per group, data derived from one experiment). g, vector genome copy number (VGNC) in HB mice injected with an AAV8-hFIX vector. All data are shown as mean ± s.d. Statistical analyses were performed by d, one-way ANOVA with Tukey’s multiple comparisons test; e, g, one-way ANOVA with Tukey’s multiple comparisons test; f, one-way ANOVA, non-parametric with Dunn’s multiple comparisons test.
Extended Data Fig. 2 IdeS treatment enhances transduction in NHPs in the presence of pre-existing anti-AAV Nabs.
a, Follow-up of digestion of circulating IgG in NHP6 (IdeS treated) and NHP2 (control) assessed by Western blot. One representative out of two independent experiments shown. b, Effect of IdeS administration on anti-AAV8 IgG and c, NAbs (n = 1 monkey per treatment group, triplicate independent measurements of each sample). The horizontal dotted lines in panels b, and c, represent the levels of anti-AAV8 IgG and NAbs in the control animal NHP2. d, hFIX transgene levels in plasma over time (n = 1 monkey per treatment group, duplicate independent measurements of each sample). e, vector genome copy number (VGCN) in liver at sacrifice (n = 1 monkey per treatment group, average VGCN in each of the four main liver sections). f, Anti-AAV8 IgG and g, anti-AAV8 NAbs at baseline and post AAV8-hFIX vector administration (n = 1 monkey per treatment group, duplicate independent measurements of each sample). All data are shown as mean ± s.d. Statistical analyses were performed by b, c, repeated measures one-way ANOVA with Dunnett’s multiple comparisons test (vs. Day-2); d, f, g, repeated measures two-way ANOVA with Sidak’s multiple comparisons test (vs. NHP2); e, two-tailed unpaired t-test.
Extended Data Fig. 3 IdeS treatment allows for AAV8 vector readministration in NHPs.
Two male Cynomolgus monkeys with an anti-AAV8 NAb titer of 1:31.6 received an AAV8-hFIX vector (5 × 1012 vg kg−1) at day 0, with (NHP4, treated) or without (NHP1, control) an intravenous infusion of IdeS (500 µg kg−1) at day -1. At day 82 and 83, both NHPs received IdeS twice (500 µg kg−1 per infusion) followed by a second administration of the AAV8-hFIX vector (5 × 1012 vg kg−1) at day 84. b, Anti-AAV8 IgG measured over time in NHP4 after the first IdeS administration (n = 1 monkey per treatment group, triplicate independent measurements of each sample). The horizontal dotted line represents the levels of anti-AAV8 IgG in the control animal NHP1 (see Supplementary Table 1). c, hFIX in plasma and c, anti-AAV8 IgG at baseline and after the first vector administration (n = 1 monkey per treatment group, duplicate independent measurements of each sample). d, hFIX in plasma following AAV8-hFIX readministration (n = 1 monkey per treatment group, duplicate independent measurements of each sample). e, Western blot follow-up of total IgG cleavage between day 82 and 105 after the second IdeS treatment in NHP1 and NHP4. One representative out of two independent experiments shown. f, Anti-AAV8 IgG and g, NAbs measured over time in NHP1 and NHP4 after the second IdeS administration (n = 1 monkey per treatment group, duplicate independent measurements of each sample). All data are shown as mean ± s.d. Statistical analyses were performed by b, repeated measures one-way ANOVA with Dunnett’s multiple comparisons test (vs. Day -1); c,d, repeated measures two-way ANOVA with Sidak’s multiple comparisons test (vs. NHP1); f, repeated measures one-way ANOVA with Dunnett’s multiple comparisons test (vs. day -82), g, repeated measures, non-parametric one-way ANOVA with Dunn’s multiple comparisons test (vs. day -82).
Extended Data Fig. 4 IdeS treatment allows for AAV8 vector readministration in NHPs.
a, hFIX in plasma following AAV8-hFIX readministration. b, Anti-hFIX IgG measured by ELISA. c, vector genome copy number (VGCN) in liver at sacrifice (n = 1 monkey per treatment group, average VGCN in each of the four main liver sections). d, Anti-AAV8 IgG and e, anti-AAV8 NAbs pre- and post- AAV8-hFIX vector readministration. f, Anti-AAV8 IgM measured over time in NHP1 and NHP4 after the second IdeS administration. a, b, d–f, n = 1 monkey per treatment group, duplicate independent measurements of each sample. All data are shown as mean ± s.d. Statistical analyses were performed by a, b, d–f repeated measures two-way ANOVA with Sidak’s multiple comparisons test (vs. NHP1); c, two-tailed unpaired t-test.
Extended Data Fig. 5 Follow up of antibody responses following vector readministration.
a, Change in anti-AAV-LK03 NAb titers following IdeS administration. b,c, kinetics of plasma anti-AAV-LK03 IgM titers in animals from the PBS (b) and IdeS (c) treatment groups. d,e, kinetics of anti-AAV-LK03 IgG titers in animals from the PBS (d) and IdeS (e) treatment groups. f,g, Kinetics of anti-hFVIII IgG titers in animals from the PBS (f) and IdeS (g) treatment groups. Individual animals are shown in each graph.
Extended Data Fig. 6 Effect of IdeS on total IgG and anti-IdeS IgG in NHPs.
A total of 6 NHPs were included in this evaluation. NHP2 and NHP3 received a course of IdeS at day 37 post vector delivery a, total IgG measured before and, two and fifteen days after IdeS injections (n = 6 animals, in the box plot, the box extends from the 25th to 75th percentiles, whiskers show minima and maxima and median is shown in the box). b, development of anti-IdeS antibodies in NHP, 15 days after IdeS injections. c, prevalence of anti-IdeS antibodies in a cohort of 52 human serum samples. Samples were divided in [anti-IdeS]<5 μg/ml (seronegative), [anti-IdeS]<50 μg/ml (seropositive), [anti-IdeS]>50μg/ml (high seropositive). Statistical analyses were performed by a, one-way ANOVA with Dunnett’s multiple comparisons test (vs. Day 0); b, two-tailed, paired Wilcoxon test.
Extended Data Fig. 7 Effect of anti-IdeS antibodies on IgG digestion.
Serum samples from 2 Cynomolgus monkeys NHP1 (a, b) and NHP6 (c, d) collected at day -7 prior to IdeS treatment and at 21 days after were incubated with IdeS for up to 24 hours (one representative out of two independent experiments shown). a.,Western blot showing degradation of IgG over time and b, quantification of the Fc fragment of IgG in NHP1. c, Western blot showing degradation of IgG over time and d, quantification of the Fc fragment of IgG in NHP6.
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Leborgne, C., Barbon, E., Alexander, J.M. et al. IgG-cleaving endopeptidase enables in vivo gene therapy in the presence of anti-AAV neutralizing antibodies. Nat Med 26, 1096–1101 (2020). https://doi.org/10.1038/s41591-020-0911-7
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DOI: https://doi.org/10.1038/s41591-020-0911-7
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