Clinical enrollment assay to detect preexisting neutralizing antibodies to AAV6 with demonstrated transgene expression in gene therapy trials

Human serum samples

Individual human serum samples were purchased from BioIVT (Westbury, NY, USA), Discovery Life Sciences (Los Osos, CA, USA), and Golden West Biologicals (Temecula, CA, USA). Serum from hemophilia A and hemophilia B donors were purchased through custom collection from HRF, Inc (Raleigh, NC, USA). Donors varied in ethnicity (Black, White, Hispanic, Asian), ages, and geographical locations in the USA. Serum from hemophilia A and hemophilia B patients were collected from consenting subjects in Sangamo Therapeutics-sponsored clinical trials. The trials were conducted according to the principles of the Declaration of Helsinki. All subjects provided written informed consent prior to inclusion in the study. The Institutional Review Board (IRB)/Independent Ethics Committee for each site and Central IRB approved the protocols. An additional 49 serum samples from hemophilia B patients in the United Kingdom (UK) were collected during a seroprevalence study conducted at the University Hospital Southampton NHS Foundation Trust with informed consent from patients with hemophilia B registered at six UK Hemophilia Comprehensive Care Centers (S. Boyce, I. James, S. Rangarajan, et al. manuscript in preparation). The study was reviewed and supported by the Proportionate Review Sub-Committee of the East Midlands – Derby Research Ethics Committee, study number 18/EM/0313.

AAV6 luciferase

The AAV6 vector construct containing a Photinus pyralis firefly luciferase reporter gene under control of the cytomegalovirus (CMV) promoter was produced in insect Sf9 cells at Virovek (Hayward, CA, USA) and formulated in buffer consisting of phosphate-buffered saline and 0.001% Pluronic F-68. AAV titers were determined by quantitative polymerase chain reaction (qPCR). Full and empty capsid ratio was determined for applicable lot using high-performance liquid chromatography [12].

Quality controls

Negative control serum (NCS) was generated by pooling serum from individuals with tested luciferase responses comparable to the measured luciferase responses in the cell culture media (normalized ratio ranged from 0.9 to 1.3). Pooled positive human serum from donors and a mouse monoclonal anti-AAV6 antibody (ADK6) from Progen (Wayne, PA, USA) were both used to generate quality control (QC) samples for the AAV6 transduction inhibition assay. For controls made using pooled positive human serum, QC1, QC2, QC3 were prepared by spiking the pooled positive control serum into the pooled NCS at different ratios. For controls made using ADK6, QC1, QC2, QC3 were prepared by spiking ADK6 at 3.5, 1.5, and 0.9 µg/mL, respectively, into the pooled NCS. These controls were aliquoted into single-use vials and stored at −65 °C to −90 °C. Three determinations of each control (in duplicate) are assessed on every plate during sample testing. All analyses were performed using data normalized to NCS and presented as normalized response (NR). QC1, QC2, and QC3 were designed to have approximate NR of < 0.1, 0.25, 0.45, respectively with QC1 and QC2 below the clinical cutoff of 0.34 and QC3 above the clinical cutoff.

AAV6 TI assay

The cell-based transduction inhibition assay utilized human U-87 MG (glioma cell line) HTB-14 (ATCC, Manassas, VA, USA) and an AAV6-CMV-luciferase vector construct as reporter. U-87 MG cells from the working cell bank were cultured in U-87 MG growth media containing minimum essential media (MEM) (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (HyClone, Logan, UT, USA), 1% Sodium Pyruvate (Gibco, Grand Island, NY, USA), and 1% Penicillin-Streptomycin (Lonza, Morristown, NJ, USA). Viable U-87 MG cells were seeded at 20,000 cells per well (100 µL per well) in white opaque 96-well plates. On the following day, QCs (positive and negative controls) and samples were initially diluted fivefold in U-87 MG dilution media (U-87 MG growth media with 1% bovine serum albumin), followed by another twofold dilution of U-87 MG dilution media containing AAV6-CMV-luciferase to yield a 10-fold dilution prior to incubation with cells. QC samples were analyzed at minimum required dilution of 1:10 (MRD 10) and clinical samples were analyzed at MRD 10 followed by four serial 2.5-fold dilutions, at 10% final serum prior to incubation with cells. Data from MRD 10 were used for reporting and the remaining dilutions were for information purposes only. Multiplicity of infection (MOI) of AAV6-CMV-luciferase used in the assay is lot-specific. Each new lot was bridged prior to use through partial validation including the evaluation of positive control titration curves, QCs precision, and a minimum 30 healthy donors to evaluate concordance in test results. MOI ranged from 4e4 to 1e5 across three lots were tested and bridged. Diluted samples and AAV6-CMV-luciferase (1:1 mix at 75 µL each to reach 150 µL in total volume) were incubated at 37 °C for 30–40 min. Following incubation, all media from the plated U-87 MG cells were removed via aspiration. 50 µL of U-87 MG growth media was added to all wells and 50 µL of AAV6-CMV-luciferase/sample or QCs mix was added to the plate in duplicate, bringing final volume to 100 µL. Plates were incubated for 24 h (±2 h) in a humidified 37 °C incubator, 5% CO2. The following day, plates were removed from the incubator and incubated at room temperature for 10–15 min. 100 µL of One-Glo reagent/buffer mixture (Promega, Sunnyvale, CA, USA) was added to each well, incubated at room temperature for 5–30 min and read via luminescence using BioTek Synergy 2 (Agilent Technologies, Santa Clara, CA, USA) plate reader.

Neutralizing antibodies and non-antibody factors against the viral capsid reduce the ability of AAV6 to transduce the cell and thereby result in lower luciferase expression. On each plate, luminescent signals from test samples are normalized to mean signal of wells transduced with AAV6-CMV-luciferase in the presence of NCS. A reduction in luciferase activity following incubation with serum dilutions indicates the presence of anti-AAV6 neutralizing activity.

AAV6 in vivo passive transfer mouse study

All animal studies were conducted at Pacific BioLabs (Hercules, CA, USA) using 79-week-old-male C57BL/6 mice. The studies were performed in compliance with all applicable sections of the Final Rules of the Animal Welfare Act Regulations (9 CFR 1–3), the Public Health Service Policy on Humane Care and Use of Laboratory Animals, the Guide for the Care and Use of Laboratory Animals, and the guidelines of the Pacific BioLabs Institutional Animal Care and Use Committee (IACUC).

Human serum samples (100 or 200 µL), either a pool or from individual donors, were administered IV to naïve C57BL/6 mice (n = 5–10/group), followed 2 h later by IV delivery of 200 µL AAV6 encoding human Factor 9 (hF9) cDNA (Virovek, Hayward, CA, USA) at a dose level of 6e10 vg/mouse. Preexisting neutralizing activity to AAV6 capsid in the serum is anticipated to impact in vivo transduction of hepatocytes and subsequent expression of the hF9 transgene.

Endpoints in these studies included plasma hFIX levels, which represent successful hepatocyte transduction and transgene expression. hFIX concentration in the plasma was measured by ELISA using VisuLize FIX® antigen kit according to the manufacturer procedure (Affinity Biologicals, Ancaster, ON, Canada).

Cutoff determination

Cutoff determination was performed by B2S Life Sciences (Franklin, IN, USA). All statistical analyses were completed using R (R version 3.3.1, 2016–06–21), JMP (Version 13; SAS Institute, Inc., Cary, NC, USA), and Microsoft Excel 2016. The logarithm base 10 transformed NR values from individual subject samples were used to estimate overall variability and intra-run analytical variability. Statistical methods used for the calculation of cutoff were consistent with procedures recommended by refs. [13] and [14] when applied to immunoassay designs described by ref. [13]. A total of 312 NR values generated from three independent experiments were included in the evaluation and six were identified as analytical outliers using a linear mixed-effects Analysis of Variance (ANOVA) model leaving 306 values for cutoff calculations. The ANOVA model included fixed effects of analyst, gender, and ethnicity and random effects for subject, run within analyst, and residual. Estimates for the parametric and nonparametric cutoff at 0.1%, 1%, and 5% error rates were determined using the log-transformed values. A parametric method with Tukey’s biweight procedure was used to calculate robust estimates of the mean and standard deviation (SD) of all log-transformed ratios [15]. The parametric cutoff calculation with the corresponding error rate was calculated as

$$Parametric\;cutoff = 10^} \right]}$$

where tα, n−1 is the α percentile of the t-distribution with degrees of freedom equal to the number of log-transformed NR values minus 1. The nonparametric cutoff calculation with the corresponding error rate was determined by the calculation of the corresponding empirical percentile for the log-transformed NR values followed by an inverse log transformation.

Cell-based assays tend to have higher variability both due to inherent biological variability and operator variability. A statistical analysis was conducted to determine how best to compare a subject test result to a fixed cutoff value. To statistically capture this variability, the MSR [16,17,18,19] approach was used to calculate the response range for each calculated cutoff, taking into account intra-run variability. To determine the MSR, after the removal of outliers, the SD of the remaining log-transformed values was calculated using restricted maximum likelihood estimates from the mixed-effects model [14]. The intra-run SD was estimated as the square root of the residual variance from the ANOVA model. The overall SD was 0.094. The MSR of 1.27 was calculated using the formula:

$$}}}}}}} = 10^}}}}}}}\;}}}}}}}]}.$$

The estimated response range for the corresponding false error rate was established by multiplying the cutoff estimate by 1.27 to define the upper end of the range, and the lower end of the range was determined by dividing the cutoff estimate by 1.27.

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