Safety Analyses in STRATOS 1 and 2

All safety summaries and ADA analyses in both trials were based on the safety analysis sets, defined as all participants in each trial who received at least one dose of placebo or tralokinumab. In the safety analysis set, participants were assigned to treatment groups based on the actual treatment they received, regardless of which group they were randomized to.

The Medical Dictionary for Regulatory Activities (MedDRA^®) version 20.0 was used to code AEs in both trials. AEs in STRATOS 1 were initially coded in MedDRA^® version 19.1, but were then recoded in MedDRA^® version 20.0 when follow-up data were included.

All AEs were reviewed by study and safety physicians on an ongoing basis. In addition, all safety information was reviewed by an independent Data Safety Monitoring Board.

Pre-dose serum samples for measuring the presence of ADAs were collected in the STRATOS 1 and 2 trials at baseline (before the first administration of tralokinumab) and at Weeks 26, 56 (6–8 weeks after the final tralokinumab dose), and 72 (22–24 weeks after the final tralokinumab dose). The ADA evaluable population included all participants in the safety population who had non–missing baseline ADA results and at least one non–missing post-baseline ADA result.

The presence and titer of ADAs in serum was detected using a Meso Scale Discovery bridging assay (Meso Scale Discovery, Gaithersburg, MD, USA). Briefly, serum samples were acid dissociated to release any ADAs complexed with free drug. Neutralized samples were mixed with biotinylated tralokinumab and Sulfo-tagged tralokinumab, to allow formation of an antibody–complex bridge. Following incubation, samples were added to a streptavidin-coated plate, and unbound material was washed away. Buffer containing tripolyamine was added, and an electric voltage applied to produce a chemiluminescent signal. Positive results were confirmed by competitive binding with excess tralokinumab to assess the specificity of binding.

For ADA titer determination, the assay was performed using serial dilutions of samples in human serum, with two replicates conducted. The titer was calculated as the reciprocal of the product of the assay’s minimum required dilution (1 in 13) multiplied by the greatest serial dilution that yielded a response greater than the assay cut-point. In low concentration samples below the assay cut-point, titer will be reported as < 13. Consistent with statistical methods mandated by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA), the cut-point and normalization factor for the ADA screening assay was set to allow a 5% false-positive rate (determined by a 90% one-sided lower confidence interval [CI] for the 95th percentile of the negative control population, with a 90% confidence level) [24, 25]. Similarly, the ADA confirmatory assay cut-point was set to a 1% false-positive rate (determined using an 80% one-sided lower CI for the 99th percentile of the negative control population, with an 80% confidence level) [24, 25].

Confirmed ADA-positive samples were also tested for the presence of neutralizing antibodies (nAbs). Murine anti-human IL-13 mAb, clone 32116, (R&D Systems, Minneapolis, MN, USA) was immobilized to Meso Scale Discovery High-Bind plates. Samples and controls were incubated in the presence of acidic conditions, and neutralized and incubated in the presence of a known ruthenylated tralokinumab concentration overnight. Recombinant human IL-13 (R&D Systems, Minneapolis, MN, USA) was added to the blocked assay plates, incubated, and washed to remove unbound material. The sample–tralokinumab complex was then added to the assay plates and unbound material was washed away. Buffer containing tripolyamine was added, and an electric voltage applied to produce a chemiluminescent signal. An inhibition of signal, relative to a non-neutralizing control, was indicative of a neutralizing sample.

Case records of participants with a positive ADA response were reviewed for patterns of AEs.

All AEs of hypersensitivity were identified using the MedDRA^® Standardized MedDRA^® Query (SMQ) for hypersensitivity, anaphylactic reaction, and anaphylactoid shock conditions. Within this group, a two-step process was used to identify and evaluate a subset comprising possible anaphylactic reactions or any AE for anaphylactoid shock or hypersensitivity. Using a programmatic approach, AEs were identified that occurred either the day of, or within 72 h of the day after, trial drug administration by a modified algorithm of the SMQ for anaphylactic reaction. The preferred terms (PTs) representing gastrointestinal events were also added to the algorithm. The PTs comprising the algorithm of this SMQ are provided in Table 1. If an AE was captured under the anaphylaxis SMQ, it was automatically considered as one possible anaphylaxis event. If two or more AEs were identified as occurring in two or more of the other categories (for respiratory, dermatological, cardiovascular, and gastrointestinal PTs), these AEs were also considered as one possible anaphylaxis event (Table 1). In addition to this programmatic approach, the blinded study physicians reviewed all AEs that occurred in STRATOS 1 and 2 individually and identified those that could possibly be anaphylaxis events.

MedDRA^® Preferred Terms included in the modified MedDRA^® SMQ ‘anaphylactic reaction’ algorithm^a

MedDRA Medical Dictionary for Regulatory Activities, SMQ Standardized MedDRA Query

^aA possible case of anaphylaxis according to the modified MedDRA^® algorithm is: any term within category A; or at least two terms within two different categories (or more) within B, C, D, and E

The clinical evaluation of hypersensitivity reactions for possible anaphylaxis events followed the Sampson criteria [26]. Briefly, the diagnosis of anaphylaxis is highly likely when one of these three clinical scenarios is observed:

The acute onset of a reaction (minutes to hours) with involvement of the skin, mucosal tissue, or both, and at least one of the following:

respiratory compromise; or

reduced blood pressure or symptoms of end-organ dysfunction.

Two or more of the following occurring rapidly after exposure: involvement of the skin/mucosal tissue, respiratory compromise, reduced blood pressure, or associated symptoms and/or persistent gastrointestinal symptoms.

Reduced blood pressure after exposure to an allergen for that participant.

The list of possible anaphylaxis AEs was reviewed by an internal study physician and then referred to an independent, external, blinded evaluator (an allergist who was otherwise not involved in the study) for a participant-level review. The external evaluator was provided with the full profile, a narrative of the event, concomitant medications, medical history, respiratory history, and the dosing data for the participant. The evaluator was asked to determine whether the AE met the Sampson criteria for anaphylaxis. If these criteria were not met, the evaluator was then asked to determine whether the AE represented a non-anaphylactic hypersensitivity event. The evaluator could answer yes, no, or undetermined.