Subcutaneous Allergen Immunotherapy With Hypoallergenic Bet v 1 Compared to Conventional Extract: Poorer Blocking Antibody Capacity Dominated by IgG1 Instead of IgG4

Abstract

Background: Hypoallergenic recombinant fold-variants of major allergens have been suggested as safer and more effective AIT candidates. The Bet v 1-fold variant BM41, with confirmed preclinical hypoallergenicity and increased immunogenicity, was proposed for the treatment of birch pollen allergy.

Methods: We performed a 6-month randomized, double-blind, placebo-controlled first-in-human clinical trial with BM41, a licensed birch pollen extract-based treatment, as the active comparator (AC), and placebo (n = 16, n = 16, and n = 15, respectively). The primary endpoint was safety. Secondary outcomes were Bet v 1-specific (s)IgE, IgG, IgG₁, and IgG₄ responses measured by ImmunoCAP, and sIgE-blocking activity using mediator release and facilitated antigen binding assays.

Results: Despite SPT-confirmed hypoallergenicity (~50% compared to natural Bet v 1), more adverse events occurred in response to BM41. Although similar sIgG and sIgG₁ levels were induced, sIgG₄ levels increased 3-fold more in AC compared to the BM41 group. In AC, the sIgG₄/sIgG₁ ratio tripled over time, whereas for BM41 it stagnated. BM41 induced efficient serum inhibitory activity for sIgE compared to placebo but was 12%-32% less efficient than AC. Both sIgG₄ and sIgG₁ contributed to the blocking effect in AC, while in BM41 both sIgG subclasses showed a lowered functional capacity.

Conclusion: Preclinically established hypoallergenicity of BM41 did not result in a lower number of adverse events. The reduced induction of sIgG₄ by the fold variant in the course of the treatment was less efficient in blocking sIgE-mediated responses. This is the first study providing evidence that, instead of a Th1-favored IgG₁-dominated response, "modified Th2"-skewed IgG₄-dominated humoral responses are beneficial in AIT vaccine design.

Keywords: AIT; Bet v 1; IgG4; birch; hypoallergen.

FIGURE 1

SCIT protocol for administration of BM41 and placebo (A), patient screening and randomization (B), and hypoallergenicity assessment in tSPT and ImmunoCAP. The concentrations of BM41 that were administered were adjusted to the Bet v 1 concentrations contained in BPE‐AC in a cluster up‐dosing regimen (C); V1 = t = 1; V7 = t = 2. Patients' IgE was significantly binding less to BM41 compared to Bet v 1 in ImmunoCAP (D), and the reactivity of patients' IgE to Bet v 1 was about twice as high as for BM41 in all three treatment groups at t = 1 (E). Data from tSPT with BM41 of all patients combined before first SCIT administration (at t = 1, n = 47) in comparison to results obtained with birch pollen extract SPT solution performed at the inclusion phase of the patients' screening (F). The median of 15 μg/mL Bet v 1 of the birch pollen SPT resulted in a similar wheal size as 100 μg/mL of BM41. The reactivity of the patients to BM41 was investigated using a tSPT at the three various time points of the treatment and the AUC of the dose–response curves were calculated per patient (G). BM41 SPT sizes remained stable in both active treatment groups throughout the trial, while for placebo the area‐under‐the‐curve (AUC) of the tSPT wheal‐and‐flare reactions increased significantly from t = 1 to t = 3 (pollen season of early flowering trees. BPE‐AC, birch pollen extract‐based active comparator; SAE, severe adverse events; sIgE, specific IgE; tSPT, titrated skin‐prick‐test. **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

FIGURE 2

BPE‐, Bet v 1‐ and BM41‐specific (s) IgE (A), IgG (B), IgG₁ (C) and IgG₄ levels (D) in sera of SCIT patients treated with either BM41, BPE‐AC or placebo. Placebo treatment showed a small but significant decrease in IgE against Bet v 1 (A). The average fold increase (t = 1 to t = 3) for BM41 sIgG and sIgG₁ (B‐C) was higher in the BM41 (4.2‐ and 3.4‐fold, respectively) compared to the BPE‐AC group (3.0‐ and 2.3‐fold, respectively), indicating either that not all BPE‐AC‐induced Bet v 1 sIgG antibodies are cross‐reacting with BM41, and vice versa, or that they are of lower affinity. Data are shown as bar charts (including individual data) with mean and standard error of the mean. Ratios of Bet v 1‐sIgG₄/IgG₁ between the different time points (E). The medians of BPE‐, Bet v 1‐ and BM41 sIgG₁, sIgG₄ and sIgG fold increase (t = 1 to t = 3) for all three treatment groups is shown (F). BPE‐AC, birch pollen extract‐based active comparator; s, specific. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

FIGURE 3

Serum inhibitory activity for Bet v 1‐specific IgE is induced in course of the active treatment with BM41 and BPE‐AC. The inhibition of IgE‐Bet v 1 binding was determined by inhibition ELISA (A). The placebo arm remained at a relatively constant level ranging between 2.5% and 9%. The inhibition of mediator release by patients' sera was investigated using a human reference serum containing high levels of Bet v 1‐specific IgE (C). FAB competition (E) assay was performed using serum from SCIT‐treated subjects. For placebo, the inhibition of complex formation remained at a constant level at around 20%–30%, reflecting at all time points the basal inhibition effect of BM41 and BPE‐AC patients' sera prior to treatment start, and around 0 in the Δ‐values (F). The data derived from all three assays were expressed as relative to the uninhibited reference (100%). Data are shown as violin plots with individual data (median is indicated by the red bar). The corresponding delta graphs (t = 2−t = 1, t = 3−t = 2, and t = 3−t = 1) are also shown (B, D and F). Of note, for all these approaches, IgE‐depleted patients' sera were used for the allergen preincubation step, and the reference IgE was derived from a human sera pool containing high Bet v 1‐sIgE to ensure experimental consistency across all samples. BPE‐AC, birch pollen extract‐based active comparator. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

FIGURE 4

Distinct response and parameter correlation pattern in BPE‐AC‐ versus BM41‐treated patients. Bet v 1‐specific IgG, IgG₁, IgG₄ and IgE, the IgG₄/IgG₁ ratio, inhibition ELISA, FAB inhibition, FAB competition, and the iMRA were used to compute the PCA (A), with the corresponding loading graph (B). Only the two PCs with the largest eigenvalue were used for the graphical representation. Treatment specific patients' clusters were highlighted in the respective colors (BPE‐AC = green, BM41 = yellow, placebo = red). Correlation matrices (Spearman's rank correlation) of all three time points for BPE‐AC (C), BM41 (D) and placebo (E) are shown as heatmaps, only significant correlations are shown. In all three groups, the FAB inhibition correlated with FAB competition, and only the actively treated groups also correlated with the iMRA. In the BM41 group, a correlation between sIgE and sIgG₄ (r = 0.54) at t = 3 was observed, which was not the case for BPE‐AC, most likely due to the higher, nonproportional induction of sIgG₄ in BPE‐AC. In BM41, there was no correlation between sIgG₁ and the inhibition assays, however, sIgG₄ and sIgG correlated with FAB competition at t = 2 (r = 0.54 both) and t = 3 (r = 0.60 and r = 0.65, respectively). Red boxes highlight the results mentioned in the text. BPE‐AC, birch pollen extract‐based active comparator; FAB, facilitated allergen binding assay; iMRA, inhibition mediator release assay; MRA, mediator release assay; s, specific; PCA, principal component analysis.

FIGURE 5

Serum inhibitory activity of IgG₁‐ and IgG₄‐depleted sera is reduced in BM41‐treated patients indicating a lack of functionality of the induced IgG antibodies. FAB inhibition assay was performed using the non‐, IgG₁‐ and IgG₄‐depleted sera (A). The depletion procedure resulted in a dilution of the samples of approximately 21%–28%. Since this dilution effect was not significant, we have decided not to correct for it. Inhibition of mediator release by depleted and nondepleted sera induced by Bet v 1‐cross‐linking of patients' serum IgE was investigated (B). The data derived from both assays were expressed as relative to the uninhibited reference (100%), the Δt = 3−t = 1 was calculated and data are shown as violin plots with individual data (median is indicated by the red bar). For BPE‐AC, Δt = 3−t = 1 showed 38.5% inhibition by IgG₁‐depleted and 35.4% by IgG₄‐depleted versus 48.5% by nondepleted control serum in the FAB inhibition assay, and 18.9% IgG₁‐depleted, 26.8% IgG₄‐depleted, and 50.9% by nondepleted control serum in iMRA. Regarding inhibition of complex formation (A) in BM41 group, 36.6% inhibition was achieved by IgG₁‐depleted, 8.2% by IgG₄‐depleted, and 32.2% by nondepleted control. Thus, IgG₁‐depleted sera were like nondepleted placebo (−11.4%). In both assays, the sum of inhibition achieved by the IgG₁‐ and the IgG₄‐depleted sera nicely resembles the percentage inhibition obtained with the nondepleted sera, as exemplified with the BPE‐AC group (C‐D). Spearman's rank correlation matrix of the Δt = 3−t = 1 values of the non‐, IgG₁‐ and IgG₄‐depleted sera of the BPE‐AC (E) and BM41 (F) group, only significant correlations are shown. Red boxes highlight the results mentioned in the text. BPE‐AC, birch pollen extract‐based active comparator; FAB, facilitated allergen binding assay; iMRA, inhibition mediator release assay; s, specific. **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.