Specificity of human anti-variable heavy (VH ) chain autoantibodies and impact on the design and clinical testing of a VH domain antibody antagonist of tumour necrosis factor-α receptor 1

Abstract

During clinical trials of a tumour necrosis factor (TNF)-R1 domain antibody (dAb™) antagonist (GSK1995057), infusion reactions consistent with cytokine release were observed in healthy subjects with high levels of a novel, pre-existing human anti-VH (HAVH) autoantibody. In the presence of HAVH autoantibodies, GSK1995057 induced cytokine release in vitro due to binding of HAVH autoantibodies to a framework region of the dAb. The epitope on GSK1995057 was characterized and dAbs with reduced binding to HAVH autoantibodies were generated; pharmacological comparability was determined in human in-vitro systems and in-vivo animal experiments. A Phase I clinical trial was conducted to investigate the safety and tolerability of the modified dAb (GSK2862277). A significant reduction in HAVH binding was achieved by adding a single alanine residue at the C-terminus to create GSK2862277. Screening a pool of healthy donors demonstrated a reduced frequency of pre-existing autoantibodies from 51% to 7%; in all other respects, GSK2862277 and the parent dAb were comparable. In the Phase I trial, GSK2862277 was well tolerated by both the inhaled and intravenous routes. One subject experienced a mild infusion reaction with cytokine release following intravenous dosing. Subsequently, this subject was found to have high levels of a novel pre-existing antibody specific to the extended C-terminus of GSK2862277. Despite the reduced binding of GSK2862277 to pre-existing HAVH autoantibodies, adverse effects associated with the presence of a novel pre-existing antibody response specific to the modified dAb framework were identified and highlight the challenge of developing biological antagonists to this class of receptor.

Trial registration: ClinicalTrials.gov NCT01818024.

Keywords: ADA; Phase I; TNF; clinical trial.

© 2015 British Society for Immunology.

Figures

Figure 1

Study schematic. A = active (GSK2862277); P = placebo; filled and empty diamonds represent interim decision points based on safety.

Figure 2

C-terminal extensions have reduced ability to inhibit human anti-variable region immunoglobulin (Ig)G heavy chain (VH) (HAVH) autoantibody binding. Binding of HAVH autoantibodies to GSK1995057 is inhibited by preincubation of HAVH-positive serum samples with free unlabelled GSK1995057 and to a varying degree by preincubation with mutated domain antibodies (dAbs). Variants with a P14A substitution or with C-terminal extensions up to +ASTKGP have reduced ability to inhibit HAVH autoantibody binding to GSK1995057. Horizontal line indicates assay cut-point for cross-reactivity with HAVH autoantibodies.

Figure 3

In-silico identification of residues in GSK1995057 which may be involved in binding of HAVH autoantibodies. Green = no predicted involvement, yellow = predicted weak involvement, light red = predicted moderate involvement, dark red = predicted strong involvement. Residues are annotated using the Kabat numbering scheme.

Figure 4

Screening for autoantibodies. (a) A panel of human serum samples was screened in assays designed to detect autoantibodies specific for GSK1995057 or GSK2862277 (horizontal line represents assay cut point). (b) Correlation analysis shows no significant correlation between autoantibodies specific for GSK1995057 and GSK2862277 in human serum.

Figure 5

In-vitro activation of tumour necrosis factor (TNF)-R1. (a) GSK2862277 was incubated with human sera that contain HAVH autoantibodies (solid lines) or those that test negative (broken lines). In-vitro activation of TNF-R1 by antibody complexes was measured by interleukin (IL)−8 release from MRC-5 cells. (b,c) Serum samples which had tested positive (solid lines) or negative (broken lines) for autoantibodies specifically to GSK2862277 were tested for TNF-R1 activation when preincubated with GSK1995057 (b) or GSK2862277 (c). Points represent mean of assay replicates ± standard deviation.

Figure 6

Cytokine release in a patient with elevated titres of anti-drug antibodies (ADAs). Plasma levels of interleukin (IL)−6, tumour necrosis factor (TNF)-α (left axis) and C-reactive protein (CRP) (right axis) were measured over time following intravenous dosing with GSK2862277 2 mg/kg once daily for 5 days.

Figure 7

GSK2862277 inhibits ex-vivo tumour necrosis factor (TNF)-α-induced interleukin (IL)-8 release in whole blood. (a) Ex-vivo TNF-α-induced IL-8 was measured as described in the Methods. TNF-α-induced IL-8 levels in whole blood from predose samples ranged from 339 to 2094pg/ml in placebo subjects (n = 4) and from 311 to 3268pg/ml in subjects dosed subsequently with GSK2862277 (n = 18). (b) Comparison of ex-vivo IL-8 data from GSK1995057 and GSK2862277. Red vertical dashed lines = in-vitro IC50 and IC90 parameters for GSK2862277 and GSK1995057. Black vertical dashed lines = lower limit of quantification (LLQ) of GSK2862277 and GSK1995057. Black horizontal dashed line = LLQ of IL-8.

Figure 8

Pre-existing GSK2862277-specific autoantibodies activate tumour necrosis factor (TNF)-R1 in vitro. Predose serum from a subject with a high titre for GSK2862277-specific antibodies was incubated with increasing concentrations of GSK2862277 and tested for TNF-R1 activation in vitro (black solid lines). For comparison, serum obtained pre- and post-dose from a subject who tested negative for pre-existing GSK2862277-specific autoantibodies but who seroconverted post-dosing was also tested (grey broken lines). Points represent mean of assay replicates ± standard deviation.