Blockade of HLA Antibody-Triggered Classical Complement Activation in Sera From Subjects Dosed With the Anti-C1s Monoclonal Antibody TNT009-Results from a Randomized First-in-Human Phase 1 Trial

Abstract

Background: Complement may play a key role in antibody-mediated rejection. A promising therapeutic approach may be classical pathway (CP) inhibition at the level of early component C1.

Methods: In this first-in-human, double-blind, randomized placebo-controlled phase 1 trial, we evaluated the safety and complement inhibitory effect of TNT009, a humanized monoclonal anti-C1s antibody. Sixty-four adult healthy volunteers received either single (n = 48; 7 consecutive cohorts, 0.3-100 mg/kg) or 4 weekly infusions (n = 16; 2 consecutive cohorts, 30 and 60 mg/kg per infusion) of TNT009 or placebo. To assess the effect of treatment on complement activity, sera from dosed subjects were analyzed in a CP activation assay evaluating C3d deposition on HLA-coated microbeads spiked with alloantibodies.

Results: Single doses of TNT009 at 3 to 100 mg/kg uniformly and profoundly inhibited HLA antibody-mediated C3d deposition (≥86% after 60 minutes), whereby the duration of CP inhibition (2-14 days) was dose-dependent. Four weekly doses persistently blocked complement for 5 to 6 weeks. Ex vivo serum CP activity was profoundly inhibited when TNT009 concentrations exceeded 20 μg/mL. Infusions were well tolerated without serious or severe adverse events.

Conclusions: Treatment with TNT009 was safe and potently inhibited CP activity. Future studies in patients are required to assess the potential of TNT009 for preventing or treating antibody-mediated rejection.

Trial registration: ClinicalTrials.gov NCT02502903.

Conflict of interest statement

The study was funded by an investigator-initiated research grant from True North Therapeutics (to B.J.). G.C.P., J.C.G., and S.P. are employees of True North Therapeutics. The other authors have no conflicts of interest.

Figures

FIGURE 1

In vitro effect of TNT009 on HLA antibody-triggered CP activation in healthy volunteer and patient sera. To assess the effect of TNT009 on HLA antibody-dependent CP activation in vitro, serum samples were spiked with TNT009 in serial 1:2 dilution steps (concentrations between 250 and 1.95 μg/mL) and incubated with (A) HLA antibody-coated (samples obtained from 8 representative healthy volunteers) or (B) unmodified screening beads (samples obtained from 4 broadly sensitized patients). The assay read-out was the level of C3d MFI (healthy volunteers: mean of normalized MFI obtained on 15 different screening bead populations; sensitized patients: mean of normalized MFI on the 3 bead populations showing the highest levels of C3d fixation). In C, the mean (±SD) percentage of baseline MFI is shown for healthy volunteers and patient sera.

FIGURE 2

Effect of intravenous infusion of single doses (0.3-100 mg/kg) of TNT009 on HLA antibody-triggered CP activation. Forty-eight volunteers (study flow: A) were randomized in 7 cohorts (B-G) to receive ascending doses of TNT009 (black lines/symbols) or placebo (blue lines/symbols). Longitudinal serum samples obtained from study participants were incubated with presensitized HLA antibody-coated beads to assess the effect of TNT009 administration on ex vivo complement activation. The IDs of the participants are indicated. The effect of treatment on % preintervention C3d MFI (mean of normalized MFI obtained on 15 different screening bead populations) was recorded over a study period of 14 days (end-of-study visit).

FIGURE 3

Effect of intravenous infusion of 4 weekly doses of TNT009 (30 or 60 mg/kg) on HLA antibody-triggered CP activation. Sixteen volunteers (study flow: A) were randomized in 2 cohorts (B, C) to receive 2 different repeated doses of TNT009 (black lines/symbols) or placebo (blue lines/symbols). Black arrows indicate the time points of infusion. The IDs of the participants are indicated. Longitudinal serum samples obtained from study participants were incubated with HLA antibody-coated beads to assess the effect of TNT009 administration on ex vivo complement activation. The effect of treatment on % preintervention C3d MFI (mean of normalized MFI obtained on 15 different screening bead populations) was recorded over a study period of 35 days (end-of-study visit).

FIGURE 4

Relationship between TNT009 serum concentration and CP inhibition. Preintervention and postintervention sera obtained from 48 volunteers (single and multiple ascending dose parts of the study) allocated to verum treatment were evaluated for TNT009 serum concentration and, in parallel, C3d MFI (mean of normalized MFI obtained on 16 different screening bead populations) (A) CH50 activity (B), and (C) alternative pathway (AP) activity, respectively.