The classical pathway (CP) of complement may contribute to the pathogenesis of antibody-mediated rejection (ABMR). Selective CP blockade may be a promising strategy to counteract rejection. The objective of this first-in-patient phase 1b trial was to evaluate the safety/tolerability and CP-blocking potential of four weekly doses (60 mg/kg) of the anti-C1s antibody BIVV009 in complement-mediated disorders. Here we describe the results in a cohort of 10 stable kidney transplant recipients (median of 4.3 years post-transplantation) with late active ABMR and features of CP activation, such as capillary C4d or complement-fixing donor-specific antibodies (DSA). During seven weeks follow-up, no severe adverse events were reported, and BIVV009 profoundly inhibited overall and DSA-triggered CP activation in serum. Five of eight C4d-positive recipients turned C4d-negative in 5-week follow-up biopsies, while another two recipients showed a substantial decrease in C4d scores. There was, however, no change in microcirculation inflammation, gene expression patterns, DSA levels, or kidney function. In conclusion, we demonstrate that BIVV009 effectively blocks alloantibody-triggered CP activation, even though short-course treatment had no effect on indices of activity in late ABMR. This initial trial provides a valuable basis for future studies designed to clarify the therapeutic value of CP blockade in transplantation. ClinicalTrials. gov NCT#02502903

Late antibody-mediated rejection (ABMR) triggered by donor-specific antibodies (DSA) is a cardinal cause of kidney allograft dysfunction and loss. Diagnostic criteria for this rejection type are well established, but effective treatment remains a major challenge. Recent randomized controlled trials (RCT) have failed to demonstrate the efficacy of widely used therapies, such as rituximab plus intravenous immunoglobulin or proteasome inhibition (bortezomib), reinforcing a great need for new therapeutic concepts. One promising target in this context may be interleukin-6 (IL-6), a pleiotropic cytokine known to play an important role in inflammation and adaptive immunity. This investigator-driven RCT was designed to assess the safety and efficacy of clazakizumab, a genetically engineered humanized monoclonal antibody directed against IL-6. The study will include 20 DSA-positive kidney allograft recipients diagnosed with ABMR ≥ 365 days after transplantation. Participants will be recruited at two study sites in Austria and Germany (Medical University of Vienna; Charité University Medicine Berlin). First, patients will enter a three-month double-blind RCT (1,1 randomization, stratification according to ABMR phenotype and study site) and will receive either clazakizumab (subcutaneous administration of 25 mg in monthly intervals) or placebo. In a second open-label part of the trial (months 4–12), all patients will receive clazakizumab at 25 mg every month. The primary endpoint is safety and tolerability. Secondary endpoints are the pharmacokinetics and pharmacodynamics of clazakizumab, its effect on drug metabolism in the liver, DSA characteristics, morphological ABMR lesions and molecular gene expression patterns in three- and 12-month protocol biopsies, serum/urinary biomarkers of inflammation and endothelial activation/injury, Torque Teno viral load as a measure of overall immunosuppression, kidney function, urinary protein excretion, as well as transplant and patient survival. Currently, there is no treatment proven to be effective in halting the progression of late ABMR. Based on the hypothesis that antagonizing the effects of IL-6 improves the outcome of DSA-positive late ABMR by counteracting DSA-triggered inflammation and B cell/plasma cell-driven alloimmunity, we suggest that our trial has the potential to provide proof of concept of a novel treatment of this type of rejection. ClinicalTrials.gov, NCT03444103 . Registered on 23 February 2018 (retrospective registration).

Antibody-mediated rejection (ABMR) is a leading cause of kidney allograft failure. While the exact mechanisms contributing to donor-specific antibody (DSA)-triggered tissue injury are still incompletely understood, complement activation via the classical pathway is believed to be one of the key players. There is now growing interest in complement blockade as an antirejection treatment. One attractive strategy may be inhibition of terminal complex formation using anti-C5 antibody eculizumab. Anecdotal reports, case series, and a unique cohort of flow crossmatch-positive live donor kidney transplant recipients subjected to eculizumab-based desensitization have demonstrated successful prevention and reversal of acute clinical ABMR. Nevertheless, maybe due to complement activation steps proximal of C5 or even complement-independent mechanisms, subclinical rejection processes that might culminate in chronic injury were found to escape inhibition. Larger studies designed to clarify the actual clinical value of terminal complement inhibition as an antirejection treatment are currently underway. In addition, alternative concepts, such as therapies that target key component C1, are currently under development, and we will see in the near future whether new strategies in the pipeline will have the potential to beneficially impact clinical practice.

Complement may play a key role in antibody-mediated rejection (ABMR). A promising therapeutic approach may be classical pathway (CP) inhibition at the level of early component C1. 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 analysed in a CP activation assay evaluating C3d deposition on HLA-coated microbeads spiked with alloantibodies. Single doses of TNT009 at 3-100 mg/kg uniformly and profoundly inhibited HLA antibody-mediated C3d deposition (≥86% after 60 min), whereby the duration of CP inhibition (2-14 days) was dose-dependent. Four weekly doses persistently blocked complement for 5-6 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. 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 ABMR. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

Background: Screening for donor-specific antibodies (DSA) has limited diagnostic value in patients with late antibody-mediated rejection (ABMR). Here, we evaluated whether biomarkers reflecting microcirculation inflammation or tissue injury—as an adjunct to DSA detection—are able to improve non-invasive ABMR monitoring. Methods: Upon prospective cross-sectional antibody screening of 741 long-term kidney transplant recipients with a silent clinical course, 86 DSA-positive patients were identified and biopsied. Serum and urine levels of E-selectin/CD62E, vascular cell adhesion molecule 1 (VCAM-1), granzyme B, hepatocyte growth factor (HGF), C-C motif chemokine ligand (CCL)3, CCL4, C-X-C motif chemokine ligand (CXCL)9, CXCL10, and CXCL11 in DSA-positive recipients were investigated applying multiplexed bead-based immunoassays. Results: Diagnosis of ABMR (50 patients) was associated with significantly higher levels of CXCL9 and CXCL10 in blood and urine and of HGF in blood. Overall, urinary CXCL9 had the highest diagnostic accuracy for ABMR (area under the receiver operating characteristic curve: 0.77; accuracy: 80%) and its combined evaluation with the mean fluorescence intensity of the immunodominant DSA (DSAmax MFI) revealed a net reclassification improvement of 73% compared to DSAmax MFI alone. Conclusions: Our results suggest urinary CXCL9 testing, combined with DSA analysis, as a valuable non-invasive tool to uncover clinically silent ABMR late after transplantation.

The classic pathway (CP) of complement is believed to significantly contribute to alloantibody‐mediated transplant injury, and targeted complement inhibition is currently considered to be a promising approach for preventing rejection. Here, we investigated the mode of action and efficacy of the humanized anti‐C1s monoclonal antibody TNT009 and its parental mouse variant, TNT003, in preclinical in vitro models of HLA antibody–triggered CP activation. In flow cytometric assays, we measured the attachment of C1 subcomponents and C4/C3 split products (C4b/d, C3b/d) to HLA antigen–coated flow beads or HLA‐mismatched aortic endothelial cells and splenic lymphocytes. Anti‐C1s antibodies profoundly inhibited C3 activation at concentrations >20 μg/mL, in both solid phase and cellular assays. While C4 activation was also prevented, this was not the case for C1 subcomponent attachment. Analysis of serum samples obtained from 68 sensitized transplant candidates revealed that the potency of inhibition was related to the extent of baseline CP activation. This study demonstrates that anti‐C1s antibodies TNT009 and TNT003 are highly effective in blocking HLA antibody–triggered complement activation downstream of C1. Our results provide the foundation for clinical studies designed to investigate the potential of TNT009 in the treatment or prevention of complement‐mediated tissue injury in sensitized transplant recipients.