Effect of the Anti-C1s Humanized Antibody TNT009 and Its Parental Mouse Variant TNT003 on HLA Antibody–Induced Complement Activation—A Preclinical In Vitro Study

Abstract

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.

Anti-C1s monoclonal antibody BIVV009 in late antibody-mediated kidney allograft rejection—results from a first-in-patient phase 1 trial

“…The relationship between CP blockade and BIVV009 serum concentrations was thereby consistent with previously experimental studies13,15 and a trial performed in healthy volunteers 17. We investigated the ability of 4 weekly doses of the antibody to block the CP in patients with late ABMR, both in peripheral blood and in the rejecting allograft.…”

“…by measuring the deposition of the C3 complement split product C3d to SAFB (patient serum as complement source) following an earlier-described protocol. 15 In brief, patient sera were incubated for 30 minutes with SAFB at room temperature and then incubated with a biotin-conjugated monoclonal antibody against human C3d (4 μg/ mL; Quidel, San Diego, CA) for another 30 minutes. Subsequently, phycoerythrin-conjugated streptavidin (1 μg/mL; eBioscience, San Diego, CA) was added for 30 minutes.…”

“…In addition, CP blockade upstream of the classical C3 convertase may, in contrast to terminal complement inhibition, shut down antibody-triggered C3 cleavage and prevent the release of C3a, a pro-inflammatory anaphylatoxin that may contribute to rejection. [13][14][15] Recently, the same antibody was evaluated in healthy volunteers, 16 where it was found to be well tolerated and effectively block CP activity in the peripheral blood. 11,12 In a randomized controlled trial, treatment with C1-INH, in addition to "standard of care treatment" plasmapheresis and intravenous immunoglobulin, did not affect the primary endpoint (day 20 pathology or graft survival), although there was a trend toward improvement in renal function and less development of transplant glomerulopathy in 6-month follow-up biopsies.…”

“…TNT003, a murine monoclonal anti-C1s antibody and its humanized variant BIVV009 (formerly TNT009), were shown to profoundly block chemotaxis of macrophages in experimental models of HLA antibody-triggered CP activation. [13][14][15] Recently, the same antibody was evaluated in healthy volunteers, 16 where it was found to be well tolerated and effectively block CP activity in the peripheral blood. 17 The present first-in-patient single-arm phase 1b trial was primarily designed to investigate the safety/tolerability profile and the complement inhibitory activity of a short course of this novel antibody in a cohort of stable kidney transplant recipients on maintenance immunosuppression.…”

Intrarenal Complement System Transcripts in Chronic Antibody-Mediated Rejection and Recurrent IgA Nephropathy in Kidney Transplantation

“…The complement system involvement was shown to play a role in both chronic antibody-mediated rejection and IgA nephropathy pathogenesis ( 7 – 11 ). Both complement and complement regulators have been studied in relation to antibody-mediated rejection, and therapies based both on C5 blockade ( 12 , 13 ) and C1 inhibition using the C1-inhibitor ( 14 ) or anti-C1 antibody ( 15 ) have been considered. However, the effect of complement activation and the significance of different components of the complement cascade are not fully understood when considering their impact on the long-term survival of the renal allograft.…”