An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA

Abstract

Antibody-mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor-specific antibodies (DSA) binding donor Class I and II HLA (HLA-I and HLA-II) expressed on endothelial cells. While F(ab′)2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti-C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA-Ab)-induced complement activation. Complement deposition induced by HLA-Ab was evaluated using novel cell- and bead-based assays. Human aortic endothelial cells (HAEC) were cultured with HLA-Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA-Ab and human complement. TNT003 inhibited HLA-Ab mediated complement deposition on HAEC in a concentration-dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI-Ab)- and Class II (HLAII-Ab)-specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA-I/II-Ab in vitro.

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

“…Reed and coworkers 22 found 2.5‐fold higher IC 50 values for TNT003 on beads compared with human AECs, and they ascribed this difference to higher levels of available C1s in the solid phase assays (37.5% vs. 25% in cellular assays) or, alternatively, to a lack of complement inhibitory receptors on bead surfaces. Our failure to detect meaningful differences in potency in head‐to‐head comparisons between assay systems when using a constant dilution of defined monospecific or polyspecific allosera (75 vol%) supports the belief that C1s levels are the primary determinant of TNT003 potency.…”

Complement inhibitors to treat IgM-mediated autoimmune hemolysis

“…33 Recently, TNT003 was successfully tested in an in vitro model for antibody-mediated graft rejection (AMR) showing its potency to reduce antibody-mediated classical pathway activation. 67 In addition, plasma-derived C1-inh is currently being tested in clinical trials for its efficacy in preventing AMR in kidney transplantation (clincialtrials.gov identifier: 01147302 and 01134510). Both inhibitors, C1-inh and TNT003, seem to be suitable to efficiently inhibit complement-deposition as well as insertion of the MAC into RBCs, and hence complement-mediated extraand intravascular hemolysis, respectively.…”

Uncoupling complement C1s activation from C1q binding in apoptotic cell phagocytosis and immunosuppressive capacity

“…Specific targeting of C1s enzymatic activity via a blocking mAb selectively inhibits the earliest step of classical pathway activation and the downstream generation of inflammatory mediators (such as C3a and C5a anaphylatoxins), while preserving a) C1q opsonization and its downstream effects, and b) the activation of alternative and lectin pathways. TNT003 has previously been shown to prevent antibody-driven complement activation on red blood cells [67] and endothelial cells [68], and its humanized version, TNT009, is currently under evaluation in healthy volunteers (ClinicalTrials.gov; ID: NCT02120482). This strategy runs the theoretical risk of impeding AC clearance [56].…”