Introduction. Considering the key role of the classical complement pathway as a trigger of inflammation and injury in antibody-mediated rejection (ABMR), targeted classical pathway inhibition may be an attractive strategy for prevention or treatment of this condition. In this experimental preclinical study, we studied the mechanism of action and the in vitro efficacy of a humanized antibody against C1s (TNT009) and its parental mouse monoclonal antibody (TNT003) in blocking classical complement activation triggered by anti-HLA antibodies.

Methods. The effect of anti-C1s antibodies on alloantibody-mediated complement binding and activation was analyzed by incubating HLA antigen-coated single antigen flow beads (SAFB) with sera from highly sensitized end stage renal disease patients spiked with various concentrations of TNT003/009 (range: 0 – 250 [micro]g/ml). Complement deposition onto SAFB was quantitated using biotinylated anti-C1q, C1r, C1s, C4d and C3d antibodies. Furthermore, we performed flow cytometric crossmatch analysis to assess the effects of TNT009 on complement C3 split product deposition (C3d) onto primary human aortal endothelial cells and splenocytes.

Results. Our analysis of complement inhibition by TNT003 and TNT009 revealed that deposition of C4 and C3 split products onto SAFB was effectively prevented while attachment of C1 subcomponents was not or only partly impeded. Additionally, TNT003 was detected on the surface of C1s-positive SAFB. Concentration-dependent inhibition of anti-HLA mediated C3 split product deposition was strongly related to the strength of anti-HLA antibody-mediated classical pathway activation, as suggested by a linear dependency of IC50 values on baseline C3d signals detected on individual SAFB. C3d flow crossmatch analysis on endothelial cells and splenocytes using poly- or mono-specific sera revealed an efficacy of TNT009 comparable to the SAFB assay, namely an excellent control over C3 split product deposition at 62.5 to 250 [micro]g/ml.

Conclusion. Our results suggest that the novel antibody TNT009 has great potential in blocking the activation of HLA antibody-triggered classical pathway downstream of C1 and could be a useful therapeutic to treat or prevent alloantibody-mediated tissue injury in allograft recipients.

CITATION INFORMATION: Wahrmann M, Mühlbacher J, Regele H, Eskandary F, Gilbert J, Panicker S, Böhmig G. TNT009 and Its Parental Mouse Monoclonal Antibody TNT003 Target Complement Component C1s and Effectively Block Alloantibody-Induced Classical Complement Pathway Activation – The Preclinical Experience. Am J Transplant. 2016;16 (suppl 3).

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