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. 2017 Sep;17(9):2300-2311.
doi: 10.1111/ajt.14256. Epub 2017 Mar 31.

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

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Free PMC article

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

M Wahrmann et al. Am J Transplant. .
Free PMC article

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.

Keywords: basic (laboratory) research/science; complement biology; flow cytometry; fusion proteins and monoclonal antibodies; histocompatibility; immunosuppressant; immunosuppression/immune modulation; major histocompatibility complex (MHC); rejection: antibody-mediated (ABMR); translational research/science.

Figures

Figure 1
Figure 1
Binding of anti‐C1s mA bs to C1s attached to single antigen flow bead (SAFB) ‐bound HLA antibodies. (A) HLA class I SAFBs (97 different microbead populations coated with defined HLA class I antigens/alleles) were incubated with a polyspecific alloserum and subsequently stained for C1s using a biotin‐conjugated polyclonal anti‐C1s antibody. In parallel, beads were stained for IgG binding after EDTA treatment of the serum to prevent complement‐dependent interference with IgG detection. Binding of anti‐C1s mAbs (B) TNT009 (filled circles) or (C) TNT003 (filled circles) and their isotype controls (open circles; nonbinding human IgG4 or mouse IgG2a, respectively; final antibody concentration 62.5 μg/mL) was also evaluated. Mean fluorescence intensity (MFI) levels obtained in the different assays were correlated by using Spearman correlation. To assess the binding of anti‐C1s antibodies, SAFBs were incubated with alloserum spiked with increasing concentrations of TNT009 (D) or TNT003 (E) versus their isotype controls (human IgG4 and mouse IgG2a, respectively) and then stained for antibody binding by applying indirect immunofluorescence. Box plots indicate the median MFI and interquartile range (outliers: open circles; extreme outliers: asterisks).
Figure 2
Figure 2
Effect of anti‐C1s mA bs on single antigen flow bead (SAFB) binding of C1 complex subcomponents. Binding of C1q (A) and C1s (B) was evaluated on SAFBs. For each assay variant, only single beads that stained positive with methylamine (MeNH 2)‐treated serum (mean fluorescence intensity thresholds, see Materials and Methods section) were included in the analysis (C1q: n = 44; C1s: n = 33). PBS or nonbinding IgG2a and IgG4 were used as the corresponding negative controls. Assays were performed by using untreated alloserum or serum preincubated with MeNH 2 to counteract complement‐dependent (C4 and C3 split product) interference. To assess the impact of MeNH 2 on C3b/d deposition as a trigger of complement interference, SAFBs were stained for C3 split product deposition in the presence or absence of MeNH 2 (C), whereby 44 SAFBs that stained positive with PBS‐treated serum were included in the analysis. Box plots indicate median and interquartile range (outliers: circles, extreme outliers: asterisks). For statistical comparisons, the Mann–Whitney U test was used.
Figure 3
Figure 3
Blockade of C3 split product deposition by anti‐C1s antibodies. The effect of increasing concentrations of TNT009 (A) or TNT003 (B), in comparison to corresponding isotype controls (human IgG4 and murine IgG2a, respectively), was evaluated by using HLA class I single antigen flow beads (SAFBs). Box plots indicate the median and interquartile range (outliers: open circles, extreme outliers: asterisks) of mean fluorescence intensity (MFI) recorded for 47 SAFBs that showed a positive baseline (PBS) C3b/d result (MFI >100). The relationship between IC 50 calculated for each individual SAFB is shown for TNT009 (C and E) and TNT003 (D and F) in relation to C3b/d MFI (C and D) or C1s MFI (E and F) detected in the absence of treatment. For statistical analysis, Spearman correlation was used.
Figure 4
Figure 4
Effect of TNT 009 on HLA antibody–triggered classic pathway activation on aortic endothelial cells ( AECs ) or lymphocytes versus single antigen flow beads (SAFBs) . Applying flow cytometry, C3b/d deposition was measured on cells obtained from three different HLA‐typed deceased organ donors (donors 1, 2, and 3), all of them expressing HLA‐A2. Donor AECs or splenic lymphocytes (separate evaluation of T vs. B cells) were incubated with a polyspecific alloserum containing C3b/d‐fixing reactivity against four to six different HLA antigens of the selected donors or with a monospecific alloserum that exclusively contained C3b/d‐fixing reactivity against HLA‐A2. C3b/d deposition was assessed in Luminex SAFB assays, and HLA specificities corresponding to the targeted donor antigens are shown. Assays were carried out in the presence of TNT009 at increasing concentrations, and results are expressed as the percentage of mean fluorescence intensity obtained in untreated assays.
Figure 5
Figure 5
The impact of increased donor cell HLA antigen expression on the inhibitory effect of TNT 009. For upregulation of HLA antigens, aortic endothelial cells (AECs) were stimulated with interleukin‐1β (5 ng/mL), tumor necrosis factor‐α (1000 units/mL), and interferon‐γ (300 units/mL) for 14 h. Cells from three different donors (1, 2, and 3) were then incubated with a polyspecific and a monospecific alloserum and C3b/d (A) and, as a measure of alloantibody binding, IgG (B) were detected by flow cytometry. (C) The IC 50 levels calculated for experiments carried out in the absence or presence of TNT009 at increasing concentrations (between 0 and 250 μg/mL). Box plots indicate the median and interquartile range of IC 50 levels on unstimulated versus stimulated AECs. For statistical comparison, the Wilcoxon signed‐rank test was used.
Figure 6
Figure 6
Effect of TNT 009 on C3 split product deposition to single antigen flow beads (SAFBs) triggered by allosera obtained from a cohort of waitlisted transplant candidates. The results obtained for 45 HLA class I– (A) and 54 HLA class II–reactive allosera (B) are summarized (HLA class I: 4365 single reactions; HLA class II: 5130 single reactions). C3b/d mean fluorescence intensity (MFI) values obtained for untreated samples are shown in relation to MFI results obtained after treatment of samples with 250 μg/mL TNT009 (filled circles) versus the same concentration of nonbinding human IgG4 (open circles).

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