Monovalent TNF receptor 1-selective antibody with improved affinity and neutralizing activity

Abstract

Selective inhibition of tumor necrosis factor (TNF) signaling through the proinflammatory axis of TNF-receptor 1 (TNFR1) while leaving pro-survival and regeneration-promoting signals via TNFR2 unaffected is a promising strategy to circumvent limitations of complete inhibition of TNF action by the approved anti-TNF drugs. A previously developed humanized antagonistic TNFR1-specific antibody, ATROSAB, showed potent inhibition of TNFR1-mediated cellular responses. Because the parental mouse antibody H398 possesses even stronger inhibitory potential, we scrutinized the specific binding parameters of the two molecules and revealed a faster dissociation of ATROSAB compared to H398. Applying affinity maturation and re-engineering of humanized variable domains, we generated a monovalent Fab derivative (13.7) of ATROSAB that exhibited increased binding to TNFR1 and superior inhibition of TNF-mediated TNFR1 activation, while lacking any agonistic activity even in the presence of cross-linking antibodies. In order to improve its pharmacokinetic properties, several Fab13.7-derived molecules were generated, including a PEGylated Fab, a mouse serum albumin fusion protein, a half-IgG with a dimerization-deficient Fc, and a newly designed Fv-Fc format, employing the knobs-into-holes technology. Among these derivatives, the Fv13.7-Fc displayed the best combination of improved pharmacokinetic properties and antagonistic activity, thus representing a promising candidate for further clinical development.

Keywords: affinity maturation; antibody humanization; inflammatory diseases; monovalent antibody; neutralizing antibody; tumor necrosis factor; tumor necrosis factor receptor 1.

Figure 1.

Receptor binding ATROSAB and H398. a) Binding of ATROSAB and H398 to immobilized TNFR1-Fc in ELISA (n = 3, mean ±SD) and QCM (b, c) using a chip of 48 Hz ligand density and a concentration range from 62.5 nM to 3.9 nM in 2-fold dilution steps. Each concentration was analyzed in triplicates.

Figure 2.

Affinity maturation and framework replacement of scFv ATROSAB (scFv IZI06.1) results in improved activity and stability. Binding of the parental scFv IZI06.1, the intermediate scFvs IG11 and T12B and the final version scFv 13.7, after affinity maturation and framework replacement was analyzed in ELISA (a, mean ± SD, n = 2). Inhibition of TNF-induced (0.1 nM TNF) IL-8 release from HT1080 cells (b) was analyzed for all scFvs (displayed are mean ±SD, n = 3). Thermal stability of scFv IZI06.1(c), scFv T12B (d) and scFv 13.7 (e) was analyzed using dynamic light scattering. T_m was determined upon visual interpretation of the displayed data points.

Figure 3.

Expression, purification and receptor binding of 13.7 derived IgG and Fab. Purity and correct assembly were analyzed by analytical size exclusion chromatography (a, Yarra SEC-2000 column, flow rate 0.5 ml/min) and SDS-PAGE (b, 12% separation gel, reducing conditions; c, 8% separation gel, non-reducing conditions). The indicated molecular weights were interpolated using a standard curve of proteins with known mass and retention time. Binding to human TNFR1 was evaluated in ELISA (d, n = 3, mean ± SD) and QCM experiments (e-h). In QCM measurements, concentrations between 64 nM and 4 nM (ATROSAB and IgG13.7) or 128 nM to 8 nM (FabATR and Fab13.7), were analyzed in triplicates. Measurements (grey, dotted line) and fit (black, solid) are shown.

Figure 4.

Bioactivity of 13.7 antibodies. The potential of IgG13.7 and Fab13.7 to stimulate TNFR1 on the surface of HT1080 and Kym-1 cells was analyzed by detection of IL-8 (a) release as well as by the induction of cell death (b). The inhibitory activity of Fab13.7 in the respective assays was analyzed (c and d) with respect to receptor stimulation by 0.1 nM TNF (c) or 0.01 nM TNF (d). ATROSAB and FabATR served as controls. Stimulatory experiments were performed twice (a and b, n = 2, mean ±SD), inhibitory experiments were performed three times (c and d, n = 3, mean ±SD).

Figure 5.

Fab13.7 lacks agonistic activity upon crosslinking. Binding of a goat anti-human Fab antibody to Fab 13.7 in ELISA (a) and (b) the effect of this goat anti-human Fab antibody (64 µg/ml) on the stimulatory activity of Fab13.7 towards TNFR1 was analyzed in an IL-8 release assay using HT1080 cells and compared to ATROSAB (n = 2, mean ±SD).

Figure 6.

Pharmacokinetic study on Fab13.7 and half-life engineered variants. PK profiles were obtained after i.v. bolus injection of 25 µg protein using C57BL/6J mice (n = 3) homozygously bearing the extracellular domain of human TNFR1 at the locus of the mouse gene. Initial and terminal half-lifes as well as the area under the curve were calculated after detection of remaining active protein in serum samples by ELISA.