Bispecific antibody derivatives with restricted binding functionalities that are activated by proteolytic processing

Abstract

We have designed bispecific antibodies that bind one target (anti-Her3) in a bivalent IgG-like manner and contain one additional binding entity (anti-cMet) composed of one V(H) and one V(L) domain connected by a disulfide bond. The molecules are assembled by fusing a V(H,Cys44) domain via flexible connector peptides to the C-terminus of one H-chain (heavy chain), and a V(L,Cys100) to another H-chain. To ensure heterodimerization during expression in mammalian cells, we introduced complementary knobs-into-holes mutations into the different H-chains. The IgG-shaped trivalent molecules carry as third binding entity one disulfide-stabilized Fv (dsFv) without a linker between V(H) and V(L). Tethering the V(H) and V(L) domains at the C-terminus of the C(H)3 domain decreases the on-rates of the dsFv to target antigens without affecting off-rates. Steric hindrance resolves upon removal of one side of the double connection by proteolysis: this improves flexibility and accessibility of the dsFv and fully restores antigen access and affinity. This technology has multiple applications: (i) in cases where single-chain linkers are not desired, dsFvs without linkers can be generated by addition of furin site(s) in the connector that are processed during expression within mammalian cells; (ii) highly active (toxic) entities which affect expression can be produced as inactive dsFvs and subsequently be activated (e.g. via PreScission cleavage) during purification; (iii) entities can be generated which are targeted by the unrestricted binding entity and can be activated by proteases in target tissues. For example, Her3-binding molecules containing linkers with recognition sequences for matrix metalloproteases or urokinase, whose inactivated cMet binding site is activated by proteolytic processing.

Fig. 1.

Composition of trivalent bispecific antibody derivatives. (A) Schematic presentation of bispecific antibodies (BsAb) containing dsFvs. (B) Magnification of Fc part with detailed composition of connector-peptides comprising recognition sequences for proteolytic processing during or after expression. (C) SPR sensorgrams with binding kinetics of BsAb containing uncleavable connectors of different length. Sequential binding to cMet and Her3 extracellular domain (cMet, Her3) is shown. Right panel presents a magnification of BsAb to visualize differences.

Fig. 2.

Expression and purification of trivalent BsAb containing dsFv. (A) Coomassie blue-stained reducing SDS-PAGE of protein preparations after Protein-A and size exclusion (SEC) purification (H = cleaved; H1 = H + V_H; H2 = H + V_L). Note that the disulfide-connected V_L domains are visible as separate bands in molecules that are expressed without second or with furin connector. BsAb which contain connectors that are recognized by the proteases Prescission, MMP or uPA are produced with extended H-chains. (B) Exemplary SEC profile of the trivalent bispecific dsFv-containing antibody with a MMP2/9 site in its connector demonstrates pure monomeric compositions free of aggregates.

Fig. 3.

Proteolytic activation of the restricted dsFv moieties. Coomassie blue-stained reducing SDS-PAGE of BsAb derivatives containing restricted dsFvs before and after proteolytic processing. (A) Protein-A elution profile of PreScission cleaved purified protein (ct.). (B) Proteins that contain furin cleavage sites are activated already during expression (cf. Fig. 2). Proteins containing MMP or uPA cleavage sites are generated as restricted molecules (cf. Fig. 2) and become activated upon exposure to MMP or uPA. (C) Exemplary SPR sensorgrams of unprocessed and processed BsAb with PreScission cleavage site. Shown are titration series with cMet extracellular domain as ligand.

Fig. 4.

Cellular binding of restricted and unrestricted trivalent BsAb. Binding of the bivalent unrestricted Her3-modules to Her3-expressing, cMet-negative T47D cells is shown on the left panels. Binding of the different restricted cMet-modules to Her3-low cMet-expressing A549 cells is shown on the right panels (gray fill = isotype control). Weak binding is observed for the restricted modules while protease-processing leads to full binding and accumulation on cells (solid line = uncleaved; dashed line = processed). Uncleaved Prescission connector BsAb was used as control for furin-connector containing BsAb.

Fig. 5.

Cellular activity of trivalent bispecific antibodies. (A) MCF7 cells were incubated with the respective antibodies (triangle: 0.1 and 1.0 µg/ml) and the Her3 ligand Heregulin. Whole-cell lysates were used to detect phosphorylated (p-Her3) and total Her3 (t-Her3). Note that the Her3 antibody induces internalization. Beta-actin was used as loading control. Reduction of phosphorylated Her3 levels is observed for all constructs and independent of previous protease treatment. (B) A549 cells were stimulated with the cMet ligand hepatocyte growth factor (HGF) and the indicated antibodies. Dose-dependent inhibition of cMet phosphorylation (p-Met) was observed. Total cMet (t-Met) and beta-actin bands were unchanged. (C) A549 cells were stimulated with HGF and treated with the indicated antibodies. The ratio of phosphorylated and total AKT was used as readout to determine efficacy of the restricted and unrestricted cMet binding moiety (triangle: 1.9, 7.5, 30 µg/ml BsAb). Consistent lack of activity was observed for unprocessed PreScission BsAb while BsAb with uncleaved MMP connector displayed activity in some instances.