Format and geometries matter: Structure-based design defines the functionality of bispecific antibodies

Abstract

Multispecific antibodies can be generated in different formats. More than two decades of R&D in the field of bispecific antibody engineering revealed that the design and choice of format can have a profound impact on the antibody functionality. This holds in particular true for entities that elicit (inter-)cellular processes such as receptor activation, receptor internalization, receptor clustering or the formation of immunological synapses between two cells. This review covers design parameters that influence the functionality of multispecific formats, with particular focus on T cell-recruiting bispecific antibodies. We describe formats that display the same size and domain sequences but a varying geometry. The structural composition of (artificial) immune synapses is reviewed and allows conclusions why some formats that share size and domain composition are more effective than others. To support the statement that the geometry matters, we present a recently designed antibody format that is characterized by its compact shape. The TriFab-Contorsbody consists of two tumor cell-targeting entities and one moiety for T cell recruitment. The unique barrel-like shape provides a 35-fold increase in potency compared to an IgG-like molecule with identical domain sequences.

Keywords: Antibody format; Geometry; Immune synapse; Protein engineering; T cell bispecific.

Fig. 1

Application areas of bispecific antibodies: 1) Receptor activation. 2) Receptor blocking and inhibition of soluble ligand (L) or membrane-bound ligand binding, e.g. of effector cells (E). 3) Receptor internalization. 4) Receptor clustering. 5) Receptor association. 6) Bicellular binding and retargeting of effector cells (E).

Fig. 2

Physiological immune synapse in comparison to TCB-induced artificial synapse formation. 1) Physiological immune synapse between effector T cell and tumor cell. The intermembrane distance is about 15 nm. 2) Short interdomain-distant (id)-antibodies induce the formation of artificial immune synapses that share common features (spanning) with the physiological condition. 3) Using short id-distant antibodies that bind to distal epitopes extends the intermembrane spacing. Molecules are less effective. 4) Long id-distant antibodies extend the intercellular spacing which might lead to decreased T cell activity.

Fig. 3

T cell-engaging antibodies with same size and domain sequences display different potencies. A) A FynomAb is either C-terminally or N-terminally fused to an IgG. B) A CD3ε-targeting scFv is either fused in cis- or trans- orientation to the light chain of an IgG-based monovalent, tumor-targeting antibody. C) A conventional tandem scFv (left) and a disulfide-stabilized DART molecule with alternating domain composition (right). D) Bispecific diabodies can be designed in four different ways, starting with either VH or VL on each chain. Depicted are VH->VL (both chains start with VH from N terminus to C terminus) and VL->VH. Not shown here are the respective hybrids.

Fig. 4

A) TriFab format with two Fab moieties recognizing the Lewis Y antigen (blue) and one Fv directed against the CD3ε antigen (yellow) according to Dickopf et al. (*). Interdomain disulfide bridges are indicated in red. B) The equivalent binders described in the TriFab format were used to design a two-chained format that is related to the Contorsbody technology by Georges et al. 2020 . Molecules were produced in HEK suspension culture expression systems by transient transfection. C) After kappa-select affinity capture, 85% of the protein presented as folded TriFab-Contorsbody. As side product a tandem-like Fab molecule (9%) and high-molecular weight species (aggregates) were also observed which could be removed by SEC. Preferential assembly of the Contors-TriFab format from these input molecules occurs because during translation and folding, intrachain assembly/and disulfides appear to form earlier than the interchain assembly of knob-into-hole CH3 and VH/VL. The final yields of purified TriFab-Contorsbodies was 8 mg per liter culture. D) Lewis Y targeting TriFab-TCB and Contorsbody-TCB were applied to co-culturing assays of LeY-positive MCF-7 cells and human PBMC (E:T ratio = 10:1). Results are expressed as mean and SD from triplicate wells and plotted as 3-parameter non-linear regression fitting using Graphpad Prism software. Representative plot of three independent experiments is shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)