A bispecific IgG format containing four independent antigen binding sites

Abstract

Bispecific antibodies come in many different formats, including the particularly interesting two-in-one antibodies, where one conventional IgG binds two different antigens. The IgG format allows these antibodies to mediate Fc-related functionality, and their wild-type structure ensures low immunogenicity and enables standard methods to be used for development. It is however difficult, time-consuming and costly to generate two-in-one antibodies. Herein we demonstrate a new approach to create a similar type of antibody by combining two different variable heavy (VH) domains in each Fab arm of an IgG, a tetra-VH IgG format. The VHs are used as building blocks, where one VH is placed at its usual position, and the second VH replaces the variable light (VL) domain in a conventional IgG. VH domains, binding several different types of antigens, were discovered and could be rearranged in any combination, offering a convenient "plug and play" format. The tetra-VH IgGs were found to be functionally tetravalent, binding two antigens on each arm of the IgG molecule simultaneously. This offers a new strategy to also create monospecific, tetravalent IgGs that, depending on antigen architecture and mode-of-action, may have enhanced efficacy compared to traditional bivalent antibodies.

Figure 1

Schematic outline for construction of tetra-VH IgGs. Binding VHs can be isolated from VHs, scFvs, Fabs, or IgGs. These VHs are used as building blocks to create either bispecific or monospecific tetra-VH IgGs.

Figure 2

Binding-analysis of monospecific antibody fragments. (a) Screening in FMAT of individual soluble scFv showing the binding to CD40 expressing cells versus binding to mock transfected cells. (b) Binding of unique clones to CD40 expressing cells in the first screening versus their binding after re-expression. (c) Binding of clones in ELISA to coated recombinant CD40 and a non-related protein carrying the same tag as the target (non-target). (d) Screening of VH-VLD Fab clones in ELISA for binding to target versus non-target protein. Yellow-marked clones were selected as binders and used for Sanger sequencing to identify unique sequences.

Figure 3

Dose response ELISA showing the binding of anti-CD40 VH-VLD Fab clones, compared to their corresponding parental clones, in a Fab format, to the CD40 target protein and a non-target protein. (a) Binding of clone a-005-A04. (b) Binding of clone a-001-A04. (c) Binding of clone a-004-B03. (d) Binding of clone a-009-A06.

Figure 4

Binding-analysis of a CD40/OX40 specific tetra-VH IgG. The antibody was evaluated for (a) binding to recombinant proteins in ELISA and for binding to (b) transfected cells or (c) endogenously expressing cells in flow cytometry. In vitro activated (IVA) CD4+ T-cells express OX40 and 4-1BB and the B-cell line Raji express CD40.

Figure 5

Binding-analysis of bispecific tetra-VH IgGs to overexpressing cells in flow cytometry. The two VHs, binding CD40 and OX40 respectively, were either linked to CH1 or CL in the antibody. In IgG #2 the CD40 specific VH is linked to CL and the OX40 specific VH is linked to CH1. In IgG #5, it is the other way around, the OX40 specific VH is linked to CL and the CD40 specific VH is linked to CH1.

Figure 6

Evaluation of antibodies effect on B-cells proliferation. (a) Wild-type parental IgGs, tetra-VH IgGs and control antibodies, were cross-linked and analyzed for induction of B-cell proliferation on B-cells from healthy donors. Proliferation was measured as the percent of live CD19+ cells that were CD86 high. Samples were run as duplicates and data from 2–4 different donors were plotted as mean with SEM using GraphPad Prism. (b) Dose response analysis of antibodies effect on B-cell proliferation showing the mean, after subtraction of values for the isotype control, from 2 different experiments using B-cells from 4 different donors.

Figure 7

Binding-analysis of bispecific tetra-VH IgGs and Fabs capability to bind two different antigens simultaneously. (a) Schematic outline of the ELISA set-up. Tetra-VH IgGs were captured on a coated anti-human-Fc specific antibody followed by addition of a non-biotinylated antingen. Binding of the same or a second antigen, in a biotinylated version, was then detected using HRP-labeled Streptavidin and a luminescent substrate. (b) ELISA analysis of an anti-CD40/OX40 tetra-VH IgG for binding to biotinylated CD40 or OX40 after blocking with non-biotinylated CD40 or OX40. The assay was run as described in A. (c) Bridging ELISA. An anti-CD40/4-1BB Fab was analyzed for binding to coated CD40 respectively 4-1BB followed by addition of biotinylated CD40, OX40 or 4-1BB antigen. Binding of biotinylated antigens was detected using Streptavidin-HRP and a luminecent substrate. (d) Binding-analysis of CD40 and 41BB to an anti-41BB/CD40 tetra-VH IgG in Biacore. Antibodies were captured on an immobilize catcher antibody followed by addition of 800 nM of the first antigen to achieve binding saturation. The second antigen was then added at 800 nM, diluted in 800 nM of the first antigen, to avoid signal loss due to dissociation of the first antigen.