The use of CrossMAb technology for the generation of bi- and multispecific antibodies

Abstract

The major challenge in the generation of bispecific IgG antibodies is enforcement of the correct heavy and light chain association. The correct association of generic light chains can be enabled using immunoglobulin domain crossover, known as CrossMAb technology, which can be combined with approaches enabling correct heavy chain association such as knobs-into-holes (KiH) technology or electrostatic steering. Since its development, this technology has proven to be very versatile, allowing the generation of various bispecific antibody formats, not only heterodimeric/asymmetric bivalent 1+1 CrossMAbs, but also tri- (2+1), tetravalent (2+2) bispecific and multispecific antibodies. Numerous CrossMAbs have been evaluated in preclinical studies, and, so far, 4 different tailor-made bispecific antibodies based on the CrossMAb technology have entered clinical studies. Here, we review the properties and activities of bispecific CrossMAbs and give an overview of the variety of CrossMAb-enabled antibody formats that differ from heterodimeric 1+1 bispecific IgG antibodies.

Keywords: 2+1, 1+1; 2+2; Ang-2; CEA TCB; CrossMAb; DAF-CrossMAb; DVD-CrossMAb; DuoMAb; EGFR; HER1; HER3; Immunoglobulin domain crossover; Kappa-Lambda-CrossMAb; MoAb; MoAb-Dimer; MonoMAb; P329G LALA; RG7221; RG7386; RG7716; RG7802; Triple A; VEGF-A; asymmetric; heterodimeric; knobs-into-holes (KiH); vanucizumab.

Figure 1.

Schematic overview about the 3 major CrossMAb formats: CrossMAb^Fab, CrossMAb^VH-VL and CrossMAb^CH1-CL and predicted side products that can be formed. Mass spectrometry following transient expression and purification via protein A confirmed the presence of the predicted antibody species. In the case of CrossMAb^Fab, a non-functional monovalent heavy chain dimer is formed; in the case CrossMAb^VH-VL, an antibody with a VL-CL Bence-Jones-like associated chain can occur. KiH technology or alternative heavy chain heterodimerization technologies applied where appropriate and indicated by colors only. Constant heavy chain domains are colored in gray, constant light chain domains in white, variable heavy chains are colored uniformly, light chain domains are colored with a line pattern.

Figure 2.

Overview of the clinical stage CrossMAbs: (A) Ang-2-VEGF CrossMAb vanucizumab (RG7112) for oncology, (B) VEGF-Ang-2 CrossMAb RG7716 for ophthalmology, (C) CEA TCB (RG7802) for CEA-positive solid tumors, (D) FAP-DR5 tetravalent CrossMAb RG7386. The black star depicts the P329G LALA (P329G, L235A, L234A) mutations to abolish FcγR and C1q binding, the green star Triple A (I253A, H310A, H435A) mutations to abolish FcRn binding. Constant heavy chain domains are colored in gray, constant light chain domains in white, variable heavy chains are colored uniformly, light chain domains are colored with a line pattern.

Figure 3.

The CrossMAb zoo: Schematic overview about different mono-, bi- and multispecific antibody formats enabled by CrossMAb technology: (A) Heterodimeric/asymmetric trivalent 2+1 IgG CrossMAbs; (B) Symmetric tetravalent 2+2 IgG CrossMAbs; (C) MoAb (MonoMAb) and MoAb-Dimer (DuoMAb; (D) Trispecific Pan-HER family DAF-CrossMAb antibody; (E) Trispecific CrossMAb-VH-VL; (F) Tri-, tetraspecific CrossMAb-scFAb fusions; (G) DVD-CrossMAb; (H) Heterodimeric/asymmetric Kappa-Lambda-CrossMAb; (I) Fc-free Tandem Fab-CrossFab antibody. (J) Fc-free bispecific Fab fusion proteins using alternative fusion partners (green, yellow) based on Fab crossover. KiH technology or alternative heavy chain heterodimerization technologies applied where appropriate and indicated by colors only. The drawings given represent only examples, since in many cases crossed and uncrossed Fabs can be assembled in various ways. Constant heavy chain domains are colored in gray, constant light chain domains in white, variable heavy chains are colored uniformly, light chain domains are colored with a line pattern.

Figure 4.

Design of CrossMAbs with CH1-CL crossover: Top: Typical structures of VH, Vk and Vλ domains and their superposition in the sense that the Cα atoms of the β-sheets adjacent to the elbow region (displayed as spheres in the structures, gray in the sequences) match in space. Middle: the same applied to the CH1, Cκ and Cλ domains. Bottom: wildtype and designed CrossMAb sequences; β-sheets adjacent to the elbow regions are colored gray. The newly designed sequence is shown in red.