Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

Abstract

We developed an IgG1 domain-tethering approach to guide the correct assembly of 2 light and 2 heavy chains, derived from 2 different antibodies, to form bispecific monovalent antibodies in IgG1 format. We show here that assembling 2 different light and heavy chains by sequentially connecting them with protease-cleavable polypeptide linkers results in the generation of monovalent bispecific antibodies that have IgG1 sequence, structure and functional properties. This approach was used to generate a bispecific monovalent antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor that: 1) can be produced and purified using standard IgG1 techniques; 2) exhibits stability and structural features comparable to IgG1; 3) binds both targets simultaneously; and 4) has potent anti-tumor activity. Our strategy provides new engineering opportunities for bispecific antibody applications, and, most importantly, overcomes some of the limitations (e.g., half-antibody and homodimer formation, light chains mispairing, multi-step purification), inherent with some of the previously described IgG1-based bispecific monovalent antibodies.

LegoBody: facile generation of bispecific and multi-specific antibodies

“…Given the modular nature of the process, our approach should be applicable to the generation of even higher order of specificities. This marks a major distinction from an approach where the IgGlike bispecific is produced as a single giant polypeptide chain before enzymatic processing 58 , which has production issues due to the sheer size of the molecule and is essentially limited to bispecific only. Given the explosion of information on the molecular mechanism of human diseases and the rapid expansion of the list of potential targets, multi-specific antibodies are likely to become an expanding class of novel therapeutics due to their unique ability to generate synergistic or synthetic interactions among targets, thus uncovering new biology and targeting opportunities.…”

Design and engineering of bispecific antibodies: insights and practical considerations

“…In addition to the assembly methods described above, other strategies more heavily relying on chemical processing have also been explored. One example is expression of an IgG-like bsAb consisting of 4 different polypeptide chains expressed as a single construct where the antibody chains had been connected by linkers that steered chain assembly and could be removed post-expression ( Dimasi et al, 2017 ). BsAb chain association has also been controlled by appending leucine zippers ( Wranik et al, 2012 ) and full-length IgG molecules have been combined through SpyTag/SpyCatcher system ( Mei et al, 2022 ) or using click chemistry ( Wagner et al, 2014 ).…”

Fab-Arm Exchange Combined with Selective Protein A Purification Results in a Platform for Rapid Preparation of Monovalent Bispecific Antibodies Directly from Culture Media

“…One of the main limitations of this technology was improper light chain pairing, which was later remedied with CrossMAb technology, whereby the CH1 region and CL1 region of one arm are swapped [29]. Additional technologies have since been developed for the generation of monovalent bsAbs with properly paired light chains [30], including tethered-variable CLBsIgG (tcBsIgG) technology, which utilizes a (G 4 S) 4 linker between the VL and VH [31], and iMab, an IgG1 domain-tethering approach to guide the correct pairing of 2 light and 2 heavy chains, derived from 2 different antibodies [32]. However, most of these bsAb technologies have structural limitations that prevent their use for high-throughput screening purposes [23].…”