A Plug-and-Play Approach for the De Novo Generation of Dually Functionalized Bispecifics

Diseases are multifactorial, with redundancies and synergies between various pathways. However, most of the antibody-based therapeutics on the market interact with only one target, thus limiting their efficacy. The targeting of multiple epitopes could improve the therapeutic index of treatment and counteract mechanisms of resistance. To this effect, a new class of therapeutics has emerged: bispecific antibodies. Bispecific formation using chemical methods is rare and low-yielding and/or requires a large excess of one of the two proteins to avoid homodimerization and heterogeneity. In order for chemically prepared bispecifics to deliver their full potential, high-yielding, modular, and reliable cross-linking technologies are required. Herein, we describe a novel approach not only for the rapid and high-yielding chemical generation of bispecific antibodies from native antibody fragments, but also for the site-specific dual functionalization of the resulting bioconjugates. Based on orthogonal clickable functional groups, this strategy enables the assembly of functionalized bispecifics with controlled loading in a modular and convergent manner

Employing defined bioconjugates to generate chemically functionalised gold nanoparticles for in vitro diagnostic applications

Novel methods for introducing chemical and biological functionality to the surface of gold nanoparticles serve to increase the utility of this class of nanomaterials across a range of applications. To date, methods for functionalising gold surfaces have relied upon uncontrollable non-specific adsorption, bespoke chemical linkers, or non-generalisable protein–protein interactions. Herein we report a versatile method for introducing functionality to gold nanoparticles by exploiting the strong interaction between chemically functionalised bovine serum albumin (f-BSA) and citrate-capped gold nanoparticles (AuNPs). We establish the generalisability of the method by introducing a variety of functionalities to gold nanoparticles using cheap, commercially available chemical linkers. The utility of this approach is further demonstrated through the conjugation of the monoclonal antibody Ontruzant to f-BSA–AuNPs using inverse electron-demand Diels–Alder (iEDDA) click chemistry, a hitherto unexplored chemistry for AuNP–IgG conjugation. Finally, we show that the AuNP–Ontruzant particles generated via f-BSA–AuNPs have a greater affinity for their target in a lateral flow format when compared to conventional physisorption, highlighting the potential of this technology for producing sensitive diagnostic tests

Modular Chemical Construction of IgG-like Mono- and Bispecific Synthetic Antibodies (SynAbs)

In recent years there has been rising interest in the field of protein−protein conjugation, especially related to bispecific antibodies (bsAbs) and their therapeutic applications. These constructs contain two paratopes capable of binding two distinct epitopes on target molecules and are thus able to perform complex biological functions (mechanisms of action) not available to monospecific mAbs. Traditionally these bsAbs have been constructed through protein engineering, but recently chemical methods for their construction have started to (re)emerge. While these have been shown to offer increased modularity, speed, and for some methods even the inherent capacity for further functionalization (e.g., with small molecule cargo), most of these approaches lacked the ability to include a fragment crystallizable (Fc) modality. The Fc component of IgG antibodies offers effector function and increased half-life. Here we report a first-in-class disulfide rebridging and click-chemistry-based method for the generation of Fc-containing, IgG-like mono- and bispecific antibodies. These are in the FcZ-(FabX)-FabY format, i.e., two distinct Fabs and an Fc, potentially all from different antibodies, attached in a homogeneous and covalent manner. We have dubbed these molecules synthetic antibodies (SynAbs). We have constructed a T cellengager (TCE) SynAb, FcCD20-(FabHER2)-FabCD3, and have confirmed that it exhibits the expected biological functions, including the ability to kill HER2+ target cells in a coculture assay with T cells