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Bispecific antibodies combine the specificity of two antibodies into one molecule. During the past two decades, advancement in protein engineering enabled the development of more than 100 bispecific formats, three of which are approved by the FDA for clinical use. In parallel to protein engineering methods, advancement in conjugation chemistries have spurred the use of chemical engineering approaches to generate bispecific antibodies. Herein, we review selected chemical strategies employed to generate bispecific antibodies that cannot be made using protein engineering methods., (Copyright © 2021. Published by Elsevier Ltd.)

In recent years, bispecific antibodies (BisAbs) have emerged as novel pharmaceutical candidates owing to their ability to engage two disease mediators simultaneously, thus providing a possible alternative therapeutic approach in complex diseases such as cancer and inflammation. Here we provide an overview of the molecular design, recombinant expression in mammalian cells and purification of BisAbs based on full-length IgG-scFv formats. Practical considerations and strategies to optimize transient expression and purification are also discussed., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.

By simultaneous binding two disease mediators, bispecific antibodies offer the opportunity to broaden the utility of antibody-based therapies. Herein, we describe the design and characterization of Bs4Ab, an innovative and generic bispecific tetravalent antibody platform. The Bs4Ab format comprises a full-length IgG1 monoclonal antibody with a scFv inserted into the hinge domain. The Bs4Ab design demonstrates robust manufacturability as evidenced by MEDI3902, which is currently in clinical development. To further demonstrate the applicability of the Bs4Ab technology, we describe the molecular engineering, biochemical, biophysical, and in vivo characterization of a bispecific tetravalent Bs4Ab that, by simultaneously binding vascular endothelial growth factor and angiopoietin-2, inhibits their function. We also demonstrate that the Bs4Ab platform allows Fc-engineering similar to that achieved with IgG1 antibodies, such as mutations to extend half-life or modulate effector functions.

In recent years, biodistribution analyses of pharmaceutical compounds in preclinical animal models have become an integral part of drug development. Here we report on the use of optical imaging biodistribution analyses in a mouse xenograft model to identify tissues that nonspecifically retained a bispecific antibody under development. Although our bispecific antibody bound both the epidermal growth factor receptor and insulin growth factor 1 receptor are expressed on H358, nonsmall-cell lung carcinoma cells, the fluorescence from labeled bispecific antibody was less intense than expected in xenografted tumors. Imaging analyses of live mice and major organs revealed that the majority of the Alexa Fluor 750 labeled bispecific antibody was sequestered in the liver within 2 h of injection. However, results varied depending on which near-infrared fluorophore was used, and fluorescence from the livers of mice injected with bispecific antibody labeled with Alexa Fluor 680 was less pronounced than those labeled with Alexa Fluor 750. The tissue distribution of control antibodies remained unaffected by label and suggests that the retention of fluorophores in the liver may differ. Given these precautions, these results support the incorporation of optical imaging biodistribution analyses in biotherapeutic development strategies.

Targeting multiple receptors with bispecific antibodies is a novel approach that may prevent the development of resistance to cancer treatments. Despite the initial promise, full clinical benefit of this technology has yet to be realized. We hypothesized that in order to optimally exploit bispecific antibody technology, thorough fundamental knowledge of their pharmacological properties compared to that of single agent combinations was needed. Therefore, we developed a mathematical model for the binding of bispecific antibodies to their targets that accounts for the spatial distribution of the binding receptors and the kinetics of binding, and is scalable for increasing valency. The model provided an adequate description of internal and literature-reported in vitro data on bispecific binding. Simulations of in vitro binding with the model indicated that bispecific antibodies are not always superior in their binding potency to combination of antibodies, and the affinity of bispecific arms must be optimized for maximum binding potency. Our results suggest that this tool can be used for the design and development of the next generation of anti-cancer bispecific compounds.

Widespread drug resistance due to empiric use of broad-spectrum antibiotics has stimulated development of bacteria-specific strategies for prophylaxis and therapy based on modern monoclonal antibody (mAb) technologies. However, single-mechanism mAb approaches have not provided adequate protective activity in the clinic. We constructed multifunctional bispecific antibodies, each conferring three mechanisms of action against the bacterial pathogen Pseudomonas aeruginosa by targeting the serotype-independent type III secretion system (injectisome) virulence factor PcrV and persistence factor Psl exopolysaccharide. A new bispecific antibody platform, BiS4, exhibited superior synergistic protection against P. aeruginosa-induced murine pneumonia compared to parent mAb combinations or other available bispecific antibody structures. BiS4αPa was protective in several mouse infection models against disparate P. aeruginosa strains and unexpectedly further synergized with multiple antibiotic classes even against drug-resistant clinical isolates. In addition to resulting in a multimechanistic clinical candidate (MEDI3902) for the prevention or treatment of P. aeruginosa infections, these antibody studies suggest that multifunctional antibody approaches may be a promising platform for targeting other antibiotic-resistant bacterial pathogens., (Copyright © 2014, American Association for the Advancement of Science.)