Prodrug-Activating Chain Exchange (PACE) converts targeted prodrug derivatives to functional bi- or multispecific antibodies

Abstract

Driven by the potential to broaden the target space of conventional monospecific antibodies, the field of multi-specific antibody derivatives is growing rapidly. The production and screening of these artificial proteins entails a high combinatorial complexity. Antibody-domain exchange was previously shown to be a versatile strategy to produce bispecific antibodies in a robust and efficient manner. Here, we show that the domain exchange reaction to generate hybrid antibodies also functions under physiological conditions. Accordingly, we modified the exchange partners for use in therapeutic applications, in which two inactive prodrugs convert into a product with additional functionalities. We exemplarily show the feasibility for generating active T cell bispecific antibodies from two inactive prodrugs, which per se do not activate T cells alone. The two complementary prodrugs harbor antigen-targeting Fabs and non-functional anti-CD3 Fvs fused to IgG-CH3 domains engineered to drive chain-exchange reactions between them. Importantly, Prodrug-Activating Chain Exchange (PACE) could be an attractive option to conditionally activate therapeutics at the target site. Several examples are provided that demonstrate the efficacy of PACE as a new principle of cancer immunotherapy in vitro and in a human xenograft model.

A pivotal decade for bispecific antibodies?

“…Finally, given the versatility of bsAbs and the potential to mediate completely novel MOAs, the field of bsAbs is poised to see novel emerging approaches and candidates enter the clinic, hopefully providing pivotal data in the years to come, both in oncology and in non-oncology indications, including applications in infection/virology, autoimmunity, metabolism, neurology and ophthalmology. These novel concepts include different approaches as described recently, 5 including the development of: 1) effector cell engagers different from TCEs, engaging, e.g., myeloid, NK or γδ-T cells, 96–98 2) in situ assembly concepts to specifically activate bsAbs on dual target-expressing cells 99 , 100 or in the tumor microenvironment, 101 3) PROTAC-like approaches resulting in internalization and degradation of membrane proteins, 102 4) antibody-based cytokine mimetics to trigger cytokine receptors, 103 , 104 and 5) unique solutions for delivery of bsAbs beyond barriers such as the blood-brain-barrier, 105 which may have applications for the treatment of neurodegenerative and other diseases. 106 …”

Antibody variable region engineering for improving cancer immunotherapy

“…Despite the progress on protease‐activating masking moieties, designing peptide sequences specific to tumor proteases remains challenging. An alternative approach is format chain exchange (ForCE) technology to the CH3 domain of bispecific antibodies, which allows the activation and arm exchange of two inactive probodies only when they are in close proximity in vitro [ 109 ]. Drug‐conjugated probodies against CD71 have entered phase I clinical trials, demonstrating the capability of probodies for pinpointing previously undruggable antigens.…”

The Role of Force Fields and Water Models in Protein Folding and Unfolding Dynamics

“…Due to their high variability in sequence and structure they can fold into a panoply of different structures and fulfill various different roles and functions, e.g., in metabolism, , signal transduction, , immunity, , muscle contraction, , or drug delivery. , Protein-based therapeutics, also known as biologics, are an increasingly important class of drugs, which has been highlighted in various previous studies. − Hence, understanding protein folding, unfolding, and misfolding is an important ongoing field of research. − It is well-known that most proteins can adopt a distinct fold, which is evolutionarily well conserved, even more than their sequence . However, it still remains elusive why a specific sequence adopts a certain fold. , The accurate prediction of protein folding and unfolding dynamics is crucial for numerous fields of research, including drug design, , enzyme engineering, and understanding the molecular basis of diseases. , After all, certain conformational rearrangements, such as misfolds, are expected to be involved in numerous diseases, like Alzheimer’s disease, Huntington disease, Parkinson’s disease, or diabetes type 2. − Additionally, refolding events resulting from point mutations have been shown to elucidate changes in specificity or even polyreactivity, providing new applications in drug design and engineering. − …”