Bispecific antibodies and antibody fragments are one of the most rapidly progressing classes of antibody-based therapeutics. Their differentiating functionalities could profit from a third antigen specificity. We have employed symmetrical bispecific parental antibodies of mAb2 format, which features a novel antigen-binding site in the CH3 domains, and engineered them by controlled Fab-arm exchange into biologically active trispecific well-expressing molecules with good biophysical characteristics.

Antibody-domain exchange reactions can be applied to generate hybrid antibodies under physiological conditions, thereby enabling prodrug functionalities. T cell bispecific antibodies (TCBs) can be assembled on target cells from two inactive prodrugs. Prodrug-Activating Chain Exchange (PACE) can thereby conditionally activate therapeutics at the target site. Examples will be provided that demonstrate potential applications of PACE as a new approach in conditional immunotherapy.

Triple-targeting formats hold great therapeutic promise but translation of concepts into active molecules is challenging both in obtaining differentiated functional activity, as well as meeting stringent developability criteria. We discuss the discovery and characterization of the components and final candidates of a trispecific antibody format referred to as Triclonics that permits high-throughput in-format repertoire screening to result in active molecules that harness the developability characteristics of regular human monoclonal antibodies.

We develop trifunctional antibody-fusion proteins composed of a tumor-directed antibody moiety and two different immunomodulatory molecules – common gamma chain receptor cytokines/ costimulatory ligands of the TNF-superfamily. Aiming for improved localization and efficacy at the tumor site, the design focus on targeted presentation and combined mode of action. Molecular properties will be discussed.

The number of combinatorial approaches in the clinic has increased exponentially with PD1/VEGF targeting combos being the most represented. While this combo has shown efficacy, dose-limiting tox is a concern. We have engineered a standard antibody targeting both PD1 & VEGFR2 to maximize potency while minimizing the significant peripheral tox associated with VEGF pathway targeting. Co-crystal structures describe a unique interaction mechanism for this 2-in-1 mAb and co-culture IO model systems suggest potential synergy in dual pathway targeting.

TrYbe is a multi-specific, Fc-free, therapeutic antibody format. The design consideration for this new fragment-based therapeutic format will be discussed, both in terms of the functional biology and the molecular properties. Data from multiple programs will be shared that exemplify a range of functional activities; demonstrate some beneficial properties of target engagement with respect to immune complex formation; show consistent in vivo PK from albumin binding; efficient and high yielding production at manufacturing scale; and long shelf-life stability.

We developed CoV-X2, a fully human, IgG-like bispecific antibody rationally engineered to bind simultaneously to two sites on the SARS-CoV-2 Spike (Nature, 2021); it potently neutralizes all variants of concern, protects from the virus both prophylactically and therapeutically and prevents formation of viral escape mutants in vivo. Bispecifics against Zika (Cell 2017) and Prion also had synergistic properties beyond those of the parental monoclonals. Simultaneous targeting of non-overlapping epitopes by IgG-like bispecific antibodies is feasible and effective against infectious diseases, combining the advantages of antibody cocktails with those of single-molecule approaches.

Biologic drugs, especially anti-TNF, are considered as the gold standard therapy in rheumatoid arthritis. However, non-uniform efficacy, incidence of infections, and high costs are major concerns. Novel tissue-specific agents may overcome the current limitations of systemic administration, providing improved potency, and safety. We developed a bispecific antibody (BsAb), combining human arthritic joint targeting, via the synovial-specific single-chain variable fragment (scFv)-A7 antibody, and TNFa neutralization, via the scFv-anti-TNFa of adalimumab, with the binding/blocking capacity comparable to adalimumab immune globulin G (IgG). Tissue-targeting capacity of the BsAb was confirmed on the human arthritic synovium in vitro and in a synovium xenograft Severe combined immune deficient (SCID) mouse model. Peak graft accumulation occurred at 48 h after injection with sustained levels over adalimumab-IgG for 7 days and increased therapeutic effect, efficiently decreasing tissue cellularity, and markers of inflammation with higher potency compared to the standard treatment. This study provides the first description of a BsAb capable of drug delivery, specifically to the disease tissue, and strong evidence of improved therapeutic effect on the human arthritic synovium, with applications to other existing biologics.