A multivalent antibody assembled from different building blocks using tag/catcher systems: a case study.

The field of therapeutic antibodies and, especially bi- or multispecific antibodies, is growing rapidly. Especially for treating cancers, multispecific antibodies are very promising, as there are multiple pathways involved and multispecific antibodies offer the possibility to interfere at two or more sites. Besides being used as therapeutic, multispecific antibodies can be helpful tools in basic research. However, the design and choice of the most appropriate multispecific antibody format are far from trivial. The generation of multispecific antibodies starts with the generation of antibodies directed against the desired targets and then combining the different antigen-binding sites in one molecule. This is a time-consuming and laborious approach since the most suitable geometry cannot be predicted. The SpyTag technology is based on a split-protein system, where a small peptide of said protein, the SpyTag, can bind to the remaining protein, the SpyCatcher. An irreversible isopeptide bond between the SpyTag and the SpyCatcher is formed. A related Tag-Catcher system is the SnoopTag-SnoopCatcher. These systems offer the opportunity to separately produce proteins fused to the tag-peptides and to the catcher-domains and assemble them in vitro. Our goal was to design and produce different antibody fragments, Fab domains and Fc-containing domains, with different tags and/or catchers as building blocks for the assembly of different multivalent antibodies. We have shown that large multivalent antibodies consisting of up to seven building blocks can be prepared. Binding experiments demonstrated that all binding sites in such a large molecule retained their accessibility to their corresponding antigens.

Pre-clinical development of a novel CD3-CD123 bispecific T-cell engager using cross-over dual-variable domain (CODV) format for acute myeloid leukemia (AML) treatment.

Novel therapies are needed for effective treatment of AML. In the relapsed setting, prognosis is very poor despite salvage treatment with chemotherapy. Evidence suggests that leukemic stem cells (LSCs) cause relapse. The cell surface receptor CD123 is highly expressed in blast cells and LSCs from AML patients and is a potential therapeutic target. CD123 cross-over dual-variable domain T-cell engager (CD123-CODV-TCE) is a bispecific antibody with an innovative format. One arm targets the CD3εδ subunit of T-cell co-receptors on the surface of T cells, while the other targets CD123 on malignant cells, leading to cell-specific cytotoxic activity. Here, we describe the preclinical activity of CD123-CODV-TCE. CD123-CODV-TCE effectively binds to human and cynomolgus monkey CD3 and CD123 and is a highly potent T-cell engager. It mediates T-cell activation and T-cell-directed killing of AML cells in vitro. In vivo, CD123-CODV-TCE suppresses AML tumor growth in leukemia xenograft mouse models, where it achieves an effective half-life of 3.2 days, which is a significantly longer half-life compared to other bispecific antibodies with no associated Fc fragment. The in vitro safety profile is as expected for compounds with similar modes of action. These results suggest that CD123-CODV-TCE may be a promising therapy for patients with relapsed/refractory AML.

Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques.

The development of an effective AIDS vaccine has been challenging because of viral genetic diversity and the difficulty of generating broadly neutralizing antibodies (bnAbs). We engineered trispecific antibodies (Abs) that allow a single molecule to interact with three independent HIV-1 envelope determinants: the CD4 binding site, the membrane-proximal external region (MPER), and the V1V2 glycan site. Trispecific Abs exhibited higher potency and breadth than any previously described single bnAb, showed pharmacokinetics similar to those of human bnAbs, and conferred complete immunity against a mixture of simian-human immunodeficiency viruses (SHIVs) in nonhuman primates, in contrast to single bnAbs. Trispecific Abs thus constitute a platform to engage multiple therapeutic targets through a single protein, and they may be applicable for treatment of diverse diseases, including infections, cancer, and autoimmunity.